Annual Fall Meeting of GBM Abstracts Symposia Authors Contact
Annual Fall Meeting of GBM Abstracts Symposia Authors Contact
Annual Fall Meeting of GBM Abstracts Symposia Authors Contact
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<strong>Annual</strong> <strong>Fall</strong> <strong>Meeting</strong> <strong>of</strong> <strong>GBM</strong><br />
Münster (Westfalen), September 19-22, 2004<br />
<strong>Abstracts</strong><br />
Complete list <strong>of</strong> abstracts<br />
<strong>Symposia</strong><br />
Cytoskeleton dynamics<br />
Development and Aging <strong>of</strong> the Neuronal Cytoskeleton<br />
ECTS Short contributions<br />
ECTS/GZG: Stem Cells<br />
Endothelial and epithelial barriers<br />
Engineering and application <strong>of</strong> aut<strong>of</strong>luorescent proteins<br />
Extracellular matrices and cell migration<br />
Ion channels and transporters<br />
Key players in RNA-silencing<br />
Mechanisms <strong>of</strong> Cellular Compartimentalization<br />
Molecular recognition mechanism in plant-microbe interactions<br />
New bioanalytical techniques and developments<br />
Nuclear structure and gene expression<br />
Plenary Lectures<br />
Small non coding RNAs<br />
Structural Basis <strong>of</strong> Electron Transport<br />
Structural organization and dynamics <strong>of</strong> protein<br />
_Other (free) topic_<br />
<strong>Authors</strong><br />
Complete list <strong>of</strong> authors<br />
<strong>Contact</strong><br />
<strong>GBM</strong> e.V.<br />
Mörfelder Landstraße 125<br />
60598 Frankfurt am Main<br />
phone: 069 / 660567-0<br />
fax: 069 / 660567-22<br />
mail: info@gbm-online.de<br />
web: www.gbm-online.de<br />
Kongressbüro <strong>GBM</strong> Münster 2004<br />
Universität Münster<br />
Institut für Biochemie<br />
Wilhelm-Klemm-Str.2<br />
D-48149 Münster<br />
Tel: +49 (0)251 83-33200<br />
Fax: +49 (0)251 83-33206<br />
mail: wiesean@uni-muenster.de
<strong>Abstracts</strong> (complete list)<br />
Julian Ollesch, Eva Zobeley, Rudi Glockshuber, Klaus Gerwert<br />
α-β-Transition Studies In Proteins And Peptides With Timeresolved FTIR [poster]<br />
Andreas Böhm, Albert Sickmann<br />
A Clustering Solution for Distributed Data Analysis in Mass Spectrometry (paOla) [poster]<br />
Bernhard Andreas Krenig<br />
A new developed biochemical process in purifikation [poster]<br />
Heike Schäfer, Jean-Pierre Chervet, Christian Bunse, Cornelia Joppich, Helmut E. Meyer, Katrin Marcus<br />
A peptide pre-concentration approach for nano-HPLC to diminish memory effects [poster]<br />
Dorothea Stahl, Walter Sibrowski<br />
A regulatory role <strong>of</strong> IgG2 in IgM-IgG immune complexes <strong>of</strong> normal human plasma [poster]<br />
Dietrich Möbest, Stephan Göttig, Bithiah Grace, Reinhard Henschler<br />
A role for Rho A is indicated in migration and hematopoietic regeneration after bone marrow<br />
transplantation demonstrated by inactivation <strong>of</strong> small GTPases with clostridial toxins [oral<br />
presentation]<br />
Heike Betat, Christiane Rammelt, Georges Martin, Mario Mörl<br />
A Twin Study: Chimeras between Poly(A) Polymerase and CCA enzyme show unexpected<br />
activities [poster]<br />
Stefan Kerscher, Ljuban Grgic, Aurelio Gar<strong>of</strong>ano, Noushin Kashani-Poor, Ulrich Brandt<br />
A yeast genetics approach to study mitochondrial complex I [oral presentation]<br />
Anja Drees, Tanja Eisenblätter, Hans-Joachim Galla<br />
ABC-Transporters at the blood-brain-barrier [poster]<br />
Markus Rüffer, Randy Kurz, Winnie Weigel, Andrée Rothermel, Jürgen Trzewik, Gerhard Artmann, Andrea A. Robitzki<br />
Active cardiomyocytes on collagen gels for biohybrid systems [poster]<br />
Angelika Bondzio, Erwin Reinwald<br />
Adhesion <strong>of</strong> Trypanosoma congolense activates signalling pathways in endothelial cells [poster]<br />
Bettina Krüger, Susanne Mühlich, Thomas Huff, Angela Graness, Margarete Goppelt-Strübe<br />
Alterations in the actin and microtubular network influence CTGF expression in endothelial cells<br />
[poster]<br />
Joerg Stetefeld<br />
Alternative mRNA-splicing in agrin- A tool for structural and functional diversity [oral presentation]<br />
Dennis Fiegen, Lars-Christian Haeusler, Lars Blumenstein, Ulrike Herbrand, Radovan Dvorsky, Ingrid R. Vetter,<br />
Mohammad R. Ahmadian<br />
Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating GTPase [poster]<br />
Sabine Riekenberg, Birte Witjes, Gundula Key, Henning Scholze<br />
Amoebasin, a chagasin-like cysteine proteinase inhibitor in trophozoites Entamoeba histolytica<br />
[poster]<br />
Ingo Neumann, Christoph Hutter, Angelika B. Reske-Kunz, Stefan Burdach, Martin S. Staege<br />
Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma<br />
cells identifies new options for immunotherapy [poster]<br />
Manfred Wuhrer, Carolien A. M. Koeleman, André M. Deelder, Cornelis H. Hokke
Analysis <strong>of</strong> protein glycosylation using normal-phase nano-scale liquid chromatography-mass<br />
spectrometry <strong>of</strong> glycopeptides and oligosaccharides [oral presentation]<br />
Gregor Witte, Claus Urbanke, Ute Curth<br />
Analytical Ultracentrifugation pinpoints the interaction <strong>of</strong> DNA polymerase III and primase with<br />
EcoSSB to its C-terminal region [poster]<br />
Olaf Zschörnig, Christ<strong>of</strong> Gutsche, Matthias Müller, Klaus Arnold<br />
Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing vesicles [oral presentation]<br />
Luitpold Miller, Astrid Tiefenbacher, Laurenz Wurzinger, Manfred Gratzl<br />
Antibody to E-selectin inhibits SCLC cell adhesion to endothelial cells [poster]<br />
Nils Kurlemann, Andreas Liese<br />
Application <strong>of</strong> immobilized benzaldehyde lyase as catalyst in the continuous synthesis <strong>of</strong> a chiral<br />
2-hydroxy ketone [poster]<br />
Daniele Penzo, Valeria Petronilli, Alessia Angelin, Claudia Cusan, Raffaele Colonna, Luca Scorrano, Francesco Pagano,<br />
Maurizio Prato, Fabio Di Lisa, Paolo Bernardi<br />
Arachidonic Acid Mediates Calcium-dependent Apoptosis through the Mitochondrial Pathway [oral<br />
presentation]<br />
Romy Kronstein, Jochen Seebach, Hans Schnittler<br />
Association <strong>of</strong> Eps15 with beta-Catenin in endothelial cells: a novel mechanism how endothelial<br />
cells might regulate barrier function [oral presentation]<br />
Martin Kahms, Corinna Schroth, Julia Fiebach, Jürgen Kuhlmann<br />
Aut<strong>of</strong>luorescent Proteins in Interaction Analysis [oral presentation]<br />
Andreas Böhm, Albert Sickmann<br />
Automatic Synchronizing and self-Updating Protein Sequence Database Management System<br />
[poster]<br />
Angelika Bondzio, Christoph Gabler, Dagmar Beier, Siegfried Risse, Ralf Einspanier<br />
Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia Virus (BLV) expression through<br />
Toll-like receptor 4 [poster]<br />
Rüdiger Adam, Tobias Tenenbaum, Hans-Joachim Galla, Horst Schroten<br />
Bacterial Meningitis: Interactions at the Blood-CSF-Barrier [poster]<br />
Verena Goebeler, Ursula Rescher, Volker Gerke<br />
Biochemical Characterization <strong>of</strong> Annexin A9 and Identification <strong>of</strong> Interacting Proteins [poster]<br />
Antje Schulte, André Schönichen, Nadine Czudnochowski, Matthias Geyer<br />
Biochemical Characterization <strong>of</strong> the Interaction between Cyclin T1 and Hexim1 and its<br />
Competition with the HIV-1 Tat protein [poster]<br />
Silke Steffens, Jens Oldendorf, Lifeng Chi, Günter Haufe, Hans-Joachim Galla<br />
Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and stearicacidethylesters [poster]<br />
Christoph Riethmüller, Joachim Wegener*, Marianne Wilhelmi, Pia Jungmann, Hans Oberleithner<br />
Bradykinin enhances vesicular transport <strong>of</strong> solutes across endothelial cell layers [poster]<br />
Andreas Böhm, Florian Grosse-Coosmann, Albert Sickmann<br />
Calculating theoretical ms spectra for given sequences [poster]<br />
Dyakonov Lev, Galnbek Tatjana, Simonova Alla, Savenko Natalia, Klenovitsky Petr, Ernst Lev, Zinovieva Natalia,<br />
Shevcova Natalia<br />
Caryological characteristics <strong>of</strong> some interspecific hybryd anymal cell cultures [poster]<br />
Cord Brakebusch, Aleksandra Czuchra, Xunwei Wu, Tanja Bosse, Klemens Rottner
Cdc42 is not required for the formation <strong>of</strong> filopodia and lamellipodia, but crucial for the uptake <strong>of</strong><br />
Listeria [oral presentation]<br />
Roland H<strong>of</strong>weber, Gudrun Horn, Hans Robert Kalbitzer<br />
cell free studies on the function <strong>of</strong> cold shock proteins [poster]<br />
John R Masters<br />
Cell line misrepresentation [oral presentation]<br />
Dessy Nikova, Volodymyr Nechyporuk-Zloy, Christian Stock, Albrecht Schwab, Hans Oberleithner, Hermann Schillers<br />
Cell membrane isolation for high resolution AFM imaging [oral presentation]<br />
Peter Fromherz<br />
Cells 'n Chips or Ionics meets Electronics [oral presentation]<br />
Sergey V. Tokalov, Herwig O. Gutzeit, Jutta Ludwig-Müller, Barbara Kind, Alexander Franz, Yvonne Henker<br />
Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism in human cells [poster]<br />
Monika Lichtenauer, Wolfgang E Thasler, Anja Gräbe, Birgit Jahn, Karl-Walter Jauch, Hans-Jürgen Schlitt, Thomas S<br />
Weiss<br />
Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic progenitor cells from surgical<br />
resected human liver tissue [poster]<br />
Alexander Teplyashin, Natalia Tchupikova, Svetlana Sharifullina, Mariya Rostovskaya, Svetlana Korjikova, Igor<br />
Abdrakhmanov, Alexander Dunaev, Sergey Petrin, Valeriy Eremenko, Irina Savchenkova<br />
Characterization <strong>of</strong> human MSC-like cells isolated from bone marrow, adipose tissue, skin and<br />
placenta. [oral presentation]<br />
Judith Austermann, Max Koltzscher, Volker Gerke<br />
Characterization <strong>of</strong> the calcium-regulated S100P-ezrin interaction [poster]<br />
Till Biskup, Uta Joos, Jana Kriegsmann, Christian Heise, Ines Westphal, Götz Pilarczyk, Claus Duschl<br />
Chemically modified glass surfaces for growth <strong>of</strong> culture cells examined by light microscopy.<br />
[poster]<br />
Nour Eddine El Gueddari, Bruno Moerschbacher<br />
Chitosan Activates Resistance Against Pathogens After eXposure (CARAPAX) [poster]<br />
Alexander Prodöhl, Thomas Volkmer, Dirk Schneider<br />
C<strong>of</strong>actor Binding to Membrane Proteins - From the Two-Stage to a Three-Stage Model? [poster]<br />
Susanne Berger, Martina Papadopoulos, Katharina Bonfig, Ulrich Schreiber, Thomas Roitsch<br />
Complex regulation <strong>of</strong> gene expression, photosynthesis and sugar levels by pathogen infection<br />
[oral presentation]<br />
Ernst Bamberg<br />
Conformational dynamics <strong>of</strong> P-type ATPases: Intraprotein signalling and subunit interaction<br />
probed by voltage fluorometry [oral presentation]<br />
Michael Spörner, Thosten Graf, Burkhard König, Hans Robert Kalbitzer<br />
Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour therapy [poster]<br />
Beate Rinner, Harald Greger, Brigitte Brem, Peter Pürstner, Veronika Siegl, Thomas Efferth, Roswitha Pfragner<br />
Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary thyroid carcinoma? [poster]<br />
Oliver Bremm, Andre Remy, Klaus Gerwert<br />
Coupling <strong>of</strong> light-induced electron transfer to proton uptake in photosynthesis [poster]<br />
Thomas Carell, Alexandra Mees, Tobias Klar, Andre P. M. Eker, Lars-Oliver Essen<br />
Crystal structure <strong>of</strong> DNA Photolyase complexed to double stranded DNA [oral presentation]
Roswitha Pfragner, A. Stift*, J. Friedl*, B. Niederle*, M. Gnant*<br />
Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent Advances in Immunotherapy [oral<br />
presentation]<br />
Hameeda Sultana, Angelika.A Noegel<br />
Cyclase associated protein CAP in the regulation <strong>of</strong> the actin cytoskeleton and cell polarity in<br />
Dictyostelium [oral presentation]<br />
Nediljko Budisa<br />
Designing novel classes <strong>of</strong> fluorescent proteins with an expanded genetic code [oral presentation]<br />
Thomas Schulenborg, Heike Schäfer, Christian Bunse, Helmut E. Meyer, Katrin Marcus<br />
Detection <strong>of</strong> phosphorylation sites using a hybrid triple quadrupole/linear ion trap mass<br />
spectrometer [poster]<br />
Monika Kogler-Voigt, Roswitha Pfragner, Sabine Supanz, Christa Nöhammer, Konrad Schauenstein, Karl-Heinz<br />
Preisegger<br />
Detection <strong>of</strong> rare tumor cells in peripheral blood for early identification <strong>of</strong> recurrent disease<br />
[poster]<br />
Wolfgang D. Schmitt, Stephan Ruhla, Steffen Hauptmann<br />
Detection <strong>of</strong> Retrotransposition in Cancer [oral presentation]<br />
Maria Iris Hermanns, Sabine Fuchs, Ron Unger, Charles James Kirkpatrick<br />
Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-Well Screening <strong>of</strong> Barrier<br />
Properties [poster]<br />
Heiko Andresen, Kim Zarse, Carsten Grötzinger, Oliver J. Kreuzer, Marc Birringer, Eva Ehrentreich-Förster, Frank F.<br />
Bier<br />
Development <strong>of</strong> Peptide Chips for Biomedical Applications [poster]<br />
Sabine Fuchs, Ron Unger, Maria Iris Hermanns, Kirsten Peters, Charles James Kirpatrick<br />
Different populations <strong>of</strong> endothelial progenitor cells for applications in tissue engineering [poster]<br />
Stefan Helling, Petra Lutter, Petra Weingarten, Christoph Hüls, Helmut Jonuleit, Edgar Schmitt, Helmut E. Meyer,<br />
Katrin Marcus<br />
Differential analysis <strong>of</strong> T cell membrane proteins [poster]<br />
Tadaomi Takenawa, Shiro Suetsugu, Daisuke Yamazaki<br />
Differential functions <strong>of</strong> WAVE1 and 2 in cell migration and spreading. [oral presentation]<br />
Buzz Baum<br />
Dissecting actin cytoskeletal organization in Drosophila using RNAi [oral presentation]<br />
Wittaya Pimtong, Raimund Wagener, Mats Paulsson, Peter Bruckner, Uwe Hansen<br />
Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage [poster]<br />
Robert Henderson, David Dryden, Michael Edwardson, Michael Waring<br />
DNA, Drugs and Proteins: Insights from the Atomic Force Microscope [oral presentation]<br />
Peter Friedl<br />
Dynamic imaging <strong>of</strong> adhesion receptor and protease function in tumor cell invasion [oral<br />
presentation]<br />
Robin Vetter, Melanie Wagner, Jürgen Kuhlmann<br />
Effects <strong>of</strong> an inhibitor <strong>of</strong> Acyl Protein Thioesterase 1 on Ras-mediated signal transduction [poster]<br />
Andreas Gorschlüter, L.H. Mak *, M. Knoll *, N. Dankbar, C. Sundermeier<br />
Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding Forces between Single Molecules<br />
[poster]
Teodora Nikolova, Jaroslaw Czyz, Alexandra Rolletschek, Przemyslaw Blyszczuk, Jürgen Schuderer, Niels Kuster, Anna<br />
M Wobus<br />
Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and apoptosis-related genes in p53deficient<br />
embryonic stem (ES) cells and ES-derived neural progenitors [poster]<br />
Jürgen Hescheler<br />
Embryonic stem cells [oral presentation]<br />
Albrecht Schwab, Volodymyr Nechyporuk-Zloy, Christian Stock<br />
Endocytic internalization <strong>of</strong> potassium channels in migrating cells [poster]<br />
Sabine Fuchs, Michael Laue, Maria Iris Hermanns, Charles James Kirkpatrick<br />
Endocytosis in an in vitro model <strong>of</strong> the human alveolar epithelial barrier: Uptake <strong>of</strong> GM-1 by<br />
human alveolar epithelial cells in primary culture [poster]<br />
Tatiana Afanasieva, Victoria Jensen, Jochen Seebach, Ute Stroeher, Heinz Feldmann, Hans-J. Schnittler<br />
Endothelial activation and change <strong>of</strong> barrier function by soluble Ebola virus glycoproteins [poster]<br />
Frank Wegmann, Klaus Ebnet, Louis Du Pasquier, Dietmar Vestweber, Stefan Butz<br />
Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and<br />
recruits it to cell contacts [poster]<br />
Markus-N. Kruse, Karim Sabrane, Larissa Fabritz, Ines Veltrup, Melanie Voß, Boris Skryabin, Michaela Kuhn<br />
Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong> atrial natriuretic peptide [poster]<br />
Henrik Brutlach, Sadasivam Jeganathan², Martin von Bergen², Eckhard Mandelkow², Heinz-Juergen Steinh<strong>of</strong>f<br />
EPR investigation on site-directed spin labeled Tau protein [poster]<br />
Enrica Brodignon, Johann Klare, Martin Engelhard, Heinz-Jürgen Steinh<strong>of</strong>f<br />
EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the photo-transducer NpHtrII [poster]<br />
Andrée Rothermel, Randy Kurz, Thomas Biedermann, Markus Rüffer, Winnie Weigel, Andrea Robitzki<br />
Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro [oral presentation]<br />
Mekky Mohammed Abouzied, Heba Mahmoud El-tahir, Volkmar Gieselmann, Sebastian Franken<br />
Evidence for a cell surface receptor <strong>of</strong> HDGF [poster]<br />
Nadja Bilko, Denys Bilko<br />
Ex vivo proliferation and differentiation <strong>of</strong> cord blood cells in gel culture system [poster]<br />
Nediljko Budisa, Prajna Paramita Pal, Sandra Lepthien, Barbara Mulinacchi, Luis Moroder<br />
Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold” class <strong>of</strong> green fluorescent<br />
proteins [poster]<br />
Ziv Reich<br />
Exploring Protein-Binding Mechanisms and Energy Landscapes by Single-Molecule Mechanical<br />
Unbinding Experiments [oral presentation]<br />
Wolfgang Baumeister, Martin Beck, Ariane Briegel, Marek Cyrklaff, Thomas Keil, Julia Kuerner, Vladan Lucic, Ohad<br />
Medalia, Stephan Nickell, Juergen Plitzko<br />
Exploring the inner space <strong>of</strong> cells by cryoelectron tomography [oral presentation]<br />
Claudia Alma Staab, Jan-Anders Nilsson, Zsolt Sarang, Jan-Olov Höög, Roland Grafström<br />
Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in human normal and transfromed oral<br />
keratinocytes [oral presentation]<br />
Gerald Bäuml, Gudrun Horn, Widmar Tanner, Hans Robert Kalbitzer<br />
Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the Saccharomyces cerevisiae cell<br />
adhesion molecule a-agglutinin [poster]
Michael Schnoor, Katrin Stolle, Jürgen Rauterberg, Paul Cullen, Stefan Lorkowski<br />
Expression <strong>of</strong> collagens by human monocyte-derived macrophages [poster]<br />
Johann Gross, Astrid Machulik, Amarjagal Nyamaa, Birgit Mazurek<br />
Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat cochlea [poster]<br />
Angela Magin, Thomas Noll<br />
Feasibility <strong>of</strong> the Use <strong>of</strong> HUVEC for Autologous Co-Culture Expansion <strong>of</strong> HSC from Umbilical Cord<br />
Blood [poster]<br />
Peter Pohl<br />
Fluid transport through epithelial barriers: the importance <strong>of</strong> ion channels and transporters [oral<br />
presentation]<br />
Matthias Gralle, Michelle Botelho, Cristiano Oliveira, Iris Torriani, Sergio Ferreira<br />
Folding and structure <strong>of</strong> the amyloid precursor protein investigated by solution scattering and<br />
spectroscopy [poster]<br />
Jan Anders Nilsson, Claudia A. Staab, Jan-Olov Höög, Roland C. Grafström<br />
Formaldehyde-related toxicity and gene expression in cultured human oral keratinocytes [oral<br />
presentation]<br />
Partha Chakrabarti, Yan Suveyzdis, Alfred Wittingh<strong>of</strong>er, Klaus Gerwert<br />
FTIR on the Rap-RapGAP reaction: GTPase activation without an arginine finger [poster]<br />
Angela Hirtreiter, Luis Figueiredo, Daniel Klunker, Bernd Haas, Debbie Ang*, Dean Naylor, Michael Kerner, Costa<br />
Georgopoulos*, Ulrich Hartl, Manajit Hayer-Hartl<br />
Functional Characterization <strong>of</strong> group I and II chaperonins in the archaeon Methanosarcina mazei<br />
[poster]<br />
Günter Gisselmann, Christian Wetzel, Hanns Hatt<br />
Functional characterization <strong>of</strong> Ih-channel splice variants in insects [poster]<br />
Oliver Schilling, Andreas Vogel*, Brenda Kostelecky, Wolfram Meyer-Klaucke<br />
Functional diversity and metal selectivity <strong>of</strong> proteins sharing the metallo-β-lactamase fold<br />
[poster]<br />
Daniel Hilger, Gunnar Jeschke, Christoph Wegener, Heinz-Jürgen Steinh<strong>of</strong>f, Etana Padan, Heinrich Jung<br />
Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli probed by EPR spectroscopy<br />
[poster]<br />
Nicholas J. Talbot, Martin J. Gilbert, Darren M. Soanes, Madhumita Barooah, Zheng Yi Wang, Sarah Ahmad, Claire<br />
Veneault-Fourrey, Joanna M. Jenkinson, Lucy J. Holcombe, Gurpreet Bhambra<br />
Functional genomics <strong>of</strong> plant infection by the rice blast fungus Magnaporthe grisea [oral<br />
presentation]<br />
Björn Reiss, Andreas Jansh<strong>of</strong>f, Claudia Steinem, Joachim Wegener<br />
Functionalized vesicles as a model for cell adhesion studies [poster]<br />
B. van den Heuvel<br />
Gene hunting and gene cloning in human OXPHOS deficiency [oral presentation]<br />
Cornelia Wiese, Alexandra Rolletschek, Ihor Zahanich#, Jürgen F. Heubach#, Jaroslaw Czyz, Anne Navarrete-Santos+,<br />
Olaf Horstmann*, Kenneth R. Boheler§, Anna M. Wobus<br />
Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell type from adult mouse and human<br />
intestinal epithelium [poster]<br />
Jonas Frisen<br />
Generation <strong>of</strong> neurons from stem cells in the adult brain [oral presentation]<br />
Frank Schnütgen, Silke De-Zolt, Petra Van Sloun, Patricia Ruiz, Thomas Floss, Wolfgang Wurst, Harald von Melchner
Genomewide targeting <strong>of</strong> secreted and transmembrane proteins using the secretory gene trap<br />
U3Ceo [poster]<br />
Lambrini Tontsidou, Konstantinos Bilbilis, Petra Pennekamp, Boris Skryabin, Hans-Joachim Galla, Jürgen Horst, Bernd<br />
Dworniczak<br />
GGT-Cre mice for tissue-specific gene ablation in proximal kidney tubules [poster]<br />
Poonam Balani, Christian Weidenfeller, Carsten Mim*, Christ<strong>of</strong> Grewer*, Thomas Rauen<br />
Glutamate transporter function in synaptic transmission [poster]<br />
Helena Schwaßmann, Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger Seelert, Norbert A. Dencher<br />
H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas reinhardtii [poster]<br />
Christoph Wegener, Anton Savitsky, Mathias Pfeiffer, Igor Grigorev, Klaus Möbius, Heinz-Jürgen Steinh<strong>of</strong>f<br />
High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled bacteriorhodopsin: A study <strong>of</strong> the<br />
polarity and the local pH in the Bacteriorhodopsins proton channel. [poster]<br />
Daniela Villone, Leena Bruckner-Tuderman, Peter Bruckner, Uwe Hansen<br />
Human dermal basement membrane zone: Characterization <strong>of</strong> functional suprastructures [poster]<br />
Roswitha Pfragner, Werner Emberger, Sigrun Sodia, Olaf Reich, Sigrid Regauer<br />
Human mucosa-associated malignant melanomas: cytogenetic and molecular genetic<br />
characterization [poster]<br />
Uwe Schulte, Jörg Oliver Thumfart, Wolfgang Bildl, Hans-Günther Knaus, Claudia Sailer, Matthias Mann, Jens<br />
Andersen, Martin Biniossek, Volker Rohde, Bernd Fakler<br />
Identification and functional characterization <strong>of</strong> protein supercomplexes associated with Kv1.1<br />
potassium channels [poster]<br />
Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger Seelert, Norbert Dencher<br />
Identification <strong>of</strong> integral thylakoid membrane proteins from Chlamydomonas reinhardtii by<br />
peptide mass fingerprinting and MALDI-MS [poster]<br />
Olesya Chayka, Jörg Kintscher, Karl-Heinz Klempnauer<br />
Identification <strong>of</strong> MYB and C/EBP responsive enhancer <strong>of</strong> the chicken mim-1 gene [poster]<br />
Barbara Sitek, Ognjan Apostolov, Kathy Pfeiffer, Kai Stühler, Helmut E. Meyer, Angelika Eggert, Alexander Schramm<br />
Identification <strong>of</strong> proteins differentially expressed upon neurotrophin receptor activation using<br />
DIGE and MALDI-MS [poster]<br />
Frank R. Neumann, M.R. Gartenberg, A. Taddei, T. Laroche, M. B•aszczyk, Susan Gasser<br />
Imaging functional chromosomes: tethers and dynamics in the yeast nucleus [oral presentation]<br />
Jerome Cavaille, Hervé Seitz, Hélène Royo, Shau-Ping Lin *, Neil Youngson *, Anne C. Ferguson-Smith *<br />
Imprinted microRNA genes at the mouse Dlk1-GTl2 domain. [oral presentation]<br />
Marion Jech, Birgit Hutter-Paier, Elisabeth Ingolic, Harald Höger, Elisabeth Grygar, Sepp Porta, Brigitte Poncza,<br />
Manfred Windisch, Roswitha Pfragner<br />
In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines - New Models for Neuroendocrine<br />
Research [oral presentation]<br />
S. Ramponi, A. Grotti, S. Vultaggio, A. Morisetti, G. Alfieri, V. Lorusso<br />
Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial permeability and morphology: an in<br />
vitro model [poster]<br />
Stefan Malcharek, Christian Schachtrup, Friedrich Spener, Hans-Joachim Galla<br />
Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong> H- and E- FABP double knock-out<br />
mice [poster]<br />
Ana Kilic, Ana Velic, Sevdalina Yurukova, Larissa Fabritz, Eberhard Schlatter, Michaela Kuhn
Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in Guanylyl cyclase-A deficient mice<br />
[poster]<br />
Robert Tampe<br />
INs and OUTs <strong>of</strong> Antigens - The Transport Machinery TAP in the Cellular Immune System [oral<br />
presentation]<br />
Udo Heinemann, Konrad Büssow, Uwe Mueller, Patrick Umbach<br />
Insight into the structure <strong>of</strong> human proteins from a structural genomics approach [oral<br />
presentation]<br />
Melina Haupt, Murray Coles, Horst Kessler, Marc Bramkamp, Karlheinz Altendorf<br />
Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC complex, a P-type ATPase, are not driven<br />
by ATP-induced conformational changes [poster]<br />
Norman Kachel, Werner Kremer, Ralph Zahn, Hans Robert Kalbitzer<br />
Intermediate states and implications for the species barrier <strong>of</strong> the human prion protein revealed<br />
by high pressure NMR [poster]<br />
Jaros•aw Czyz, Katarzyna Miekus, Marta Czernik, Jolanta Sroka, Zbigniew Madeja<br />
Interrelation between rat prostate carcinoma cell coupling and motility in the determination <strong>of</strong><br />
their invasiveness and metastatic activity [poster]<br />
Viktoria Kukhtina, Denise Kottwitz, Chris Weise, Ferdinand Hucho<br />
Intracellular domain - terra incognita <strong>of</strong> the nicotinic acetylcholine receptor [poster]<br />
Gaby-Fleur Böl, Regina Brigelius-Flohé<br />
Intracellular trafficking <strong>of</strong> the Interleukin-1 receptor (IL-1RI) associated kinase IRAK depends<br />
on its kinase-activity [poster]<br />
Vincent Lemaitre, Anthony Watts, Wolfgang Fischer<br />
Ion channels with minimalist design – from viruses. [poster]<br />
Iris G. Altug, Harro J. Bouwmeester, Wilfried A. König<br />
Isolation and functional expression <strong>of</strong> cDNAs encoding sesquiterpene synthases, including the<br />
enantiomeric (+)- and (-)-Germacrene D-Synthases from Solidago canadensis L. [poster]<br />
Claus Wasternack, Irene Stenzel, Bettina Hause, Gerd Hause, Otto Miersch<br />
Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong> tomato [poster]<br />
Daniela Rehder, Dietmar Vestweber, Klaus Ebnet<br />
Junctional Adhesion Molecule-A (JAM-A) participates in tight junction formation and the<br />
establishment <strong>of</strong> cell polarity in epithelial cells [poster]<br />
Joachim Wegener, Charles Keese*, Ivar Giaever*<br />
Kinetics <strong>of</strong> cell spreading monitored by electric cell-substrate impedance sensing [oral<br />
presentation]<br />
Peter Becker<br />
Lifting a chromosome - Dosage Compensation in Drosophila [oral presentation]<br />
Mieke Sprangers, Hui Wang, Niklas Feldhahn, Florian Klein, Markus Müschen<br />
Lineage infidelity in pre-B acute lymphoblastic leukemia cells carrying an MLL-AF4 gene<br />
rearrangement [poster]<br />
Lubov Rochlina, Thomas Linn, Reinhard Bretzel, Eugen Kolossov, Juergen Hescheler, Irina Drobinskaya<br />
Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the Murine Embryonic Stem Cell<br />
System [poster]<br />
Randy Kurz, Markus Rüffer, Christin Urban, Andrée Rothermel, Winnie Weigel, Andrea A. Robitzki
Living cells on a chip – the use <strong>of</strong> electric properties for the development <strong>of</strong> a cardiomyocyte<br />
based biosensor [poster]<br />
Alexander Bubikat, Bernd Zetsche, Larissa Fabritz, Axel Gödecke, Michaela Kuhn<br />
Local ANP prevents cardiac remodeling in hypertensive eNOS-deficient mice [poster]<br />
Franz Hillenkamp<br />
MALDI Mass Spectrometry <strong>of</strong> Nucleic Acids [oral presentation]<br />
Alejandro Heredia, C. C. Bui, Ueli Suter, Peter Young, Tilman Schäffer<br />
Mechanical properties <strong>of</strong> myelinated and de-myelinated peripheral axons with the atomic force<br />
microscope [poster]<br />
Marco Schmeer, Thomas Seipp, Sergej Kakorin, Eberhard Neumann<br />
Mechanism for the conductivity changes caused by membrane electroporation <strong>of</strong> CHO cell -<br />
pellets [poster]<br />
Blaga Popova, Markus Kuhlmann, Markus Kaller, Wolfgang Nellen<br />
Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene silencing [oral presentation]<br />
Carsten Kötting, Katrin Beckmann, Marco Blessenohl, Sven Brucker, Partha Chakrabarti, Carolin Eichholz, Angela<br />
Kallenbach, Yan Suveyzdis, Klaus Gerwert<br />
Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved FTIR-Spectroscopy [poster]<br />
Marc Bramkamp, Jeff Errington<br />
Membrane bound proteins <strong>of</strong> the division machinery in bacteria [poster]<br />
Christiane Wiegand, Marco Hagedorn, Harald Jockusch<br />
Misfolded TMV Coat Proteins: New Mutants and Pathogenicity in Plant and Animal Cells [poster]<br />
Erik Hauzman, Annette Staebler, Martin Götte, Kathrin Tausch, Igor B. Buchwalow, Olaf Buchweitz, Ludwig Kiesel,<br />
Robert R. Greb<br />
Molecular analysis <strong>of</strong> the angiogenic status in endometriotic lesions and eutopic endometrium<br />
[poster]<br />
Lars Hemsath, Patricia Stege, Mohammad Reza Ahmadian<br />
Molecular basis <strong>of</strong> Cdc42 recognition by Wiskott-Aldrich Syndrome Proteins [poster]<br />
Anja Bruehl, Arnd Baumann<br />
Molecular characterization <strong>of</strong> rat calcium-activated chloride channels [poster]<br />
Kenji Irie, T. Sakisaka, W. Ikeda, H. Ogita, Yoshimi Takai<br />
Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and polarity [oral presentation]<br />
Tanja Meyer, Christian Schwöppe, Antje von Schaewen<br />
Molecular properties <strong>of</strong> a new G6PD-like is<strong>of</strong>orm from Arabidopsis [poster]<br />
Gabriele Bixel, Stephan Kloep, Stefan Butz, Björn Petri, Britta Engelhardt, Dietmar Vestweber<br />
Mouse CD99 is required for transendothelial migration <strong>of</strong> T cells [poster]<br />
Gabriele Bixel, Björn Petri, Stephan Kloep, Stefan Butz, Engelhardt Britta, Vestweber Dietmar<br />
Mouse CD99 participates in T cell recruitment into inflamed skin [poster]<br />
Oliver Schmidt, Helmut E. Meyer, Katrin Marcus<br />
Multi-dimensional chromatography <strong>of</strong> proteins [poster]<br />
Heinz-Jürgen Steinh<strong>of</strong>f<br />
Multi-frequency EPR reveals the conformational dynamics <strong>of</strong> membrane proteins: colcin A and<br />
the sensory rhodopsin-transducer complex [oral presentation]
Jürgen Alves, Wolfgang Küster, Imke Peters<br />
Mutants <strong>of</strong> the EcoRI Restriction Endonuclease Exhibit Changes <strong>of</strong> Specificity [poster]<br />
Kerstin Brands, Ferda Cevikbas, Frank Echtermeyer<br />
My<strong>of</strong>ibroblast differentiation and TGF-b1 signalling is affected in syndecan-4 deficient fibroblasts<br />
[poster]<br />
Alexandra Rolletschek, Cornelia Wiese, Gabriela Kania, Przemyslaw Blyszczuk, Barbara Steinfarz*, Ihor Zahanich+,<br />
Thomas Braun#, Oliver Brüstle*, Kenneth R. Boheler~, Anna M. Wobus<br />
Nestin-positive progenitor cells generated from adult mouse intestinal epithelium differentiate<br />
into cells expressing neural, hepatic and pancreatic properties [oral presentation]<br />
Markus Rüegg<br />
Neuromuscular diseases: Concepts and new approaches for therapies [oral presentation]<br />
Carlos Dotti<br />
Neuronal membrane cholesterol loss results in low plasmin activity yet higher beta-secretase<br />
cleavage <strong>of</strong> APP [oral presentation]<br />
Sebastian Schrot, Hans-Joachim Galla<br />
Neutrophile Granulocytes Transendothelial Migration in vitro [poster]<br />
Imre Berger, Daniel Fitzgerald, Timothy Richmond<br />
New Baculovirus Expression Tools for Multiprotein Applications [poster]<br />
Mohammad Reza Ahmadian<br />
New insights into structure-function relationships in GTPase-effector interaction [oral<br />
presentation]<br />
Astrid Blume, Andrew J. Benie, Stephan Hinderlich2, Werner Reutter2, Richard R. Schmidt3, Thomas Peters<br />
New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic acid biosynthesis, UDP-Nacetylglucosamine<br />
2-epimerase/N-acetylmannosamine kinase, with different ligands [poster]<br />
M. Tharwat Ghoneim*, A.M. Baraka*, M.M . Matta**, H. Kamal**<br />
New insights into the modulatory role <strong>of</strong> serotonin reuptake inhibitors in myocardial ischemia<br />
reperfusion injury in the rabbit [poster]<br />
Rico Czaja, Marc Struhalla, Katja Höschler, Wolfram Saenger, Norbert Sträter, Uli Hahn<br />
New structural aspects for substrate specificity <strong>of</strong> RNase T1 [oral presentation]<br />
Eva C. Woenne, Cathrine Schmidt, Nick Mirancea, Roswitha Nischt, Neil Smyth, U. Werner, Hans-Jürgen Stark, Norbert<br />
E. Fusenig, M. Gerl, Dirk Breitkreutz<br />
Nidogen-1 or -2 Promote Basement Membrane Formation in Skin -Type 3D-Cocultures [poster]<br />
Ton Bisseling, Rene Geurts, Caroline Franken, Erik Limpens, Patrick Smit<br />
Nod factor perception and transduction [oral presentation]<br />
Timm Schroeder, Hella Kohlh<strong>of</strong>, Nikolaus Rieber, Satomi Nishikawa, Georg Bornkamm, Shin-Ichi Nishikawa, Tasuku<br />
Honjo, Ursula Just<br />
Notch signalling in embryonic and adult hematopoietic stem cells [oral presentation]<br />
Konstantin Lukyanov, Dmitry Chudakov, Vladislav Verkhusha, Mikhail Matz, Dmitry Shagin, Yurii Yanushevich,<br />
Ekaterina Barsova, Sergey Lukyanov<br />
Novel fluorescent proteins: diversity, mutagenesis and applications [oral presentation]<br />
Günter Gisselmann, Hermann Pusch, Hanns Hatt<br />
Novel ligand gated channels in D. melanogaster: GABA-gated cation channels and gating by<br />
multiple neurotransmitters [poster]<br />
Marcia von Zeska Kress Fagundes, Marcela Savoldi, Joel Fernandes, Roy Larson, Maria Helana Goldman, Gustavo<br />
Goldman<br />
NpkA, a cdc2-related kinase from Aspergillus nidulans, interacts with the UvsBATR kinase [poster]
Tamas Kiss<br />
Nuclear organization <strong>of</strong> human modification guide RNA machinery [oral presentation]<br />
Marian Farkasovsky, Peter Herter, Beate Voß, Alfred Wittingh<strong>of</strong>er<br />
Nucleotide binding and filament assembly <strong>of</strong> recombinant yeast septin complexes. [poster]<br />
S. Wings, C. Pullen, T. Boettcher, E. Leistner<br />
Observations on the erratic occurrence <strong>of</strong> maytansinoids in plants and microorganisms [poster]<br />
Frank Möhrlen, Sebastian Gornik, Melanie Mertens, Ina Rattmann, Irene Yiallouros<br />
Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong> metalloproteinases [poster]<br />
Ulrich Sentner, Sebastian Fettig<br />
P1/HcPro: viral suppressors <strong>of</strong> gene silencing in wheat [oral presentation]<br />
Ester Martín-Villar, Maria Marta Yurrita, Carlos Gamallo, Miguel Quintanilla<br />
PA2.26 antigen (T1alpha, podoplanin), a membrane mucin associated with cancer which is<br />
involved in epithelial-mesenchymal transitions [oral presentation]<br />
Tina Jordan, Annett Meyer, Kristin Peter, Patrick Römer, Sebastian Schornack, Ulla Bonas, Thomas Lahaye<br />
Pepper Bs3 and tomato Bs4 - distinct molecular principles for perception <strong>of</strong> highly-related<br />
Xanthomonas AvrBs3 and AvrBs4 proteins [oral presentation]<br />
Josef H. Wissler<br />
Physiologic Roles for Receptors for Advanced Glycosylation End Products [RAGE] <strong>of</strong> Endothelial<br />
Cells: RNA-, Redox- and Metalloregulated Non-Mitogenic Angio-Morphogenesis in Health and<br />
Disease. [poster]<br />
Manfred Schliwa<br />
Portrait <strong>of</strong> a molecular motor [oral presentation]<br />
Ralph Goethe, Katrin Hübner, Loc Phi-van<br />
Possible involvement <strong>of</strong> Ca2+/calmodulin dependent protein kinase II (CaMKII) in lysozyme premRNA<br />
splicing [poster]<br />
Christina Rommel, Jan Endell, Ralf Wehrspohn, Petra Göring, Claudia Steinem, Andreas Jansh<strong>of</strong>f, Hans-Joachim Galla,<br />
Joachim Wegener<br />
Probing transepithelial substrate permeation with sub-cellular lateral resolution. [poster]<br />
Carsten Wermter, Markus Höwel, Vera Hintze, Irene Yiallouros, Walter Stöcker<br />
Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition and Specificity [oral presentation]<br />
Vera Hintze, Markus Höwel, Carsten Wermter, Irene Yiallouros, Walter Stöcker<br />
Procollagen C-proteinase: Substrate recognition by C-terminal domains [poster]<br />
Anna Kantola, Jorma Keski-Oja, Katri Koli<br />
Promoter characterization <strong>of</strong> the latent transforming growth factor (TGF)-β binding protein 3<br />
(LTBP-3) [poster]<br />
Holger Seelert, Ansgar Poetsch, Sascha Rexroth, Norbert A. Dencher, Daniel J. Müller, Jinshu Yu, Werner Kühlbrandt,<br />
Janet Vonck<br />
Properties <strong>of</strong> the proton translocating oligomer in chloroplast FOF1 ATP synthase [poster]<br />
Christian Ungermann<br />
Protein palmitoylation during membrane fusion [oral presentation]<br />
Anke Rattenholl, Stephan Seeliger, Jörg Buddenkotte, Astrid Grevelhörster, Martin Steinh<strong>of</strong>f<br />
Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor in Skin Carcinogenesis [poster]
Steffen Amme, Twan Rutten, Bernhard Schlesier, Michael Melzer, Hans-Peter Mock<br />
Proteome analysis <strong>of</strong> tobacco trichomes [poster]<br />
Roland C. Grafström, Claudia A Staab, Jan Anders Nilsson<br />
Quantitative Genomics <strong>of</strong> Cultured Normal and transformed Human Oral Keratinocytes [oral<br />
presentation]<br />
Romano Hebeler, P. P. Dijkwel, H. E. Meyer, B. Warscheid<br />
Quantitative proteomics for the investigation <strong>of</strong> leaf senescence in Arabidopsis thaliana [poster]<br />
Sonja Hess, Xiaoli Chen, Samuel W. Cushman, Lewis K. Pannell<br />
Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong> adipocytes [oral presentation]<br />
Markus Knop, Elin Aareskjold, Volker Gerke<br />
Rab3d and annexin A2 play a role in regulated secretion <strong>of</strong> vWF but not tPA from human<br />
endothelial cells [poster]<br />
Jochen Seebach, Beata Wojciak-Stothard, Hans-Joachim Schnittler<br />
Rac mediates shear stress induced increase in endothelial barrier-function [poster]<br />
Jost Seibler, Birgit Küter-Luks, Heidrun Kern, Frieder Schwenk<br />
Rapid method for reproducible, shRNA-mediated gene silencing in vivo [oral presentation]<br />
Hermann Schillers, Hans-Georg Koch, Astrid Stumpf, Kerstin Wenners-Epping, Mike Waelte, Dessy Nikova, Hans<br />
Oberleithner<br />
Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis [poster]<br />
Günter Müller, Susanne Wied, Christian Jung, Andrea Schulz, Norbert Tennagels, Wendelin Frick<br />
Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-Talk to the Insulin Signaling<br />
Cascade by the Antidiabetic Drug Glimepiride [poster]<br />
Ulf Schulze Topph<strong>of</strong>f, Patrick Zeni, Hans-Joachim Galla<br />
Regulation <strong>of</strong> matrix metalloproteases and their endogenous inhibitors at the choroid plexus<br />
epithelium in vitro [poster]<br />
Birgit Scharf, Christine Rotter, Rüdiger Schmitt, Maria de Soto<br />
Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for host association are coordinated in<br />
S. meliloti [poster]<br />
Dirk Bald, Toru Hisabori, Georg Groth, Holger Lill<br />
Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and activation <strong>of</strong> F1-ATPase by tentoxin<br />
[poster]<br />
Bernd Lepenies, Bernhard Fleischer, Thomas Jacobs<br />
Regulation <strong>of</strong> T cell responses by BTLA: Functional analysis using a soluble BTLAIg fusion protein<br />
[poster]<br />
Oliver Hantschel, Bhushan Nagar, John Kuriyan, Giulio Superti-Furga<br />
Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases [oral presentation]<br />
Thorsten Hoppe, Giuseppe Cassata, José M. Barral, Wolfdieter Springer, Alex H. Hutagalung, Henry F. Epstein, Ralf<br />
Baumeister<br />
Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a Novel E3/E4-Multiubiquitylation<br />
Complex [poster]<br />
Bas van Steensel, Maartje Vogel, Martin Loden, Elzo de Wit, Frauke Greil, Ben Abbas<br />
Regulatory networks revealed by genomic maps <strong>of</strong> heterochromatin protein binding in flies and<br />
humans [oral presentation]<br />
Alexandre Fedotov, Ivan Ovcharuk<br />
Research <strong>of</strong> the mechanism <strong>of</strong> self-assembly fibronectins in modelling system. [poster]
Vania Braga<br />
Rho GTPases and cell-cell adhesion [oral presentation]<br />
Radovan Dvorsky, Lars Blumenstein, Patricia Stege, Mohhamad Reza Ahmadian<br />
RhoA-p160Rock communication [poster]<br />
Witold Filipowicz, Caroline Artus, Lukasz Jaskiewicz, Fabrice Kolb, Ramesh Pillai, Haidi Zhang<br />
RNAi and microRNA machineries in mammalian cells [oral presentation]<br />
Eva-Maria Mandelkow, J. Biernat, T. Timm, E. Thies, X.-Y. Li, E. Mandelkow<br />
Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite growth and degeneration [oral<br />
presentation]<br />
Pia Wülfing, Martin Götte, Christian Kersting, Joke Tio, Christopher Poremba, Raihanatou Diallo, Werner Böcker, Ludwig<br />
Kiesel<br />
Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis [poster]<br />
Matthias Böcker, Pia Heidenreich, Harald Fuchs, Tilman Schäffer<br />
Scanning ion conductance microscope with shear-force control [poster]<br />
Pia M. Heidenreich, Sebastian Schrot, Hans-Joachim Galla, Harald Fuchs, Tilman E. Schäffer<br />
Scanning Ion Conductance Microscopy - Topographical non-contact imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
[poster]<br />
Keiichi Namba<br />
Self-assembly and switching <strong>of</strong> the bacterial flagellum [oral presentation]<br />
Stefan Kreusch, Heidrun Rhode, Horst Hoppe, Thomas Moore, Renate Bublitz, Joachim Misselwitz, Margarete Schulze,<br />
Gerd Cumme<br />
Separation and analysis <strong>of</strong> native human proteomes using parallel chromatography with<br />
microplates: Alport syndrome versus healthy serum [poster]<br />
Georg Reiser, Mohan Tulapurkar, Theo Hanck<br />
Sequestration and recycling pathway <strong>of</strong> P2Y2 nucleotide receptor: Clathrin-and actin<br />
cytoskeleton-dependent receptor endocytosis, live cell visualization <strong>of</strong> green fluorescent protein<br />
tagged receptor [poster]<br />
Karl Matter<br />
Signalling at tight junctions in the regulation <strong>of</strong> epithelial proliferation and differentiation [oral<br />
presentation]<br />
Jan Scheffer, Yvonne Rolke, Paul Tudzynski<br />
Signalling in early stages <strong>of</strong> pathogenic development <strong>of</strong> Claviceps purpurea. [oral presentation]<br />
Andreas Krieger, Marcel Kamp, Margit Henry, Alexey Pereverzev, Marco Weiergräber, Jürgen Hescheler, Toni Schneider<br />
Signalling through the E-type voltage-gated calcium channel. Identification <strong>of</strong> interaciton<br />
partners. [poster]<br />
Hermann Gaub<br />
Single Molecule Force Spectroscopy- A Bettter Understanding <strong>of</strong> Biomolecules with Newton? [oral<br />
presentation]<br />
Heinrich Hörber<br />
Single molecule mechanics [oral presentation]<br />
Gerhard Schütz, Manuel Mörtelmaier, Mario Brameshuber, Karel Drbal, Hannes Stockinger<br />
Single Molecule Microscopy for the study <strong>of</strong> Living T Cells [oral presentation]<br />
Johannes A. Eble, Ulrike Mayer, Jörg Haier
Snake venom inhibitors <strong>of</strong> collagen- and laminin-binding integrins suppress tumor cell migration<br />
and invasion [poster]<br />
Peter Geyer, Rolf Döker, Xiaodong Zhao, Werner Kremer, Jürgen Kuhlmann, Alfred Wittingh<strong>of</strong>er, Hans Robert Kalbitzer<br />
Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2 and its interaction with Ran [poster]<br />
R. Gerke, M. Schmeer, T. Seipp, U. Pliquett, E. Neumann<br />
Spatial Resolution <strong>of</strong> Gene Expression by Membrane Electroporation <strong>of</strong> CHO Cell Layers [poster]<br />
Gert Schwach, M. Tschemmernegg, E. Schreiner, M. Windisch, R. Pfragner, E. Masliah, E. Rockenstein, E. Ingolic<br />
Stably transfected rat neuronal cell lines expressing alpha-Synuclein GFP Fusion Proteins as an<br />
in vitro model <strong>of</strong> Parkinson's Disease [poster]<br />
Anna M. Wobus<br />
Stammzellforschung: Potenzial, Perspektiven und Probleme [oral presentation]<br />
Bernd Schmeck, Ralph Gross, Phillipe Dje N´Guessan, Andreas C. Hocke, Sven Sven Hammerschmidt, Tim J. Mitchell,<br />
Simone Rosseau, Norbert Suttorp, Stefan Hippenstiel<br />
Streptococcus pneumoniae induced caspase 6-deptendent apoptosis in lung epithelium [poster]<br />
Michael Lammers, Rolf Wagner, Atsushi Shimada, Alfred Wittingh<strong>of</strong>er<br />
Structural and biochemical characterisation <strong>of</strong> the Rho effector mDia [poster]<br />
Claudia Hoemme, Jens Schwamborn, Alexander Antipenko, Dimitar B. Nikolov, Andreas W. Püschel<br />
Structural and functional analysis <strong>of</strong> the axon guidance molecule Sema3A and its receptor<br />
[poster]<br />
Martin Kühn, Lakshmi Pulagam, Denis Duché, Anton Savitsky, Klaus Möbius, Heinz-Juergen Steinh<strong>of</strong>f<br />
Structural Aspects <strong>of</strong> Membrane Bound Colicin A [poster]<br />
Oliver Einsle, Peter MH Kroneck<br />
Structural Basis <strong>of</strong> Denitrification [oral presentation]<br />
Anke Vahrmann, Anna Szkodowska, Christoph Linke, Monika C. M Mueller, Tilly Bakker-Grunwald, Henning Scholze<br />
Structural characterization and localization <strong>of</strong> annexin E1 in trophozoites <strong>of</strong> Giardia lamblia<br />
[poster]<br />
Birgit von Janowsky, Karin Röttgers, Martin Krayl, Wolfgang Voos<br />
Structural properties <strong>of</strong> the substrate protein determine degradation by the mitochondrial AAA+<br />
protease PIM1 [poster]<br />
Daniel Hilger, Jeschke Gunnar, Christoph Wegener, Heinz-Jürgen Steinh<strong>of</strong>f, Heinrich Jung<br />
Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP <strong>of</strong> E. coli: a protein chemical and<br />
EPR spectroscopic analysis [poster]<br />
Tom Rapoport<br />
Structure and function <strong>of</strong> a protein-conducting channel [oral presentation]<br />
Prasad Gajula, Igor Borovykh*, Christian Beier, Peter Gast*, Heinz-Juergen Steinh<strong>of</strong>f<br />
Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled photosynthetic reaction centers from<br />
Rhodobacter sphaeroides based on molecular dynamics simulations and EPR spectroscopy.<br />
[poster]<br />
Evelyn Hollnack, Tanja Eisenblätter, Hans-Joachim Galla<br />
Substrate and inhibitor specificity <strong>of</strong> the brain multidrug resistance protein (BMDP) at he bloodbrain<br />
barrier [poster]<br />
Angela Magin, Heike Partenheimer, Claudia Lange, Michael Lietz, Thomas Noll<br />
Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC [poster]<br />
Rüdiger Hampp, Mika Tarkka, Uwe Nehls
Symbiotic interactions at the root level <strong>of</strong> forest trees: receive, deliver and get protected [oral<br />
presentation]<br />
Ferda Cevikbas, Kerstin Brands, Frank Echtermeyer<br />
Syndecan-4 Deficiency leads to an upregulation <strong>of</strong> syndecan-1 in skin fibroblasts [poster]<br />
Matthias F. Melzig, Philipp Hebestreit<br />
Synergistic action <strong>of</strong> saponins with type I Ribosime-inactivating proteins agrostin and saporin<br />
[poster]<br />
Caroline E. Chwieralski, Ingo Schnurra, Lars Thim, Werner H<strong>of</strong>fmann<br />
Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-2B bronchial epithelial cells<br />
depend on different signaling cascades [poster]<br />
Markus von Nickisch-Rosenegk, Eva Ehrentreich-Förster, Berit Umann, Frank F. Bier<br />
Synthetic and recombinant zinc fingers as a nano-addressable probe to doublestranded DNA<br />
structures [poster]<br />
Christiane Schaffitzel, Imre Berger, Jan Postberg, Jozef Hanes, Hans J. Lipps, Andreas Plückthun<br />
Telomeric Guanine Quadruplex DNA in Ciliate Nuclei [poster]<br />
Thomas E Scholzen<br />
Terminating the stress: proteolytic processing <strong>of</strong> proopiomelanocortin-derived stress and antiinflammatory<br />
hormones by dermal microvascular endothelial cell (EC) extracellular peptidases<br />
[poster]<br />
Christina Schreier, Werner Kremer, Bernd Fakler, Hans Robert Kalbitzer<br />
Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type voltage gated potassium channels<br />
[poster]<br />
Katrin Stolle, Michael Schnoor, Jürgen Rauterberg, Paul Cullen, Stefan Lorkowski<br />
TGF-β1 inducible genes in coronary smooth muscle cells [poster]<br />
Brian Anderton<br />
The ageing neuronal cytoskeleton and neurodegenerative disease [oral presentation]<br />
Claus Kerkh<strong>of</strong>f, Wolfgang Nacken, Malgorzata Czarny, Marie Claire Dagher, Claudia Sopalla, Jacques Doussiere<br />
The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation in intact<br />
cells [poster]<br />
Niklas Feldhahn, Florian Klein, Markus Müschen<br />
The BCR-ABL1 kinase bypasses selection for the expression <strong>of</strong> a pre-B cell receptor in pre-B<br />
acute lymphoblastic leukemia cells [poster]<br />
David Denis S<strong>of</strong>eu Feugaing, Hans Kresse, Martin Götte<br />
The dermatan sulfate proteoglycans Decorin and Biglycan follow partially diverging endocytic<br />
routes [poster]<br />
Jörg Andrä, Sylvie E. Blondelle, Roman Jerala, Guillermo Martinez de Tejada, Michel H. J. Koch, Karl Lohner, Klaus<br />
Brandenburg<br />
The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and endotoxin neutralization [poster]<br />
Frank Dietz, Friedericke Lehmann, Heiko Gäthje, Maija Slaidina, Daniel Daraban, Sörge Kelm<br />
The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new splice variant [poster]<br />
Daniel Braas, Holger Gundelach, Karl-Heinz Klempnauer<br />
The glioma amplified sequence 41 gene (GAS41) is a direct Myb target gene [poster]<br />
Martin Götte, Laura Rossi, Robert Greb, Dorothe Spillmann, Antonia Joussen, Merton Bernfield, Ludwig Kiesel<br />
The heparan sulfate proteoglycan syndecan-1 is a novel regulator <strong>of</strong> leukocyte-endothelial cell<br />
adhesion and leukocyte transmigration [oral presentation]
Christoph Becker-Pauly, Markus-N. Kruse, Daniel Lottaz*, Irene Yiallouros, Hans-Willi Krel**, Erwin E. Sterchi*, Walter<br />
Stöcker<br />
The human Metalloprotease Meprin - A Candidate for epithelial differentiation [poster]<br />
María Jose Gómez-Lechón, Alfonso Serralta, Teresa Donato, Enrique O´Connor, Jose Vicente Castell, Jose Mir<br />
The immunusupresive drug FK506 prevents Fas-induced apoptosis and mitochondrial dysfunction<br />
in human hepatocytes. [poster]<br />
Lutz Hilterhaus, Stefan Malcharek, Hans-Joachim Galla<br />
The Influence <strong>of</strong> Cholesterol and POPE on mono- and multilayers conatining surfactant protein C<br />
[poster]<br />
Ute Klenz, Hans-Joachim Galla<br />
The Influence <strong>of</strong> Membrane Fluidity on Vesicle Insertion in a SP-B or SP-C containing Monolayer<br />
[poster]<br />
Pariya Na Nakorn, Carol Flach, Rich Mendelsohn, Hans-Joachim Galla<br />
The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C17±palm ) on the surface properties <strong>of</strong> lipid<br />
monolayers at the air/water-interface [poster]<br />
S<strong>of</strong>ia Depner, Wibke Schwarzer, Nicole Daum, Jean Kadathukalam, Norbert E. Fusenig, Dirk Breitkreutz<br />
The insulin-receptor signaling complex in epidermal keratinocytes [poster]<br />
Regine Willumeit, Mont Kumpugdee, Sebastian Linser, Thomas Hauss, Sergio S. Funari, Jörg Andrä<br />
The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes: Insights by Neutron Diffraction and<br />
X-ray Scattering [poster]<br />
Raiko Stephan, C. Klämbt, S. Bogdan<br />
The Kette/Abi/Sra-1 complex regulates actin dynamics by inhibting wave and activating wasp<br />
function in Drosophila [poster]<br />
Jürgen Meyer zu Tittingdorf, Sascha Rexroth, Eva Schäfer, Ralf Schlichting, Christoph Giersch, Nobert A. Dencher,<br />
Holger Seelert<br />
The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not affect the stoichiometry <strong>of</strong> its<br />
chloroplast ATP synthase oligomer III [poster]<br />
Hans Schöler, Luca Gentile, James Kehler, Karin Hübner, Michele Boiani<br />
The Oocyte – Embryonic Stem Cell Cycle [oral presentation]<br />
Christina Zechel, Ingrid Koziollek-Drechsler, Ulrike Sattler, Marek Samochocki<br />
The orphan receptor GCNF in neuronal differentiation [poster]<br />
Sven Huelsmann, Christina Hepper, Rolf Reuter<br />
The PDZ-GEF Dizzy regulates cell form and cell adhesion via rap1 and integrins in macrophages<br />
<strong>of</strong> the Drosophila embryo. [poster]<br />
Heike Bruhn, Matthias Leippe<br />
The pore-forming mechanism <strong>of</strong> amoebapore A characterized by mutational analysis [poster]<br />
A. Markert, S. Kucht, J. Gross, M. Ahimsa-Mueller, Y. Hussein, U.* Steiner, E. Leistner<br />
The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline alkaloids in Ipomoea asarifolia<br />
(Convolvulaceae) [poster]<br />
Yanfeng Li, Jan Dudek, Bernard Guiard, Nikolaus Pfanner, Peter Rehling, Wolfgang Voos<br />
The presequence translocase-associated protein import motor <strong>of</strong> mitochondria: Pam16 functions<br />
in an antagonistic manner to Pam18 [poster]<br />
Florian Garczarek, Klaus Gerwert<br />
The Proton Release Mechanism <strong>of</strong> Bacteriorhodopsin’s WT, E204D and E194D revealed with Time-<br />
Resolved Step-Scan and H/D-Exchange FTIR Measurements [poster]
Beat A. Imh<strong>of</strong>, Michel Aurrand-Lions, Chrystelle Lamagna<br />
The role <strong>of</strong> junctional adhesion molecule-C (JAM-C) in angiogenesis and inflammation [oral<br />
presentation]<br />
O. Bergner, C. Brendel, Claudia Koch-Brandt<br />
The role <strong>of</strong> the lipoprotein receptor LRP1 in connective tissue factor (CTGF) induced signal<br />
transduction: Implications for the pathogenesis <strong>of</strong> atherosclerosis [poster]<br />
Jan Barnikow, Ron Tynes, Maik Kindermann, Stefan Steurer, Andreas Brecht<br />
The SNAP-tagTM - a unique tool for covalent labeling and immobilization <strong>of</strong> proteins [poster]<br />
Stefan Harjes, Peter Bayer, Axel Scheidig<br />
The structure <strong>of</strong> human 3'-phosphoadenosine-5'-phosphosulfate synthetase 1 - A molecular<br />
pendulum? [poster]<br />
Kristina Beck, Karl-Heinz Klempnauer<br />
The transcription factor C/EBP&beta triggers phosphorylation <strong>of</strong> the coactivator p300: A new<br />
mechanism <strong>of</strong> cross-talk between transcription factors and coactivators? [oral presentation]<br />
Maret Böhm, Nadja Bitomsky, Karl-Heinz Klempnauer<br />
The transformation supressor Protein Pdcd4 binds to RNA and is involved in the regulation <strong>of</strong> c-<br />
Jun activity. [poster]<br />
Christoph Hutter, Ingo Neumann, Stefan Burdach, Martin S. Staege<br />
The tumor specific chimeric transcription factor EWS-FLI1 down-regulates gene expression<br />
[poster]<br />
Andrea Schulze, Sybille Standera, Elke Bürger, Marjolein Kikkert, Emmanuel Wiertz, Peter-Michael Kloetzel, Michael<br />
Seeger<br />
The ubiquitin domain protein HERP is stabilised upon interaction with a component <strong>of</strong> the<br />
ubiquitin-proteasome system and promotes degradation <strong>of</strong> an ERAD substrate [poster]<br />
Nicole Reifschneider, S. Goto, Norbert A. Dencher, Frank Krause<br />
Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong> Rattus norvegicus studied by<br />
peptide mass fingerprinting [poster]<br />
Peter Hinterdorfer<br />
Topography and Recognition Imaging using AFM [oral presentation]<br />
Henriette Brüggemann, Antje Berken, Alfred Wittingh<strong>of</strong>er<br />
Towards the function <strong>of</strong> plant Rop GTPases in actin reorganisation [poster]<br />
Jana Mooster, Florian Klein, Niklas Feldhahn, Mieke Sprangers, Markus Müschen<br />
Tracing the pre-B to immature B cell transition in human leukemia cells reveals a coordinated<br />
sequence <strong>of</strong> primary and secondary IGK gene rearrangement, IGK deletion and IGL gene<br />
rearrangement [poster]<br />
Krzyszt<strong>of</strong> Wandzik, Katrin Dassler, Matthias Kaup, Hendrik Fuchs<br />
Transferrin receptor expression and shedding during megakaryopoiesis [poster]<br />
Peter Rehling, Agnieszka Chacinska, Maria Lind, Ann E. Frazier, Jan Dudek, Nikolaus Pfanner<br />
Transport <strong>of</strong> nuclear encoded proteins across and into the inner mitochondrial membrane [poster]<br />
Andrea Scrima, Ingrid Vetter, Alfred Wittingh<strong>of</strong>er<br />
TrmE, a GNBP involved in tRNA-modification [poster]<br />
Martin W. Volmer, Yvonne Radacz, Stephan A. Hahn, Susanne Klein-Scory, Kai Stühler, Marc Zapatka, Wolff<br />
Schmiegel, Helmut E. Meyer, Irmgard Schwarte-Waldh<strong>of</strong>f<br />
Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the anti-adhesive invasion-promoting<br />
matricellular protein SPARC [poster]
Heidrun Rhode, M. Schulze, E. Mitre, G.A. Cumme, T. Rausch, D. Philipp, A. Horn<br />
Turbo-mixing in microplates [poster]<br />
Sven Gathmann, Dorothée Walter, Dirk Schneider<br />
Uncovering the mechanisms for protein sorting in cyanobacteria [poster]<br />
Antje Burse, Sabrina Discher, Jürgen Kuhn, Wilhelm Boland<br />
Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in Leaf Beetle Larvae [poster]<br />
Volker Kroehne, Ingo Heschel*, Frank Schügner*, Jörg W. Bartsch, Harald Jockusch<br />
Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for Muscle Cell Differentiation in<br />
Culture and in Grafts [oral presentation]<br />
Masamitsu Futai, Ge-Hong Sun-Wada, Yoh Wada<br />
Vacuolar-type proton pump ATPases (V-ATPases) and inside acidic organelles/compartments:<br />
rotational mechanism and diverse physiological roles. [oral presentation]<br />
Klaus-Peter Vogel, Monique Karl, Heinz-Jürgen Steinh<strong>of</strong>f, Wolfgang H. Ziegler<br />
Vinculin phospholipid interaction studied by EPR utilizing site-directed spin labeling [poster]<br />
Maria Heiser, Birgit Hutter-Paier, Lidia Jerkovic, Michael Becker-Andre, Hans Dieplinger, Roswitha Pfragner, Manfred<br />
Windisch<br />
Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical chicken neurons in vitro [poster]<br />
Marten Wikstrom<br />
Warburg's Atmungsferment: A molecular energy transducer [oral presentation]<br />
Karen Strenge, Paul Crocker, Nicolai Bovin<br />
What are the functions <strong>of</strong> siglecs on human monocytes and macrophages? [poster]<br />
Tatjana M. E. Schwabe, Daniela Schlüsener, Jochen Kruip<br />
Ycf3 from Synechocystis sp. PCC 6803: Localisation, Oligomerisation and Role in Photosystem I<br />
Biogenesis [poster]
Julian Ollesch, Eva Zobeley, Rudi Glockshuber, Klaus Gerwert<br />
α-β-Transition Studies In Proteins And Peptides With<br />
Timeresolved FTIR<br />
FTIR-spectroscopy is a versatile tool to examine the secondary structure <strong>of</strong> a protein<br />
looking upon its amide-I-band, representing the conformationally sensitive backbone<br />
C=O-stretching vibration. An interesting question is how prion propagation takes place.<br />
Understanding the folding mechanism <strong>of</strong> prion protein is a key to understanding prion<br />
diseases such as BSE, CJD, GSS etc. The reduction <strong>of</strong> the full-length prion protein's only<br />
disulfide bond by addition <strong>of</strong> DTT leads to a slow refolding process which was<br />
investigated using time resolved FTIR implementing a new aspect in data analysis.<br />
We present a continuous-flow mixing device, a microchip etched on a silicon wafer [1].<br />
The α-β-refolding process and vice versa <strong>of</strong> β-Lactoglobulin A in different concentrations<br />
<strong>of</strong> 2,2,2-trifluoroethanol (TFE) was observed time resolved. The dead time <strong>of</strong> continuousflow-mixing<br />
could thus be reduced down to approx. 400 µs. The structural changes <strong>of</strong><br />
synthetic peptides during membrane fusion are investigated in solutions <strong>of</strong> different<br />
polarity aiming towards time-resolved measurements <strong>of</strong> conformational changes during<br />
the fusion process.<br />
Literature<br />
1) Kauffmann, E; Darnton, N. C.; Austin, R. H.; Batt, C.; Gerwert, K. (2001) PNAS, (12),<br />
6646-9<br />
contact:<br />
Dipl. Biol. Julian Ollesch<br />
Ruhr-Universität Bochum<br />
LS Biophysik<br />
ollesch@bph.rub.de<br />
Universitätsstr. 150<br />
44780 Bochum (Deutschland)
Andreas Böhm, Albert Sickmann<br />
A Clustering Solution for Distributed Data Analysis in Mass<br />
Spectrometry (paOla)<br />
In mass spectrometry huge amounts <strong>of</strong> data are acquired each day. This data must be<br />
analyzed in less or maximal equal time it is recorded. Otherwise the volume <strong>of</strong> data will<br />
grow over time will need more and more time to be processed. Reducing the processing<br />
time can be achieved by three ways: By applying high-performance hardware or by<br />
increasing the degree <strong>of</strong> parallelism. The third possibility is tuning <strong>of</strong> the used<br />
algorithms, but this can seldom be done in case <strong>of</strong> commercial s<strong>of</strong>tware. We decided to<br />
combine the first two possibilities and designed a clustered computer environment<br />
named "protein analysis on linux architecture" (paOla). This design implements a<br />
queuing architecture with a task submitting interface. The queue is implemented in a<br />
way ensuring every submitted task will have its turn. Using paOla, the analyzing tasks<br />
can be distributed easily and in a transparent way over independent CPUs and machines,<br />
so these tasks do not interfere each other. Actually in our lab the cluster consists <strong>of</strong> four<br />
computers with two CPUs each, allowing a maximum <strong>of</strong> eight analyzing tasks running at<br />
the same time. The job types are configurable, so adding new analyzing algorithms or<br />
even new task types can be done with nearly no effort. The key administration features<br />
<strong>of</strong> the queue like task submission, killing, stopping, viewing progress and status are all<br />
implemented in an easy-to-use web-based service that can be used even by<br />
inexperienced users.<br />
Literature<br />
[1] Boehm, A.M., F. Grosse-Coosmann, and A. Sickmann. Bioinformatics, 2004. in press.<br />
[2] Perkins, D.N., et al. Electrophoresis, 1999. 20(18): p. 3551-3567.<br />
[3] Eng, J.K., A.L. McCormack, and I. Yates, John R. Journal <strong>of</strong> the American Society for<br />
Mass Spectrometry, 1994. 5(11): p. 976-989.<br />
[4] Wall, L., T. Christiansen, and R.L. Schwartz, Programming Perl. 1996: O´Reilly.<br />
[5] Pedrioli, P., The SASHIMI Project. http://sashimi.sourceforge.net/ 2003.<br />
contact:<br />
Andreas Böhm<br />
Universität Würzburg<br />
Rudolf-Virchow-Center for Experimental Biomedicine<br />
andreas.boehm@virchow.uni-wuerzburg.de<br />
Versbacher Str. 9<br />
97078 Würzburg (D)
Bernhard Andreas Krenig<br />
A new developed biochemical process in purifikation<br />
Purification <strong>of</strong> monoclonal antibodies is a key process in biomedical research. One <strong>of</strong> the<br />
most common used techniques to separate proteins is ion exchange chromatography,<br />
but this typically involves time consuming procedures and expensive equipment. The<br />
rapid purification protocol is based on electrodialysis. We developed a<br />
microelectrodyalisis (MED)process to desalt proteins. Usage <strong>of</strong> this tool resulted in very<br />
high purity <strong>of</strong> the proteins as shown by subsequent mass spectrometry. So establisched<br />
MED process with special ion exchange membranes is more efficient than standard<br />
dialysis. Rapid purification periods are needed for handling unstable proteins. Very fast<br />
purification <strong>of</strong> electrolyte ions up to 95%<br />
in a volume <strong>of</strong> 10µl is possible with MED. Purification results are strongly dependend on<br />
membranes quality. We developed membranes with permselectivities up to 95%. The<br />
MED membranes can be used topurify concentrations >1µmol/10µl. So MED is the<br />
preferred method for protein purification. In case <strong>of</strong> too strong membrane fouling<br />
processes, the transport depletion has to be preferred. Both techniques are predominant<br />
compared to the standard dialysis procedure and therefore should be the future for<br />
purification and concentration <strong>of</strong> recombinant proteins.<br />
contact:<br />
Dipl. chem, Bernhard Andreas Krenig<br />
Universitätsklinikum Frankfurt<br />
Virologie<br />
bernhard-andreas.krenig@kgu.de<br />
Theodor-Stern Kai 7<br />
60950 Frankfurt a.M. (Deutschland)
Heike Schäfer, Jean-Pierre Chervet, Christian Bunse, Cornelia Joppich, Helmut E.<br />
Meyer, Katrin Marcus<br />
A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Most LC-based proteomic studies today deal with the analysis <strong>of</strong> peptides rather than<br />
proteins due to their higher solubility in a wide variety <strong>of</strong> solvents and their improved<br />
separation properties. However, the sample complexity <strong>of</strong> digested proteins is<br />
enormously increased and a tremendous number <strong>of</strong> different peptides has to be<br />
separated. To ensure reproducibility and reliability <strong>of</strong> the achieved results, the sample<br />
analysis should be done repeatedly and highly standardised. Memory effects caused by<br />
incomplete peptide elution <strong>of</strong> previous analyses have to be minimised as such artefacts<br />
falsify results.<br />
We developed a method for the analysis <strong>of</strong> complex protein mixtures by nano-reversed<br />
phase HPLC coupled to mass spectrometry. The occurrence <strong>of</strong> memory effects was<br />
prevented by employing a parallel set <strong>of</strong> two pre-columns for simultaneous separation<br />
and washing procedures. The system was tested extensively analysing tryptic digests <strong>of</strong><br />
three single standard proteins. Additional tests using more complex protein mixtures<br />
were done by separation <strong>of</strong> ribosomal proteins.<br />
The described pre-column washing procedure shows several convincing advantages:<br />
• time-saving as no blank gradients are needed.<br />
• more effective than running blank gradients as even highly hydrophobic peptides are<br />
eliminated.<br />
• no memory effect even when huge quantities <strong>of</strong> complex peptide mixtures are<br />
analysed.<br />
With this approach a reliable analysis <strong>of</strong> a complex peptide mixture is possible, ensuring<br />
reproducible results.<br />
contact:<br />
Dipl. Biochem. Heike Schäfer<br />
Ruhr-Universität Bochum<br />
Medizinisches Proteom-Center<br />
heike.schaefer@rub.de<br />
Universitätsstr. 150<br />
44801 Bochum (Deutschland)
Dorothea Stahl, Walter Sibrowski<br />
A regulatory role <strong>of</strong> IgG2 in IgM-IgG immune complexes <strong>of</strong><br />
normal human plasma<br />
Antibodies <strong>of</strong> the isotype IgG2 in humans are associated with the protective immune<br />
response toward carbohydrate antigens in bacterial infections. In the present study, we<br />
identify IgG2 as the predominant IgG subclass in IgM-IgG immune complexes <strong>of</strong> normal<br />
human plasma. Peripheral blood mononuclear cells (PBMNC) obtained from healthy<br />
individuals bind to IgM containing IgM-IgG immune complexes <strong>of</strong> normal human plasma<br />
significantly better than to IgM devoid <strong>of</strong> IgM-IgG immune complexes (p < 0.0293). IgM-<br />
IgG immune complexes containing predominantly IgG2 mediate IgM-PBMNC interaction<br />
more efficiently than IgM-IgG immune complexes containing IgG3, as shown using IgM-<br />
IgG immune complexes derived from plasma <strong>of</strong> patients with warm autoimmune<br />
hemolytic anemia (WAIHA) (p < 0.05). The predominant presence <strong>of</strong> IgG2 in IgM-IgG<br />
immune complexes under physiological conditions defines a so far unrecognized function<br />
<strong>of</strong> IgG2 in the normal immune system <strong>of</strong> humans. Our data suggest an influence <strong>of</strong> IgG2<br />
on the immunoglobulin-mediated regulation <strong>of</strong> activation <strong>of</strong> peripheral blood cells<br />
involved in antigen presentation and processing. The disturbed IgG subclass distribution<br />
in IgM-IgG immune complexes <strong>of</strong> patients with warm autoimmune hemolytic anemia<br />
may influence activation <strong>of</strong> self-reactive B cells in WAIHA.<br />
contact:<br />
Dr. med. Dorothea Stahl<br />
University <strong>of</strong> Muenster<br />
Institute for Transfusion Medicine<br />
stahld@uni-muenster.de<br />
Domagkstrasse 11<br />
48149 Muenster (Germany)
Dietrich Möbest, Stephan Göttig, Bithiah Grace, Reinhard Henschler<br />
A role for Rho A is indicated in migration and hematopoietic<br />
regeneration after bone marrow transplantation demonstrated<br />
by inactivation <strong>of</strong> small GTPases with clostridial toxins<br />
We investigated the effect <strong>of</strong> different clostridial toxins which specifically inhibit Rho<br />
family small GTPases on the migratory behaviour <strong>of</strong> hematopoietic progenitor cells (HPC)<br />
in vitro and after transplantation in mice. C2I-C3, a cell permeable C3 transferase was<br />
used to target Rho, lethal toxin (LT) from C. sordellii to inactivate Rac/Cdc42, and Toxin<br />
B from C. difficile to inactivate Rho, Rac and Cdc42. Whereas inhibition <strong>of</strong> Rho by C2I-C3<br />
resulted in increased migration speed <strong>of</strong> multipotent FDCP-mix and lin- HPC, treatment<br />
with LT and Toxin B which primarily affect Rac/Cdc42 resulted in a loss <strong>of</strong> HPC adhesion<br />
and inhibited migration in vitro. When HPC pretreated with LT or Toxin B were<br />
transplanted into mice, homing <strong>of</strong> HPC to the bone marrow and spleen was impaired,<br />
whereas C2I-C3 treatment did not alter HPC homing. However, in a competitive<br />
repopulation model, HPC pretreated with all inhibitors including C2I-C3 showed inferior<br />
recovery <strong>of</strong> donor hematopoiesis compared with untreated controls. These data show<br />
that clostridial toxins can modulate HPC migration, whereby Rac/Cdc42 inhibition results<br />
in defective adhesion and impaired homing to hematopoietic organs and Rho inactivation<br />
by C2I-C3 increases spontaneous migration but induces a hematopoietic repopulation<br />
defect which is located distal to transendothelial passage.<br />
contact:<br />
Dr. Dietrich Möbest<br />
German Red Cross<br />
Institute <strong>of</strong> Transfusion Medicine and Immune Hematology<br />
moebest@gmx.net<br />
Sandh<strong>of</strong>str. 1<br />
60528 Frankfurt (Deutschland)
Heike Betat, Christiane Rammelt, Georges Martin, Mario Mörl<br />
A Twin Study: Chimeras between Poly(A) Polymerase and CCA<br />
enzyme show unexpected activities<br />
The striking similarity between bacterial poly(A) polymerase (PAP) and tRNA<br />
nucleotidyltransferase (CCA enzyme) is a biological mystery. Based on a conserved Nterminal<br />
catalytic domain, both proteins are members <strong>of</strong> the polymerase b superfamily.<br />
At present, it is not possible to distinguish them by their primary structure - instead, the<br />
identity can only be determined by activity, which differs dramatically: PAP adds<br />
stretches <strong>of</strong> A residues to mRNAs without length restriction, while the CCA enzyme<br />
incorporates a single CCA triplet to the 3’-end <strong>of</strong> tRNA precursors.<br />
Due to the high sequence similarity, it has been speculated that these enzymes not only<br />
have a common ancestor, but may have interconverted during evolution. Using domain<br />
substitution experiments, we show that these enzymes indeed follow a modular concept<br />
with individual domains that function in both CCA enzyme as well as PAP contexts:<br />
replacement <strong>of</strong> N- and C-terminal regions leads to chimeric enzymes with new and<br />
unexpected activities, indicating that the C-terminus <strong>of</strong> tRNA nucleotidyltransferase<br />
carries an “anchor domain” that restricts polymerization to three nucleotides.<br />
We could also identify a region <strong>of</strong> 27 amino acids in the CCA enzyme that converts the<br />
specificity <strong>of</strong> one chimera: although this enzyme carries the catalytic core <strong>of</strong> PAP, it now<br />
acts like a bona-fide tRNA nucleotidyltransferase, adding CCA to the 3’-end <strong>of</strong> tRNAs.<br />
Sequence alignments suggest that the catalytic cores <strong>of</strong> both enzymes carry identical<br />
components involved in recognition and incorporation <strong>of</strong> CTP and ATP. This seems to be<br />
a prerequisite for the observed reprogramming <strong>of</strong> the catalytic center <strong>of</strong> PAP to<br />
incorporate a sequence <strong>of</strong> defined length and composition instead <strong>of</strong> long stretches <strong>of</strong> A<br />
residues.<br />
Literature<br />
contact:<br />
Dr. Heike Betat<br />
MPI Evolutionary Anthropology<br />
betat@eva.mpg.de<br />
Deutscher Platz 6<br />
04103 Leipzig (Germany)
Stefan Kerscher, Ljuban Grgic, Aurelio Gar<strong>of</strong>ano, Noushin Kashani-Poor, Ulrich Brandt<br />
A yeast genetics approach to study mitochondrial complex I<br />
We have established the obligate aerobic yeast Yarrowia lipolytica as a powerful new<br />
model for the structural and functional analysis <strong>of</strong> mitochondrial complex. The Y.<br />
lipolytica enzyme consists <strong>of</strong> about 40 subunits; 37 <strong>of</strong> these could be identified by<br />
molecular cloning and sequencing, and/or MALDI-TOF MS, combined with homology<br />
searches in the recently finished genomic sequence. Attachment <strong>of</strong> a hexa-histidin tag to<br />
the carboxy-terminus <strong>of</strong> the 30 kDa subunit allows highly efficient purification <strong>of</strong> the<br />
multisubunit assembly. Attachment <strong>of</strong> yellow fluorescent protein to the same position<br />
allows in-vivo localization <strong>of</strong> complex within Y. lipolytica mitochondria. Complex is an<br />
essential enzyme in Y. lipolytica. Nevertheless, complex deletion strains could be<br />
generated by attaching the targeting sequence <strong>of</strong> the complex 75 kDa subunit to the<br />
external “alternative” NADH-dehydrogenase (NDH2) present in this yeast. Thereby, we<br />
could complement complex deficiency by redirecting NDH2 to the matrix side. Deletion<br />
strains for several complex I subunits have been constructed and complemented by<br />
shuttle plasmids carrying the deleted gene. This allows for efficient site-directed<br />
mutagenesis <strong>of</strong> individual subunits <strong>of</strong> Y. lipolytica complex I and was used to reconstruct<br />
and characterize several pathogenic mutations in the 49 kDa, 30 kDa, 24 kDa, PSST and<br />
TYKY subunits <strong>of</strong> complex I that cause severe neuromuscular disorders in man. The<br />
effects <strong>of</strong> the 49 kDa and PSST mutations will be discussed in the light <strong>of</strong> a model for the<br />
catalytic core <strong>of</strong> complex I that is based on the known structures <strong>of</strong> water-soluble [NiFe]<br />
hydrogenases.<br />
contact:<br />
Dr. Stefan Kerscher<br />
Universitätsklinikum Frankfurt<br />
Biochemie I<br />
Kerscher@zbc.kgu.de<br />
Theodor-Stern-Kai 7<br />
60590 Frankfurt a. M. (Deutschland)
Anja Drees, Tanja Eisenblätter, Hans-Joachim Galla<br />
ABC-Transporters at the blood-brain-barrier<br />
The blood-brain-barrier (BBB), constituted by endothelial cells <strong>of</strong> brain capillaries, plays<br />
the predominat role in controlling the passage <strong>of</strong> endogenous and xenobiotic substances<br />
between the circulating blood and the extracellular fluid <strong>of</strong> the brain. The presence <strong>of</strong><br />
several specific transporters like P-gp (ABCB1), Mrpl (ABCC1) and ABCG2 (BCRP),<br />
belonging to the family <strong>of</strong> energy dependent ABC-transporters (ATP binding cassettetransporters),<br />
leads to multidrug resitance, crucially determines brain drugs access.<br />
In our cell culture model based on porcine brain capillary endothelial cells (PBCEC),<br />
which form up BBB properties, the expression <strong>of</strong> an ABC-transporter, named BMDP<br />
(brain multidrug resistance protein), was recently shown [1, 2]. Its aa sequence has a<br />
86% identity to human BCRP.<br />
We generated an anti-porcine BMDP antibody to show the apical localisation <strong>of</strong> BMDP. In<br />
western blot analysis <strong>of</strong> membrane fractions a 72 kDa monomer as well as a cross-linked<br />
dimer about 170 kDa could be detected. Comparing BMDP expression levels in different<br />
cerebral cell types low expression was found in pericytes, whereas in actrozytes no<br />
expression was provided.<br />
Additionally, we demonstrated the expression <strong>of</strong> further ABC-transporters, Mrp1, Mrp4<br />
and Mrp5, in PBCEC, which conciliate multidrug resistance. The quantification <strong>of</strong> their<br />
expression level was analyzed by RealTime-PCR in PBCEC and different cell types.<br />
Literature<br />
Eisenblätter, T. and Galla, H.-J. (2002)Biochem Biophys Res Commun 293, 1273-1278<br />
Eisenblätter, T. and Galla, H.-J. (2002)Res 971, 221-231<br />
contact:<br />
Anja Drees<br />
WWU Münster<br />
Institut für Biochemie<br />
dreesanja@gmx.de<br />
Wilhelm-Klemm-Str. 2<br />
48149 Münster (Deutschland)
Markus Rüffer, Randy Kurz, Winnie Weigel, Andrée Rothermel, Jürgen Trzewik,<br />
Gerhard Artmann, Andrea A. Robitzki<br />
Active cardiomyocytes on collagen gels for biohybrid systems<br />
Cardiomyocytes show inherent contractile activity. Under the influence <strong>of</strong> stimulating<br />
agents like angiotensin-II the frequency <strong>of</strong> these contractions rises. Determination <strong>of</strong><br />
contraction frequency can be carried out optically, electrophysiologically (using Multi-<br />
Electrode-Arrays) or by a force sensor. For use with a force sensor cells are cultivated as<br />
a monolayer in a collagen-sandwich that serves as bottom <strong>of</strong> the cultivation vessel.<br />
Changes in tension caused by contracting and relaxing cardiomyocytes are detected by<br />
recording the degree <strong>of</strong> sagging <strong>of</strong> the gel. For use with MEAs a monolayer <strong>of</strong> cells is<br />
cultivated on top <strong>of</strong> a reinforced collagen gel. The gel is placed upside-down on top <strong>of</strong><br />
the electrodes during measurements. Since cells are not grown directly on the MEA the<br />
chips can be reused in shorter intervals. Conventional cell cultivation techniques force<br />
cells to cope with rather artificial conditions. Collagen is a vital component <strong>of</strong> the extracellular<br />
matrix in connective tissues and provides a more natural surface for cells to<br />
adhere to. Being a hydrated gel it can deliver nutrients so that cells can either be<br />
cultivated as a monolayer on top <strong>of</strong> or between layers <strong>of</strong> collagen or even as threedimensional<br />
cultures in the collagen-matrix itself. Cardiomyocytes grown on collagen<br />
gels appear more viable and show coordinated contractile activity at an earlier time<br />
during cultivation than their counterparts grown on standard plasticware. Further studies<br />
to optimize and characterize cardiomyocyte growth on collagen substrates for biosensors<br />
are carried out.<br />
contact:<br />
Dipl. Biologe Markus Rüffer<br />
University <strong>of</strong> Leipzig<br />
Biotechnological Biomedical Center<br />
markus.rueffer@bbz.uni-leipzig.de<br />
Deutscher Platz 5<br />
04103 Leipzig (Germany)
Angelika Bondzio, Erwin Reinwald<br />
Adhesion <strong>of</strong> Trypanosoma congolense activates signalling<br />
pathways in endothelial cells<br />
Contrary to other African trypanosomes Trypanosoma congolense bloodstream forms<br />
specifically adhere to endothelial cells. Nothing is known about the receptors involved<br />
and the biological significance <strong>of</strong> this phenomen. In this study we show that signal<br />
transduction pathways are activated in human umbilical vein endothelial cells (HUVECs)<br />
during trypanosomes adhesion.<br />
Adhesion between Trypanosoma congolense and HUVECs is only transient, with a<br />
maximum effect at 2 hours incubation time. After that the adhesion is declined. In<br />
parallel to the increase and decrease <strong>of</strong> adhesion NF-κB is activated and IL-8 is secreted<br />
into the medium. In addition, HUVECs produce reactive oxygen species (ROS) during<br />
adhesion, as measured with pholasin. Preincubation <strong>of</strong> HUVECs with the antioxidants Nacetyl-L-cysteine<br />
(NAC) and pyrrolidine dithiocarbamate (PDTC) and the NAD(P)H<br />
oxidase inhibitor apocynin completely abolish NF-κB activation, IL-8 secretion and<br />
Trypanosoma congolense adhesion. Thus, either ROS or NF-κB or both may be involved<br />
in the regulation <strong>of</strong> the activity <strong>of</strong> the adhesion receptor.<br />
contact:<br />
Dr.rer.nat. Angelika Bondzio<br />
Freie Universität Berlin<br />
Veterinärbiochemie<br />
dienstlich:bondzio@vetmed.fu-berlin.de ;privat:rbondzio@gmx.de<br />
Oertzenweg 19b<br />
14163 Berlin (Deutschland)
Bettina Krüger, Susanne Mühlich, Thomas Huff, Angela Graness, Margarete Goppelt-<br />
Strübe<br />
Alterations in the actin and microtubular network influence<br />
CTGF expression in endothelial cells<br />
CTGF (connective tissue growth factor) is widely recognized as a fibrogenic factor, but<br />
was originally described as a protein released from serum-stimulated endothelial cells. It<br />
mediates adherence and migration <strong>of</strong> monocytes, vascular smooth muscle cells, and<br />
activated platelets suggesting a functional role in the development <strong>of</strong> atherosclerotic<br />
lesions and plaque rupture. Shear stress was shown to induce CTGF suggesting that<br />
changes in cell morphology and alterations <strong>of</strong> the cytoskeleton influence CTGF<br />
expression.<br />
CTGF expression was investigated in primary cultures <strong>of</strong> HUVEC and in a glomerular<br />
microvascular endothelial cell line. Treatment <strong>of</strong> these cells with colchicine disassembled<br />
the microtubular network, which resulted in RhoA activation and stress fibre formation<br />
accompanied by a dramatic up-regulation <strong>of</strong> CTGF. Inhibition <strong>of</strong> the RhoA-associated<br />
Kinase (ROCK) by Y27632 prevented stress fibre formation and CTGF expression,<br />
indicative <strong>of</strong> a role for RhoA in both processes. Disassembly <strong>of</strong> F-actin by latrunculin B,<br />
which increases G-actin levels, decreased CTGF expression suggesting a reverse<br />
relationship between them. This was supported by incubation <strong>of</strong> endothelial cells with<br />
thymosin beta 4, which induced CTGF expression by sequestering G-actin, or by<br />
yasplakinolide, which reduced G-actin levels by stabilization <strong>of</strong> F-actin.<br />
Disruption <strong>of</strong> microtubules by colchicine was also accompanied by a fragmentation <strong>of</strong> the<br />
golgi apparatus. A comparable staining pattern was also observed, when the cells were<br />
treated with brefeldin A, which directly disrupts golgi transport. However, brefeldin A did<br />
not induce CTGF mRNA and de novo biosynthesis. Taken together our data are in line<br />
with the hypothesis that CTGF expression is related to G-actin levels, thereby linking<br />
CTGF expression and morphological changes <strong>of</strong> endothelial cells.<br />
contact:<br />
Dr. Bettina Krüger<br />
Universität Erlangen-Nürnberg<br />
Nephrologie<br />
bettina.krueger@fd42.de<br />
Loschgestr. 8<br />
91054 Erlangen (Deutschland)
Joerg Stetefeld<br />
Alternative mRNA-splicing in agrin- A tool for structural and<br />
functional diversity<br />
The aggregation <strong>of</strong> acetylcholine receptors on postsynaptic membranes is a key step in<br />
neuromuscular junction development. This process is induced by alternative splicing <strong>of</strong><br />
agrin that include ‘B-inserts’ <strong>of</strong> 8, 11, or 19 residues in the protein’s globular C-terminal<br />
domain, G3. Structures <strong>of</strong> the neural B8 and B11 inserts <strong>of</strong> agrin-G3 were determined by<br />
X-ray crystallography. The structure <strong>of</strong> G3-B0, which lacks inserts, was determined by<br />
NMR. The agrin-G3 domain adopts a b-jellyroll fold. The B-insert site is flanked by four<br />
loops on one edge <strong>of</strong> the b-sandwich, which forms a versatile recognition surface in LNS<br />
proteins. NMR and X-ray data indicate that this interaction interface is flexible, and that<br />
the flexibility is reduced by Ca2+ binding. The plasticity <strong>of</strong> the interaction interface could<br />
enable different splice forms <strong>of</strong> agrin to select between multiple binding partners.<br />
contact:<br />
PD Dr rer nat Joerg Stetefeld<br />
Universität Basel<br />
Bozentrum<br />
joerg.stetefeld@unibas.ch<br />
Kingelbergstr.70<br />
4056 Basel (Swtzerland)
Dennis Fiegen, Lars-Christian Haeusler, Lars Blumenstein, Ulrike Herbrand, Radovan<br />
Dvorsky, Ingrid R. Vetter, Mohammad R. Ahmadian<br />
Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
Rac1b was recently identified in malignant colorectal tumors as an alternative splice<br />
variant <strong>of</strong> Rac1 containing a 19 amino acid insertion next to the switch II region. The<br />
structures <strong>of</strong> Rac1b in the GDP- and the GppNHp-bound form, determined at a resolution<br />
<strong>of</strong> 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly<br />
mobile switch II. As a consequence, Rac1b has an accelerated GEFindependent GDP/GTPexchange<br />
and an impaired GTP-hydrolysis, which is restored partially by GAPs.<br />
Interestingly, Rac1b is able to bind the GTPase-binding domain <strong>of</strong> PAK but not full-length<br />
PAK in a GTP-dependent manner, suggesting that the insertion does not completely<br />
abolish effector interaction. The presented study provides insights into the structural and<br />
biochemical mechanism <strong>of</strong> a selfactivating GTPase.<br />
contact:<br />
Diplom Biochem. Dennis Fiegen<br />
MPI Dortmund<br />
dennis.fiegen@mpi-dortmund.mpg.de<br />
Otto-Hahn Str. 11<br />
44227 Dortmund (Deutschland/NRW)
Sabine Riekenberg, Birte Witjes, Gundula Key, Henning Scholze<br />
Amoebasin, a chagasin-like cysteine proteinase inhibitor in<br />
trophozoites Entamoeba histolytica<br />
Entamoeba histolytica, the causative agent <strong>of</strong> amoebiasis, is equipped with a range <strong>of</strong><br />
cysteine proteinases, some <strong>of</strong> which are supposed to be involved in different aspects <strong>of</strong><br />
pathogenesis. However, little is known about the regulation <strong>of</strong> their enzymatic activity.<br />
We identified a gene encoding a putative cysteine proteinase inhibitor by screening <strong>of</strong><br />
the data base <strong>of</strong> the Entamoeba genome project (TIGR) showing significant homology to<br />
chagasin, a cysteine proteinase inhibitor <strong>of</strong> Trypanosoma cruzi. We amplified the full<br />
coding sequence by polymerase chain reaction from genomic DNA <strong>of</strong> E. histolytica and<br />
overexpressed it in Escherichia coli. The recombinant protein inhibited the protease<br />
activity <strong>of</strong> papain as well as that <strong>of</strong> a trophozoite lysate with equilibrium dissociation<br />
constants, K i , in the picomolar range. Western blot analysis <strong>of</strong> a soluble trophozoite<br />
extract using polyclonal antibodies raised against recombinant protein revealed a single<br />
protein band <strong>of</strong> about 13 kD, suggesting that the inhibitor designated amoebasin is<br />
really expressed in trophozoites <strong>of</strong> E. histolytica. Immunocytochemical localization<br />
studies reveal a granular distribution <strong>of</strong> the protein in the trophozoites. A synthetic<br />
heptapeptide having the sequence GNPTTGF corresponding to a conserved motif <strong>of</strong><br />
chagasin (1) inhibited the protease activity <strong>of</strong> both papain (K i 1.5 mM) and trophozoite<br />
extract (K i in sub-mM range), suggesting that this motif is indeed directly involved in<br />
protease inhibition.<br />
Literature<br />
1. Ridgen, D.J., Monteiro, A.C., and Grossi de Sa, M.F. 2001. The protease inhibitor<br />
chagasin <strong>of</strong> Trypanosoma cruzi adopts an immunoglobulin-type fold and may have arisen<br />
by horizotal gene transfer. FEBS Lett. 267: 13-15<br />
contact:<br />
Sabine Riekenberg<br />
University <strong>of</strong> Osnabrueck<br />
Department <strong>of</strong> Biology/Chemistry<br />
Riekenberg@biologie.uni-osnabrueck.de<br />
Barbarastr. 11<br />
49076 Osnabrueck (Germany)
Ingo Neumann, Christoph Hutter, Angelika B. Reske-Kunz, Stefan Burdach, Martin S.<br />
Staege<br />
Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene<br />
expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma cells identifies new<br />
options for immunotherapy<br />
Retinoids like all-trans retinoic acid (ATRA) have been shown to inhibit proliferation and<br />
induce differentiation <strong>of</strong> different tumor cell types and are currently used clinically for<br />
treatment <strong>of</strong> selected neoplasias including neuroblastoma, the most common malignant<br />
solid tumor in early childhood. We analysed the influence <strong>of</strong> ATRA on proliferation, cell<br />
cycle progression and gene expression in the MYCN-negative neuroblastoma cell line SH-<br />
SY5Y. ATRA treatment inhibited proliferation <strong>of</strong> SH-SY5Y cells in a dose dependent<br />
manner. Cell cycle analysis revealed an ATRA induced arrest in G1. Using high-density<br />
DNA-microarrays (Affymetrix HG-U133A) we analyzed the gene expression pr<strong>of</strong>ile <strong>of</strong><br />
ATRA treated SH-SY5Y cells in comparison to untreated control cells and identified a<br />
great number <strong>of</strong> up- and down-regulated genes. Expression <strong>of</strong> selected genes was<br />
validated using RT-PCR and monoclonal antibodies. Up-regulated genes include several<br />
genes involved in the metabolism <strong>of</strong> ATRA, for instance cytochrome P450RAI1<br />
(CYP26A1) and cytochrome P450RAI2 (CYP26B1). In addition, we observed an increased<br />
expression <strong>of</strong> CD54 (intercellular adhesion molecule 1, ICAM-1). Kinetic analysis showed<br />
a rapid up-regulation <strong>of</strong> CD54 on the surface <strong>of</strong> ATRA stimulated cells. CD54 is involved<br />
in the interaction <strong>of</strong> cytotoxic T cells and NK cells with their target cells. We therefore<br />
tested whether treatment <strong>of</strong> SH-SY5Y cells with ATRA increases the capacity <strong>of</strong> these<br />
cells to serve as targets for cytotoxic T cells. Using an established cytotoxic T cell clone<br />
with known bystander lytic activity (10BK.1) we found that ATRA stimulated SH-SY5Y<br />
cells are more sensitive to bystander lysis then untreated cells. These data suggest that<br />
the combination <strong>of</strong> ATRA with immunotherapy may be an interesting treatment option<br />
for neuroblastoma.<br />
contact:<br />
Diplom-Biochemiker Ingo Neumann<br />
Martin-Luther-Universität Halle-Wittenberg<br />
Forschungslabor für Kinder- und Jugendmedizin<br />
ingo.neumann@medizin.uni-halle.de<br />
Weinbergweg 22<br />
06120 Halle (Saale) (Germany)
Manfred Wuhrer, Carolien A. M. Koeleman, André M. Deelder, Cornelis H. Hokke<br />
Analysis <strong>of</strong> protein glycosylation using normal-phase nanoscale<br />
liquid chromatography-mass spectrometry <strong>of</strong><br />
glycopeptides and oligosaccharides<br />
Reverse-phase nano-scale online-liquid chromatography (LC) electrospray ionization<br />
mass spectrometry (MS) is widely used for the analysis <strong>of</strong> enzymatic digests in<br />
proteomics. We here show that in the normal-phase mode using an amide-column, nano-<br />
LC-MS has a big potential in glycoproteomics. Firstly, oligosaccharides released from<br />
glycoproteins can be analyzed at low femtomole sensitivity with [1] or without [2]<br />
derivatization. Oligosaccharide mixtures are separated over a one-hour range which<br />
permits the detailed characterization <strong>of</strong> the different species by MS-MS. Secondly, (glyco-<br />
)peptides derived from glycoproteins by enzymtic digestion can likewise be analysed by<br />
normal-phase nano-LC-MS, allowing the separation <strong>of</strong> various glyc<strong>of</strong>orms and the<br />
characterization <strong>of</strong> both glycan and peptide moiety by multiple ion selection and<br />
fragmentation steps using ion trap MS. Taken together, normal-phase nano-LC-MS is<br />
useful for the sensitive and quick analysis <strong>of</strong> glycosylation patterns from various<br />
biological samples.<br />
Literature<br />
[1] Wuhrer, M., Koeleman, C. A. M., Hokke, C. H., Deelder, A. M., (2004) Int. J. Mass<br />
Spec., 232, 51-57.<br />
[2] Wuhrer, M., Koeleman, C. A. M., Deelder, A. M., Hokke, C. H. (2004) Anal. Chem.,<br />
76, 873-878.<br />
contact:<br />
Dr Manfred Wuhrer<br />
Leiden University Medical Center<br />
Biomol. Mass Spectrometry Unit<br />
m.wuhrer@lumc.nl<br />
P. O. Box 9600<br />
2300RC Leiden (The Netherlands)
Gregor Witte, Claus Urbanke, Ute Curth<br />
Analytical Ultracentrifugation pinpoints the interaction <strong>of</strong> DNA<br />
polymerase III and primase with EcoSSB to its C-terminal<br />
region<br />
Single-stranded DNA binding (SSB) proteins are involved in DNA replication, repair and<br />
recombination. They bind without sequence specificity to single-stranded DNA (ssDNA)<br />
but not to double-stranded DNA [1]. This results in stabilizing the ssDNA, preventing<br />
hairpin formation and holding it in a suitable conformation for the action <strong>of</strong> other<br />
proteins involved in e.g. DNA replication. The E.coli SSB protein (EcoSSB) is composed<br />
<strong>of</strong> three regions. The DNA-binding region comprises the N-terminal 2/3 <strong>of</strong> the protein<br />
and is succeeded by a flexible, glycine-rich linker. The negatively charged region <strong>of</strong> the<br />
last 10 amino acids <strong>of</strong> EcoSSB is highly conserved among bacterial SSB proteins. Though<br />
this highly conserved C-terminus is not involved in DNA binding it is essential for the<br />
survival <strong>of</strong> the bacterial cell [2].<br />
It has previously been shown that a mutation in this C-terminus weakens the interaction<br />
between EcoSSB and the χ subunit <strong>of</strong> DNA-polymerase III, the main replication enzyme<br />
in E.coli [3]. Using sedimentation velocity experiments we show that a mutant protein<br />
lacking the last 26 amino acids <strong>of</strong> EcoSSB does not interact with χ. Therefore the Cterminus<br />
<strong>of</strong> EcoSSB is essential for the interaction with χ. At low salt concentrations we<br />
demonstrate by analytical ultracentrifugation that the affinity <strong>of</strong> χ towards EcoSSB is<br />
enhanced approximately 20-fold in the presence <strong>of</strong> ssDNA. DNA melting experiments<br />
show that the affinity <strong>of</strong> EcoSSB towards ssDNA is enhanced specifically in the presence<br />
<strong>of</strong> χ. Thus the interaction <strong>of</strong> EcoSSB and χ prevents premature dissociation <strong>of</strong> EcoSSB at<br />
the lagging strand <strong>of</strong> the replication fork, thereby enhancing the processivity <strong>of</strong> DNA<br />
polymerase III [4].<br />
At the lagging strand <strong>of</strong> the replication fork EcoSSB also interacts with primase, thereby<br />
preventing a premature dissociation <strong>of</strong> the RNA primer synthesized by the primase [5].<br />
While the sedimentation constant <strong>of</strong> a complex <strong>of</strong> wild-type EcoSSB and ssDNA is shifted<br />
from 27 S to 39 S by the presence <strong>of</strong> primase, a complex <strong>of</strong> C-terminally shortened<br />
EcoSSB and ssDNA is barely affected. Thus, we can demonstrate for the first time that<br />
the interaction <strong>of</strong> EcoSSB and primase is also mediated by the C-terminus <strong>of</strong> EcoSSB.<br />
Literature<br />
1. Greipel, J., C. Urbanke, and G. Maass. in Protein-Nucleic Acid Interaction, W. Saenger<br />
and U. Heinemann, Editors. 1989, Macmillan: London. p. 61-86.<br />
2. Curth, U., J. Genschel, C. Urbanke, and J. Greipel. Nucleic Acids Res, 1996. 24(14): p.<br />
2706-11.<br />
3. Kelman, Z., A. Yuzhakov, J. Andjelkovic, and M. O'Donnell. Embo J, 1998. 17(8): p.<br />
2436-49.<br />
4. Witte, G., C. Urbanke, and U. Curth. Nucleic Acids Res, 2003. 31(15): p. 4434-40.<br />
5. Yuzhakov, A., Z. Kelman, and M. O'Donnell. Cell, 1999. 96(1): p. 153-63.<br />
contact:<br />
Dr. Ute Curth<br />
Medizinische Hochschule Hannover<br />
Institut für Biophysikalische Chemie<br />
curth.ute@mh-hannover.de<br />
Carl-Neuberg-Str. 1<br />
30625 Hannover (Germany)
Olaf Zschörnig, Christ<strong>of</strong> Gutsche, Matthias Müller, Klaus Arnold<br />
Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing<br />
vesicles<br />
Annexin II belongs to a class <strong>of</strong> Ca2+-binding proteins for which different functions in<br />
the cell have proposed, e. g. involvement in exocytosis and in the coagulation process.<br />
All these functions are related to the ability <strong>of</strong> the annexins to bind to acidic<br />
phospholipids. In this study the interaction <strong>of</strong> monomeric and tetrameric annexin II with<br />
small and large unilamellar vesicles (SUV, respectively LUV) prepared from<br />
phosphatidylserine (PS) or from phosphatidic acid (PA) was investigated at neutral and<br />
acidic pH. Annexin II strongly binds to either lipid at acidic pH, whereas at neutral pH<br />
only weak binding to pure PA and no binding to pure PS LUV occured. Addition <strong>of</strong> 40 µM<br />
Ca2+ leads to a strong binding to the lipids at neutral pH as shown by fluorescence<br />
resonance energy transfer from Trp 212 to pyrene PC. Using fluorescence correlation<br />
spectroscopy (FCS) tetramethylrhodamine labeled tetrameric annexin II binding to PC<br />
LUV containing 20 mol% PS, PA, lyso-bis-PA, PI, PIP, PIP2 was measured in the<br />
presence <strong>of</strong> µM Ca2+ concentrations. The binding is regulated by the different electric<br />
charge <strong>of</strong> the protein below and above its isoelectric point and the netto charge <strong>of</strong><br />
different phospholipids. As shown for other annexins, the binding <strong>of</strong> annexin II induces<br />
dehydration <strong>of</strong> the phospholipid vesicle surface and a decrease <strong>of</strong> the phospholipid<br />
lateral diffusion within the bilayer. The strength <strong>of</strong> both effects is much greater at pH 4<br />
than at pH 7.4. Monomeric Annexin II promotes the Ca2+-induced fusion <strong>of</strong> PA LUV by<br />
diminishing the Ca2+ threshold concentration at pH 7.4. Fusion <strong>of</strong> PS LUV is not affected<br />
by monomeric annexin II at pH 7.4. At pH 4 annexin II induces membrane fusion <strong>of</strong><br />
either vesicles without Ca2+. Despite the low binding extents at neutral pH annexin II<br />
induces a Ca2+ independent leakage <strong>of</strong> PS- / PA- LUV. The leakage extent is increased<br />
at acidic pH.<br />
contact:<br />
Dr. Olaf Zschörnig<br />
University <strong>of</strong> Leipzig<br />
Institute for Medical Physics and Biophysics<br />
zsco@medizin.uni-leipzig.de<br />
Liebigstr. 27<br />
04103 Leipzig (Germany)
Luitpold Miller, Astrid Tiefenbacher, Laurenz Wurzinger, Manfred Gratzl<br />
Antibody to E-selectin inhibits SCLC cell adhesion to<br />
endothelial cells<br />
Introduction: The attachment <strong>of</strong> tumour cells to endothelial cells is an important step<br />
during cell evasion <strong>of</strong> tumour cells. We hypothesize that the cell adhesion molecule Eselectin<br />
is involved in this process. We investigated the expression <strong>of</strong> E-selectin on the<br />
surface <strong>of</strong> human endothelial cells and examined whether E-selectin is involved in the<br />
adhesion <strong>of</strong> small cell lung carcinoma (SCLC) cells to human endothelial cell monolayers.<br />
Methods: Human umbilical vein endothelial cells (HUVEC) were isolated and then<br />
cultured for two passages. HUVECs were seeded into 6-well plates and grown to a<br />
confluent monolayer. The endothelial monolayer was stimulated with TNF (10ng/ml) to<br />
increase E-selectin expression. Two types <strong>of</strong> SCLC cells (H 24, H 69) were fluorescently<br />
labelled with Quantum Dots QTracker (Quantum Dots, California, USA) two days prior<br />
to the adhesion experiments. Approximately 50.000 labelled SCLC cells were added to<br />
each <strong>of</strong> the wells containing the endothelial monolayer. After stringent wash steps,<br />
adherent SCLC cells in each well were counted in 15 fields <strong>of</strong> view and expressed as<br />
mean number <strong>of</strong> cells per high power field <strong>of</strong> view (hpf). E-selectin mediated adhesion<br />
was investigated through the addition <strong>of</strong> a monoclonal E-selectin blocking antibody<br />
(2µg/ml).<br />
Results: Immunhistochemical stainings showed that stimulation <strong>of</strong> HUVEC monolayers<br />
with TNF resulted in an increased expression <strong>of</strong> E-selectin. H 69 SCLC cell adhesion<br />
increased from 6.5(± 0.4) cells/hpf on monolayers without TNF stimulation to 26.4 (±<br />
0.7, p
Nils Kurlemann, Andreas Liese<br />
Application <strong>of</strong> immobilized benzaldehyde lyase as catalyst in<br />
the continuous synthesis <strong>of</strong> a chiral 2-hydroxy ketone<br />
Enantiopure 2?hydroxy ketones are important building blocks in the synthesis <strong>of</strong><br />
biologically active compounds. Both (R)?2-hydroxy-1?phenyl-propanone ((R)?HPP) and<br />
its derivatives are applied as pharmaceutical intermediates. An efficient approach is the<br />
stereospecific condensation <strong>of</strong> aldehydes catalyzed by the thiamine diphosphate (ThDP)dependent<br />
enzymes benzaldehyde lyase and benzoyl formate decarboxylase yielding<br />
enantio-complementary 2?hydroxy ketones. Hexahistidine-tagged benzaldehyde lyase<br />
from E. coli SG13009 / BALHIS was immobilized by means <strong>of</strong> metal ion affinity binding<br />
to a nickel(II)-nitrilotriacetic acid derivatized carrier. The binding is reversible and allows<br />
a regeneration <strong>of</strong> the solid carrier from exhausted enzyme. The immobilized lyase was<br />
applied as heterogeneous biocatalyst in the synthesis <strong>of</strong> (R)-2?hydroxy-1?phenylpropanone<br />
out <strong>of</strong> benzaldehyde and acetaldehyde. The applicability <strong>of</strong> the immobilization<br />
by metal ion affinity binding was proven in repetitive batch reactions and in a<br />
continuously operated plug flow reactor.<br />
Literature<br />
Dünkelmann, P. et al. Journal <strong>of</strong> the American Chemical Society 2002, 124, 12084-<br />
12085.<br />
Kihumbu, D.; Stillger, T.; Hummel, W.; Liese, A. Tetrahedron: Asymmetry 2002, 13,<br />
1069-1072.<br />
Demir, A. S. et al. Advanced Synthesis and Catalysis 2002, 344, (1), 96-103.<br />
Müller, T. and Bahrmann, H. Journal <strong>of</strong> Molecular Catalysis: Chemical 1997, 116, 39-42.<br />
Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations. Weinheim: Wiley-<br />
VCH: 2000.<br />
Zhang, K. Z. and Cass, A. E. G. Analytical Biochemistry 2001, 292, 307-310.<br />
Nahalka, J.; Liu, Z.; Chen, X.; Wang, P. G., Chem. Eur. J. 2003, 2, 372-377<br />
Iding, H. et al. Chemistry 2000, 6, 1483-1495.<br />
contact:<br />
Nils Kurlemann<br />
Universität Münster<br />
Biochemin<br />
nkurlemann@web.de<br />
Wilhelm-Klemm-Str. 2<br />
48149 Münster (Deutschland)
Daniele Penzo, Valeria Petronilli, Alessia Angelin, Claudia Cusan, Raffaele Colonna, Luca<br />
Scorrano, Francesco Pagano, Maurizio Prato, Fabio Di Lisa, Paolo Bernardi<br />
Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Mitochondria are central to apoptotic Ca2+ signalling. A key switch between cell survival<br />
and death is the mitochondrial Permeability Transition Pore (PTP), a high conductance<br />
channel that may open in response to mitochondrial Ca2+ uptake (1). Addition <strong>of</strong> the<br />
divalent cation ionophore A23187 should mimic the cellular Ca2+ overload taking place<br />
under pathological conditions, yet we found that addition <strong>of</strong> A23187 caused death <strong>of</strong><br />
about 30% <strong>of</strong> cells, which is inconsistent with the expected consequences <strong>of</strong> cellular<br />
Ca2+ overload (2). Here we will show that addition <strong>of</strong> A23187 causes a fast but only<br />
transient rise <strong>of</strong> [Ca2+]c, which is sequentially followed by a rapid increase <strong>of</strong> cPLA2<br />
activity and then by PTP opening, the latter event occurring when [Ca2+]c had already<br />
returned to nearly basal levels. We identified the proapoptotic signal triggered by<br />
A23187 as free arachidonic acid, whose levels could be further increased by treatment<br />
with the lipoxygenase inhibitor MK886 plus the cyclooxygenase inhibitor indomethacin.<br />
Cell death was preceded by cytochrome c release, cleavage <strong>of</strong> caspase 9 and 3 but not<br />
<strong>of</strong> caspase 8, all these events being prevented by the PLA2 inhibitor aristolochic acid and<br />
by the PTP inhibitor cyclosporin A (3). Thus, A23187 triggers the apoptotic cascade<br />
through the Ca2+-dependent release <strong>of</strong> arachidonic acid by cPLA2 in a process that is<br />
amplified when transformation <strong>of</strong> arachidonic acid into prostaglandins and leukotrienes is<br />
inhibited. These findings identify cPLA2 as the causal link between A23187-dependent<br />
perturbation <strong>of</strong> Ca2+ homeostasis and the effector mechanisms <strong>of</strong> cell death. The role <strong>of</strong><br />
arachidonic acid and <strong>of</strong> PTP opening in relevant models <strong>of</strong> apoptosis in vivo is being<br />
actively investigated.<br />
Literature<br />
1. Bernardi P (1999) Physiol Rev 79:1127-1155<br />
2. Petronilli V et al. (2001) J Biol Chem 276:12030-12034<br />
3. Penzo D et al. (2004) J Biol Chem 279:25219-25225<br />
contact:<br />
Pr<strong>of</strong>essor Paolo Bernardi<br />
University <strong>of</strong> Padova<br />
Department <strong>of</strong> Biomedical Sciences<br />
bernardi@bio.unipd.it<br />
Viale Giuseppe Colombo 3<br />
35121 Padova (Italy)
Romy Kronstein, Jochen Seebach, Hans Schnittler<br />
Association <strong>of</strong> Eps15 with beta-Catenin in endothelial cells: a<br />
novel mechanism how endothelial cells might regulate barrier<br />
function<br />
Eps15 is a protein that is important in regulation <strong>of</strong> endocytic processes in epithelial<br />
cells. The function <strong>of</strong> eps15 in endothelium is not elucidated yet. We characterized the<br />
expression <strong>of</strong> Eps15 in endothelial cells by Western blotting and RT-PCR.<br />
Immunochemical staining showed localisation <strong>of</strong> the protein in a punctuated pattern<br />
within the cytoplasm and at the junctions. We found for the first time, that Eps15 coprecipitates<br />
with b-catenin, a crucial structural and regulatory molecule <strong>of</strong> the junctional<br />
VE-cadherin/catenin complex. To further characterize the Eps15/b-catenin-association<br />
we used the inflammatory mediator thrombin. Within 2 minutes <strong>of</strong> thrombin stimulation<br />
the endothelial barrier function increased and that was accompanied by the strong<br />
recruitment <strong>of</strong> Eps15 to the junctions. At this time point the fraction <strong>of</strong> b-catenin<br />
associated with Eps15 remained unchanged. 20 to 30 minutes after thrombin treatment<br />
cells dissociate from each other and both Eps15 and a fraction <strong>of</strong> b-catenin translocated<br />
into the cytosol. At these time points Eps15-associated b-catenin increased 2-3 fold.<br />
Interestingly, both Eps15 and Eps15-bound b-catenin were not tyrosine phosphorylated.<br />
These results suggest that endocytosis <strong>of</strong> b-catenin is mediated by Eps15. This<br />
association can provide an alternative mechanism <strong>of</strong> decreasing endothelial cell-cell<br />
adhesion and, correspondingly, barrier function in vascular endothelial cells.<br />
contact:<br />
dipl.-biol. Romy Kronstein<br />
Medizinische Fakultät der TU-Dresden<br />
Institut für Physiologie<br />
romy.kronstein@gmx.net<br />
Fetscherstr. 74<br />
01307 Dresden (Deutschland)
Martin Kahms, Corinna Schroth, Julia Fiebach, Jürgen Kuhlmann<br />
Aut<strong>of</strong>luorescent Proteins in Interaction Analysis<br />
Molecular engineering <strong>of</strong> the Green Fluorescent Protein (GFP) has produced several<br />
variants with altered spectral characteristics. These variants allow not only simultaneous<br />
visualisation <strong>of</strong> the distribution <strong>of</strong> different proteins in the living cell but also interaction<br />
analysis via Fluorescence Resonance Energy Transfer (FRET) [1]. This technique is<br />
widely used in cell biology but has been hardly investigated in vitro using recombinant<br />
fusion proteins. Taking the interaction <strong>of</strong> the small GTPase Ras with the Ras Binding<br />
Domain <strong>of</strong> the Raf kinase (RafRBD) as a model interaction [2] we have investigated the<br />
potential and drawbacks <strong>of</strong> conventional FRET analysis using purified recombinant<br />
CFP/YFP fusion proteins.<br />
These results are compared with a bimolecular fluorescence complementation assay.<br />
This approach is based on complementation <strong>of</strong> two non-fluorescent fragments <strong>of</strong> the<br />
Yellow Fluorescent Protein (YFP) when they are brought together by interactions<br />
between proteins fused to each fragment [3].<br />
Literature<br />
1. Erijman and Jovin Nature Cell Biology 21, 1385-1397 (2003)<br />
2. Herrmann et al. JBC 270, 2901-2905 (1995)<br />
3. Hu et al. Molecular Cell 9, 789-798 (2002)<br />
contact:<br />
Martin Kahms<br />
MPI-Dortmund<br />
martin.kahms@mpi-dortmund.mpg.de<br />
Otto-Hahn-Strasse 11<br />
44227 Dortmund (Deutschland)
Andreas Böhm, Albert Sickmann<br />
Automatic Synchronizing and self-Updating Protein Sequence<br />
Database Management System<br />
A common problem in proteomics is the deficient quality <strong>of</strong> some available sequence<br />
databases, especially concerning accessibility and redundancy, for example the nonredundant<br />
sequence database (nrdb), which is not truly non-redundant. Often they are<br />
available online only for a short period <strong>of</strong> time, then disappear from the internet and<br />
cannot be obtained in other ways. Common proteomic or genomic "database" formats<br />
(omic-databases) like FASTA or geneDB are neither designed for efficient maintenance<br />
nor practical for data management purposes in environments where millions <strong>of</strong><br />
sequences must be handled. Therefore we decided to develop our own sequence<br />
database system and to keep it up to date by integrating a practicable automatism for<br />
maintenance purposes. This system queries each online database for available updates<br />
and merges the differences in our system called the Automatic Syncing and Updating<br />
Protein Sequence Database (ASUP-SDB). Making the database non-redundant by<br />
applying known and our own algorithms, the key feature is, that the classes <strong>of</strong> mistakes<br />
which may emerge while making the database non-redundant are known and thus we<br />
can react appropriately to these circumstances. ASUP-SDB is capable <strong>of</strong> storing<br />
sequence data as well as the appropriate information concerning splice-variants, posttranslational<br />
modifications, is<strong>of</strong>orms, localization and taxonomy. Of course it allows<br />
precise retrieval <strong>of</strong> this information and exports in common omic formats, too. As a<br />
remarkable feature ASUP-SDB is capable <strong>of</strong> downloading external online databases by<br />
looking for updates and synchronizes with these data automatically using several<br />
algorithms. The ASUP-SDB supports common import and export omic formats such as<br />
FASTA or swissprot, for instance. Other importing or exporting formats can be added<br />
easily. As ASUP-SDB uses an underlying relational database management system<br />
(RDBMS), maintenance <strong>of</strong> sequence information is centralized and thus omic-databases<br />
used for sequence analysis such as mass spectrometry are kept up to date simply by<br />
exporting them from the central ASUP-SDB on a regularly basis.<br />
Literature<br />
[1] Pearson, W.R. and D.J. Lipman. PNAS, 1988. 85(8): p. 2444-2448.<br />
[2] Altschul, S.F., et al. JMB, 1990. 215(3): p. 403-410.<br />
[3] http://www.ncbi.nih.gov/, 1988.<br />
[4] http://us.expasy.org/sprot/, 2004<br />
contact:<br />
Andreas Böhm<br />
Universität Würzburg<br />
Rudolf-Virchow-Center for Experimental Biomedicine<br />
andreas.boehm@virchow.uni-wuerzburg.de<br />
Versbacher Str. 9<br />
97078 Würzburg (D)
Angelika Bondzio, Christoph Gabler, Dagmar Beier, Siegfried Risse, Ralf Einspanier<br />
Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia<br />
Virus (BLV) expression through Toll-like receptor 4<br />
BLV is an oncogenic retrovirus and the causative agent for enzootic bovine leukosis. BLV<br />
infection results in a large asymptomatic period, followed by development <strong>of</strong> persistent<br />
lymphocytosis. Since the major target cell for this virus is the B-lymphocyte, the<br />
expression <strong>of</strong> BLV should respond to agents that activating these cells. LPS is one <strong>of</strong> the<br />
agents that can stimulate BLV expression in bovine peripheral blood mononuclear cells<br />
(PBMCs). However, the signalling mechanisms utilized by LPS to stimulate BLV<br />
expression are still incompletely understood. In this study we showed by RT-PCR that<br />
the initiation <strong>of</strong> PBMC response to LPS involves Toll-like receptor 4 (TLR4). Stimulation <strong>of</strong><br />
PBMCs with LPS resulted in an significant increase in the expression level <strong>of</strong> TLR4 mRNA<br />
in PBMCs in a time-dependent manner, but the expression <strong>of</strong> TLR2 mRNA was not<br />
affected. In parallel, we showed that LPS activated NF-κB in bovine PBMCs and enhanced<br />
the secretion <strong>of</strong> IL-8 in a time- and dose-dependent manner. As demonstrated by ELISA<br />
the expression <strong>of</strong> viral antigens BLVp24 and gp51 in PBMCs was enhanced by LPS<br />
compared to the unstimulated controls. These effects were accompanied by free radical<br />
formation as measured with pholasin, giving further evidence for the role <strong>of</strong> reactive<br />
oxygen species (ROS) in activation <strong>of</strong> BLV expression, that we have previously reported<br />
(Bondzio et al., 2003). In summary, our results indicate that TLR4 may mediate LPSstimulated<br />
BLV expression via ROS.<br />
Literature<br />
Angelika Bondzio, Petra Blankenstein, Siegfried Risse (2003). Effects <strong>of</strong> hydrogen<br />
peroxide on Bovine Leukemia Virus expression. Biol. Chem. 384, 7; 1063-1072.<br />
contact:<br />
Angelika Bondzio<br />
Freie Universität Berlin<br />
Veterinärbiochemie<br />
bondzio@vetmed.fu-berlin.de<br />
Oertzenweg 19b<br />
14163 Berlin (Deutschland)
Rüdiger Adam, Tobias Tenenbaum, Hans-Joachim Galla, Horst Schroten<br />
Bacterial Meningitis: Interactions at the Blood-CSF-Barrier<br />
Background: Despite therapeutic advances bacterial meningitis continues to be an<br />
important cause <strong>of</strong> morbidity and mortality. In the pathogenesis <strong>of</strong> bacterial meningitis<br />
the replication <strong>of</strong> microorganisms represents a crucial step as CSF is principally devoid <strong>of</strong><br />
relevant protective factors (complement, immunoglobulins, leukocytes) 1 .<br />
We wanted to elucidate the role <strong>of</strong> the choroid plexus during bacterial meningitis.<br />
Material and Methods: Primary porcine choroid plexus epithelial cells (PCPEC) were used<br />
as an established model <strong>of</strong> the blood-CSF-barrier 2 . After prestimulation with<br />
proinflammatory cytokines, the PCPEC were challenged with Streptococcus suis, an<br />
important agent <strong>of</strong> meningitis both in swine and humans. Bacterial growth rates were<br />
determined in the supernatant and possible antibacterial mechanisms were determined<br />
(production <strong>of</strong> nitric oxide NO, generation <strong>of</strong> reactive oxygen species, depletion <strong>of</strong><br />
tryptophan). In addition the PCPEC were challenged with bacterial stimuli (live, UVinactivated,<br />
sonicated, supernatant) and the production <strong>of</strong> IL-6 and IL-8 was<br />
determined. Barrier properties <strong>of</strong> the PCPEC monolayers were determined with TEER and<br />
paracellular [ 3 H]-mannitol flux.<br />
Results: pCPEC showed a marked bacteriostatic effect. Repletion <strong>of</strong> tryptophan<br />
demonstrated an activation <strong>of</strong> indoleamine 2,3-dioxygenase (IDO) to be responsible 3 .<br />
Furthermore the PCPEC produced relevant amounts <strong>of</strong> IL-6 and IL-8. Barrier properties<br />
were substantially altered after bacterial challenge.<br />
Conclusion: The choroid plexus seems to play a major role in the pathogenesis <strong>of</strong><br />
bacterial meningitis. It displays antibacterial properties and is able to produce cytokines<br />
relevant in the course <strong>of</strong> the disease.<br />
Literature<br />
1 Kim KS: Nat Rev Neurosci. 2003,4:376-385.<br />
2 Gath U, Hakvoort A, Wegener J, Decker S, Galla HJ: Eur J Cell Biol. 1997,74:68-78.<br />
3 Adam RA, Tenenbaum T, Valentin-Weigand P, Laryea M, Schwahn B, Angelow S, Galla<br />
HJ, Daubener W, Schroten H: Infect Immun. 2004,72:3084-3087.<br />
contact:<br />
Dr. med. Rüdiger Adam<br />
Universitätsklinikum Düsseldorf<br />
Klinik für Allgemeine Pädiatrie<br />
adam@med.uni-duesseldorf.de<br />
Moorenstraße 5<br />
40225 Düsseldorf (Deutschland)
Verena Goebeler, Ursula Rescher, Volker Gerke<br />
Biochemical Characterization <strong>of</strong> Annexin A9 and Identification<br />
<strong>of</strong> Interacting Proteins<br />
Annexins are a multigene family <strong>of</strong> evolutionarily conserved proteins characterized by<br />
their ability to interact with phospholipids in a Ca2+ dependent manner.1 They are<br />
expressed in a variety <strong>of</strong> cells and tissues and are thought to participate in a number <strong>of</strong><br />
membrane-related events.<br />
Central to the annexin action is their Ca2+ dependent binding to negatively charged<br />
phospholipids <strong>of</strong> cellular membranes. This property is mediated through the structurally<br />
highly conserved annexin protein core which has the form <strong>of</strong> a slightly curved disc and<br />
contains two types <strong>of</strong> Ca2+ binding sites, the so-called type II and type III sites. Binding<br />
to cellular membranes seems to depend on the integrity <strong>of</strong> the high affinity type II Ca2+<br />
binding sites.<br />
Annexin A9 represents a special annexin subclass ins<strong>of</strong>ar as all type II Ca2+ binding<br />
sites in annexin A9 are inactivated by replacement <strong>of</strong> the critical amino acid residues.2<br />
Although annexin A9 is able to bind phosphatidylserine containing vesicles this binding<br />
only occures at a Ca2+ concentration exceeding 2 mM and appears to be irreversible.3<br />
These binding characteristics suggest some conformational changes in the annexin A9-<br />
Ca2+-liposome complex that may lead to an exposure <strong>of</strong> Ca2+-insensitive hydrophobic<br />
surfaces in the protein. This hydrophobicity increase was further characterized by phase<br />
separation using Triton X-114.<br />
In contrast to the highly conserved tetrad annexin core the N-terminal domains <strong>of</strong><br />
different annexins are highly divergent and are believed to confer specifity to the<br />
individual members <strong>of</strong> the protein family. Although showing limited sequence homology,<br />
the N-terminal regions <strong>of</strong> annexin A2 and A9 share the potential <strong>of</strong> forming an<br />
amphiphatic •-helix. In annexin A2 this helix is essential for binding <strong>of</strong> the intracellular<br />
ligand S100A10. Therefore we started to identify possible binding partners <strong>of</strong> human<br />
annexin A9 by using an affinity chromatography approach and subsequent MALDI-MS<br />
analysis. Results <strong>of</strong> these approaches will be discussed.<br />
Literature<br />
1 Gerke V and Moss SE (2002) Physiol. Rev., 331-371<br />
2 Morgan RO and Fernandez MP(1998) FEBS Lett. 434, 300-304<br />
3 Goebeler et al. (2003) FEBS Lett. 546, 359-364<br />
contact:<br />
Verena Goebeler<br />
Centre for Molecular Biology <strong>of</strong> Inflammation<br />
Institute for Medical Biochemistry<br />
goebele@uni-muenster.de<br />
Von-Esmarch-Str. 56<br />
48149 Münster (Germany)
Antje Schulte, André Schönichen, Nadine Czudnochowski, Matthias Geyer<br />
Biochemical Characterization <strong>of</strong> the Interaction between<br />
Cyclin T1 and Hexim1 and its Competition with the HIV-1 Tat<br />
protein<br />
The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive<br />
elongation <strong>of</strong> nascent RNA molecules by RNA polymerase II. Core active P-TEFb is<br />
composed <strong>of</strong> the kinase CDK9 and Cyclin T1. The cellular protein Hexim1 inhibits the<br />
kinase activity <strong>of</strong> P-TEFb to suppress RNA pol II transcriptional elongation in a process<br />
that specifically requires the small nuclear RNA 7SK. We have identified a C-terminal<br />
domain within Hexim1 that specifically interacts with the cyclin boxes <strong>of</strong> Cyclin T1.<br />
Isothermal titration calorimetry and fluorescence spectroscopy reveal the binding<br />
kinetics and thermodynamic parameters for this interaction in vitro. As we show by size<br />
exclusion chromatography full length Hexim1 as well as the C-terminal domain appear<br />
as homodimers, while in complex with Cyclin T1 the Hexim1 molecule is monomeric.<br />
Importantly, the viral transcription activator protein HIV-1 Tat binds to the same surface<br />
<strong>of</strong> Cyclin T1 as Hexim1 and competes with Hexim1 to connect the transactivation<br />
response (TAR) element RNA to the productive transcription <strong>of</strong> the human<br />
immunodeficiency virus genome.<br />
contact:<br />
Antje Schulte<br />
MPI for Molecular Physiology<br />
Dept. <strong>of</strong> Physical Biochemistry<br />
antje.schulte@mpi-dortmund.mpg.de<br />
Otto-Hahn-Str. 11<br />
44227 Dortmund (D)
Silke Steffens, Jens Oldendorf, Lifeng Chi, Günter Haufe, Hans-Joachim Galla<br />
Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters<br />
The eucariotic cell membrane consists essentially <strong>of</strong> phospholipids, cholesterol,<br />
sphingolipids and proteins. An enormous number <strong>of</strong> naturally occurring lipids, like<br />
ceramides or sphingomyeline, have sphingosine or dihydrosphingosine (sphinganine) as<br />
a backbone. 1 They also play an important role in cell-communication, signalling and<br />
apoptosis.<br />
The novel synthesis <strong>of</strong> fluorinated dihydroceramides, 2 which include structure elements<br />
<strong>of</strong> sphinganine, lead to different fluorinated derivatives <strong>of</strong> stearicacidethylester as<br />
diastereomeric and racemic/diastereomeric mixtures. 3 Fluorination <strong>of</strong> the 4-position <strong>of</strong><br />
these compounds might induce interesting effects on the free hydroxyl function in 3position<br />
as the formation <strong>of</strong> a hydrogen bond should cause the hydroxyl- and the<br />
fluorine-substituent to align in gauche conformation which has been observed in the case<br />
<strong>of</strong> 2-fluoroethanol. 4 Intermolecular interactions are investigated at the air/water<br />
interface with biophysical methods like Film Balance Measurements and Brewster Angle<br />
Microscopy (BAM). Additionally the domain structure <strong>of</strong> the monolayer films in the phase<br />
transition area was transferred via Langmuir-Blodgett and displayed by Scanning Force<br />
Microscopy (SFM). All compounds form stable monolayer films and show surface activity.<br />
The dihydroceramides show extreme rigid monolayer behaviour underlined by BAM<br />
measurements. Film Balance Measurements <strong>of</strong> the fluorinated derivatives <strong>of</strong><br />
stearicacidesters showed the correlation <strong>of</strong> the stability <strong>of</strong> the monomolecular films with<br />
the polarity <strong>of</strong> the head groups. The different behaviour <strong>of</strong> the diastereomeric and<br />
racemic / diastereomeric mixtures in the phase transition area was imaged by BAM<br />
measurements. The SFM images underline the shape and structure <strong>of</strong> domains observed<br />
in BAM and demonstrated the formation <strong>of</strong> three-dimensional aggregates.<br />
Literature<br />
[1] R. M. Bell, Y. A. Hannun, A. H. Merrill Jr. in „Advances in Lipid Research:<br />
Sphingolipids and their metabolites“, Vol. 25 & 26, Academic Press, Olando FL, 1993.<br />
[2] J. Oldendorf, Dissertation, University <strong>of</strong> Muenster, 2002<br />
[3] J. Oldendorf, G. Haufe, J. Prakt. Chem. 2000, 342, 52-57.<br />
[4] J. M. Bakke, J. H. Bjerkeseth, T. E. C. L. Ronnow, K. Steinsvoll, J. Mol. Struct. 1994,<br />
321, 205-214v<br />
contact:<br />
Silke Steffens<br />
Muenster<br />
Biochemie<br />
ssteffe@uni-muenster.de<br />
Wilhelm-Klemm Str. 2<br />
48147 Muenster (Germany)<br />
additional information<br />
Financial Support: SFB 424
Christoph Riethmüller, Joachim Wegener*, Marianne Wilhelmi, Pia Jungmann, Hans<br />
Oberleithner<br />
Bradykinin enhances vesicular transport <strong>of</strong> solutes across<br />
endothelial cell layers<br />
Bradykinin (BK) is a mediator <strong>of</strong> inflammatory reactions like vasodilation, pain<br />
sensitization and increased vascular permeability. The increased volume shift from from<br />
blood plasma across the endothelium to interstitial space facilitates the formation <strong>of</strong><br />
edema. Water and solutes are assumed to take the paracellular route, but contribution<br />
<strong>of</strong> transcellular vesicular transport has also been discussed. We used electrical cell<br />
impedance spectroscopy (ECIS) to continuously monitor the resistance <strong>of</strong> a cell layer<br />
which is a measure <strong>of</strong> intercellular distance. As a cellular system we used isolated<br />
human umbilical vein endothelial cells (HUVEC). BK (1µM) led to a biphasic response in<br />
terms <strong>of</strong> electrical resistance: an initial short drop (30min, 29+5%, n=12)<br />
with a maximum after 10 min. These phases could be assigned to rises in intracellular<br />
calcium and cAMP, respectively. Computational modulation <strong>of</strong> data clearly demonstrated<br />
that BK leads to reduction <strong>of</strong> intercellular distances. To assess solute permeability in our<br />
cell culture model, the diffusion rate <strong>of</strong> 40 kD FITC-Dextran across HUVEC-monolayers<br />
grown on filter membranes was measured. Solute permeability was raised by 71%<br />
through the action <strong>of</strong> BK. This permeability increase shrinks down to 27% when the<br />
experiments were performed at 28°C. This votes for a vesicular mechanism <strong>of</strong> transport<br />
because vesicle formation is inhibited by temperature reduction. Conclusion: In contrast<br />
to the initial assumption, BK leads to a transient tightening <strong>of</strong> endothelial junctions,<br />
paralleled by upregulated vesicular transport <strong>of</strong> fluids.<br />
contact:<br />
Dr. Christoph Riethmüller<br />
Universität Münster<br />
Physiologie II, UKM<br />
chrth@uni-muenster.de<br />
Robert-Koch-Straße 27b<br />
48149 Münster (Deutschland)<br />
additional information<br />
* Institute <strong>of</strong> Biochemistry, University <strong>of</strong> Münster, Wilhelm-Klemm-Strasse 10, 48149 Münster
Andreas Böhm, Florian Grosse-Coosmann, Albert Sickmann<br />
Calculating theoretical ms spectra for given sequences<br />
Scientists usually want to verify the ion matching process <strong>of</strong> algorithms that look up<br />
peptide sequences in DNA or protein databases. The verification step is <strong>of</strong>ten done<br />
numerically or visually. Not all search algorithms present the appropriate theoretical<br />
spectrum information within their results. Thus, the theoretical spectrum for each result<br />
should be calculated from the sequence <strong>of</strong> the matched peptide. We present an<br />
operating-system-independent command line tool for this purpose that can be integrated<br />
easily into complex as well as existing environments, and can be used to present the<br />
theoretical spectrum to the user in either graphical or tabular format by third party<br />
products. The source code and binaries for windows and linux can be downloaded from<br />
http://www.protein-ms.de<br />
Literature<br />
Kernighan, B. W. and Ritchie, D. M. (1990) The C Programming Language<br />
National Institute for Standards and Technology (2004) The Nist Reference on<br />
Constants, Units and Uncertainty, http://physics.nist.gov/cuu/Constants<br />
Perkins, D. N., Pappin, D. J. C., Creasy, D. M., Cottrell, J. S. (1999) Probability-Based<br />
Protein Identification by Searching Sequence Databases Using Mass Spectrometry Data,<br />
Electrophoresis, 20, 3551-3567<br />
Yates, J. R., Eng, J. K., Clauser, K. R. and Burlingame, A. L. (1996) Search <strong>of</strong> Sequence<br />
Databases with Uninterpreted High-Energy Collision-Induces Dissociation Spectra <strong>of</strong><br />
Peptides, J Am Soc Mass Spectom, 7, 1089-1098<br />
contact:<br />
Andreas Böhm<br />
Universität Würzburg<br />
Rudolf-Virchow-Center for Experimental Biomedicine<br />
andreas.boehm@virchow.uni-wuerzburg.de<br />
Versbacher Str. 9<br />
97078 Würzburg (D)
Dyakonov Lev, Galnbek Tatjana, Simonova Alla, Savenko Natalia, Klenovitsky Petr,<br />
Ernst Lev, Zinovieva Natalia, Shevcova Natalia<br />
Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Hybridization <strong>of</strong> animal cells is a promising method <strong>of</strong> cell engineering which allows to<br />
obtain cell cultures with useful features (sensitivity to viruses, the production <strong>of</strong><br />
physiologically active substances, and others). The cultivation <strong>of</strong> hybrid cell cultures<br />
causes certain problems, such as instability <strong>of</strong> caryotype, the segregation <strong>of</strong><br />
chromosomes and as a result, the loss <strong>of</strong> some biological features. The following<br />
interspecific hybrid cell cultures have been obtained in VIEV: pig kidney x cow<br />
lymphocytes (SPEV TKxLK-D5), pig kidney x horse lymphocytes (SPEV TK- x HL – A4 x<br />
L), sheep kidney x rabbit lymphocytes and splenocytes (SK TK- x RL, SK TK- x RS),<br />
sheep kidney x beta cells <strong>of</strong> rabbit pancreas (SK TK- x BRPC) and others. It was <strong>of</strong><br />
certain interest for us to study the condition <strong>of</strong> the caryotype <strong>of</strong> the cell cultures A4 X L<br />
(pig and horse) which had been kept for 17 years (- 196o°C) in liquid nitrogen, and<br />
recently obtained cell cultures SK TK- x RL (sheep x rabbit). Differential coloring <strong>of</strong><br />
chromosomes was carried out according to the method <strong>of</strong> Romanovsky-Guimza.<br />
Metaphase plates were studied in the computer system. The analysis made it clear that<br />
the proportion <strong>of</strong> chromosomes in A4xL culture is variable. None <strong>of</strong> the metaphase plates<br />
contained a full set <strong>of</strong> the chromosomes <strong>of</strong> the partners (38+64). The number <strong>of</strong> the<br />
chromosomes varied from 35 up to 78. The modal class <strong>of</strong> 50 metaphase plates<br />
accounted 39 chromosomes. Pig chromosomes in metaphase plate with 78 chromosome<br />
accounted for about 50% and horse chromosome accounted for 50%. 24 akrocentrical<br />
chromosomes were lost. Pig chromosomes dominated in metaphase plates containing 35-<br />
39 chromosomes. Horse chromosomes only accounted for 3-5%. As cultivation in GAT<br />
medium showed, some cells only contained pig chromosomes; however they<br />
corresponded to hybrid cells. In the population <strong>of</strong> cells growing in the suspension the<br />
number <strong>of</strong> the chromosomes accounted for 51-82. Culturally and morphologically hybrid<br />
culture A4 xL retained initial features and was represented both by lymphocytolike cells<br />
typical for horse lymphocytes and by epithelium like cells which have much in common<br />
in morphology with SPEV cells. Populations <strong>of</strong> cultures with the domination <strong>of</strong><br />
lymphocyto like cells with reduced adhesion were obtained by directed selection. Sheep<br />
x rabbit cell culture (SK TK- x RL) which had 29 passages after hybridization had more<br />
stable cerotype, i.e. the majority <strong>of</strong> cells had even number <strong>of</strong> the chromosomes <strong>of</strong> the<br />
partners. The number <strong>of</strong> the chromosomes accounted for 88-120. We were able to<br />
identify the existence <strong>of</strong> two-shouldered chromosomes <strong>of</strong> pig and sheep. The specific<br />
differentiation <strong>of</strong> akrocentrical chromosomes turned out to be impossible because <strong>of</strong> the<br />
small size and similarity <strong>of</strong> sheep and rabbit chromosomes <strong>of</strong> this type. The hybrid cell<br />
culture SK TK- x RL at the level <strong>of</strong> 29th passage retained genetic information received<br />
from cells <strong>of</strong> two kinds. Cultural and morphological hybrid culture SK TK- x RL is<br />
represented by lymphocyto like and epithelium like cells.<br />
contact:<br />
Dr, Pr<strong>of</strong>.,PhD BS Dyakonov Lev<br />
VIEV<br />
Dep. <strong>of</strong> Cell Biothehnology<br />
levdyakonov@mail.ru<br />
Rjazansky pr.24/1<br />
109428 MOSCOW (Russija)
Cord Brakebusch, Aleksandra Czuchra, Xunwei Wu, Tanja Bosse, Klemens Rottner<br />
Cdc42 is not required for the formation <strong>of</strong> filopodia and<br />
lamellipodia, but crucial for the uptake <strong>of</strong> Listeria<br />
Rho GTPases regulate the reorganisation <strong>of</strong> the actin cytoskeleton, but also other<br />
processes such as cell polarity, proliferation, apoptosis, and secretion. On the cellular<br />
level, many effects <strong>of</strong> Rho GTPases have been studied by inhibiting the function <strong>of</strong> Rho<br />
GTPases using dominant negative mutant forms. This approach, however, lacks<br />
specificity since it blocks not only a given Rho GTPase, but also certain activation<br />
pathways <strong>of</strong> other Rho GTPases. To investigate specifically the functions <strong>of</strong> Cdc42 we<br />
generated murine embryonic stem cells, fibroblastoid cell lines and mice with an<br />
inducible deletion <strong>of</strong> the Cdc42 gene using the cre-loxP system. In fibroblastoid cells,<br />
inactivation <strong>of</strong> the Cdc42 gene was induced by transient, adenoviral transduction <strong>of</strong> Cre<br />
recombinase. Viable Cdc42-null cell lines were obtained and analysed in various assays.<br />
Cdc42-deficient cells showed a more elongated morphology and impaired cell-cell<br />
contacts in a confluent monolayer. Surprisingly, filopodia and lamellipodia formation was<br />
not affected by the loss <strong>of</strong> Cdc42. Similarly, proliferation and activation <strong>of</strong> Erk, Jnk, p38,<br />
GSK and Akt were not significantly altered. Internalin B-mediated uptake <strong>of</strong> Listeria,<br />
however, was strongly impaired in the absence <strong>of</strong> Cdc42.<br />
contact:<br />
Dr Cord Brakebusch<br />
MPI für Biochemie<br />
Nachwuchsgruppe<br />
brakebus@biochem.mpg.de<br />
Am Klopferspitz 18<br />
82152 Martinsried (Deutschland)
Roland H<strong>of</strong>weber, Gudrun Horn, Hans Robert Kalbitzer<br />
cell free studies on the function <strong>of</strong> cold shock proteins<br />
When bacteria are exposed to a cold shock, the mainly induced protein among the cold<br />
induced proteins are the so called cold shock proteins (CSP). This is a family <strong>of</strong> small<br />
(7,5 kDa) and highly conserved proteins capable <strong>of</strong> RNA binding. CSP´s have the<br />
putative function <strong>of</strong> RNA chaperones. That means, they can bind to RNA and inhibit the<br />
formation <strong>of</strong> thermodynamic stable RNA secondary structures like intramolecular hairpin<br />
loops. We tested cold shock proteins from the mesophilic bacterium Bacillus subtilis, the<br />
thermophilic Bacillus caldolyticus and the hyperthermophilic Thermotoga maritima in an<br />
E.coli cell free transcription / translation system on their capability <strong>of</strong> enhancing protein<br />
expression due to the lack <strong>of</strong> mRNA secondary structures. As test systems we used the<br />
expression <strong>of</strong> Chloramphenicol acetyltransferase (CAT) and <strong>of</strong> H- Ras. Furthermore we<br />
examined the influence <strong>of</strong> cold shock proteins on transcription and translation<br />
respectively.<br />
contact:<br />
Roland H<strong>of</strong>weber<br />
Uni Regensburg<br />
Institut für Biophysik und physikalische Biochemie<br />
roland.h<strong>of</strong>weber@biologie.uni-regensburg.de<br />
Universitätsstrasse 31<br />
93053 Regensburg (Deutschland)
John R Masters<br />
Cell line misrepresentation<br />
The first continuous cell line derived from a human cancer, HeLa, was developed in 1952<br />
from a glandular cancer <strong>of</strong> the cervix (Gey et al, 1952). In 1967, it was shown that most<br />
continuous cell lines developed since 1952 were HeLa cells (Gartler, 1967), indicating<br />
cross-contamination. The false cell lines include KB, HEp-2, Int407, Chang liver and<br />
WISH. Despite many publications showing these cell lines are indistinguishable from<br />
HeLa genetically, they continue to be used as models <strong>of</strong> skin and head and neck cancer,<br />
fetal intestine, hepatocytes and amnion. Similarly, ECV304 is used as a model <strong>of</strong> normal<br />
endothelium, despite being the T24 bladder cancer cell line (Dirks et al, 1999). Crosscontaminating<br />
cell lines are frequently claimed to show characteristics <strong>of</strong> the tissue from<br />
which they are thought to be derived. Whether these reports are due to wishful thinking<br />
or indicate some more serious misrepresentation is not apparent. Since there are many<br />
publications demonstrating the false nature <strong>of</strong> cross-contaminated cell lines, they should<br />
not be used unless it is made clear that the model system being used is HeLa or one <strong>of</strong><br />
many other cross-contaminating cell lines. Nevertheless, it appears from searches <strong>of</strong><br />
databases that false use <strong>of</strong> cross-contaminated cell lines is on the increase. Where the<br />
cell line being used is not derived from the tissue from which it is claimed to be derived,<br />
the results and conclusions are misleading and publications making such claims should<br />
be withdrawn.<br />
Literature<br />
Dirks, W.G., Drexler, H.G. & MacLeod, R.A.F. (1999) In Vitro Cell Devl. Biol. 35, 558-<br />
559.<br />
Gartler, S.M. (1967) Natl. Cancer Inst. Monogr. 26, 167-195.<br />
Gey, G.O., C<strong>of</strong>fman, W.D. & Kubicek, M.T. (1952) Cancer Res. 12, 264-265.<br />
contact:<br />
Pr<strong>of</strong>essor John R Masters<br />
University College London<br />
The Prostate Cancer Research Centre<br />
J.Masters@ucl.ac.uk<br />
67 Riding House Street<br />
W1W 7EJ London (UK)
Dessy Nikova, Volodymyr Nechyporuk-Zloy, Christian Stock, Albrecht Schwab, Hans<br />
Oberleithner, Hermann Schillers<br />
Cell membrane isolation for high resolution AFM imaging<br />
Membrane proteins are important components <strong>of</strong> the cellular machinery and involved in<br />
many cellular processes. To study membrane proteins in their natural environment they<br />
must be embedded in lipid membrane. This limits the 3D crystallization <strong>of</strong> proteins.<br />
There is only a small number <strong>of</strong> resolved 3D structures <strong>of</strong> proteins. Atomic Force<br />
Microscopy (AFM) is a good alternative for determining the structure <strong>of</strong> proteins such as<br />
ion channels in their physiological environment with nanometer resolution. AFM was<br />
used to study membrane proteins in human melanoma cells (potassium channel IK1)<br />
and human airway epithelial airway cells (CFTR). The larger parts <strong>of</strong> CFTR and IK1<br />
channels protrude from the inner surface <strong>of</strong> the plasma membrane into the cytoplasm.<br />
In order to visualize the three-dimensional structure <strong>of</strong> these proteins by AFM an<br />
effective method was developed for inside-out membrane isolation. Cells were<br />
sandwiched between two poly-L-lysine treated mica surfaces. The “sandwich” was<br />
rapidly frozen in liquid nitrogen. Two mica surfaces were fractured apart while still<br />
frozen. AFM imaging <strong>of</strong> thus prepared samples revealed single cell membrane patches<br />
with diameters <strong>of</strong> up to 30 µm. The lipid bilayer with its typical height <strong>of</strong> 5 nm and a<br />
great number <strong>of</strong> membrane proteins are clearly detectable. This sets the basis for<br />
identifying single CFTR or IK1 channel proteins.<br />
contact:<br />
Mr Volodymyr Nechyporuk-Zloy<br />
University <strong>of</strong> Muenster<br />
Institute <strong>of</strong> Physiology II<br />
zloy@uni-muenster.de<br />
Robert-Koch-Str. 27b<br />
48149 Münster (Germany)
Peter Fromherz<br />
Cells 'n Chips or Ionics meets Electronics<br />
Electrical information processing in brains and computers relies on different charge<br />
carrier, ions and electrons. A development <strong>of</strong> hybrid systems must rely on a direct<br />
noninvasive interfacing on the three levels <strong>of</strong> ion channels, nerve cells and brain tissue.<br />
In the first area, potassium and sodium channels are coupled to silicon chips with<br />
transistors for recording and with capacitors for stimulation. On the second level,<br />
individual neurons from snail and rat brain as well as small neuronal networks are<br />
electrically coupled to silicon chips. Potentiation <strong>of</strong> chemical synapses is induced and<br />
recorded by the chip. In the third area acute and cultured brain slices are joined to the<br />
chips to enable spatially resolved studies <strong>of</strong> neuronal memory.<br />
contact:<br />
Pr<strong>of</strong>. Peter Fromherz<br />
Abteilung Membran- und Neurophysik<br />
Max-Planck-Institut für Biochemie<br />
fromherz@biochem.mpg.de<br />
Am Klopferspitz 18<br />
82152 Martinsried (Deutschland)
Sergey V. Tokalov, Herwig O. Gutzeit, Jutta Ludwig-Müller, Barbara Kind, Alexander<br />
Franz, Yvonne Henker<br />
Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Despite the wealth <strong>of</strong> information concerning biological effects <strong>of</strong> flavonoids a systematic<br />
approach to analyse the molecular targets is still lacking and, for this reason, a rational<br />
evaluation <strong>of</strong> the risks or benefits <strong>of</strong> flavonoid- containing food or <strong>of</strong> possible<br />
pharmaceutical applications has not been possible. We have exploited the property <strong>of</strong><br />
quercetin to elicit fluorescence when bound to specific target proteins. The effect was<br />
studied in detail using BSA and insulin as model proteins. Furthermore, cellular target<br />
proteins in the polyploid nuclei and in cytoplasmic structures <strong>of</strong> Drosophila follicles were<br />
visualised in living cells by fluorescence microscopy. Nuclear target proteins were also<br />
present in human leukaemia (HL-60) cell cultures. However, vital HL-60 cells quickly lost<br />
their fluorescence while apoptotic cells were characterised by a strong and persisting<br />
signal (analysis by microscopy and flow cytometry). This observation indicated rapid<br />
metabolic conversion <strong>of</strong> quercetin in vital human cells. The observed dynamics <strong>of</strong><br />
decreasing fluorescence was confirmed and quantified by HPLC analysis. While apoptotic<br />
cells still contained considerable amounts <strong>of</strong> quercetin, vital cells rapidly metabolised the<br />
flavonoid (e.g. methylation or glycosylation). Of several known metabolites which were<br />
tested with BSA and insulin for their fluorogenic properties, only isorhamnetin but no<br />
other metabolite elicited strong fluorescence.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Herwig O. Gutzeit<br />
TU Dresden<br />
Zoologie<br />
Herwig.Gutzeit@mailbox.tu-dresden.de<br />
Helmholtzstr. 10<br />
01069 Dresden (Deutschland)
Monika Lichtenauer, Wolfgang E Thasler, Anja Gräbe, Birgit Jahn, Karl-Walter Jauch,<br />
Hans-Jürgen Schlitt, Thomas S Weiss<br />
Characterisation <strong>of</strong> non parenchymal cell fractions with<br />
hepatic progenitor cells from surgical resected human liver<br />
tissue<br />
The regeneration <strong>of</strong> the liver is mainly based on the ability <strong>of</strong> hepatocytes to proliferate<br />
upon stimulation. Additionally, it was shown that under certain circumstances liver<br />
resident cells with progenitor capabilities either <strong>of</strong> haematopoietic or hepatic origin are<br />
involved in liver cell proliferation and differentiation. Using immunohistochemistry as<br />
well as FACS analysis we investigated and characterised fractions <strong>of</strong> isolated non<br />
parenchymal cells (NPC) from human normal and diseased livers for expression <strong>of</strong><br />
markers for haematopoietic and hepatic progenitor cells. NPC fractions were obtained by<br />
a multi-step perfusion technique as part <strong>of</strong> the isolation procedure <strong>of</strong> primary<br />
hepatocytes. After digestion <strong>of</strong> surgical resected tissue with EGTA/collagenase solution<br />
followed by a collagenase/DNAse/Pronase treatment, NPC fractions were purified by<br />
several centrifugation steps including a Percoll gradient. Different NPC fractions were<br />
characterised by FACS analysis for expression <strong>of</strong> CD31 (endothelial cells), HEA125 (bile<br />
duct cells), CD45 (haematopoietic cells) and progenitor cell markers CD34, CD90 (Thy-<br />
1), CD117 (c-kit). Markers were confirmed performing immunohistochemistry studies, in<br />
which most <strong>of</strong> the CD34, CD90 and CD117 positive cells were found closed by or around<br />
the portal field. Additional, the detected cells were mainly positive for the oval cell<br />
marker OV-6, too. FACS analysis revealed a NPC fraction with significant levels <strong>of</strong> CD34<br />
(5.95 ± 4.71%, n = 5), CD 90 (5.75 ± 1.53%, n = 5) and CD117 (6.89 ± 7.32%, n =<br />
5) cells. Within this fraction CD45 positive cells were also positive for CD34 (17.96 ±<br />
25.42%, n = 4), CD 90 (10.22 ± 7.47%, n = 4) and CD117 (8.82 ± 9.81%, n = 4).<br />
Furthermore cells were identified being double positive for CD34/CD90 (1.56 ± 0.43%)<br />
and CD34/CD117 (1.11 ± 0.24%). These results indicate a significant pool <strong>of</strong> progenitor<br />
cells suitable to be isolated from NPC fractions. Progenitor cell fractions will be further<br />
purified and analyzed for their proliferation and differentiation potential.<br />
contact:<br />
Monika Lichtenauer<br />
Uniklinik Regensburg<br />
Chirurgie Forschung<br />
monika.lichtenauer@klinik.uni-regensburg.de<br />
Franz-Josef-Strauss Allee 11<br />
93053 Regensburg (Deutschland)
Alexander Teplyashin, Natalia Tchupikova, Svetlana Sharifullina, Mariya Rostovskaya,<br />
Svetlana Korjikova, Igor Abdrakhmanov, Alexander Dunaev, Sergey Petrin, Valeriy<br />
Eremenko, Irina Savchenkova<br />
Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Cells with properties <strong>of</strong> mesenchymal stem cells (MSC) were derived and characterized<br />
from human bone marrow, adipose tissue, skin and placenta.<br />
Results <strong>of</strong> morphological analysis showed that isolated cells possessed fibroblast-like<br />
phenotype. Cells were stained by antibodies to surface antigenes CD10, CD13, CD31,<br />
CD34, CD44, CD45, CD90, CD105, CD117 (Becton Dickinson) and assayed by flow<br />
cytometer (Beckman Coulter) using indirect fluorescence. Immun<strong>of</strong>luorescent analysis<br />
revealed the presence <strong>of</strong> MSC-specific surface markers. Cells were found to be<br />
homogenously positive for CD13, CD44, CD90 and CD105, dimly positive for CD10 and<br />
negative for CD31, CD34, CD45 and CD117. Cytogenetic analysis <strong>of</strong> cells (G-banding)<br />
detected a normal diploid karyotype at least after 35 cell doublings. Cultivation in the<br />
osteogenic medium demonstrated the ability <strong>of</strong> MSC-like cells to differentiate to<br />
osteoblasts. While culturing in the adipogenic medium all these cells differentiated to<br />
adipocytes. Dynamic <strong>of</strong> distribution in tissues <strong>of</strong> cells intravenously injected to females <strong>of</strong><br />
nude mice and immunosuppressed mice was determined by PCR analysis with specific<br />
primers for human Y-chromosome. Preliminary results showed the ability <strong>of</strong> these cells<br />
to engraft primarily to lungs and then liver.<br />
Our findings suggest that cells isolated from human bone marrow, adipose tissue, skin<br />
and placenta possessed characteristics <strong>of</strong> MSC which could be used for fundamental<br />
research and clinical application.<br />
contact:<br />
doctor <strong>of</strong> medical science Alexander Teplyashin<br />
Institute <strong>of</strong> Stem Cell<br />
irinasavtchenkova@rambler.ru<br />
Sadovaya Kudrinskaya<br />
103001 Moscow (Russia)
Judith Austermann, Max Koltzscher, Volker Gerke<br />
Characterization <strong>of</strong> the calcium-regulated S100P-ezrin<br />
interaction<br />
S100 proteins are small dimeric proteins which comprise a large subfamily within the EFhand<br />
superfamily <strong>of</strong> Ca2+ binding proteins. They are thought to participate in mediating<br />
intracellular Ca2+ signals by binding to and thereby regulating target proteins in a Ca2+<br />
dependent manner.<br />
Affinity chromatographory studies with the biologically active S100P dimer identified<br />
ezrin, a membrane/ F-actin crosslinking protein, as a dimer-specific S100P ligand. The<br />
formation <strong>of</strong> the S100P-ezrin complex is Ca2+ dependent and occurs via the N-terminal<br />
domain <strong>of</strong> ezrin (N-ERMAD). This domain is accessible for interaction in dormant ezrin, in<br />
which the membrane and F-actin-binding-sites are masked through interactions between<br />
the N-terminal and C-terminal domain. Interestingly F-actin cosedimentation assays with<br />
purified ezrin and WT S100P showed that the binding <strong>of</strong> S100P unmasks the F-actin<br />
binding site in the C-terminal domain <strong>of</strong> ezrin thereby activating the ezrin molecule. This<br />
identifies S100P as a novel activator <strong>of</strong> ezrin and indicates that an activation <strong>of</strong> ezrin`s<br />
membrane/ cytoskeleton crosslinking funktion can occur directly in response to Ca 2+<br />
transients.<br />
In order to identify specific amino acid residues involved in S100P-ezrin complex<br />
formation we also generated a series <strong>of</strong> C-terminal truncated N-ERMAD and S100P<br />
derivates and analyzed their interactions with WT S100P and WT N-ERMAD respectively.<br />
Results dilineating the sites <strong>of</strong> complex formation will be discussed.<br />
contact:<br />
Judith Austermann<br />
ZMBE<br />
Institut für Med. Biochemie<br />
judithaustermann@gmx.de<br />
Von-Esmarch-Str. 56<br />
48149 Muenster (Germany)
Till Biskup, Uta Joos, Jana Kriegsmann, Christian Heise, Ines Westphal, Götz Pilarczyk,<br />
Claus Duschl<br />
Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Adherent cells can react in different ways to the coating <strong>of</strong> the surface they are cultured<br />
on. Either they adhere, spread, or migrate or they are repelled and show anoikis and<br />
apoptosis. Chemically defined surfaces can be designed in order to influence the<br />
physiology <strong>of</strong> cells. This is <strong>of</strong> high importance for the production <strong>of</strong> functional templates<br />
for cell culturing and tissue engineering.<br />
We use microcontact printing to pattern FITC conjugated poly-L-lysine (pLl). The pLl is a<br />
polycation and has cell attractive properties. Printing pLl onto a negatively charged glass<br />
surface results in a surface pattern with +/- charge contrast. Alternatively if printed onto<br />
polyHEMA, a cell repellent hydrophilic poly mer, a pattern <strong>of</strong> cell attractive and cell<br />
repellent areas results.<br />
To increase the pLl density on the polyHEMA coating oxygen plasma activation is used.<br />
Alternatively the glass surface is precoated by 3-glyoxy-propyl-trimethoxy-silane<br />
(GOPS). The resulting epoxides react covalently with the amino groups <strong>of</strong> the pLl.<br />
Murine fibroblasts (L929) are cultivated on patterned substrates, fixed with<br />
formaldehyde and stained for the f-actin cytoskeleton with phalloidin-alexa 546<br />
conjugate and for the nucleus with DAPI. Results are presented that show the influence<br />
<strong>of</strong> the surface patterns to the cell adhesion.<br />
contact:<br />
Till Biskup<br />
Fraunh<strong>of</strong>er<br />
IBMT<br />
till.biskup@ibmt.fraunh<strong>of</strong>er.de<br />
Invalidenstrasse 42<br />
10115 Berlin (Deutschland)
Nour Eddine El Gueddari, Bruno Moerschbacher<br />
Chitosan Activates Resistance Against Pathogens After<br />
eXposure (CARAPAX)<br />
Chitosan, the fully or partially N-acetylated, derivative <strong>of</strong> chitin is a natural product <strong>of</strong><br />
many phytopathogenic fungi. Chitosan released in plant/fungal interactions is a powerful<br />
inducer <strong>of</strong> certain plant genes called pathogenesis-related (PR) genes. In view <strong>of</strong> the<br />
biological activity <strong>of</strong> this crab shell waste product, its use as an agriculture chemical is<br />
desirable both because it is a natural biodegradable agent and can be utilized<br />
economically to substitute for some fungicides possessing health risks. However the<br />
Chitosans produced today are greatly variable in purity and solubility, and ill defined in<br />
chemical composition, molecular weight, and their biological effects. In the CARAPAX<br />
project extensive research has been focussed toward understanding the influence <strong>of</strong><br />
degree <strong>of</strong> polymerisation (DP) and degree <strong>of</strong> N-acetylation (DA) on biological activities <strong>of</strong><br />
chitosans. In wheat plant we measured the induction <strong>of</strong> enzymes causally involved in the<br />
hypersensitive resistance reaction against fungi. We present evidence that the different<br />
aspects <strong>of</strong> the hypersensitive reaction are elicited optimally by different chitosans.<br />
contact:<br />
Dr. Nour Eddine El Gueddari<br />
WWU<br />
Institut für Biochemie und Biotechnologie der Pflanzen<br />
guedari@uni-muenster.de<br />
Hindenburgplatz 55<br />
48143 Münster (Deutschland)
Alexander Prodöhl, Thomas Volkmer, Dirk Schneider<br />
C<strong>of</strong>actor Binding to Membrane Proteins - From the Two-Stage<br />
to a Three-Stage Model?<br />
Folding <strong>of</strong> polytopic membrane proteins can be conceptually reduced to a few steps<br />
encapsulated in the Two Stage Model. In this model, insertion <strong>of</strong> individual helices into a<br />
membrane (stage I) occurs prior to helix-helix association (assembly) to form the native<br />
protein (Stage II). It has been shown in the recent years that this simple concept is very<br />
helpful to analyze the folding <strong>of</strong> membrane proteins in vivo and in vitro. But what about<br />
bound c<strong>of</strong>actors? Little is known about the importance <strong>of</strong> c<strong>of</strong>actors for membrane protein<br />
folding and stabilization. Since more insight into the role <strong>of</strong> specific factors for folding<br />
and stability <strong>of</strong> membrane proteins is sorely needed we have started to analyze the<br />
necessity <strong>of</strong> c<strong>of</strong>actor binding to a transmembrane b-type cytochrome for the interaction<br />
<strong>of</strong> the individual helices. Helix-helix interactions were analyzed in vivo using a genetic<br />
system (GALLEX) which was recently developed by us. The degree <strong>of</strong> homo-dimerization<br />
was measured and it was tested how the helix-helix interactions are affected by the<br />
presence <strong>of</strong> c<strong>of</strong>actor or by mutations. The concentration <strong>of</strong> heme available for the<br />
formation <strong>of</strong> the b-type cytochrome was modulated by exogenous heme, heme<br />
precursors, or inhibitors <strong>of</strong> the heme biosynthesis. The role <strong>of</strong> individual amino acids in<br />
cytochrome formation was studied by site-directed mutagenesis. Additionally, we have<br />
developed a method to refold the protein for studying helix-helix interaction and c<strong>of</strong>actor<br />
binding to the cytochrome in vitro. The obtained results indicate that helix-helix<br />
interactions occur prior to c<strong>of</strong>actor binding and that heme binding is not essential for<br />
mediating specific contacts, though the bound c<strong>of</strong>actor can contribute to stabilize specific<br />
interactions.<br />
contact:<br />
Dr. Dirk Schneider<br />
Albert-Ludwigs-Universität Freiburg<br />
Institut für Biochemie und Molekularbiologie<br />
Dirk.Schneider@biochemie.uni-freiburg.de<br />
Hermann-Herder-Straße 7<br />
79104 Freiburg (Germany)
Susanne Berger, Martina Papadopoulos, Katharina Bonfig, Ulrich Schreiber, Thomas<br />
Roitsch<br />
Complex regulation <strong>of</strong> gene expression, photosynthesis and<br />
sugar levels by pathogen infection<br />
Infection <strong>of</strong> plants with pathogens results in the induction <strong>of</strong> defense reactions as well as<br />
changes in carbohydrate metabolism. On one hand the pathogen attempts to manipulate<br />
the carbohydrate metabolism <strong>of</strong> the plant for its own advantage. On the other hand the<br />
plant has to reorganize carbon fluxes to ensure fight against the pathogen. In order to<br />
further investigate the connection between pathogen infection and carbohydrate<br />
metabolism, the effect <strong>of</strong> two types <strong>of</strong> pathogens, biotrophic and necrotrophic, on gene<br />
expression, endogenous sugar levels and photosynthesis <strong>of</strong> tomato plants was analysed.<br />
Photosynthetic gene expression was downregulated upon infection with P. syringae and<br />
B. cinerea. In contrast, expression <strong>of</strong> a sink specific gene encoding a cell wall invertase<br />
and <strong>of</strong> defense genes was induced by both pathogens. These results provide evidence for<br />
a coregulation <strong>of</strong> defense, sink and photosynthetic gene expression in planta in response<br />
to both types <strong>of</strong> pathogens. The brassinosteroid-containing plant restorative ComCat<br />
enhanced resistance against B. cinerea and counterregulated the repression <strong>of</strong> RbcS.<br />
Endogenous sugar levels decreased and the hexose to sucrose ratio increased upon<br />
treatment with B. cinerea. The application <strong>of</strong> chlorophyll fluorescence imaging revealed<br />
the spatio-temporal heterogenity <strong>of</strong> the pathogen response. At 24 h after infection<br />
inhibition <strong>of</strong> photosynthetic electron transport was restricted to the direct vicinity <strong>of</strong> the<br />
infection site, which was surrounded by a ring <strong>of</strong> increased photosynthetic activity. The<br />
photosynthesis <strong>of</strong> the remaining leaf was not affected at this stage. These results show<br />
the usefulness <strong>of</strong> chlorophyll fluorescence imaging for assessment <strong>of</strong> the complex spatiotemporal<br />
changes and for definition <strong>of</strong> the areas relevant for other types <strong>of</strong><br />
determinations, as e.g. gene expression.<br />
contact:<br />
Katharina Bonfig<br />
Universität Würzburg<br />
Lehrstuhl für Pharmazeutische Biologie<br />
Bonfig@biozentrum.uni-wuerzburg.de<br />
Julius-von-Sachs-Platz 2<br />
97082 Würzburg (Deutschland)
Ernst Bamberg<br />
Conformational dynamics <strong>of</strong> P-type ATPases: Intraprotein<br />
signalling and subunit interaction probed by voltage<br />
fluorometry<br />
No abstract submitted<br />
contact:<br />
Pr<strong>of</strong>. Dr. Ernst Bamberg<br />
MPI für Biophysik<br />
Abt. Biophysikalische Chemie<br />
ernst.bamberg@mpibp-frankfurt.mpg<br />
Marie-Curie-Str. 13-15<br />
60439 Frankfurt a.M. (Deutschland)
Michael Spörner, Thosten Graf, Burkhard König, Hans Robert Kalbitzer<br />
Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour<br />
therapy<br />
The small GTPase Ras is involved in cellular signal transduction. There it acts as a<br />
molecular switch cycling between an active GTP-bound (on) state and an inactive GDPbound<br />
(<strong>of</strong>f) state. Effector proteins like Raf-kinase bind with high affinity to the active<br />
conformation <strong>of</strong> Ras. It is estimated that over 30% <strong>of</strong> all human tumors contain an<br />
activating mutation <strong>of</strong> Ras. In contrast to the X-ray structure Ras(wt) exists in solution<br />
in at least two different conformational states when it is bound to guanosine tri<br />
phosphates. These are in a dynamical equilibrium as shown by 31P NMR studies. One <strong>of</strong><br />
these states (state 2) seems to be very similar to Ras in complex with effectors. In the<br />
other state (state 1) Ras shows a drastically reduced affinity to effector proteins. The<br />
stabilization <strong>of</strong> state 1 looks like a promising approach for the suppression <strong>of</strong> Ras<br />
dependent tumor growth. Cyclene binds to the active site <strong>of</strong> the protein only when Ras is<br />
in state 1 and is thus stabilizing this state. With relaxation time measurements we were<br />
able to estimate the distance <strong>of</strong> Cu2+-Cyclene to the phosphate groups <strong>of</strong> the bound<br />
nucleotide. Cyclene is a magnificent lead structure for pharmacological inhibition <strong>of</strong> the<br />
Ras dependent signal transduction. By structure based modelling we will increase the<br />
affinity between Cyclene and Ras to shift the equilibrium predominantly towards state 1<br />
and thus inhibit Ras-effector interaction.<br />
contact:<br />
Dr. Michael Spörner<br />
Universität Regensburg<br />
Institut für Physikalische Biochemie und Biophysik<br />
michael1.spoerner@biologie.uni-regensburg.de<br />
Universitätsstraße 31<br />
93053 Regensburg (Germany)
Beate Rinner, Harald Greger, Brigitte Brem, Peter Pürstner, Veronika Siegl, Thomas<br />
Efferth, Roswitha Pfragner<br />
Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Medullary thyroid carcinoma (MTC) is a rare calcitonin-producing tumor, derived from<br />
the parafollicular C-cells <strong>of</strong> the thyroid. MTC is known to be relatively insensitive to<br />
conventional chemotherapy. Based on this fact, we tested the effects <strong>of</strong> extracts <strong>of</strong><br />
selected plants in seven MTC cell lines. Each cell line showed an upregulation <strong>of</strong> bcl-2.<br />
We investigated eleven agents from plants <strong>of</strong> the genera Stemona (Stemonaceae),<br />
Aglaia (Meliaceae) and Artemisia (Asteraceae) for their effects on proliferation and<br />
apoptotic rates. Growth kinetics and viability were examined using the Casy-1 Cell<br />
Counter and Analyzer, the WST-based cytotoxicity assay and furthermore, the lactate<br />
dehydrogenase assay. Apoptosis was studied with DAPI staining, DNA fragmentation and<br />
by measuring caspase-3 activity and bcl-2 expression. Two breast carcinoma cell lines<br />
were also tested. To compare the effects in tumor cells with non-cancer cells, human<br />
skin fibroblasts were taken as controls. All tumor cell lines were sensitive towards the<br />
drugs. Even very low concentrations <strong>of</strong> Aglaia extracts and Artesunate reduced<br />
proliferation and provoked necrosis; however, apoptosis could not be identified. Aglaia<br />
produced less sensitive effects on proliferation <strong>of</strong> human skin fibroblasts. Treatment with<br />
Stemona extracts reduced proliferation <strong>of</strong> MTC and breast carcinoma cells. Apoptotic<br />
signals could be detected by DNA fragmentation, a positive DAPI-stain, flow cytometric<br />
analysis, and caspase-3 activity. In comparison to tumor cells, when human skin<br />
fibroblasts were treated with Stemona, these normal cells were less impaired. The new<br />
anti-cancer botanical possibly <strong>of</strong>fers a new approach towards successful chemotherapy<br />
<strong>of</strong> the so far chemoresistant medullary thyroid carcinoma.<br />
contact:<br />
Mag. Dr. Beate Rinner<br />
Medizinische Universität Graz<br />
Pathophysiologie Institut<br />
beate.rinner@meduni-graz.at<br />
Heinrichstraße 31<br />
8010 Graz (Austria)
Oliver Bremm, Andre Remy, Klaus Gerwert<br />
Coupling <strong>of</strong> light-induced electron transfer to proton uptake in<br />
photosynthesis<br />
Light energy is transformed into chem. energy in photosynthesis by coupling<br />
a light-induced electron transfer to proton uptake. The resulting proton<br />
gradient drives ATP synthesis. In this study, we monitored the light-induced reactions in<br />
a 100-kDa photosynthetic protein from 30 ns to 35 s by FTIR difference spectroscopy.<br />
The results provide detailed mechanistic<br />
insights into the electron and proton transfer reactions <strong>of</strong> the QA to QB<br />
transition: redn. <strong>of</strong> QA in picoseconds induces protonation <strong>of</strong> histidines,<br />
probably <strong>of</strong> His126 and His128 in the H subunit at the entrance <strong>of</strong> the proton<br />
uptake channel, and <strong>of</strong> Asp210 in the L subunit inside the channel at 12 •s<br />
and 150 •s. This seems to be a prerequisite for the reduction <strong>of</strong> QB, mainly at 150 •s.<br />
QA- is reoxidized at 1.1 ms, and a proton is transferred from<br />
Asp210 to Glu212 in the L subunit, the proton donor to QB-. Notably, our<br />
data indicate that QB is not reduced directly by QA- but presumably through<br />
an intermediary electron donor.<br />
Literature<br />
[1] Remy, A., Boers, R.B., Egorova-Zachernyuk, T., Gast, P., Lugtenburg, J., Gerwert,<br />
K., European Journal <strong>of</strong> Biochemistry, 270(17), 3603-3609 (2003)<br />
[2] Remy, A., Gerwert, K., Nature Structural Biology, 10(8), 637-644 (2003)<br />
contact:<br />
Oliver Bremm<br />
Ruhr Universität Bochum<br />
Lehrstuhl für Biophysik<br />
oliver.bremm@rub.de<br />
Universitätsstr. 150<br />
44780 Bochum (Deutschland)
Thomas Carell, Alexandra Mees, Tobias Klar, Andre P. M. Eker, Lars-Oliver Essen<br />
Crystal structure <strong>of</strong> DNA Photolyase complexed to double<br />
stranded DNA<br />
Life in sun light encounters genome damage by ultraviolet radiation. The most abundant<br />
UV-induced DNA lesions are cis-syn cyclobutane pyrimidine dimers (CPD) which are<br />
generated by a photochemical [2+2] cycloaddition. For DNA repair photolyases exploit<br />
the energy <strong>of</strong> blue light to pump an electron from a catalytic FAD c<strong>of</strong>actor onto the CPDdamage<br />
so that the cyclization can be reversed via a radical intermediate. So far, only<br />
theoretical models for light-driven electron transfer were available due to a lack <strong>of</strong><br />
structural information about the substrate complex.<br />
Cocrystals <strong>of</strong> a cyanobacterial photolyase were grown in complex with duplex DNA that<br />
contained a synthetic CPD lesion. The X-ray structure was solved at 1.8 Å resolution and<br />
showed four photolyase complexes which adopt two alternative states in the crystals: In<br />
two complexes the enzyme is specifically bound to dsDNA, whereas in two other<br />
complexes only short stretches <strong>of</strong> ssDNA were observed. In the complexes with dsDNA,<br />
DNA bending is accompanied by a flip <strong>of</strong> the thymine dimer into the active site. As<br />
potential electron transfer route the C4-carbonyl groups <strong>of</strong> the thymines form direct Hbonds<br />
with the N6-adenine <strong>of</strong> the FADH c<strong>of</strong>actor. Interestingly, the synchrotron radiation<br />
generated a cryo-trapped cleavage intermediate <strong>of</strong> the CPD lesion. As photolyases<br />
catalyze the repair <strong>of</strong> thymine dimers at room temperature in less than two nanoseconds<br />
we have now a view on a very early step <strong>of</strong> light-driven DNA repair.<br />
Literature<br />
Mees, A., Klar, T., Gnau, P., Eker, A. P. M., Carell, T., Essen, L.-O. (submitted). Crystal<br />
structure <strong>of</strong> a photolyase-DNA complex after in situ repair <strong>of</strong> a CPD-like DNA lesion.<br />
Science.<br />
contact:<br />
Pr<strong>of</strong>. Lars-Oliver Essen<br />
Philipps-University<br />
Department <strong>of</strong> Chemistry<br />
essen@chemie.uni-marburg.de<br />
Hans-Meerwein-Strasse<br />
35032 Marburg (Germany)<br />
additional information<br />
Alexandra Mees, Thomas Carell: Department <strong>of</strong> Chemistry and Pharmacy, Butenandt-Strasse 5-13,<br />
Ludwig-Maximilians-University, 81377 Munich, Germany<br />
Andre P. M. Eker: Department <strong>of</strong> Cell Biology and Genetics, Medical Genetics Centre, Erasmus-<br />
University, PO Box 1738, 3000 DR Rotterdam, The Netherlands
Roswitha Pfragner, A. Stift*, J. Friedl*, B. Niederle*, M. Gnant*<br />
Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent<br />
Advances in Immunotherapy<br />
Surgery is the only effective treatment for medullary thyroid carcinoma (MTC). A<br />
promising approach in anti-tumor therapy is the activation <strong>of</strong> the patient’s immune<br />
system against malignant cells. One <strong>of</strong> these strategies deals with the stimulation <strong>of</strong><br />
resting T-cells by dendritic cells (DC), the most potent antigen-presenting cells for naïve<br />
T-cell activation. Autologous DCs were derived from peripheral blood monocytes by<br />
using CD14 magnetic bead selection, and subsequent cultivation in the presence <strong>of</strong><br />
granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4).<br />
The immature DCs were further induced to mature by tumor necrosis factor alpha (TNF<br />
alpha). Autologous tumor lysate was used to prime DCs, and fully mature pulsed DCs<br />
were injected into patients’ lymph nodes in 4-weekly intervals. We have so far treated<br />
10 patients with MTC. Treatment was well tolerated in all cases. Tumor marker<br />
responses were seen in 7 out <strong>of</strong> 10 patients, and 3 out <strong>of</strong> 10 patients showed formal<br />
WHO response criteria. These results <strong>of</strong> our pilot trial are promising, and the options <strong>of</strong><br />
clinical immunotherapy for MTC patients are further explored. However, the therapeutic<br />
success <strong>of</strong> this approach is sometimes critically limited by the small size <strong>of</strong> available<br />
tumors, resulting in too little tumor lysate for repeated treatment <strong>of</strong> the patients. This<br />
can particularly be a problem when earlier stages <strong>of</strong> the disease will be treated. An<br />
alternative is the cultivation and enrichment <strong>of</strong> autologous tumor cells. The aim is to get<br />
a minimum <strong>of</strong> 1x10 7 autologous tumor cells within a few weeks. We have developed<br />
optimized culture conditions for MTC, <strong>of</strong>fering a limitless availability <strong>of</strong> autologous tumor<br />
lysate for repeated treatment <strong>of</strong> patients.<br />
contact:<br />
Pr<strong>of</strong> Dr Roswitha Pfragner<br />
Medizinische Universität Graz<br />
Institut für Pathophysiologie<br />
roswitha.pfragner@meduni-graz.at<br />
Heinrichstrasse 31<br />
A 8010 Graz (Österreich)<br />
additional information<br />
*) Department <strong>of</strong> Surgery, Medical University Vienna, Austria
Hameeda Sultana, Angelika.A Noegel<br />
Cyclase associated protein CAP in the regulation <strong>of</strong> the actin<br />
cytoskeleton and cell polarity in Dictyostelium<br />
The link between signal transduction and subsequent alterations in the cytoskeleton is a<br />
central theme in cytoskeleton research. A protein that may provide this link is the<br />
cyclase associated protein, CAP, first found in Saccharomyces cerevisiae. The CAP<br />
homologue <strong>of</strong> Dictyostelium discoideum is a PIP 2 regulated G-actin sequestering protein<br />
which is present in the cytosol and shows enrichment at the plasma membrane regions.<br />
For the better understanding <strong>of</strong> CAP in the regulation <strong>of</strong> the actin cytoskeleton and <strong>of</strong><br />
cell polarity in Dictyostelium we have made use <strong>of</strong> mutants affected in signaling<br />
pathways like mutants deficient for ACA, G-protein α2 and β subunit, the ACA-regulator<br />
Pianissimo (PIA), cAMP receptor CAR1/3, temperature sensitive mutant <strong>of</strong> PIA knocked<br />
out for ACA and ACR (adenylyl cyclases), PI3-kinase1/2 and the central player <strong>of</strong> cAMP<br />
signal transduction the protein kinase A (PKA). The localisation and functioning <strong>of</strong> CAP<br />
was analysed in these mutants to see if these signaling molecules are important for<br />
targeting <strong>of</strong> CAP to the cell cortex, and also in the relocalisation <strong>of</strong> CAP upon a stimulus<br />
during the events <strong>of</strong> actin involvement processes (phagocytosis, pinocytosis).<br />
Our studies revealed that ACA, Gα2, Gβ, ACA regulator pianissimo (PIA), PI3-kinase 1/2,<br />
CAR1/3, and piaA with double mutants <strong>of</strong> ACs are not required for targeting <strong>of</strong> CAP to<br />
the cell cortex and for correct functioning <strong>of</strong> CAP. However, we found that PKA mutant<br />
showed an aberrant localization <strong>of</strong> CAP where CAP was preferentially accumulated in the<br />
crown shaped structures <strong>of</strong> the cell. Furthermore, we found that expression <strong>of</strong> CAP has a<br />
positive effect on phagocytosis <strong>of</strong> the Gβ mutant and completely rescued the severe<br />
impairment in pinocytosis <strong>of</strong> the PI3-kinase1/2 mutant. We also observed that<br />
aggregation deficient aca null cells expressing GFP-CAP were able to form EDTA sensitive<br />
cell contacts and develop into aggregating streams and mounds. We conclude that CAP<br />
is a mutifunctional protein with many activities at the cellular level, where it shows an<br />
involvement in the actin dependent processes and developmemt. Taken together, all our<br />
analysis and future studies are aiming at unraveling the link <strong>of</strong> CAP with the<br />
cytoskeleton and signaling pathways in the cell.<br />
contact:<br />
M.Sc. Hameeda Sultana<br />
University <strong>of</strong> Cologne<br />
Institute for Biochemistry I<br />
ake57@uni-koeln.de<br />
Joseph-Stelzmann-Str. 52<br />
50931 Cologne (Germany)<br />
additional information<br />
co-author: Pr<strong>of</strong>. Dr. Angelika A. Noegel
Nediljko Budisa<br />
Designing novel classes <strong>of</strong> fluorescent proteins with an<br />
expanded genetic code<br />
Fluorescence methods are now well established and powerful tools to study biological<br />
macromolecules. The canonical amino acid tryptophan (Trp) encoded by a single UGG<br />
triplet is the main reporter <strong>of</strong> intrinsic fluorescence properties <strong>of</strong> most natural proteins<br />
and peptides and is thus an attractive target for tailoring their spectral properties.<br />
Recent advances in research have provided substantial evidences that the natural<br />
protein translational machinery can be reprogrammed to genetically encode a large<br />
number <strong>of</strong> non-coded (i.e. noncanonical) Trp analogues and surrogates in various<br />
proteins. Especially attractive targets for such an engineering approach are fluorescent<br />
proteins in which the chromophore is formed post-translationally from an amino acid<br />
sequence like the green fluorescent protein from Aequorea victoria. With the currently<br />
available translationally active fluoro-, hydroxy-, amino-, halogen-, and chalcogencontaining<br />
Trp analogues and surrogates, the traditional methods for protein engineering<br />
and design can be supplemented or even fully replaced by these novel approaches.<br />
Future research will provide a further increase in the number <strong>of</strong> Trp-like amino acids that<br />
are available for redesign (by engineering <strong>of</strong> the genetic code) <strong>of</strong> native Trp-residues<br />
and enable novel strategies to generate proteins with tailored spectral properties.<br />
Literature<br />
Bae, J.H., et.al. (2003) Journal <strong>of</strong> Molecular Biology, 328, 1071-1081.<br />
Budisa, N. (2004) Angewandte Chemie, 45, In press.<br />
Budisa, N., et.al.(2004)Biological Chemistry, 385, 191-202.<br />
Budisa, N., et.al. (2002) Angewandte Chemie-International Edition, 41, 4066-4069.<br />
Budisa, N. (2003) In Swartz, J.R. (ed.), Cell Free Protein Expression. Springer Verlag,<br />
Berlin, Heidelberg, New York, pp. 89-98.<br />
contact:<br />
Dr. Nediljko Budisa<br />
Max-Planck-Institut für Biochemie<br />
Abt. Strukturforschung<br />
budisa@biochem.mpg.de<br />
Am Klopferspitz 18A<br />
82152 Martinsried (Germany)
Thomas Schulenborg, Heike Schäfer, Christian Bunse, Helmut E. Meyer, Katrin Marcus<br />
Detection <strong>of</strong> phosphorylation sites using a hybrid triple<br />
quadrupole/linear ion trap mass spectrometer<br />
Phosphorylation is a very prominent post-translational modification (PTM) <strong>of</strong> proteins.<br />
PTM, especially phosphorylation, plays an important role in regulation <strong>of</strong> many cellular<br />
processes, folding and localisation <strong>of</strong> proteins and the interaction <strong>of</strong> proteins (Kurosawa,<br />
1994). One expects, that every second protein has modifications like phosphorylation or<br />
glycosylation (Sickmann et al., 2003). In general phosphorylations in signalling cascades<br />
are accompanied by dephosphorylation. Hence, the absolute quantity <strong>of</strong> phosphorylated<br />
protein species is less than 1% <strong>of</strong> the whole protein amount. Assuming one 2D-gel spot<br />
contains about 1-10 pmol protein just 10-100 fmol <strong>of</strong> the phosphorylated species is<br />
available for the proteomic analysis, what means, that identification and localisation <strong>of</strong><br />
phosphorylation sites is still a challenge in proteome analysis today. In the presented<br />
work the localisation <strong>of</strong> phosphorylation sites <strong>of</strong> different proteins, e.g. αA-Crystallin,<br />
PKA, β-casein is shown using the 4000 Q Trap® – a new hybrid triple quadrupole/linear<br />
ion trap mass spectrometer. For analysis, the proteins were separated by 2D-PAGE or<br />
1D-PAGE, respectively. Protein spots were excised and tryptically digested. Resulting<br />
peptides were separated by nanoRP-HPLC and the phosphorylation sites were localised<br />
after a database search using Proteinscape and the PTM-Explorer (both Protagen AG,<br />
Dortmund).<br />
Literature<br />
Kurosawa, M: Phosphorylation and dephosphorylation <strong>of</strong> protein in regulating cellular<br />
function, J Pharmacol Toxicol Methods, 1994, 31, 135-9<br />
Sickmann, A, Mreyen, M, Meyer, HE: Mass spectrometry - a key technology in proteome<br />
research, Adv Biochem Eng Biotechnol, 2003, 83, 141-76<br />
contact:<br />
Dipl.-Biochem. Thomas Schulenborg<br />
Ruhr-Universität Bochum<br />
Medizinisches Proteom-Center<br />
thomas.schulenborg@rub.de<br />
Universitätsstr. 150<br />
44801 Bochum (Deutschland)
Monika Kogler-Voigt, Roswitha Pfragner, Sabine Supanz, Christa Nöhammer, Konrad<br />
Schauenstein, Karl-Heinz Preisegger<br />
Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease<br />
In breast cancer, only a rare, phenotypically distinct subpopulation <strong>of</strong> all tumor cells is<br />
committed to form metastases. One <strong>of</strong> the cell surface markers by which this<br />
subpopulation can be identified, CD44 v6, is an adhesion molecule with hyaluronan as its<br />
main ligand <strong>of</strong> the extracellular matrix. CD44 v6 is involved in many biological processes<br />
like migration <strong>of</strong> cells, hematopoiesis and activation <strong>of</strong> lymphocytes, but also in<br />
migration <strong>of</strong> tumor cells and metastases. Tumor cells disseminate from the primary<br />
tumor to secondary organs at an early stage <strong>of</strong> primary tumor developement and are<br />
determinants <strong>of</strong> subsequent metastases formation. Early detection <strong>of</strong> recurrent disease<br />
by screening peripheral blood on the presence <strong>of</strong> disseminated tumor cells by<br />
immunocytochemical identification <strong>of</strong> those cells could be a helpful tool for determining<br />
the prognosis <strong>of</strong> patients with breast cancer. For validation <strong>of</strong> a reliable method for early<br />
detection <strong>of</strong> tumor cells in the blood we created a model system spiking peripheral blood<br />
<strong>of</strong> healthy donors with different numbers <strong>of</strong> CD44 v6 expressing tumor cells to<br />
determine the recovery rate in peripheral blood by comparing two density gradient<br />
centrifugation methods. Preliminary results with three different cell lines revealed a<br />
recovery rate <strong>of</strong> 92,8% <strong>of</strong> the tumor cells spiked into peripheral blood, suggesting that<br />
CD44v6 is a clinically useful marker for tumor cell detection.<br />
contact:<br />
Mag. Monika Kogler-Voigt<br />
EccoCell Biotechnologie GmbH<br />
monika.voigt@meduni-graz.at<br />
Keplerstr. 105/1<br />
8020 Graz (Österreich)<br />
additional information<br />
R.P., S.S. & K.S.: Center <strong>of</strong> Molecular Medicine, Medical University Graz, Austria<br />
C.N.: Seibersdorf Research GmbH, Seibersdorf, Austria
Wolfgang D. Schmitt, Stephan Ruhla, Steffen Hauptmann<br />
Detection <strong>of</strong> Retrotransposition in Cancer<br />
More than 45% <strong>of</strong> the human genome consists <strong>of</strong> transposable elements, which may be<br />
classified in transposons (´cut and paste´) and retrotransposons (´copy and paste´).<br />
Among the latter ones are human endogenous retroviruses (HERV), ´long intersperced<br />
nucleotide elements´ (LINE) and ´Alu´-Elements. It seems that only LINE´s are still<br />
active in human beings, DNA transposons and HERV are presumed to be remnants <strong>of</strong> a<br />
past area, and Alu elements are dependent on the enzymatic machinery <strong>of</strong> other<br />
transposable elements.<br />
Until today, constitutive activity <strong>of</strong> retrotransposons was only detected in reproductive<br />
organs, lymphatic tissue, and in some cancer cell lines. This activity leads to<br />
rearrangements <strong>of</strong> the genome, but may also influence the activity <strong>of</strong> distant genes. This<br />
function <strong>of</strong> retrotransposons was first described by Barbara McClintock - she was<br />
honoured for this discovery with the nobel price in 1983.<br />
It is remarkable that most cancers show genetic alterations and also a high variance in<br />
gene expression compared to their tissue <strong>of</strong> origin. Both observations might be caused<br />
by retrotransposition. So we propose a connection between activation <strong>of</strong> transposable<br />
elements and cancer.<br />
In this work, we present some possibilities to detect activity <strong>of</strong> the LINE-machinery at<br />
the level <strong>of</strong> mRNA and protein. Furthermore, we examined complete retrotransposition<br />
events in a number <strong>of</strong> cancer and non-cancer cell lines and finally investigated tissue<br />
samples obtained from specimen <strong>of</strong> routine surgical pathology.<br />
Literature<br />
McClintock, B.: Controlling elements and the gene, Cold Spring Harb. Symp. Quant. Biol.<br />
21: 197-216 (1956)<br />
Ostertag, E.M. and Kazazian, H.H., Jr.: Biology <strong>of</strong> mammalian L1 retrotransposons,<br />
Annu.Rev.Genet. 35: 501-538 (2001)<br />
contact:<br />
Wolfgang D. Schmitt<br />
Martin-Luther-Universität Halle-Wittenberg<br />
Institut für Pathologie<br />
wolfgang.schmitt@medizin.uni-halle.de<br />
Magdeburger Straße 14<br />
06097 Halle (Saale) (Germany)
Maria Iris Hermanns, Sabine Fuchs, Ron Unger, Charles James Kirkpatrick<br />
Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-<br />
Well Screening <strong>of</strong> Barrier Properties<br />
Introduction In order to study barrier properties at the human alveolo-capillary barrier,<br />
we have developed a co-culture system based on a human lung cell line with<br />
characteristics <strong>of</strong> type II pneumocytes and Clara cells (NCI H441) (1, 2) and primary<br />
isolated human pulmonary microvascular endothelial cells (HPMEC) (3). Methods NCI<br />
H441 in co-culture with HPMEC on opposite sides <strong>of</strong> a permeable 24-well filter plate<br />
constitute the in vitro bilayer model. Co-cultures were treated with dexamethasone on<br />
day 3 <strong>of</strong> co-cultivation to generate a polarized epithelial cell monolayer. Functional<br />
barrier properties were investigated by measuring trans-bilayer electrical resistance<br />
(TER) and paracellular transport <strong>of</strong> sodium-fluorescein. Immun<strong>of</strong>luorescence was<br />
performed using standard techniques. Results NCI H441 in co-culture with HPMEC<br />
established contact inhibited monolayers, showing a continuous, circumferential<br />
immunostaining <strong>of</strong> the tight junctional protein, ZO-1 and the adherens junction proteins,<br />
E-Cadherin, alpha- and ß-Catenin. Maximum TER-values were found after 10 to 12 days<br />
<strong>of</strong> co-cultivation and averaged 500 Ohm*cm2 correlated with Papp-values <strong>of</strong> 0.181 ±<br />
0.021 10-6 cm*sec-1. This time schedule was chosen for exposure to the<br />
proinflammatory cytokine TNF-a from the endothelial side (basolateral) and the epithelial<br />
side (apical). Basolateral TNF-a exposure caused a significant reduction <strong>of</strong> TER-values<br />
after 24 h. This decrease in TER could be re-established to control level after removal <strong>of</strong><br />
the cytokine within 24 h. Conclusions Our co-culture system <strong>of</strong> NCI H441 with HPMEC is<br />
a suitable in vitro model to examine barrier function at the distal lung including the<br />
interaction <strong>of</strong> both epithelium and microvascular endothelium.<br />
Literature<br />
1. Gazdar et al. (1990) Am Rev Resp Dis, 131, 786-788.<br />
2. Pryhuber et al. (1998) Am J Physiol Lung, 274, L289-295.<br />
3. Hermanns et al. (2004) Lab Invest, 84, 736-752.<br />
contact:<br />
Maria Iris Hermanns<br />
Johannes Gutenberg University <strong>of</strong> Mainz<br />
Institute <strong>of</strong> Pathology<br />
hermanni@uni-mainz.de<br />
Langenbeckstr. 1<br />
55101 Mainz (Germany)
Heiko Andresen, Kim Zarse, Carsten Grötzinger, Oliver J. Kreuzer, Marc Birringer, Eva<br />
Ehrentreich-Förster, Frank F. Bier<br />
Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Miniaturisation <strong>of</strong> standard analytical techniques is a prominent task <strong>of</strong> biomedical<br />
research and development. Special attention is given to the microarray technology<br />
whose diagnostic potential enhanced and complemented genome and transcriptome<br />
analysis in the human genome project. The human proteome, however, is a multiple<br />
more complex than the human genome, emphasizing the need <strong>of</strong> an analytic tool on<br />
protein level that is comparable in efficiency and performance to the DNA microarray<br />
technology. Synthetic low-molecular weight peptides have outstanding purposes for use<br />
as variable probes in chip-based analysis, since they can be assembled in defined<br />
orientation and high density. Furthermore, fully automated synthesis makes them an<br />
economically attractive alternative to recombinant proteins. We developed a peptide chip<br />
for assaying the binding reaction <strong>of</strong> specific monoclonal antibodies to linear peptide<br />
epitopes. For the prototypic version, 13mer peptides mimicking linear epitopes <strong>of</strong> model<br />
proteins were synthesized by standard Fmoc-synthesis, equipped with a biotin linker and<br />
printed on NeutrAvidin-coated glass slides. The peptide probes were used to capture<br />
specific monoclonal antibodies with known linear epitopes. A fluorescently labelled<br />
secondary antibody was used for array readout. The peptide chip assay showed highly<br />
specific signals for all tested antibodies and sensitivity in the range <strong>of</strong> ng•ml -1 .<br />
contact:<br />
Dipl.-Ing. Heiko Andresen<br />
Fraunh<strong>of</strong>er Institut f. Biomedizinische Technik<br />
Molekulare Bioanalytik und Bioelektronik<br />
heiko.andresen@ibmt.fhg.de<br />
Arthur-Scheunert-Allee<br />
14558 Nuthetal (Deutschland)
Sabine Fuchs, Ron Unger, Maria Iris Hermanns, Kirsten Peters, Charles James<br />
Kirpatrick<br />
Different populations <strong>of</strong> endothelial progenitor cells for<br />
applications in tissue engineering<br />
Recent studies underline that EPC from peripheral blood resemble a heterogeneous cell<br />
population with different capacities to assume a differentiated and functional endothelial<br />
phenotype in vitro or with respect to proliferation. In animal studies it was shown that<br />
different populations within EPC can improve the process <strong>of</strong> neovascularization in<br />
ischemic tissues, although the underlying mechanisms are still unclear.<br />
In this study, we tested different populations in EPC for the endothelialization <strong>of</strong> silk<br />
fibroin scaffolds recently described by Unger et. al. 2004.<br />
Different populations <strong>of</strong> EPC, early EPC with an undifferentiated phenotype and<br />
outgrowing endothelial cells (OEC) with typical cobblestone like morphology and a higher<br />
proliferation capacity, were first tested for endothelial markers. Both populations showed<br />
endothelial marker such as CD31 and VE-Cadherin, whereas vWF was restricted to the<br />
OEC and CD45 to the early EPC.<br />
Furthermore, we tested the interaction <strong>of</strong> early EPC with mature endothelial cells. In cocultures<br />
some EPC integrated into endothelial cell layers. In these co-cultures EPC<br />
formed typical endothelial cell contacts with HUVECs or the endothelial cell line Isohas-1<br />
as indicated by immun<strong>of</strong>luorescent staining for CD31 and VE-Cadherin. In co-cultures<br />
they also showed other endothelial markers such as vWF but also stained positive for<br />
CD45.<br />
OEC cultured on the fibroin nets, adhered to the surface and spread along and between<br />
the fibers. The endothelial cells also showed typical endothelial cell-cell contacts by<br />
immun<strong>of</strong>luorescent staining for CD31 and VE-Cadherin. Our studies indicate that the<br />
OEC fraction in EPC are an attractive source <strong>of</strong> endogenous cells for the<br />
endothelialization <strong>of</strong> biomaterials.<br />
Literature<br />
Unger RE, Peters K, Wolf M, Motta A, Migliaresi C, Kirkpatrick CJ (2004)<br />
Endothelialization <strong>of</strong> a non-woven silk fibroin net for use in tissue engineering: growth<br />
and gene regulation <strong>of</strong> human endothelial cells. Biomaterials 25:5137-5146<br />
contact:<br />
Dr. Sabine Fuchs<br />
Johannes Gutenberg University<br />
Institute <strong>of</strong> Pathology<br />
fuchss@uni-mainz.de<br />
Langenbeckstr.<br />
55101 Mainz (Germany)
Stefan Helling, Petra Lutter, Petra Weingarten, Christoph Hüls, Helmut Jonuleit, Edgar<br />
Schmitt, Helmut E. Meyer, Katrin Marcus<br />
Differential analysis <strong>of</strong> T cell membrane proteins<br />
Cellular immunity is mainly regulated by different types <strong>of</strong> T cells, which are able to<br />
stimulate or suppress an immune response against antigens. T cells differentiate through<br />
induction by metabolites like cytokines and antigens or by cell-cell contacts. The<br />
initiating factor <strong>of</strong> this signal transduction process is the binding <strong>of</strong> cell surface proteins<br />
to their counterparts.<br />
To analyze the different cell states and molecular changes on the cell surface with<br />
proteomic tools the enrichment and the purification <strong>of</strong> the membrane proteins is<br />
necessary. The classical isolation <strong>of</strong> the plasma membrane (PM) by cell lysis followed by<br />
a combination <strong>of</strong> differential and density gradient centrifugation leads to losses <strong>of</strong> the PM<br />
material and the enclosed proteins. Incomplete lysis or the co-sedimentation with nuclei<br />
and other organelles are the main reasons for this phenomenon. Analyses with freshly<br />
isolated and highly purified T cells are limited by the available cell amounts. Therefore<br />
complex purification strategies are hard to realize.<br />
In the present study, a detergent based isolation method for membrane proteins<br />
optimized to minimal losses is described. This method was optimized on resting and<br />
stimulated Jurkat T cells and transferred to the analysis <strong>of</strong> freshly isolated human and<br />
murine T cells.The enriched proteins were separated by gel-based methods optimized for<br />
membrane proteins and identified by LC-ESI-MS or immunodetection <strong>of</strong> selected PM<br />
specific proteins.<br />
Literature<br />
Bordier C (1981) Phase separation <strong>of</strong> integral membrane proteins in Triton X-114<br />
solution. J Biol Chem 256:1604-1607<br />
contact:<br />
Dipl. Biol. Stefan Helling<br />
Ruhruniversität Bochum<br />
Medizinisches Proteom-Center<br />
stefan.helling@rub.de<br />
Universitätsstr. 150<br />
44780 Bochum (Deutschland)
Tadaomi Takenawa, Shiro Suetsugu, Daisuke Yamazaki<br />
Differential functions <strong>of</strong> WAVE1 and 2 in cell migration and<br />
spreading.<br />
WASP family proteins are known to activate Arp2/3 complex through VCA regions,<br />
leading to a new actin polymerization. WASP proteins act for filopodium formation<br />
downstream <strong>of</strong> Cdc42. WAVE proteins play crucial roles in membrane ruffle and<br />
lamellipodium formation downstream <strong>of</strong> Rac. There are three types <strong>of</strong> WAVEs, WAVE1, 2<br />
and 3. Among these WAVEs, WAVE1 and 2 are expressed in mouse fibroblasts.<br />
To study the differential roles <strong>of</strong> WAVE1 and WAVE2 in ruffling formation, cell migration<br />
and cell spreading, we prepared WAVE1- and WAVE2-deficient MEF cells from WAVE1<br />
and WAVE2 KO mice. When cells were stimulated with PDGF, two types <strong>of</strong> ruffles,<br />
peripheral ruffles and dorsal ruffles are formed. WAVE 1 is dominantly present in dorsal<br />
ruffles, while WAVE2 is present in the tip <strong>of</strong> lamellipodia. Interestingly, WAVE2deficiency<br />
impaired peripheral ruffle formation and WAVE1-deficiency impaired dorsal<br />
ruffle formation. On the other hand, when cells migrate toward chemoattractants, only<br />
peripheral ruffle, so called lamellipodia are formed at the leading edge. WAVE2-deficient<br />
MEF cells could not form marked lamellipodia, while WAVE1-deficient MEFs could. All<br />
these data suggest that WAVE1 and WAVE2 have different functions.<br />
Literature<br />
1. Suetsugu, S., Yamazaki, D., Kurisu, S., and Takenawa, T.: Differential roles <strong>of</strong> WAVE1<br />
and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration., Dev.<br />
Cell. 5, 595-609 (2003)<br />
2. Oikawa, T., Yamaguchi, H., Itoh, T., Kato, M., Ijuin, T., Yamazaki, D., Suetsugu, S.,<br />
and Takenawa, T. :PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation <strong>of</strong><br />
lamellipodia., Nature Cell Biol. 6, 420-426 (2004).<br />
contact:<br />
Pr<strong>of</strong>essor Tadaomi Takenawa<br />
University <strong>of</strong> Tokyo<br />
Institute <strong>of</strong> Medical Science<br />
takenawa@ims.u-tokyo.ac.jp<br />
4-6-1 Shirokanedai, Minato-ku<br />
108-8639 Tokyo (Japan)
Buzz Baum<br />
Dissecting actin cytoskeletal organization in Drosophila using<br />
RNAi<br />
The flexible surface <strong>of</strong> an animal cell is given its dynamic form by the underlying actin<br />
cytoskeleton. Force is generated through actin polymerisation and by the action <strong>of</strong><br />
Myosin motors on the actin scaffold. Until recently, our understanding <strong>of</strong> actin<br />
cytoskeletal organisation in cells and embryos was hindered by the difficulty <strong>of</strong><br />
generating loss <strong>of</strong> function phenotypes in models <strong>of</strong> animal cell biology. With the<br />
discovery <strong>of</strong> dsRNA-mediated interference, or RNAi, it is now possible to identify<br />
regulators <strong>of</strong> morphogenesis in cell culture using a systematic functional genomic<br />
approach. We have adapted RNAi in Drosophila cell culture for use in genome-wide<br />
screens and have shown how RNAi and gene over-expression can be used place the<br />
morphological regulators identified within pathways. We are now carrying out modifier<br />
screens to refine this analysis. Our latest findings will be presented.<br />
Literature<br />
Kunda, P., Craig, G., Dominguez V., Baum B.Abi. Current Biology, 2003,(13) 21.<br />
Kiger, A., Baum, B., Jones, S., Jones, M., Coulson, A., Echeverri, C., Perrimon N. Journal<br />
<strong>of</strong> Biology 2003, (2)<br />
contact:<br />
Dr Buzz Baum<br />
LICR<br />
UCL branch<br />
b.baum@ucl.ac.uk<br />
91 Riding House St<br />
W1W7BS London (UK)
Wittaya Pimtong, Raimund Wagener, Mats Paulsson, Peter Bruckner, Uwe Hansen<br />
Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Background: Matrilin-1 is the prototype member <strong>of</strong> the matrilins which are a novel<br />
extracellular matrix protein family. It has been shown to bind collagens [1] and<br />
aggrecan [2]. Furthermore, it has been suggested that matrilin-1 is a link protein<br />
between different matrix components in order to form network like structures [3].<br />
However, the function <strong>of</strong> matrilin-1 is still unclear. Therefore, the aim <strong>of</strong> this study is the<br />
investigation <strong>of</strong> its distribution in articular bovine cartilage by using immuno-electron<br />
microscopy to approach the role <strong>of</strong> matrilin-1.<br />
Hypothesis: We concluded that matrilin-1 interacts with different extracellular matrix<br />
proteins in order to function as link protein.<br />
Experimental design: Small slices <strong>of</strong> articular bovine cartilage were homogenized with a<br />
Polytron in 15 volumes <strong>of</strong> PBS containing EDTA and proteinase inhibitor. In order to<br />
remove fibril associated proteins the fibrillar extract was treated with Gu-HCl. For further<br />
analysis <strong>of</strong> fibrillar extracts we used immunomagnetic beads in order to separate<br />
collagen VI containing suprastructures. The composition <strong>of</strong> the fibrillar extracts was<br />
examined by transmission- and immuno-electron microscopy, respectively.<br />
Conclusions: We found that matrilin-1 colocalised with COMP on collagen II-containing<br />
fibrils. But after a treatment with Gu-HCl the colocalisations decreased. In addition, we<br />
found that matrilin-1 colocalised with biglycan and decorin on collagen II-containing<br />
fibrils without Gu-HCl treatment. We concluded that the small leucine-rich proteins<br />
(SLRPs), biglycan and decorin are involved in the interaction between matrilin-1 and<br />
COMP with collagen II-containing fibrils.<br />
Furthermore, we found a colocalisation between matrilin-1 and COMP in non-fibrillar<br />
suprastructures. Interestingly, we found matrilin-1 and COMP on filamentous structures<br />
in a periodic pattern. We presumed that these filamentous structures are probably<br />
collagen VI micr<strong>of</strong>ibrils because we also found a strong labeling for collagen VI in close<br />
vicinity to matrilin-1 and COMP. It is already known that matrilin-1 binds near Nterminus<br />
<strong>of</strong> reconstituted collagen VI via biglycan or decorin [4]. In preliminary<br />
experiments, we separated collagen VI containing suprastructures out <strong>of</strong> crude extracts<br />
by using immunomagnetic beads. In such isolated suprastructures we found matrilin-1<br />
and COMP in close vicinity.<br />
Literature<br />
1. Winterbottom N. et al., Dev Dyn 1992,193;266-76<br />
2. Hauser N. et al., J Biol Chem 1996,271(50);32247-52<br />
3. Chen Q. et al., Mol Biol Cell 1999,10;2149-62<br />
4. Wiberg C. et al., J Biol Chem 2003,278;37698-704<br />
contact:<br />
Wittaya Pimtong<br />
University Hospital Münster<br />
Dept. Physiol. Chem. & Pathobiochem.<br />
pimtong@uni-muenster.de<br />
Waldeyerstr.15<br />
48149 Münster (Germany)
Robert Henderson, David Dryden, Michael Edwardson, Michael Waring<br />
DNA, Drugs and Proteins: Insights from the Atomic Force<br />
Microscope<br />
Since its adoption by the biomedical community in the early 1990s, the atomic force<br />
microscope has been widely used to study structure <strong>of</strong> and interaction between biological<br />
macromolecules. It has the advantage over other techniques that images can be<br />
obtained at a high resolution <strong>of</strong> molecules under fluid and in real time. One <strong>of</strong> the most<br />
fruitful areas <strong>of</strong> study has been in the field <strong>of</strong> DNA. To demonstrate the breadth <strong>of</strong><br />
information that can be obtained from this technique, this presentation will focus on the<br />
use in our laboratory <strong>of</strong> atomic force microscopy to investigate: (1) the mechanism <strong>of</strong><br />
action <strong>of</strong> at type I restriction enzyme, EcoKI, that binds to and cleaves DNA, and (2) the<br />
structural effects upon DNA <strong>of</strong> anti-cancer drugs that intercalate into the nucleic acid<br />
molecule.<br />
contact:<br />
Dr Robert Henderson<br />
University <strong>of</strong> Cambridge<br />
Department <strong>of</strong> Pharmacology<br />
rmh1003@cam.ac.uk<br />
Tennis Court Road<br />
CB2 1PD Cambridge (United Kingdom)
Peter Friedl<br />
Dynamic imaging <strong>of</strong> adhesion receptor and protease function<br />
in tumor cell invasion<br />
Cancer cell interactions with the extracellular matrix and the migration therein involve<br />
the function <strong>of</strong> adhesion receptors <strong>of</strong> the integrin family as well as proteolytic<br />
mechanisms to overcome tissue barriers. Using time-resolved bright field, confocal, and<br />
multiphoton microscopy, we have reconstructed molecular live-cell dynamics in tumor<br />
and immune cells. Cell interactions with collagen fibers in 3D tissue culture models and<br />
the mouse dermis were obtained by time-lapse confocal reflection and multiphoton<br />
second harmonic generation imaging, showing the adhesive traction and remodeling <strong>of</strong><br />
matrix structures by cancer cells (proteolytic migration mechanisms) as well as cellular<br />
shape changes adapting to tissue scaffolds (physical migration mechanisms). Timeresolved<br />
semiquantitative fluorescence resconstruction was used to monitor (i)<br />
cytoskeletal dynamics <strong>of</strong> filamentous actin by invading cancer cells, (ii) the subcellular<br />
topography <strong>of</strong> proteolytic cleavage <strong>of</strong> the extracellular matrix structures, and (iii)<br />
integrin-dependent (mesenchymal) as well as integrin –independent (amoeboid)<br />
migration modes. Using cocktails <strong>of</strong> specific protease and integrin inhibitors, we have<br />
obtained the following insight into cancer cell migration and cell-cell communication: the<br />
difference between individual and collective cancer cell migration; the belt-cleavage<br />
concept <strong>of</strong> pericellular proteolysis in migrating tumor cells; the mesenchymal-amoeboid<br />
transition as a novel escape strategy in tumor cell migration after inhibition <strong>of</strong> matrixdegrading<br />
proteases as well as after the inhibition <strong>of</strong> ß1 integrins; and the collectiveamoeboid<br />
transition after blocking <strong>of</strong> ß1 integrin-mediated traction and cell-cell cohesion<br />
in invasive cancer collectives. Together, these adaptation responses suggest an<br />
unexpected degree <strong>of</strong> ‘supramolecular’ plasticity and migratory ‘escape’ strategies that<br />
sustain persistent cancer cell dissemination in tissues despite interference with<br />
pharmacotherapeutic integrin and protease inhibitors.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Ph.D. (CDN) Peter Friedl<br />
Univ. Wuerzburg<br />
Rudolf-Virchow Center and Dept. <strong>of</strong> Dermatology<br />
peter.fr@mail.uni-wuerzburg.de<br />
Josef-Schneider-Str. 2<br />
97980 Wuerzburg (Germany)
Robin Vetter, Melanie Wagner, Jürgen Kuhlmann<br />
Effects <strong>of</strong> an inhibitor <strong>of</strong> Acyl Protein Thioesterase 1 on Rasmediated<br />
signal transduction<br />
Correct localisation to its target membranes is essential for the biological function <strong>of</strong> the<br />
small GTPase Ras. Membrane binding is mediated by post translational modifications,<br />
including farnesylation and palmitoylation for the H- and N-Ras is<strong>of</strong>orms. Recent<br />
investigations revealed that the acylprotein thioesterase 1 (APT1) might be an important<br />
enzyme in this context, because it is supposed to work bifunctionally in catalyzing both<br />
the depalmitoylation and the palmitoylation <strong>of</strong> Ras lipoproteins.<br />
We have developed a set <strong>of</strong> fluorescent labelled Ras lipoproteins, which allow to analyse<br />
the localisation. Here, effects <strong>of</strong> a synthetic APT1 inhibitor on the localisation <strong>of</strong> Ras<br />
lipoproteins in living cells and on gene expression level are presented. Incubation with<br />
the inhibitor BL282 prevented the membrane localisation <strong>of</strong> N-Ras and also resulted in a<br />
reduction <strong>of</strong> the biological activity <strong>of</strong> the Ras lipoproteins as analysed by differentiation<br />
<strong>of</strong> PC12 cells.<br />
The expression analysis was performed with DNA-microarrays, indicating a large number<br />
<strong>of</strong> differentially expressed genes. From these the expression <strong>of</strong> some Ras target genes<br />
was examined by real-time RT-PCR analysis. Results are discussed in respect <strong>of</strong> putative<br />
mechanisms for APT1 function on Ras mediated signal transduction.<br />
Literature<br />
Bader et al. Bioorganic synthesis <strong>of</strong> lipid-modified proteins for the study <strong>of</strong> signal<br />
transduction, Nature 403 (2000) 223-226<br />
contact:<br />
Robin Vetter<br />
Max-Planck-Institut für molekulare Physiologie<br />
robin.vetter@mpi-dortmund.mpg.de<br />
Otto-Hahn-Strasse 11<br />
44202 Dortmund (Deutschland)
Andreas Gorschlüter, L.H. Mak *, M. Knoll *, N. Dankbar, C. Sundermeier<br />
Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding<br />
Forces between Single Molecules<br />
We report on the development <strong>of</strong> a high-sensitive biosensor combining an electrical<br />
detection <strong>of</strong> microbead labelled analytes with a magnetical system for binding strength<br />
control. The new sensor technology allows the determination <strong>of</strong> specific binding forces<br />
between analyte and the corresponding capture molecules (e.g. proteins, antibodies,<br />
DNA). The sensor chip comprises many biochemically functionalised ultramicroelectrodes<br />
to which one end <strong>of</strong> the ligand-receptor pairs is attached. By labelling the other end <strong>of</strong><br />
these molecular pairs with superparamagnetic microparticles a magnetically generated<br />
force can be applied to the molecular bonds and an unbinding can be induced. This<br />
unbinding event is electrochemically detected. With knowledge <strong>of</strong> the applied force, the<br />
binding force can be determined. We present such binding force measurements on the<br />
system neutravidin-biotin and on the antibody-antigen system BSA and monoclonal anti-<br />
BSA. Because the sensor system allows a binding strength determination at many<br />
individual molecular pairs simultaneously, it seems promising that a fast investigation <strong>of</strong><br />
environmental conditions on ligand-receptor interaction will be possible in the future.<br />
contact:<br />
Dr. Andreas Gorschlüter<br />
Universität Münster<br />
Physikalisches Institut<br />
a.gorschlueter@uni-muenster.de<br />
Wilhelm Klemm-Str. 10<br />
48149 Münster (Deutschland)<br />
additional information<br />
*) Institut für Physikalische Chemie, Münster<br />
A.G., L.M., N.D. & C.S.: former address: ICB GmbH, Münster
Teodora Nikolova, Jaroslaw Czyz, Alexandra Rolletschek, Przemyslaw Blyszczuk,<br />
Jürgen Schuderer, Niels Kuster, Anna M Wobus<br />
Electromagnetic fields affect transcript levels <strong>of</strong> regulatory<br />
and apoptosis-related genes in p53-deficient embryonic stem<br />
(ES) cells and ES-derived neural progenitors<br />
The widespread diffusion <strong>of</strong> electromagnetic fields (EMF) in human environment raised<br />
concerns about potential hazardous effects on human health, especially on the neural<br />
system. We used the model <strong>of</strong> mouse pluripotent embryonic stem (ES) cells, which have<br />
the capacity to develop in vitro into cells <strong>of</strong> all lineages, to analyse effects <strong>of</strong><br />
radi<strong>of</strong>requency (RF) and extremely low frequency (ELF) electromagnetic fields (EMF).<br />
Long-term RF-EMF exposure <strong>of</strong> p53 deficient ES cells during ‘embryoid body’ (EB)<br />
development resulted in the up-regulation <strong>of</strong> transcript levels <strong>of</strong> regulatory and<br />
housekeeping genes c-jun, p21, c-myc and hsp70 in p53-deficient ES cells. Intermittent<br />
ELF-EMF exposure also resulted in a transient up-regulation <strong>of</strong> c-jun, p21 and egr-1<br />
mRNA levels in p53-deficient cells. These observations indicate that the genetic<br />
constitution <strong>of</strong> cells determined by the p53 function affected cellular responsiveness to<br />
EMF.<br />
RF-EMF exposure <strong>of</strong> ES-derived neural progenitor cells resulted in down-regulation <strong>of</strong><br />
transcript levels <strong>of</strong> the neural-specific Nurr1 gene at early differentiation stage, whereas<br />
the cell cycle regulatory 'growth arrest DNA damage inducible' GADD45 gene was upregulated<br />
at the terminal stage. RF-EMF exposure for 6 h resulted in a transient low<br />
induction <strong>of</strong> DNA double-strand breaks. ELF-EMF exposure to neural progenitors<br />
significantly affected transcript levels <strong>of</strong> apoptosis-related bcl-2, bax and GADD45 genes,<br />
whereas mRNA levels <strong>of</strong> several neural-specific genes were not altered. Our data<br />
indicate that EMF exposure to neural progenitors may influence, at least transiently,<br />
fundamental cellular processes including programmed cell death and cell cycle<br />
regulation, thereby affecting differentiation.<br />
contact:<br />
Dr. Teodora Nikolova<br />
Institute <strong>of</strong> Plant Genetics and Crop Plant Research (IPK)<br />
In Vitro Differentiation Group<br />
nikolova@ipk-gatersleben.de<br />
Correns-Strasse 3<br />
06466 Gatersleben (Germany)
Jürgen Hescheler<br />
Embryonic stem cells<br />
No abstract submitted<br />
contact:<br />
Pr<strong>of</strong>. Dr. Jürgen Hescheler<br />
Universität Köln<br />
Neurophysiologie<br />
j.hescheler@uni-koeln<br />
Robert-Koch-Str. 39<br />
50931 Köln (Deutschland)
Albrecht Schwab, Volodymyr Nechyporuk-Zloy, Christian Stock<br />
Endocytic internalization <strong>of</strong> potassium channels in migrating<br />
cells<br />
Cell migration plays a crucial role in many (patho-)physiological processes such as<br />
immune defense, wound healing or formation <strong>of</strong> tumor metastases. On a cellular level,<br />
migration is based among others on rapid recycling <strong>of</strong> the plasma membrane and on the<br />
activity <strong>of</strong> calcium-sensitive potassium channels (IK1). The present study addresses the<br />
question <strong>of</strong> whether and how IK1 channels keep pace with membrane recycling in<br />
migrating cells. We transfected migrating MDCK-F cells with hIK1 channels containing an<br />
extracellular HA-tag. Internalization <strong>of</strong> HA-antibodies which can be visualized with<br />
immun<strong>of</strong>luorescence and quantitated with ELISA techniques thereby mirrors endocytosis<br />
<strong>of</strong> hIK1 channels. Internalization <strong>of</strong> hIK1 channels is inhibited at 4°C, so that the<br />
antibodies label only hIK1 channels in the plasma membrane. At 37°C, endocytosed<br />
hIK1 channel-containing vesicular structures moving towards the cell front are visible.<br />
Mutating a dileucin motif in the C-terminus <strong>of</strong> hIK1 channels largely abolishes their<br />
endocytosis, and relative membrane expression <strong>of</strong> hIK1 channels is increased. Moreover,<br />
hIK1 channels are colocalized with adapter proteins (AP1/2) <strong>of</strong> clathrin-mediated<br />
endocytosis. Our results are consistent with clathrin-dependent endocytosis and<br />
recycling <strong>of</strong> hIK1 channnels in migrating cells.<br />
contact:<br />
Pr<strong>of</strong>. Albrecht Schwab<br />
Universität Münster<br />
Physiologisches Institut II<br />
aschwab@uni-muenster.de<br />
Robert-Koch-Str. 27b<br />
48149 Münster (D)
Sabine Fuchs, Michael Laue, Maria Iris Hermanns, Charles James Kirkpatrick<br />
Endocytosis in an in vitro model <strong>of</strong> the human alveolar<br />
epithelial barrier: Uptake <strong>of</strong> GM-1 by human alveolar epithelial<br />
cells in primary culture<br />
Morphological characterization and marker protein synthesis <strong>of</strong> human alveolar cells in<br />
primary culture (HAEpC) showed the differentiation <strong>of</strong> isolated alveolar type II cells into<br />
tight (TEER 1-2 kΩcm2) epithelial monolayers consisting <strong>of</strong> alveolar type I-like and<br />
alveolar type II-like cells, which corresponds to the composition <strong>of</strong> the alveolar<br />
epithelium <strong>of</strong> the donor tissue (Fuchs et al. 2003). Early cultures were characterized by<br />
low caveolin-1 and high Sp-C levels. In comparison, the protein biosynthesis <strong>of</strong> alveolar<br />
cells switched with time <strong>of</strong> culture to high caveolin-1 and low Sp-C levels. This was<br />
accompanied by the formation <strong>of</strong> caveolae in later cultures. Caveolae have been shown<br />
to provide a pathway for the cell entry <strong>of</strong> bacterial toxins, viruses and bacteria but also<br />
seem to be involved in the uptake <strong>of</strong> proteins such as albumin or glycosphingolipids such<br />
as GM-1. In this study we tested the endocytotic activity <strong>of</strong> HAEpC. We compared the<br />
uptake <strong>of</strong> Bodipy-GM-1 in HAEpC which reveal a high caveolin-1 expression and caveolae<br />
formation, with the uptake <strong>of</strong> GM-1 in A549 an ATII-like cell line showing low levels <strong>of</strong><br />
caveolin-1 and no caveolae formation. The internalization <strong>of</strong> Bodipy-GM-1 (5 µM) was<br />
investigated after 15 minutes at 37°C and compared to the controls at 4°C. These<br />
studies resulted in a high uptake capacity <strong>of</strong> bodipy-GM-1 in HAEpC showing green and<br />
red (high concentrations <strong>of</strong> Bodipy-GM-1) fluorescence within the cytoplasm <strong>of</strong> HAEpC,<br />
whereas in A549 the uptake was low as indicated by the fluorescence restricted to the<br />
cell membranes.<br />
The high uptake for GM-1 capacity <strong>of</strong> HAEpC compared to A549 correlated with the<br />
ability <strong>of</strong> these cells to form caveolae and suggest an important role <strong>of</strong> caveolae in the<br />
endocytotic pathways in alveolar epithelial cells.<br />
Literature<br />
Fuchs et al.Cell Tissue Res. 2003 Jan;311(1):31-45.<br />
contact:<br />
Dr. Sabine Fuchs<br />
Johannes Gutenberg Universtity<br />
Institute <strong>of</strong> Pathology<br />
fuchss@uni-mainz.de<br />
Langenbeckstr. 1<br />
55101 Mainz (Germany)
Tatiana Afanasieva, Victoria Jensen, Jochen Seebach, Ute Stroeher, Heinz Feldmann,<br />
Hans-J. Schnittler<br />
Endothelial activation and change <strong>of</strong> barrier function by<br />
soluble Ebola virus glycoproteins<br />
Ebola virus (EBOV) is a ‘Category A List’ agent and causes a severe form <strong>of</strong> hemorrhagic<br />
fever in humans and nonhuman primates. A dysfunction <strong>of</strong> the endothelium is a hallmark<br />
<strong>of</strong> the disease. The different glycoproteins encoded by gene four <strong>of</strong> the linear genome<br />
are thought to be major pathogenic determinants <strong>of</strong> the virus. While the transmembrane<br />
glycoprotein GP1,2 has been shown to cause endothelial destruction, the role <strong>of</strong> the<br />
soluble forms (sGP, delta peptide, GP1,2D, GP1) is largely unknown. It is hypothesized<br />
that EBOV soluble glycoproteins are able to modulate the endothelial barrier function and<br />
are therefore <strong>of</strong> biological relevance in pathogenesis.<br />
We investigated the influence <strong>of</strong> EBOV soluble glycoproteins on endothelial cell activation<br />
(as judged by expression <strong>of</strong> activation markers E-selectin, ICAM-1, VCAM-1) and barrier<br />
function (by impedance spectroscopy). We found that the major soluble glycoproteins<br />
(sGP, delta peptide), unlike the virus-like particles (VLPs) generated as a model for the<br />
virion-associated glycoprotein, were not able to activate endothelial cells or decrease the<br />
endothelial barrier function. However, we discovered an unexpected effect <strong>of</strong> the major<br />
EBOV soluble glycoprotein (sGP). sGP seems to be able to protect endothelial cell layers<br />
from TNF-a-induced changes in barrier function. We hypothesize that sGP might act as a<br />
viral anti-immunity weapon and suppresses the innate immune response <strong>of</strong> the host by<br />
“sealing” the endothelium.<br />
contact:<br />
Dr. Tatiana Afanasieva<br />
TU Dresden<br />
Physiology<br />
tatiana.afanasieva@mailbox.tu-dresden.de<br />
Fiedlerstrasse 42<br />
01307 Dresden (Germany)
Frank Wegmann, Klaus Ebnet, Louis Du Pasquier, Dietmar Vestweber, Stefan Butz<br />
Endothelial adhesion molecule ESAM binds directly to the<br />
multidomain adaptor MAGI-1 and recruits it to cell contacts<br />
Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like<br />
transmembrane protein associated with endothelial tight junctions. Based on a yeast two<br />
hybrid screen we have identified the membrane-associated guanylate kinase protein<br />
MAGI-1 as an intracellular binding partner <strong>of</strong> ESAM. MAGI-1 is a multidomain adaptor<br />
protein, which binds to transmembrane, cytoskeletal and signaling molecules and has<br />
been localized to tight junctions in epithelial cells. MAGI-1 associates with the very Cterminal<br />
sequence <strong>of</strong> ESAM most likely through a PDZ domain-mediated interaction. The<br />
direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments.<br />
The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO)<br />
cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell<br />
contacts in human umbilical vein endothelial cells (HUVEC) and in mouse endothelium,<br />
where it colocalizes with ESAM. In CHO cells, recruitment <strong>of</strong> MAGI-1 to cell contacts<br />
required the presence <strong>of</strong> ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at<br />
endothelial tight junctions.<br />
contact:<br />
Frank Wegmann<br />
Universität Münster<br />
Institut für Zellbiologie (ZMBE)<br />
wegmanf@uni-muenster.de<br />
Von-Esmarch-Str. 56<br />
48149 Münster (Germany)
Markus-N. Kruse, Karim Sabrane, Larissa Fabritz, Ines Veltrup, Melanie Voß, Boris<br />
Skryabin, Michaela Kuhn<br />
Endothelium-mediated hypotensive and hypovolemic actions<br />
<strong>of</strong> atrial natriuretic peptide<br />
Atrial natriuretic peptide (ANP) has an important physiological role in the maintenance <strong>of</strong><br />
arterial blood pressure and volume via its vasodilating and diuretic effects. Its guanylyl<br />
cyclase-A (GC-A) receptor is also highly expressed in vascular endothelium, but the<br />
functional relevance is controversial. To dissect the endothelium-mediated actions <strong>of</strong><br />
ANP in vivo, we established a new mouse model with conditional, Tie2-Cre/LoxP<br />
mediated, endothelium-restricted deletion <strong>of</strong> GC-A (EC GC-A KO mice). Notably, despite<br />
full preservation <strong>of</strong> the direct vasodilating and diuretic effects <strong>of</strong> ANP, EC GC-A KO mice<br />
are markedly hypertensive. Echocardiographic and Doppler flow evaluations showed that<br />
cardiac output and stroke volume as well as the mean pressure gradient and maximal<br />
flow velocity across the aortic valve are increased in EC GC-A KO as compared to control<br />
mice with normal GC-A expression levels. Application <strong>of</strong> the Evan´s Blue dilution<br />
technique demonstrated that the total plasma volume <strong>of</strong> EC GC-A KO mice is increased<br />
by 11-15%, even under conditions <strong>of</strong> normal salt intake. Even more, intravenous<br />
infusion <strong>of</strong> synthetic ANP markedly increased hematocrit in control but not in EC GC-A<br />
KO mice, indicating that endothelial disruption <strong>of</strong> GC-A completely prevents the acute<br />
reduction <strong>of</strong> plasma volume by ANP. Taken together, our observations in this new mouse<br />
model suggest that increases in endothelial permeability are critically involved in the<br />
hypovolemic, hypotensive actions <strong>of</strong> ANP.<br />
contact:<br />
Dr. rer. nat. Markus-Nicolas Kruse<br />
Universität Münster<br />
Institut für Pharmakologie und Toxikologie<br />
krusmar@uni-muenster.de<br />
Domagkstr. 12<br />
48149 Münster (Deutschland)
Henrik Brutlach, Sadasivam Jeganathan², Martin von Bergen², Eckhard Mandelkow²,<br />
Heinz-Juergen Steinh<strong>of</strong>f<br />
EPR investigation on site-directed spin labeled Tau protein<br />
Tau is a microtubule-associated protein (MAP) found in neuronal axons where its<br />
predominant function is to stabilize microtubules, the tracks <strong>of</strong> axonal transport.<br />
Because <strong>of</strong> its role in certain neurodegenerative diseases, especially in Alzheimer’s<br />
disease (AD), it is <strong>of</strong> increasing interest. Hyperphosphorylation followed by aggregation<br />
into paired helical filaments (PHFs) <strong>of</strong> the usually well soluble Tau protein are early<br />
neuropathological symptoms <strong>of</strong> AD [1]. However, mechanisms that cause attachment<br />
and detachment <strong>of</strong> Tau to microtubules (MTs) or that can lead to PHFs are still unclear.<br />
We applied electron paramagnetic resonance (EPR) spectroscopy in combination with<br />
site-directed spin labeling (SDSL, for review see [2]) to study conformational changes <strong>of</strong><br />
Tau. Two different Tau-constructs that comprise the domain which binds to microtubules<br />
and also forms the core <strong>of</strong> PHFs were spin labelled at their native cysteines close to a<br />
hexapeptid interaction motif which supports Tau aggregation [3]. Usually formation in<br />
vitro is initiated by adding polyanions [4]. Spin-labeling prevents formation <strong>of</strong> PHFs in<br />
case <strong>of</strong> the two Tau-constructs considered up to now. Nevertheless incubation with<br />
heparin leads to a variation <strong>of</strong> spin label dynamics in both cases revealing a<br />
conformational change in the domain <strong>of</strong> tau that is responsible for its physiological and<br />
pathological functions.<br />
Literature<br />
[1] E.-M. Mandelkow, E. Mandelkow: 1998, Trends in Cell Biology 8, 425-427<br />
[2] H.-J. Steinh<strong>of</strong>f, B. Suess: 2003, Methods 29, 188-195<br />
[3] M. v. Bergen et al: 2000, PNAS 97, 5129-5134<br />
[4] P. Friedh<strong>of</strong>f et al: 1998, PNAS 95, 15712-15717<br />
contact:<br />
Henrik Brutlach<br />
Universität Osnabrück,<br />
Macromolecular Structure<br />
Henrik.Brutlach@uos.de<br />
Barbarastr. 7<br />
49069 Osnabrück (Germany)<br />
additional information<br />
²Max-Planck-Unit for Structural Molecular Biology, Hamburg, Germany
Enrica Brodignon, Johann Klare, Martin Engelhard, Heinz-Jürgen Steinh<strong>of</strong>f<br />
EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the phototransducer<br />
NpHtrII<br />
Sensory rhodopsin II, the photophobic receptor from N. pharaonis (NpSRII) forms a<br />
complex with its cognate transducer (NpHtrII) in a 2:2 stoichiometry. Light activation <strong>of</strong><br />
NpSRII leads to a movement <strong>of</strong> helix F which triggers a rotation <strong>of</strong> TM2 in NpHtrII. The<br />
signal is then transmitted, with an unknown mechanism, to the cytoplasmic domain <strong>of</strong><br />
the transducer which harbours the binding site for the protein CheW and the His-kinase<br />
CheA. In order to obtain structural information about the linker region between the<br />
transmembrane and the cytoplasmic domain, EPR experiments with site-directed spin<br />
labelled NpHtrII and NpSRII have been carried out.<br />
Residues at the end <strong>of</strong> the transducer helix TM2 (Val78 - Leu82) as well as those<br />
succeeding this helix (Gly83 – Gly101) were mutated to cysteine and subsequently<br />
coupled to the methanethiosulfonate spin label. EPR spectra <strong>of</strong> these mutants in complex<br />
with NpSRII were recorded and analyzed. Furthermore, the accessibilities <strong>of</strong> the<br />
nitroxides towards hydrophobic and hydrophilic paramagnetic quenchers revealed<br />
environmental information about the side chains. Intra and inter- molecular distances<br />
were determined to gain insights into the structural features <strong>of</strong> the linker domain, thus<br />
enabling the proposal <strong>of</strong> a topological model for the 2:2 complex. The model is discussed<br />
with regard to the photo-transduction activity <strong>of</strong> this transducer-linker-region.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Heinz-Jürgen Steinh<strong>of</strong>f<br />
Universität Osnabrück<br />
Physik<br />
hsteinho@uos.de<br />
Barbarastrasse 7<br />
49069 Osnabrück (Deutschland)<br />
additional information<br />
J.Klare, M. Engelhard, MPI für Molekulare Physiologie, Dortmund, Germany
Andrée Rothermel, Randy Kurz, Thomas Biedermann, Markus Rüffer, Winnie Weigel,<br />
Andrea Robitzki<br />
Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Cell models are <strong>of</strong>ten used to understand cellular and molecular processes involved in<br />
the formation <strong>of</strong> neuronal diseases like Retinitis pigmentosa, macular degeneration,<br />
Alzheimer's, Parkinson's, and Hirschsprung's disease.In this study, we applied the<br />
retinospheroid technology to produce three-dimensional sphere-like in vitro retina from<br />
one-day-old neonatal rats. After dissociation <strong>of</strong> the retina, cells were transferred into<br />
medium containing culture dishes where they can reaggregate under rotation. After the<br />
formation <strong>of</strong> small clusters cells begin to<br />
proliferate and to differentiate, whereby spheres grow up to a size <strong>of</strong> 200 µm. As<br />
revealed by viability staining with fluorescein diacetat rat retinospreoids can be<br />
maintained in culture at least for a period <strong>of</strong> 30 days in vitro. Immunohistochemical<br />
analysis showed that mature retinospheroids are composed <strong>of</strong> all retinal compartments<br />
also found in the in vivo retina. Thus, we were able to identify Müller cells,amacrine<br />
cells, ganglion cells and photoreceptors in cryosections <strong>of</strong> rat retinospheroids. Beside<br />
studying aspects <strong>of</strong> retinogenesis, retinosphereoids can be used in combination with a<br />
biochip to monitor the effects <strong>of</strong> several drugs for therapeutic applications under<br />
physiological conditions. Futhermore, this novel 3D neuronal tissue model can be used<br />
as a tool to test the strategies for the treatment <strong>of</strong> various neuronal diseases.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Andrea Robitzki<br />
University <strong>of</strong> Leipzig<br />
Biotechnological Biomedical Center<br />
andrea.robitzki@bbz.uni-leipzig.de<br />
Deutscher Platz 5<br />
04103 Leipzig (Germany)
Mekky Mohammed Abouzied, Heba Mahmoud El-tahir, Volkmar Gieselmann,<br />
Sebastian Franken<br />
Evidence for a cell surface receptor <strong>of</strong> HDGF<br />
Hepatoma derived growth factor (HDGF) is a stimulator <strong>of</strong> proliferation and<br />
angiogenesis. It was isolated initially from the supernatant <strong>of</strong> an human hepatoma cell<br />
line and exhibits its mitogenic activity when applied to cell culture supernatants as well<br />
as after transfection. A possible role <strong>of</strong> this growth factor in the development and<br />
progression <strong>of</strong> different kinds <strong>of</strong> cancers is currently under discussion. In this study, we<br />
investigated the interaction <strong>of</strong> HDGF with the surface <strong>of</strong> NIH 3T3 fibroblasts. This<br />
interaction is mediated only in part by heparan sulfate. The part not mediated by<br />
heparan sulfate can be localised to a peptide <strong>of</strong> 20 amino acids derived from the Nterminal<br />
part <strong>of</strong> HDGF. We identified a single lysine residue inside <strong>of</strong> this 20 amino acids<br />
that is important for cell surface interaction <strong>of</strong> HDGF. Mutation <strong>of</strong> this lysine to alanine<br />
also decreases the proliferation activity <strong>of</strong> HDGF significantly.<br />
Taken together our results point to a binding site for HDGF on the surface <strong>of</strong> NIH 3T3<br />
fibroblasts in addition to heparan sulfate and suggests a dual mechanism for the<br />
proliferation activity developed by the growth factor.<br />
Literature<br />
Dietz F, Franken S, Yoshida K, Nakamura H, Kappler J, Gieselmann V.<br />
Biochem J. 2002 366: 491-500.<br />
Abouzied MM, Baader SL, Dietz F, Kappler J, Gieselmann V, Franken S.<br />
Biochem J. 2004 Feb 15;378(Pt 1):169-76.<br />
contact:<br />
Mekky Mohammed Abouzied<br />
Universität Bonn<br />
Physiologische Chemie<br />
mekky@institut.physiochem.uni-bonn.de<br />
Nussallee 11<br />
53115 Bonn (Germany)
Nadja Bilko, Denys Bilko<br />
Ex vivo proliferation and differentiation <strong>of</strong> cord blood cells in<br />
gel culture system<br />
Cord blood (CB) cells are being used increasingly as a source <strong>of</strong> hematopoietic stem cells<br />
(HSC) for transplantation. It is known that ex vivo expansion <strong>of</strong> HSC depends on many<br />
factors and is not achievable, because addition <strong>of</strong> the major growth factors to the growth<br />
media in vitro does not fulfill all requirements for successful long-term cell maintenance.<br />
Often cultures are supplied with factor-producing feeder layers. It has been suggested,<br />
that stromal presence is important for hematopoiesis in vitro and in vivo, but the<br />
question remains: whether diffusible factors produced by stromal cells are sufficient for<br />
the regeneration <strong>of</strong> primitive haematopoietic cells, or whether direct cell-to-cell contacts<br />
would be required. Meantime, the culture <strong>of</strong> complex undefined cell populations makes<br />
the implementation <strong>of</strong> some molecular diagnostic methods non-consistent, and at times<br />
impossible. As for the potential use <strong>of</strong> cultured haematopoietic cells for cytotherapeutic<br />
purposes, contamination <strong>of</strong> the transplant material with stromal cells <strong>of</strong> the donor should<br />
be minimised, or eliminated. During present studies, influence <strong>of</strong> various feeder layers<br />
and feeder layer condition media on AC133+ cells derived from human umbilical cord<br />
blood was investigated and their proliferative, differentiative and clonogenic activity was<br />
studied. Original patented model <strong>of</strong> gel diffusion capsules (DC) was implemented.<br />
Purified cells were injected in to the inner cavity <strong>of</strong> the DC in DMEM culture media with<br />
15% FCS, that were cultured in 6-well plates supplemented with 7ml <strong>of</strong> DMEM growth<br />
media in absolute humidity at 370C and 5% CO2 for the duration <strong>of</strong> two weeks. Feeder<br />
layers were prepared from 4-6 weeks old human embryos (FLh) and co-cultured feeder<br />
cells (FCh). Effects <strong>of</strong> the condition media (FL-CMh) were also determined. Culture and<br />
feeder layer media was at first supplemented with LIF and later with IL-3 and GM-CSF.<br />
Estimation <strong>of</strong> functional properties <strong>of</strong> AC133+ suspension cell cultures were performed in<br />
secondary subculture experiments using semisolid culture media. After 14 days<br />
subcultured material was analysed and its clonogenic activity was determined by direct<br />
and indirect analysis and quantification <strong>of</strong> the colony forming units. Multipotential CFU-<br />
MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were<br />
observed and analysed using morphological and cytochemical methods. Our data<br />
indicated deffinite ex vivo expansion and myeloid differentiation <strong>of</strong> cord blood AC 133+<br />
cells in the presence <strong>of</strong> FCh or FL-CMh feeder layer supported culture models. Prolonged<br />
support <strong>of</strong> primitive haematopoietic cells (undifferentiated cells such as promyelocytes,<br />
myelocytes and metamyelocytes) and their clonogenic capacity and functional<br />
characteristics in feeder layer positive cultures, indicates that diffusible factors are<br />
sufficient for haematopoiesis and that direct cell-to-cell contacts may not be exclusively<br />
required for successful long term in vitro haematopoiesis.<br />
contact:<br />
PhD, DSci Nadja Bilko<br />
National University \<br />
Centre <strong>of</strong> Molecular and cell investigations<br />
nadja@bilko.kiev.ua<br />
G.Skovoroda 2<br />
04070 Kiev (Ukraine)
Nediljko Budisa, Prajna Paramita Pal, Sandra Lepthien, Barbara Mulinacchi, Luis<br />
Moroder<br />
Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold”<br />
class <strong>of</strong> green fluorescent proteins<br />
Much effort has been dedicated to the design <strong>of</strong> significantly red shifted variants <strong>of</strong> the<br />
green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been<br />
based on classical engineering with the twenty canonical amino acids. We report here an<br />
expansion <strong>of</strong> these efforts by incorporation <strong>of</strong> an amino substituted variant <strong>of</strong> tryptophan<br />
into the “cyan” GFP mutant, which turned it into a “gold” variant. This variant possesses<br />
a red shift in emission unprecedented for any avFP, similar to “red” FPs, but with<br />
enhanced stability and a very low aggregation tendency. An increasing number <strong>of</strong> nonnatural<br />
amino acids are available for chromophore redesign (by engineering <strong>of</strong> the<br />
genetic code) and enable new general strategies to generate novel classes <strong>of</strong> tailormade<br />
GFP proteins.<br />
contact:<br />
Dr. Nediljko Budisa<br />
Max-Planck-Institut für Biochemie<br />
Abt. Strukturforschung<br />
budisa@biochem.mpg.de<br />
Am Klopferspitz 18A<br />
82152 Martinsried (Germany)
Ziv Reich<br />
Exploring Protein-Binding Mechanisms and Energy Landscapes<br />
by Single-Molecule Mechanical Unbinding Experiments<br />
Single-molecule experiments provide fascinating possibilities for studying systems in<br />
which molecular individuality matters. Here, I describe two applications <strong>of</strong> singlemolecule<br />
dynamic force spectroscopy (DFS) to study mechanisms and energy landscapes<br />
<strong>of</strong> protein binding. In the first example, DFS is used to discriminate between alternative<br />
modes <strong>of</strong> protein binding and activation – namely induced-fit and population-shift (1). In<br />
the second application, we used DFS to derive the energy landscape roughness <strong>of</strong> a bimolecular<br />
protein complex (2). This work represents the first measurement <strong>of</strong> energy<br />
landscape roughness <strong>of</strong> biomolecules.<br />
Literature<br />
1. Nevo, R., Brumfeld, V., Elbaum, M., Hinterdorfer, P. & Reich Z. Direct discrimination<br />
between models <strong>of</strong> protein activation by single-molecule force measurements. Biophys.<br />
J. In press.<br />
2. Nevo, R., Brumfeld, V., Hinterdorfer, P, & Reich, Z. Direct measurement <strong>of</strong> protein<br />
energy landscape roughness. Manuscript submitted.<br />
contact:<br />
Associate Pr<strong>of</strong>. Ziv Reich<br />
Weizmann Institute <strong>of</strong> Science<br />
ziv.reich@weizmann.ac.il<br />
Ha'nasi Ha'rishon<br />
76100 Rehovot (Israel)
Wolfgang Baumeister, Martin Beck, Ariane Briegel, Marek Cyrklaff, Thomas Keil, Julia<br />
Kuerner, Vladan Lucic, Ohad Medalia, Stephan Nickell, Juergen Plitzko<br />
Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Electron Tomography (ET) is uniquely suited to obtain 3-D images <strong>of</strong> large pleiomorphic<br />
structures, such as whole cells. While the principles <strong>of</strong> ET have been known for decades,<br />
its use has gathered momentum only in recent years. Technological advances have<br />
made it possible to develop automated data acquisition procedures; this, in turn, allowed<br />
to reduce the total electron dose to levels low enough for studying radiation sensitive<br />
biological materials embedded in vitreous ice. As a result, we are now poised to combine<br />
the power <strong>of</strong> high-resolution 3-D imaging with the best possible preservation <strong>of</strong> the<br />
specimen [1,2,3].<br />
High resolution tomograms <strong>of</strong> frozen-hydrated organelles or cells [4,5,6] contain vast<br />
amounts <strong>of</strong> information; essentially they are 3-D images <strong>of</strong> the cell’s entire proteome<br />
and they should ultimately enable us to map the spatial relationships <strong>of</strong> macromolecules<br />
in a cellular context. However, it is no trivial task to retrieve this information because <strong>of</strong><br />
the poor signal-to-noise ratio <strong>of</strong> such tomograms and the crowded nature <strong>of</strong> cells.<br />
Advanced pattern recognition methods are needed for detecting and identifying specific<br />
macromolecules based on their structural signature. Provided that high- or mediumresolution<br />
structures <strong>of</strong> the molecules <strong>of</strong> interest are available, they can be used as<br />
templates for a systematic interrogation <strong>of</strong> the tomograms [7,8,9]. Experiments with<br />
phantom cells have shown that such a template-matching approach is feasible. Once the<br />
challenges <strong>of</strong> obtaining sufficiently good resolution are met, and comprehensive libraries<br />
<strong>of</strong> template structures become available, we will be able to map the supramolecular<br />
landscape <strong>of</strong> cells in a systematic and comprehensive manner.<br />
Literature<br />
1. A. Koster, R. Grimm, D. Typke, R. Hegerl, A. Stoschek, J. Walz and W. Baumeister, J.<br />
Struct. Biol. 120 (1997), p. 276.<br />
2. W. Baumeister, R. Grimm and J. Walz, Trends Cell Biol. 9 (1999), p. 81.<br />
3. W. Baumeister, Curr. Opin. Struct. Biol. 12 (2002), p. 679.<br />
4. R. Grimm, H. Singh, R. Rachel, D. Typke, W. Zillig and W. Baumeister, Biophys. J. 74<br />
(1998), p. 1031.<br />
5. K. Grünewald, P. Desai, D.C. Winkler, J.B. Heymann, D.M. Belnap, W. Baumeister and<br />
A.C. Steven, Science 302 (2003), p. 1396.<br />
6. O. Medalia, I. Weber, A.S. Frangakis, D. Nicastro, G. Gerisch and W. Baumeister,<br />
Science 298 (2002),<br />
p. 1209.<br />
7. J. Böhm, A.S. Frangakis, R. Hegerl, S. Nickell, D. Typke and W. Baumeister, P. Natl.<br />
Acad. Sci. USA 97 (26) (2000), p. 14245.<br />
8. A. S. Frangakis, J. Böhm, F. Förster, S. Nickell, D. Nicastro, D. Typke, R. Hegerl and<br />
W. Baumeister,<br />
P. Natl. Acad. Sci. USA 99 (2002), p. 14153.<br />
9. A. Sali, R. Glaeser, T. Earnest and W. Baumeister, Nature 422 (2003), p. 216.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Wolfgang Baumeister<br />
Max-Planck Institute <strong>of</strong> Biochemistry<br />
Dept. <strong>of</strong> Structural Biology<br />
baumeist@biochem.mpg.de<br />
Am Klopferspitz 18<br />
82152 Martinsried (Germany)
Claudia Alma Staab, Jan-Anders Nilsson, Zsolt Sarang, Jan-Olov Höög, Roland<br />
Grafström<br />
Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in<br />
human normal and transfromed oral keratinocytes<br />
Carbonyl-metabolising enzymes (CMEs) are involved in a variety <strong>of</strong> detoxification steps,<br />
including for carcinogenic agents, e.g. N-nitrosamines and formaldehyde. As a portal <strong>of</strong><br />
entry the human oral epithelium is an obvious target for such compounds. In efforts to<br />
mimic the complete phenotype <strong>of</strong> an oral mucosa in vitro basal-like and terminally<br />
differentiated, suprabasal-like human normal oral keratinocytes (NOK) can be selectively<br />
grown by modifying serum levels in the medium. Furthermore, SV40 T antigenimmortalised<br />
(SVpgC2a) and malignant (SqCC/Y1) human oral keratinocyte lines, both<br />
differentiation-deficient, can be grown under identical conditions in efforts to model<br />
premalignant and malignant tissue. On this basis, the various human oral keratinocyte<br />
lines were cultured to investigate expression <strong>of</strong> CMEs in normal and transformed oral<br />
epithelium. Transcript pr<strong>of</strong>iling by microarray identified CMEs in all cell lines. Relative to<br />
NOK, SVpgC2a and SqCC/Y1 showed mostly similar transcript levels; however, levels<br />
differed more than two-fold for nine enzymes. In NOK, induction <strong>of</strong> a suprabasal<br />
phenotype caused changed levels <strong>of</strong> nine transcripts, respectively, whereas SqCC/Y1 and<br />
SVpgC2a failed to regulate most <strong>of</strong> these genes. The overall results implicate that CMEs<br />
are expressed in the human oral epithelium, and that suprabasal differentiation<br />
influences their expression levels. The differences noted in immortalised and malignant<br />
cells implicate that deregulated CME expression might be a feature <strong>of</strong> cell<br />
transformation. These findings emphasise the need <strong>of</strong> further studies <strong>of</strong> CMEs to<br />
elucidate their functional role in human oral epithelium as well as their implication in<br />
cancer development.<br />
contact:<br />
Claudia Staab<br />
Karolinska Institute<br />
Department for Medical Biochenistry and Biophysics<br />
Claudia.Staab@mbb.ki.se<br />
Scheeles väg 2, Solna<br />
17177 Stockholm (Sweden)
Gerald Bäuml, Gudrun Horn, Widmar Tanner, Hans Robert Kalbitzer<br />
Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the<br />
Saccharomyces cerevisiae cell adhesion molecule a-agglutinin<br />
a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on<br />
the surface <strong>of</strong> cells <strong>of</strong> a mating type and consists <strong>of</strong> an anchorage subunit Aga1 and a<br />
receptor binding subunit Aga2. In order to perform NMR-studies on Aga2 to get its threedimensional<br />
structure we have to produce soluble protein in high concentration. Aga2<br />
expressed alone in E.coli is insoluble. Denaturating purification with subsequent refolding<br />
results in an unstable protein. Co-expression <strong>of</strong> Aga1 an Aga2 yields in complete<br />
unsoluble target protein. To achieve an improvement <strong>of</strong> its solubility we successfully<br />
performed a co-renaturation <strong>of</strong> separately purified Aga2 and Aga1. Currently we conduct<br />
experiments with an in vitro expression system because <strong>of</strong> its advantages concerning<br />
NMR-studies.<br />
contact:<br />
Gerald Bäuml<br />
Universität Regensburg<br />
Biophysik und physikalische Biochemie<br />
gerald.baeuml@biologie.uni-regensburg.de<br />
Frankenstrasse 6<br />
93059 Regensburg (Deutschland)
Michael Schnoor, Katrin Stolle, Jürgen Rauterberg, Paul Cullen, Stefan Lorkowski<br />
Expression <strong>of</strong> collagens by human monocyte-derived<br />
macrophages<br />
Several collagens are expressed in every section <strong>of</strong> the arterial wall, mainly by smooth<br />
muscle cells. We recently showed that macrophages also produce collagen type VIII in<br />
vitro and in the atherosclerotic plaque. Following from this result, we screened human<br />
monocyte-derived macrophages in vitro for the expression <strong>of</strong> other collagens using the<br />
reverse transcription polymerase chain reaction and mRNA-specific primers. Surprisingly,<br />
we found that both monocytes and macrophages express mRNAs for a wide variety <strong>of</strong><br />
collagens. We selected collagen type VI for further studies because this type <strong>of</strong> collagen<br />
has been shown to anchor cells and extracellular matrix components, suggesting that it<br />
may help to stabilize the atherosclerotic plaque. Western blot analysis showed that<br />
secretion <strong>of</strong> collagen type VI by macrophages occurs in a time-dependent manner. While<br />
in the medium <strong>of</strong> freshly isolated monocytes no protein could be detected, the amount <strong>of</strong><br />
secreted collagen type VI increased during differentiation to mature macrophages. The<br />
secretion occurs via a classical pathway and is regulated by TGFβ and PDGF. We found<br />
that the function <strong>of</strong> collagen type VI for the macrophage in vitro is β1-integrin-mediated<br />
cell adhesion. Taken together with our previous results, these findings indicate that in<br />
addition to their widely assumed degradative role, macrophages in the atherosclerotic<br />
plaque may also be able to stabilize the lesion by means <strong>of</strong> collagen production.<br />
contact:<br />
Dr. Stefan Lorkowski<br />
Universität Münster<br />
Institut für Arterioskleroseforschung<br />
stefan.lorkowski@uni-muenster.de<br />
Domagkstraße 3<br />
48149 Münster (Deutschland)
Johann Gross, Astrid Machulik, Amarjagal Nyamaa, Birgit Mazurek<br />
Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat<br />
cochlea<br />
The sensitivity <strong>of</strong> hearing depends strongly on the function <strong>of</strong> special mechanosensory<br />
cells, outer hair cells(OHCs), which amplify sound-induced vibrations in the organ <strong>of</strong><br />
Corti. This amplification is mainly the result <strong>of</strong> the protein prestin, highly expressed in<br />
OHCs and not expressed in inner hair cells. The hearing threshold is elevated by 40- 50<br />
dB, when OHCs are damaged or lost. To quantitate in absolute terms the prestin mRNA<br />
level in the explant culture <strong>of</strong> rat cochlea, we used competitive RT-PCR with a synthetic<br />
internal cRNA standard. Prestin gene expression at levels <strong>of</strong> 100 fg specific mRNA/µg<br />
total RNA on postnatal days 3-5, which corresponds to about 300 copies per outer hair<br />
cell (OHC), were found. The prestin mRNA levels during in vitro development increase<br />
and form an apical-basal gradient between in vitro days 3-5 and 5-7. To elucidate the<br />
variations the prestin mRNA levels undergo as a result <strong>of</strong> damage to the organ <strong>of</strong> Corti,<br />
we exposed the explant cultures to ischemia and hypoxia. While total RNA was observed<br />
to remain unchanged, the numbers <strong>of</strong> OHCs and the prestin mRNA levels were found to<br />
decrease by about 20% and 35% respectively compared to normoxia. Ischemia results<br />
in a decrease <strong>of</strong> the prestin mRNA level in parallel with OHC loss. The prestin mRNA level<br />
can therefore be used as marker <strong>of</strong> damage to or loss <strong>of</strong> OHCs. The organotypic culture<br />
seems to be a useful model to study the factors regulating the expression <strong>of</strong> prestin<br />
mRNA.<br />
Literature<br />
Zheng, J., Shen, W., He, D.Z., Long, K.B., Madison, L.D., Dallos, P., 2000. Prestin is the<br />
motor protein <strong>of</strong> cochlear<br />
outer hair cells. Nature 405, 149-155.<br />
contact:<br />
Pr<strong>of</strong>. Dr. med. Johann Gross<br />
Charité<br />
HNO, Forschungslabor<br />
johann.gross@charite.de<br />
Spandauer Damm 130<br />
14050 Berlin (Germany)
Angela Magin, Thomas Noll<br />
Feasibility <strong>of</strong> the Use <strong>of</strong> HUVEC for Autologous Co-Culture<br />
Expansion <strong>of</strong> HSC from Umbilical Cord Blood<br />
As the number <strong>of</strong> hematopoietic stem cells (HSC) in umbilical cord blood (UCB) is<br />
limited, it is currently still difficult to use these cells for transplantations in adults. Trying<br />
to find an autologous source <strong>of</strong> feeder cells for expansion <strong>of</strong> HSC from umbilical cord<br />
blood, we investigated the possibility to use human umbilical vein endothelial cells<br />
(HUVEC) in a co-culture system. It was shown that HUVEC could be obtained from the<br />
same umbilical cord as the UCB, but not in all cases due to thrombus formation or<br />
perforation <strong>of</strong> the umbilical vein.<br />
HUVEC were cultivated for a short-time interval (7 days) in a medium optimised for<br />
cultivation <strong>of</strong> HSC. Growth, viability, nutrient consumption (glutamine, glucose) and<br />
metabolite accumulation (glutamate, lactate) were analysed and compared to HUVEC<br />
kept in a specialised endothelial cell growth medium. While HUVEC grew rapidly in<br />
endothelial cell medium, they showed neither growth nor excessive nutrient consumption<br />
in HSC-medium. For this reason, they seem suitable for use in co-culture with slowly<br />
growing HSC.<br />
When using a direct co-cultivation system it is unavoidable that the transplant also<br />
contains feeder cells. To investigate whether HUVEC might have an effect on the<br />
recipient's immune system, we analysed their influence on allogeneic T-cells in a setup<br />
similar to a mixed-lymphocyte-reaction and found an activation <strong>of</strong> T-cells by FACSanalysis<br />
<strong>of</strong> CD25, 69 and 71. These findings prove the necessity <strong>of</strong> a good donorrecipient<br />
matching when using HUVEC for co-culture expansion <strong>of</strong> HSC.<br />
contact:<br />
Angela Magin<br />
Research Center Juelich<br />
Institute <strong>of</strong> Biotechnology 2<br />
a.magin@fz-juelich.de<br />
Leo-Brandt-Straße<br />
52425 Juelich (Germany)
Peter Pohl<br />
Fluid transport through epithelial barriers: the importance <strong>of</strong><br />
ion channels and transporters<br />
The coupling <strong>of</strong> ion and water flow through membrane channels and transporters is in<br />
dispute. A hypothesised secondary function <strong>of</strong> water channel proteins to act as cyclic<br />
nucleotide gated channels was tested by aquaporin-1 reconstitution into planar bilayers<br />
and overexpression in HEK293 cells. The lack <strong>of</strong> significant ion channel activity rules out<br />
a secondary role <strong>of</strong> aquaporin-1 water channels in cellular signal transduction (1). Vice<br />
versa, ion channels may provide an efficient water pathway as suggested from a<br />
comparison <strong>of</strong> single channel water permeabilities gathered for ion channels and<br />
aquaporins. The KcsA-potassium channel, for example, exhibits the highest water<br />
transport rate ever obtained for a membrane channel (2). Cotransport <strong>of</strong> water and ions<br />
was suggested also for a variety <strong>of</strong> carrier proteins, e.g. the Na + -glucose or the K + -Cl -<br />
cotransporter. Using primary cultured epithelial cells from renal inner medulla (3), we<br />
have found evidence that cotransporter activity modulates aquaporin mediated transport<br />
but we did not observe significant volume flow mediated by cotransporters. In contrast,<br />
the fraction <strong>of</strong> absorbed fluid that crosses epithelial ion channels still remains to be<br />
determined.<br />
Literature<br />
1. Satoshi P. Tsunoda, Burkhard Wiesner, Dorothea Lorenz, Walter Rosenthal, and P.<br />
Pohl. Aquaporin-1, Nothing but a Water Channel. J.Biol.Chem. 279:11364-11367, 2004.<br />
2. S. M. Saparov and P. Pohl. Beyond the diffusion limit: Water flow through the empty<br />
bacterial potassium channel. Proc.Natl.Acad.Sci.U.S.A 101:4805-4809, 2004.<br />
3. D. Lorenz, A. Krylov, D. Hahm, V. Hagen, W. Rosenthal, P. Pohl, and K. Maric. Cyclic<br />
AMP is sufficient for triggering the exocytic recruitment <strong>of</strong> aquaporin-2 in renal epithelial<br />
cells. EMBO Rep. 4:88-93, 2003.<br />
contact:<br />
Dr. Peter Pohl<br />
Forschungsinstitut fuer Molekulare Pharmakologie<br />
pohl@fmp-berlin.de<br />
Robert-Roessle-Str. 10<br />
13125 Berlin (Germany)
Matthias Gralle, Michelle Botelho, Cristiano Oliveira, Iris Torriani, Sergio Ferreira<br />
Folding and structure <strong>of</strong> the amyloid precursor protein<br />
investigated by solution scattering and spectroscopy<br />
The β-amyloid peptide (Aβ), implicated in the genesis <strong>of</strong> Alzheimer’s disease, is derived<br />
from the transmembrane amyloid precursor protein (APP). Human APP is cleaved by<br />
secretases, which release extracellular soluble fragments (sAPPα and sAPPβ). sAPPα may<br />
play important roles <strong>of</strong> its own in both normal and pathological processes in the brain.<br />
We have investigated the three-dimensional structure and stability <strong>of</strong> APP by a<br />
spectroscopic and solution scattering approach, using the recombinant human is<strong>of</strong>orms<br />
sAPPα695 and sAPPα770 expressed in Pichia pastoris. An initial SAXS-derived model <strong>of</strong><br />
sAPPα695 was calculated from measurements at low protein concentrations (Ref. 1).<br />
Both is<strong>of</strong>orms were then shown to be elongated monomers, with MW <strong>of</strong> 67 and 74 kDa<br />
and Rg <strong>of</strong> 42.5 and 46.5 Å, respectively. A combination <strong>of</strong> intrinsic and bis-ANS<br />
fluorescence, circular dichroism and SAXS measurements on the denaturation <strong>of</strong> sAPP<br />
α695 and sAPPα770 by GdnHCl and urea permitted us to propose a multi-step folding<br />
pathway for both is<strong>of</strong>orms (Ref. 2). Furthermore, higher protein concentrations<br />
permitted SAXS measurements up to q>0.8. From these measurements, we obtained<br />
higher-resolution models <strong>of</strong> the solution structure <strong>of</strong> both sAPPα695 and sAPPα770. The<br />
stepwise denaturation process may be correlated to the domain structure observed in<br />
the three-dimensional models.<br />
Supported by LNLS, FINEP, FAPERJ, HHMI and JSGMF.<br />
Literature<br />
1: Gralle, M., Botelho, M.M., Oliveira, C.L.P., Torriani, I. and Ferreira, S.T. (2002)<br />
Solution studies and structural model <strong>of</strong> the extracellular domain <strong>of</strong> human amyloid<br />
precursor protein. Biophys. J. 83:3513-24<br />
2: Botelho, M.G., Gralle, M., Oliveira, C.L.P., Torriani, I. and Ferreira, S.T. (2003)<br />
Folding and stability <strong>of</strong> the extracellular domain <strong>of</strong> the human amyloid precursor protein.<br />
J. Biol. Chem. 278:34259-67<br />
contact:<br />
Dipl. Biochem. Matthias Gralle<br />
Federal University Rio de Janeiro<br />
Medical Biochemistry<br />
gralle@bioqmed.ufrj.br<br />
Av. Brig. Trompowski s/n, CCS, Bl. H2, Sl. 19<br />
21941 Rio de Janeiro (Brasilien)<br />
additional information<br />
C.L.P.O. & I.T.: Institute <strong>of</strong> Physics, Unicamp, Campinas,Brasil
Jan Anders Nilsson, Claudia A. Staab, Jan-Olov Höög, Roland C. Grafström<br />
Formaldehyde-related toxicity and gene expression in cultured<br />
human oral keratinocytes<br />
Mouth inhalation <strong>of</strong> formaldehyde, a known respiratory carcinogen, even below<br />
recommended acceptable levels induces genotoxic damage in human buccal epithelium.<br />
On this basis, normal (NOK), SV40 T antigen-immortalized (SVpgC2a) and malignant<br />
(SqCC/Y1) buccal keratinocytes were cultured and the toxicity <strong>of</strong> formaldehyde studied.<br />
Formaldehyde caused dose-dependent cytotoxicity in the cell lines, NOK and SVpgC2a<br />
being more sensitive than SqCC/Y1. Formaldehyde induced primarily terminal<br />
differentiation in NOK, whereas SVpgC2a and SqCC/Y1 showed apoptosis and necrotic<br />
death without detectable terminal differentiation. Two NOK cultures failed to undergo<br />
transformation from repeated formaldehyde exposure. In contrast, repeated<br />
formaldehyde exposure <strong>of</strong> SVpgC2a resulted in the formation <strong>of</strong> a novel line (termed<br />
SVpgC3a) that exhibited multi-focal growth differently to its parental line. Preliminary<br />
characterization <strong>of</strong> the SVpgC3a genotype relative to SVpgC2a by microarray indicated a<br />
number <strong>of</strong> changes. We conclude that altered expression <strong>of</strong> several gene families may<br />
couple to formaldehyde-induced keratinocyte transformation, and that susceptibility to<br />
formaldehyde toxicity may vary with the stage <strong>of</strong> transformation. Cell death induction by<br />
different pathways likely cooperates in the protection against cancer development from<br />
formaldehyde exposure.<br />
contact:<br />
MSc Jan Anders Nilsson<br />
Karolinska Institutet<br />
Institute <strong>of</strong> Environmental Medicine<br />
jan.anders.nilsson@imm.ki.se<br />
BOX 210<br />
17177 Stockholm (Sweden)
Partha Chakrabarti, Yan Suveyzdis, Alfred Wittingh<strong>of</strong>er, Klaus Gerwert<br />
FTIR on the Rap-RapGAP reaction: GTPase activation without<br />
an arginine finger<br />
GTPase activating proteins (GAPs) down-regulate Ras-like proteins by stimulating their<br />
GTP hydrolysis, and malfunction <strong>of</strong> this reaction leads to disease formation. In most<br />
cases, the molecular mechanism <strong>of</strong> activation involves stabilization <strong>of</strong> a catalytic<br />
glutamine and insertion <strong>of</strong> a catalytic Arg into the active side by GAP. Neither Rap1<br />
possesses a glutamine nor does its cognate RapGAP employ an arginine. Recently it was<br />
proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all<br />
other Ras-like proteins (1). Here RapGAP mediated activation has been investigated by<br />
time resolved Fourier transform infrared (trFTIR) spectroscopy. While the intrinsic<br />
hydrolysis reactions <strong>of</strong> Rap and Ras are very similar, the GAP catalyzed reaction shows<br />
unique features. RapGAP binding induces a GTP* conformation, in which the three<br />
phosphate groups are oriented such that they are vibrationally coupled to each other, in<br />
contrast to what is seen in the intrinsic and the Ras-RasGAP reactions. However, the<br />
charge shift towards beta-phosphate observed with RasGAP is also observed for RapGAP.<br />
A GDP•P i intermediate accumulates in the GAP-catalyzed reaction since release <strong>of</strong> P i is<br />
eight times slower than the cleavage reaction and significant GTP synthesis from GDP•P i<br />
is observed. Partial steps <strong>of</strong> the cleavage reaction are correlated with structural changes<br />
<strong>of</strong> protein side groups and backbone. Thus, the Rap-RapGAP catalytic machinery<br />
compensates for the absence <strong>of</strong> a cis-Gln by a trans-Asn and for the catalytic arginine by<br />
inducing a different GTP conformation that is more prone to be attacked by a water<br />
molecule.<br />
Literature<br />
1. Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittingh<strong>of</strong>er, A. (2004)<br />
Nature 429, 197-201<br />
2. Bos, J. L., de Rooij, J., and Reedquist, K. A. (2001) Nat. Rev. Mol. Cell. Biol. 2, 369-<br />
377<br />
3. Brinkmann, T., Daumke, O., Herbrand, U., Kühlmann, D., Stege, P., Ahmadian, M. R.<br />
and Wittingh<strong>of</strong>er, A., (2002) J. Biol. Chem. 277, 12525-12531<br />
4. Allin, C., Ahmadian, M. R., Wittingh<strong>of</strong>er, A. and Gerwert, K. (2001) Proc. Natl. Acad.<br />
Sci. USA 98, 7754-7759<br />
contact:<br />
Partha Chakrabarti<br />
MPI Dortmund<br />
chakraba@mpi-dortmund.mpg.de<br />
Otto-Hahn-Str. 11<br />
44227 Dortmund (Germany)
Angela Hirtreiter, Luis Figueiredo, Daniel Klunker, Bernd Haas, Debbie Ang*, Dean<br />
Naylor, Michael Kerner, Costa Georgopoulos*, Ulrich Hartl, Manajit Hayer-Hartl<br />
Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Two distantly related classes <strong>of</strong> cylindrical chaperonin complexes assist in the folding <strong>of</strong><br />
newly-synthesized and stress-denatured proteins in an ATP-dependent manner. Group I<br />
chaperonins are thought to be restricted to the cytosol <strong>of</strong> bacteria and to mitochondria<br />
and chloroplasts, whereas the group II chaperonins are found in the archaeal and<br />
eukaryotic cytosol. Here we show that members <strong>of</strong> the archaeal genus Methanosarcina<br />
co-express both the complete group I (GroEL/GroES) and group II<br />
(thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea<br />
have acquired between 20 to 35% <strong>of</strong> their genes by lateral gene transfer from bacteria.<br />
In Methanosarcina mazei Gö1 both chaperonins are similarly abundant and are<br />
moderately induced under heat stress. The thermosome contains three paralogous<br />
subunits, α, β and γ, which assemble preferentially at a molar ratio <strong>of</strong> 2:1:1. As shown in<br />
vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory<br />
role <strong>of</strong> the β subunit.<br />
The M. mazei (Mm) GroEL/GroES proteins have the structural features <strong>of</strong> their bacterial<br />
counterparts but the groESgroEL operon <strong>of</strong> M. mazei is unable to functionally replace<br />
groESgroEL in E. coli (Ec). However, the MmGroES protein can largely complement a<br />
mutant EcGroES protein in vivo. The ATPase rate <strong>of</strong> MmGroEL is very low and the<br />
dissociation <strong>of</strong> MmGroES from MmGroEL is 15-times slower than for the EcGroEL/GroES<br />
system. Interestingly, optimal functionality <strong>of</strong> MmGroEL/GroES and its ability to<br />
encapsulate proteins, such as malate dehydrogenase, requires the presence <strong>of</strong><br />
ammonium sulfate in vitro. In the absence <strong>of</strong> ammonium sulfate, malate dehydrogenase<br />
fails to be encapsulated by GroES and rather cycles on and <strong>of</strong>f the GroEL trans ring in a<br />
non-productive reaction. These results indicate that the archaeal GroEL/GroES system<br />
has preserved the basic encapsulation mechanism <strong>of</strong> bacterial GroEL and suggest that it<br />
has adjusted the length <strong>of</strong> its reaction cycle to the slower growth rates <strong>of</strong> archaea.<br />
Additionally, the release <strong>of</strong> only folded protein from the GroEL/GroES cage may prevent<br />
adverse interactions <strong>of</strong> the GroEL substrates with the thermosome, which is not normally<br />
located within the same compartment.<br />
The co-existence <strong>of</strong> both chaperonin systems in the same cellular compartment suggests<br />
the Methanosarcina species as a useful model system in studying the differential<br />
substrate specificity <strong>of</strong> the group I and II chaperonins and in elucidating how newlysynthesized<br />
proteins are sorted from the ribosome to the proper chaperonin for folding.<br />
contact:<br />
Angela Hirtreiter<br />
MPI f. Biochemie<br />
hirtreit @ biochem.mpg.de<br />
Am Klopferspitz 18<br />
82152 Martinsried (Deutschland)<br />
additional information<br />
*Dèpartèment de Biochimie Mèdicale, Centre Mèdical Universitaire, Genève, Switzerland
Günter Gisselmann, Christian Wetzel, Hanns Hatt<br />
Functional characterization <strong>of</strong> I h -channel splice variants in<br />
insects<br />
The hyperpolarization-activated cation current (I h ) is widely distributed in excitable cells.<br />
I h -channels have been shown to play important roles in regulation <strong>of</strong> cellular excitability,<br />
rhythmic activity, and synaptic function. We isolated cDNAs for AMIH and DMIH encoding<br />
members <strong>of</strong> the I h -channel family from Drosophila melanogaster and Apis mellifera by<br />
means <strong>of</strong> polymerase chain reaction and homology screening. In Drosophila, splicing at<br />
four different sites generates a manifold variety <strong>of</strong> different channel transcripts. The<br />
found variants code for ion channel proteins with long or short N-termini and variations<br />
in the length <strong>of</strong> the interloop regions between the membrane spanning domains S3-S4<br />
and S4-S5. Comparable variants were also found in Apis mellifera. Functional expression<br />
<strong>of</strong> AMIH variants in HEK293 cells revealed that the incorporation <strong>of</strong> 32 extra amino acids<br />
between S4 and S5 shifts the activation curve by + 25 mV. In vertebrates, functional<br />
diversity <strong>of</strong> I h -channels is generated by four different genes (HCN1-HCN4). Our data<br />
suggest that in invertebrates, splice variants coded by a single gene could generate a<br />
similar diversity.<br />
Literature<br />
Gisselmann G, Warnstedt M, Gamerschlag B, Bormann A, Marx T, Neuhaus EM,<br />
Stoertkuhl K, Wetzel CH, Hatt H.<br />
Characterization <strong>of</strong> recombinant and native Ih-channels from Apis mellifera.<br />
Insect Biochem Mol Biol. 2003 Nov;33(11):1123-34<br />
Zhang Y, Oliva R, Gisselmann G, Hatt H, Guckenheimer J, Harris-Warrick RM.<br />
Overexpression <strong>of</strong> a hyperpolarization-activated cation current (Ih) channel gene<br />
modifies the firing activity <strong>of</strong> identified motor neurons in a small neural network.<br />
J Neurosci. 2003 Oct 8;23(27):9059<br />
2004)<br />
contact:<br />
Günter Gisselmann<br />
Ruhr-Uni-Bochum<br />
Lehrstuhl für Zellphysiologie<br />
guenter.gisselmann@rub.de<br />
Universitätsstr. 150<br />
44780 Bochum (Deutschland)
Oliver Schilling, Andreas Vogel*, Brenda Kostelecky, Wolfram Meyer-Klaucke<br />
Functional diversity and metal selectivity <strong>of</strong> proteins sharing<br />
the metallo-β-lactamase fold<br />
The metallo-β-lactamase fold allows for remarkably different catalytic activities, including<br />
hydrolysis <strong>of</strong> thioesters, arylsulfates, phosphodiesters, and redox activities. Typical<br />
features <strong>of</strong> the metallo-β-lactamase fold are external α-helices, two internal layers <strong>of</strong> βsheets<br />
and a bimetal active site, commonly containing zinc for hydrolytic and iron for<br />
redox activities. The Escherichia coli ElaC protein was initially a putative member <strong>of</strong> the<br />
metallo-β-lactamase superfamily with a predicted sulfatase activity. Rational and random<br />
substrate screens refuted this assumption and ElaC was shown to be an effective, zinc<br />
dependent phosphodiesterase (ZiPD). ZiPD-like proteins are found in all three domains<br />
<strong>of</strong> life and define a new class <strong>of</strong> conserved metallohydrolases. ZiPD homologs from<br />
numerous organisms are involved in 3’tRNA maturation but there is growing doubt<br />
concerning an involvement <strong>of</strong> E. coli ZiPD in this process. Analysis <strong>of</strong> both the<br />
transcriptome and proteome <strong>of</strong> an E. coli elaC gene deletion mutant confirmed these<br />
previous doubts and suggested functional diversity within the ZiPD / ElaC protein family.<br />
The ZiPD zinc coordination was shown to be identical to the thiolesterase glyoxalase II,<br />
another member <strong>of</strong> the metallo-β-lactamase superfamily. For the first time in this<br />
protein family, identical metal coordination was found for functionally diverse enzymes.<br />
Despite their highly similar metal binding sites, ZiPD and glyoxalase unexpectedly<br />
differed in their metal-activity correlation: ZiPD was exclusively activated by zinc<br />
whereas plant glyoxalase was isolated with iron, manganese, and zinc and had<br />
comparable catalytic activities with different ratios <strong>of</strong> these three metals. In sharp<br />
contrast, ZiPD and redox active metallo-β-lactamase domain proteins specifically require<br />
either zinc (ZiPD) or iron (redox enzymes) for catalytic activity. Surprisingly, both<br />
enzymes had comparable zinc dissociation constants in the low •M range. Redox active,<br />
iron selective metallo-β-lactamase domain proteins have an atypical glutamate metal<br />
ligand which was previously thought to trigger metal selectivity. Mutational studies on<br />
both hydrolytic and redox active metallo-β-lactamase domain proteins however now<br />
strongly suggest that this atypical metal ligand does not guide metal selectivity but<br />
serves to prevent potential hydrolytic cross reactivity.<br />
contact:<br />
Oliver Schilling<br />
EMBL Outstation Hamburg<br />
c/o DESY<br />
schilling@embl-hamburg.de<br />
Notkestrasse 85<br />
22603 Hamburg. (Deutschland)<br />
additional information<br />
*) present address: MPI für Kohlenforschung, Mülheim, Germany
Daniel Hilger, Gunnar Jeschke, Christoph Wegener, Heinz-Jürgen Steinh<strong>of</strong>f, Etana<br />
Padan, Heinrich Jung<br />
Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Being involved in homeostasis <strong>of</strong> Na + and pH in all cells, many Na + /H + antiporters share<br />
a unique property; their activity is directly regulated by pH. This property has been<br />
extensively studied in NhaA, the main Na + /H + antiporter <strong>of</strong> E. coli (1). NhaA is activated<br />
time trace <strong>of</strong> the DEER signal <strong>of</strong> NhaA-H225C and –V254C. This observation reflected an<br />
unaltered interspin distance but a higher number <strong>of</strong> spins interacting at this distance at<br />
alkaline pH. The observed effects were completely reversible. The findings are discussed<br />
in at alkaline pH by pH-induced conformational changes (2). Amino acid residues<br />
participating in the pH response <strong>of</strong> NhaA were found clustered in loops VII-VIII and VIII-<br />
IX. Here we used site-directed spin labeling in combination with EPR spectroscopy to<br />
probe pH-induced changes <strong>of</strong> NhaA structure and interactions within a dimer. The<br />
studies revealed pH-dependent alterations <strong>of</strong> the mobility and accessibility <strong>of</strong> spin label<br />
attached to positions 225 (loop VII-VIII) or 254 (loop VIII-IX). Furthermore, four pulse<br />
DEER measurements <strong>of</strong> NhaA-H225C detected spin-spin interactions corresponding to a<br />
mean distance <strong>of</strong> 4.1 nm at pH 5.8. Similar analyses <strong>of</strong> NhaA-V254C hint at an interspin<br />
distance smaller than 2.0 nm. The distances were explained by monomer-monomer<br />
interactions within a dimer. A pH shift from 5.8 to 8.0 increased the modulation depth <strong>of</strong><br />
the view <strong>of</strong> a possible effect <strong>of</strong> pH on the monomer-dimer equilibrium.<br />
Literature<br />
1. Padan, E., Rimon, A., Tzubery, T., Muller, M., Herz, K., and Galili, L. (2003) NhaA<br />
Na + /H + antiporter: structure, mechanism and function in homestasis <strong>of</strong> Na + and pH. In<br />
Karmazyn, M., Avkiran, M., and Fliegel, L., editors. The sodium-hydrogen exchange,<br />
from molecule to its role in disease , Kluwer, London<br />
2. Gerchman, Y., Rimon, A., and Padan, E. (1999) J.Biol.Chem 274, 24617-24624<br />
contact:<br />
apl. Pr<strong>of</strong>. Dr. Heinrich Jung<br />
LMU München<br />
Mikrobiologie<br />
heinrich.jung@lrz.uni-muenchen.de<br />
Maria-Ward-Strasse 1a<br />
80638 München (Deutschland)<br />
additional information<br />
G. Jeschke: MPI für Polymerforschung,Mainz<br />
C. Wegener, H.-J. Steinh<strong>of</strong>f: Universität Osnabrück, Fachbereich Physik<br />
E. Padan: Hebrew University, Institute <strong>of</strong> Life Sciences, Jerusalem, Israel
Nicholas J. Talbot, Martin J. Gilbert, Darren M. Soanes, Madhumita Barooah, Zheng Yi<br />
Wang, Sarah Ahmad, Claire Veneault-Fourrey, Joanna M. Jenkinson, Lucy J. Holcombe,<br />
Gurpreet Bhambra<br />
Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
The rice blast fungus Magnaporthe grisea causes one <strong>of</strong> the most serious diseases <strong>of</strong><br />
cultivated rice, and understanding the early events <strong>of</strong> the infection is <strong>of</strong> paramount<br />
importance if durable control measures are to be developed. M. grisea forms a<br />
specialised infection structure called an appressorium which is used to penetrate the<br />
tough outer cuticle <strong>of</strong> rice leaves, allowing the fungus entry to the underlying tissues<br />
(Talbot, 2003). Appressoria are melanin-pigmented, dome shaped cells, which form in<br />
response to the hydrophobic leaf surface and generate massive turgor pressure. Turgor<br />
is translated into mechanical force and a narrow penetration hypha is formed at the base<br />
<strong>of</strong> the appressorium, puncturing the cuticle We are using a multi-disciplinary approach,<br />
involving gene functional analysis, cell biology and analytical biochemistry, to investigate<br />
the biology <strong>of</strong> appressorium-mediated plant infection. In the course <strong>of</strong> these studies we<br />
have identified a number <strong>of</strong> key determinants <strong>of</strong> appressorium morphogenesis and<br />
function.<br />
Appressorium turgor is generated by accumulation <strong>of</strong> a compatible solute within the<br />
appressoria to very high concentrations. M. grisea appressoria accumulate glycerol as a<br />
major compatible solute during appressorial turgor generation, and understanding the<br />
mechanisms by which glycerol is synthesised within appressoria and how this process is<br />
genetically regulated is one <strong>of</strong> the primary aims <strong>of</strong> our research. Appressoria <strong>of</strong><br />
Magnaporthe grisea form in dew drops on the surface <strong>of</strong> rice leaves in the absence <strong>of</strong><br />
exogenous nutrients. Therefore, glycerol is synthesised from precursors that are present<br />
within un-germinated spores <strong>of</strong> the fungus. We have been examining the role <strong>of</strong><br />
trehalose, glycogen and lipids as sources for glycerol biosynthesis and progress in the<br />
analysis <strong>of</strong> glycerol biosynthetic pathways and their genetic regulation will be presented.<br />
Literature<br />
Foster, A.J., Jenkinson, J.M., Talbot, N.J. (2003) EMBO J. 22: 225-235.<br />
Wang, Z.Y., Thornton, C.R., Kershaw, M.J., Debao, L. and Talbot, N.J. (2003) Molecular<br />
Microbiology 47: 1601-12<br />
Talbot, N.J. (2003) <strong>Annual</strong> Review <strong>of</strong> Microbiology 57: 177-202<br />
contact:<br />
Dr. Nicholas Talbot<br />
University <strong>of</strong> Exeter<br />
School <strong>of</strong> Biological Sciences<br />
N.J.Talbot@exeter.ac.uk<br />
Perry Road<br />
Exeter (U.K.)
Björn Reiss, Andreas Jansh<strong>of</strong>f, Claudia Steinem, Joachim Wegener<br />
Functionalized vesicles as a model for cell adhesion studies<br />
The time course <strong>of</strong> adhesion and spreading <strong>of</strong> mammalian cells to an oscillating quartz<br />
resonator can be monitored by recording its resonance frequency . This technique<br />
referred to as quartz crystal microbalance (QCM) is well established to follow adsorption<br />
processes at the solid-liquid interface. However, the complexity <strong>of</strong> cellular systems <strong>of</strong>ten<br />
interferes with the direct evaluation <strong>of</strong> QCM-readings with respect to the physical origin<br />
<strong>of</strong> the detected frequency shifts. To get a better understanding about the contribution <strong>of</strong><br />
certain subcellular structures to the overall QCM response, we have studied a simple cell<br />
model consisting <strong>of</strong> unilamellar vesicles (d = 100 nm) doped with biotin-labeled lipids.<br />
The biotin headgroup was either directly attached to the lipid or via a hexyl-spacer.<br />
Introduction <strong>of</strong> the C6-spacer served to model different distances between membrane<br />
and resonator surface. The adhesion and spreading <strong>of</strong> these functionalized vesicles onto<br />
an avidin-coated quartz resonator was monitored by an extended QCM-setup, short D-<br />
QCM, that provides the resonance frequency <strong>of</strong> the quartz together with the disspation<br />
factor D. D mirrors the disspation <strong>of</strong> motional energy and is formally the inverse <strong>of</strong> the<br />
quality factor Q.<br />
When identical vesicle numbers were injected into the measuring chamber, the shifts in<br />
resonance frequency and dissipation factor decreased with increasing concentrations <strong>of</strong><br />
biotin-labeled dopant (0 mol % to 30 mol %). This phenomenon was consistently<br />
observed for both distances <strong>of</strong> the biotin- group from the vesicle shell and independently<br />
supported by impedance analysis <strong>of</strong> the loaded resonators. Scanning force microscopy<br />
studies on these systems revealed a gradually increasing degree <strong>of</strong> vesicle spreading<br />
with increasing amounts <strong>of</strong> biotin-labeled lipids. For the highest dopant concentrations<br />
we observed extended regions covered with simple lipid bilayers. Thus, the large<br />
frequency shifts observed for vesicles doped with only small amounts <strong>of</strong> biotinylated<br />
lipids is very likely to originate from the water enclosed inside the vesicle or between<br />
vesicle membrane and surface. The variation <strong>of</strong> vesicle-substrate distance showed a<br />
significant effect only for a low concentrations <strong>of</strong> biotin-labeled lipid. The poster will<br />
compare these vesicle studies with QCM readings on the attachment and spreading <strong>of</strong><br />
mammalian cells.<br />
Literature<br />
Wegener et al., Cell Biochem. Biophys. 34/1(2001)121-151.<br />
contact:<br />
Björn Reiss<br />
Universität Münster<br />
Institut für Biochemie<br />
nudge@uni-muenster.de<br />
Wilhelm-Klemm Str 2<br />
48149 Münster (Deutschland)
B. van den Heuvel<br />
Gene hunting and gene cloning in human OXPHOS deficiency<br />
no abstract submitted<br />
contact:<br />
Dr. B. van den Heuvel<br />
University Medical Center Nijmegen<br />
Department <strong>of</strong> Pediatrics<br />
b.vandenheuvel@cukz.umcn<br />
P.O.Box 9101<br />
6500 Nijmegen (NL)
Cornelia Wiese, Alexandra Rolletschek, Ihor Zahanich#, Jürgen F. Heubach#, Jaroslaw<br />
Czyz, Anne Navarrete-Santos+, Olaf Horstmann*, Kenneth R. Boheler§, Anna M. Wobus<br />
Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
We describe the derivation <strong>of</strong> nestin-positive cells from adult mouse and human<br />
intestinal epithelium (INPs, intestinal epithelium-derived nestin-positive progenitors).<br />
The formation <strong>of</strong> INPs requires cultivation on mouse embryonic fibroblast feeder layers<br />
(FL). Continuous culture on FL resulted in the formation <strong>of</strong> nestin-positive clusters. When<br />
transferred in suspension culture, the clusters formed nestin-positive spheroids and<br />
neurosphere-like aggregates. RT-PCR analysis <strong>of</strong> mouse intestinal epithelial tissue and <strong>of</strong><br />
INP-derived clusters revealed Cdx1 and nestin transcripts. In human intestinal tissue<br />
and INPs after culture, nestin transcripts were also detected. Transcripts <strong>of</strong> the<br />
pluripotency factor nanog, the endodermal marker genes, HNF3b and a-fetoprotein, and<br />
the intestinal progenitor marker gene, math1, were found in mouse intestinal tissue, but<br />
were absent in INPs in vitro. Nestin-positive cells proliferated in vitro and partially coexpressed<br />
desmin suggesting an immature cell type <strong>of</strong> high plasticity.<br />
Electrophysiological analysis <strong>of</strong> mouse derived INPs revealed K+ and low voltageactivated<br />
Ca2+ and Ba2+ currents indicating a neural immature phenotype. Na+<br />
currents were absent in the nestin-positive cells. We conclude that nestin expression is a<br />
property <strong>of</strong> intestinal epithelial-derived progenitor cells after reprogramming by signals<br />
released from mouse embryonic fibroblast FL cells.<br />
contact:<br />
Cornelia Wiese<br />
IPK Gatersleben<br />
In Vitro Differentiation Group<br />
wiese@ipk-gatersleben.de<br />
Corrensst. 3<br />
06466 Gatersleben (Deutschland)<br />
additional information<br />
#) Department <strong>of</strong> Pharmacology and Toxicology, Dresden University <strong>of</strong> Technology,<br />
+) Institute <strong>of</strong> Anatomy and Cell Biology University <strong>of</strong> Halle-Wittenberg,<br />
*) Surgery University Clinics, University <strong>of</strong> Göttingen<br />
§) National Institute on Aging, NIH, Baltimore
Jonas Frisen<br />
Generation <strong>of</strong> neurons from stem cells in the adult brain<br />
No abstract submitted<br />
contact:<br />
Dr. Jonas Frisen<br />
Karolinska Institute<br />
Dept. Cell and Molecular Biology<br />
jonas.frisen@cmb.ki<br />
Box 285<br />
17165 Solna (Sweden)
Frank Schnütgen, Silke De-Zolt, Petra Van Sloun, Patricia Ruiz, Thomas Floss,<br />
Wolfgang Wurst, Harald von Melchner<br />
Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
A retroviral gene trap containing a human CD2 cell surface<br />
antigen/neomycinphosphotransferase fusion gene in the U3 region <strong>of</strong> its LTR (U3Ceo)<br />
was developed recently and used to screen the mammalian genome for genes encoding<br />
secreted and/or transmembrane proteins. From an integration library in murine<br />
fibroblasts, a collection <strong>of</strong> neomycin resistant (NeoR) clones was obtained; 86% also<br />
expressed the CD2 cell surface antigen. Molecular analysis <strong>of</strong> a random sample <strong>of</strong> NeoR<br />
clones revealed that the U3Ceo gene trap preferentially disrupted genes coding for<br />
secreted and transmembrane proteins. In each case, the signal sequence <strong>of</strong> the<br />
endogenous gene was fused in frame to the CD2/ neomycinphosphotransferase reporter<br />
gene due to a cryptic splice acceptor site embedded in the coding region <strong>of</strong> the CD2<br />
cDNA. To investigate whether U3Ceo would also effectively disrupt secretory pathway<br />
genes expressed in ES cells, we isolated several U3Ceo infected clones by selecting in<br />
G418 and recovered their GTSTs by 5’RACE. Sequencing <strong>of</strong> RACE products revealed that<br />
only one out <strong>of</strong> ten U3Ceo integrations occurred in a gene without a signal sequence .<br />
Thus, as in murine fibroblasts, signal sequence capture efficiency <strong>of</strong> U3Ceo in EScells is<br />
quite high and therefore makes it suitable for a large-scale approach. Based on this, we<br />
have produced and analyzed 5000 U3Ceo containing ES-cell clones, which are available<br />
for the scientific community via the german gene trap consortium website<br />
(http://www.genetrap.de/).<br />
contact:<br />
Dr. Frank Schnütgen<br />
University <strong>of</strong> Frankfurt, Medical School<br />
Molecular Hematology<br />
schnuetgen@em.uni-frankfurt.de<br />
Theodor Stern Kai 7<br />
60596 Frankfurt am Main (Germany)
Lambrini Tontsidou, Konstantinos Bilbilis, Petra Pennekamp, Boris Skryabin, Hans-<br />
Joachim Galla, Jürgen Horst, Bernd Dworniczak<br />
GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Autosomal dominant polycystic kidney disease (ADPKD) is one <strong>of</strong> the most common<br />
monogenic diseases in humans with an incidence <strong>of</strong> 1:1000. The main characteristic <strong>of</strong><br />
the disease is the formation <strong>of</strong> fluid filled cysts in both kidneys (originating from the<br />
proximal tubules), frequently resulting in end stage renal failure. In the vast majority <strong>of</strong><br />
cases the disease is caused by a mutation in the PKD1 or PKD2 gene. In the last couple<br />
<strong>of</strong> years numerous conventional Pkd1 and Pkd2 knockout mouse models have been<br />
generated. All <strong>of</strong> them are prenatal lethal, thus making it impossible to analyze the<br />
pathogenesis <strong>of</strong> ADPKD after birth. These limitations can be circumvented by conditional<br />
mutagenesis in which the loss <strong>of</strong> gene function is placed under temporal and/or spatial<br />
control. In order to establish this system we generated a transgenic mouse line<br />
expressing the Cre-recombinase under the control <strong>of</strong> a truncated mouse type II GGT<br />
promoter. We expected that this promoter allows the spatial excision <strong>of</strong> loxP flanked<br />
genes in proximal tubular cells.<br />
The recombinase activity was demonstrated by crossing these mice with Cre-reporter<br />
lines (Z/AP and ROSA26). Both lines carry a marker gene whose expression requires<br />
excision <strong>of</strong> loxP flanked stop sequences. Using alkaline phosphatase histochemistry in<br />
Z/AP mice as well as Beta-Galactosidase histochemistry in ROSA26 mice we observed a<br />
strong and specific expression <strong>of</strong> Cre-recombinase in proximal tubular renal cells.<br />
This GGT-Cre transgenic mouse line, which induces efficient Cre-mediated excision <strong>of</strong><br />
DNA in proximal tubules, should provide a useful genetic resource to elucidate the role <strong>of</strong><br />
loxP manipulated genetic targets in cyst formation <strong>of</strong> ADPKD.<br />
Literature<br />
1) Igarashi & Somlo (2002) J Am Soc Nephrol;13(9):2384-98<br />
2) Stec & Sigmund (1998) exp Nephrol; Nov-Dec;6(6):568-75<br />
3) Sepulveda et al (1997) J Biol Chem; May,272(18):11959-67<br />
4) Lobe et al. (1999) Dev Biol; Apr 15;208(2):281-92<br />
5) Soriano (1999) Nat Genet; Jan, 21(1):70-1<br />
contact:<br />
Dipl.-Biol. Lambrini Tontsidou<br />
Westfälische Wilhelms Universität Münster<br />
Institut für Humangenetik<br />
tontsid@uni-muenster.de<br />
Vesaliusweg 12-14<br />
48149 Münster (NRW Deutschland)
Poonam Balani, Christian Weidenfeller, Carsten Mim*, Christ<strong>of</strong> Grewer*, Thomas<br />
Rauen<br />
Glutamate transporter function in synaptic transmission<br />
Five different subtypes <strong>of</strong> glutamate transporters (GLAST1, GLT1, EAAC1, EAAT4,<br />
EAAT5) have been cloned to date. Here, we compared the retinal localization, the<br />
kinetics, the pharmacology, and the electrophysiological properties <strong>of</strong> the major<br />
glutamate transporters subtypes including two splicing variants <strong>of</strong> GLT1. These<br />
transporters are differentially expressed in the retina. Native and heterologously<br />
expressed transporters appear to assemble as homotrimers. Heterologously expressed<br />
transporters exhibited no significant differences in their substrate affinity, but<br />
demonstrated a unique pharmacological pr<strong>of</strong>ile, suggesting structural differences in their<br />
binding sites. Comparing the steady-state cycle time <strong>of</strong> the highly anion-conducting<br />
EAAT4 with that <strong>of</strong> GLAST1, GLT1 and EAAC1, indicated 5-10 times larger cycle time<br />
constants for EAAT4. This suggests that EAAT4, in contrast to the other subtypes, is a<br />
high-affinity, low-capacity transporter. The transient kinetic experiments in combination<br />
with the differential localization <strong>of</strong> glutamate transporter (pre-, post-, extra-synaptic and<br />
glial) shed new light onto their complex function in regulating the glutamate<br />
concentration in the CNS. Our results suggest that rapid binding <strong>of</strong> glutamate to synaptic<br />
transporter binding sites, that act as a glutamate buffer, could provide a discriminating<br />
mechanism to temporally control the activation <strong>of</strong> AMPA and NMDA receptors by<br />
glutamate.<br />
CG and TR are grateful for financial support from the DFG , and PB from GSC-MS<br />
contact:<br />
Dr. Thomas Rauen<br />
Universität Münster<br />
Biochemie<br />
rauen@uni-muenster.de<br />
Wilhelm Klemm Str.2<br />
48149 Müpnster (Germany)<br />
additional information<br />
*University <strong>of</strong> Miami, USA,
Helena Schwaßmann, Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause,<br />
Holger Seelert, Norbert A. Dencher<br />
H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
H + -ATP synthase is the dominant ATP production site in mitochondria and chloroplasts.<br />
So far dimerization <strong>of</strong> ATP synthase has been observed only in mitochondria by<br />
biochemical and electron microscopic investigations. Although the physiological<br />
relevance remains still enigmatic, dimerization was proposed to be a unique feature <strong>of</strong><br />
the mitochondrion [Schägger, H., Biochim. Biophys. Acta 2002, 1555, 154-159.]. It is<br />
hard to imagine, however, that closely related protein complexes <strong>of</strong> mitochondria and<br />
chloroplast should show such severe differences in structural organization. We present<br />
the first evidences for dimerization <strong>of</strong> chloroplast ATP synthases within the thylakoid<br />
membrane [1].<br />
By investigation <strong>of</strong> the thylakoid membrane <strong>of</strong> Chlamydomonas reinhardtii by bluenative<br />
polyacrylamide gel electrophoresis, dimerization <strong>of</strong> the chloroplast ATP synthase<br />
was detected. Chloroplast ATP synthase dimer dissociates into monomers upon<br />
incubation with vanadate or phosphate but not by incubation with molybdate, while the<br />
mitochondrial dimer is completely unaffected. This suggests a distinct dimerization<br />
mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and<br />
phosphate bind to the active sites, contact sites located on the hydrophilic CF 1 part are<br />
suggested for the chloroplast ATP synthase dimer. As the degree <strong>of</strong> dimerization varies<br />
with phosphate concentration, dimerization might be a response to low phosphate<br />
concentrations.<br />
Literature<br />
[1] Rexroth, S., Meyer zu Tittingdorf, J.M.W., Schwaßmann, H.J., Krause, F., Seelert, H.,<br />
Dencher, N.A. (2004), H+-ATP Synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii . BBA Bioenergetics (submitted).<br />
contact:<br />
Helena Schwaßmann<br />
TU Darmstadt<br />
Physikalische Biochemie<br />
Helena@schwassmann.de<br />
Petersenstr. 22<br />
64287 Darmstadt (Deutschland)
Christoph Wegener, Anton Savitsky, Mathias Pfeiffer, Igor Grigorev, Klaus Möbius,<br />
Heinz-Jürgen Steinh<strong>of</strong>f<br />
High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed<br />
spin labeling (SDSL) has emerged as a powerful tool to obtain information on the<br />
structure and conformational dynamics <strong>of</strong> membrane proteins [1-3].<br />
Here we report on high-field high-frequency (95 GHz, W-band) EPR spectroscopy studies<br />
on site-directed spin labeled Bacteriorhodopsin (BR), an integral membrane protein<br />
which acts as a light driven proton pump. According to the enhanced Zeeman resolution<br />
in the frequency regime <strong>of</strong> the W-band the EPR spectra <strong>of</strong> spin labeled proteins recorded<br />
at low temperature (T
Daniela Villone, Leena Bruckner-Tuderman, Peter Bruckner, Uwe Hansen<br />
Human dermal basement membrane zone: Characterization <strong>of</strong><br />
functional suprastructures<br />
Cutaneous basement membrane zone consists <strong>of</strong> several adhesive suprastructures<br />
tethering the epidermis to the dermis. They are part <strong>of</strong> different separated but<br />
interconnected networks. We want to characterize their components, and especially the<br />
molecular composition <strong>of</strong> adhesive suprastructures, such as anchoring fibrils containing<br />
collagen VII. In addition, we are interested in morphological and compositional<br />
alterations occuring in dystrophic epidermolysis bullosa.<br />
Authentic suprastructural fragments were obtained by mechanical disruption <strong>of</strong><br />
deepithelialized human skin. The crude skin extract contains components <strong>of</strong> the<br />
basement membrane and the papillary dermis. The separation <strong>of</strong> the crude skin extract<br />
into fibrillar and network-like fractions were performed by using magnetic immunobeads.<br />
Morphology and composition within suprastructures were examined by immunogoldelectron<br />
microscopy.<br />
In double-labeling experiments using crude skin extracts collagen VII and laminin 5<br />
occurred as part <strong>of</strong> the same supramolecular structure. Similarly, we found a partial<br />
colocalisation <strong>of</strong> collagen VII with collagen XVII. By using magnetic immunobeads<br />
directed against collagen I we achieved the separation <strong>of</strong> D-periodically banded fibrils<br />
containing collagen I from the entangled networks <strong>of</strong> the basement membrane.<br />
Furthermore, the specific isolation <strong>of</strong> morphologically distinct network-like fractions<br />
containing as a major component laminin 5 on the one hand and collagen IV on the<br />
other hand could be achieved. The laminin 5 enriched fraction did not seem to contain<br />
collagen IV. Treatment with collagenase did not affect the network-like appearence <strong>of</strong><br />
the laminin 5 fraction in contrast to the completely digested collagen IV fraction.<br />
contact:<br />
Daniela Villone<br />
University Hospital Muenster<br />
Dept. Physiol. Chem. & Pathobiochem.<br />
dvillone@uni-muenster.de<br />
Waldeyerstraße 15<br />
48143 Münster (Germany)<br />
additional information<br />
L. Bruckner-Tuderman: Dept. Dermatol. University Hospital Freiburg, Germany
Roswitha Pfragner, Werner Emberger, Sigrun Sodia, Olaf Reich, Sigrid Regauer<br />
Human mucosa-associated malignant melanomas:<br />
cytogenetic and molecular genetic characterization<br />
Mucosa-associated malignant melanomas are very rare neoplasms, representing 2.5% <strong>of</strong><br />
all malignant melanomas. While cutaneous melanomas are common UV-light- induced<br />
neoplasms, the mucosal MM arise from unexposed mucosal areas. Mucosal MM are<br />
characterized by a highly aggressive clinical course with early hematogenous<br />
metastases. The etiology and pathophysiology are poorly investigated and no cell culture<br />
model was available . Comparative genomic hybridization <strong>of</strong> sinunasal MM*) described<br />
complex cytogenetic aberrations involving chromosomes # 1, 6, and 8, however,<br />
cytogenetic analyses for vulvar MM were lacking. We cultured three cases <strong>of</strong> vulvar MM<br />
and two cases <strong>of</strong> sinunasal MM and characterized them cytogenetically. Molecular<br />
cytogenetic analyses using multicolor in situ hybridization (M-FISH)were performed for<br />
detailed characterization. The results <strong>of</strong> combined analyses <strong>of</strong> G-banded metaphases and<br />
M-FISH showed complex chromosonmal aberrations in all cases. A high grade <strong>of</strong><br />
similarity <strong>of</strong> breakpoints gains and losses, with hotspots on chromosomes #1, 6, 8, 10,<br />
12, 13 and 21 was identified in mucosal MM. This pattern is distinct from cutaneous MM.<br />
It is hoped that molecular characterization <strong>of</strong> mucusal MM may indicate a pattern that<br />
can be used in diagnosis and treatment.<br />
Literature<br />
*)Van Dijk M., et al. Distinct chromosomal aberrations in sinunasal mucosal melanoma<br />
as detected by comparative genomic hybridization; Genes Chromosomes Cancer<br />
2003;36:151-158<br />
contact:<br />
Dr Roswitha Pfragner<br />
Medizinische Universität Graz<br />
Institut für Pathophysiologie<br />
roswitha.pfragner@meduni-graz.at<br />
Heinrichstrasse 31<br />
A 8010 Graz (Österreich)<br />
additional information<br />
Departments <strong>of</strong> 1)Pathophysiology 2)Medical Biology and Human Genetics 3)Gynecology<br />
4)Pathology; Medical University Graz, Austria
Uwe Schulte, Jörg Oliver Thumfart, Wolfgang Bildl, Hans-Günther Knaus, Claudia<br />
Sailer, Matthias Mann, Jens Andersen, Martin Biniossek, Volker Rohde, Bernd Fakler<br />
Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Voltage-gated potassium (K v ) channels are key determinants <strong>of</strong> neuronal excitability that<br />
control action potential repolarization, interspike membrane potential, and action<br />
potential frequency. K v 1.1 is one <strong>of</strong> the most abundant K v subunits in the brain and<br />
forms a non-inactivating delayed rectifier channel if assembled homomerically. However,<br />
interaction <strong>of</strong> K v 1.1 with other proteins considerably affects its functional properties.<br />
Thus, association with K v 1.4 or with certain K v beta subunits endows K v 1.1 with rapid<br />
voltage-dependent inactivation, and interaction with Caspr2 targets K v 1.1 to the node <strong>of</strong><br />
Ranvier.<br />
We have isolated K v 1.1 containing protein complexes from rat brain and identified their<br />
constituents with nano-liquid chromatography MS/MS mass spectrometry. As expected,<br />
these analyses showed that K v 1.1 may be associated with other K v alpha subunits<br />
(K v 1.2, K v 1.4, K v 1.6), with K v beta subunits (beta1, beta2, beta3), as well as with<br />
Caspr2 and the PDZ protein PSD95. In addition, however, a number <strong>of</strong> further proteins<br />
were determined that control subcellular localization <strong>of</strong> the K v 1.1 channel complexes as<br />
well as their functional characteristics including activation and inactivation gating.<br />
contact:<br />
Dr. Jörg Oliver Thumfart<br />
University <strong>of</strong> Freiburg<br />
Physiology II<br />
joerg.thumfart@physiologie.uni-freiburg.de<br />
Hermann-Herder-Str. 7<br />
79104 Freiburg i. Br. (Deutschland)<br />
additional information<br />
U. Schulte, W. Bildl, V. Rohde: Complexio-GmbH, March-Buchheim, Germany.<br />
H.G. Knaus, C. Sailer: Dept. <strong>of</strong> Biochemical Pharmacology, University <strong>of</strong> Innsbruck, Austria.<br />
M. Mann, J. Andersen: Dept. <strong>of</strong> Biochemistry & Molecular Biology, USD Odense, Denmark.<br />
M.Biniossek: Dept. <strong>of</strong> Molcular Medicine, University <strong>of</strong> Freiburg, Germany.<br />
B. Fakler: Dept. <strong>of</strong> Physiology II, University <strong>of</strong> Freiburg, Germany.
Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger Seelert, Norbert<br />
Dencher<br />
Identification <strong>of</strong> integral thylakoid membrane proteins from<br />
Chlamydomonas reinhardtii by peptide mass fingerprinting<br />
and MALDI-MS<br />
Analysis <strong>of</strong> the membrane integral proteome is mainly dependent in the ability <strong>of</strong> protein<br />
separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique, so<br />
far mainly applied to mitochondrial respiratory chain, capable <strong>of</strong> efficient membrane<br />
protein separation. Applying BN-PAGE to thylakoid membranes after mild solubilisation<br />
with digitonin, we identified extremely hydrophobic subunits <strong>of</strong> the photosystem<br />
complexes with 5 - 11 transmembrane helices by trypsination and subsequent matrix<br />
assisted laser desorption / ionization - mass spectronomy (MALDI-MS). All<br />
photophosphorylation complexes, as well as their supercomplexes, from thylakoids <strong>of</strong> C.<br />
reinhardtii were resolved in a single gel, providing an analytical tool suitable to<br />
characterize composition <strong>of</strong> membrane protein supercomplexes in different physiological<br />
states. Using this technique significant differences in supercomplex composition from C.<br />
reinhardtii grown under photoautotrophic and photomixothrophic conditions can be<br />
observed. While under photoautotrophic conditions the organism is dependent on<br />
chloroplastidic energy production, under photomixotrophic growth the relevance <strong>of</strong> the<br />
mitochondrial oxidative phosphorylation is increased noticeably. In response to the<br />
physiological state the stability <strong>of</strong> thylakoid membrane protein complexes changes<br />
significantly, in addition to changes in protein expression level.<br />
Literature<br />
Rexroth, S., Meyer zu Tittingdorf, J.M.W., Krause, F., Dencher, N.A. and Seelert, H.<br />
Thylakoid membrane proteome at altered metabolic state: Challenging the forgotten<br />
realms <strong>of</strong> the proteome. Electrophoresis 24, 2814-2823 (2003).<br />
contact:<br />
Sascha Rexroth<br />
TU Darmstadt<br />
Physikalische Biochemie<br />
srex@gmx.de<br />
Petersenstr. 22<br />
64287 Darmstadt (Deutschland)
Olesya Chayka, Jörg Kintscher, Karl-Heinz Klempnauer<br />
Identification <strong>of</strong> MYB and C/EBP responsive enhancer <strong>of</strong> the<br />
chicken mim-1 gene<br />
Myb and C/EBP transcription factors play crucial roles in activating a number <strong>of</strong> genes<br />
that are specifically expressed in myelomonocytic cells. The best studied <strong>of</strong> these genes<br />
is the chicken mim-1 gene, which also provides a paradigm for how Myb activates a<br />
target gene in a lineage specific manner within the hematopoietic system.<br />
Previous work has shown that the mim-1 promoter contains Myb as well as C/EBP<br />
binding sites and is activated synergistically by both transcription factors. We have now<br />
explored the regulation <strong>of</strong> the mim-1 gene by Myb and C/EBP• in more detail. We have<br />
used mapping <strong>of</strong> DNase I hypersensitive sites in the chromatin <strong>of</strong> a number <strong>of</strong> chicken<br />
cell lines to identify a cell specific cis-acting sequence elements in the upstream region<br />
<strong>of</strong> the mim-1 gene. Using transient and stable transfection experiments we show that<br />
this region corresponds to a Myb-dependent and myelomonocytic-specific enhancer.<br />
Sequence analysis <strong>of</strong> the mim-1 enhancer region shows the presence <strong>of</strong> a number <strong>of</strong><br />
binding sites for Myb, C/EBP and other proteins.<br />
A variety <strong>of</strong> techniques, such as in vivo footprinting, Chromatin Immunoprecipitation,<br />
Electrophoretic Mobility-Shift and reporter gene assays, were performed to further<br />
characterize the proteins involved in regulation <strong>of</strong> the enhancer activity.<br />
The results <strong>of</strong> these experiments have shown that mim-1 enhancer is activated through<br />
interaction with Myb, C/EBPß and p300 proteins. Thus the activation <strong>of</strong> the mim-1 gene<br />
by Myb is more complex than currently thought and involves a potent cell-specific<br />
enhancer in addition to the promoter <strong>of</strong> the gene.<br />
contact:<br />
Olesya Chayka<br />
Universität Muenster<br />
Institut für Biochemie<br />
tchaikao@uni-muenster.de<br />
Wilhelm-Klemm 2<br />
48149 Muenster (Germany)
Barbara Sitek, Ognjan Apostolov, Kathy Pfeiffer, Kai Stühler, Helmut E. Meyer,<br />
Angelika Eggert, Alexander Schramm<br />
Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
The tyrosine kinases TrkA and TrkB are members <strong>of</strong> the family <strong>of</strong> neurotrophin<br />
receptors, which mediate survival, differentiation, growth and apoptosis <strong>of</strong> neurons in<br />
response to stimulation by the neurotrophins NGF (nerve growth factor) and BDNF (brain<br />
derived neurotrophic factor), respectively.<br />
Activation <strong>of</strong> TrkA by NGF causes differentiation <strong>of</strong> neurons and neuroblastoma cells,<br />
whereas it induces proliferation <strong>of</strong> fibroblasts and apoptosis <strong>of</strong> medulloblastoma cells.<br />
Similarly, TrkB is associated with a favorable biology and good prognosis in medullary<br />
thyroid carcinoma, which is in contrast to the situation found in neuroblastoma. Since<br />
TrkA/TrkB exhibit high level <strong>of</strong> sequence similarity and use overlapping pathways for<br />
signal transduction there have to be specific effectors critical for receptor and cell-type<br />
specific response.<br />
In order to analyze biological effects <strong>of</strong> TrkA und TrkB in a defined model, we have<br />
chosen neuroblastoma cell line SY5Y stably transfected with TrkA or TrkB, respectively.<br />
We performed a proteome analysis <strong>of</strong> these cell lines in the absence or presence <strong>of</strong> their<br />
respective ligands in a time course from 0 to 24 h. The use <strong>of</strong> the recently introduced<br />
DIGE (fluorescence 2-D difference gel electrophoresis) system (Amersham Biosciences)<br />
allowed us to monitor differences in protein expression between samples in one gel.<br />
Following image analysis we have detected 22 BDNF-dependent regulated proteins<br />
(expression change > 1,5 fold compared to unstimulated cells, p < 0,05) in TrkB SY5Y<br />
cells. The analysis <strong>of</strong> TrkA SY5Y cells resulted in 9 NGF-dependent regulated proteins<br />
(ratio > 1,3 compared to unstimulated cells, p < 0,05). Differentially expressed proteins<br />
were subsequently identified by MALDI-PMF/PFF-MS analysis. Functional assignment<br />
revealed, that the majority <strong>of</strong> these proteins is involved in organisation and maintenance<br />
<strong>of</strong> cellular structures (e.g. lamin A/C).<br />
contact:<br />
Barbara Sitek<br />
Ruhr-Universität Bochum<br />
Medizinisches Proteom-Center<br />
barbara.sitek@rub.de<br />
Universitätsstr. 150<br />
44801 Bochum (Deutschland)
Frank R. Neumann, M.R. Gartenberg, A. Taddei, T. Laroche, M. B•aszczyk, Susan<br />
Gasser<br />
Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
We have analysed the dynamics <strong>of</strong> chromosomal domains in interphase yeast nuclei by<br />
tagging different types <strong>of</strong> chromosomal regions with a GFP- lac repressor fusion. We<br />
monitor the absolute movement and subnuclear position <strong>of</strong> specific sites in the yeast<br />
genome in real-time, sampling at very short time intervals (1.5 s) with either 2D or 3D<br />
imaging techniques. Internal transcribed regions are highly mobile in G1 phase,<br />
frequently moving more than 0.5 microns/10 s, in an energy-dependent fashion. The<br />
rapid diffusive movement <strong>of</strong> chromatin detected in G1 becomes constrained in S phase<br />
through a mechanism dependent on active DNA replication. In contrast, telomeres and<br />
centromeres provide replication-independent constraint on chromatin movement.<br />
The silent mating-type loci <strong>of</strong> Saccharomyces cerevisiae are transcriptionally repressed<br />
by a heterochromatin-like structure. Autonomous positions <strong>of</strong> these silent chromosomal<br />
domains within the nucleus are masked by their tight linkage to telomeres, which are<br />
also silenced and held together in clusters at the nuclear periphery by Ku. To determine<br />
the intrinsic localization <strong>of</strong> silent chromatin, the HMR mating-type locus was uncoupled<br />
from the chromosome by inducible site-specific recombination, and the position and<br />
dynamics <strong>of</strong> the excised fragment was determined. We find that silenced rings<br />
containing HMR associate with the nuclear periphery in a SIR-dependent manner, while<br />
non-silent rings move without detectable constraint throughout the nucleoplasm. This<br />
movement is similar for a second excised locus, LYS2, which is also transcriptionally<br />
active.The same mobility can be detected for silent rings if the anchorage proteins at the<br />
nuclear periphery are deleted from the yeast genome. Thus we identify the necessary<br />
anchors and show that while anchoring is important for repression in wildtype cells, once<br />
SIR proteins are distributed throughout the nucleoplasm, there is no need for anchorage<br />
to keep chromatin repressed.<br />
The relationship <strong>of</strong> the striking ATP-dependent movement <strong>of</strong> expressed chromatin<br />
sequences has been examined in relation to transcriptional activity. We find that the<br />
targeting <strong>of</strong> a strong activator such as VP16 increases chromatin mobility, while histone<br />
acetlyases do not. VP167 can even overcome the anchorage <strong>of</strong> telomeres, probably by<br />
recruitment <strong>of</strong> chromatin remodellers such as SWI/SNF2. Inhibition <strong>of</strong> transcriptional<br />
elongation, on the other hand, does not affect chromatin mobility. We conclude that<br />
chromatin positioning and movement is tightly linked to transcriptional potential and<br />
local chromatin structure.<br />
Literature<br />
1. Heun, P., Laroche, T., Shimada, K., Furrer, P., and Gasser, S.M. (2001) Chromosomal<br />
dynamics in the<br />
yeast interphase nucleus.<br />
Science, 293, 2181 - 2186<br />
2. Gasser, SM (2002) Visualizing Chromatin Dynamics in Interphase Nuclei. Science,<br />
296, 1412 – 1416<br />
3. Hediger, F, Dubrana, K and Gasser, SM (2002) Myosin-like proteins 1 and 2 are not<br />
required for silencing or telomere anchoring but act in the Tel1 pathway <strong>of</strong> telomere<br />
length control J Struct Biol, 140, 79 - 91<br />
4. Hediger, F, Neumann, FR,Van Houwe, G, Dubrana, K and Gasser, SM (2002) Live<br />
imaging <strong>of</strong> yeast telomeres: yKu and Sir mutations define independent but redundant<br />
anchoring pathways Curr Biology, 12, 2076-2089<br />
5. Taddei, A, Hediger, F, Neumann, FR, Bauer, C and Gasser, SM (2004) Separation <strong>of</strong><br />
silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins<br />
EMBO J, 23, 1301-1312<br />
contact:<br />
Pr<strong>of</strong>. Susan Gasser<br />
University <strong>of</strong> Geneva<br />
Dept. <strong>of</strong> Molecular Biology<br />
susan.gasser@molbio.unige.ch<br />
30, Quai Ernest Ansermet<br />
1211 Geneva 4 (Switzerland)
Jerome Cavaille, Hervé Seitz, Hélène Royo, Shau-Ping Lin *, Neil Youngson *, Anne C.<br />
Ferguson-Smith *<br />
Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
microRNAs (or miRNAs) represent a class <strong>of</strong> eucaryotic small non-coding RNAs (~21-23<br />
nt) that can silence gene expression by either repressing translation or by triggering<br />
mRNA degradation (RNA interference or RNAi). Here we show that the human imprinted<br />
domain at 14q32 encodes for many potential microRNA genes (~ 46), with most <strong>of</strong> them<br />
arranged in tandem arrays <strong>of</strong> closely related sequences. In the mouse, this miRNA gene<br />
cluster is conserved at the homologous distal chromosome 12. In vivo all the miRNAs<br />
that we have detected are expressed in the developing embryo (both in the head and in<br />
the trunk) and in the placenta while in the adult their expression is mainly restricted to<br />
the brain. We also show that the miRNA genes are only expressed from the maternallyinherited<br />
chromosome and that their imprinted expression is regulated by an intergenic<br />
germline-derived differentially-methylated region (IG-DMR) located about 200 kb<br />
upstream from the miRNA cluster. The functions <strong>of</strong> these miRNAs, which seem only<br />
conserved in mammals, will be discussed both in terms <strong>of</strong> epigenetic control and gene<br />
regulation during development.<br />
Literature<br />
Seitz et al., 2003 Nature Genetics.<br />
Lin et al., 2003 Nature Genetics.<br />
Seitz et al, 2004 Genome research, in press.<br />
contact:<br />
Dr Jerome Cavaille<br />
CNRS<br />
Laboratoire de Biologie Moleculaire des Eucaryotes<br />
cavaille@ibcg.biotoul.fr<br />
118 Route de Narbonne<br />
31062 Toulouse (France)<br />
additional information<br />
*) Department <strong>of</strong> Anatomy, University <strong>of</strong> Cambridge, UK.
Marion Jech, Birgit Hutter-Paier, Elisabeth Ingolic, Harald Höger, Elisabeth Grygar,<br />
Sepp Porta, Brigitte Poncza, Manfred Windisch, Roswitha Pfragner<br />
In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Pheochromocytomas are rare tumors which arise from chromaffin cells <strong>of</strong> the adrenal<br />
medulla. They can occur sporadically or as part <strong>of</strong> Multiple Endocrine Neoplasia type 2<br />
and in patients with neur<strong>of</strong>ibromatosis type 1. Two pheochromocytoma cell lines, KNA*,<br />
established from a Caucasian female with MEN2A-2 syndrome and MPC 862L, derived<br />
from a neur<strong>of</strong>ibromatosis knockout mouse**, were analyzed. The tumorigenicity <strong>of</strong> the<br />
cell lines was investigated by injecting the cells into nude and SCID mice. Both lines<br />
turned out to be tumorigenic. The cell lines as well as tumor tissues derived from<br />
transplanted mice were tested by immunocyto- and immunohistochemistry for<br />
neuroendocrine markers (synaptophysin, tyrosine hydroxylase, S100, neur<strong>of</strong>ilament and<br />
vimentin). Both cell lines showed immunoreactivity for synaptophysin, tyrosine<br />
hydroxylase, neur<strong>of</strong>ilament and S100. Immunoreactivity to vimentin was observed in<br />
KNA cells only. The tissues showed immunoreactivity for synaptophysin, tyrosine<br />
hydroxylase and S100. However, neur<strong>of</strong>ilament and vimentin were positive in KNA tissue<br />
only. Furthermore, as investigated by electron microscopy, both cell lines and tumor<br />
tissues, showed granules containing catecholamines. Neurite-like processes were<br />
detected on MPC 862L cells and analyses with HPLC showed large quantities <strong>of</strong><br />
catecholamines in MPC tumor tissue and KNA cells. In vitro models <strong>of</strong> neuroendocrine<br />
tumors prove to be valuable models for investigations in neuronal disease like Morbus<br />
Alzheimer or Morbus Parkinson.<br />
Literature<br />
*)Weinhäusel A et al., Endocrine Pathology, 14(4):375-382, 2003<br />
**) Powers JG et al., Cell Tissue Research 302:309-320, 2000<br />
contact:<br />
Mag. Marion Jech<br />
Medizinische Universität Graz<br />
Pathophysiologie<br />
marion.jech@meduni-graz.at<br />
Heinrichstraße 31a<br />
8010 Graz (Österreich)
S. Ramponi, A. Grotti, S. Vultaggio, A. Morisetti, G. Alfieri, V. Lorusso<br />
Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
The structural and functional integrity <strong>of</strong> the Endothelial Cell (EC) monolayer is essential<br />
to allow the exchange <strong>of</strong> water, solutes and the cell migration, to prevent platelet<br />
deposition and thrombus formation and to regulate the Vascular Permeability (VP) in the<br />
different districts. MultiHance, Magnevist and ProHance are Gadolinium based Contrast<br />
Media (CM) used clinically in Magnetic Resonance Imaging. After iv injection they rapidly<br />
distribute in plasma, cross the Vascular Endothelium (VE) and diffuse in the interstitial<br />
space, being then eliminated mainly via the kidneys. Reduced renal excretion increases<br />
the concentration and the time <strong>of</strong> VE exposure to CM, possibly resulting in direct toxic<br />
effects, functional alterations and activation <strong>of</strong> EC or more subtle changes (junctional<br />
reorganization, cytoskeleton modification and EC retraction).<br />
CM were tested for their effects on VP, using Transwell unit that measures the transendothelial<br />
flux (luminal to abluminal) <strong>of</strong> a tracer macromolecule (FITC dextran) at<br />
different time points. Results from this functional assay were compared to EC<br />
morphological changes by Scansion Electron Microscopy and to cytoskeleton<br />
modification/ reorganization by F-actin staining.<br />
This approach, which mimics the pathological vascular persistence <strong>of</strong> high levels <strong>of</strong> CM<br />
and their effects on vessel permeability, can be applied to comparatively study the<br />
toxicity and the VE permeability <strong>of</strong> different CM, thus predicting their distribution in vivo.<br />
contact:<br />
Stefania Vultaggio<br />
Bracco Imaging S.p.A<br />
beatrice.brunella@bracco.com<br />
via Folli 50<br />
20134 Milano (Italy)
Stefan Malcharek, Christian Schachtrup, Friedrich Spener, Hans-Joachim Galla<br />
Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong><br />
H- and E- FABP double knock-out mice<br />
Since lung surfactant contains 90 % lipids endogenous and exogenous fatty acids are<br />
nessecary for the synthesis <strong>of</strong> lung surfactant. Fatty-acid-binding-proteins (FABPs) are<br />
required to transport and to provide these fatty acids to the lipid metabolism. The<br />
isolated hydrophobic extract from epidermal (E) and heart (H) FABP knock-out mice<br />
show a decreased lipid/protein ratio. Film balance (FB) and scanning force microscopy<br />
(SFM) measurements <strong>of</strong> this lipid/protein mixtures demonstrate a dysfunction in<br />
formation <strong>of</strong> multilayers at the air-water interface. Hydrophobic extract obtained from<br />
wildtype and double knock-out mice was investigated by fluorescence microscopy (FM).<br />
We are able to show a difference in size and structure <strong>of</strong> domains, which corresponds to<br />
high and low values <strong>of</strong> the lipid/protein ratio <strong>of</strong> wildtype and double knock-out mice.<br />
Double knock-out were treated by oral administration <strong>of</strong> pioglitazone, a drug that<br />
intensifies lipidsynthesis. An increase <strong>of</strong> the lipid/protein ratio was observed coming<br />
close to a value till <strong>of</strong> wildtype mice. Additionally the formation <strong>of</strong> multi-layers in the<br />
plateau region <strong>of</strong> hydrophobic extract was regenerated by pioglitazone treatment. The<br />
isotherms and SFM images indicate an increasing formation <strong>of</strong> multilayers. FM<br />
measurements show comparable size and structure <strong>of</strong> domains. In conclusion we<br />
demonstrate that the lipid metabolism is strongly correlated to the structural<br />
organisation and proper function <strong>of</strong> the alveolar surfactant.<br />
contact:<br />
Dipl. Chem. Stefan Malcharek<br />
Universität Münster<br />
Institut für Biochemie<br />
schlange@uni-muenster.de<br />
Wilhelm Klemmstraße 2<br />
48149 Münster (Deutschland)
Ana Kilic, Ana Velic, Sevdalina Yurukova, Larissa Fabritz, Eberhard Schlatter, Michaela<br />
Kuhn<br />
Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Mice with global deletion <strong>of</strong> the guanylyl cyclase-A receptor for ANP (GC-A -/- mice)<br />
have chronical arterial hypertension, hypervolemia and global cardiac hypertrophy.<br />
Clinical and experimental studies indicate that increased expression or activity <strong>of</strong> the<br />
Na+-H+ exchanger (NHE-1) might contribute to cardiac hypertrophy. Our studies on<br />
isolated adult cardiomyocytes from wild type (WT) and GC-A -/- mice with fluorimetric<br />
dyes BCECF and Indo-1 showed that GC-A -/- cadiomyocytes have increased intracellular<br />
pH (pHi) (7.41±0.11 vs. 8.23 ± 0.17; p< 0.05) as well as systolic Ca2+ levels (Indo-1<br />
ratio, 1.60 ± 0.02 vs. 1.69 ± 0.03; p< 0.05). The increased pHi suggests that NHE-1 is<br />
more active in GC-A -/- cardiomyocytes thereby leading to a decrease in [H+]i and an<br />
increase in [Na+]i. Rise in [Na+]i might be responsible for an increase in [Ca2+]i levels<br />
via Na+-Ca2+ exchange (NCX) which could contribute to cardiac hypertrophy. To study<br />
this hypothesis GC-A -/- mice were treated with a food containing Cariporide (Aventis<br />
Pharmaceuticals), an inhibitor <strong>of</strong> NHE-1. Cariporide had no effect on arterial<br />
hypertension <strong>of</strong> GC-A -/- mice. Despite <strong>of</strong> persistent hypertension, cardiac hypertrophy<br />
was significantly inhibited by Cariporide treatment and pHi and Ca2+ amplitudes<br />
returned on WT levels. Conclusions: The cardiac NHE-1 represents one <strong>of</strong> the heart’s key<br />
components to maintain physiological intracellular pH. Increased activation <strong>of</strong> the NHE-1<br />
contributes to development <strong>of</strong> cardiac hypertrophy in GC-A -/- mice, probably by<br />
increasing intracellular sodium load, which finally results in elevated intracellular calcium<br />
via the NCX. Pharmacological inhibition <strong>of</strong> the cardiac NHE-1 might prove useful for the<br />
treatment <strong>of</strong> hypertensive cardiac hypertrophy.<br />
Literature<br />
Engelhardt S., Circ. Res. 2002; 90: 814-819<br />
Cingolani HE., Circ. Res. 2002;90: 751-753<br />
contact:<br />
Dipl Biol Ana Kilic<br />
Universitätklinikum Münster<br />
Institut <strong>of</strong> Pharmacology and Toxicology<br />
kilicana@uni-muenster.de<br />
Domagkstr. 12<br />
48194 Münster (Germany)
Robert Tampe<br />
INs and OUTs <strong>of</strong> Antigens - The Transport Machinery TAP<br />
in the Cellular Immune System<br />
The immune system has evolved to defend the vertebrate organism against numerous<br />
bacteria, viruses, toxins and parasites, which it is daily confronted with. The transporter<br />
associated with antigen processing (TAP) plays a pivotal role in the adaptive immune<br />
response. The ABC transporter TAP translocates peptides derived mainly from<br />
proteasomal degradation from the cytosol into the endoplasmic reticulum, where these<br />
peptide are loaded on MHC I molecules. At the cell surface, MHC I complexes display<br />
their antigenic cargo to cytotoxic T-lymphocytes, which eventually will eliminate infected<br />
or tumorigenic cells. Due to its key function in the compartmentalization <strong>of</strong> antigens<br />
(connecting the inside with the outside), the TAP transport machinery is targeted by<br />
sophisticated inhibition mechanism, by which tumors and viruses can evade immune<br />
surveillance. The function and structural organization <strong>of</strong> the TAP complex as well as viral<br />
inhibition strategies are discussed.<br />
contact:<br />
Pr<strong>of</strong>.Dr. Robert Tampé<br />
Biozentrum Frankfurt<br />
Institut für Biochemie<br />
tampe@em.uni-frankfurt.de<br />
Marie-Curie-Str. 9<br />
60439 Frankfurt a.M. (Germany)
Udo Heinemann, Konrad Büssow, Uwe Mueller, Patrick Umbach<br />
Insight into the structure <strong>of</strong> human proteins from a structural<br />
genomics approach<br />
Structural genomics or structural proteomics are terms used to describe a concerted<br />
world-wide effort to determine the three-dimensional structures <strong>of</strong> all proteins or other<br />
biological macromolecules encoded by the genomes <strong>of</strong> key organisms. The Berlin-based<br />
Protein Structure Factory consortium focusses on the structure analysis <strong>of</strong> medically<br />
relevant human proteins at reduced cost and elevated throughput (1). The Factory has<br />
implemented high-throughput innovations in the fields <strong>of</strong> semi-automated expression<br />
cloning and protein purification, NMR spectral assignment, protein crystallization, and<br />
synchrotron-based protein crystallography. A number <strong>of</strong> NMR and crystal structures have<br />
been determined that both validate the approach taken by the Factory towards sample<br />
preparation and structure analysis at increased throughput and provide important insight<br />
into the molecular basis <strong>of</strong> disease. The human protein hp14.5, for example, is a<br />
representative <strong>of</strong> the large family <strong>of</strong> YjgF/YER057c/UK114 proteins conserved from<br />
bacteria to man. The crystal structure shows hp14.5 to adopt a chorismate mutase-like<br />
subunit structure and to bind benzoate molecules in inter-subunits clefts at the surface<br />
<strong>of</strong> its trimeric arrangement which are likely to serve as sites <strong>of</strong> enzyme activity (2).<br />
Gankyrin, to use a second example, is the product <strong>of</strong> the first human oncogene linked to<br />
hepatocellular carcinoma. Crystallographic analysis shows gankyrin to be composed <strong>of</strong><br />
five complete and two incomplete ankyrin repeats and provides a structural framework<br />
to understand its documented interactions with the retinoblastoma protein, Rb, the<br />
cyclin-dependent protein kinases Cdk4 and Cdk6, the melanoma antigen MAGE-A4, and<br />
the 26S proteasome (3).<br />
Literature<br />
1. Heinemann, U., Büssow, K., Mueller, U. & Umbach, P. (2003) Acc. Chem. Res. 36,<br />
157-163.<br />
2. Manjasetty, B.A., et.al. (2004) Proteins 54, 797-800.<br />
3. Manjasetty, B.A., Quedenau, C., Sievert, V., Büssow, K., Niesen, F., Delbrück, H. &<br />
Heinemann, U. (2004) Proteins 55, 214-217.<br />
contact:<br />
Pr<strong>of</strong>. Udo Heinemann<br />
Max-Delbrück-Centrum für Molekulare Medizin Berlin-Buch<br />
FG Kristallographie<br />
heinemann@mdc-berlin.de<br />
Robert-Rössle-Str. 10<br />
13125 Berlin (Deutschland)
Melina Haupt, Murray Coles, Horst Kessler, Marc Bramkamp, Karlheinz Altendorf<br />
Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC<br />
complex, a P-type ATPase, are not driven by ATP-induced<br />
conformational changes<br />
P-type ATPases are involved in the active transport <strong>of</strong> ions across biological membranes.<br />
The KdpFABC complex (P-type ATPase) <strong>of</strong> E. coli is a high-affinity K + uptake system that<br />
operates only when the cell experiences osmotic stress or K + limitation [1]. Here, we<br />
present the solution structure <strong>of</strong> the nucleotide binding domain <strong>of</strong> KdpB (backbone RMSD<br />
0.25 Å) and a model <strong>of</strong> the AMP-PNP binding mode based on intermolecular distance<br />
restraints. The calculated AMP-PNP binding mode shows the purine ring <strong>of</strong> the nucleotide<br />
to be 'clipped' into the binding pocket via a pi-pi-interaction to F377 on one side and a<br />
cation-pi-interaction to K395 on the other. This binding mechanism seems to be<br />
conserved in all P-type ATPases, except the heavy metal transporting ATPases (type IB).<br />
Thus, we conclude that the Kdp-ATPase (currently type IA[2]) is misgrouped and has<br />
more similarities to type III ATPases. The KdpB N-domain is the smallest and simplest<br />
known for a P-type ATPase, and represents a minimal example <strong>of</strong> this functional unit. No<br />
evidence <strong>of</strong> significant conformational changes was observed within the N-domain upon<br />
nucleotide binding, thus ruling out a role for ATP-induced conformational changes in the<br />
reaction cycle.<br />
Literature<br />
[1] Altendorf, K., Gassel, M., Puppe, W., Mollenkamp, T., Zeeck, A., Boddien, C.,<br />
Fendler, K., Bamberg, E. & Drose, S. (1998). Structure and function <strong>of</strong> the Kdp-ATPase<br />
<strong>of</strong> Escherichia coli. Acta Phys. Scand. 163, 137-146.<br />
[2] Axelsen, K.B. and Palmgren, M.G. (1998). Evolution <strong>of</strong> substrate specificities in the Ptype<br />
ATPase superfamily. J. Mol. Evol. 46, 84-101.<br />
contact:<br />
Dipl. Chem. Melina Haupt<br />
Technische Universität München<br />
Organische Chemie & Biochemie II<br />
Melina.Haupt@ch.tum.de<br />
Lichtenbergstr. 4<br />
85747 Garching (Germany)
Norman Kachel, Werner Kremer, Ralph Zahn, Hans Robert Kalbitzer<br />
Intermediate states and implications for the species barrier <strong>of</strong><br />
the human prion protein revealed by high pressure NMR<br />
Prions as causative agents <strong>of</strong> transmissible spongiform encephalopathies in humans and<br />
animals are principally composed <strong>of</strong> the infectious isomer, PrPSc, <strong>of</strong> the cellular prion<br />
protein, PrPc. The conversion and thus the propensity <strong>of</strong> PrPc to adopt alternative folds<br />
leads to the species-specific propagation <strong>of</strong> the disease. High pressure is a powerful tool<br />
to study the physico-chemical properties <strong>of</strong> proteins, especially folding, as well as the<br />
dynamics and structure <strong>of</strong> folding intermediates[1;2]. Here we combine pressure with<br />
multidimensional NMR spectroscopy to characterise the physico-chemical properties <strong>of</strong><br />
the human prion protein PrPc(121-230) and PrPc(23-230). The application <strong>of</strong> pressure is<br />
reversible and we see virtually no difference between huPrPc(121-230) and huPrPc(23-<br />
230). The most pressure-sensitive region is the loop between strand 1 and helix 1,<br />
indicating that this region is the first entry point for the infectious conformer to convert<br />
the cellular protein. Importantly residues I139, H140, and F141 exhibit a cluster <strong>of</strong> very<br />
low dG values and seem to be the most unstable part <strong>of</strong> the protein. Interestingly I139<br />
was shown to be the residue responsible for the mouse/hamster species barrier. The<br />
highest dG values are observed in helix III very close to the disulfide bridge in<br />
accordance with known hydrogen protection patterns. The results indicate that the<br />
species barrier as well as the first entry-point <strong>of</strong> the scrapie isomer is determined in<br />
addition to surface charge differences through contributions <strong>of</strong> residue-biased and thus<br />
species-specific subpopulations <strong>of</strong> conformers as seen in prion strains with differing<br />
infectivity.<br />
Literature<br />
[1] Kuwata,K., Li,H., Yamada,H., Legname,G., Prusiner,S.B., Akasaka,K., & James,T.L.<br />
(2002) Biochemistry, 41, 12277-12283.<br />
[2] Inoue,K., Yamada,H., Akasaka,K., Herrmann,C., Kremer,W., Maurer,T., Doker,R., &<br />
Kalbitzer,H.R. (2000) Nat. Struct. Biol., 7, 547-550.<br />
contact:<br />
Norman Kachel<br />
Universität Regensburg<br />
Institut für Biophysik und physikalische Biochemie<br />
norman.kachel@biologie.uni-regensburg.de<br />
Universitätsstr. 31<br />
93053 Regensburg (D)
Jaros•aw Czyz, Katarzyna Miekus, Marta Czernik, Jolanta Sroka, Zbigniew Madeja<br />
Interrelation between rat prostate carcinoma cell coupling and<br />
motility in the determination <strong>of</strong> their invasiveness and<br />
metastatic activity<br />
While the role <strong>of</strong> cell motility in the determination <strong>of</strong> tumour cell invasion and metastasis<br />
is documented, the involvement <strong>of</strong> gap junctional coupling in this process remains a<br />
controversial matter. Here, the motility and gap junctional coupling <strong>of</strong> two sub-clones <strong>of</strong><br />
rat prostate cancer cells differing in their metastatic activity in vivo was compared. We<br />
demonstrate that both cell lines are characterised by similar motile activity when moving<br />
on the surface <strong>of</strong> normal fibroblasts, while highly metastatic cells were previously shown<br />
to be more mobile on plastic surface. The metastatic clone was, however, characterised<br />
by high level <strong>of</strong> homo- and heterologic intercellular coupling mediated by connexin43,<br />
one <strong>of</strong> the most abundant proteins <strong>of</strong> connexin family. These data indicate that connexin<br />
function, although not sufficient, seems necessary for tumour cell metastasis. Apigenin,<br />
a plant flavonoid shown to inhibit tumour metastases in vivo, inhibited the motility <strong>of</strong><br />
both carcinoma sub-clones but induced the gap junctional coupling <strong>of</strong> highly metastatic<br />
cells suggesting that apigenin may exert its anti-metastatic potential in vivo<br />
predominantly via the effect on cell motility. On the other hand, the correlation between<br />
induced coupling in metastatic cell populations and the inhibition <strong>of</strong> mutual cell<br />
translocations by apigenin may result from apigenin-induced stabilisation <strong>of</strong> gap<br />
junctions thus introducing a new variable into the discussion <strong>of</strong> data obtained from dye<br />
transfer experiments.<br />
contact:<br />
Dr. Jaroslaw Czyz<br />
Jagiellonian University<br />
Department <strong>of</strong> Cell Biology, Faculty <strong>of</strong> Biotechnology<br />
Jaro@mol.uj.edu.pl<br />
ul. Gronostajowa 7<br />
30-387 Cracow (Poland)
Viktoria Kukhtina, Denise Kottwitz, Chris Weise, Ferdinand Hucho<br />
Intracellular domain - terra incognita <strong>of</strong> the nicotinic<br />
acetylcholine receptor<br />
There are quite detailed structural data on the extracellular ligand-binding domain and<br />
the intramembrane channel-forming domain <strong>of</strong> the nicotinic acetylcholine receptors<br />
(nAChR). However, the structure <strong>of</strong> the intracellular domain, which has variable amino<br />
acid sequences in different nAChR subunits, remains unknown. We expressed in E.coli<br />
the intracellular loops (between transmembrane fragments TM3 and TM4) <strong>of</strong> the dsubunits<br />
from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate<br />
purification, (His) 6 -tags were attached with or without linkers, and the effects <strong>of</strong> protein<br />
truncations at C-or N-termini were examined. The proteins were purified from inclusion<br />
bodies under denaturing conditions by Ni-NTA-chromatography. Molecular weight and<br />
peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion<br />
chromatography revealed that the Torpedo intracellular d-loop refolded in an aqueous<br />
buffer was present in solution as a dimer. Phosphorylation <strong>of</strong> this protein with protein<br />
kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as<br />
in the native receptor. According to CD spectra, the secondary structure was not<br />
sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the<br />
presence <strong>of</strong> nonionic detergents or lipids. CD spectra indicate that the Torpedo and rat<br />
proteins have differences in their secondary structure. In the presence <strong>of</strong><br />
dodecylphosphocholine, high concentrations (up to 6 mg/ml) <strong>of</strong> the Torpedo and rat<br />
intracellular loops were achieved. The results suggest that the spatial structure <strong>of</strong> the<br />
intracellular loops is dependent on environment and species, but is not changed<br />
significantly upon enzymatic phosphorylation.<br />
Literature<br />
[1] F. Hucho, C. Weise, Ligand-gated ion channels, Angew. Chem. Int. Ed. 40 (2001)<br />
3100-3116.<br />
[2] D. Kottwitz, V. Kukhtina, N. Dergousova, T. Alexeev, Y. Utkin, V. Tsetlin, F. Hucho,<br />
Intracellular domains <strong>of</strong> the d-subunits <strong>of</strong> Torpedo and rat acetylcholine receptors -<br />
expression, purification and characterization, Protein Express. Purif. submitted.<br />
contact:<br />
Dr Viktoria Kukhtina<br />
Freie Universität Berlin<br />
Institut für Chemie/Biochemie<br />
kukhtina@chemie.fu-berlin.de<br />
Thielallee 63<br />
14195 Berlin (Germany)
Gaby-Fleur Böl, Regina Brigelius-Flohé<br />
Intracellular trafficking <strong>of</strong> the Interleukin-1 receptor (IL-1RI)<br />
associated kinase IRAK depends on its kinase-activity<br />
The proinflammatory cytokine Interleukin-1 (IL-1) induces a fine tuned signaling cascade<br />
consisting <strong>of</strong> various serine/threonine kinases and adapter proteins finally leading to<br />
nuclear gene activation. Upon stimulation <strong>of</strong> cells with IL-1 the IL-1 receptor type I (IL-<br />
1RI) associated kinase IRAK transiently associates to and dissociates from the IL-1RI.<br />
Thereafter the kinase translocates into the nucleus. Here we show that nuclear<br />
translocation <strong>of</strong> IRAK depends on its kinase activity as translocation was not observed in<br />
EL-4 cells overexpressing a kinase negative IRAK mutant. IRAK itself, an endogenous<br />
substrate with an apparent molecular weight <strong>of</strong> 24 kDa (pp24) and exogenous<br />
substrates like histon H1 and myelin basic protein are phosphorylated by nuclear located<br />
IRAK in the murine T cell line EL-4. pp24 is exclusively associated to IRAK but not to the<br />
IL-1RI. Phosphorylation <strong>of</strong> pp24 takes also place in the cytoplasm, but cannot be<br />
detected in wild type EL-4 cells or those overexpressing a kinase negative mutant <strong>of</strong><br />
IRAK. Therefore, phosphorylation <strong>of</strong> pp24 depends on a high kinase activity <strong>of</strong> IRAK. As<br />
IL-1 signaling is accompanied by oxidative processes, phosphorylation and nuclear<br />
translocation <strong>of</strong> IRAK and pp24 are inhibited by preincubation <strong>of</strong> the cells with the thiol<br />
modifying agent menadione. It is therefore concluded that intracellular localization <strong>of</strong><br />
IRAK depends on its kinase activity and that IRAK functions also as a kinase in the<br />
nucleus with pp24 being a new endogenous substrate in the nuclear compartment and in<br />
the cytoplasm.<br />
contact:<br />
Dr. rer. nat. Gaby-Fleur Böl<br />
German Institute <strong>of</strong> Human Nutrition, Potsdam-Rehbrücke<br />
Dept. <strong>of</strong> Vitamins and Atherosclerosis<br />
boel@mail.dife.de<br />
Arthur-Scheunert-Allee 114-116<br />
14558 Nuthetal (Germany)
Vincent Lemaitre, Anthony Watts, Wolfgang Fischer<br />
Ion channels with minimalist design – from viruses.<br />
Ion flux across the lipid membrane is a prerequisite for the function <strong>of</strong> the cell. Ion<br />
channel proteins have been evolved to enable a controlled flux. Viral genomes also<br />
encode this type <strong>of</strong> membrane protein which works for the benefit <strong>of</strong> viral replication.<br />
Interestingly, ion channels found so far in animal and human viruses, are very much<br />
smaller than those in the host cell. Little is known about the structure – function<br />
correlation <strong>of</strong> these membrane proteins: how do they assemble, is there a gating, do<br />
these channels discriminate between ions, and finally, can we use them? Currently these<br />
questions are addressed by studying the ion channel forming protein Vpu from HIV-1 in<br />
a combined computational and experimental approach.<br />
Vpu is a small 81 amino acid integral type I membrane protein with two phosphorylation<br />
sites [1,2]. It is involved in the down-regulation <strong>of</strong> the receptor protein CD4 via its<br />
cytoplasmic domain and the enhancement <strong>of</strong> particle release from the plasma membrane<br />
via its transmembrane (TM) domain [1,3,4].<br />
Non-equilibrium molecular dynamics simulations are used to address ion selectivity and<br />
the state <strong>of</strong> oligomerisation <strong>of</strong> the ion conducting bundle based on the TM domain. In<br />
addition models <strong>of</strong> the full length Vpu generated by merging computationally derived TM<br />
structure with structures <strong>of</strong> the cytoplasmic domain based on NMR spectroscopic data<br />
will be presented. Bilayer recordings complement the investigations.<br />
Literature<br />
[1] Fischer W. B.; FEBS Lett. (2003) 552, 39-46<br />
[2] Cordes F. S., Tustian, A. D., Sansom M. S. P., Watts A., Fischer W. B.; Biochemistry<br />
(2002) 41, 7359 – 7365.<br />
[3] Sramala I., Lemaitre V., Faraldo-Gómez J. D., Vincent S., Watts A., Fischer W. B.;<br />
Biophys. J. (2003) 84, 3276-3284<br />
[4] Lemaitre V., Ali R., Kim C. G., Watts A., Fischer W. B.; Interaction FEBS Lett. (2004)<br />
563, 75-81<br />
contact:<br />
PD Dr. Wolfgang Fischer<br />
Oxford<br />
Physics<br />
wolfgang.fischer@bioch.ox.ac.uk<br />
Parks Road<br />
OX1 3PU Oxford (UK)
Iris G. Altug, Harro J. Bouwmeester, Wilfried A. König<br />
Isolation and functional expression <strong>of</strong> cDNAs encoding<br />
sesquiterpene synthases, including the enantiomeric (+)- and<br />
(-)-Germacrene D-Synthases from Solidago canadensis L.<br />
The sesquiterpene germacrene D is common in the essential oils <strong>of</strong> many higher plants.<br />
While in most higher plants germacrene D is present as its (-)-enantiomer, Solidago<br />
species are exceptional by producing both enantiomers in large amounts. Germacrene D<br />
is well known in plant-insect interaction, giving antennal responses and showing host<br />
plant recognition effects. Enantioselective analysis <strong>of</strong> germacrene D enantiomers with<br />
receptor neurons <strong>of</strong> Helicoverpa armigera showed response to both enantiomers, but (-)germacrene<br />
D gave a 10 times stronger effect [1]. Plant-microbe interaction are not<br />
analyzed yet for germacrene D, though many terpenes protect plants againt microbial<br />
infection.<br />
As shown in prior investigations S. canadensis possesses two individual germacrene Dsynthases<br />
catalyzing the enantiospecific formation <strong>of</strong> (+)- or (-)-germacrene D [2,3].<br />
Screening a cDNA library made out <strong>of</strong> leaf material from S. canadensis resulted in<br />
several full-length clones. The function <strong>of</strong> the isolated full-length clones was tested by<br />
heterologous expression in E. coli. After incubation with the substrate farnesyl<br />
diphosphate, product formation was analyzed by GC-MS and GC-FID with<br />
enantioselective column. The analysis revealed one enzyme producing exclusively (+)germacrene<br />
D and another producing exclusively (-)-germacrene D. Furthermore a αgurjunene<br />
synthase and a cascarilladiene synthase were identified.<br />
The encoding sequences <strong>of</strong> the both germacrene D-synthases show very high homology<br />
with sequence identity <strong>of</strong> 85%. This gave the possibility to identify amino acid residues<br />
possibly involved in the enantiospecific catalysis <strong>of</strong> the (+)- and (-)-germacrene Dsynthase<br />
by protein homology modeling. Comparative homology modeling was<br />
performed on the known structure <strong>of</strong> epi-aristolochene synthase [4], followed by<br />
substrate docking experiments. In this way five amino acid residues were determined,<br />
which presumably are responsible in the enantiospecific catalysis <strong>of</strong> both germacrene Dsynthases<br />
[5].<br />
Literature<br />
[1] M. Stranden, A.-K. Borg-Karlson, H. Mustaparta, Chemical Senses 2002, 27:143-152<br />
[2] C. O. Schmidt, H. J. Bouwmeester, J.-W. de Kraker, W. A. König, Angew. Chem. Int.<br />
Ed. Engl. 1998,<br />
37: 1400-1402.<br />
[3] C. O. Schmidt, H. J. Bouwmeester, S. Franke, W. A. König, Chirality 1999, 11: 353-<br />
362.<br />
[4] C.M. Starks, K. Back, J. Chappell, J.P. Noel, Science 1997, 277:1815-1820<br />
[5] I. Prosser, I.G. Altug, A.L. Phillips, W.A. König, H.J. Bouwmeester, M.H. Beale,<br />
Archiv. Biochem. Biophys. 2004, in press<br />
contact:<br />
Dr. Iris Altug<br />
Universität Hamburg<br />
Institut für Biochemie und Molekularbiologie<br />
iris.altug@chemie.uni-hamburg.de<br />
Martin-Luther-King-Platz 6<br />
20146 Hamburg (Germany)
Claus Wasternack, Irene Stenzel, Bettina Hause, Gerd Hause, Otto Miersch<br />
Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong><br />
tomato<br />
The allene oxide cyclase (AOC), an essential enzyme in jasmonate (JA) biosynthesis, is<br />
localized in vascular bundles (1). Detailed inspection <strong>of</strong> vascular bundles revealed<br />
occurrence <strong>of</strong> AOC and the preceeding enzymes allene oxide synthase and lipoxygenase<br />
in companion cells and sieve elements (SE) suggesting formation <strong>of</strong> JA even in SE(2).<br />
Upon wounding AOC is activated leading to JA formation preferentially in main veins.<br />
Inspection <strong>of</strong> the corresponding wild types, 35S:AOCsense and 35S::AOCantisense<br />
plants for levels <strong>of</strong> AOC mRNA, AOC protein, AOC activity, for tissue-specific location <strong>of</strong><br />
AOC protein as well as for levels <strong>of</strong> JA, 12-oxophytodienoic acid and proteinase inhibitor2<br />
mRNA suggests a systemin-dependent AOC expression and an AOC-dependent<br />
amplification <strong>of</strong> the local wound response via vascular bundle-specific generation <strong>of</strong><br />
jasmonates (3). In constrast to leaves <strong>of</strong> 35S::AOCsense plants, where the substrate <strong>of</strong><br />
JA biosynthesis has to be generated for JA formation e.g. by wounding, constitute<br />
overexpression <strong>of</strong> AOC leads to dramatically elevated levels and altered<br />
ratios <strong>of</strong> JA and other oxylipins (4). Role <strong>of</strong> JA in plant defense is discussed.<br />
Literature<br />
1 Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R., Ziegler, J., Wasternack,<br />
C. Plant J. 24: 113-126 (2000)<br />
2 Hause, B., Hause, G., Kutter, C., Miersch, O., Wasternack, C. Plant and Cell Physiol.<br />
44: 643-648 (2003)<br />
3 Stenzel, I., Hause, B., Maucher, M., Pitzschke, A., Miersch, O., Ziegler, J., Ryan, C.A.,<br />
Wasternack, C. Plant J. 33: 577-589 (2003)<br />
4 Miersch, O., Weichert, H., Stenzel,I., Hause, B., Maucher, H., Feussner, I.,<br />
Wasternack, C. Phytochem. 65: 847-856 (2004)<br />
contact:<br />
Pr<strong>of</strong>. Dr. Claus Wasternack<br />
Leibniz-Insitut für Pflanzenbiochemie<br />
cwastern@ipb-halle.de<br />
Weinberg 3<br />
06120 Halle/Saale (Sachen-Anhalt)
Daniela Rehder, Dietmar Vestweber, Klaus Ebnet<br />
Junctional Adhesion Molecule-A (JAM-A) participates in tight<br />
junction formation and the establishment <strong>of</strong> cell polarity in<br />
epithelial cells<br />
Tight junctions (TJ) play a critical role in the establishment <strong>of</strong> cell polarity in vertebrate<br />
epithelial cells. Several integral membrane proteins have been identified in TJs <strong>of</strong><br />
epithelial cells, including occludin, the claudins, CRB-3, and several immunoglobulinsuperfamily<br />
members such as JAM-A, CAR, and JAM4. We have recently identified the<br />
cell polarity protein PAR-3 as a cytoplasmic protein associated with JAM-A. PAR-3 is part<br />
<strong>of</strong> a ternary protein complex consisting <strong>of</strong> PAR-3, aPKC, and PAR-6, which is implicated<br />
in the formation <strong>of</strong> TJs and cell polarity. These findings suggested a role for JAM-A in TJ<br />
formation. To address the functional relevance <strong>of</strong> this interaction, we have generated<br />
MDCK II Tet-Off cell lines stably expressing dominant-negative JAM-A mutants lacking<br />
the extracellular domain <strong>of</strong> JAM-A. The overexpression <strong>of</strong> JAM-A mutants resulted in a<br />
delayed formation <strong>of</strong> transepithelial electrical resistance (TER), indicating a defect in TJ<br />
formation. In addition, cells overexpressing JAM-A mutants were severely compromised<br />
in their ability to form cysts when grown in three-dimensional collagen matrices. The<br />
overexpression <strong>of</strong> JAM-A mutants lacking the PDZ binding motif required for the<br />
association <strong>of</strong> JAM-A with PAR-3 had no or only weak effects on TER development and<br />
cyst formation. These findings suggest that JAM-A plays an important role in the<br />
assembly <strong>of</strong> TJs and in the formation <strong>of</strong> cell polarity.<br />
contact:<br />
Daniela Rehder<br />
Westfälische Wilhelms-Universität<br />
Institute for Cell Biology/ZMBE<br />
rehderd@uni-muenster.de<br />
von-Esmarch-Str. 56<br />
48149 Münster (Germany)<br />
additional information<br />
Additional affiliation for Dietmar Vestweber:<br />
Max-Planck-Institute for Molecular Biomedicine, Münster, Germany
Joachim Wegener, Charles Keese*, Ivar Giaever*<br />
Kinetics <strong>of</strong> cell spreading monitored by electric cell-substrate<br />
impedance sensing<br />
Electric cell-substrate impedance sensing (ECIS) is a novel experimental technique to<br />
monitor the attachment and spreading <strong>of</strong> mammalian cells on artificial surfaces in a<br />
highly quantitative and computer-controlled manner. The method is based on measuring<br />
changes in the electrical impedance <strong>of</strong> small gold film electrodes (d = 250 µm) deposited<br />
on a culture dish by means <strong>of</strong> photolithography and used as growth substrate.<br />
Based on experimental data and theoretical considerations we demonstrate that<br />
impedance readings above a threshold frequency <strong>of</strong> 10 kHz return a direct measure for<br />
the degree <strong>of</strong> cell spreading on the electrode surface. Interpretation <strong>of</strong> ECIS data was<br />
validated by means <strong>of</strong> established microscopic techniques.<br />
The excellent time resolution <strong>of</strong> the ECIS device allows an in-depth analysis <strong>of</strong> cellspreading<br />
kinetics. From ECIS data we extracted both the time necessary to achieve halfmaximum<br />
cell spreading and the apparent spreading rate for various experimental<br />
conditions. For instance, we compared the attachment and spreading <strong>of</strong> epithelial MDCK<br />
cells on different protein coatings and investigated the impact <strong>of</strong> divalent cations on<br />
spreading kinetics. We quantified the inhibitory effect <strong>of</strong> soluble peptides that mimic the<br />
recognition sequence <strong>of</strong> fibronectin (RGDS). We also applied the ECIS technique to<br />
monitor the detachment <strong>of</strong> confluent fibroblastic cell layers by means <strong>of</strong> these peptides.<br />
contact:<br />
Dr. Joachim Wegener<br />
Universität Münster<br />
Institut für Biochemie<br />
wegenej@uni-muenster.de<br />
Wilhelm-Klemm Str 2<br />
48149 Münster (Deutschland)<br />
additional information<br />
*) Rensselaer Polytechnic Institute, School <strong>of</strong> Science, Troy (NY) 12180, USA
Peter Becker<br />
Lifting a chromosome - Dosage Compensation in Drosophila<br />
Dosage compensation in fruit flies involves doubling the transcription <strong>of</strong> genes on the<br />
single male X chromosome effectively adjusting their ex-pres-sion to the levels originating<br />
from the two active X chromo-somes in females. Such fine-tuning <strong>of</strong> gene<br />
expression is vital for fruit flies since failure leads to male-specific lethality.<br />
Dosage compensation is regulated at the level <strong>of</strong> chromatin organization. Acety-lation <strong>of</strong><br />
histone H4 at lysine 16 by the acetylase MOF is crucial. MOF is targeted to the X<br />
chromosome as part <strong>of</strong> a dosage compen-sation complex (DCC) consisting <strong>of</strong> at least<br />
four other male-specific lethal (MSL) proteins and non-coding roX RNA. The determinants<br />
for efficient interaction <strong>of</strong> DCC with X chromosomal targets are not known.<br />
Using a variety <strong>of</strong> biochemical, cell biological and genetic methods we are character-ising<br />
the protein-protein, protein-DNA and protein-RNA interactions that define DCC and its<br />
association with chromatin in general as well as with the X chromosome in particular.<br />
Literature<br />
Morales, V., Straub, T., Neumann, M. F., Mengus, G., Akhtar, A. & Becker, P.B. (2004).<br />
EMBO J, in press<br />
Gilfillan, G., Dahlsveen, I. & Becker, P.B. (2004). FEBS Letters in press<br />
Brehm, A., Tufteland, K.R., Aasland, R. & Becker, P.B. (2004). BioEssays, 26, 133-140<br />
Park, Y., Mengus, G., Bai, X., Kageyama, Y., Meller, V.H., Becker, P.B. & Kuroda. M.I.<br />
(2003). Cell 11, 977-986.<br />
Akhtar, A. & Becker, P.B. (2001). EMBO Reports 2, 113-118.<br />
Akhtar, A., Zink, D. & Becker, P.B. (2000). Nature 407, 405-409.<br />
Akhtar, A. & Becker, P.B. (2000). Molecular Cell 5, 367-375<br />
contact:<br />
Pr<strong>of</strong>. Peter Becker<br />
Ludwig-Maximilians-Universität<br />
Adolf-Butanandt-Institut<br />
pbecker@med.uni-muenchen.de<br />
Schillerstr. 44<br />
80336 München (Germany)
Mieke Sprangers, Hui Wang, Niklas Feldhahn, Florian Klein, Markus Müschen<br />
Lineage infidelity in pre-B acute lymphoblastic leukemia cells<br />
carrying an MLL-AF4 gene rearrangement<br />
B cell precursor leukemia cells carrying an MLL-AF4 fusion gene, frequently exhibit a<br />
mixed B lymphoid/ myeloid phenotype. Using MLL-AF4+ leukemia cells as a model for<br />
the B lymphoid vs. myeloid cell fate decision, we identified MLL-related effector<br />
molecules, which interfere with B cell lineage commitment in the human and are<br />
implicated in phenotypic ‘lineage infidelity’. This was done by comparing genome-wide<br />
gene expression pr<strong>of</strong>iles generated by SAGE. SAGE-data were confirmed by semiquantitative<br />
RT-PCR, Western blot and FACS analysis. The data show that MLL-AF4+<br />
leukemia cells exhibit traces <strong>of</strong> myeloid differentiation in that they aberrantly express<br />
c/EBPa, CD13 and IL3Rα (myeloid lineage) together with genes implicated in hemoglobinsynthesis:<br />
porphobilinogen deaminase (PBG-D), d-Aminolevulinate-synthase (ALAS2)<br />
and T-cell acute lymphocytic leukemia1(TAL1). They also do not express IKAROS, a<br />
transcription factor that initiates lymphoid lineage commitment. The pre-B cell receptor<br />
(pre-BCR) signal transduction is severely impaired in MLL-AF4+ leukemia cells: signaling<br />
molecules including SYK, BLNK, BTK, SLP76 and PLCg2 either are not expressed or are<br />
not tyrosine-phosphorylated in response to pre-BCR engagement in the leukemic cells.<br />
Consistent with this, the SRC kinase LYN, which represents an important component <strong>of</strong><br />
the pre-BCR-signal transduction cascade, is inactivated by inhibitory phosphorylation at<br />
tyrosine 507.<br />
These data collectively indicate that pre-BCR signaling in MLL-AF4+ leukemia cells is<br />
inactivated either by i) lack <strong>of</strong> expression <strong>of</strong> signaling molecules, ii) lack <strong>of</strong> tyrosine<br />
phosphorylation or iii) inhibitory tyrosine phosphorylation.<br />
contact:<br />
Mieke Sprangers<br />
Universität Düsseldorf<br />
Lab. for Molecular Stem Cell Biology<br />
sprangers@itz.uni-duesseldorf.de<br />
Moorenstr. 5<br />
40225 Düsseldorf (D)
Lubov Rochlina, Thomas Linn, Reinhard Bretzel, Eugen Kolossov, Juergen Hescheler,<br />
Irina Drobinskaya<br />
Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in<br />
the Murine Embryonic Stem Cell System<br />
The overall goal <strong>of</strong> the present study is to generate in vitro endoderm–like cells with<br />
biological features close to the native endodermal cells. A methodical approach applied is<br />
based on the use <strong>of</strong> expression vectors containing genes <strong>of</strong> the reporter “live”<br />
fluorescent protein and antibiotic resistance cassette, both driven by tissue–specific gene<br />
promoters <strong>of</strong> interest. This allows to trace the differentiation <strong>of</strong> the targeted cell types in<br />
a “live” mode and to perform a lineage selection and purification <strong>of</strong> the differentiating<br />
cell cultures. We have generated a recombinant expression vector containing a<br />
green–fluorescent protein (GFP) gene and a puromycin resistance cassette, both driven<br />
by the common mouse endoderm–specific α–fetoprotein (AFP) gene promoter. In ES cell<br />
aggregates (embryoid bodies, EBs) produced from the transgenic ES clones possessing<br />
this gene construct, we have detected a high efficiency <strong>of</strong> development <strong>of</strong> the puromycin<br />
resistant GFP–expressing cells under optimised culturing conditions. This data<br />
demonstrate the activation <strong>of</strong> the AFP promoter, thus suggesting the differentiation <strong>of</strong><br />
the ES cells into endoderm–like cells. It was proven by the results <strong>of</strong> the fluorescent<br />
antibody staining <strong>of</strong> the EB–derived single GFP–labelled cells for AFP. Thus, the<br />
GFP–positive cell population <strong>of</strong> the presumably endoderm–like cells can be selected and<br />
purified from other emerging cell types by an application <strong>of</strong> the selective drug.<br />
contact:<br />
Dr. Irina Drobinskaya<br />
University <strong>of</strong> Cologne<br />
Institute for Neurophysiology<br />
irina.drobinskaya@uni-koeln.de<br />
Robert-Koch-Str. 39<br />
50931 Cologne (Germany)<br />
additional information<br />
T.L. & R.B.: University <strong>of</strong> Giessen, Medical Clinic III, Germany<br />
E.K.: Cell Centre Cologne, Axiogenesis AG, Cologne, Germany
Randy Kurz, Markus Rüffer, Christin Urban, Andrée Rothermel, Winnie Weigel, Andrea<br />
A. Robitzki<br />
Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
The non-destructive realtime monitoring <strong>of</strong> cellular conditions is essential for the<br />
development <strong>of</strong> cell-based biohybrid sensors. For this purpose, cells are placed in an<br />
electric circuit on a microchip in order to determine their electric properties (e.g. electric<br />
activity). The field-potential fluctuation caused by spontaneously beating cardiomyocytes<br />
is an example for such an activity. We used specific multi-electrode arrays (MEAs) with a<br />
defined surface topology mediated by an immobilisation with laminin to record the fieldpotential.<br />
Therefore this modified MEA enables us to determine the contraction<br />
frequency <strong>of</strong> cardiomyocytes derived from neonatal rats. The interaction between the<br />
angiotensin II AT1 receptor and most <strong>of</strong> its ligands has an influence on the contraction<br />
frequency <strong>of</strong> cardiomyocytes. Therefore the developed cardiomyocyte based multielectrode<br />
array sensor can be used as a fast monitoring system for detecting extreme<br />
low concentration <strong>of</strong> the ligands within a few minutes.Angiotensin II represents such a<br />
ligand and can be detected online by the biosensor in a concentration range <strong>of</strong> 10-8 M<br />
via the changed cellular motility. The goal <strong>of</strong> this study is the development <strong>of</strong> a<br />
microchip-based biosensor for high-throughput screening <strong>of</strong> drugs and autoantibodies<br />
directed to the angiotensin II AT1 receptor and their role in preeclampsia and transplant<br />
rejection.<br />
Literature<br />
Kurz, R., Rothermel, A., Rüffer, M., Urban, C., Jahnke, H.-G., Weigel, W. and Robitzki,A.<br />
A. (2004) A functional cardiomyocyte-based biosensor for prediagnostic monitoring: an<br />
angiotensin II study. IFMBE Proceedings in press.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Andrea A. Robitzki<br />
University <strong>of</strong> Leipzig<br />
Biotechnological Biomedical Center<br />
andrea.robitzki@bbz.uni-leipzig.de<br />
Deutscher Platz 5<br />
04103 Leipzig (Germany)
Alexander Bubikat, Bernd Zetsche, Larissa Fabritz, Axel Gödecke, Michaela Kuhn<br />
Local ANP prevents cardiac remodeling in hypertensive eNOSdeficient<br />
mice<br />
The crucial endocrine vs local roles <strong>of</strong> atrial natriuretic peptide (ANP) and endothelial<br />
nitric oxide/NO in the regulation <strong>of</strong> arterial blood pressure (ABP) were shown by the<br />
hypertensive phenotype <strong>of</strong> mice with systemic inactivation <strong>of</strong> either the ANP receptor<br />
(GC-A-/-) or endothelial NO synthase (eNOS-/-). Whereas GC-A-/- mice show cardiac<br />
hypertrophy, similar levels <strong>of</strong> hypertension in eNOS-/- mice barely affect cardiac<br />
morphology. Using mice with cardiomyocyte-restricted deletion <strong>of</strong> the ANP receptor (CM<br />
GC-A KO) we could show that ANP locally inhibits cardiomyocyte growth. Increased<br />
cardiac ANP levels in eNOS-/- mice suggest that enhanced activation <strong>of</strong> cardiac GC-A can<br />
prevent hypertensive heart disease. To test this, we generated double knockout mice<br />
with systemic inactivation <strong>of</strong> eNOS and cardiomyocyte-selective deletion <strong>of</strong> GC-A. ABP<br />
levels were similar in both eNOS-/- and eNOS-/-;CM GC-A KO mice, but significantly<br />
enhanced compared to wildtype mice. Heart-to-body weight ratios, cardiomyocyte<br />
diameter, and interstitial fibrosis were significantly increased in eNOS-/-;CM GC-A KO<br />
compared to eNOS-/- mice. Hypertrophy markers ANP and β-MHC were upregulated in<br />
hearts <strong>of</strong> eNOS-/-;CM GC-A KO, whereas α-MHC mRNA levels were decreased.<br />
Phosphorylation <strong>of</strong> the MAPK ERK1/2 was significantly increased, suggesting that<br />
ANP/GC-A signaling functions as a myocyte intrinsic pathway which counter-regulates<br />
MAPK/ERK - dependent pathways <strong>of</strong> cardiac myocyte growth in hypertensive eNOS-/-<br />
mice.<br />
contact:<br />
Dipl. Biol. Alexander Bubikat<br />
Universitätsklinikum Münster<br />
Institut für Pharmakologie und Toxikologie<br />
bubikat@ uni-muenster.de<br />
Domagkstr. 12<br />
48149 Münster (Germany)
Franz Hillenkamp<br />
MALDI Mass Spectrometry <strong>of</strong> Nucleic Acids<br />
Over the past decade Mass Spectrometry (MS) has become an indispensable tool for the<br />
analysis and identification <strong>of</strong> biological macromolecules. Besides Electrospray Ionization<br />
(ESI) Matrix-Assisted Laser Desorption/Ionization (MALDI) is one <strong>of</strong> the key technologies<br />
used for the ionization <strong>of</strong> such non-volatile and thermally labile molecules.<br />
The presentation will review the basic mechanisms as well as the current status <strong>of</strong><br />
instrumentation <strong>of</strong> MALDI-MS. Some strategies for the analysis <strong>of</strong> different classes <strong>of</strong><br />
biological macromolecules will be discussed as well as techniques for sample<br />
preparation. While the analysis <strong>of</strong> proteins is largely routine these days, nucleic acids<br />
still pose considerable difficulties. Emphasis will, therefore, be placed on the MALDI-MS<br />
<strong>of</strong> oligonucleotides.<br />
contact:<br />
Pr<strong>of</strong>. Franz Hillenkamp<br />
Universität Münster<br />
Institut für Medizinische Physik und Biophysik<br />
hillenk@uni-muenster.de<br />
Robert-Koch-Str. 31<br />
D-48149 Münster (Germany)
Alejandro Heredia, C. C. Bui, Ueli Suter, Peter Young, Tilman Schäffer<br />
Mechanical properties <strong>of</strong> myelinated and de-myelinated<br />
peripheral axons with the atomic force microscope<br />
Schwann cells are the principal glial cells <strong>of</strong> the peripheral nervous system (PNS). They<br />
envelop the axons in the PNS <strong>of</strong> vertebrates by forming insulating myelin sheaths,<br />
thereby providing saltatory conduction and axonal regeneration following nerve injury.<br />
Saltatory conduction is achieved by reducing the depolarization regions to only the<br />
Ranvier nodes, which are the small gaps that are directly exposed to the extracellular<br />
environment between adjacent Schwann cells. Conversely, axons regulate the<br />
differentiation, survival and/or proliferation <strong>of</strong> Schwann cells at several stages during<br />
their development. Several human diseases (multiple sclerosis, Pelizeaus-Merzbacher<br />
disease, etc) result from a deficiency or disruption <strong>of</strong> the myelin sheaths.<br />
We used the atomic force microscopy (AFM) to image myelinated and de-myelinated<br />
mouse peripheral nerve axons. We furthermore established a method <strong>of</strong> measuring<br />
locally resolved, quantitative elastic properties <strong>of</strong> axons with the AFM, and acquired twodimensional<br />
images <strong>of</strong> their local elastic modulus. Typical elastic moduli <strong>of</strong> re-hydrated<br />
axons at physiological conditions were in the 100-900 kPa range. We further present<br />
initial results <strong>of</strong> investigations linking the mechanical properties <strong>of</strong> the axons to the ionic<br />
concentration <strong>of</strong> the solution.<br />
contact:<br />
Alejandro Heredia<br />
Universität Münster<br />
Center for Nanotechnology<br />
heredia@fisica.unam.mx<br />
Gievenbecker Weg 11<br />
48149 Münster (Germany)<br />
additional information<br />
A.H.: Universität Münster and Instituto de Ciencias Nucleares, UNAM, México.<br />
C.C.B. & P.Y.: Klinik und Poliklinik für Neurologie, Universitätsklinikum Münster<br />
U.S.: Institute <strong>of</strong> Cell Biology, ETH Hönggerberg Zürich, Switzerland.
Marco Schmeer, Thomas Seipp, Sergej Kakorin, Eberhard Neumann<br />
Mechanism for the conductivity changes caused by membrane<br />
electroporation <strong>of</strong> CHO cell - pellets<br />
Densely-packed pellets <strong>of</strong> chinese hamster ovary (CHO) cells, mean cell radius(standard<br />
deviation) a c = 7.5 •m, serve as models for the electrotransfer by membrane<br />
electroporation (MEP) <strong>of</strong> bioactive substances such as gene DNA and oligonucleotides in<br />
the medical disciplines <strong>of</strong> electrotherapy and gene therapy. MEP is indicated by the<br />
increased electric conductivity <strong>of</strong> the pellet, in rectangular electric field (E) pulses. The<br />
conductivity relaxations exhibit up to 3 in-field normal modes and post-field relaxations.<br />
The rapid mode, Y1, probably reflects Wien effects <strong>of</strong> ionic atmosphere perturbations<br />
and ion pair dissociations on the cell surfaces.<br />
The second (Y2) and the third (Y3) mode, both are analyzed in terms <strong>of</strong> electric pore<br />
formation (MEP), according to a minimum scheme for the structural transitions from<br />
closed (C, C1) states to, at least, two porous states P2 and P3 <strong>of</strong> largely different life<br />
times. Electrothermodynamic analysis yields mean pore radii and mean pore life times at<br />
zero applied field:<br />
Pore type P2: r2 = 1.0 nm, τ2 0 = 1.0 ms; P3: r3 = 1.5 nm, τ3 0 = 45 s.<br />
The post-field relaxation reflects resealing <strong>of</strong> the long-lived P3 pore states.<br />
contact:<br />
Dipl. Chem. Marco Schmeer<br />
Universität Bielefeld<br />
Fakultät Chemie<br />
marco.schmeer1@uni-bielefeld.de<br />
Postfach 100 131<br />
33501 Bielefeld (Deutschland)
Blaga Popova, Markus Kuhlmann, Markus Kaller, Wolfgang Nellen<br />
Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene<br />
silencing<br />
RNA interference and antisense mediated gene silencing have been implied to be<br />
essentially based on the same mechanisms. However, the comparison <strong>of</strong> RNAi and<br />
antisense efficiency in various knock-out strains <strong>of</strong> Dictyostelium revealed that both<br />
mechanisms have similar though distinct requirements.<br />
Dictyostelium has three genes encoding RNA directed RNA polymerases (rrpA, rrpB and<br />
rrpC). Only rrpA is essential for RNAi (Martens et al., 2002) but all three are necessary<br />
for efficient silencing by antisense. Furthermore, we have shown that RNAi constructs<br />
(that generate a fold-back hairpin RNA) and antisense constructs (that generate a single<br />
stranded antisense transcript) work synergistically in gene silencing.<br />
Screening the Dictyostelium genome, we have detected a candidate gene (helF) that<br />
displays high homology to “DICER related helicases” and we have examined the function<br />
<strong>of</strong> HelF in silencing mechanisms. Disruption <strong>of</strong> the helF gene results in a significant<br />
increase in RNAi mediated silencing but has no effect on antisense RNA. HelF thus<br />
functions in vivo as an inhibitor <strong>of</strong> RNAi. Surprisingly, the HelF protein has a distinct<br />
localization in multiple nuclear foci even though most <strong>of</strong> the RNAi machinery is believed<br />
to be cytosolic.<br />
We will present a model that combines our results with those <strong>of</strong> other findings and that<br />
may provide further insight into antisense and RNAi mechanisms (Martens and Nellen,<br />
2002).<br />
Literature<br />
Martens, H. and Nellen, W. (2002) Genesilencing durch RNAi und antisense RNA.<br />
Biospektrum, 4, 351-354.<br />
Martens, H., Novotny, J., Oberstrass, J., Steck, T.L., Postlethwait, P. and Nellen, W.<br />
(2002) RNAi in Dictyostelium: the role <strong>of</strong> RNA-directed RNA polymerases and doublestranded<br />
RNase. Mol Biol Cell, 13, 445-453.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Wolfgang Nellen<br />
Universität Kassel<br />
Genetik<br />
nellen@uni-kassel.de<br />
Heinrich-Plett-Str. 40<br />
34132 Kassel (Germany)
Carsten Kötting, Katrin Beckmann, Marco Blessenohl, Sven Brucker, Partha<br />
Chakrabarti, Carolin Eichholz, Angela Kallenbach, Yan Suveyzdis, Klaus Gerwert<br />
Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Point mutations <strong>of</strong> Ras-genes are found in 30% <strong>of</strong> the human tumors [1]. Ras plays a<br />
central role in cell signaling pathways transducing growth signals from the plasma<br />
membrane to the nucleus [2]. Ras acts as a switch, transmitting the signal in an active<br />
GTP-bound form and turning the signal <strong>of</strong>f in an inactive GDP-bound form. The switch <strong>of</strong>f<br />
is accomplished by GTP hydrolysis, which is catalyzed by Ras and can be further<br />
accelerated by GTPase activating proteins (GAPs). In oncogenic Ras this hydrolysis<br />
reaction is perturbed. If one wants to understand, how oncogenic Ras can be activated,<br />
the fundament will be the understanding <strong>of</strong> the detailed mechanism <strong>of</strong> the hydrolysis<br />
reaction on the atomic level.<br />
We studied this reaction mechanism by time resolved Fourier transform infrared (FTIR)<br />
difference spectroscopy [3-5]. The reaction was initiated by means <strong>of</strong> a photolabile<br />
trigger. Detailed information were revealed utilizing isotopic labels <strong>of</strong> the nucleotide, the<br />
GTPase or the GAP. Furthermore, mutants <strong>of</strong> Ras and GAP were investigated. From the<br />
spectra we gained information on charge distribution, protein structure, involved amino<br />
acids and kinetics. The reaction proceeds in two steps, the bond breakage and the Pi<br />
release. We show, that the catalysis is entirely due to enthalpic effects and are caused<br />
by a charge shift towards the β-phosphate <strong>of</strong> GTP [6]. This shift can be correlated with<br />
the hydrolysis rate. Further, we compare the hydrolysis reactions <strong>of</strong> other GNBPs, Rap,<br />
Rho and Ran with Ras. Whereas the intrinsic reactions are similar, we find significant<br />
differences for the GAP catalyzed reactions.<br />
Literature<br />
[1] Barbacid, M. Annu. Rev. Biochem. 56, 779-827 (1987)<br />
[2] Wittingh<strong>of</strong>er, A. Biol. Chem. 379, 933-937 (1998)<br />
[3] Cepus, V.; Scheidig, A. J.; Goody, R. S.; Gerwert, K. Biochemistry 37, 10263-10271<br />
(1998)<br />
[4] Allin, C.; Gerwert K. Biochemistry 40, 3037-3046 (2001)<br />
[5] Allin, C.; Ahmadian, M.R.; Wittingh<strong>of</strong>er, A.; Gerwert, K. Proc. Natl. Acad. Sci. USA<br />
98, 7754-7759 (2001)<br />
[6] Kötting, C.; Gerwert, K. Chemical Physics, in press<br />
contact:<br />
Dr. Carsten Kötting<br />
Ruhr-Universität Bochum<br />
Biophysik ND 04/352<br />
carsten.koetting@rub.de<br />
Universitätsstr. 150<br />
44801 Bochum (Germany)
Marc Bramkamp, Jeff Errington<br />
Membrane bound proteins <strong>of</strong> the division machinery in<br />
bacteria<br />
Cell division (cytokinesis) is one <strong>of</strong> the pivotal biological processes in prokaryotic cells.<br />
The site <strong>of</strong> cell division is usually marked by the assembly <strong>of</strong> a so called Z-ring which is<br />
composed out <strong>of</strong> the bacterial tubulin homologue FtsZ. Proteins involved in division<br />
localise in a defined hierarchical order to the contractile Z-ring, forming a multisubunit<br />
complex, named divisome or septosome. Most <strong>of</strong> the division proteins contain at least<br />
one transmembrane span or are integral membrane proteins. Due to advances in<br />
sophisticated fluorescence microscopy methods, recent studies <strong>of</strong> these proteins have<br />
given a detailed picture <strong>of</strong> the localisation and temporal organisation <strong>of</strong> the division<br />
proteins. Genetic approaches have given insights into some <strong>of</strong> the properties <strong>of</strong> the<br />
different proteins. However a detailed biochemical characterisation <strong>of</strong> most <strong>of</strong> the<br />
division proteins is still lacking. This broad and pr<strong>of</strong>ound lack <strong>of</strong> knowledge is in some<br />
part due to the fact that most division proteins are membrane bound and therefore not<br />
easily amenable to overexpression, purification and biochemical characterisation. We<br />
present studies on the membrane integral proteins FtsW, FtsL and DivIC <strong>of</strong> Bacillus<br />
subtilis. Biochemical and genetic approaches were used to address possible functions to<br />
these proteins or to their functional modules.<br />
contact:<br />
Dr. Marc Bramkamp<br />
University <strong>of</strong> Oxford<br />
Sir William Dunn School<br />
marc.bramkamp@path.ox.ac.uk<br />
South Parks Road<br />
OX1 3RE Oxford (United Kingdom)
Christiane Wiegand, Marco Hagedorn, Harald Jockusch<br />
Misfolded TMV Coat Proteins: New Mutants and Pathogenicity<br />
in Plant and Animal Cells<br />
Misfolded proteins have been identified as causes <strong>of</strong> human neurodegenerative diseases.<br />
In plants, the accumulation <strong>of</strong> misfolded viral coat proteins (CPs) may cause chloroplast<br />
destruction as with "yellow strains" <strong>of</strong> tobacco mosaic virus (TMV). Many but not all<br />
temperature-sensitive (ts) mutant CPs <strong>of</strong> TMV are cytopathic [1]. There are 8 proline (P)<br />
residues in the 158 amino acid TMV CP that may be important for conformational<br />
stability. We have replaced, by in vitro mutagenesis, P by leucine (L) at different<br />
positions x, resulting in mutants PxL. P7L, P78L and P102L were "lethal", i. e. they could<br />
not be propagated in the host plant. P20L (as in the nitrous acid mutant Ni 118 [1, 2]),<br />
P54L, and P56L were ts to varying degrees, as is P63S (= Ni 1196), whereas P156L ( =<br />
Ni 1927) is tr. In the host plant, the ts mutant "flavum", D19A, which has lost a negative<br />
charge, is highly cytopathic. Chloroplast destrucion by PxL mutants increases in the<br />
order P20L, P56L, P54L, but all are milder than D19A. Misfolded ts but not tr TMV CPs<br />
are ubiquitylated in the plant cell [2]. In order to find out whether these observations<br />
can be generalized to animal systems, immortalized simian kidney cells (COS7) were<br />
transfected with vectors coding for wildtype (WT) and mutant TMV CPs and incubated at<br />
31 and 37°C. The distribution and the processing <strong>of</strong> these foreign proteins depended on<br />
their folding status, as determined by the type <strong>of</strong> mutation (tr or ts) and incubation<br />
temperature. The most thermolabile proteins accumulated in lumpy aggregates and<br />
were highly cytopathic, causing cell death within hours. Ubiquitin immunoreactivity<br />
reactivity colocalized with the aggregates <strong>of</strong> misfolded TMV CP.<br />
Supported by Fonds der Chemischen Industrie.<br />
Literature<br />
[1] Jockusch, H. (1968) Naturwissenschaften 55, 514-518 (review)<br />
[2] Jockusch, H., & Wiegand, C. (2003) FEBS Letters 545, 229-232<br />
contact:<br />
Christiane Wiegand<br />
Universität Bielefeld<br />
Entwicklungsbiologie und Molekulare Pathologie<br />
Christiane.Wiegand@uni-bielefeld.de<br />
Universitätsstr. 25<br />
33615 Bielefeld (Deutschland)
Erik Hauzman, Annette Staebler, Martin Götte, Kathrin Tausch, Igor B. Buchwalow, Olaf<br />
Buchweitz, Ludwig Kiesel, Robert R. Greb<br />
Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Endometriosis is a major cause <strong>of</strong> abdominal pain and infertility in women. Angiogenesis<br />
facilitates establishment, growth and invasion <strong>of</strong> endometriotic implants (1-3). We<br />
investigated the angiogenic status in eutopic and ectopic endometrium by<br />
immunohistochemistry and quantitative RT-PCR.The vessel proliferation index (VPI) was<br />
determined as the percentage <strong>of</strong> microvessels containing proliferating endothelial cells<br />
(Ki67+). In normal endometrium, the VPI in specimens from the proliferative phase<br />
[15%] was significantly higher than in the secretory phase [5%] (P
Lars Hemsath, Patricia Stege, Mohammad Reza Ahmadian<br />
Molecular basis <strong>of</strong> Cdc42 recognition by Wiskott-Aldrich<br />
Syndrome Proteins<br />
As molecular switches, the members <strong>of</strong> the Rho family <strong>of</strong> small GTPases control a variety<br />
<strong>of</strong> cellular processes. Beside cell polarity, gene expression and vesicular trafficking their<br />
main target <strong>of</strong> regulation is the actin cytosceleton. Cdc42, one <strong>of</strong> the best-investigated<br />
member <strong>of</strong> the Rho GTPases, induces the formation <strong>of</strong> filopodia through the activation <strong>of</strong><br />
Wiskott-Aldrich syndrome proteins (WASp and N-WASp). In its inactive form WASp<br />
exhibits an autoinhibitory conformation masking its functional VCA domain. This<br />
intramolecular interaction can be countered by the binding <strong>of</strong> Cdc42 to WASp, which sets<br />
the VCA domain free to bind G-actin and to activate the Arp2/3 complex, leading to actin<br />
polymerization. In addition to Cdc42, several other members <strong>of</strong> the Rho family (Rac1,<br />
TC10, TCL and Chp) have been reported to interact with WASp and to be involved in<br />
WASp-mediated remodeling <strong>of</strong> actin filaments. To examine the specificity <strong>of</strong> the WAS<br />
proteins towards the different Rho GTPases we studied the kinetics <strong>of</strong> their interaction<br />
using fluorescence spectroscopic techniques. We will present data which provide insights<br />
into the crucial initial steps <strong>of</strong> WASp binding that is exclusively specific to Cdc42 but not<br />
to other Rho GTPases.<br />
contact:<br />
Dipl. Biochem. Lars Hemsath<br />
MPI Dortmund<br />
lars.hemsath@mpi-dortmund.mpg.de<br />
Otto-Hahn Str. 11<br />
44227 Dortmund (Deutschland)
Anja Bruehl, Arnd Baumann<br />
Molecular characterization <strong>of</strong> rat calcium-activated chloride<br />
channels<br />
Chloride channels subserve important functions including transepithelial transport,<br />
regulation <strong>of</strong> cell volume, and neuronal excitability. Electrophysiological as well as<br />
molecular cloning approaches revealed that chloride channels are encoded by at least<br />
four gene families: the voltage-gated chloride channels (ClC), the ligand-gated chloride<br />
channels (Glycin and GABA receptors), the CFTR, and the calcium-activated chloride<br />
channels (ClCa).<br />
In olfactory signal transduction, ClCa channels participate in the generation <strong>of</strong> action<br />
potentials. Binding <strong>of</strong> an odorant to an olfactory receptor located in the cilia membrane<br />
<strong>of</strong> an olfactory sensory neuron, initiates a cascade <strong>of</strong> biochemical reactions leading to<br />
the synthesis <strong>of</strong> the intracellular messenger cAMP. Binding <strong>of</strong> cAMP to cyclic nucleotidegated<br />
(CNG) ion channels leads to an inward current <strong>of</strong> sodium and calcium ions through<br />
the opened CNG channels. The rise <strong>of</strong> the intracellular calcium concentration then causes<br />
ClCa channels to open, giving way to chloride efflux from the cell. Both, the inward<br />
current through CNG channels and the chloride efflux through ClCa channels depolarizes<br />
the cell and generates an action potential.<br />
A candidate gene assumed to encode functional ClCa channels, is the bestrophin gene.<br />
We have cloned four members <strong>of</strong> this gene family (Best 1-4) from rattus norvegicus. The<br />
Bestrophin proteins share similar N-terminal halves but differ in the C-terminal halves.<br />
The four cDNA clones were C-terminally modified by a hemagglutinin (HA)-Tag to<br />
facilitate protein detection after heterologous expression. Best 1-4 were expressed in<br />
HEK 293 cells as shown by anti HA-immunostaining. Currently, we are testing whether<br />
the Bestrophins form functional channels by electrophysiological recordings.<br />
contact:<br />
Anja Bruehl<br />
Forschungszentrum Juelich<br />
Institut fuer biologische Informationsverarbeitung I<br />
a.bruehl@fz-juelich.de<br />
Leo-Brandt-Str.<br />
52428 Juelich (Deutschland)
Kenji Irie, T. Sakisaka, W. Ikeda, H. Ogita, Yoshimi Takai<br />
Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and<br />
polarity<br />
In polarized epithelial cells, cell-cell adhesion is mediated through a junctional complex<br />
comprised <strong>of</strong> tight junctions (TJs) and adherens junctions (AJs), which are typically<br />
aligned from the apical to the basal side. The formation and maintenance <strong>of</strong> TJs are<br />
dependent on the formation and maintenance <strong>of</strong> AJs. At AJs, cadherins are key Ca2+dependent<br />
cell adhesion molecules (CAMs), whereas claudins are key Ca2+-independent<br />
CAMs at TJs. Junctional adhesion molecules (JAMs), Ca2+-independent immunoglobulin<br />
(Ig)-like CAMs, also localize at TJs and are involved in formation <strong>of</strong> cell polarity by<br />
directly binding Par-3, which forms a ternary complex with Par-6 and atypical protein<br />
kinase C (aPKC). Our series <strong>of</strong> studies have revealed that nectins, Ca2+-independent Iglike<br />
CAMs, localize at AJs, and play key roles in the organization <strong>of</strong> the junctional<br />
complex in epithelial cells. Nectins constitute a family consisting <strong>of</strong> four members, nectin-<br />
1, -2, -3, and -4. Nectins first form cell-cell adhesion where cadherins are recruited,<br />
resulting in the formation <strong>of</strong> AJs. Nectins then recruit JAMs and claudins to the apical<br />
side <strong>of</strong> AJs, resulting in the formation <strong>of</strong> TJs. In addition, nectins induce activation <strong>of</strong><br />
Cdc42 and Rac small G proteins. Cdc42 then increases the number <strong>of</strong> filopodia and cellcell<br />
contact sites, whereas Rac induces formation <strong>of</strong> lamellipodia to fill up gaps between<br />
the filopodia and to zip up the opposed plasma membranes like a “zipper”. On the other<br />
hand, nectins regulate formation <strong>of</strong> cell polarity through directly binding Par-3 and<br />
activating Cdc42, which binds to Par-6 and induces the formation <strong>of</strong> the Par-3/Par-<br />
6/aPKC complex. We describe here the roles and modes <strong>of</strong> actions <strong>of</strong> nectins in the<br />
formation <strong>of</strong> cell junctions and polarity.<br />
contact:<br />
Dr. Kenji Irie<br />
Osaka University Graduate School <strong>of</strong> Medicine<br />
Department <strong>of</strong> Molecular Biology and Biochemistry<br />
ytakai@molbio.med.osaka-u.ac.jp<br />
2-2 Yamada-oka<br />
565-0871 Suita, Osaka (JAPAN)
Tanja Meyer, Christian Schwöppe, Antje von Schaewen<br />
Molecular properties <strong>of</strong> a new G6PD-like is<strong>of</strong>orm from<br />
Arabidopsis<br />
Usually, Glucose-6-Phosphate Dehydrogenase (G6PDH, EC 1.1.1.49) catalyses the rate<br />
limiting step <strong>of</strong> the irreversible part <strong>of</strong> the oxidative pentose-phosphate pathway (OPPP),<br />
generating 2 moles <strong>of</strong> NADPH and 1 mole <strong>of</strong> ribulose-5-phosphate from 1 mole <strong>of</strong><br />
glucose-6-phosphate (on expense <strong>of</strong> 1 mole CO2). We described a cytosolic and two<br />
plastidic G6PDH is<strong>of</strong>orms at the molecular level. New data support the existence <strong>of</strong><br />
another G6PDH in higher plants. We used the cDNA <strong>of</strong> this new G6PD is<strong>of</strong>orm from<br />
Arabidopsis for first molecular studies in order to gain insight into biochemical<br />
properties, subcellular localisation, and way <strong>of</strong> regulation. To study the subcellular<br />
localization we created different proteinfusions, in which GFP is fused either to the C- or<br />
the N-terminus. We expressed different constructs <strong>of</strong> the new is<strong>of</strong>orm (with varying Nterminal<br />
lengths and different tags) in a g6pd-deficient E. coli strain. However, all<br />
attempts to detect G6PDH activity failed. Western-blot analysis <strong>of</strong> A. thaliana extracts<br />
against derived antiserum <strong>of</strong> the new is<strong>of</strong>orm and <strong>of</strong> its N-terminal sequence revealed<br />
that the protein migrates at ~ 72 kDa. Furthermore we wanted to gain insights into the<br />
possible role <strong>of</strong> the new is<strong>of</strong>orm. Therefore we made Y-2-H Screens in which we had to<br />
divide the protein in two halves because the whole protein was lethal to yeasts.<br />
contact:<br />
Tanja Meyer<br />
Universität Münster<br />
Institut für Botanik<br />
meyertan@uni-muenster.de<br />
Schloßgarten 3<br />
48149 Münster (Deutschland)
Gabriele Bixel, Stephan Kloep, Stefan Butz, Björn Petri, Britta Engelhardt, Dietmar<br />
Vestweber<br />
Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Human CD99 is a small highly O-glycosylated cell surface protein with a unique structure<br />
without resemblance to any known protein family. It is expressed on most leukocytes<br />
and on endothelial cells where it is found at endothelial cell contacts. Human CD99 was<br />
recently found to participate in the transendothelial migration (TEM) <strong>of</strong> monocytes in<br />
vitro. Diapedesis involved homophilic interaction <strong>of</strong> CD99 on monocytes with CD99 on<br />
endothelial cells (1).<br />
We cloned a mouse cDNA coding for a protein 45% identical in its sequence with human<br />
CD99. Based on the cDNA we generated antibodies against this mouse homolog <strong>of</strong><br />
CD99, which detected its antigen on most leukocytes, on endothelia <strong>of</strong> various tissues<br />
and at cell contacts <strong>of</strong> cultured endothelial cells. In cell aggregation assays mouse CD99<br />
was able to mediate homotypic aggregation <strong>of</strong> transfected CHO cells, a function that was<br />
completely blocked by anti-CD99 antibodies (2). To analyse whether CD99 is involved in<br />
the interactions <strong>of</strong> T cells with endothelial cells, we used an antigen specific T cell line in<br />
adhesion and transmigration assays. Although anti-CD99 antibodies did not affect<br />
adhesion <strong>of</strong> T cells to cultured endothelial cells, the same antibodies efficiently blocked<br />
transmigration <strong>of</strong> T cells through a monolayer <strong>of</strong> cultured endothelial cells. TEM <strong>of</strong> T cells<br />
was inhibited by anti-CD99 antibodies, independent <strong>of</strong> whether T cells or endothelial<br />
cells were preincubated with antibodies (2).<br />
This is the first description <strong>of</strong> a protein likely to be the homolog and functional analog <strong>of</strong><br />
human CD99. Here we report that mouse CD99 can support homotypic cell adhesion and<br />
that it participates in the TEM <strong>of</strong> lymphocytes in vitro.<br />
Literature<br />
(1) Schenkel AR, Mamdouh Z, Chen X, Liebman RM, Muller WA. CD99 plays a major role<br />
in the migration <strong>of</strong> monocytes through endothelial junctions. Nat.Immunol. 2002;3:143-<br />
150<br />
(2) Bixel G, Kloep S, Butz S, Petri B, Engelhardt B, Vestweber D. Mouse CD99<br />
participates in T cell recruitment into inflamed skin. Blood; in press.<br />
contact:<br />
Diplom Biologe Stephan Kloep<br />
Westfälische Wilhelms Universität Münster<br />
Institut für Zellbiologie<br />
kloep@uni-muenster.de<br />
Von Esmarch Straße 56<br />
48149 Münster (Deutschland)
Gabriele Bixel, Björn Petri, Stephan Kloep, Stefan Butz, Engelhardt Britta, Vestweber<br />
Dietmar<br />
Mouse CD99 participates in T cell recruitment into inflamed<br />
skin<br />
Recruitment <strong>of</strong> leukocytes into inflamed tissue requires emigration <strong>of</strong> leukocytes from<br />
the blood stream and migration through the endothelial lining and the basement<br />
membrane <strong>of</strong> the blood vessels. Although docking and tight adhesion <strong>of</strong> extravasating<br />
leukocytes on the endothelial cell surface have been well studied to date, much less is<br />
known about the last step in this process, the diapedesis <strong>of</strong> leukocytes through the blood<br />
vessel wall.<br />
Human CD99, a small highly O-glycosylated cell surface protein expressed on most<br />
leukocytes, was recently found to participate in transendothelial migration <strong>of</strong> monocytes<br />
in vitro (1). We cloned a mouse cDNA coding for a protein 45% identical in its sequence<br />
with human CD99 and generated antibodies against this mouse homolog <strong>of</strong> CD99 (2).<br />
Anti-CD99 antibodies, which detected its antigen on most leukocytes, on endothelia <strong>of</strong><br />
various tissues and at cell contacts <strong>of</strong> cultured endothelial cells, efficiently inhibited the<br />
transmigration <strong>of</strong> T cells in vitro (2). In a cutaneous delayed-type hypersensitivity<br />
reaction (DTH), anti-CD99 antibodies inhibited the recruitment <strong>of</strong> in vivo activated T cells<br />
into inflamed skin as well as edema formation (2).<br />
Our results establish for the first time that the endothelial cell contact associated<br />
membrane protein CD99 is involved in T lymphocyte extravasation. We conclude that<br />
mouse CD99 participates in the transendothelial migration <strong>of</strong> lymphocytes and in their<br />
recruitment to inflamed skin in vivo. This establishes CD99 as a valid target for<br />
interference with cutaneous inflammatory processes.<br />
Literature<br />
(1) Schenkel AR, Mamdouh Z, Chen X, Liebman RM, Muller WA. CD99 plays a major role<br />
in the migration <strong>of</strong> monocytes through endothelial junctions. Nat.Immunol. 2002;3:143-<br />
150<br />
(2) Bixel G, Kloep S, Butz S, Petri B, Engelhardt B, Vestweber D. Mouse CD99<br />
participates in T cell recruitment into inflamed skin. Blood; in press.<br />
contact:<br />
Dr. Gabriele Bixel<br />
Max-Planck-Institut für molekulare Biomedizin<br />
Institut für Zellbiologie<br />
mgbixel@uni-muenster.de<br />
Von-Esmarch-Str. 56<br />
48149 Münster (Deutschland)
Oliver Schmidt, Helmut E. Meyer, Katrin Marcus<br />
Multi-dimensional chromatography <strong>of</strong> proteins<br />
Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used<br />
as a standard approach. Its drawbacks have limited its widespread use and applications<br />
in proteomics. In the meantime, various liquid-phase based separation techniques have<br />
been explored. The ability to characterise a complex protein sample (e.g. the brain<br />
proteome) by mass spectrometry (MS) depends on the power and sensitivity <strong>of</strong> the<br />
separation technique employed prior to the MS analysis. The high complexity <strong>of</strong> such<br />
samples and the large dynamic range involved in analysing protein mixtures, make a<br />
multi-dimensional separation necessary. Because the separation <strong>of</strong> complex samples on<br />
peptide basis is insufficient for comprehensive protein identification, new techniques<br />
based on protein separation should be established.<br />
In our approach we have tried to separated the Proteins <strong>of</strong> the Serva Proteome Standard<br />
(a mixture <strong>of</strong> 8 proteins) with 2D HPLC using chromat<strong>of</strong>ocusing in the first and reversed<br />
phase chromatography in the second dimension.The proteins were identified by nano-LC-<br />
ESI-MS/MS after tryptic digestion <strong>of</strong> the RP fractionated Proteome Standard.<br />
contact:<br />
LM-Chem. Oliver Schmidt<br />
Universität Bochum<br />
Medizinisches Proteom Center<br />
oliver.schmidt@ruhr-uni-bochum.de<br />
Universitätsstraße 150<br />
44780 Bochum (Deutschland)<br />
additional information<br />
www.medizinisches-proteom-center.de
Heinz-Jürgen Steinh<strong>of</strong>f<br />
Multi-frequency EPR reveals the conformational dynamics <strong>of</strong><br />
membrane proteins: colcin A and the sensory rhodopsintransducer<br />
complex<br />
X- , Q- and W-band electron paramagnetic resonance (EPR) spectroscopy in combination<br />
with site-directed spin labeling (SDSL) and molecular dynamics (MD) simulations has<br />
emerged as a powerful method to study the structure and conformational dynamics <strong>of</strong><br />
membrane proteins. Analyses <strong>of</strong> the dynamics and accessibility <strong>of</strong> the spin label side<br />
chains as well as the determination <strong>of</strong> inter-spin distances and <strong>of</strong> the polarity in the<br />
vicinity <strong>of</strong> the spin label binding site provide valuable information for restraint modeling<br />
<strong>of</strong> protein structures and conformational changes. The talk presents details <strong>of</strong> the<br />
structure and conformational dynamics <strong>of</strong> the halobacterial phototaxis receptor sensory<br />
rhodopsin (pSRII) in complex with the transducer pHtrII as determined from a set <strong>of</strong> 26<br />
pairs <strong>of</strong> interacting nitroxide spin labels introduced into the pSRII-pHtrII complex. Time<br />
resolved detection <strong>of</strong> inter-spin distance changes after light activation reveals<br />
conformational changes <strong>of</strong> pSRII and uncovers the mechanism <strong>of</strong> the signal transfer<br />
from pSRII to the associated transducer pHtrII [1-2].<br />
The outlook presents an application <strong>of</strong> high-field EPR spectroscopy to site-directed spin<br />
labeled colicin A, a channel forming toxin. The high-field high-frequency EPR data<br />
provide evidence for a distinct model <strong>of</strong> the membrane associated closed state <strong>of</strong> the<br />
channel at neutral pH [3].<br />
Literature<br />
1. Wegener, A., Klare, J., Engelhard, M., Steinh<strong>of</strong>f, H.-J. 2001. EMBO J. 20, 5312-5319<br />
2. Klare, J., Gordeliy, V.I., Labahn, J., Büldt, G., Steinh<strong>of</strong>f, H.-J., and Engelhard, M.<br />
2004. FEBS Letters 564:219-224<br />
3. Wegener, C., Savitsky, A., Pfeiffer, M., Möbius, K., and Steinh<strong>of</strong>f, H.-J. 2002. Appl.<br />
Mag. Res. 21:441-452<br />
contact:<br />
Pr<strong>of</strong>. Dr. Heinz-Jürgen Steinh<strong>of</strong>f<br />
Universität Osnabrück<br />
Physik<br />
hsteinho@uos.de<br />
Barbarastrasse 7<br />
49069 Osnabrück (Deutschland)
Jürgen Alves, Wolfgang Küster, Imke Peters<br />
Mutants <strong>of</strong> the EcoRI Restriction Endonuclease Exhibit<br />
Changes <strong>of</strong> Specificity<br />
The restriction endonuclease EcoRI binds the sequence G/AATTC with very high<br />
specificity and cleaves the DNA in the presence <strong>of</strong> magnesium ions as indicated by the<br />
slash. This is based on the formation <strong>of</strong> numerous hydrogen bonds, ionic and<br />
hydrophobic interactions with bases and phosphates <strong>of</strong> the recognition sequence.<br />
According to the co-crystal structure, Met137 was predicted to form a hydrophobic<br />
contact to the cytosine <strong>of</strong> the recognition sequence. Various substitutions replacing this<br />
amino acid confirm its importance for DNA recognition and result in a decrease in<br />
specific activity by a factor <strong>of</strong> 100 at least.<br />
An unexpected exception is the M137Q mutant. This variant exhibits a specific activity<br />
comparable to the wild-type enzyme. In addition M137Q does not discriminate against or<br />
even prefers a methylated cytosine depending on the sequence context.<br />
Based on this preference for a methyl group at the 3´-end <strong>of</strong> the recognition sequence<br />
we created additional mutations <strong>of</strong> those residues contacting the complementary<br />
guanine. However, only the loss <strong>of</strong> recognition <strong>of</strong> this base pair towards NAATTN´ with a<br />
preference for CAATTG could be achieved.<br />
contact:<br />
Pr<strong>of</strong>. Jürgen Alves<br />
Medizinische Hochschule Hannover<br />
Biophysikalische Chemie<br />
alves@bpc.mh-hannover.de<br />
Carl-Neuberg-Str. 1<br />
30625 Hannover (Deutschland)
Kerstin Brands, Ferda Cevikbas, Frank Echtermeyer<br />
My<strong>of</strong>ibroblast differentiation and TGF-b1 signalling is affected<br />
in syndecan-4 deficient fibroblasts<br />
Mice with a deletion <strong>of</strong> syndecan-4, a multifunctional cell surface proteoglycan, exhibit a<br />
delayed wound closure rate and an absence <strong>of</strong> wound contraction during wound healing.<br />
In order to elucidate the cellular mechanisms causing the wound healing problems <strong>of</strong><br />
syndecan-4 deficient mice, we analysed the abilities <strong>of</strong> syndecan-4 -/- fibroblasts.<br />
Contractile forces were measured in a collagen gel contraction assay after induction with<br />
TGF-b1. The acceleration <strong>of</strong> contraction by TGF-b1 is significantly slower in syndecan-4<br />
deficient fibroblasts than in wildtype cells.<br />
Second we investigated if the TGF-b1 induced differentiation <strong>of</strong> my<strong>of</strong>ibroblasts is<br />
affected in syndecan-4 -/- fibroblasts. My<strong>of</strong>ibroblasts, which derive from fibroblasts in<br />
the wound tissue, can generate strong contractile forces and are responsible for the<br />
contraction <strong>of</strong> a wound. Differentiation <strong>of</strong> fibroblasts into my<strong>of</strong>ibroblasts in vitro was<br />
achieved by the addition <strong>of</strong> TGF-b1 to cells grown in attached collagen gels. We could<br />
demonstrate, that the number <strong>of</strong> my<strong>of</strong>ibroblasts was clearly reduced in case <strong>of</strong> the<br />
syndecan-4 -/- fibroblasts.<br />
Third we investigated the signalling <strong>of</strong> TGF-b1 in syndecan-4 knockout fibroblasts since<br />
the first two experiments revealed a reduced response to the growth factor TGF-b1 in<br />
the absence <strong>of</strong> syndecan-4. We examined the activation <strong>of</strong> the smad proteins, which are<br />
the direct mediators <strong>of</strong> the TGF-b1 signal. So far we could not detect any differences in<br />
the activation, expression level or nuclear translocation for any <strong>of</strong> these smad proteins<br />
between wild type and syndecan-4 deficient fibroblasts. Other TGF-b1 affected signalling<br />
pathways are under investigation.<br />
contact:<br />
Dipl. Biol. Kerstin Brands<br />
Muenster University Hospital<br />
Department <strong>of</strong> Physiological Chemistry and Pathobiochemistry<br />
brandsk@uni-muenster.de<br />
Waldeyerstr. 15<br />
48149 Münster (Germany)
Alexandra Rolletschek, Cornelia Wiese, Gabriela Kania, Przemyslaw Blyszczuk,<br />
Barbara Steinfarz*, Ihor Zahanich+, Thomas Braun#, Oliver Brüstle*, Kenneth R.<br />
Boheler~, Anna M. Wobus<br />
Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
We have generated nestin-positive cells from adult mouse intestine (intestinal epitheliumderived<br />
nestin-positive progenitors = INPs) by culture on embryonic fibroblast feeder<br />
layers (FL). INPs were generated by continuous cultivation. Additionally, compacted<br />
nestin-positive clusters appeared on FL giving rise to nestin-positive spheroids and<br />
neurosphere-like aggregates.<br />
After induction <strong>of</strong> neuronal differentiation, transcripts <strong>of</strong> the neural-specific genes en-1,<br />
synaptophysin, NFM and GFAP were detected. Immunostaining revealed ß-III-tubulin<br />
and GFAP at high levels. Co-culture <strong>of</strong> EGFP-expressing INPs with PA6 stromal cells<br />
resulted in a high amount <strong>of</strong> b-III-tubulin- and synaptophysin-expressing neurons. After<br />
implantation into hippocampal brain slices, EGFP-positive INPs showed extensive<br />
migration into the host tissue and developed b-III-tubulin- and GAP 43-positive cells<br />
after 2-3 weeks <strong>of</strong> differentiation in situ. Electrophysiological analysis revealed<br />
expression <strong>of</strong> K+ outward currents, but no Na+ currents.<br />
After pancreatic differentiation, INPs showed pancreas-specific transcripts <strong>of</strong> islet-1 and<br />
insulin. Immunocytochemistry revealed insulin- and C-peptide-expressing cells. Hepatic<br />
differentiation <strong>of</strong> INPs resulted in the differentiation <strong>of</strong> cells expressing HNF3b, afetoprotein,<br />
albumin and a1-antitrypsin. Immunocytochemistry revealed albumin-, a1antitrypsin-<br />
and cytokeratin 18-positive cells.<br />
We conclude that signals released from mouse embryonic fibroblast FL induce a nestinpositive<br />
progenitor cell type (= INP) with high plasticity. INPs differentiate into cells with<br />
neuroectodermal and endodermal properties, however the in vitro conditions do not fully<br />
support the development <strong>of</strong> mature cell types.<br />
contact:<br />
Dr. Alexandra Rolletschek<br />
IPK Gatersleben<br />
In Vitro Differentiation Group<br />
rolleta@ipk-gatersleben.de<br />
Corrensstr. 3<br />
06466 Gatersleben (Deutschland)<br />
additional information<br />
*Institute <strong>of</strong> Reconstructive Neurobiology, University <strong>of</strong> Bonn; +Department <strong>of</strong> Pharmacology and<br />
Toxicology, TU Dresden; #Institute <strong>of</strong> Physiological Chemistry, University <strong>of</strong> Halle-Wittenberg;<br />
~National Institute on Aging, NIH, Baltimore, USA
Markus Rüegg<br />
Neuromuscular diseases: Concepts and new approaches for<br />
therapies<br />
A proper functioning <strong>of</strong> the connection between motor neurons and skeletal muscle<br />
(called neuromuscular junction) is requisite for coordinated movement including<br />
breathing. Diseases that affect the neuromuscular junction or the capability <strong>of</strong> muscle to<br />
withstand the mechanical forces during contraction, cause muscle wasting. Both<br />
situations lead to decreased muscle force and the inability to properly control movement.<br />
In my presentation, I will discuss experiments that aim at understanding the molecular<br />
principles used in the formation <strong>of</strong> the neuromuscular junction. In particular, I will<br />
discuss the role <strong>of</strong> agrin and its splice variants as key organizers for postsynaptic<br />
differentiation. I will continue to show that the principles and molecules used for the<br />
formation <strong>of</strong> the neuromuscular junction also have the potential to be effective for the<br />
treatment <strong>of</strong> neuromuscular diseases. For example, a specifically designed, miniaturized<br />
version <strong>of</strong> one particular agrin splice variant has been shown to greatly improve the<br />
health in an animal model for MDC1A, the most frequent form <strong>of</strong> congenital muscular<br />
dystrophy in Europe. MDC1A is caused by mutations in the alpha2-chain <strong>of</strong> the<br />
extracellular matrix molecule laminin and is <strong>of</strong>ten lethal within the first decade <strong>of</strong> life. I<br />
will also show that an analogously designed version <strong>of</strong> perlecan exerts the same<br />
beneficial effect as the miniaturized version <strong>of</strong> agrin. These experiments set the basis for<br />
our current efforts to evaluate the potential <strong>of</strong> such approaches for the treatment <strong>of</strong><br />
MDC1A or other muscular dystrophies in human patients.<br />
contact:<br />
Pr<strong>of</strong>. Markus Rüegg<br />
University <strong>of</strong> Basel<br />
Biozentrum<br />
markus-a.ruegg@unibas.ch<br />
Klingelbergstrasse 70<br />
4056 Basel (Switzerland)
Carlos Dotti<br />
Neuronal membrane cholesterol loss results in low plasmin<br />
activity yet higher beta-secretase cleavage <strong>of</strong> APP<br />
The human hippocampus <strong>of</strong> ApoE4 bearers, that developed and died from Alzheimer’s<br />
disease, present a 20% reduction in membrane cholesterol than age-matched non<br />
ApoE4 Alzheimer’s sufferers (Ledesma et al. 2003). Consistent with the deficit in<br />
membrane cholesterol content, membrane and membrane-associated proteins that<br />
normally partition in the so-called raft domains appeared reduced from this fraction in<br />
the AD ApoE4 bearers and displaced to the lighter fractions. This was evident for the<br />
canonical raft component flotilin1, for the amyloid degrading enzyme plasmin (Ledesma<br />
et al 2000) and its activating receptor uPA, and also for the enzyme BACE1 (Ledesma et<br />
al. 2003 Abad-Rodriguez, J., Ledesma, M.D et al, submitted). Interestingly, the amyloid<br />
precursor protein (APP) that is a substrate for BACE1 is not present in rafts in the<br />
hippocampal plasma membrane, <strong>of</strong> neither AD nor non AD individuals, nor in the<br />
hippocampi <strong>of</strong> human APP-expressing mice. Quite on the contrary, APP floats in the light<br />
fractions where BACE1 enriches in the ApoE4-low membrane cholesterol samples.<br />
Consistently with a direct relationship between cholesterol-loss and presence <strong>of</strong> plasmin<br />
and BACE1 in the light, detergent-soluble, fractions, the induction <strong>of</strong> membrane<br />
cholesterol loss, in a chronic and mild form, in rodent hippocampal neurons growing in<br />
culture conditions resulted in the redistribution <strong>of</strong> these proteins to lighter fractions and<br />
also increased spatial colocalization. More importantly, this type <strong>of</strong> treatment resulted in<br />
the loss <strong>of</strong> the proteolytic activity <strong>of</strong> plasmin yet a gain <strong>of</strong> function <strong>of</strong> BACE1. These<br />
results suggest that the gradual loss <strong>of</strong> cholesterol from the plasma membrane can have<br />
deleterious consequences to neuronal function, such as the loss <strong>of</strong> extracellular matrix<br />
degradation needed for synaptic plasticity and remodeling that take place during<br />
learning and memory processes, as well as increase in the amount <strong>of</strong> APP primed for the<br />
action <strong>of</strong> the gamma complex.<br />
Literature<br />
Ledesma M.D. EMBO Rep. 2000<br />
Ledesma M.D. EMBO Rep. 2003<br />
contact:<br />
PhD Carlos Dotti<br />
Univ. <strong>of</strong> Turin<br />
Fondazione Cavalieri Ottolenghi, AO S.Luigi<br />
carlos.dotti@unito.it<br />
Regione Gonzole 10<br />
10043 Orbassano (TO) (Italy)
Sebastian Schrot, Hans-Joachim Galla<br />
Neutrophile Granulocytes Transendothelial Migration in vitro<br />
Scanning force microscopy and confocal laser scanning microscopy have become<br />
fundamental tools in several fields <strong>of</strong> research, particularly for the visualisation <strong>of</strong><br />
biological processes under physiological conditions. The blood-brain-barrier accomplishes<br />
predominantly the regulation <strong>of</strong> the transfer <strong>of</strong> compounds between the circulating<br />
bloodstream and the brain interstitial fluid. In case <strong>of</strong> inflammation the blood-brainbarrier<br />
allows leukocytes to emigrate from the bloodstream into inflamed tissues by<br />
sequential intercellular interactions at the endothelium. In particular the neutrophile<br />
granulocytes endothelial transmigration process is classified into multiple steps initiated<br />
by tethering and concluding at the traversing stage. The scanning force microscopy<br />
technique visualises this transmigration process at high resolution. After application <strong>of</strong><br />
neutrophile granulocytes the adhesion, the diapedesis process and the accompanying<br />
morphological rearrangements have been observed after TNF-α stimulation <strong>of</strong> confluent<br />
endothelial cell layer in vitro.<br />
contact:<br />
Sebastian Schrot<br />
Universität Münster<br />
Institut für Biochemie<br />
schrot@uni-muenster.de<br />
Wilhelm-Klemm-Straße 2<br />
48149 Münster (Bundesrepublik Deutschland)
Imre Berger, Daniel Fitzgerald, Timothy Richmond<br />
New Baculovirus Expression Tools for Multiprotein<br />
Applications<br />
The discovery in recent years <strong>of</strong> an ever increasing number <strong>of</strong> multiprotein complexes in<br />
the cell necessitates improved heterologous protein production techniques for studying<br />
their structure and function at the molecular level. Here we describe a straightforward<br />
and versatile system for generating recombinant baculovirus DNA for expression <strong>of</strong><br />
protein complexes comprising a large number <strong>of</strong> subunits. Our method utilizes transfer<br />
vectors which contain a nested multiplication module to facilitate combination <strong>of</strong><br />
polycistronic expression cassettes, thereby minimizing requirements for unique<br />
restriction sites. The transfer vectors access a modified baculovirus DNA by cre-lox sitespecific<br />
recombination or Tn7 transposition. This baculovirus has improved protein<br />
expression characteristics due to elimination <strong>of</strong> specific viral genes. Insertion reactions<br />
are carried out in E. coli either sequentially or concomitantly in a rapid, one-step<br />
procedure. Our system is useful for both recombinant multiprotein production and<br />
multigene transfer applications.<br />
contact:<br />
Dr Imre Berger<br />
ETH Zürich<br />
Institut für Molekularbiologie und Biophysik<br />
iberger@mol.biol.ethz.ch<br />
ETH Hönggerberg<br />
8093 Zürich (Schweiz)
Mohammad Reza Ahmadian<br />
New insights into structure-function relationships in GTPaseeffector<br />
interaction<br />
Signaling functions <strong>of</strong> small GTPases are based on the formation <strong>of</strong> distinct proteinprotein<br />
complexes. The large number <strong>of</strong> complex structures affords a unique opportunity<br />
to comprehend binding and specificity determining sites <strong>of</strong> the GTPases. Our<br />
investigation has been focused on interacting surfaces <strong>of</strong> the Rho GTPases and the<br />
quantitative analysis <strong>of</strong> their structures in complex with regulators and effectors. We<br />
found striking evidence <strong>of</strong> common `hot spots´ for GTPase interaction with diverse<br />
partners. In contrast to the regulators (GDIs, GEFs, GAPs), whose mechanisms <strong>of</strong> action<br />
are well defined, the structural features <strong>of</strong> effectors, their binding mode with the GTPase<br />
and the underlying principles <strong>of</strong> activation are still poorly understood. We have been<br />
using a combination <strong>of</strong> X-ray crystallography and fluorescence spectroscopy to study<br />
GTPase-effector interaction in detail, using RhoA as a model. The characterization <strong>of</strong> a<br />
novel second and third RhoA binding domain found in Rho kinase, describing the<br />
structure-function relationship <strong>of</strong> RhoA-effector interaction and shedding new light on<br />
possible activation mechanisms, involving scaffold proteins and protein kinases, will also<br />
be discussed.<br />
contact:<br />
Dr. Mohammad Reza Ahmadian<br />
MAx-Planck-Institut für molekulare Physiologie<br />
Abteilung Strukturelle Biologie<br />
reza.ahmadian@mpi-dortmund.mpg-de<br />
Otto-Hahn 11<br />
44227 Dortmund (Germany)
Astrid Blume, Andrew J. Benie, Stephan Hinderlich2, Werner Reutter2, Richard R.<br />
Schmidt3, Thomas Peters<br />
New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Sialic acids comprise a family <strong>of</strong> terminal sugars essential for a variety <strong>of</strong> biological<br />
processes including inflammation and metastasis. Increased sialic acid expression masks<br />
antigenic structures and protects cells against recognition by the immune system. The<br />
bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (UDP-<br />
GlcNAc 2-epimerase/ManNAc kinase) catalyses the first two steps <strong>of</strong> sialic acid<br />
biosynthesis and is the key regulator <strong>of</strong> this pathway [1]. Loss <strong>of</strong> UDP-GlcNAc 2epimerase<br />
leads to a drastic decrease <strong>of</strong> cellular sialylation, influencing several sialicacid-dependent<br />
functions.<br />
Using saturation transfer difference nuclear magnetic resonance (STD-NMR) methods<br />
[2,3] it is possible to obtain information about interactions between a protein and<br />
different ligands. These experiments allow not only the detection <strong>of</strong> binding but also the<br />
determination <strong>of</strong> binding epitopes for each ligand at an atomic resolution. The binding<br />
epitope <strong>of</strong> the natural substrate for the epimerase reaction, UDP-GlcNAc, and fragments<br />
revealed that the protons <strong>of</strong> the nucleotide moiety are closer to the protein surface than<br />
protons <strong>of</strong> the GlcNAc residue. Furthermore it was shown that at least one phosphate is<br />
required for binding. Titrations performed with UDP, one <strong>of</strong> the products <strong>of</strong> the<br />
epimerase reaction and a known in vivo inhibitor, show that UDP binds more strongly to<br />
the enzyme than UDP-GlcNAc. Taking into account the intracellular concentrations <strong>of</strong> the<br />
two ligands the UDP-GlcNAc 2-epimerase seems to be in an almost permanently<br />
inhibited state .<br />
ManNAc is a product <strong>of</strong> the epimerase reaction and simultaneously an educt <strong>of</strong> the<br />
ManNAc kinase reaction <strong>of</strong> the bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase.<br />
Our data show that the epimerase reaction exclusively generates the ?-anomer, and that<br />
the kinase exclusively binds ?-ManNAc. The educts (ManNAc, ATP) <strong>of</strong> the ManNAc kinase<br />
show a higher affinity to the active site <strong>of</strong> the enzyme than the products (ManNAc-6phosphate<br />
and ADP). Binding epitopes reveal that this is due to a change in the binding<br />
mode <strong>of</strong> the products after the phosphorylation reaction.<br />
On the basis <strong>of</strong> these results a new insight into the action <strong>of</strong> the active centers and<br />
mechanistic processes <strong>of</strong> the key enzyme <strong>of</strong> sialic acid biosynthesis will be presented.<br />
These conclusions also provide significant new information for the design <strong>of</strong> inhibitors for<br />
the UDP-GlcNAc 2-epimerase.<br />
Literature<br />
[1] Kornfeld, S., Kornfeld, R., Neufeld, E. and O’Brien, P.J. (1964) Proc. Natl. Acad. Sci.<br />
USA 52, 371-379<br />
[2] Mayer, M. and Meyer, B. (1999), Angew. Chem. Int. Ed. 38, 1784-1787<br />
[3] Meyer, B. and Peters, T. (2003), Angew. Chem. Int. Ed. 42, 864-890<br />
contact:<br />
Dr. Astrid Blume<br />
Universität zu Lübeck<br />
Institut für Chemie<br />
ablume@chemie.mu-luebeck.de<br />
Ratzeburger Allee 160<br />
23538 Lübeck (Germany)<br />
additional information<br />
2) Institut für Molekularbiologie und Biochemie, FU Berlin<br />
3) Fachbereich Chemie, Universität Konstanz
M. Tharwat Ghoneim*, A.M. Baraka*, M.M . Matta**, H. Kamal**<br />
New insights into the modulatory role <strong>of</strong> serotonin reuptake<br />
inhibitors in myocardial ischemia reperfusion injury in the<br />
rabbit<br />
The present study was designed to test the hypothesis <strong>of</strong> decreased risk <strong>of</strong> myocardial<br />
infarction (MI) associated with the use <strong>of</strong> Selective Serotonin Reuptake Inhibitors<br />
(SSRIs) in rabbits exposed to myocardial ischemic reperfusion injury (IRI). Also the work<br />
was designed to evaluate the possible protective effect <strong>of</strong> SSRIs in animals exposed to<br />
tobacco smoking and also exposed to IRI. Myocardial ischemia was induced by coronary<br />
artery ligation and reperfusion. The animals were classified into five groups. The first<br />
group was the sham operated animals, the second group was the coronary ligated and<br />
reperfused group. The third group was exposed to tobacco smoking daily for three weeks<br />
and then coronary ligated and reperfused. The forth group treated with fluoxetin for<br />
three weeks before coronary ligation and reperfusion. The fifth group was exposed to<br />
smoking and then treated with fluoxetin for three weeks before coronary ligation and<br />
reperfusion. IRI produced a significant attenuation <strong>of</strong> the endothelium dependent<br />
relaxation <strong>of</strong> the left anterior coronary artery (-37.3 %). Smoking produced a similar<br />
attenuation (-37%). Fluoxetin treatment significantly improved the endothelium<br />
dependent relaxation in the IRI and also in IRI with smoking (+ 37 % and +23 %).<br />
Fluxetin treatment resulted in a significant decrease in plasma cardiac troponin (cTnT) (-<br />
53.4 %) and plasma Homocysteine (Hyc) (- 19.3 %) and in percent platelet aggregation.<br />
The data <strong>of</strong> the present study support the notion that SSRI could play a beneficial role.<br />
Platelets also play a role in IRI. It is concluded that SSRIs could be protective against<br />
myocardial infarction. Further investigations are needed to explain the possible<br />
mechanism(s) <strong>of</strong> the protective effect.<br />
contact:<br />
Pr<strong>of</strong>. Dr. M. Tharwat Ghoneim<br />
Alexandria University<br />
Department <strong>of</strong> Pharmacology<br />
ghoneimt@lycos.com<br />
Faculty <strong>of</strong> Medicine<br />
21521 Alexandria (Egypt.)<br />
additional information<br />
(*) Department <strong>of</strong> Pharamacology,and (**) Department <strong>of</strong> Biochemistry, Faculty <strong>of</strong> Medicine,<br />
Alexandria University, 21521 El-Messalla, Alexandria, Egypt
Rico Czaja, Marc Struhalla, Katja Höschler, Wolfram Saenger, Norbert Sträter, Uli Hahn<br />
New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Ribonuclease (RNase) T1 represents a suitable model for studying protein-nucleic acid<br />
interactions. It cleaves single-stranded RNA specifically after Guanosine. Due to the<br />
knowlwdge <strong>of</strong> numerous structures the guanine recognition is well understood. Many sitedirected<br />
mutagenesis studies have been reported but all <strong>of</strong> these did not lead to new<br />
specificity. Substitutions <strong>of</strong> Glu46, a highly conserved residue within the RNase T1<br />
family, led to variants with extremly decreased catalytic activity. A semirational<br />
approach by randomizing the guanine binding site led to the variant 9/5 with the<br />
guanine recognition segment K41E-Y42F-N43R-N44N-Y45W-E46Q. In this variant the<br />
glutamine at position 46 forms bidentate hydrogen bonds to the backbone similar as the<br />
inactive variant E46Q. In contrast to variant E46Q, 9/5 is able to bind 2‘-GMP at its<br />
primary guanine recognition site presumably due to the increased aromatic stacking with<br />
the 2’-GMP caused by the Tyr45Trp substitution. We constructed the variant K41E-Y42F-<br />
N43R-N44N-Y45W-E46N which exhibits an altered nucleotide specificity. The crystal<br />
structure <strong>of</strong> this new purine-specific variant gives new structural insights into the<br />
recognition mechanism <strong>of</strong> the enzyme and is also the basis for further experiments.<br />
contact:<br />
Pr<strong>of</strong>essor Uli Hahn<br />
Universität Hamburg<br />
Biochemie<br />
uli.hahn@hamburg.de<br />
Martin-Luther-King-Platz 6<br />
20146 Hamburg (Deutschland)
Eva C. Woenne, Cathrine Schmidt, Nick Mirancea, Roswitha Nischt, Neil Smyth, U.<br />
Werner, Hans-Jürgen Stark, Norbert E. Fusenig, M. Gerl, Dirk Breitkreutz<br />
Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
General basement membrane (BM) components are polymeric collagen-IV, laminin-10,<br />
and associated nidogen and perlecan, while laminin-5 is epidermis-specific. Since BMdefects<br />
in mice are mostly lethal during development, we have studied the role <strong>of</strong><br />
nidogen in 3D-cocultures <strong>of</strong> human keratinocytes (HK or HaCaT cells) and fibroblasts<br />
(human/ mouse, HF/MF) by either blocking interactions or deleting nidogen. HK or<br />
HaCaT were grown on collagen gels harboring HF or MF, BM-formation was analyzed by<br />
immuno-fluorescence (IF), regular (EM), immuno-electron microscopy (ImEM), and<br />
Western blots <strong>of</strong> the epithelial and ‘dermal’ protein fractions. (i) Nidogen-laminin<br />
interaction was inhibited by the laminin-fragment (gamma1-III3-5, Lgf) binding nidogen<br />
with high affinity. In 3D-cocultures <strong>of</strong> HK and HF Lgf-treatment blocked mainly<br />
deposition <strong>of</strong> nidogen, laminin-10, and perlecan. Although other BM-zone components<br />
(laminin-5, BP180, integrin alpha6beta4) were still detectable (IF), by EM and ImEM also<br />
any BM-structures or hemidesmosomes (insertion <strong>of</strong> keratins) were absent*. (ii) Nidogen-<br />
1,-2 (ND1/2, made by fibroblasts) were eliminated by employing MF from ND1/2 komice.<br />
In 3D-cocultures use <strong>of</strong> ND1/2 (--/++)-MF abolished ND1 staining, whereas (--/+-<br />
)-MF reduced additionally ND2, collagen-IV, and laminin-10. Complete absence <strong>of</strong> ND1/2<br />
(--/--) further abolished collagen-IV and laminin-5, integrins (alpha6beta4) appearing<br />
fairly normal (IF). (iii) BM-formation was restored and even accelerated with<br />
recombinant ND1 or ND2 (IF, EM). (iv) By contrast, perlecan could be provided either by<br />
fibroblasts or keratinocytes for regular BM-structures. Thus, in this closed system BMcomponents<br />
are efficiently guided to their assembly site.<br />
Literature<br />
* Breitkreutz D, Mirancea N, Schmidt C, Beck R, Werner U, Stark HJ, Gerl M, Fusenig NE.<br />
J Cell Sci. 2004 May 15;117:2611-2622.<br />
contact:<br />
Eva C. Woenne<br />
Deutsches Krebsforschungszentrum Heidelberg<br />
e.woenne@dkfz.de<br />
Im Neuenheimer Feld 280<br />
69120 Heidelberg (Deutschland)
Ton Bisseling, Rene Geurts, Caroline Franken, Erik Limpens, Patrick Smit<br />
Nod factor perception and transduction<br />
The formation <strong>of</strong> legume root nodules involves 2 major processes. On one hand the<br />
nodule organ has to be formed from root cortical cells and on the other hand the<br />
rhizobia have to enter plant cells in a manner strictly controlled by the host.<br />
The rhizobial Nod factors are pivotal in the induction <strong>of</strong> all (early) steps in legume nodule<br />
formation. Recently, several genes involved in Nod factor perception or signaling have<br />
been cloned and this provided insight in the underlying molecular mechanisms<br />
mechanism. Further, these cloned genes are tools to address new questions concerning<br />
Nod factor perception mechanisms.<br />
During this talk an overview will be given <strong>of</strong> our studies on Nod factor perception at the<br />
root epidermis and it will be discussed whether the same Nod factor<br />
perception/transduction machinery is involved in the induction <strong>of</strong> cortical cell division<br />
Further the mechanism <strong>of</strong> Nod factor perception/transduction active in root nodules will<br />
be discussed<br />
contact:<br />
Dr. Ton Bisseling<br />
Wageningen University<br />
Department <strong>of</strong> Plant Sciences<br />
Ton.Bisseling@wur<br />
Dreijenplaan 3<br />
6703 Wageningen (NL)
Timm Schroeder, Hella Kohlh<strong>of</strong>, Nikolaus Rieber, Satomi Nishikawa, Georg Bornkamm,<br />
Shin-Ichi Nishikawa, Tasuku Honjo, Ursula Just<br />
Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Signalling by activated Notch receptors plays an important role in controlling the<br />
development <strong>of</strong> many different cell types during embryogenesis and in the adult. Here<br />
we investigate the role <strong>of</strong> Notch signalling in murine embryonic and adult hematopoiesis.<br />
To do this, we have generated embryonic stem (ES) and adult hematopoietic stem cell<br />
lines in which Notch signalling can be activated in an inducible and reversible manner.<br />
Under differentiation conditions, activation <strong>of</strong> Notch1 in ES cells increases the percentage<br />
<strong>of</strong> cells with an undifferentiated phenotype and inhibits the generation <strong>of</strong> Flk1+ lateral<br />
plate mesodermal cells and further <strong>of</strong> hematopoietic cells and cardiomyocytes. Activation<br />
<strong>of</strong> Notch1 at a later time, in hematopoietic progenitors generated from ES cells in vitro,<br />
does not inhibit the generation <strong>of</strong> mature myeloid, megakaryocytic or erythroid cells.<br />
However, the colony forming efficiency and maintenance <strong>of</strong> hematopoietic colonies on<br />
stroma cells is decreased, suggesting a reduction <strong>of</strong> self renewal <strong>of</strong> hematopoietic<br />
progenitors by activated Notch. In line with the results on embryonic hematopoietic stem<br />
cells, activation <strong>of</strong> Notch1 in the adult hematopoietic stem cell line FDCP-mix irreversibly<br />
reduces self renewal <strong>of</strong> multipotent progenitor cells accompanied by increased and<br />
accelerated differentiation along the myeloid lineage. Activated Notch1 directly increases<br />
PU.1 transcription leading to a high concentration <strong>of</strong> PU.1 protein which has been shown<br />
to direct myeloid differentiation. These findings identify Notch as an extrinsic regulator <strong>of</strong><br />
myeloid commitment and the lineage-affiliated transcription factor PU.1 as a specific<br />
direct target gene <strong>of</strong> Notch.<br />
Literature<br />
Schroeder et al., J. Immunol. 170, 5538-5548, 2003<br />
Schroeder et al., Proc. Natl. Acad. Sci. USA 100, 4018-4023, 2003<br />
contact:<br />
Pr<strong>of</strong>. Dr. Ursula Just<br />
Christian-Albrechts-Universität zu Kiel<br />
Institut für Biochemie<br />
ujust@biochem.uni-kiel.de<br />
Olshausenstr. 40<br />
24098 Kiel (Germany)<br />
additional information<br />
Please note the address affiliations for the authors: Timm Schroeder1,3, Hella Kohlh<strong>of</strong>1, Nikolaus<br />
Rieber1, Satomi Nishikawa3, Georg W. Bornkamm1, Shin-Ichi Nishikawa3, Tasuku Honjo4 and<br />
Ursula Just1,2<br />
1 GSF - National Research Centre for Environment and Health, Institute <strong>of</strong> Clinical Molecular Biology<br />
and Tumorgenetics, Munich, Germany<br />
2 Department <strong>of</strong> Biochemistry, University <strong>of</strong> Kiel, Kiel, Germany<br />
3 Laboratory for Stem Cell Biology, Riken Center for Developmental Biology, Kobe, Japan<br />
4 Department <strong>of</strong> Medical Chemistry, Kyoto University, Kyoto, Japan
Konstantin Lukyanov, Dmitry Chudakov, Vladislav Verkhusha, Mikhail Matz, Dmitry<br />
Shagin, Yurii Yanushevich, Ekaterina Barsova, Sergey Lukyanov<br />
Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Green Fluorescent Protein (GFP) from jellyfish Aequorea victoria and its mutants became<br />
an integral part <strong>of</strong> modern methodology in cell and molecular biology. GFP-like proteins<br />
represent the only genetically encoded fluorescent tag that can be widely used to label<br />
organisms, cells, organelles, and proteins. Last years a number <strong>of</strong> GFP homologues were<br />
cloned from marine animals. We characterized GFPs in planktonic copepods (phylum<br />
Arthropoda). Thus, GFP-like proteins evolved before separation <strong>of</strong> Bilateria and Cnidaria<br />
and therefore may be responsible for fluorescence and/or coloration in virtually any<br />
animal. We found a broad spectral diversity <strong>of</strong> GFP-like proteins in jellyfishes (class<br />
Hydrozoa), including green and yellow fluorescent proteins, non-fluorescent purple<br />
chromoprotein and colorless protein. Novel GFP homologues are a good source <strong>of</strong> useful<br />
mutants. In particular, a red fluorescent mutant suitable for protein labeling was<br />
generated from the novel chromoprotein. Also, a photoactivatable fluorescent mutant<br />
was created on the base <strong>of</strong> the colorless wild type protein.<br />
contact:<br />
Dr Konstantin Lukyanov<br />
Institute <strong>of</strong> Bioorganic Chemistry<br />
kluk@ibch.ru<br />
Miklukho-Maklaya 16/10<br />
117997 Moscow (Russia)
Günter Gisselmann, Hermann Pusch, Hanns Hatt<br />
Novel ligand gated channels in D. melanogaster: GABA-gated<br />
cation channels and gating by multiple neurotransmitters<br />
Ligand gated ion channels mediate the fast responses <strong>of</strong> neuronal and muscle cells to<br />
neurotransmitters. They form a large family <strong>of</strong> cation channels that can be activated by<br />
acetylcholine, serotonin and anion channels which are opened by gamma-aminobutyric-acid<br />
(GABA) and glycine. In addition glutamate, histamine and serotonine gated<br />
anion channels exist which can only be found in invertebrates. Recently there was<br />
considerable progress in the characterization <strong>of</strong> ligand-gated chloride channels in<br />
invertebrates due to the cloning <strong>of</strong> histamine and serotonine gated channels from<br />
Drosophila and C. elegans, but the molecular basis <strong>of</strong> several other types <strong>of</strong> ion channels<br />
known from electrophysiological studies, like ACh- and multitransmitter-gated anion<br />
channels is unknown. The wealth <strong>of</strong> accumulating information from the D. melanogaster<br />
genome sequencing project has given us the possibility to describe all members <strong>of</strong> the<br />
superfamily <strong>of</strong> ligand gated ion channels used by an individual species. A systematic<br />
analysis reveals that this superfamily consits <strong>of</strong> 23 genes. The corresponding cDNAs<br />
cloned by RT-PCR, were expressed in Xenopus oocytes and the funtion was investiged by<br />
two-electrode voltage clamp. We identified the first insect GABA-gated cation channel<br />
genes and give further evidence <strong>of</strong> the existance <strong>of</strong> ion channels gated by multiple<br />
neurotransmitters. The pharmacological properties <strong>of</strong> the recombinant heteromeric GABAgated<br />
cation channel match those <strong>of</strong> native channels found in different invertebrates.<br />
Therefore such channels are probably the molecular basis for the excitatory action <strong>of</strong><br />
GABA in insects.<br />
Literature<br />
Gisselmann G, Plonka J, Pusch H, Hatt H. 2004 Jun;142(3):409-13. Epub 2004 May 17<br />
Gisselmann G, Pusch H, Hovemann BT, Hatt H. Nat Neurosci. 2002 Jan;5(1):11-2.<br />
Gisselmann G., Plonka J., Pusch H., Hatt H. (submitted 2004)<br />
contact:<br />
Dr. Günter Gisselmann<br />
Ruhr-Uni-Bochum<br />
Lehrstuhl für Zellphysiologie<br />
guenter.gisselmann@rub.de<br />
Universitätsstr. 150<br />
44780 Bochum (Deutschland)
Marcia von Zeska Kress Fagundes, Marcela Savoldi, Joel Fernandes, Roy Larson,<br />
Maria Helana Goldman, Gustavo Goldman<br />
NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
interacts with the UvsB ATR kinase<br />
The DNA damage response is a protective mechanism that ensures the maintenance <strong>of</strong><br />
genomic integrity. We have been using Aspergillus nidulans as a model system to<br />
characterize the DNA damage response caused by the anti-topoisomerase I drug,<br />
camptothecin. We report the molecular characterization <strong>of</strong> a p34Cdc2-related gene,<br />
npkA, from A. nidulans. The npkA gene is transcriptionally induced by camptothecin and<br />
other DNA-damaging agents, and its induction in the presence <strong>of</strong> camptothecin is<br />
dependent on the uvsB ATR gene. There were no growth defects, changes in<br />
developmental patterns, increased sensitivity to DNA damaging agents, septation, nor<br />
growth rate in the A. nidulans npkA deletion strain. However, the ΔnpkA mutation can<br />
partially suppress HU-sensitivity caused by the ΔuvsB ATR and uvsD153 ATRIP checkpoint<br />
mutations. We demonstrated that A. nidulans uvsB ATR gene is involved in DNA<br />
replication and the intra-S-phase checkpoints, and that the ΔnpkA mutation can<br />
suppress its intra-S-phase checkpoint deficiency. There is a defect in both the intra-Sphase<br />
and DNA replication checkpoints due to the npkA inactivation when DNA<br />
replication is slowed at 6 mM HU. Our results suggest that npkA gene plays a role in cell<br />
cycle progression during S phase as well as in a DNA-damage signal transduction<br />
pathway in A. nidulans.<br />
Financial Support: FAPESP e CNPq<br />
Literature<br />
Brown, E. J., and D. Baltimore, 2003 Genes Dev. 17: 615-628.<br />
Kafer, E., 1977 Adv. Genet. 19: 33-131.<br />
Ko, T.K., E. Kelly and J. Pines, 2001 J. Cell Sci. 114: 2951-2603.<br />
Kress Fagundes, M.Z., et al., 2003 Genetics 164: 935-945<br />
Osmani, S. A., and X. S. Ye, 1996 Biochem. J. 317: 633-641.<br />
contact:<br />
PhD student Marcia von Zeska Kress Fagundes<br />
University <strong>of</strong> São Paulo<br />
Faculdade de Ciências Farmacêuticas de Ribeirão Preto<br />
kress@fcfrp.usp.br<br />
Café s/n<br />
14040-903 Ribeirão Preto - SP (Brazil)
Tamas Kiss<br />
Nuclear organization <strong>of</strong> human modification guide RNA<br />
machinery<br />
Eukaryotic stable cellular RNAs, such as rRNAs, snRNAs and snoRNAs, carry a large<br />
number <strong>of</strong> 2’-O-methylated nucleotides and pseudouridines. These modified nucleotides<br />
contribute to the correct function <strong>of</strong> the ribosome and spliceosome. The posttranscriptional<br />
synthesis <strong>of</strong> most 2’-O-methylated nucleotides and pseudouridines occurs<br />
in the nucleus shortly after synthesis <strong>of</strong> the substrate RNA and it is directed by sequencespecific<br />
modification guide RNAs. While the box C/D guide RNAs direct 2’-O-methylation,<br />
the pseudouridylation guide RNAs share the conserved H and ACA box elements. Both<br />
classes <strong>of</strong> modification guide RNAs select the target residues by forming direct basepairing<br />
interactions with the appropriate target sequences. To avoid undesirable RNA<br />
modification, the modification machinery is strictly organized in the nucleus. Guide RNAs<br />
involved in rRNA modification are sequestered into the nucleolus and guides directing<br />
modification <strong>of</strong> spliceosomal snRNAs localize to Cajal bodies. Accumulation <strong>of</strong><br />
modification guide RNAs in Cajal bodies (scaRNAs) is directed by a cis-acting targeting<br />
signal, called the CAB box. The molecular machinery responsible for intranuclear<br />
localization <strong>of</strong> scaRNAs is also utilized by the human telomerase RNA that contains a CAB<br />
box element and accumulates in the Cajal body.<br />
contact:<br />
Dr Tamas Kiss<br />
Universite Paul Sabatier<br />
Laboratoire de Biologie Moleculaire Eucaryote du CNRS<br />
tamas@ibcg.biotoul.fr<br />
118 route de Narbonne<br />
31062 Toulouse (France)
Marian Farkasovsky, Peter Herter, Beate Voß, Alfred Wittingh<strong>of</strong>er<br />
Nucleotide binding and filament assembly <strong>of</strong> recombinant<br />
yeast septin complexes.<br />
Septins are filament-forming GTPases involved in cytokinesis and cortical organization.<br />
In yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and<br />
CDC12 are localized at the cytoplasmatic face <strong>of</strong> the plasma membrane in the mother<br />
bud neck. High molecular weight septin complex forms a scaffold for the localized<br />
assembly <strong>of</strong> various other proteins and serves as diffusion barrier for membrane and<br />
membrane-associated proteins. While the septin function at the cellular level is good<br />
understood, the molecular function <strong>of</strong> these proteins is still in its infancy. Progress on<br />
septin biochemistry and structural biology has been limited by the lack <strong>of</strong> the larger<br />
amount <strong>of</strong> pure complex. We show here that stable complexes <strong>of</strong> two, three or four<br />
septins can be produced by co-expession from bicistronic vectors in E. coli. These<br />
complexes show differences in saturation with guanine nucleotides. Only the fourcomponent<br />
septin complex is fully coordinated with GTP/GDP.<br />
Heterotetramer consisting <strong>of</strong> two essential yeast septin (Cdc3p and Cdc12p) expressed<br />
and isolated from E.coli was nucleotide free, but could bind guanosine triphosphate and<br />
diphosphate analogs. Bacterially expressed three-septin heterohexamer (Cdc3-Cdc10-<br />
Cdc12) or heteroheptamer (Cdc3-Cdc11-Cdc12) complexes contained approximately one<br />
nucleotide per complex and could bind further three (Cdc3-Cdc10-Cdc12) or two<br />
nucleotides (Cdc3-Cdc11-Cdc12). We used negative stain EM and immun<strong>of</strong>luorescence<br />
microscopy to visualize recombinant septin complex at different salt concentration. At<br />
low salt condition, trimeric and quarternary complexes form a bundles <strong>of</strong> paired<br />
filaments in a GTP-depending manner.<br />
contact:<br />
Dr. Marian Farkasovsky<br />
MPI fuer molekulare Physiologie<br />
marian.farkasovsky@mpi-dortmund.mpg.de<br />
Otto-Hahn-Str. 11<br />
44227 Dortmund (Deutschland)
S. Wings, C. Pullen, T. Boettcher, E. Leistner<br />
Observations on the erratic occurrence <strong>of</strong> maytansinoids in<br />
plants and microorganisms<br />
Maytansine is a maytansinoid ansa antibiotic with a strong antineoplastic activity (1) .<br />
Maytansinoids are constituents <strong>of</strong> taxonomically unrelated organisms such as a gram<br />
positive bacterium (Actinosynnema pretiosum; Streptomycetaceae) and higher plants<br />
(Putterlickia verrucosa, Maytenus serrata; Celastraceae).<br />
We have investigated the occurrence <strong>of</strong> maytansinoids in individual Putterlickia plants in<br />
their natural habitats in South Africa and found that some plants contain maytansinoids<br />
but that maytansinoids were not detectable in all plants (2) . This led to the assumption<br />
that plants are colonized by a microorganism responsible for the accumulation <strong>of</strong><br />
maytansinoids in plants.<br />
Indeed, we discovered a hitherto unknown gram positive bacterium in the rhizosphere <strong>of</strong><br />
Putterlickia plants which was newly described, characterized and named Kitasatospora<br />
putterlickiae (3) . This bacterium harbours genes (4) which are required for maytansinoid<br />
biosynthesis. Thus, the erratic occurrence <strong>of</strong> maytansinoids in nature very likely can be<br />
explained by the ability <strong>of</strong> Putterlickia verrucosa plants to harbour a maytansinoid<br />
producing microorganism.<br />
Literature<br />
(1) Cassady, Chem Pharm Bull.(Tokyo).2004 Jan; 52(1):1-26<br />
(2) Pullen, Phytochemistry. 2003 Feb; 6(3): 377-87<br />
(3) Groth, Int J Syst Evol Microbiol. 2003 Nov;53(Pt 6):2033-40.<br />
(4) Tin-Wein, Proc Natl Acad Sci USA. Jun 11; 99(12): 7968-73<br />
contact:<br />
Susanne Wings<br />
Rheinische Friedrich-Wilhelms-Universität Bonn<br />
Institut für Pharmazeutische Biologie<br />
S.Wings@uni-bonn.de<br />
Nußallee 6<br />
53115 Bonn (Deutschland)
Frank Möhrlen, Sebastian Gornik, Melanie Mertens, Ina Rattmann, Irene Yiallouros<br />
Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong><br />
metalloproteinases<br />
The astacin family belongs to the metzincin superfamily and comprises extracellular<br />
proteases found in vertebrates, invertebrates and prokaryotes. They occur as membrane<br />
bound and secreted enzymes and participate in processes like digestion (crayfish<br />
astacin), hatching (e.g. Xenopus hatching enzyme) or embryonic development and<br />
tissue differentiation (e.g. tolloid / Bmp1-like proteases).<br />
Screening human and mouse genomic databases we identified a new member <strong>of</strong> this<br />
enzyme family, characterized by the conserved motifs <strong>of</strong> the zinc binding region<br />
HExxHxxGxxHE and the Met-turn (SVMHY). The catalytic domain, following the potential<br />
signal sequence and the prodomain, shows high sequence similarity to the astacin<br />
hatching enzymes. The C-terminal domain shows no sequence similarity to known<br />
pattern.<br />
We performed in situ hybridization to analyse the expression pattern <strong>of</strong> the enzyme in<br />
different tissues <strong>of</strong> mouse organs. Signals were only detected in the ovary, i.e. in the<br />
oocytes and in the follicle cells. In the meantime, this new member was termed<br />
ovastacin by Quesada et al. (2004) JBC, 279, 26627-26634.<br />
To characterize ovastacin in respect to structure and function, we cloned the mouse full<br />
length cDNA sequence with either a C-terminal His-tag or a Strep-tag, starting from an<br />
EST-clone, and expressed the protein in Hi five insect cells using the Bac-to-Bac<br />
expression systems.<br />
contact:<br />
Dr Irene Yiallouros<br />
University <strong>of</strong> Muenster<br />
Institute <strong>of</strong> Zoophysiology<br />
yiallou@uni-muenster.de<br />
Hindenburgplatz 55<br />
48143 Muenster (Germany)<br />
additional information<br />
F.M. and S.G. Institute <strong>of</strong> Zoology, University <strong>of</strong> Heidelberg, Germany
Ulrich Sentner, Sebastian Fettig<br />
P1/HcPro: viral suppressors <strong>of</strong> gene silencing in wheat<br />
Often expression <strong>of</strong> transgenes in wheat is weak and unstable. This is attributed to the<br />
capability <strong>of</strong> wheat to switch <strong>of</strong>f expression <strong>of</strong> introduced genes by the mechanism <strong>of</strong><br />
RNA silencing. It was investigated whether transformation efficiency and/or expression<br />
<strong>of</strong> the reporter gene gfp (green fluorescent protein) could be enhanced by the action <strong>of</strong><br />
viral proteins.<br />
The proteins P1/HcPro (protein1/helper component proteinase) <strong>of</strong> wheat streak mosaic<br />
virus are possible suppressors <strong>of</strong> RNA silencing. The cDNA sequence was subcloned in a<br />
plant expression vector and constitutively expressed under control <strong>of</strong> the ubiquitin<br />
promoter from maize. To investigate whether P1/HcPro make an impact on silencing in<br />
wheat the dying out <strong>of</strong> gfp-expression was measured after cotransformation <strong>of</strong> immature<br />
wheat embryos with gfp and viral suppressors. In presence <strong>of</strong> P1/HcPro a prolonged<br />
transient expression <strong>of</strong> gfp was observed with high significancy.<br />
The production <strong>of</strong> a stable P1/HcPro-transgenic wheat plant by particle bombardment<br />
has not been possible up to now. One reason might be that the ubiquitous expression <strong>of</strong><br />
P1/HcPro could lead to detrimental effects that might be so severe in wheat that<br />
regeneration <strong>of</strong> transgenic plants is hindered totally or at least to a high degree.<br />
contact:<br />
Ulrich Sentner<br />
University <strong>of</strong> Bayreuth<br />
Institute <strong>of</strong> Plant Physiology<br />
ulrich.sentner@uni-bayreuth.de<br />
Universitätsstr. 30<br />
95447 Bayreuth (Germany)
Ester Martín-Villar, Maria Marta Yurrita, Carlos Gamallo, Miguel Quintanilla<br />
PA2.26 antigen (T1alpha, podoplanin), a membrane mucin<br />
associated with cancer which is involved in epithelialmesenchymal<br />
transitions<br />
PA2.26 is a membrane mucin-like glycoprotein originally identified as a cell-surface<br />
antigen induced in keratinocytes during mouse skin carcinogenesis and wound healing<br />
(1). Sequence analysis <strong>of</strong> the mouse and human cDNAs revealed that PA2.26 antigen is<br />
identical to T1alpha, a protein expressed in alveolar type I epithelial cells, and<br />
podoplanin, a specific marker for the lymphatic endothelia. In cultured cells, PA2.26 is<br />
concentrated at cell-surface extensions (microvilli, filopodia, lamellipodia and ruffles),<br />
where it interacts with F-actin through the association with ezrin and moesin (2),<br />
members <strong>of</strong> the ERM (ezrin, radixin, moesin) protein family that regulate cell adhesion<br />
and motility. We have recently found that PA2.26 is induced in a subset <strong>of</strong> human early<br />
oral squamous cell carcinomas (SCCs). Expression <strong>of</strong> PA2.26 in the tumours is<br />
heterogenous, <strong>of</strong>ten restricted to the invasive front, and associated with downregulation<br />
<strong>of</strong> membrane E-cadherin expression (3). Ectopic expression <strong>of</strong> PA2.26 in cultured human<br />
and mouse keratinocytes induces a partial or full epithelial-mesenchymal transition<br />
(EMT), associated with loss <strong>of</strong> cell-cell cohesion and stimulation <strong>of</strong> migration and<br />
invasiveness (3,4), depending on the particular stage <strong>of</strong> neoplastic progression. These<br />
results suggest that PA2.26 cooperates with other genetic or epigenetic events to<br />
promote malignant progression <strong>of</strong> SCCs. The mechanism for PA2.26-induced EMTs<br />
appears to involve a redistribution <strong>of</strong> ezrin to the cell edges, a reorganization <strong>of</strong> the actin<br />
cytoskeleton, and the induction <strong>of</strong> cell-surface extensions, indicating that linkage <strong>of</strong> the<br />
PA2.26 cytoplasmic domain with the actin cytoskeleton is a crucial event for the function<br />
<strong>of</strong> this glycoprotein in cell migration.<br />
Literature<br />
1. Gandarillas A et al. Mol. Carcinog. 20: 10-18, 1997.<br />
2. Scholl FG et al. J. Cell Sci. 112: 4601-4613, 1999.<br />
3. Martín-Villar et al. Submitted.<br />
4. Scholl FG et al. Lab. Invest. 80: 1749-1759, 2000.<br />
contact:<br />
Ph. D. Miguel Quintanilla<br />
Spanish Research Council (CSIC)<br />
Instituto de Investigaciones Biomédicas<br />
mquintanilla@iib.uam.es<br />
Arturo Duperier 4<br />
28029 Madrid (Spain)
Tina Jordan, Annett Meyer, Kristin Peter, Patrick Römer, Sebastian Schornack, Ulla<br />
Bonas, Thomas Lahaye<br />
Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Elucidation <strong>of</strong> the resistance (R) gene-mediated plant defense has been the "Holy Grail"<br />
<strong>of</strong> plant pathologists in the last decade 1,2 . Comparative characterization <strong>of</strong> the pepper<br />
Bs3 and tomato Bs4 R genes promises insights into the principles <strong>of</strong> recognition<br />
specificity since Bs3 and Bs4 perceive specifically the almost identical Xanthomonas type<br />
III effector proteins AvrBs3 and AvrBs4, respectively 3 . Genetic and biochemical studies<br />
have shown that nuclear localization signals (NLSs) in AvrBs3 and AvrBs4 mediate<br />
nuclear targeting <strong>of</strong> both Xanthomonas proteins 4 . Mutations in the NLS motives <strong>of</strong><br />
AvrBs3 and AvrBs4 abolished Bs3- but not Bs4-mediated recognition 3,5 , indicating that<br />
Bs3 and Bs4 act in the nucleus and cytoplasm, respectively 6 . Isolation <strong>of</strong> tomato Bs4<br />
revealed that it is a novel member <strong>of</strong> the TIR-NBS-LRR class <strong>of</strong> resistance proteins 7<br />
being most homologous to the tobacco N gene which mediates resistance against<br />
tobacco mosaic virus. We generated GFP- and MYC-tagged Bs4-derivatives in order to<br />
localize Bs4 at the subcellular level. In accordance with the analysis <strong>of</strong> AvrBs4 NLSdeletion<br />
derivatives, confocal microscopy and fractionation studies localized Bs4 in the<br />
cytoplasm. Analysis <strong>of</strong> several other gene-for-gene interactions indicates that R-protein<br />
localization is generally dictated by the Avr protein destination 1,8,9 . However, given that<br />
Bs4 and AvrBs4 are located in the cytoplasm and nucleus respectively, the Bs4-avrBs4<br />
gene-for-gene interaction might be the first documented example in which cognate R<br />
and Avr proteins are not targeted to identical cellular compartments.<br />
Literature<br />
1) Bonas, U. and Lahaye, T. (2002) Curr Opin Microbiol 5 (1), 44-50<br />
2) Jones, D. and Takemoto, D. (2004) Curr Opin Immunol 16 (1), 48-62<br />
3) Ballvora, A. et al. (2001) Mol Plant-Microbe Interact 14 (5), 629–638<br />
4) Szurek, B. et al. (2002) Mol Microbiol 46 (1), 13–23<br />
5) Van den Ackerveken, G. et al. (1996) Cell 87 (7), 1307-1316<br />
6) Lahaye, T. and Bonas, U. (2001)Trends Plant Sci 6 (10), 479-485<br />
7)Schornack, S. et al. (2004) Plant J 37 (1), 46-60<br />
8) Martin, G.B. et al. (2003) Annu Rev Plant Biol 54 (1), 23-61<br />
9) Nimchuk, Z. et al. (2003) Annu Rev Genet 37 (1), 579-609<br />
contact:<br />
Dr Thomas Lahaye<br />
Martin Luther Universität<br />
Institut für Genetik<br />
lahaye@genetik.uni-halle.de<br />
Weinbergweg 10<br />
06120 Halle (Saale) (Germany)<br />
additional information<br />
http://www.biologie.uni-halle.de/genet/plant/index.html
Josef H. Wissler<br />
Physiologic Roles for Receptors for Advanced Glycosylation<br />
End Products [RAGE] <strong>of</strong> Endothelial Cells: RNA-, Redox- and<br />
Metalloregulated Non-Mitogenic Angio-Morphogenesis in<br />
Health and Disease.<br />
RAGE are central surface receptors on endothelial and other cells and are members <strong>of</strong><br />
scavenger receptor and immunoglobulin superfamilies. Extracellular ligands for RAGE are<br />
misfolded, amyloidogenic, glycosyl- and otherwise modified proteins in competition with<br />
so-called EN-RAGE [Extracellular Newly identified RAGE-binding] S100A12-EF-hand and<br />
amphoterin proteins. Thus, RAGE got known to be causually involved in various<br />
pathological mechanisms and chronic diseases. Since any physiological role for RAGE<br />
escaped consideration, so far, should RAGE have been evolved as a molecular guide to<br />
diseases?<br />
Here is shown that RAGE have physiological functions in non-mitogenic angiomorphogenesis.<br />
It is basic to all processes in health and disease since being a key<br />
reaction in development and maintenance <strong>of</strong> blood capillary networks. This was<br />
investigated by angiotropins [AT]. The bioactive metalloregulated RNA-containing AT<br />
cytokines [ribokines] are described elsewhere. In short, AT are leukocytic extracellular<br />
copper-ribonucleoprotein [CuRNP] angio-morphogens for endothelial cell vascularization.<br />
They consist <strong>of</strong> metalloregulated AT-related protein [ARP] and small extracellular eRNA<br />
bioaptamers [ARNA] complexed together to CuRNP by copper ions. ARP is a RAGE ligand<br />
in competition with other metabolic products and redox reactions, e.g. those emerging<br />
from inositol epimerase/ D-xylose:NADP dehydrogenase [E.C.1.1.1.179] at high<br />
glucose/galactose levels: The sequence <strong>of</strong> ARP is homologous to RAGE-binding S100A12-<br />
EF-hand proteins [calgranulins]. ARNA are sequence-defined Fenton-type OH* radical<br />
redox reaction-regulated, highly modified and edited 5'-end-phosphorylated eRNA [72-<br />
78n] with intrinsic angio-morphogen activity which, in contrast, ARP has not per se.<br />
RAGE has now found to have nucleic acid-binding domains which are suggested as<br />
targets for the RNA moiety <strong>of</strong> CuRNP. The domains are considered as lid <strong>of</strong> RNA<br />
chaperone-shaped assemblies <strong>of</strong> metalloregulated ARP hexamers. These are formed<br />
upon Ca/Cu ion-mediated multivalent binding reactions <strong>of</strong> the ARP moiety <strong>of</strong> CuRNP to<br />
RAGE by which downstream regulation <strong>of</strong> nuclear factor NF-(kappa)B-dependent<br />
transduction pathways in transcription mechanisms are mediated by RAGE. From the<br />
extracellular CuRNP, the bioactive eRNA is presented to intracellular RNA-binding RAGE<br />
domains as reformed ssRNA. From there, in balance with RAGE, hyoxia/ radical, glucose/<br />
galactose/ xylose/ inositol and Cu/ Ca ion-mediated NF-(kappa)B and metalloregulated<br />
redox pathways, it may display non-mitogenic angio-morphogen activity via ribosomal<br />
proteins in translation mechanisms. The evaluated balanced RAGE-mediated multiple<br />
pathways suggest diagnostic and therapeutic potentials in health care and disease<br />
management.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Josef H. Wissler<br />
ARCONS Applied Research & Didactics<br />
jhw@arcons-research.de<br />
Postfach 1327<br />
D-61231 Bad Nauheim (Germany)
Manfred Schliwa<br />
Portrait <strong>of</strong> a molecular motor<br />
No abstract submitted<br />
contact:<br />
Pr<strong>of</strong>. Dr. Manfred Schliwa<br />
Universität München<br />
Inst. für Zelbiologie<br />
schliwa@bio.med.uni-muenchen.de<br />
Schillerstr. 42<br />
80036 München (Deutschland)
Ralph Goethe, Katrin Hübner, Loc Phi-van<br />
Possible involvement <strong>of</strong> Ca2+/calmodulin dependent protein<br />
kinase II (CaMKII) in lysozyme pre-mRNA splicing<br />
In contrast to most RNA transcripts in eukaryotic cells, the lysozyme primary transcript<br />
is processed very slowly, particularly in LPS-activated myelomonocytic HD11 cells (1). In<br />
this study, we investigated the influence <strong>of</strong> protein kinase inhibitors on the lysozyme premRNA<br />
splicing. Northern blot analysis shows that the splicing <strong>of</strong> lysozyme pre-mRNA in<br />
LPS-activated cells is significantly delayed by treatment with KN-93 or KN-62, two<br />
selective inhibitors <strong>of</strong> Ca2+/calmodulin-dependent protein kinase II (CaMKII), but not<br />
with Gö 6976 and herbimycin A, inhibitors <strong>of</strong> calcium-dependent protein kinase C (PKC)<br />
and protein tyrosine kinase (PTK), respectively. In vitro kinase assay using autocamtide<br />
2 as specific substrate for CaMKII demonstrates that KN-62, when added to the extract<br />
from HD11 cells, inhibits selectively CaMKII activity. Treatment <strong>of</strong> HD11 cells with<br />
cycloheximide, a potent inhibitor <strong>of</strong> protein synthesis, also results in transient increase in<br />
lysozyme pre-mRNA levels. Moreover, neither cycloheximide nor KN-62 has any effect<br />
on the glyceraldehyde-3-phophate dehydrogenase pre-mRNA splicing. Together, our<br />
results indicate that phosphorylation by CaMKII, and probably new protein synthesis<br />
may be required for the lysozyme pre-mRNA processing.<br />
Literature<br />
(1) Goethe, R., and Phi-van, L. (1998) J. Immunol. 160, 4970-4978<br />
contact:<br />
Dr. Loc Phi-van<br />
Federal Agricultural Research Center<br />
Institute for Animal Welfare & Husbandry<br />
Loc.phi-van@fal.de<br />
Dörnbergstr. 25-27<br />
29223 Celle (Germany)
Christina Rommel, Jan Endell, Ralf Wehrspohn, Petra Göring, Claudia Steinem,<br />
Andreas Jansh<strong>of</strong>f, Hans-Joachim Galla, Joachim Wegener<br />
Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Measuring substrate permeation across epithelial cell layers is important for both,<br />
fundamental research addressing the molecular mechanisms <strong>of</strong> epithelial barrier function<br />
as well as for applied studies <strong>of</strong> drug absorption and drug targeting. In current in vitro<br />
approaches cell layers are grown to confluence on permeable filter membranes, the<br />
compound <strong>of</strong> interest is applied to one side <strong>of</strong> the cell layer and its concentration in the<br />
opposite compartment is determined as a function <strong>of</strong> time. Calculating the area specific<br />
transfer rate provides an integral measure for the permeation characteristics <strong>of</strong> the<br />
compound. However, this approach is integral by nature and does neither report on<br />
possible “hot spots” <strong>of</strong> the cell layer’s permeability nor on the permeation pathway:<br />
transcellular or paracellular. By using nano-porous silicon chips as growth substrates for<br />
epithelial cell layers we have developed a microscopic assay to determine transepithelial<br />
substrate permeation with lateral resolution. The pores in the substrate (d = 900 nm)<br />
serve as a lateral array <strong>of</strong> cuvettes in which the substrate is collected after passage<br />
across the cell layer. The size <strong>of</strong> the pores is small enough to distinguish paracellular<br />
from transcellular permeation.<br />
contact:<br />
Christina Rommel<br />
Universität Münster<br />
Institut für Biochemie<br />
rommelc@uni-muenster.de<br />
Wilhelm-Klemm Str. 2<br />
48149 Münster (Deutschland)
Carsten Wermter, Markus Höwel, Vera Hintze, Irene Yiallouros, Walter Stöcker<br />
Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition<br />
and Specificity<br />
The procollagen C-proteinase (PCP; i.e. BMP1, bone morphogenetic protein 1) is a<br />
secreted, glycosylated zinc metalloendoproteinase <strong>of</strong> the astacin family and the<br />
metzincin superfamily. It converts fibrillar procollagens to mature collagens and<br />
regulates the rigidity <strong>of</strong> collagen fibers by activating the prolysyl oxidase. In addition,<br />
the PCP processes other matrix proteins like laminin 5 or probiglycan. During<br />
embryogenesis, PCP is needed for dorsal-ventral patterning.<br />
The PCP consists <strong>of</strong> a pro-domain, an astacin-like catalytic domain (containing the zinc<br />
binding motif HExxHxxGxxHE), CUB-domains (Complement factor C1r/C1s, sea urchin<br />
protein Uegf and BMP1), and EGF-like domains. The overall domain composition is Pro-<br />
Catalytic-CUB1-CUB2-EGF-CUB3-C-tail.<br />
To study the substrate specificity <strong>of</strong> the PCP we have produced the full length PCP in<br />
insect and mammalian cells. Additionally, we have expressed the catalytic domain alone<br />
as a recombinant protein in E. coli .<br />
We could show that the cleavage pattern <strong>of</strong> the astacin-like catalytic domain differs from<br />
that <strong>of</strong> the full length PCP using gelatine or a truncated construct <strong>of</strong> procollagen VII. And<br />
the catalytic domain alone shows a broader specificity if other ECM proteins (e.g.<br />
collagen IV or fibronectin) are used as substrates. These results implicate that the<br />
substrate specificity <strong>of</strong> the full length enzyme is determined by the CUB/EGF domains.<br />
Furthermore, the processing <strong>of</strong> thrombospondin-1 by the full length PCP could be shown<br />
for the first time.<br />
Supported by the Deutsche Forschungsgemeinschaft (SFB492 A5).<br />
contact:<br />
Dipl. Biol. Carsten Wermter<br />
WWU Münster<br />
Institute <strong>of</strong> Zoophysiology<br />
wermtec@uni-muenster.de<br />
Hindenburgplatz 55<br />
48143 Münster (Germany)<br />
additional information<br />
new address: Institute <strong>of</strong> Zoology, Department I, Johannes Gutenberg University, Müllerweg 6, D-<br />
55099 Mainz; stoecker@uni-mainz.de
Vera Hintze, Markus Höwel, Carsten Wermter, Irene Yiallouros, Walter Stöcker<br />
Procollagen C-proteinase: Substrate recognition by C-terminal<br />
domains<br />
The procollagen C-proteinase (PCP) is a member <strong>of</strong> the astacin protein family and the<br />
superfamily <strong>of</strong> zinc peptidases. The enzyme plays a key role in the metabolism <strong>of</strong> the<br />
extracellular matrix, since it removes the C-terminal propeptides <strong>of</strong> procollagens, and<br />
activates a series <strong>of</strong> other matrix proteins. Beside its catalytic astacin-protease domain<br />
the PCP contains several C-terminal CUB-modules (complement factors C1s and C1r, in<br />
the sea urchin Uegf protein and in BMP1), interrupted by EGF-like domains. The two<br />
major splice forms PCP1/BMP1 and PCP2/mTld have the overall domain composition<br />
PROTEASE-CUB1-CUB2-EGF-like I-CUB3 and PROTEASE-CUB1-CUB2-EGF-like I-CUB3-<br />
EGF-like II-CUB4-CUB5, respectively. CUB-EGF-like domains are thought to mediate<br />
protein-protein interactions.<br />
To investigate the interaction properties and the structure <strong>of</strong> PCP-subdomains we<br />
produced several modules like CUB1, EGF-like I/CUB3, CUB 3/EGF-like II, EGF-like<br />
II/CUB 4, and the CUB4/CUB5 domains as recombinant proteins in E. coli. The human<br />
PCP cDNA was used as a template for PCR-amplification. Products were successfully<br />
cloned into the pET3a-vector and overexpressed in E. coli . The solubilized inclusion body<br />
proteins were purified by Ni-NTA-affinity chromatography under denaturing and reducing<br />
conditions. The domains were successfully renatured via dialysis against urea-free<br />
buffer. The CD spectra confirmed the predicted antiparallel b-sheet structure <strong>of</strong> these<br />
domains. The functionality <strong>of</strong> refolded EGF-like I/CUB3 and CUB3/EGF-like II constructs<br />
could be shown by surface plasmon resonance (BiaCore) via a concentration-dependent<br />
binding to procollagen I and collagen I.<br />
Supported by the Deutsche Forschungsgemeinschaft (SFB 492 A5).<br />
contact:<br />
Dipl. Biol. Vera Hintze<br />
University <strong>of</strong> Münster<br />
Institute <strong>of</strong> Zoophysiology<br />
hintzev@uni-muenster.de<br />
Hindenburgplatz 55<br />
48143 Münster (Germany)<br />
additional information<br />
new address: Institute <strong>of</strong> Zoology, Department I, Johannes Gutenberg University, Müllerweg 6, D-<br />
55099 Mainz, Germany, stoecker@uni-mainz.de
Anna Kantola, Jorma Keski-Oja, Katri Koli<br />
Promoter characterization <strong>of</strong> the latent transforming growth<br />
factor (TGF)-β binding protein 3 (LTBP-3)<br />
Transforming growth factor-βs (TGF-βs) are important regulators <strong>of</strong> cell growth and<br />
differentiation. They are secreted as latent inactive complexes containing the 25 kDa<br />
mature TGF-β, its propeptide and a latent TGF-β-binding protein (LTBP). LTBPs are<br />
important for the folding, secretion and activation <strong>of</strong> TGF-β. In addition they target the<br />
TGF- β complex to the extracellular matrix. LTBP-3 is expressed mainly in hearth,<br />
skeletal muscle, prostate, ovaries and in certain osteosarcoma and fibroblastic cell lines<br />
(Penttinen et al., 2002). Severe bone abnormalities in LTBP-3 knock-out mice suggest a<br />
central role for LTBP-3 in the regulation <strong>of</strong> the bioavailability <strong>of</strong> TGF-β in bone (Dabovic<br />
et al., 2002). To understand the regulation <strong>of</strong> LTBP-3 expression we have isolated a 3 kb<br />
fragment <strong>of</strong> the 5´-flanking sequence <strong>of</strong> the LTBP-3 gene and studied the transcriptional<br />
activity in MG-63 and SaOs-2 osteosarcoma cells. Subsequently several promoter<br />
constructs <strong>of</strong> different lengths were produced. The basal promoter activities showed a<br />
putative negative regulatory element in the region between –2149 and -3014 upstream<br />
from the translation initiation site. We analysed the effects <strong>of</strong> bone derived growth<br />
factors on transcriptional activity and noticed that TGF-β-1 stimulated LTBP-3 promoter<br />
activity 5-fold. In order to find the TGF-β responsive region we analysed the<br />
transcriptional activity <strong>of</strong> the deletion constructs in TGF-β treated and control cells. We<br />
found a putative TGF-β responsive element located at -2738. This region contains a<br />
Smad binding element. Our findings suggest that TGF-β is an important regulator <strong>of</strong> its<br />
binding protein, LTBP-3, in bone derived cells.<br />
Literature<br />
Dabovic B., Chen Y., Colarossi C., Zambuto L., Obata H. and Rifkin D.B. (2002).Bone<br />
defects in latent TGF-β binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-β<br />
presentation. Journal <strong>of</strong> Endocrinology, 175, 129-141<br />
Penttinen, C., Saharinen J., Weikkolainen, K., Hyytiäinen, M. and Keski-Oja J. (2002).<br />
Secretion <strong>of</strong> human latent TGF-β binding protein-3 (LTBP-3) is dependent on coexpression<br />
<strong>of</strong> TGF-β. Journal <strong>of</strong> Cell Science, 115, 3457-3468<br />
contact:<br />
PhD student Anna Kantola<br />
University <strong>of</strong> Helsinki<br />
Departments <strong>of</strong> Virology and Pathology, Haartman Institute<br />
anna.k.kantola@helsinki.fi<br />
Haartmaninkatu 8<br />
00014 Helsingin yliopisto (Finland)
Holger Seelert, Ansgar Poetsch, Sascha Rexroth, Norbert A. Dencher, Daniel J. Müller,<br />
Jinshu Yu, Werner Kühlbrandt, Janet Vonck<br />
Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
The ATP synthase, the enzyme responsible for energy conversion in all living organisms,<br />
includes the two smallest rotary motors in nature. The membrane integral rotor <strong>of</strong> the<br />
ATP synthase, which couples the proton translocation to a rotational motion, is an<br />
oligomer <strong>of</strong> identical subunits, called III in chloroplasts and c in bacteria and<br />
mitochondria. The number <strong>of</strong> subunits in the oligomer determines the H + /ATP ratio and<br />
therefore the effiency <strong>of</strong> energy conversion. To exclude the possibility that a previously<br />
found stoichiometry <strong>of</strong> 14 subunits III in the chloroplast oligomer (1) could be influenced<br />
by the isolation procedure, we reconstituted three different ATP synthase subcomplexes<br />
into lipid bilayers. By imgaging the topography <strong>of</strong> the 2D crystals obtained with atomic<br />
force microscopy the 14-fold symmetry was confirmed (2). Additionally, cryoelectron<br />
microscopy resolved the 28 α-helices in this proton turbine at 7Å resolution.<br />
The predominantly α-helical secondary structure was confirmed by spectroscopy (CD and<br />
FTIR) (3). With spectroscopy, a conformational change was observed upon diluting SDSsolubilized<br />
monomer III. In contrast, the oligomer did not show such changes.<br />
Literature<br />
(1) Seelert, H., Poetsch, A., Dencher, N.A., Engel, A., Stahlberg, H. and Müller, D.J.,<br />
Nature 405, 418-419 (2000).<br />
(2) Seelert, H., Dencher, N. A. and Müller, D. J., Journal <strong>of</strong> Molecular Biology 333,<br />
337–344 (2003)<br />
(3) Poetsch, A., Rexroth, S., Heberle, J., Link, T. A., Dencher, N. A. and Seelert, H.,<br />
Biochimica et Biophysica Acta 1618, 59– 66 (2003)<br />
contact:<br />
Dr. Holger Seelert<br />
Technische Universität Darmstadt<br />
Physikalische Biochemie<br />
seelert@hrzpub.tu-darmstadt.de<br />
Petersenstr. 22<br />
64287 Darmstadt (Deutschland)<br />
additional information<br />
A. Poetsch, Ruhr-University Bochum, Biochemistry <strong>of</strong> Plants<br />
J. Yu, D. J. Müller, MPI <strong>of</strong> Molecular Cell Biology and Genetics, Dresden<br />
W. Kühlbrandt, J. Vonck, MPI <strong>of</strong> Biophysics, Frankfurt
Christian Ungermann<br />
Protein palmitoylation during membrane fusion<br />
Intracellular transport <strong>of</strong> secreted proteins depends on vesicles that bud from a donor<br />
compartment and fuse with an acceptor organelle. Fusion <strong>of</strong> vesicles requires a<br />
coordinated cascade that depends on a conserved set <strong>of</strong> proteins. One class are the<br />
SNARE proteins found on vesicles and organelles. Most SNAREs contain a<br />
transmembrane anchor. Defined complexes between SNAREs from both vesicle and<br />
organelle form at docking and lead to fusion. Several essential fusion factors do not<br />
contain a transmembrane domain, but are bound to membranes by lipid anchors.<br />
Palmitoylation is the only reversible lipid modification, thus providing a possibility for a<br />
regulated cycle <strong>of</strong> protein binding (and palmitoylation) or protein release (via<br />
depalmitoylation) during the fusion reaction. We use the yeast vacuole as a model<br />
system to analyze factors that are required for membrane fusion and are subject to<br />
palmitoylation. During our studies we identified the Vac8 fusion factor as a palmitoylated<br />
protein (Veit et al., 2001; 2003). In addition, we identified Ykt6 as a factor mediating<br />
Vac8 acylation on vacuoles (Dietrich et al., 2004). I will discuss the role <strong>of</strong> both factors<br />
in fusion and the implication for other fusion reactions during my lecture.<br />
Literature<br />
Dietrich, L.E.P., Gurezka, R., Veit, M., and Ungermann, C. (2004) EMBO J. 23, 45-53.<br />
Veit, M., Dietrich, L.E.P., and Ungermann, C. (2003) FEBS letters, 540, 101-105.<br />
Veit, M. , Laage, R., Dietrich, L., Wang, L., and Ungermann, C. (2001) EMBO J. 20, 3145-<br />
3155.<br />
contact:<br />
PD Dr. Christian Ungermann<br />
Universität Heidelberg<br />
Biochemie Zentrum der Universität Heidelberg<br />
cu2@ix.urz.uni-heidelberg.de<br />
Im Neuenheimer Feld 328<br />
69120 Heidelberg (Deutschland)
Anke Rattenholl, Stephan Seeliger, Jörg Buddenkotte, Astrid Grevelhörster, Martin<br />
Steinh<strong>of</strong>f<br />
Proteinase-Activated Receptor-2 (PAR-2): A Tumor<br />
Suppressor in Skin Carcinogenesis<br />
Proteinase activated receptors are G protein coupled receptors that show a unique<br />
activation mechanism which requires N-terminal cleavage by a specific proteinase. The<br />
newly formed N-terminus then interacts with a conserved extracellular loop, leading to<br />
conformational changes and subsequent signal transduction. So far, four members <strong>of</strong><br />
this receptor class are known. PAR-1, -3 and –4 are activated by thrombin whereas PAR-<br />
2 is cleaved by trypsin and mast cell derived tryptase. In the skin, epidermal<br />
keratinocytes as well as several other cell types produce PAR-2. Malignant keratinocytederived<br />
skin tumors (squamous cell carcinoma) show considerably diminished<br />
immunoreactivity for PAR-2. This indicates a protective role <strong>of</strong> this receptor in the<br />
development <strong>of</strong> epidermal skin tumors. To further analyze this, we established a mouse<br />
skin tumor model using chemically induced carcinogenesis. PAR-2-deficient mice showed<br />
a significantly increased number <strong>of</strong> skin tumors (mostly benign papilloma-type tumors)<br />
in contrast to the wild type. Analysis <strong>of</strong> possible signal transduction pathways activated<br />
upon PAR-2 stimulation in HaCaT keratinocytes showed an involvement <strong>of</strong> ERK1/2 and<br />
pr<strong>of</strong>ound EGF receptor transactivation. Interestingly, phosphorylation <strong>of</strong> ERK1/2 did not<br />
lead to keratinocyte proliferation, possibly due to retention <strong>of</strong> ERK1/2 in the cytosol.<br />
Moreover, stimulation <strong>of</strong> PAR-2 in HaCaT keratinocytes led to secretion <strong>of</strong> the tumorsuppressing<br />
factor TGF-β1.<br />
Literature<br />
1) Derian, C. K., Eckardt, A. J., and Andrade-Gordon, P. (1997) Cell Growth Differ.<br />
8:743–749<br />
2) Rattenholl, A., and Steinh<strong>of</strong>f, M. (2003) Drug Dev. Res. 59:408-416<br />
3) Steinh<strong>of</strong>f, M., Buddenkotte, J., Shpacovitch, V., Moormann, C., Rattenholl, A.,<br />
Vergnolle, N., Luger, T. A., and Hollenberg, M. D. (2004) Endocr. Rev., in press<br />
contact:<br />
Dr. Anke Rattenholl<br />
Universität Münster<br />
Universitäts-Hautklinik<br />
rattenho@uni-muenster.de<br />
Von-Esmarch-Str. 58<br />
48149 Münster (NRW/Deutschland)
Steffen Amme, Twan Rutten, Bernhard Schlesier, Michael Melzer, Hans-Peter Mock<br />
Proteome analysis <strong>of</strong> tobacco trichomes<br />
The leaf surface <strong>of</strong> most terrestrial plants is covered with trichomes. Trichomes are<br />
epidermal appendages and comprise a diverse set <strong>of</strong> structures in a multitude <strong>of</strong> forms<br />
and size. In addition intraspezific variation <strong>of</strong> trichome types and density is known in<br />
many species. Functionally, trichomes play a role in the protection against biotic and<br />
abiotic stresses and may also complement the chemical defense <strong>of</strong> a plant by possessing<br />
glands to exude toxic secondary metabolites such as alkaloids. We attempted a<br />
proteome approach to better define the cellular mechanisms operating in plant trichomes<br />
in parallel to metabolic pr<strong>of</strong>iling. Leaves <strong>of</strong> tobacco were chosen as a source <strong>of</strong> trichomes<br />
due to the well-known occurrence <strong>of</strong> several classes <strong>of</strong> secondary compounds playing a<br />
role in plant defence mechanisms against pathogens. Plants were cultivated in the<br />
greenhouse until 12 to 14 leaves had developed. The morphological characterisation <strong>of</strong><br />
trichomes from Nicotiana tabacum was accomplished by scanning electron microscopy<br />
and showed several types as previously described [1,2]: short trichomes with an<br />
unicellular stalk and a multicellular head and tall trichomes with a multicellular stalk<br />
possessing uni- or multicellular heads. Fluorescence microscopy demonstrated that<br />
chlorophyll is absent in short trichomes; however small quantities <strong>of</strong> this pigment are<br />
accumulated in the heads <strong>of</strong> the tall trichomes indicating residual photosynthetic activity.<br />
For biochemical analysis trichomes were mechanically abraded from leaves using glass<br />
beads <strong>of</strong> appropriate size and purified from contaminating leaf tissue by filtration.<br />
Trichomes from young and older leaves were separately harvested to investigate the<br />
influence <strong>of</strong> leaf development on the metabolite and proteome pr<strong>of</strong>ile <strong>of</strong> trichomes. The<br />
purity <strong>of</strong> isolated trichomes was assessed using the amount <strong>of</strong> chlorophyll as a relative<br />
measure for contamination by leaf tissue.<br />
Analysis <strong>of</strong> the alkaloid nicotine demonstrated several-fold increased contents <strong>of</strong> this<br />
toxic compound in trichomes, relative to leaves. HPLC phenylpropanoid pr<strong>of</strong>iling showed<br />
a pronounced increase <strong>of</strong> the main compounds chlorogenic acid, the flavonoid rutin as<br />
well as <strong>of</strong> three other yet unknown compounds. As a global approach for the<br />
characterization <strong>of</strong> plant trichomes we performed a proteomic study on trichomes using<br />
2-D gel electrophoresis to separate proteins <strong>of</strong> leaves and trichomes. For IEF, pHgradients<br />
from 3-10 as well as from 4-7 were used. Comparative image analysis <strong>of</strong> the<br />
protein patterns indicated a number <strong>of</strong> spots which were highly enriched in trichomes<br />
relative to leaves. These proteins represented potential candidates contributing to<br />
trichome-specific functions and were excised for identification by mass spectrometry.<br />
Trichome-specific expression <strong>of</strong> identified proteins is now confirmed by Western and<br />
Northern blotting as well as by measuring enzymic activities where applicable. Future<br />
work will make use <strong>of</strong> transgenic approaches to further investigate the role <strong>of</strong> identified<br />
proteins in the network <strong>of</strong> cellular functions in trichomes with a focus on stress defense.<br />
Literature<br />
[1] Akers CP, Weybrew JA, Long RC (1978) Amer J Bot 65: 282-292<br />
[2] Meyberg M, Krohn S, Brühmer B, Kristen U (1991) Flora 185: 357-363<br />
contact:<br />
Dipl. Biol. Steffen Amme<br />
IPK-Gatersleben<br />
amme@ipk-gatersleben.de<br />
Corrensstrasse 3<br />
06466 Gatersleben (Deutschland)
Roland C. Grafström, Claudia A Staab, Jan Anders Nilsson<br />
Quantitative Genomics <strong>of</strong> Cultured Normal and transformed<br />
Human Oral Keratinocytes<br />
Our laboratory is engaged in the development <strong>of</strong> models for exploration <strong>of</strong> the genetics<br />
underlying malignant transformation. Normal (NOK), SV40 T antigen-immortalized<br />
(SVpgC2a) and malignant (SqCC/Y1) oral keratinocytes were therefore cultured under a<br />
standardized condition in both monolayer and organotypic states. Transcript pr<strong>of</strong>iling by<br />
Affymetrix microarray indicated expression <strong>of</strong> 4-5 x 103 genes in the cell lines. The<br />
changes from in vitro-immortalization (SVpgC2a versus NOK) outnumbered those<br />
associated to malignant transformation (SqCC/Y1 versus NOK). Observed alterations<br />
related to cyto-skeleton, cell adhesion, differentiation and oncogenesis. enzymes and<br />
Enzymes involved in xenobiotic metabolism and detoxification <strong>of</strong> reactive oxygen were<br />
variably detected. Development <strong>of</strong> retinoid insensitivity from malignant transformation<br />
was also implied from the expression analysis. Microarray results were mostly confirmed<br />
by other methods, including for mRNA (Northern, in situ hybridization and standardized<br />
quantitative RT-PCR) and protein (immunochemistry, 2D gel electrophoresis and enzyme<br />
activity). For example, immunochemical pr<strong>of</strong>iling demonstrated that NOK expressed the<br />
typical keratins <strong>of</strong> normal tissue whereas the expression pattern in SVpgC2a and<br />
SqCC/Y1 mimicked severe epithelial dysplasia and well-differentiated squamous cell<br />
carcinoma, respectively. Normal and transformed oral keratinocytes are useful tools for<br />
investigating the genetics <strong>of</strong> malignant transformation.<br />
Literature<br />
Vondracek, M., Weaver, D., Sarang, Z., Hedberg, J.J., Willey, J., Wärngård, L. and<br />
Grafström, R.C. Int. J. Cancer, 99: 776-782, 2002<br />
Dressler, D., Sarang, Z., Vondracek, M., Engelhart, K., Zsondy, Z., and Grafström, R.C.<br />
Int. J. Oncology, 20: 897-903, 2002<br />
Grafström, R.C. In: Culture <strong>of</strong> Epithelial Cells, Second edition, John Wiley and Sons, Inc.,<br />
New York, pp. 195-255, 2002<br />
Hansson, A., Bloor, B., Sarang, Z., Haig, Y., Morgan, P., Stark, H.-J., Fusenig, N. E.,<br />
Ekstrand, J. and Grafström, R.C Eur. J. Oral Sci., 111: 34-41, 2003<br />
Sarang, Zs., Haig, Y., Hansson A., Wärngård, L. and Grafström R.C. ATLA-Altern. Lab.<br />
Anim., 31:575-585, 2003<br />
Nilsson, J.-A., Hedberg, J.J., Vondracek, M., Staab, C.A., Hansson A., Höög, J.-O. and<br />
Grafström, R. C. CMLS, Cell. Mol. Life Sci., 61: 610-617, 2004<br />
Willey, J.C., Crawford, E.L., Warner, K.A., Knight, C., Motten, C., Herness, E.,<br />
Zahorchak, R.J., Graves, T., Harr, M., Weaver, D.A., Khuder, S., Vondracek, M. and<br />
Grafström R.C. In: A-Z <strong>of</strong> quantitative PCR (SA Bustin, Ed.), IUL, La Jolla, Ca, in press<br />
contact:<br />
Pr<strong>of</strong>essor Roland C. Grafström<br />
Institute <strong>of</strong> Environmental Medicine<br />
Karolinska Institute<br />
roland.grafstrom@imm.ki.se<br />
Box 210<br />
SE-17177 Stockholm (Sweden)
Romano Hebeler, P. P. Dijkwel, H. E. Meyer, B. Warscheid<br />
Quantitative proteomics for the investigation <strong>of</strong> leaf<br />
senescence in Arabidopsis thaliana<br />
Arabidopsis thaliana is used as a model organism to study the onset <strong>of</strong> leaf senescence.<br />
Onset <strong>of</strong> leaf death (old) mutants <strong>of</strong> A. thaliana have been studied by senescence<br />
associated gene (SAG) expression, ion leakage, and chlorophyll degradation. [1].<br />
For proteome analysis <strong>of</strong> A. thaliana with normal and altered leaf senescence, plant<br />
tissues were processed under liquid N 2 and in the presence <strong>of</strong> protease inhibitors. After<br />
centrifugation <strong>of</strong> the cell extract, the soluble sample fraction was collected. Membrane<br />
and DNA associated proteins were extracted from the pellet by additional homogenation<br />
and centrifugation [2]. Cytosolic proteins were separated via two-dimensional gel<br />
electrophoresis (2D-PAGE) following the protocol by Klose et al. [3], and subsequently<br />
identified using MALDI-MS combined with searches in the NCBI database. To overcome<br />
limitations regarding differential protein analysis by 2D-PAGE, the wild type and mutants<br />
<strong>of</strong> A. thaliana were labeled in vivo using 14 N/ 15 N- ammonium sulphate. Upon harvest,<br />
plants labeled with ‘light’ and ‘heavy’ isotopes were directly pooled in a 1:1 ratio and<br />
jointly separated by 2D-PAGE. Protein expression levels were determined via relative<br />
signal intensities <strong>of</strong> protonated tryptic peptide pairs detected by nHPLC/ESI-qTOF MS. A<br />
customized in house s<strong>of</strong>tware was employed for advanced quantitative data evaluation.<br />
Literature<br />
[1] Jing, H.C., et al., Plant J, 2002. 32(1): p. 51-63<br />
[2] Giavalisco, P., et al., Electrophoresis, 2003. 24(1-2): p. 207-16<br />
[3] Klose, J. and U. Kobalz, Electrophoresis, 1995. 16(6): p. 1034-59<br />
contact:<br />
Romano Hebeler<br />
Universität Bochum<br />
Medizinisches Proteom-Center<br />
romano.hebeler@ruhr-uni-bochum.de<br />
Universitätsstr. 150<br />
44780 Bochum (D)
Sonja Hess, Xiaoli Chen, Samuel W. Cushman, Lewis K. Pannell<br />
Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong><br />
adipocytes<br />
In the past adipose cells have generally been regarded as energy storage sites. Only<br />
recently, the critical endocrine function <strong>of</strong> adipocytes to release signaling molecules has<br />
been recognized. It is now known that adipose cells and their regulation play a central<br />
role in the pathogenesis <strong>of</strong> obesity and related diseases. However, the signaling<br />
cascades are not well understood. Therefore, we have developed a 2D-LC-MS/MS and<br />
18O proteolytic labeling strategy to identify and compare levels <strong>of</strong> secretory proteins<br />
with low abundance in the conditioned medium <strong>of</strong> adipose cells w/o or w ith insulin<br />
stimulation. This approach allowed us to catalog a large number <strong>of</strong> secretory proteins<br />
and to detect the different levels <strong>of</strong> many secreted proteins in the basal versus insulin<br />
treatment.<br />
Adipose cells were isolated from rats and cultured in a serum free DMEM medium for<br />
48h. Culture medium was concentrated and separated on a Zorbax C3 column using a<br />
gradient from an aqueous solution <strong>of</strong> 0.05%TFA to acetonitrile. Eight combined fractions<br />
were collected. Each fraction was reduced, alkylated, digested with Lys-C and trypsin.<br />
The samples were separated on a C18 column. The peptides were characterized by LC-<br />
MS/MS using a QTOF2 mass spectrometer. Data were analyzed using a MASCOT search<br />
engine. For 18O proteolytic labeling, 16O-to-18O exchange in the digested peptides from<br />
eight individual fractions was carried out in parallel in H216O and H218O with<br />
immobilized trypsin and the ratios <strong>of</strong> isotopically distinct peptides were measured by LC-<br />
MS.<br />
We have set up proteomics experiments using a 2D LC-MS/MS and 18O proteolytic<br />
labeling strategy to identify and quantify secretory proteins <strong>of</strong> adipose cells without or<br />
with insulin treatment. Proteins were identified by using MASCOT searches <strong>of</strong> the NCBInr<br />
database. A minimum criterion <strong>of</strong> one positive peptide identification was set. In addition,<br />
all secretory proteins were checked by SignalP predictions for their identity as secretory<br />
proteins. From comparison <strong>of</strong> stro mal-vascular cells that are also known as<br />
preadiopcytes, we could clearly show that mature adipocytes function as secretory cells.<br />
A total <strong>of</strong> 70 secreted proteins have been identified using this approach. When<br />
comparing the proteins identified in this study with those found in earlier studies, it is<br />
evident that our approach is extremely powerful in identifying secretory proteins <strong>of</strong><br />
adipose cells since the number <strong>of</strong> secretory proteins identified has been considerably<br />
increased in our study. To those few proteins that are already known to be secreted by<br />
adipose cells, we have compiled a group <strong>of</strong> known secretory proteins previously not<br />
associated with adipose cells and a few unknown proteins. Comparative proteomics <strong>of</strong><br />
18O proteolytic labeling allowed us to detect the different levels <strong>of</strong> many secreted<br />
proteins in the basal versus insulin treatment.<br />
18O proteolytic labeling was chosen because <strong>of</strong> its accuracy, versatility and applicability<br />
in animal and human studies. Taken together, our proteomic approach is able to identify<br />
and quantify the secretory proteome <strong>of</strong> adipose cells that is applicable to animal and<br />
human studies. This will certainly lead to an improved understanding <strong>of</strong> the signaling<br />
cascades <strong>of</strong> adipose cells and thus, pathogenesis <strong>of</strong> obesity and its related diseases.<br />
Literature<br />
1. Ahima, R.S., & Flier, J.S. (2000) Trends Endocrinol. Metab. 11, 327-332.<br />
2. Kratchmarova, I. et al. (2002) Mol. Cell. Proteomics 1, 213-222.<br />
3. Washburn, M.P., Wolters, D., & Yates, J.R. 3rd (2001) Nat. Biotechnol. 19, 242-247.<br />
4. Hess, S., van Beek, J., & Pannell, L.K. (2002) Anal Biochem. 311, 19-26.<br />
contact:<br />
Dr. Sonja Hess<br />
National Institutes <strong>of</strong> Health<br />
NIDDK<br />
Sonja_Hess@nih.gov<br />
9000 Rockville Pike<br />
20892 Bethesda, MD (USA)
Markus Knop, Elin Aareskjold, Volker Gerke<br />
Rab3d and annexin A2 play a role in regulated secretion <strong>of</strong><br />
vWF but not tPA from human endothelial cells<br />
Ca2+-triggered exocytosis <strong>of</strong> pro- and anti-thrombogenic factors from human<br />
endothelial cells is critically involved in the regulated secretion <strong>of</strong> blood clotting and local<br />
immune response. Von-Willebrand-Factor (vWF) and tissue-type plasminogen activator<br />
(tPA) are two such factors which are acutely released into the vasculature following<br />
stimulation, but exhibit opposing physiological effects: vWF induces coagulation and tPA<br />
triggers fibrinolysis. In HUVEC these factors are stored in morphologically distinguishable<br />
granules indicating that their release could be controlled differentially. To search for<br />
components potentially involved in regulating in a differential manner the secretion <strong>of</strong><br />
vWF and tPA, we concentrate on factors previously implicated in exocytotic events in<br />
other systems, rab3D and the annexin A2/S100A10 (p11) complex. In HUVEC rab3D<br />
colacalizes with Weibel-palade bodies (WPb), the principal storage granules <strong>of</strong> multimeric<br />
vWF. Mutant rab3D proteins interfere with the formation <strong>of</strong> bona-fide WPbs and<br />
consequently the acute, histamine-induced release <strong>of</strong> vWF. In contrast, neither the<br />
appearance nor the exocytosis <strong>of</strong> tPA-granules are affected. Annexin A2 and S100A10<br />
are not found on either (vWF or tPA) storage granules but localizes to the<br />
plamamembrane <strong>of</strong> HUVEC. Nontheless, siRNA-mediated downregulation <strong>of</strong> annexin<br />
A2/S100A10 and disruption <strong>of</strong> the complex by microinjection <strong>of</strong> peptide competitors<br />
results in a marked reduction in vWF but not tPA secretion without affecting the<br />
appearance <strong>of</strong> WPbs. Thus, rab3D and annexin A2/S100A10 are two components<br />
involved in the regulated secretion <strong>of</strong> pro- (vWF) but not anti-thrombogenic (tPA)<br />
factors.<br />
contact:<br />
Markus Knop<br />
ZMBE / WWU Münster<br />
med. Biochemie<br />
knopm@uni-muenster.de<br />
Von-Esmarch-Str. 56<br />
48143 Münster (Germany)
Jochen Seebach, Beata Wojciak-Stothard, Hans-Joachim Schnittler<br />
Rac mediates shear stress induced increase in endothelial<br />
barrier-function<br />
Endothelial cells adapt to enhanced laminar fluid shear stress with an alignment in the<br />
direction <strong>of</strong> flow, cell elongation and a reorganisation <strong>of</strong> the cytoskeleton. These<br />
processes are in part regulated by small GTPases <strong>of</strong> the Rho-family. In earlier studies we<br />
showed by determination <strong>of</strong> transendothelial electrical resistance (TER) that shear stress<br />
induced an early and transient increase in endothelial barrier function. This increase was<br />
accompanied by transient tyrosine phosphorylation <strong>of</strong> the vascular endothelial (VE)cadherin/catenin<br />
complex.<br />
Here we show that the shear stress induced increase in barrier function was<br />
accompanied by a reorganisation <strong>of</strong> the VE-cadherin from a discontinuous to a<br />
condensed and continuous pattern and a recruitment <strong>of</strong> actin fibers to the cell-cell<br />
contacts. This rearrangement as well as the shear stress induced tyrosine<br />
phosphorylation <strong>of</strong> the VE-cadherin/catenin complex could be blocked by expression <strong>of</strong><br />
dominant negative Rac (N17Rac). Moreover application <strong>of</strong> laminar shear stress on<br />
endothelial cells expressing N17Rac no longer induced an increase but a continuous<br />
decrease in TER. In contrast expression <strong>of</strong> GFP, dominant negative Rho or dominant<br />
negative Cdc42 did not alter shear stress induced changes in TER.<br />
We conclude that the observed quick rearrangement <strong>of</strong> VE-cadherin and actin leads to a<br />
strengthening <strong>of</strong> the endothelial cell-cell contacts that is mediated by tyrosine<br />
phosphorylation and Rac activation. This early activation seems to be important for<br />
coordinated endothelial rearrangement without loss <strong>of</strong> barrier function under shear<br />
stress.<br />
contact:<br />
Dr Jochen Seebach<br />
TU-Dresden<br />
Physiologie<br />
Jochen.Seebach@mailbox.tu-dresden.de<br />
Fiedlerstr 42<br />
01307 Dresden (Germany)
Jost Seibler, Birgit Küter-Luks, Heidrun Kern, Frieder Schwenk<br />
Rapid method for reproducible, shRNA-mediated gene<br />
silencing in vivo<br />
Recent reports have demonstrated that shRNA-mediated gene knock down in mice is<br />
feasible. Since these experiments employed random transgenesis, however, each<br />
individual mouse line showed a unique, irreproducible shRNA expression pattern. In<br />
addition, screening <strong>of</strong> multiple embryonic stem cell or mouse lines for appropriate siRNA<br />
expression was required, which is laborious and time-consuming. Furthermore, the<br />
organ-specific activities <strong>of</strong> the most commonly used promoters for shRNA expression, U6<br />
and H1, have not been analyzed systematically and the requirements with respect to<br />
copy number and integration site <strong>of</strong> transgenes remained unclear. Taken together, an<br />
established protocol for simple and reproducible RNAi in mice was not presented.<br />
To address these issues, we have performed a side-by-side comparison <strong>of</strong> a firefly<br />
luciferase-specific shRNA transgene under the control <strong>of</strong> the human H1 and U6<br />
promoter, respectively. A single copy <strong>of</strong> either <strong>of</strong> these constructs was inserted into the<br />
rosa26 locus to exclude variable position effects. We have chosen rosa26 for this<br />
purpose, since it has been described as being accessible for transcription in all tissues.<br />
In parallel a dual reporter system was established by the expression <strong>of</strong> both, the firefly<br />
luciferase as a test substrate and the Renilla luciferase as a reference to quantify the<br />
degree <strong>of</strong> shRNA-mediated silencing. Our results establish that a single copy shRNAtransgene<br />
placed into a defined locus is appropriate to mediate ubiquitous gene knock<br />
down in mice. We further demonstrate the usefulness <strong>of</strong> our approach by the expression<br />
<strong>of</strong> a leptin receptor specific shRNA. Using the tetraploid blastocysts complementation we<br />
show that our strategy allows the rapid production <strong>of</strong> stable RNAi knockdown mice within<br />
4 months.<br />
contact:<br />
Dr. Jost Seibler<br />
Artemis Pharmaceuticals GmbH<br />
J.Seibler@artemispharma.de<br />
Neurather Ring 1<br />
51063 Koeln (Deutschland)
Hermann Schillers, Hans-Georg Koch, Astrid Stumpf, Kerstin Wenners-Epping, Mike<br />
Waelte, Dessy Nikova, Hans Oberleithner<br />
Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
We developed a functional blood test for the diagnosis <strong>of</strong> cystic fibrosis using hemoglobin<br />
release as the detection parameter. This test is based on triggering cell lysis by<br />
gadolinium (Gd 3+ ) and finally measurement <strong>of</strong> released hemoglobin. Hemolysis is the<br />
final result <strong>of</strong> a mechanism described as RBC apoptosis. This process involves the<br />
activation <strong>of</strong> phospholipids scramblase which increases annexin V binding, leads to the<br />
rupture <strong>of</strong> membrane-to-skeleton bridges and the induction <strong>of</strong> transglutaminasemediated<br />
protein cross-linking.<br />
Washed human red blood cells (RBC) were incubated in high potassium buffer with 100<br />
µM Gd 3+ for 75 min at 37°C. Quantification <strong>of</strong> hemolysis with OD 546 shows a hemolysis<br />
degree <strong>of</strong> 4.75 % (s.e.m. 0.355 n= 82) <strong>of</strong> non-CF donors RBC and 1.46 % (s.e.m. 0.458<br />
n= 48) <strong>of</strong> CF patients RBC.<br />
It was shown that Gd 3+ enters the cell by a still unknown pathway and displaces Ca 2+<br />
from the cytoskeleton which represents the RBC calcium store. Increasing free<br />
intracellular Ca 2+ activates the RBC apoptosis which is detected by increased Annexin V<br />
binding. We assume that the Gd 3+ entry pathway is CFTR regulated and therefore RBC<br />
from CF-patients, in which CFTR is defect, exhibit a reduced hemolysis.<br />
Conclusion: This is a simple and reliable method for the functional diagnosis <strong>of</strong> cystic<br />
fibrosis.<br />
contact:<br />
Dr. Hermann Schillers<br />
University <strong>of</strong> Muenster<br />
Physiology<br />
schille@uni-muenster.de<br />
Robert-Koch-Str. 27b<br />
48149 Muenster (Germany)
Günter Müller, Susanne Wied, Christian Jung, Andrea Schulz, Norbert Tennagels,<br />
Wendelin Frick<br />
Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and<br />
Cross-Talk to the Insulin Signaling Cascade by the Antidiabetic<br />
Drug Glimepiride<br />
The insulin receptor-independent insulin-mimetic signaling provoked by the antidiabetic<br />
sulfonylurea drug, glimepiride, is accompanied by the redistribution and concomitant<br />
activation <strong>of</strong> lipid raft-associated signaling components, such as the acylated tyrosine<br />
kinase, pp59Lyn, and some glycosylphosphatidylinositol-anchored proteins (GPIproteins).<br />
We now found that impairment <strong>of</strong> glimepiride-induced lipolytic cleavage <strong>of</strong> GPIproteins<br />
in rat adipocytes by the novel inhibitor <strong>of</strong> glycosylphosphatidylinositol-specific<br />
phospholipase C (GPI-PLC), GPI-2350, led to almost complete blockade <strong>of</strong> (i)<br />
dissociation from caveolin-1 <strong>of</strong> pp59Lyn and GPI-proteins, (ii) their redistribution from<br />
high cholesterol- (hcDIGs) to low cholesterol-containing (lcDIGs) lipid rafts, (iii) tyrosine<br />
phosphorylation <strong>of</strong> pp59Lyn and insulin receptor substrate-1 (IRS-1), (iv) stimulation <strong>of</strong><br />
glucose transport and (v) inhibition <strong>of</strong> lipolysis in response to glimepiride. In contrast,<br />
blockade <strong>of</strong> the moderate insulin activation <strong>of</strong> GPI-PLC and lipid raft protein<br />
redistribution by GPI-2350 caused a slight reduction in insulin signaling, only.<br />
Importantly, in response to both insulin and glimepiride, lipolytically cleaved GPIproteins<br />
remain associated with hcDIGs rather than redistribute to lcDIGs as do their<br />
uncleaved versions. In conclusion, GPI-PLC controls the localization within lipid rafts and<br />
thereby the activity <strong>of</strong> certain GPI-anchored and acylated signaling proteins. Its<br />
stimulation by glimepiride (but not insulin) may be sufficient for insulin-mimetic crosstalking<br />
to IRS-1 via redistributed and activated pp59Lyn.<br />
Literature<br />
Nosjean, O., Briolay, A., and Roux, B. (1997) Biochim. Biophys. Acta 1331, 153-186.<br />
Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol. Cell. Biol.<br />
21, 4553-4567.<br />
Müller, G. (2002) FEBS Lett. 531, 81-87.<br />
Smart, E.J., Graf, G. A., McNiven, M. A., Sessa, W. C., Engelman, J. A., Scherer, P.E.,<br />
Okamoto, T., and Lisanti, M. P. (1999) Mol. Cell. Biol. 19, 7289-7304.<br />
contact:<br />
Dr Günter Müller<br />
Aventis Pharma Germany<br />
DG Metabolic Diseases<br />
guenter.mueller@aventis.com<br />
Industrial Park Höchst<br />
65926 Frankfurt am Main (Germany)
Ulf Schulze Topph<strong>of</strong>f, Patrick Zeni, Hans-Joachim Galla<br />
Regulation <strong>of</strong> matrix metalloproteases and their endogenous<br />
inhibitors at the choroid plexus epithelium in vitro<br />
The epithelial cells <strong>of</strong> the choroid plexus (CP) are the structural basis <strong>of</strong> the barrier<br />
between the blood and the cerebrospinal fluid (CSF). Modifications <strong>of</strong> this system, for<br />
example a greater permeability during neuroinflammatory processes, can partly be<br />
explained by alterations <strong>of</strong> protease activity. The interplay between matrix<br />
metalloproteases (MMPs) and their tissue inhibitors (TIMPs) is known to be important for<br />
the regulation <strong>of</strong> physiological and pathological extracellular matrix reconstruction. It is<br />
the major objective <strong>of</strong> this study to characterise our porcine in vitro model <strong>of</strong> the blood-<br />
CSF-barrier focussing on MMP and TIMP expression by the CP epithelium under basal<br />
and inflammatory conditions. MMP and TIMP levels were monitored in time course <strong>of</strong> cell<br />
culture by means <strong>of</strong> zymography and quantitative real-time polymerase chain reaction.<br />
MMP-2 and MMP-9 levels were found to be reduced during cell culture. When the cells<br />
were transferred to serum-free medium, they exhibited much higher electrical<br />
transepithelial resistances (TERs), accompanied by a drastic drop in MMP-9 secretion. Readdition<br />
<strong>of</strong> serum led to a sudden decrease <strong>of</strong> TER and to a rise <strong>of</strong> the MMP-9 level. MMP-<br />
2, TIMP-1, and TIMP-2 were all up-regulated under serum-free conditions, while TIMP-3<br />
was not affected. Furthermore, when the cells were cultured on filter systems, MMP-2<br />
and MMP-9 were secreted to a greater extend to the apical, CSF mimicking cell side than<br />
to the basolateral, blood-facing compartment. Applying tumour necrosis factor – alpha<br />
(TNF-α) to the cultured cells, an in vitro inflammation <strong>of</strong> the CP epithelium was<br />
established. Levels <strong>of</strong> ICAM-1 and VCAM-1 mRNA were drastically raised after addition <strong>of</strong><br />
the cytokine. Interestingly, MMP-9 was down-regulated by TNF-α, while TIMP-1 mRNA<br />
expression was significantly increased. TNF-α lowered the TIMP-3 level but had no visible<br />
effect on MMP-2 and TIMP-2 mRNA, respectively. Two other cytokines, IL-1β and IL-6,<br />
were also tested concerning their effects on the expression level <strong>of</strong> MMP and TIMP<br />
genes. Only IL-6 was found to up-regulate TIMP-1 expression. Combinations <strong>of</strong> either IL-<br />
1β or IL-6 with TNF-α were found to show weaker effects on MMP-9, TIMP-1, and TIMP-<br />
3, respectively, compared to sole addition <strong>of</strong> TNF-α. In summary, we found that both<br />
different culture conditions and application <strong>of</strong> various cytokines modulate the expression<br />
and secretion <strong>of</strong> MMPs and TIMPs by the CP epithelium in vitro. Our studies support the<br />
assumption that changes in protease activity can account for neuroinflammatory<br />
alterations.<br />
Literature<br />
Haselbach M. et al. (2001) Microsc. Res. Tech. 52: 137-152<br />
contact:<br />
Patrick Zeni<br />
Westf. Wilhelms-Universität<br />
Institut für Biochemie / AK Galla<br />
azerwan@uni-muenster.de<br />
Wilh.-Klemm-Str. 2<br />
48149 Münster (Deutschland)
Birgit Scharf, Christine Rotter, Rüdiger Schmitt, Maria de Soto<br />
Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for<br />
host association are coordinated in S. meliloti<br />
Motility and chemotaxis play an important role in microbial ecology, allowing optimum<br />
adaptation to changing environments. However, the high energetic cost <strong>of</strong> motility has<br />
led to the evolution <strong>of</strong> strict regulatory mechanisms that control both the synthesis and<br />
the function <strong>of</strong> the motility apparatus. For the soil bacterium Sinorhizobium meliloti, all<br />
genes essential for chemotaxis and flagellar synthesis are organized in 13 transcriptional<br />
units on the flagellar regulon. Their expression is hierarchically regulated in a three-step<br />
cascade. Products <strong>of</strong> the class I-genes, visN and visR, regulate the expression <strong>of</strong> all class<br />
II and class III-genes. This cascade reflects the temporal order <strong>of</strong> flagellar synthesis,<br />
biosynthesis <strong>of</strong> the proton channel, and components <strong>of</strong> the signalling chain. S. meliloti<br />
can establish symbiotic nitrogen-fixing associations with legume plants. We have<br />
investigated the role <strong>of</strong> the master flagellar gene regulator in the establishment <strong>of</strong><br />
symbiosis with alfalfa plants. Deletions <strong>of</strong> visNR caused a reduction <strong>of</strong> competitiveness<br />
by 40%, indicating an integration <strong>of</strong> motility and symbiosis. The level <strong>of</strong> expression <strong>of</strong><br />
essential genes in the establishment <strong>of</strong> the symbiotic interaction like nodC (nodulation<br />
protein C) and expE (a structural gene <strong>of</strong> EPSII) in both wild type and visNR deletion<br />
strains were analyzed. While the nodC promoter activity was identical in both strains,<br />
the expE promoter activity was increased by a factor <strong>of</strong> 50 for the deletion strain. This<br />
result proves the function <strong>of</strong> VisNR as a direct or indirect repressor <strong>of</strong> the exp genes. In<br />
conclusion, the flagellar regulator is not only involved in regulation <strong>of</strong> motility, but also<br />
in regulation <strong>of</strong> bacterial-plant interaction.<br />
Literature<br />
V. Sourjik, Muschler P., Scharf B., Schmitt R. (2000) VisN and VisR are global regulators<br />
<strong>of</strong> chemotaxis, flagellar, and motility genes in Sinorhizobium (Rhizobium) meliloti.<br />
Journal <strong>of</strong> Bacteriology 182, 782-788.<br />
contact:<br />
Dr Birgit Scharf<br />
Universität Regensburg<br />
Lehrstuhl für Genetik<br />
birgit.scharf@biologie.uni-r.de<br />
Universitätsstr. 30<br />
93040 Regensburg (Deutschland)
Dirk Bald, Toru Hisabori, Georg Groth, Holger Lill<br />
Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and<br />
activation <strong>of</strong> F1-ATPase by tentoxin<br />
F1-ATPase, the hydrophilic part <strong>of</strong> FoF1 ATP synthase, hydrolysis ATP into ADP and<br />
phosphate. It acts as a molecular motor: during catalysis one part <strong>of</strong> the enzyme, the<br />
gamma subunit, rotates within a ring <strong>of</strong> three alpha and three beta subunits [1].<br />
Tentoxin is a cyclic tetra-peptide produced by fungi, which acts as a phytophathogenic<br />
toxin, inhibiting F1-ATPase <strong>of</strong> sensitive plant species [2]. Whereas low concentrations<br />
(~10-8 M) <strong>of</strong> the toxin block the ATP hydrolysis activity, higher concentrations (~10-4<br />
M) surprisingly re-activate the enzyme [3]. Here we investigate individual molecules <strong>of</strong><br />
engineered, tentoxin-sensitive F1-ATPase from the thermophilic Bacillus PS3 in the<br />
presence <strong>of</strong> tentoxin using the polystyrene bead detection method which allow very long<br />
observation times (>1h) [4]. We show that while low tentoxin concentrations suppress<br />
rotation, under reactivating conditions the rotary movement <strong>of</strong> the gamma subunit<br />
commences again, but with new characteristics compared to the enzyme in the absence<br />
<strong>of</strong> tentoxin indicating that the importance <strong>of</strong> reaction intermediates is changed [5]. The<br />
mechanism <strong>of</strong> inhibition and re-activation will be discussed.<br />
Literature<br />
[1] Noji et al. (1997) Nature 299-302<br />
[2] Steele et al. (1976) Proc. Natl. Acad. Sci. USA 73, 2245- 2248<br />
[3] Santolini et al. (1999) J. Biol. Chem. 274, 849-858<br />
[4] Yasuda et al. (2001) Nature 410, 898-904<br />
[5] Pavlova et al. (2004) J. Biol. Chem. 279, 9685-9688<br />
contact:<br />
Dr. Dirk Bald<br />
Free University Amsterdam<br />
Structural Biology<br />
dirk.bald@falw.vu.nl<br />
De Boelelaan 1085<br />
1081HV Amsterdam (Niederlande)<br />
additional information<br />
T.Hisabori: Chemical Resources Laboratory, Tokyo Institute <strong>of</strong> Technology, Japan<br />
G.Groth: Plant Biochemistry, University <strong>of</strong> Duesseldorf, Germany<br />
H.Lill: Structural Biology, Free University Amsterdam, Netherlands
Bernd Lepenies, Bernhard Fleischer, Thomas Jacobs<br />
Regulation <strong>of</strong> T cell responses by BTLA: Functional analysis<br />
using a soluble BTLAIg fusion protein<br />
Activation <strong>of</strong> T lymphocytes through TCR signalling takes place within the context <strong>of</strong><br />
numerous other cell surface proteins. To prevent unnecessary activation <strong>of</strong> T cells the<br />
immune system has developed an intricate balance between positive and negative<br />
costimulatory signals. Positive costimulatory signals determine whether antigen<br />
recognition by T lymphocytes leads to full activation or to anergy/death. In contrast the<br />
expression <strong>of</strong> inhibitory costimulatory molecules by T cells (such as CTLA-4 and PD-1)<br />
seems to mediate the regulation <strong>of</strong> immune response and thus play a pivotal role in the<br />
maintenance <strong>of</strong> peripheral tolerance. Recently, BTLA (B and T lymphocyte attenuator)<br />
was described as a novel costimulatory molecule on T cells. In the present work we<br />
demonstrate that in an experimental malaria model using Plasmodium berghei BTLA<br />
mRNA expression was upregulated in different organs, such as liver, kidney or brain. To<br />
determine the function <strong>of</strong> BTLA a fusion protein between the extracellular domain <strong>of</strong><br />
BTLA and the immunoglobulin domain was constructed. BTLA-Ig was purified from the<br />
culture supernatant <strong>of</strong> transiently transfected COS cells. The BTLA-Ig fusion protein was<br />
used for detection <strong>of</strong> the BTLA-ligand B7H4, which was found to be expressed on mature<br />
dendritic cells. BTLA-Ig as a soluble fusion protein blocks the binding <strong>of</strong> BTLA to B7H4<br />
and thus could be used to determine the function <strong>of</strong> BTLA. To study the influence <strong>of</strong><br />
BTLA on T cell activation in more detail purified dendritic cells were used to stimulate T<br />
cells. Detection <strong>of</strong> secreted cytokines allows to monitor the consequence <strong>of</strong> BTLAligation.<br />
BTLA-Ig also <strong>of</strong>fers the opportunity to investigate the role <strong>of</strong> BTLA in vivo. Our<br />
results indicate that BTLA is a costimulatory molecule with inhibitory properties and thus<br />
BTLA:B7H4 interaction might prevent severe immune pathology under inflammatory<br />
conditions as occuring during infectious disease.<br />
Literature<br />
(1) Han P; Goularte OD; Rufner K; Wilkinson B; Kaye J: An inhibitory Ig superfamily<br />
protein expressed by lymphocytes and APCs is also an early marker <strong>of</strong> thymocyte<br />
positive selection. J Immunol. 2004 May 15; 172 (10): 5931-39.<br />
(2) Jacobs T; Gräfe SE; Niknafs S; Gaworski I; Fleischer B: Murine malaria is<br />
exacerbated by CTLA-4 blockade. J Immunol. 2002 Sep 1; 169 (5): 2323-29.<br />
(3) Watanabe N et al.: BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-<br />
4 and PD-1. Nat Immunol. 2003 Jul; 4 (7): 670-79.<br />
contact:<br />
Bernd Lepenies<br />
Bernhard-Nocht-Institut für Tropenmedizin<br />
berndlepenies@gmx.de<br />
Bernhard-Nocht-Str. 74<br />
20359 Hamburg (Deutschland)
Oliver Hantschel, Bhushan Nagar, John Kuriyan, Giulio Superti-Furga<br />
Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases<br />
The c-Abl tyrosine kinase is a prototypic non-receptor tyrosine kinase that is thought to<br />
participate in a wide variety <strong>of</strong> cellular processes. Tight temporal and spatial control <strong>of</strong><br />
the kinase activity <strong>of</strong> c-Abl is essential, as aberrant activity associated with the<br />
oncogenic fusionprotein Bcr–Abl leads to different forms <strong>of</strong> leukemia in humans.<br />
We study regulatory mechanisms <strong>of</strong> c-Abl using a complementary approach <strong>of</strong> structurefunction<br />
analysis, X-ray crystallography and NMR spectroscopy. Our recent work<br />
uncovered unexpected mechanisms <strong>of</strong> Abl Ib autoinhibition by a novel autoregulatory<br />
myristoyl/phosphotyrosine switch, which functionally replaces the SH2 domainphosphorylated<br />
tail interaction in Src kinases: Intramolecular engagement <strong>of</strong> the Nterminal<br />
myristoyl modification with the kinase domain induces conformational changes<br />
in the kinase domain that allow for the assembly <strong>of</strong> the regulatory SH3-SH2 domain<br />
clamp. The observations shed new light onto the role <strong>of</strong> the SH2 domain and the first<br />
exon region <strong>of</strong> c-Abl. The findings provide a foundation for interpreting the observed<br />
intracellular mobility and cellular activation <strong>of</strong> c-Abl by tyrosine-phosphorylated proteins<br />
and provide new insights into the mechanism <strong>of</strong> action <strong>of</strong> the anti-cancer drug<br />
STI571/Glivec/Imatinib.<br />
An unsuspected link between mutations conferring resistance to the Abl inhibitor STI-<br />
571 and enzyme activation indicates that c-Abl-like regulatory constraints are probably<br />
also operational in the oncoprotein Bcr–Abl.<br />
Literature<br />
Hantschel, O., Nagar, B., Guettler, S., Kretzschmar, J., Dorey, K., Kuriyan, J., and<br />
Superti-Furga, G. (2003) Cell 112, 845-857.<br />
Nagar, B., Hantschel, O., Young, M. A., Scheffzek, K., Veach, D., Bornmann, W.,<br />
Clarkson, B., Superti-Furga, G., and Kuriyan, J. (2003) Cell 112, 859-871.<br />
Hantschel, O. and Superti-Furga, G. (2004) Nature Rev. Mol. Cell Biol. 5(1), 33-44.<br />
contact:<br />
Dr. Oliver Hantschel<br />
European Molecular Biology Laboratory<br />
Developmental Biology Programme<br />
hantschel@embl.de<br />
Meyerh<strong>of</strong>str. 1<br />
69117 Heidelberg (Germany)
Thorsten Hoppe, Giuseppe Cassata, José M. Barral, Wolfdieter Springer, Alex H.<br />
Hutagalung, Henry F. Epstein, Ralf Baumeister<br />
Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Organization <strong>of</strong> the motor protein myosin into structures that perform essential<br />
processes such as cell division, cell motility, vesicular traffic and muscle development is<br />
the result <strong>of</strong> a regulated multi-step assembly pathway. Caenorhabditis elegans UNC-45<br />
binds myosin and Hsp90 simultaneously and thereby functions both as a molecular<br />
chaperone and as an Hsp90 co-chaperone for myosin during thick filament assembly.<br />
Here, we have characterized a novel E3/E4 complex, formed by UFD-2 and the CHIP<br />
ortholog CHN-1, which is necessary and sufficient to multiubiquitylate UNC-45 in vitro.<br />
In vivo, the myosin assembly defects <strong>of</strong> unc-45 temperature-sensitive animals are<br />
partially suppressed by chn-1 loss-<strong>of</strong>-function, while UNC-45 overexpression in worms<br />
deficient for chn-1 results in severely disorganized muscle cells. Our findings support a<br />
model in which CHN-1 and UFD-2 form a functional E3/E4 complex that regulates UNC-<br />
45 levels, thereby permitting proper assembly <strong>of</strong> myosin into muscle thick filaments.<br />
Literature<br />
Barral, J. M., Hutagalung, A. H., Brinker, A., Hartl, F. U., and Epstein, H. F. (2002).<br />
Science 295, 669-671.<br />
Rape, M., Hoppe, T., Gorr, I., Kalocay, M., Richly, H., and Jentsch, S. (2001). Cell 107,<br />
667-677.<br />
Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H.D., and Jentsch, S.<br />
(2000). Cell 102, 577-586.<br />
Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D., Mayer, T.U., and Jentsch, S. (1999).<br />
Cell 96, 635-644.<br />
contact:<br />
Dr. Thorsten Hoppe<br />
Uni Hamburg<br />
ZMNH<br />
thorsten.hoppe@zmnh.uni-hamburg.de<br />
Falkenried 94<br />
20251 Hamburg (Deutschland)
Bas van Steensel, Maartje Vogel, Martin Loden, Elzo de Wit, Frauke Greil, Ben Abbas<br />
Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
We have employed our DamID technology to generate genomic maps <strong>of</strong> the in vivo<br />
target loci <strong>of</strong> heterochromatin proteins in Drosophila and human cells, yielding many<br />
surprising new results.<br />
We previously identified more than 100 target genes for HP1 and Su(var)3-9 in<br />
Drosophila Kc cells. These data revealed that HP1 and Su(var)3-9 can either bind<br />
together or independently <strong>of</strong> each other. Linking <strong>of</strong> these data to a large developmental<br />
expression database (Arbeitman et al, Science, 2002) revealed that the distinct<br />
complexes preferentially associate with genes belonging to specific developmental<br />
programs.<br />
We have adapted the DamID technology for use in mammalian cells and generated<br />
binding pr<strong>of</strong>iles <strong>of</strong> CBX1 (HP1beta) in MCF7 human breast carcinoma cells, probing<br />
~25,000 genes. Strikingly, among the more than 300 CBX1 targets we find at least 50<br />
genes encoding zinc-finger transcription factors (ZNF). These ZNF genes are<br />
concentrated on chromosome 19 and occur in large tandem arrays, which can be more<br />
than 1Mb in size. Interestingly, most <strong>of</strong> these ZNF genes encode proteins with a KRAB<br />
box, which is a repressive domain that forms a complex with HP1 proteins. Preliminary<br />
results indicate that CBX5 (HP1alpha) and CBX3 (HP1gamma) also associate with the<br />
ZNF genes. Expression pr<strong>of</strong>iling and and datamining <strong>of</strong> public databases show that the<br />
ZNF genes bound by the HP1 proteins are components <strong>of</strong> a distinct large regulatory<br />
network. The possible roles and evolutionary implications <strong>of</strong> HP1 association with this<br />
large set <strong>of</strong> ZNF genes will be discussed.<br />
contact:<br />
Dr. Bas van Steensel<br />
Netherlands Cancer Institute<br />
Dept <strong>of</strong> Molecular Genetics<br />
b.v.steensel@nki.nl<br />
Plesmanlaan 121<br />
1066 CX Amsterdam (the Netherlands)
Alexandre Fedotov, Ivan Ovcharuk<br />
Research <strong>of</strong> the mechanism <strong>of</strong> self-assembly fibronectins in<br />
modelling system.<br />
Earlier we had been showed an opportunity <strong>of</strong> modulation <strong>of</strong> mobility <strong>of</strong> the fixed red<br />
blood cells covered by gelatin (FRBCG) in conditions <strong>of</strong> aggregation in modeling<br />
conditions. (1). For research <strong>of</strong> the mechanism fibril genesis we create model in which<br />
cooperates soluble fibronectin (sFn) dissolved from 1000 up to 10 mkg / ml in plastic<br />
tablets <strong>of</strong> firm Linbro with FRBCG d in quantity 2-3 • 103 on everyone volume. In<br />
system 0,01% solution ATP on PBS c pH 7,4 is added, and also 0,3 ml fetal serum <strong>of</strong> calf<br />
(FSC) and all volume <strong>of</strong> system in everyone volume is finished up to 2 ml cultivation by<br />
Egla MEM environment. Tablets placed in term boxing at 12-24 o'clock at 370°• or left<br />
on a cold at 250°• for day. Self-fibril took into account at regular intervals. On the given<br />
model it has been established, that process self-fibril will consist <strong>of</strong> two stages: slow<br />
from 1 up to 14 - 18 hours and actually fibril genesis, coming to the end by 24 o'clock.<br />
The Fibril bunch is strictly directed from a flat bottom volume to a surface <strong>of</strong> a liquid and<br />
makes about 2-3 mm in diameter, and the periphery <strong>of</strong> a bunch is surrounded with a<br />
free liquid in which in strictly vertical direction move FRBCG under a microscope. It is<br />
paradoxical, that in each <strong>of</strong> volume fibril-organization a bunch it is not dependent on<br />
concentration sFn had the identical sizes. Such original cells with matrix we do not<br />
connect «dynamic dialogue «with no-enzymatic process and, most likely it is the pure<br />
physical and chemical mechanism no-enzymatic the nature (2). The model created by us<br />
can find application in research interaction <strong>of</strong> cells with surfaces covered sFn -<br />
bioimplantants. And also at research interaction artificial vesicles with molecules <strong>of</strong> selfassembly<br />
- mechanisms <strong>of</strong> self-assembly <strong>of</strong> bimolecular and in other areas <strong>of</strong> researches<br />
where sFn it is used as object for research <strong>of</strong> modulation <strong>of</strong> interaction biosygnals. (3)<br />
Literature<br />
1.Fedotov A.V., Dykonov L.P. JECB v.74, p.67, 1997.<br />
2. Hynes R.O. PNAS, v.96, •6, p.2588-90, 1999.<br />
3. Bustanji Y., and al. PNAS, Vol.100, •23, Nov.11, p.13292-97.2003.<br />
contact:<br />
Assoc.Pr<strong>of</strong>. Alexandre Fedotov<br />
Moscow St.Uni. Appl. Biothech.<br />
Biotechnics<br />
avfedotov@yandex.ru<br />
Talalichina 33<br />
109383 MOSCOW (Russija)
Vania Braga<br />
Rho GTPases and cell-cell adhesion<br />
During establishment <strong>of</strong> cell-cell contacts, a tight coordination between cell adhesion and<br />
cytoskeletal remodelling is essential to ensure the morphology and integrity <strong>of</strong> epithelial<br />
sheets. These changes in cytoarchitecture are accompanied by the acquisition <strong>of</strong><br />
functional and signalling properties that are intrinsic to epithelial function. The<br />
organization <strong>of</strong> actin filaments in polarized epithelia has been known for many years.<br />
However, during establishment <strong>of</strong> cell-cell adhesion, the temporal and spatial changes in<br />
actin dynamics and remodelling are poorly understood. During keratinocyte polarization,<br />
we identified two distinct actin pools: junctional-actin and thin actin bundles. These two<br />
actin populations differ in their actin dynamics, mechanism <strong>of</strong> formation and<br />
interestingly, have distinct roles during epithelial polarization. While junctional-actin<br />
stabilizes clustered cadherin receptors at cell-cell contacts, peripheral actin bundle<br />
reorganization is essential for increase in cell height during polarization. The contribution<br />
<strong>of</strong> Rho GTPases to the formation <strong>of</strong> each actin population will be discussed in a model<br />
that integrates the adhesion-dependent changes in actin dynamics and specific signalling<br />
pathways to form a polarized cell shape.<br />
contact:<br />
Dr. Vania Braga<br />
Imperial College London - Faculty <strong>of</strong> Medicine<br />
Cell and Molecular Biology Section - Div. Biomedical Sciences<br />
v.braga@imperial.ac.uk<br />
Sir Alexander Fleming Building<br />
SW7 2 AZ London (U.K.)
Radovan Dvorsky, Lars Blumenstein, Patricia Stege, Mohhamad Reza Ahmadian<br />
RhoA-p160Rock communication<br />
The GTP-binding proteins <strong>of</strong> the Rho-family (or Rho-GTPases) regulate a variety <strong>of</strong><br />
cellular processes in all eukaryotic cells, ranging from cytoskeletal reorganization and<br />
cell motility to gene transcription in response to external stimuli. To date, 19 different<br />
mammalian Rho-GTPases have been identified from which Cdc42, Rac1 and RhoA are<br />
the most extensively characterised members <strong>of</strong> the Rho-family. The function <strong>of</strong> Rho-<br />
GTPases depends on the guanine nucleotide-bound state. As molecular switches Rho-<br />
GTPases cycle between an inactive GDP-bound state and an active GTP-bound state,<br />
which is controlled by numerous cellular proteins. Activated GTP-bound form <strong>of</strong> Rho-<br />
GTPases interact with their downstream targets, so-called effector proteins, that are<br />
responsible for the diverse biological effects <strong>of</strong> Rho-GTPases. Several effector molecules<br />
for RhoA have been identified which are involved in activation <strong>of</strong> entirely different<br />
pathway such as Rhophilin, Rhotekin, PRK/PKN, Dia, Kinectin, citron and ROCK. RhoAinduced<br />
activation <strong>of</strong> the Ser/Thr kinase p160ROCK, a central components <strong>of</strong><br />
actin–myosin filament assembly, have been implicated in tumor progression and<br />
metastasis. In order to characterize the interaction <strong>of</strong> p160ROCK with RhoA we<br />
determined the structure <strong>of</strong> its RBD (Rho-binding domain) in complex with RhoA in<br />
active GTP analogue bound state and measured the equilibrium dissociation constants <strong>of</strong><br />
several Rho binding domains <strong>of</strong> three different effector proteins (Rhotekin,<br />
ROCKI/ROKb/p160ROCK, PRK1/PKNa) with both forms <strong>of</strong> RhoA using fluorescence<br />
spectroscopy. We will present the results from these biochemical and structural<br />
investigations that provide an insight into the molecular mechanism <strong>of</strong> communication<br />
between ROCK1, PRK1 and RhoA. In addition, we have identified two novel Rhointeracting<br />
domains in ROCKI, which bind with high affinity to RhoA, but not to Cdc42 or<br />
Rac1. Our findings together with recent structural observations support the notion <strong>of</strong><br />
multiple effector-binding sites in RhoA and strongly indicate the existence <strong>of</strong> a<br />
cooperative binding mechanism for PRK1 and ROCK1 that may be the molecular basis <strong>of</strong><br />
Rho-mediated effector activation.<br />
contact:<br />
PhD. Radovan Dvorsky<br />
Institute for Molekular Physiology<br />
Max-Plnack Institute<br />
radovan.dvorsky@mpi-dortmund.mpg.de<br />
Otto-Hahn Str. 11<br />
44227 Dortmund (Germany)
Witold Filipowicz, Caroline Artus, Lukasz Jaskiewicz, Fabrice Kolb, Ramesh Pillai, Haidi<br />
Zhang<br />
RNAi and microRNA machineries in mammalian cells<br />
In eukaryotes, dsRNA induces sequence-specific inhibition <strong>of</strong> gene expression at the<br />
level <strong>of</strong> mRNA degradation, known as RNAi. During RNAi, dsRNA is processed to ~21-bp<br />
siRNAs, which are incorporated into the RNA Induced Silencing Complex (RISC) to guide<br />
the cleavage <strong>of</strong> mRNA. miRNAs are ~21-nt RNA regulators interacting with 3’-UTRs <strong>of</strong><br />
mRNAs and arresting translation by unknown mechanism. ~300 miRNAs are predicted to<br />
function in mammals. RNAi and miRNA pathways involve many proteins, either<br />
participating in siRNA and miRNA biogenesis, or functioning as components <strong>of</strong> RISC and<br />
miRNPs targeting mRNAs for degradation or translational repression.<br />
Dicer is a multidomain nuclease responsible for biogenesis <strong>of</strong> siRNAs and miRNAs. Dicer<br />
domains include an RNA helicase/ATPase, DUF283 and PAZ and dsRBD domains, and two<br />
RNase III-like domains. Studies <strong>of</strong> human Dicer revealed its many interesting properties.<br />
In contrast to Drosophila and C. elegans enzymes, cleavage <strong>of</strong> dsRNA by the human<br />
Dicer is ATP-independent. The enzyme has a strong preference for cutting <strong>of</strong>f siRNAs and<br />
miRNAs from substrate ends. Dicer is known to interact with Argonaute (Ago) proteins,<br />
established components <strong>of</strong> RISC. In collaboration with T. Hobman’s group, we mapped<br />
domains responsible for the interaction. Dicer functions as a monomer, with its two<br />
RNase III domains associating together to form a pseudo-dimer, which contains a single<br />
processing center, generating siRNAs and miRNAs.<br />
Nearly all metazoan miRNAs control gene expression by imperfectly base-pairing with<br />
the 3’-UTR <strong>of</strong> target mRNAs and repressing protein synthesis. It is unknown whether<br />
miRNA-mRNA duplexes containing mismatches and bulges provide specific features that<br />
are recognized by factors mediating the repression. Ago proteins are the best<br />
characterized protein components <strong>of</strong> miRNPs. We found that effects <strong>of</strong> miRNAs on<br />
translation can be mimicked in human HeLa cells by the miRNA-independent tethering <strong>of</strong><br />
Ago proteins to the 3’-UTR <strong>of</strong> a reporter mRNA. These findings indicate that a primary<br />
function <strong>of</strong> miRNAs is to guide their associated proteins to the mRNA.<br />
contact:<br />
Dr. Witold Filipowicz<br />
Friedrich Miescher Institute<br />
filipowi@fmi<br />
PO Box 2543<br />
4002 Basel (Switzerland)
Eva-Maria Mandelkow, J. Biernat, T. Timm, E. Thies, X.-Y. Li, E. Mandelkow<br />
Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth<br />
and degeneration<br />
The microtubule-associated protein tau fulfills several functions in neurons, such as<br />
microtubules stabilization, regulation <strong>of</strong> intracellular transport, promotion <strong>of</strong> cell-process<br />
outgrowth and establishment <strong>of</strong> cell polarity. The functions are regulated by<br />
phosphorylation. In Alzheimer's disease, tau is pathologically phosphorylated and<br />
aggregated. The protein kinase MARK (alias Par-1)was originally discovered because <strong>of</strong><br />
its ability to phosphorylate the KXGS motifs in tau which are enhanced in Alzheimer's<br />
disease, and because phosphorylation at these motifs causes the detachment <strong>of</strong> tau<br />
from microtubules. Related kinases (Par-1, Kin1) occur in various organisms and are<br />
involved in the establishment and maintenance <strong>of</strong> cell polarity. MARK/Par-1 is able to<br />
control the differentiation and outgrowth <strong>of</strong> cell processes from neuroblastoma and other<br />
cell models. The formation <strong>of</strong> neurites in N2a cells is blocked if MARK is inactivated or if<br />
the KXGS motifs on tau are mutated. Since one <strong>of</strong> the effects <strong>of</strong> MARK is the<br />
destabilization <strong>of</strong> microtubules, the results suggest that microtubules must become labile<br />
at a certain point in space and time, to allow neurites to grow. MARK/Par-1 is activated<br />
by phosphorylation. We recently identified the activating kinase, MARKK, a member <strong>of</strong><br />
the Ste20-like kinases (Timm et al., EMBO J. 2003). Since tau is involved not only in<br />
microtubule stabilization but also in intracellular transport we studied its effect on the<br />
trafficking <strong>of</strong> vesicles and organelles in primary cortical neurons. When differentiated<br />
neurons are transfected by CFP-tau(via adenovirus), kinesin-dependent transport<br />
towards the synapse is inhibed, and retrograde transport by dynein dominates. As a<br />
consequence the axon is depleted <strong>of</strong> vesicles and organelles, which leads to starvation <strong>of</strong><br />
the axon, growth inhibition, and vulnerability against oxidative stress. This effect can be<br />
relieved by activating the MARKK-MARK pathway.<br />
Supported by DFG.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Eva-Maria Mandelkow<br />
MPG Arbeitsgruppen für strukturelle Molekularbiologie<br />
am DESY<br />
mand@mpasmb.desy<br />
Nottkestr. 85<br />
22607 Hamburg (Deutschland)
Pia Wülfing, Martin Götte, Christian Kersting, Joke Tio, Christopher Poremba,<br />
Raihanatou Diallo, Werner Böcker, Ludwig Kiesel<br />
Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
The endothelin-1 (ET-1) peptide and its G-protein coupled receptors ETAR and ETBR (ETaxis)<br />
modulate vasomotor tone, tissue differentiation, development, cell proliferation and<br />
hormone production (1). We have previously demonstrated an overexpression <strong>of</strong> the ETaxis<br />
in mammary carcinoma, which is associated with more aggressive tumors and<br />
poorer clinical outcome (2-4). To clarify if expression <strong>of</strong> the ET-axis participates in<br />
angiogenesis <strong>of</strong> breast carcinoma, we analyzed expression <strong>of</strong> ET-1, ETAR, ETBR and<br />
vascular endothelial growth factor (VEGF) in 600 breast carcinoma specimens by tissue<br />
microarray technology. Moderate or strong immunostaining was observed for ET-1 in<br />
25.4%, for ETAR in 43.7% and for ETBR in 22.2% <strong>of</strong> breast carcinomas. Of all cases,<br />
44.7% showed significant expression <strong>of</strong> VEGF. Microvessel density (MVD) and ET<br />
expression status were positively correllated, with higher MVD in ET-positive tumors.<br />
VEGF expression was positively correlated with MVD and was more frequent in tumors<br />
overexpressing the ET axis (P
Matthias Böcker, Pia Heidenreich, Harald Fuchs, Tilman Schäffer<br />
Scanning ion conductance microscope with shear-force control<br />
We have built a scanning ion conductance microscope (SICM) that measures the local<br />
ion conductance <strong>of</strong> surfaces. A pulled micropipette, having an opening diameter <strong>of</strong> less<br />
than 100nm and filled with electrolyte, acts as local ion conductance probe and is<br />
scanned over a porous sample surface. To obtain a meaningful interpretation <strong>of</strong> the<br />
measured ion conductance, the pipette-sample distance needs to be kept constant. This<br />
is achieved by implementing a complementary shear-force distance control: The<br />
micropipette is mechanically oscillated at a fixed frequency (3-10kHz), in a direction<br />
parallel to the surface. When the pipette is approached to the surface, the oscillation<br />
amplitude decreases due to arising shear forces. This amplitude decrease is detected<br />
optically by focusing a laser beam onto the thin end <strong>of</strong> the micropipette and measuring<br />
the modulation <strong>of</strong> the transmitted or reflected light. A feedback loop uses this signal to<br />
keep the pipette at constant distance to the surface while scanning, thereby<br />
simultaneously measuring two complementary surface properties: topography and ion<br />
conductance. We have used this microscope for imaging gratings, CDs and cells.<br />
contact:<br />
Dr. Tilman Schäffer<br />
Universität Münster<br />
Center for Nanotechnology<br />
tilman.schaeffer@uni-muenster.de<br />
Gievenbecker Weg 11<br />
48149 Münster (Germany)<br />
additional information<br />
http://bi<strong>of</strong>orce.centech.de
Pia M. Heidenreich, Sebastian Schrot, Hans-Joachim Galla, Harald Fuchs, Tilman E.<br />
Schäffer<br />
Scanning Ion Conductance Microscopy - Topographical noncontact<br />
imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
We have developed a scanning ion conductance microscope (SICM) that can obtain<br />
topographical images <strong>of</strong> s<strong>of</strong>t and biological surfaces such as cells, with a lateral<br />
resolution <strong>of</strong> 50 nm. SICM is a non-contact scanning probe method which is based on<br />
the measurement <strong>of</strong> an ion current that flows through the tip opening <strong>of</strong> a micropipette.<br />
The ion current generally decreases when the tip approaches the surface. This “current<br />
squeezing” effect is used as a contrast mechanism in SICM. It is possible to control the<br />
tip-sample distance with this effect while scanning across the sample surface. In this<br />
case, the topography <strong>of</strong> the sample is imaged. Our experimental setup also can be<br />
operated in a spectroscopic mode. The measured current-distance curves do not only<br />
show the current squeezing when the tip is approached to a sample. They contain a lot<br />
<strong>of</strong> information about sample and tip properties, but not all effects are fully understood<br />
yet.<br />
contact:<br />
Pia Heidenreich<br />
Universität Münster<br />
Center for NanoTechnology & Physikalisches Institut<br />
heidenre@uni-muenster.de<br />
Gievenbecker Weg 11<br />
48149 Münster (Germany)
Keiichi Namba<br />
Self-assembly and switching <strong>of</strong> the bacterial flagellum<br />
The bacterial flagellum is made <strong>of</strong> a rotary motor and a long helical filament by means <strong>of</strong><br />
which bacteria swim. The flagellar motor rotates at around 300 Hz and drives the rapid<br />
rotation <strong>of</strong> each flagellum to propel the cell movements. The long helical filament, which<br />
is a tubular structure with a diameter <strong>of</strong> about 20 nm, is made <strong>of</strong> a single protein<br />
flagellin. The filament can switch between left-handed or right-handed helical forms in<br />
response to the twisting force produced by reversal <strong>of</strong> the motor rotation, allowing<br />
bacteria to alternate their swimming pattern between running and tumbling for taxis.<br />
The flagellum also has a short, highly curved segment called hook, which connects the<br />
motor and the helical propeller. Its bending flexibility makes it work as a universal joint,<br />
while the filament is relatively more rigid to be a propeller. A very short segment made<br />
<strong>of</strong> HAP1 and HAP3 connects the two mechanically distinct structures. The flagellum is<br />
constructed by self-assembly <strong>of</strong> proteins translocated from the cytoplasm to the distal<br />
end <strong>of</strong> the growing structure through its narrow central channel, where cap complexes<br />
help efficient self-assembly.<br />
We have solved some parts <strong>of</strong> these structures by X-ray crystallography, fiber diffraction<br />
and electron cryomicroscopy. All these structures present interesting implications for the<br />
function <strong>of</strong> each molecule, demonstrating the importance <strong>of</strong> dual nature <strong>of</strong> protein<br />
molecules, flexibility and precision.<br />
contact:<br />
Pr<strong>of</strong>essor Keiichi Namba<br />
Graduate School <strong>of</strong> Frontier Biosciences, Osaka University<br />
keiichi@fbs.osaka-u.ac.jp<br />
1-3 Yamadaoka<br />
565-0871 Suita, Osaka (Japan)
Stefan Kreusch, Heidrun Rhode, Horst Hoppe, Thomas Moore, Renate Bublitz, Joachim<br />
Misselwitz, Margarete Schulze, Gerd Cumme<br />
Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
The Alport syndrome is a hereditary nephropathy caused by mutations in type IV<br />
collagen genes. It results in hematuria and/or proteinuria and, therewith, alteration <strong>of</strong><br />
serum proteins. We analyzed human serum proteomes <strong>of</strong> 3 Alport syndrome patients<br />
versus 3 healthy probands. 0.5 ml serum (about 30 mg protein) were separated by size<br />
exclusion chromatography and subsequent anion exchange chromatography. A parallel<br />
chromatography device in the 8*12 microplate format was used together with the liquid<br />
handling device CyBiä-Well. Protein concentrations within the resulting 2400 fractions <strong>of</strong><br />
each serum were quantified by UV-absorbance measurements at 205, 215, and 280 nm.<br />
Normalized protein concentration differences, syndrome minus healthy, are shown as<br />
two-dimensional pattern according to both the separations performed. In liquid aliquots<br />
<strong>of</strong> the native serum fractions obtained, activities <strong>of</strong> alkaline phosphatase, acetylcholine<br />
esterase, and GPI-phospholipase D were determined. Triglycerides and cholesterol as<br />
lipoprotein markers and the immunoreactivity <strong>of</strong> C-reactive protein were determined.<br />
Semiquantitative MALDI-MS using mean peak intensity sums <strong>of</strong> tryptic peptides<br />
normalized by internal standard peptide peak heights was applied to validate alterations<br />
<strong>of</strong> selected protein levels. It turned out that Alport syndrome patients partially<br />
compensate for loss <strong>of</strong> lower molecular weight proteins by a higher serum concentration<br />
<strong>of</strong> alpha-2-macroglobulin.<br />
contact:<br />
Dr. Stefan Kreusch<br />
Klinikum der FSU Jena<br />
Inst. f. Biochemie I<br />
skre@mti.uni-jena.de<br />
Nonnenplan 2<br />
07743 Jena (D)
Georg Reiser, Mohan Tulapurkar, Theo Hanck<br />
Sequestration and recycling pathway <strong>of</strong> P2Y2 nucleotide<br />
receptor: Clathrin-and actin cytoskeleton-dependent receptor<br />
endocytosis, live cell visualization <strong>of</strong> green fluorescent protein<br />
tagged receptor<br />
Nucleotides exert a large number <strong>of</strong> physiological effects through activation <strong>of</strong> G proteincoupled<br />
receptors. We investigated the trafficking <strong>of</strong> the P2Y2 receptor, which was<br />
tagged to GFP and stably expressed in HEK-293 cells. The GFP tag at the C-terminus <strong>of</strong><br />
the receptor allows visualization <strong>of</strong> receptor translocation in live cells. Functional<br />
expression <strong>of</strong> the GFP tagged receptor was confirmed by determining [Ca2+]i increase<br />
after stimulation <strong>of</strong> the cells with the agonists ATP or UTP (1). Stimulation with ATP or<br />
UTP also caused receptor internalization, which was visible as time-dependent<br />
translocation <strong>of</strong> the receptor from the plasma membrane to the cytoplasm using confocal<br />
laser scanning microscopy. The internalization was dependent on concentration <strong>of</strong><br />
agonist and duration <strong>of</strong> stimulation. There was no internalization <strong>of</strong> the receptor at 40C<br />
in the presence <strong>of</strong> agonist, ruling out constitutive receptor internalization. The<br />
internalization <strong>of</strong> the receptor involves the actin cytoskeleton and myosin. A massive<br />
rearrangement <strong>of</strong> the actin cytoskeleton was observed which facilitates internalization.<br />
Colocalization <strong>of</strong> the internalized receptor with early endosomes, clathrin and lysosomes<br />
was seen. The inhibition <strong>of</strong> receptor internalization by 0.45-M sucrose in the presence <strong>of</strong><br />
UTP indicates that the receptor could be internalized by the clathrin-mediated pathway.<br />
Inhibition <strong>of</strong> internalization in the presence <strong>of</strong> chlorpromazine confirms that<br />
internalization occurs by a clathrin-dependent pathway. However, the caveolin-mediated<br />
pathway was not involved since internalization <strong>of</strong> the receptor was not inhibited in the<br />
presence <strong>of</strong> filipin-III. Depending on the duration <strong>of</strong> stimulation, the receptor is either<br />
recycled back to the membrane or degraded. The targeting <strong>of</strong> the receptor from<br />
endosomes to lysososmes possibly also involves the proteasome pathway, because the<br />
proteasomal inhibitor MG-132 blocked targeting <strong>of</strong> the receptor to lysosomes. This<br />
inhibition facilitated the recycling <strong>of</strong> the receptor back to the plasma membrane.<br />
Literature<br />
(1) Tulapurkar, M.E., Laubinger W., Nahum V., Fischer B. and Reiser, G. Subtype specific<br />
internalization <strong>of</strong> P2Y1 and P2Y2 receptors induced by novel adenosine 5`-O-(1boranotriphosphate)<br />
derivatives. Br. J. Pharmacol. (2004) advance online publication,<br />
June 14, doi:10.1038/sj.bjp.0705859<br />
contact:<br />
Pr<strong>of</strong>. Georg Reiser<br />
Otto-von-Guericke Universität Magdeburg<br />
Institut für Neurobiochemie<br />
georg.reiser@medizin.uni-magdeburg.de<br />
Leipziger Str. 44<br />
39120 Magdeburg (Germany)
Karl Matter<br />
Signalling at tight junctions in the regulation <strong>of</strong> epithelial<br />
proliferation and differentiation<br />
Polarised epithelial cells form barriers consisting <strong>of</strong> cellular sheets that separate<br />
compartments <strong>of</strong> different composition. The formation and maintenance <strong>of</strong> polarised<br />
epithelia requires careful timing <strong>of</strong> cell growth, differentiation, and gene expression.<br />
Tight junctions are the most apical structure <strong>of</strong> the epithelial cell-cell junctional complex.<br />
They regulate the diffusion <strong>of</strong> molecules through the paracellular pathway and act as<br />
apical/basolateral intramembrane diffusion barriers. Recent evidence suggests that tight<br />
junctions also participate in the regulation <strong>of</strong> epithelial differentiation and proliferation.<br />
We have identified tight junction-associated signalling proteins that include a<br />
transcription factor, a cell cycle kinase and a Rho activator. Our data suggest that these<br />
proteins are part <strong>of</strong> a tight junction associated signalling system that participates in the<br />
regulation <strong>of</strong> epithelial proliferation and differentiation.<br />
contact:<br />
Dr. Karl Matter<br />
University College London<br />
Div. <strong>of</strong> Cell Biology, Institute <strong>of</strong> Opthalmology<br />
k.matter@ucl.ac.uk<br />
Bath Street<br />
EC1V 9EL London (UK)
Jan Scheffer, Yvonne Rolke, Paul Tudzynski<br />
Signalling in early stages <strong>of</strong> pathogenic development <strong>of</strong><br />
Claviceps purpurea.<br />
Claviceps purpurea is a biotrophic phytopathogenic ascomycete which shows a high<br />
organ specificity: it attacks exclusively florets <strong>of</strong> grasses, growing along the pollen tube<br />
path to the ovarian basis and tapping the vascular tissue. Defense reactions <strong>of</strong> the host<br />
plant are confined to strictly localized areas. Genes conferring resistance against this<br />
economically important pathogen are so far unknown. Detailed analyses <strong>of</strong> this system<br />
have presented evidence that degradation <strong>of</strong> pectin by the fungus is essential for<br />
colonization <strong>of</strong> host tissue. In analogy to pollen tube growth, also adhesion <strong>of</strong> spores and<br />
oriented growth <strong>of</strong> the fungus in the plant tissue could involve a pectin compound. Two<br />
different mitogen-activated-protein (MAP) kinase cascades obviously are involved in<br />
pathogenicity-related signalling processes, since loss-<strong>of</strong>-function experiments (via<br />
targeted gene inactivation) have shown that they both are essential for early steps in<br />
pathogenicity.<br />
We are interested in the signalling events triggering early infection steps and oriented<br />
growth in the infected plant tissue in this specialized interaction system, with special<br />
focus on mechanisms <strong>of</strong> establishing polarity and orientation. For the identification <strong>of</strong><br />
genes involved in early steps <strong>of</strong> infection (including the polarity dependent penetration<br />
phase and e.g. the basis <strong>of</strong> host and organ specificity) an SSH approach is used to find<br />
target genes for the two MAPK cascades involved.<br />
Two different approaches are used to find genes involved in oriented growth in planta:<br />
an insertional library based on the Agrobacterium T-DNA transfer system is currently<br />
screened for tagged mutants showing disturbed/ disoriented growth in planta. In<br />
addition, homologues <strong>of</strong> genes known to be involved in polar/oriented growth in other<br />
systems (cdc42, cot-1, cla4) are directly investigated for their role in this system by<br />
targeted inactivation.<br />
Literature<br />
Tudzynski, P. and Scheffer, J. (2004) Claviceps purpurea: molecular aspects <strong>of</strong> a unique<br />
pathogenic lifestyle. Mol Plant Pathol. DOI: 10.1111/J.1364-3703.2004.00237.X<br />
contact:<br />
Jan Scheffer<br />
Westfälische Wilhelms-Universität<br />
Institut für Botanik<br />
scheffj@uni-muenster.de<br />
Schlossgarten 3<br />
48149 Münster (Deutschland)
Andreas Krieger, Marcel Kamp, Margit Henry, Alexey Pereverzev, Marco Weiergräber,<br />
Jürgen Hescheler, Toni Schneider<br />
Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Multiple types <strong>of</strong> voltage-activated Ca2+ channels (T, L, N, P, Q, R/E type) coexist in<br />
excitable cells and participate in Ca2+-dependent processes as synaptic differentiation<br />
and plasticity, and neurotransmitter release. E-type Ca2+ channels are lacking a typical<br />
synprint site which is responsible for the interaction with synaptic vesicle proteins. But<br />
the II-III linker from neuronal splice variants <strong>of</strong> Cav2.3 confers a novel Ca2+ sensitivity<br />
to E-type channels when expressed in HEK-293 cells which is linked to protein kinase C<br />
activation and might be involved in synaptic plasticity <strong>of</strong> hippocampal mossy fibers.<br />
To understand the mechanism <strong>of</strong> the Ca2+ and phorbolester mediated facilitation in<br />
Cav2.3 / E-type Ca2+ channels, protein partners <strong>of</strong> the II-III linker were isolated. We<br />
screened by overexpression <strong>of</strong> the FLAG-tagged II-III – loop from Cav2.3 in HEK-293<br />
cells for interacting proteins. The full length II-III – loop present in neuronal splice<br />
variants <strong>of</strong> Cav2.3 / E-type Ca2+ channels led to the coimmunoprecipitation <strong>of</strong> a protein<br />
partner which is known to interact with protein kinase C (PKC). The immunoaffinitypurified<br />
II-III – loop proteins from neuronal and endocrine splice variants stimulate<br />
autophosphorylation <strong>of</strong> exogeneous added and endogenous present protein kinase C, but<br />
do not get phosphorylated themselves leading to the conclusion that adaptor proteins<br />
are targeting PKC to the Cav2.3 / R-type Ca2+ channel via determinants within the<br />
cytosolic II-III linker domain.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Toni Schneider<br />
Klinikum der Universität zu Köln<br />
Institut für Neurophysiologie<br />
toni.schneider@uni-koeln.de<br />
Robert-Koch-Str. 39<br />
50931 Köln (NRW)
Hermann Gaub<br />
Single Molecule Force Spectroscopy- A Bettter Understanding<br />
<strong>of</strong> Biomolecules with Newton?<br />
Local interactions between bio-molecules regulate the complex diversity <strong>of</strong> life. With the<br />
development <strong>of</strong> AFM-based techniques, which allow control and measurement <strong>of</strong><br />
interaction forces between individual molecules at physiological conditions, a multitude<br />
<strong>of</strong> essential processes such as molecular recognition and protein folding have become<br />
accessible at unparalleled resolution and sensitivity. Moreover, the description <strong>of</strong><br />
molecular devices and machines based on forces rather than thermodynamic variables<br />
has provided novel insight into their biological function. This lecture will highlight the<br />
advances <strong>of</strong> the recent years.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Hermann Gaub<br />
Universität München<br />
Angewandte Physik<br />
gaub@physik.uni-muenchen.de<br />
Amalienstr. 54<br />
80799 München (Deutschland)
Heinrich Hörber<br />
Single molecule mechanics<br />
In the context <strong>of</strong> evolution, nature has found many elegant nano-technological concepts.<br />
In the cells <strong>of</strong> our body innumerable examples <strong>of</strong> multi-functional, self-organizing and<br />
self-reproducing molecular structures are found. All cellular functions are performed by<br />
groups <strong>of</strong> highly specialized molecules carrying out exact programs. These components<br />
possess an extremely high efficiency level and their functions are regulated stably and<br />
precisely, both by networking with other functional groups as well as by reacting to<br />
changing environmental conditions. Thus biologists talk about proteins like machines for<br />
good reason: proteins function like machines with specific tasks equipped with built-in<br />
programming and <strong>of</strong>ten with a chemical on/<strong>of</strong>f switch. It is possible to use and in some<br />
cases change the functions <strong>of</strong> proteins for technological applications. Such modifications<br />
need to be monitored carefully and for certain molecules their mechanical properties are<br />
the most relevant features. The three-dimensional analysis <strong>of</strong> thermal position<br />
fluctuations can reveal mechanical properties, for instance <strong>of</strong> molecular motor proteins<br />
like Kinesin and Myosin. This became possible by a novel three-dimensional scanning<br />
probe microscope, the Photonic Force Microscope (PFM). Such measurements can<br />
complement AFM studies <strong>of</strong> protein unfolding revealing at higher forces information<br />
about internal forces determining the three-dimensional structure <strong>of</strong> these molecules. A<br />
double detection scheme has been developed to provide the necessary stability <strong>of</strong> the<br />
AFM doing experiments on short polymer chains and to work in a “force-clamp” mode.<br />
Literature<br />
J. K. H. Horber and Mervin Miles, 2003 Science 302, 1002-1005<br />
Hörber J.K.H. 2002 Methods in Cell Biology, Vol. 68, 1-31<br />
Altmann, S. M., R. G. Grunberg, P. F. Lenne, J. Ylanne, A. Raae, K. Herbert, M. Saraste,<br />
M. Nilges, and J. K. H. Horber, 2002 Structure 10, 1085-1096.<br />
Pralle, A., P. Keller, E. L. Florin, K. Simons, and J. K. H. Horber, 2000 Journal <strong>of</strong> Cell<br />
Biology 148, 997-1007<br />
contact:<br />
Pr<strong>of</strong>. Dr. Heinrich Hörber<br />
Wayne State University<br />
Physiology<br />
hhoerber@med.wayne.edu<br />
540 E. Canfield Ave.<br />
48201 Detroit (USA)
Gerhard Schütz, Manuel Mörtelmaier, Mario Brameshuber, Karel Drbal, Hannes<br />
Stockinger<br />
Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
A detailed understanding <strong>of</strong> molecular processes is the basic requirement for the<br />
description <strong>of</strong> cellular function. These processes typically involve the interplay <strong>of</strong><br />
different proteins, but also <strong>of</strong> proteins and the lipids in the cell membrane. While largescale<br />
rearrangements <strong>of</strong> the protein distribution proceed on seconds up to minutes time<br />
scales, local changes are much faster. New ultra-sensitive methodologies for imaging<br />
single molecules in living cells allow the direct observation <strong>of</strong> molecular rearrangements<br />
on millisecond time scales. Such rearrangements are <strong>of</strong> particular importance during the<br />
stimulation <strong>of</strong> immune cells. We followed the motion <strong>of</strong> individual molecules during<br />
different phases <strong>of</strong> T-cell stimulation in the area <strong>of</strong> the immunological synapse. The<br />
mobility <strong>of</strong> different proteins, as they pass the synapse, reveals information upon the<br />
microstructure <strong>of</strong> such cell-to-cell contact areas. Such studies shed further light on the<br />
structural relevance <strong>of</strong> lipid microdomains for T-cell stimulation.<br />
(supported by the Austrian Research Funds and the Austrian Federal Ministry <strong>of</strong><br />
Education, Science and Culture.)<br />
contact:<br />
Dr. Gerhard Schütz<br />
Johannes Kepler Universität Linz<br />
Institut für Biophysik<br />
gerhard.schuetz@jku.at<br />
Altenbergerstr.69<br />
4040 Linz (Österreich)<br />
additional information<br />
Karel Drbal, Hannes Stockinger: Institut für Immunologie, Medizinische Universität Wien
Johannes A. Eble, Ulrike Mayer, Jörg Haier<br />
Snake venom inhibitors <strong>of</strong> collagen- and laminin-binding<br />
integrins suppress tumor cell migration and invasion<br />
Introduction: Disintegrins <strong>of</strong> snake venoms usually inhibit the platelet integrin<br />
&alphaIIb&beta3 in an RGD-dependent manner. Only recently, RGD-independent<br />
integrin inhibitors have been identified, e.g. rhodocetin, which blocks the collagenbinding<br />
integrin &alpha2&beta1. Yet, it has been unclear whether snake venom<br />
components exist which inhibit laminin-binding integrins.<br />
Methods: Binding <strong>of</strong> a recombinant soluble integrins to laminin were tested in a cell-free<br />
binding assay. In this assay, almost 40 snake venoms were screened for integrin<br />
inhibitors. Thus, two novel inhibitors <strong>of</strong> laminin-binding b1 integrins, called lebein-1 and<br />
–2, were isolated from the venom <strong>of</strong> Vipera lebetina. We studied their biological<br />
activities both in vitro and in situ, e.g. on attachment and invasion <strong>of</strong> fluorescently<br />
labeled hepatocarcinoma cells into rat liver by vital microscopy.<br />
Results: (1) Lebein-1 and -2 are among the first known disintegrins to block lamininbinding<br />
&beta1-integrins. Only the homodimeric lebein-1, but not the heterodimeric<br />
lebein-2, contains RGD-sequences. (2) In an RGD-independent manner, both lebeins<br />
bind avidly to their integrin targets thus blocking their binding to laminin. (3) Especially<br />
lebein-1 prevents hepatocarcinoma cells from adhering to and penetrating through the<br />
laminin-rich basement membrane. In contrast, rhodocetin, a snake venom inhibitor<br />
directed against the collagen-binding &alpha2&beta1 integrin, does not reduce tumor cell<br />
attachment onto the basement membrane, but inhibits tumor cell invasion into the type<br />
I collagen-rich interstitial connective tissue. Thus, snake venom-derived integrin<br />
anatgonists can suppress tumor progression and metastasis both at the basement<br />
membrane and the interstitial connective tissue.<br />
Literature<br />
Eble J.A., Bruckner, P. Mayer, U.; J. Biol. Chem. 278: 26488-26496 (2003)<br />
Eble, J. A., Niland, S., Dennes, A., Schmidt-Hederich, A., Bruckner, P., Brunner, G.;<br />
Matrix Biol. 21: 545-556 (2002)<br />
contact:<br />
PD Dr. Johannes A. Eble<br />
Universitätsklinikum Münster<br />
Inst. f. Physiologische Chemie<br />
eble@uni-muenster.de<br />
Waldeyerstr. 15<br />
48149 Münster (D)<br />
additional information<br />
U. Maier, Wellcome Trust Centre for Cell-Matrix Research, University <strong>of</strong> Manchester, UK<br />
J. Haier, Klinik und Poliklinik für Allgemeine Chirurgie, Universitätsklinikum Münster
Peter Geyer, Rolf Döker, Xiaodong Zhao, Werner Kremer, Jürgen Kuhlmann, Alfred<br />
Wittingh<strong>of</strong>er, Hans Robert Kalbitzer<br />
Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
The termination <strong>of</strong> export processes from the nucleus to the cytoplasm in higher<br />
eukaryotes is mediated by binding <strong>of</strong> the small GTPase Ran as part <strong>of</strong> the export<br />
complexes to the Ran binding domains (RanBD) <strong>of</strong> Ran binding protein 2 (RanBP2) that<br />
is part <strong>of</strong> the nuclear pore complex. So far the structure <strong>of</strong> the first RanBD <strong>of</strong> RanBP2 in<br />
complexes with Ran has been known from x-ray crystallographic studies (1,2). Here we<br />
report the NMR solution structure <strong>of</strong> the uncomplexed second RanBD <strong>of</strong> RanBP2. Like<br />
RanBD1 the structure shows a pleckstrin-homology (PH) fold featuring two almost<br />
orthogonal β-sheets consisting <strong>of</strong> three and four strands and an α-helix sitting on top.<br />
This is in contrast to the crystal structures <strong>of</strong> the complexed RanBD1 that are missing<br />
one β-strand. This is probably due to the binding <strong>of</strong> the C-terminal α-helix <strong>of</strong> Ran to the<br />
RanBD in these complexes. To analyze the interaction between RanBD2 and the Cterminus<br />
<strong>of</strong> Ran, NMR-titration studies with peptides comprising the 6 and 28 terminal<br />
residues <strong>of</strong> Ran were performed. While the 6 residue peptide alone does not bind to<br />
RanBD2 in a specific manner, the 28 residue peptide including the entire C-terminal helix<br />
<strong>of</strong> Ran binds to RanBD2 in a manner analogous to the crystal structures. By solving the<br />
solution structure <strong>of</strong> the 28mer peptide alone we confirmed that it adopts an α-helical<br />
structure like in native Ran and therefore serves as a valid model <strong>of</strong> the Ran C-terminus.<br />
These results support current models that assume recognition <strong>of</strong> the transport<br />
complexes by the RanBDs through the Ran C-terminus that is exposed in these<br />
complexes.<br />
Literature<br />
(1) Seewald, M. J., Korner, C., Wittingh<strong>of</strong>er, A., & Vetter, I. R. (2002). Nature 415, 662-<br />
666.<br />
(2) Vetter, I. R., Nowak, C., Nishimoto, T., Kuhlmann, J., & Wittingh<strong>of</strong>er, A. (1999).<br />
Nature 398, 39-46.<br />
contact:<br />
Peter Geyer<br />
Universität Regensburg<br />
Institut für Biophysik und physikalische Biochemie<br />
peter.geyer@biologie.uni-regensburg.de<br />
Universitätsstrasse 31<br />
93053 Regensburg (Deutschland)
R. Gerke, M. Schmeer, T. Seipp, U. Pliquett, E. Neumann<br />
Spatial Resolution <strong>of</strong> Gene Expression by Membrane<br />
Electroporation <strong>of</strong> CHO Cell Layers<br />
Membrane electroporation (MEP) is a powerful technique that uses electric field pulses to<br />
render cell membranes transiently porous: electroporated cell membranes become<br />
permeable to otherwise impermeable substances. The various applications <strong>of</strong><br />
electroporation include the direct transfer <strong>of</strong> genes, proteins, ionic dyes and drugs into<br />
the cell interior. Clinical applications <strong>of</strong> this method gain increasing importance for the<br />
electrochemotherapy <strong>of</strong> skin tumors and gene therapy.<br />
The method <strong>of</strong> MEP is applicable only in small ranges <strong>of</strong> the applied electric field and<br />
pulse duration. Strong electric fields and long durations cause irreversible cell damage.<br />
When an oligo-layer <strong>of</strong> adherently-grown chinese hamster ovary (CHO) cells are<br />
electroporated in presence <strong>of</strong> green-fluorescence protein (GFP) plasmids, gene<br />
expression can be spatially visualized and quantitatively characterized. Knowledge <strong>of</strong> the<br />
spatial resolution <strong>of</strong> gene expression between the electrodes is the basis to improve not<br />
only electric gene transfer parameters but also the design <strong>of</strong> optimized electrodes<br />
minimizing unavoidable cell damage required for efficient electrochemotherapy and<br />
electrogenetherapy. The electric parameters for efficient electrotransfer, however, have<br />
to be experimentally optimized in each case. This refers to isolated cells as well as for<br />
the densely packed cells in tissue or oligo-layers <strong>of</strong> adherent cells in culture dishes.<br />
Literature<br />
Miklav•i•, D. et.al., Biophys. J., 74, 1998, 2152-2158.<br />
Mir, L. M., Bioelectrochem., 53, 2000, 1-10.<br />
Neumann, E., Bioelectrochem. Bioenerg., 28, 1992, 247-267.<br />
Neumann, E., Schaefer-Ridder, M., Wang, Y., H<strong>of</strong>schneider, P. H., EMBO J., 1982, 841-<br />
845.<br />
contact:<br />
Dipl. Chem. Robert Gerke<br />
Universität Bielefeld<br />
Fakultät Chemie<br />
robert.gerke@uni-bielefeld.de<br />
Postfach 100 131<br />
33501 Bielefeld (Deutschland)
Gert Schwach, M. Tschemmernegg, E. Schreiner, M. Windisch, R. Pfragner, E. Masliah,<br />
E. Rockenstein, E. Ingolic<br />
Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Background: Human alpha-synuclein, a 140 aminoacid polypeptide was originally<br />
isolated from plaques <strong>of</strong> Alzheimer´s disease (AD) brain as a protein precursor <strong>of</strong> the<br />
nonamyloid component (NAC) <strong>of</strong> plaques. The NAC peptide can self-aggregate and<br />
induces aggregation <strong>of</strong> the ß-amyloid peptide. alpha-synuclein is abundant in<br />
presynaptic terminals and Lewy bodies found in a variant <strong>of</strong> AD, diffuse Lewy body<br />
disease and Parkinson´s disease (PD). Studies with transgenic mice expressing human<br />
wild-type (wt) alpha-synuclein also suggest that accumulation <strong>of</strong> this protein may play a<br />
potential role in synaptic function and neural plasticity.<br />
Methods: Plasmids expressing the wt and a mutant human alpha-synuclein regulated by<br />
either the CMV (cytomegalovirus) or the PDGF-ß (platelet derived growth factor-ß)<br />
promoter were prepared and used for creating stably transfected neuronal rat cell lines.<br />
RNA expression levels <strong>of</strong> the different targets were analyzed with ribonuclease protection<br />
assays. Using selected clones a preliminary characterisation was done with Western blot<br />
technique and cell culture experiments.<br />
Results and Interpretation: A great number <strong>of</strong> neuronal clones were prepared for<br />
analysing their levels <strong>of</strong> alpha-synuclein expression depending on the different regulating<br />
promoter sequences and target genes used. Specific clones were selected to test<br />
substances interesting in PD.<br />
Conclusions: Such in vitro model systems could represent rapid and valuable tools for<br />
testing new compounds for the treatment <strong>of</strong> Parkinson´s disease and similar disorders<br />
especially in early stages <strong>of</strong> drug development.<br />
contact:<br />
Mag Gert Schwach<br />
JSW Research/ Medizinische Universität Graz<br />
Institut für Pathophysiologie<br />
gschwach@jswreseach.com<br />
Rankengasse 28<br />
8020 Graz (Austria)<br />
additional information<br />
E.R, E.M.: Department <strong>of</strong> Neurosciences, University <strong>of</strong> California San Diego, USA<br />
R.P.: Departments <strong>of</strong> General and Experimental Pathology, University <strong>of</strong> Graz, Austria<br />
E.I.: Department <strong>of</strong> Electron Microscopy, Technical University Graz, Austria
Anna M. Wobus<br />
Stammzellforschung: Potenzial, Perspektiven und Probleme<br />
Im Organismus höherer Lebewesen haben Stammzellen die Funktion, Gewebe und<br />
Organe des Körpers zu bilden und zu erhalten. Pluripotente embryonale Stammzellen<br />
(ES-Zellen), etabliert aus frühen Embryonalstadien von Maus und Mensch, haben das<br />
Potenzial, sich in Kultur nahezu unbegrenzt vermehren und in zahlreiche Zellen des<br />
Körpers entwickeln zu können. Während der Differenzierung in Kultur rekapitulieren ES-<br />
Zellen das Programm der frühen Embryonalentwicklung. Mit Hilfe genetischer Verfahren,<br />
spezifischer Selektionsmethoden und dem Einsatz von Differenzierungsfaktoren können<br />
funktionell aktive Herz-, Nerven-, Muskel-, glatte Gefäßmuskel-, Leber- oder<br />
pankreatische Beta-Zellen generiert werden. Mit der Etablierung humaner ES-Zellen aus<br />
in vitro befruchteten Embryonen verknüpfen sich nun H<strong>of</strong>fnungen auf eine Nutzung<br />
dieser Zellen für eine Zell- und Gewebetherapie beim Menschen.<br />
Neben embryonalen Stammzellen existieren im fötalen und erwachsenen Organismus<br />
auch gewebespezifische (adulte) Stammzellen, die für die Regeneration der einzelnen<br />
Gewebesysteme verantwortlich sind. Adulte Stammzellen haben im Vergleich zu ES-<br />
Zellen ein geringeres Vermehrungs- und Entwicklungspotenzial. Obwohl adulte<br />
Stammzellen über eine gewisse Flexibilität ihrer Entwicklungseigenschaften verfügen,<br />
konnten Berichte, wonach sie sich stabil in Zellen und Gewebe anderer (fremder) Linien<br />
entwickeln und neue gewebespezifische Funktionen ausprägen können, in der Regel<br />
nicht bestätigt werden. Die Phänomene wurden u. a. auf Fusionen der Spenderzellen mit<br />
Zellen des Empfängerorganismus zurückgeführt. Inwieweit es in der Zukunft gelingen<br />
wird, adulte Stammzellen stabil in verschiedenste Zelltypen zu entwickeln, ist derzeit<br />
noch Gegenstand der Grundlagenforschung. Möglicherweise sind adulte Stammzellen, z.<br />
B. aus dem Knochenmark, jedoch indirekt an der Induktion von Regenerationsprozessen<br />
im erwachsenen Organismus beteiligt.<br />
Der Vortrag wird am Beispiel der Entwicklung von Insulin-produzierenden Zellen der<br />
Bauchspeicheldrüse einen Überblick über den gegenwärtigen Stand der<br />
Stammzellforschung geben. Es werden Methoden vorgestellt, die es in der Zukunft<br />
ermöglichen sollen, pankreatische Zellen in vitro zu generieren, Vorläuferzellen zu<br />
isolieren, oder zu aktivieren, um sie für zukünftige Therapien einzusetzen. Dabei werden<br />
auch die <strong>of</strong>fenen Fragen und Probleme behandelt, die mit dem Einsatz embryonaler bzw.<br />
adulter Stammzellen verbunden sind, und die gelöst werden müssen, ehe Stammzellen<br />
in zelltherapeutischen Verfahren eingesetzt werden können.<br />
Literature<br />
Czyz, J., C. Wiese, A. Rolletschek, P. Blyszczuk, M. Cross, and A.M. Wobus; Biol Chem.,<br />
384, 1391-1409. (2003)<br />
Blyszczuk, P., J. Czyz, G. Kania, M. Wagner, U. Roll, L. St Onge, and A.M. Wobus; PNAS,<br />
100, 998- 1003. (2003)<br />
Blyszczuk, P. and A.M. Wobus; J. Biotech. (2004, in press)<br />
Kania, G., P. Blyszczuk, J. Czyz, A. Navarrete-Santos, and A.M. Wobus; Methods<br />
Enzymol., 365, 287-303. (2003)<br />
Wobus, A.M., Kontinuität und Plastizität im Organismus. Nova Acta Leopoldina NF 86,<br />
Nr. 324: 49-76 (2002)<br />
contact:<br />
Pr<strong>of</strong>. Anna M. Wobus<br />
In Vitro Differentiation Group, Institut für Pflanzengenetik (IPK), Leibniz-Institut<br />
wobusam@ipk-gatersleben.de<br />
Corrensstraße 3<br />
06466 Gatersleben (Deutschland)
Bernd Schmeck, Ralph Gross, Phillipe Dje N´Guessan, Andreas C. Hocke, Sven Sven<br />
Hammerschmidt, Tim J. Mitchell, Simone Rosseau, Norbert Suttorp, Stefan Hippenstiel<br />
Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Background: Streptococcus pneumoniae is the major pathogen <strong>of</strong> community acquired<br />
pneumonia and one <strong>of</strong> the most common causes <strong>of</strong> death due to infectious diseases in<br />
industrialized countries. Lung epithelium lines the airways and constitutes the first line <strong>of</strong><br />
innate defense against respiratory pathogens. Little is known about the molecular<br />
interaction <strong>of</strong> pneumococci with lung epithelial cells. Apoptosis <strong>of</strong> lung epithelium is<br />
involved in some bacterial lung infection.<br />
Results: Different pneumococcal strains specifically induced either apoptotic or necrotic<br />
death <strong>of</strong> human alveolar and bronchial epithelial cells. Pneumococci-induced apoptosis<br />
did not depend on the virulence factors pneumolysin or H2O2. Apoptotic cells showed<br />
increased activity <strong>of</strong> caspase 6, 8, and 9, but not 3. Moreover, programmed cell death<br />
could be strongly reduced by a caspase 6 inhibitor and a pan-caspase inhibitor.<br />
Inhibitors <strong>of</strong> calpain, chymotrypsin- and trypsin-like proteases also reduced pneumococciinduced<br />
apoptosis. Furthermore, pneumococci-infected human alveolar epithelial cells<br />
showed Bid cleavage and reduced levels <strong>of</strong> Bcl2 and Bax. Overexpression <strong>of</strong> Bcl2 in<br />
these cells reduced apoptosis significantly.<br />
Conclusion: Pneumococci induced apoptosis <strong>of</strong> human alveolar and bronchial epithelial<br />
cells. Programmed cell death was executed by caspase 6 and non-caspase proteases,<br />
but not caspase 3, and could be blocked by overexpression <strong>of</strong> Bcl2.<br />
Literature<br />
contact:<br />
Dr. Bernd Schmeck<br />
Charité - Universitätsmedizin Berlin<br />
Medizinische Klinik m.S. Infektiologie<br />
Bernd.Schmeck@charite.de<br />
Augustenburger Platz 1<br />
13353 Berlin (Deutschland)
Michael Lammers, Rolf Wagner, Atsushi Shimada, Alfred Wittingh<strong>of</strong>er<br />
Structural and biochemical characterisation <strong>of</strong> the Rho<br />
effector mDia<br />
GTP-binding proteins <strong>of</strong> the Rho subfamily cycle between an inactive GDP-bound and an<br />
active GTP-bound state [1]. In the GTP-bound form, these proteins interact with<br />
effectors, which are <strong>of</strong>ten involved in regulating the polymerisation and organisation <strong>of</strong><br />
the actin cytoskeleton [2]. Here, we report on the structural and biochemical<br />
characterisation <strong>of</strong> the downstream Rho effector protein mDia which belongs to the<br />
family <strong>of</strong> formin proteins. These have a poly-proline-rich formin homology-1 (FH1)<br />
domain in common, which binds pr<strong>of</strong>ilin and SH3 or WW domain containing proteins. The<br />
structurally defined FH2 domain is the catalytic domain <strong>of</strong> the protein, catalysing the<br />
formation <strong>of</strong> unbranched actin-filaments [3]. The FH3 domain is the least conserved<br />
formin homology domain with undefined structure and length. In the diaphanous related<br />
formins (Drfs) this domain overlaps the N-terminal GTPase binding domain (GBD). The<br />
Drfs are regulated by the C-terminal diaphanous autoregulatory domain (DAD). Binding<br />
to the GBD inhibits its biological activity [4] [5], whereas binding <strong>of</strong> active Rho to this<br />
domain abolishes this inhibition thus enabling mDia to mediate its effect concerning the<br />
reorganisation <strong>of</strong> the actin-cytoskeleton [6] and microtuble alignment [7] [8]. The<br />
structure <strong>of</strong> the FH2 domain <strong>of</strong> mouse mDia1 was determined showing that it is an αhelical<br />
crescent-shaped molecule. In biochemical studies the interaction between<br />
RhoA*GTP and the GBD was characterised. The bicistronically expressed GBD-DAD<br />
complex was purified, the affinity between the GBD and the DAD was examined and the<br />
dissociative effect <strong>of</strong> active Rho protein on the GBD-DAD complex was determined.<br />
Literature<br />
[1] Etienne-Manneville and Hall (2002) Nature 420:629-635<br />
[2] Bishop and Hall (2000) Biochem. J. 348 Pt2:241-255<br />
[3] Shimada et al. (2004) Mol, Cell 13: 511-522<br />
[4] Watanabe et al.(1999) Nat. Cell Biol. 1: 136-143<br />
[5] Alberts (2001) J. Biol. Chem. 276: 2824-2830<br />
[6] Watanabe et al. (1997) Embo J. 16: 3044-3056<br />
[7] Ishizaki et al. (2001) Nat. Cell. Biol. 3: 8-14<br />
[8] Palazzo et al. (2001) Nat. Cell. Biol. 3: 723-729<br />
contact:<br />
Michael Lammers<br />
Max-Planck-Institut für molekulare Physiologie<br />
michael.lammers@mpi-dortmund.mpg.de<br />
Otto-Hahn-Str. 11<br />
44227 Dortmund (Germany)
Claudia Hoemme, Jens Schwamborn, Alexander Antipenko, Dimitar B. Nikolov, Andreas<br />
W. Püschel<br />
Structural and functional analysis <strong>of</strong> the axon guidance<br />
molecule Sema3A and its receptor<br />
The secreted semaphorin 3A (Sema3A) belongs to a large protein family that is involved<br />
in the patterning <strong>of</strong> neuronal connections in the developing nervous system <strong>of</strong> both<br />
vertebrates and invertebrates. Sema3A acts as a chemorepellant for many types <strong>of</strong><br />
axons by activating a receptor complex that contains neuropilin-1 (Nrp-1) as the ligand<br />
binding and an A-type plexin as the signal-transducing subunit. In addition to its<br />
repulsive effects, Sema3A also acts as an attractant for cortical dendrites.<br />
To elucidate the molecular mechanisms underlying Sema3A-mediated chemoattraction,<br />
we used PC12 cells as a model system for neuronal differentiation. Sema3A is as<br />
effective as NGF in stimulating the extension <strong>of</strong> neurites by PC12 cells and this response<br />
depends on Nrp-1. Inhibition <strong>of</strong> cGMP production or MAPK activity completely block<br />
extension <strong>of</strong> neurites in response to Sema3A but not to NGF. The repulsive effects <strong>of</strong><br />
Sema3A are mediated in part by regulating the balance <strong>of</strong> Rho and Rac activity.<br />
Therefore, we analyze the involvement <strong>of</strong> different signaling pathways and GTPases in<br />
neurite extension <strong>of</strong> PC12 cells in response to Sema3A.<br />
Semaphorins are defined by the presence <strong>of</strong> a conserved domain <strong>of</strong> 500 amino acids at<br />
their amino termini that determines their receptor specificity. The crystal structure <strong>of</strong> a<br />
65 kDa form <strong>of</strong> Sema3A revealed the semaphorin fold as a variation <strong>of</strong> the beta propeller<br />
topology. Structure-based mutagenesis identified a dimerization and Nrp-1 binding site<br />
in the semaphorin domain. In addition, the structure suggested the presence <strong>of</strong> a second<br />
potential site <strong>of</strong> interaction between semaphorin domains. Here we report on the<br />
progress in the functional characterization <strong>of</strong> these binding sites based on the crystal<br />
structure.<br />
contact:<br />
Claudia Hoemme<br />
Universität Münster<br />
Institut für Allgemeine Zoologie und Genetik<br />
claudia.hoemme@gmx.de<br />
Schlossplatz 5<br />
48149 Münster (Germany)
Martin Kühn, Lakshmi Pulagam, Denis Duché, Anton Savitsky, Klaus Möbius, Heinz-<br />
Juergen Steinh<strong>of</strong>f<br />
Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
The study <strong>of</strong> the structure and structural changes <strong>of</strong> proteins is the key to a deeper<br />
understanding <strong>of</strong> protein function. Although NMR and crystallographic methods are<br />
powerful tools for exploring protein structures, they may not be applicable to membrane<br />
proteins in general. In this case EPR spectroscopy in combination with site-directed spin<br />
labeling (SDSL) is emerging as a powerful method to get specific information about<br />
secondary structure, polarity, mobility and intramolecular distances at selected sites <strong>of</strong><br />
the protein [1, 2, 3]. This method is applied to the bacterial toxin colicin A. One <strong>of</strong> its<br />
three domains forms a voltage gated ion channel in the cytoplasmic membrane <strong>of</strong> E.coli.<br />
Although the water soluble structure <strong>of</strong> colicin A is known by crystallographic methods,<br />
detailed models for the membrane bound conformations are still missing. By<br />
examination <strong>of</strong> the polarity pr<strong>of</strong>ile and accessibility for paramagnetic quenchers for spin<br />
labels bound at different sites <strong>of</strong> helix 9, the orientation <strong>of</strong> this prominent helix with<br />
respect to the membrane can be revealed for the closed channel. Low temperature EPR<br />
spectra at 95GHz extend the polarity pr<strong>of</strong>ile by determination <strong>of</strong> the g tensor component<br />
gxx, that could recently be correlated with the proticity <strong>of</strong> the binding site environment<br />
[4]. Although studies <strong>of</strong> the related colicin E1 seem to confirm a transmembrane<br />
orientation <strong>of</strong> helix 9, our results reveal a parallel orientation in case <strong>of</strong> colicin A [5].<br />
Literature<br />
[1] Hubbell, W.L., Gross, A., Langen, R., Lietzow, M.A. Current Opinion in<br />
Structural Biology 8:649-56(1998)<br />
[2] Pfeiffer, M., Rink, T., Gerwert, K., Oesterhelt, D., Steinh<strong>of</strong>f, H.-J.<br />
Journal <strong>of</strong> Molecular Biology 287:163-72(1999)<br />
[3] Wegener, A. A., Klare, J. P., Engelhard, M., Steinh<strong>of</strong>f, H.-J. EMBO J. 20:5312-9<br />
(2001)<br />
[4] Steinh<strong>of</strong>f, H.-J., Savitsky, A., Wegener, C., Pfeiffer, M., Plato, M.<br />
Möbius, K. Biochemica et Biophysica Acta 1457:253-62(2000)<br />
[5] Savitsky, A., Kühn, M., Duché, D., Möbius, K., Steinh<strong>of</strong>f, H.-J. Journal<br />
<strong>of</strong> Physical Chemistry B, in press.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Heinz-Juergen Steinh<strong>of</strong>f<br />
Universität Osnabrück<br />
Physik<br />
hsteinho@uos.de<br />
Barbarastrasse 7<br />
49069 Osnabrück (Deutschland)
Oliver Einsle, Peter MH Kroneck<br />
Structural Basis <strong>of</strong> Denitrification<br />
Denitrification represents an important part <strong>of</strong> the biogeochemical cycle <strong>of</strong> nitrogen. It<br />
constitutes the predominant pathway <strong>of</strong> the reductive dissimilation <strong>of</strong> nitrate in our<br />
environment. Via four enzymatic reactions, nitrate is transformed stepwise to nitrite,<br />
nitric oxide, nitrous oxide to finally produce dinitrogen gas(1). All four steps are<br />
catalyzed by complex multi-site metalloenzymes with unique structural and<br />
spectroscopic features. In recent years, high resolution X-ray structures have been<br />
published with the exception <strong>of</strong> the structure for the membrane-bound NO reductase (2).<br />
In this contribution we will review the structural, catalytic and spectroscopic properties<br />
<strong>of</strong> the enzymes <strong>of</strong> the dentrification pathway.<br />
Literature<br />
(1) W.G. Zumft, Microbiol. Mol. Rev., 61, 533-616 (1997)<br />
(2) M. Rudolf and P.M.H. Kroneck, Metal Ions in Biological Systems (A. Sigel, H. Sigel,<br />
and R.K.O. Sigel, eds), Vol. 43, in press (2005)<br />
contact:<br />
Pr<strong>of</strong>. Dr. Peter MH Kroneck<br />
Universitaet Konstanz<br />
Fachbereich Biologie<br />
peter.kroneck@uni-konstanz.de<br />
Universitaetsstrasse 10<br />
78457 Konstanz (Germany)<br />
additional information<br />
Pr<strong>of</strong>. Dr. Oliver Einsle<br />
Universität Göttingen, Molekulare Strukturbiologie, Göttingen, Germany
Anke Vahrmann, Anna Szkodowska, Christoph Linke, Monika C. M Mueller, Tilly Bakker-<br />
Grunwald, Henning Scholze<br />
Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia<br />
Annexins represent a multigene family <strong>of</strong> eukaryotic proteins with various, but in detail<br />
mostly unknown functions. We have identified a novel annexin, ANXE1, in a cell extract<br />
<strong>of</strong> trophozoites <strong>of</strong> Giardia lamblia (1). The annexin has a true type II Ca 2+ -binding motif<br />
(GXGTD{38}D) in the second annexin repeat and a further rudimentary one in the<br />
fourth repeat. Furthermore, in its C-terminal region, some elements typically occurring<br />
in α-giardins are conspicuous, such as an ITG/AM- and a KXXYK-motif, and a Trp residue<br />
followed by a charged residue next to the C terminus. All these elements are not<br />
conserved in the annexins <strong>of</strong> higher eukaryotes. According to molecular modelling <strong>of</strong> the<br />
sequence based on known annexin structures, these residues are positioned next to the<br />
concave side <strong>of</strong> the molecule, which in other annexins is orientated to the cytoplasmic<br />
face <strong>of</strong> the molecule. Western blot analyses <strong>of</strong> isolated giardial flagella revealed a strong<br />
cross reaction with anti-ANXE1 antibodies. This finding was confirmed by<br />
immunocytochemical analyses which show that ANXE1 is a constitutive part <strong>of</strong> the eight<br />
giardial flagella as well as <strong>of</strong> the median body <strong>of</strong> the trophozoites. Electron microscopy <strong>of</strong><br />
the purified recombinant annexin E1 suggests that the protein is able to self assemble<br />
into fibrils. Taken together, we hypothesize that ANXE1 may function as a Ca 2+ -<br />
regulated structural element linking the phospholipid bilayer to the underlying axoneme.<br />
Literature<br />
(1) Szkodowska, A., Müller, M. C. M., Linke, C. and Scholze, H.(2002) Annexin XXI<br />
(ANX21) <strong>of</strong> Giardia lamblia has sequence motifs uniquely shared by giardial annexins<br />
and is specifically localized in the flagella. J. Biol. Chem.277, 25703-25706<br />
contact:<br />
Anke Vahrmann<br />
University <strong>of</strong> Osnabrueck<br />
Department <strong>of</strong> Biology/Chemistry<br />
Vahrmann@biologie.uni-osnabrueck.de<br />
Barbarastr. 11<br />
49076 Osnabrueck (Germany)
Birgit von Janowsky, Karin Röttgers, Martin Krayl, Wolfgang Voos<br />
Structural properties <strong>of</strong> the substrate protein determine<br />
degradation by the mitochondrial AAA+ protease PIM1<br />
Mitochondrial protein-quality control requires the specific recognition and removal <strong>of</strong><br />
damaged or misfolded protein molecules. Protein degradation in the matrix is carried out<br />
by the protease Pim1/LON. Although Pim1 has a preference for misfolded proteins, the<br />
basis for the specific recognition <strong>of</strong> substrate proteins has not been elucidated. It was<br />
shown that Pim1/LON cooperates with mitochondrial chaperone proteins <strong>of</strong> the Hsp70<br />
family in the degradation <strong>of</strong> soluble proteins that were imported into the matrix. To<br />
study the properties <strong>of</strong> substrate proteins that lead to their removal by Pim1, we<br />
employed an in organello assay system for proteolysis. The assay is based on the<br />
degradation <strong>of</strong> reporter proteins that are preloaded into intact mitochondria by an in<br />
vitro import reaction. We analyzed the dependence <strong>of</strong> the degradation on the size and<br />
folding state <strong>of</strong> the substrate proteins under in vivo conditions. Proteolysis <strong>of</strong> the<br />
imported substrate proteins was dependent on the mitochondrial level <strong>of</strong> ATP.<br />
Mitochondria from hsp78∆ cells exhibited a significant defect in the degradation<br />
efficiency. The proteolysis defect in the hsp78∆ mitochondria did not correlate with the<br />
aggregation behavior <strong>of</strong> the substrate proteins. Hsp78 is most likely a targeting<br />
component <strong>of</strong> the degradation system, enhancing the efficiency <strong>of</strong> the proteolysis<br />
reaction. Pim1 was not able to degrade proteins that contained an amino-terminal<br />
unstructured segment <strong>of</strong> less than 60 amino acid residues and lacked a robust<br />
endogenous unfolding activity. We conclude that an unstructured protein segment<br />
exceeding a certain minimal length is a major determinant <strong>of</strong> the substrate recognition<br />
process <strong>of</strong> Pim1/LON and governs the selectivity for damaged proteins.<br />
Literature<br />
Voos, W. & Röttgers, K. (2002). BBA, 1592, p. 51<br />
Röttgers, K. et al. (2002). J. Biol. Chem. 277, p. 45829<br />
contact:<br />
PD Dr. Wolfgang Voos<br />
Universität Freiburg<br />
Inst. für Biochemie und Molekularbiologie<br />
wolfgang.voos@biochemie.uni-freiburg.de<br />
Hermann-Herder-Str. 7<br />
79104 Freiburg (Baden-Württemberg)
Daniel Hilger, Jeschke Gunnar, Christoph Wegener, Heinz-Jürgen Steinh<strong>of</strong>f, Heinrich<br />
Jung<br />
Structure and dynamics <strong>of</strong> the sodium/proline transporter<br />
PutP <strong>of</strong> E. coli: a protein chemical and EPR spectroscopic<br />
analysis<br />
Sodium is a common coupling ion used to drive solute uptake into pro- and eukaryotic<br />
cells. To obtain insights into the molecular mechanism <strong>of</strong> function <strong>of</strong> corresponding<br />
transport systems we utilize the sodium/proline transporter PutP <strong>of</strong> E. coli as a model<br />
system. PutP is an integral protein <strong>of</strong> the cytoplasmic membrane and catalyzes the<br />
sodium-coupled uptake <strong>of</strong> proline with a 1:1 stoichiometry. As other secondary<br />
transporters, PutP is thought to undergo a series <strong>of</strong> conformational transitions such that<br />
the ligand-binding sites are alternatively accessible to one side <strong>of</strong> the membrane or the<br />
other. We used site-directed labeling techniques in combination with electron<br />
paramagnetic resonance (EPR) spectroscopy to obtain information on tertiary<br />
interactions and conformational alterations related to PutP function. Crosslinking was<br />
observed between pairs <strong>of</strong> Cys residues located at various positions along helices II and<br />
IX and adjacent loops. In addition, distances between amino acid positions were<br />
estimated by continuous wave and pulsed EPR measurements. The studies confirm, e.g.,<br />
a proximity between transmembrane helices II and IX, a finding consistent with the<br />
proposed participation <strong>of</strong> both domains in the formation <strong>of</strong> a solute translocation<br />
pathway. Furthermore, the results demonstrate a large conformational flexibility <strong>of</strong> the<br />
protein regions investigated.<br />
Literature<br />
1. Pirch, T., Landmeier, S., and Jung, H. (2003) J.Biol.Chem. 278, 42942-42949.<br />
2. Jeschke, G., Wegener, C., Nietschke, M., Jung, H., and Steinh<strong>of</strong>f, H. J. (2004)<br />
Biophys.J. 86, 2551-2557.<br />
contact:<br />
apl. Pr<strong>of</strong>. Dr. Heinrich Jung<br />
LMU München<br />
Mikrobiologie<br />
heinrich.jung@lrz.uni-muenchen.de<br />
Maria-Ward-Strasse 1a<br />
80638 München (Deutschland)<br />
additional information<br />
G. Jeschke: MPInstitut für Polymerforschung, Mainz<br />
C. Wegener, H.-J. Steinh<strong>of</strong>f: Universität Osnabrück, Fachbereich Physik
Tom Rapoport<br />
Structure and function <strong>of</strong> a protein-conducting channel<br />
A conserved heterotrimeric membrane protein complex (Sec61/ SecYcomplex) forms a<br />
protein-conducting channel, allowing polypeptides to be transferred across or integrated<br />
into membranes. The channel associates with different partners in different translocation<br />
pathways: the ribosome in cotranslational translocation, the Sec62/63p complex and BiP<br />
in posttranslational translocation in eukaryotes, and SecA in posttranslational<br />
translocation in bacteria. We have recently solved the crystal structure <strong>of</strong> the SecY<br />
complex from Methanococcus jannaschii at 3.2 Å resolution. The structure suggests<br />
mechanisms for how the membrane barrier can be maintained during translocation, how<br />
the signal sequence is recognized, and how TMs <strong>of</strong> nascent membrane proteins exit into<br />
lipid.<br />
contact:<br />
Pr<strong>of</strong>essor Tom Rapoport<br />
HHMI/Harvard Medical School<br />
tom_rapoport@hms.harvard.edu<br />
240 Longwood Avenue<br />
02115 Boston (USA)
Prasad Gajula, Igor Borovykh*, Christian Beier, Peter Gast*, Heinz-Juergen Steinh<strong>of</strong>f<br />
Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled<br />
photosynthetic reaction centers from Rhodobacter<br />
sphaeroides based on molecular dynamics simulations and<br />
EPR spectroscopy.<br />
To understand the photosynthetic machinery it is essential to perform a detailed analysis<br />
<strong>of</strong> the involved proteins. The functionality <strong>of</strong> these bio molecules is strongly related to<br />
the arrangement and dynamics <strong>of</strong> the embedded chromophore groups, a study <strong>of</strong> the<br />
dynamical behavior <strong>of</strong> the environmental protein regions is required for a general<br />
understanding <strong>of</strong> the electron transfer process. The electron paramagnetic spectroscopy<br />
<strong>of</strong> spin labels (SDSL-EPR) [1,2] is a powerful method to analyse local structural dynamics<br />
over a wide time range. In addition a newly developed EPR spectra calculation<br />
algorithm [3] allows to directly compare EPR spectra obtained from experiment with the<br />
results <strong>of</strong> specific molecular dynamics simulations.<br />
The Reaction Center (RC) <strong>of</strong> the purple bacteria Rhodobacter sphaeroides contains five<br />
native cysteine molecules, out <strong>of</strong> which three cysteines are bound to the L and two to<br />
the H subunit (positions 92, 108, 247 and 156, 234 respectively) [4] . Molecular dynamics<br />
simulations are performed with virtual spin labels bound to each native cysteine to<br />
analyse the reorientational dynamics at those sites. EPR spectra calculated on the basis<br />
<strong>of</strong> these MD simulations reveal that the predicted spectrum <strong>of</strong> position 156 is most<br />
consistent with the measured spectrum <strong>of</strong> the spin labeled wild type RC. Comparison <strong>of</strong><br />
simulated and experimental spectra at different temperatures give insights into the role<br />
<strong>of</strong> possible conformational changes <strong>of</strong> RC.<br />
Literature<br />
[1] Steinh<strong>of</strong>f, H.-J.(2002) Frontiers in Bioscience 7:c97-110.<br />
[2] Hubbell, W.L., Mchaourab, H., Altenbach, C., Lietzow, M.(1996) Structure 4:779-783.<br />
[3] Steinh<strong>of</strong>f, H.-J., Müller, M., Beier, C. and Pfeiffer M. (2000) J.Molecular Liquids 84:17-<br />
27<br />
[4] Stowell, M.H.B., McPhillips, T. M., Rees, D.C., Soltis, S.M., Abresch, E. and Feher, G.<br />
(1997) Science 276:812-816<br />
contact:<br />
Prasad Gajula<br />
University <strong>of</strong> Osnabrueck<br />
Institute for Macromolecular Structure<br />
prasad.gajula@uos.de<br />
Barbarastr-7<br />
49069 Osnabrueck (Germany)<br />
additional information<br />
*) Department <strong>of</strong> Biophysics, Huygens Laboratory, Leiden, The Netherlands
Evelyn Hollnack, Tanja Eisenblätter, Hans-Joachim Galla<br />
Substrate and inhibitor specificity <strong>of</strong> the brain multidrug<br />
resistance protein (BMDP) at he blood-brain barrier<br />
The blood-brain barrier (BBB) controls the passage <strong>of</strong> endogenous and xenobiotic<br />
substances between the blood and the interstitial fluid <strong>of</strong> the brain. The transfer <strong>of</strong><br />
compounds is strictly regulated by brain capillary endothelial cells, which are<br />
interconnected by tight intercellular junctions. Porcine brain capillary endothelial cells<br />
(PBCEC) cultured in serum-free and hydrocortisone supplemented medium closely mimic<br />
the BBB phenotype in vitro . A new multidrug resistance protein named brain multidrug<br />
resistance protein (BMDP) was found at the BBB. It shows a 86% amino acid identity to<br />
the human breast cancer resistance protein (BCRP) [1, 2].<br />
It is our objective to elucidate the physiological role <strong>of</strong> BMDP at the BBB in comparison<br />
to other well-known drug transporters like P-Gp and MRP1. We try to establish a<br />
substrate and inhibitor spectrum <strong>of</strong> BMDP and compare it with those <strong>of</strong> the BCRP.<br />
Several radioactive labelled substances in combination with different inhibitors are tested<br />
in active transport studies. While daunomycin transport shows a high rate and could be<br />
inhibited by e. g. GF 120918, PSC 833 and cyclosporin, vincristine is less transported<br />
and inhibited only weakly by PSC 833. Methotrexate is not accepted and hydrocortisone<br />
is hardly transported. In cytotoxicity-tests using daunomycin as substrate a fourfold<br />
decrease <strong>of</strong> the EC 50 -value could be observed in the case <strong>of</strong> GF 120918 and the PBCEC<br />
respond threefold more sensitively if no hydrocortisone is added.<br />
Literature<br />
(1) Eisenblätter, T. and Galla, H.-J. (2002) A new multidrug resistance protein at the<br />
blood-brain barrier Biochem Biophys Res Commun 293, 1273-1278<br />
(2) Eisenblätter, T. and Galla, H.-J. (2002) Characterisation <strong>of</strong> the brain multidrug<br />
resistance protein (BMDP) expressed at the blood-brain-barrier Brain Res 971, 221-231<br />
contact:<br />
Evelyn Hollnack<br />
Westfälische Wilhelms-Universität Münster<br />
Institut für Biochemie/AK Galla<br />
evelynh@uni-muenster.de<br />
Wilhelm-Klemm-Str.2<br />
48149 Münster (Deutschland)
Angela Magin, Heike Partenheimer, Claudia Lange, Michael Lietz, Thomas Noll<br />
Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
To identify cells obtained from the Wharton's Jelly <strong>of</strong> human umbilical cords (WJC), we<br />
compared morphology and surface marker expression pr<strong>of</strong>iles <strong>of</strong> these cells with other<br />
human primary cells. Our reference cells included mesenchymal stem cells from bone<br />
marrow (MSC), arterial smooth muscle cells (SMC) and umbilical cord vein endothelial<br />
cells (HUVEC). MSC and SMC both showed a spindle-shaped morphology, whereas<br />
HUVEC had the typical appearance <strong>of</strong> cultured endothelial cells. WJC had a fibroblastlike,<br />
spindle-shaped morphology.<br />
For phenotypical characterisation, a panel <strong>of</strong> fluorochrome-conjugated monoclonal<br />
antibodies directed against cell surface antigens was used. The investigated antigens<br />
were grouped into markers typically expressed on cells <strong>of</strong> different hematopoietic<br />
lineages (CD3, 4, 8, 11b, 14, 16, 38, 45), on endothelial cells (CD31, 105, 106, 144) or<br />
on fibroblasts and stem cells (CD34, 54, 90, 166). Furthermore, the expression <strong>of</strong><br />
adhesion molecules (CD31, 54, 106, 144, 166) and cytokine / chemokine receptors<br />
(CD71, 95, 114, 117, 123, CCR7, CXCR4) was characterised.<br />
Both in morphological and surface marker analysis, WJC do not exhibit typical<br />
characteristics <strong>of</strong> endothelial cells (e.g. no expression <strong>of</strong> CD144 and only weak<br />
expression <strong>of</strong> CD105). They do much more resemble to MSC and SMC suggesting WJC<br />
might be cells <strong>of</strong> mesenchymal origin evolving into smooth muscle cells.<br />
contact:<br />
Angela Magin<br />
Research Center Juelich<br />
Institute for Biotechnology 2<br />
a.magin@fz-juelich.de<br />
Leo-Brandt-Straße<br />
52425 Jülich (Deutschland)<br />
additional information<br />
Claudia Lange: Department Hematology / Oncology, University Hospital Hamburg-Eppendorf<br />
Michael Lietz: Department <strong>of</strong> Molecular Cardiovascular Research, RWTH Aachen
Rüdiger Hampp, Mika Tarkka, Uwe Nehls<br />
Symbiotic interactions at the root level <strong>of</strong> forest trees: receive,<br />
deliver and get protected<br />
Roots constitute important plant organs for water and nutrient uptake. They, however,<br />
also release a wide range <strong>of</strong> carbon compounds <strong>of</strong> low molecular weight which are called<br />
exudates. These compounds form the basis for an environment rich in diversified<br />
microbiological populations, the rhizosphere. Bacteria are an important part <strong>of</strong> these<br />
populations. In addition, roots <strong>of</strong> most terrestrial plants develop symbiotic structures<br />
(mycorrhiza) with soil-born fungi. In these interactions, the fungal partner provides the<br />
plant with improved access to water and nutrients in the soil, due to more or less<br />
complex hyphal structures that emanate from the root surface and extend far into the<br />
soil. The plant, in return, supplies carbohydrates for fungal growth and maintenance.<br />
Owing to leakage and the turnover <strong>of</strong> mycorrhizal structures, these solutes are also<br />
released into the rhizosphere where they can be accessed by other microogranisms.<br />
With this background, the presentation will deliver a current view about the molecular<br />
biology <strong>of</strong> interactions between soil bacteria and symbiotic fungi (both negative and<br />
positive), and with regard to the functioning <strong>of</strong> the plant/fungus symbiosis.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Rüdiger Hampp<br />
Universität Tübingen<br />
Physiologische Ökologie der Pflanzen<br />
ruediger.hampp@uni-tuebingen.de<br />
Auf der Morgenstelle 1<br />
72076 Tübingen (Deutschland)
Ferda Cevikbas, Kerstin Brands, Frank Echtermeyer<br />
Syndecan-4 Deficiency leads to an upregulation <strong>of</strong> syndecan-1<br />
in skin fibroblasts<br />
Deletion <strong>of</strong> the gene coding for the multifunctional transmembrane proteoglycan<br />
syndecan-4 in mice leads to a significant delayed wound closure rate between day 3 and<br />
6 after wounding. But on day 15 after wounding the wounds from synd-4 knockout and<br />
wild type mice are closed equally.<br />
Therefore we investigated whether the loss <strong>of</strong> synd-4 might be compensated by other<br />
members <strong>of</strong> the syndecan-family. We could show that the amounts <strong>of</strong> synd-1 mRNA and<br />
protein are elevated in synd-4 deficient fibroblasts whereas synd-2 expression remained<br />
unchanged.<br />
Furthermore, we analysed the influence <strong>of</strong> TGF-b1 on the expression <strong>of</strong> synd-1 in synd-4<br />
deficient fibroblasts since recent results from our lab showed that TGF-b1 induced<br />
differentiation is altered in synd-4 -/- fibroblasts. Stimulation by TGF-b1 resulted in a<br />
downregulation <strong>of</strong> the synd-1 expression in synd-4 -/- but not in wild type cells.<br />
contact:<br />
Ferda Cevikbas<br />
Universitätsklinikum Münster<br />
Physiologische Chemie und Pathobiochemie<br />
ferdacev@uni-muenster<br />
Waldeyerstr. 15<br />
48149 Münster (Deutschland)
Matthias F. Melzig, Philipp Hebestreit<br />
Synergistic action <strong>of</strong> saponins with type I Ribosimeinactivating<br />
proteins agrostin and saporin<br />
In the course <strong>of</strong> our investigation <strong>of</strong> Agrostemma githago L. var. githago (1), a well<br />
known toxic member <strong>of</strong> the Caryophyllaceae family, we tested Saponinum album from<br />
Gypsophila paniculata likewise with gypsogenin (3beta-hydroxy-olean-12-en-23-al-28oic<br />
acid) as aglycone. A combination <strong>of</strong> these particular saponin derivatives with a formyl<br />
function in triterpene position 4 (3 µg/ml) together with RIPs and other natural toxins<br />
revealed a co-operative toxicity against ECV-304 cells. Ribosome-inactivating proteins<br />
(RIPs; EC 3.2.2.22) are a heterogeneous family <strong>of</strong> structurally and evolutionary related<br />
plant proteins sharing a common functional domain that catalytically removes a specific<br />
adenine residue from a highly conserved, surface-exposed stem-loop structure found in<br />
the large rRNA <strong>of</strong> prokaryotic and eukaryotic ribosomes (2,3). The combined<br />
administration <strong>of</strong> individually non-toxic concentrations <strong>of</strong> a RIP type 1 and a saponin<br />
presented in this study leads to a potent cytotoxic effect on tumor cells. An analogy<br />
could be drawn between the observed induction <strong>of</strong> RIP-toxicity <strong>of</strong> Agrostin and Saporin<br />
from Saponaria <strong>of</strong>ficinalis by Gypsophilasaponin. Obviously these proteins, both obtained<br />
from the seeds <strong>of</strong> Caryophyllaceae species, use a similar mechanism to penetrate<br />
through the cell membrane in vitro by support <strong>of</strong> saponins suggesting a similar defence<br />
mechanism <strong>of</strong> these plants against pathogenic organisms.<br />
Literature<br />
1. Hebestreit P, Melzig MF (2003) Planta Med 69: 921-5<br />
2. Endo Y, Tsurugi K (1987) J Biol Chem 262: 8128-30<br />
3. Stirpe F, Gasperi-Campani A, Barbieri L, Falasca A, Abbondanza A, Stevens WA<br />
(1983) Biochem J 216: 617–25<br />
contact:<br />
Pr<strong>of</strong>. Dr. Matthias F. Melzig<br />
Freie Universität Berlin<br />
Institut für Pharmazie<br />
melzig@zedat.fu-berlin.de<br />
KöniginLuise-Str. 2+4<br />
14195 Berlin (Deutschland)
Caroline E. Chwieralski, Ingo Schnurra, Lars Thim, Werner H<strong>of</strong>fmann<br />
Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-<br />
2B<br />
bronchial epithelial cells depend on different signaling<br />
cascades<br />
A process termed "restitution" enables rapid repair <strong>of</strong> the respiratory epithelium by<br />
migration <strong>of</strong> neighbouring cells, e.g. in the course <strong>of</strong> diseases such as asthma. Epidermal<br />
growth factor (EGF) and trefoil factor family (TFF) peptides are both typical luminal<br />
surveillance peptides which are thought to regulate epithelial restitution due to their<br />
motogenic effects 1,2 . Rapid re-epithelialization <strong>of</strong> damaged areas is also promoted by<br />
interactions between migrating cells and extracellular matrix proteins. Here, migration <strong>of</strong><br />
the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used<br />
as a model system for airway restitution. The motogenic effect <strong>of</strong> TFF2 was previously<br />
demonstrated to depend on ERK1/2 and protein kinase C (PKC) activation but not on<br />
p38 or phosphoinositide 3-kinase (PI3K) 2 . In contrast, it is shown here that the EGFtriggered<br />
motogenic response is independent <strong>of</strong> ERK1/2 activation, but sensitive to the<br />
inhibition <strong>of</strong> PI3K, p38, PKC or NF-κB. However, the motogenic effects <strong>of</strong> EGF and TFF2<br />
are synergistic and are both strictly dependent upon a haptotactic substrate. Taken<br />
together, the data suggest that EGF and TFF peptides can act synergisticly in the human<br />
respiratory epithelium to enhance rapid repair processes but they use different signalling<br />
cascades 3 .<br />
Supported by the BMBF (NBL3), the Land Sachsen-Anhalt and the "Fonds der<br />
Chemischen Industrie".<br />
Literature<br />
1 Oertel, M., A. Graness, L. Thim, f. Bühling, H. Kalbacher, and W. H<strong>of</strong>fmann. 2001. Am.<br />
J. Respir. Cell Mol. Biol. 25:418-424.<br />
2 Graness, A., C.E. Chwieralski, D. Reinhold, L. Thim, and W. H<strong>of</strong>fmann. 2002. J. Biol.<br />
Chem. 277:18440-18446.<br />
3 Chwieralski, C.E., I. Schnurra, L. Thim, and W. H<strong>of</strong>fmann. 2004. Am. J. Respir. Cell Mol.<br />
Biol. in press<br />
contact:<br />
Pr<strong>of</strong>. Dr. Werner H<strong>of</strong>fmann<br />
Universitätsklinikum Magdeburg<br />
Institut für Molekularbiologie und Medizinische Chemie<br />
Werner.H<strong>of</strong>fmann@Medizin.Uni-Magdeburg.de<br />
Leipziger Str. 44<br />
39120 Magdeburg (Deutschland)
Markus von Nickisch-Rosenegk, Eva Ehrentreich-Förster, Berit Umann, Frank F. Bier<br />
Synthetic and recombinant zinc fingers as a nano-addressable<br />
probe to doublestranded DNA structures<br />
In bionanotechnology nanostructures based on DNA have been constructed.<br />
Generally it is a prerequisite to generate single stranded DNA (ssDNA) molecules to open<br />
the possibility <strong>of</strong> probing with oligonucleotides. The production <strong>of</strong> long ssDNA,which is<br />
suitable to address a sufficient amount <strong>of</strong> short sites with oligonucleotides, is not an<br />
easy task.<br />
Our experiments describe an alternative method to bind to dsDNA specifically and with a<br />
high affinity i. e. zinc finger (ZF) molecules. The aim is to develope zinc finger probes<br />
that are able to ligate at arbitrary sites <strong>of</strong> dsDNA molecules. We cloned two different zinc<br />
finger recognition sites (SP1, LexA) each in a different standard plasmids (pBluescript).<br />
The same plasmid in its unligated form was used as a control. In a second approach we<br />
used two hybridized Oligonucleotides. The resulting dsDNA molecule containig the SP1<br />
motive, served as an zinc finger recognition site and an alternative binding partner. One<br />
(SP1) <strong>of</strong> the two zinc finger probes was commercially synthezised and modified with a<br />
Dy-633 fluorophore (Dyomics, Jena). The SP1 peptide was folded into a functional zinc<br />
finger using zinc chloride. The second ZF probe (LexA) was co-expressed with a<br />
recombinant modified sticking proteine that is called SnapTag (Covalys, Witterswil, data<br />
not shown). SnapTag is able to fix visualizing dyes by a secondary labeling procedure.<br />
The addressable dsDNA was immobilized either on a biochip surface or on an optical<br />
fibre, where the kinetics and binding rates <strong>of</strong> the artifical zinc finger probes are<br />
measured by fluorescence detecting devices (chip-scanner, photomultiplying tube).<br />
The sucessful experiments show a possible way to create dsDNA-binding probes by<br />
variation <strong>of</strong> synthezised zinc finger binding domains <strong>of</strong> the diverse existing natural zinc<br />
fingers. This might raise the chance to address dsDNA used in a nanoarray format.<br />
Literature<br />
Kwang-Hee Bae et al. 2003. Human zinc fingers as building blocks in the construction <strong>of</strong><br />
artifical transcription factors. Nature Biotech. 21:275-280<br />
Harvey A. Greisman and Carl O. Pabo 1997. A general strategy for selecting high-affinity<br />
zinc finger proteins for diverse DNA target sites. Science 275:657-661<br />
contact:<br />
Dr. Markus von Nickisch-Rosenegk<br />
Fraunh<strong>of</strong>er IBMT<br />
Molekulare Bioanalytik und Bioelektronik<br />
nickisch@ibmt.fhg.de<br />
Arthur-Scheunert-Allee 114-116<br />
14558 Nuthetal (Deutschland)
Christiane Schaffitzel, Imre Berger, Jan Postberg, Jozef Hanes, Hans J. Lipps, Andreas<br />
Plückthun<br />
Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Most eukaryotic telomeres contain a repeating motif with stretches <strong>of</strong> guanine residues<br />
that form a 3' terminal overhang extending beyond the telomeric duplex region. The<br />
telomeric repeat <strong>of</strong> hypotrichous ciliates, d(T4G4), forms a 16 nucleotide 3’-overhang.<br />
Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex<br />
conformations in vitro. While it has been proposed that guanine-quadruplex<br />
conformations may have important cellular roles including telomere function,<br />
recombination and transcription, evidence for the existence <strong>of</strong> this DNA structure in vivo<br />
has been elusive. We have generated high affinity antibody scFv probes for the guaninequadruplex<br />
formed by the Stylonychia telomeric repeat, by ribosome display from a<br />
combinatorial library. Of the scFvs selected, one (Sty3) had an affinity <strong>of</strong> 125 pM for the<br />
parallel-stranded guanine-quadruplex and could discriminate with at least 1000-fold<br />
specificity between parallel or antiparallel quadruplex conformations formed by the same<br />
sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel<br />
quadruplex with similar (3-5 nM) affinity. Indirect immun<strong>of</strong>luorescence studies show that<br />
Sty49 reacts specifically with the macronucleus but not the micronucleus <strong>of</strong> Stylonychia<br />
lemnae. The replication band, the region where replication and telomere elongation<br />
takes place, was also not stained, suggesting that the guanine-quadruplex is resolved<br />
during replication. Our results provide for the first time experimental evidence that the<br />
telomeres <strong>of</strong> Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure<br />
indicating that this structure may have an important role for telomere functioning.<br />
Literature<br />
Schaffitzel C., Berger I., Postberg J., Hanes J., Lipps H.J. & Plückthun A. (2001). In vitro<br />
generated antibodies specific for telomeric guanine-quadruplex DNA react with<br />
Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. U.S.A. 98, 8572-8577.<br />
contact:<br />
Dr. Christiane Schaffitzel<br />
ETH Zürich<br />
Institut für Molekularbiologie und Biophysik<br />
schaffitzel@mol.biol.ethz.ch<br />
ETH Hönggerberg<br />
8093 Zürich (Schweiz)
Thomas E Scholzen<br />
Terminating the stress: proteolytic processing <strong>of</strong><br />
proopiomelanocortin-derived stress and anti-inflammatory<br />
hormones by dermal microvascular endothelial cell (EC)<br />
extracellular peptidases<br />
Dermal microvascular endothelial cells (EC) are both source and target <strong>of</strong> the<br />
proopiomelanocortin (POMC) peptides adrenocorticotropin (ACTH) and α-melanocytestimulating<br />
hormone (α-MSH) (1-2). The availability <strong>of</strong> neuropeptides as modulators <strong>of</strong><br />
innate and adaptive immune responses is controlled by neuropeptide-specific zinc<br />
metalloproteases such as neutral endopeptidase (NEP) or angiotensin-converting<br />
enzyme (ACE) (3). Here, we have tested the possibility that NEP or ACE expressed by EC<br />
may influence the local bioavailability <strong>of</strong> POMC peptides. As determined by flow<br />
cytometry, NEP and ACE expression is inversely correlated in primary dermal EC<br />
(HDMEC; ACEhigh /NEPlow ) and in the EC cell line HMEC-1 (ACElow /NEPhigh ). Cell<br />
membranes prepared from EC degraded ACTH1-39 over time resulting in temporary<br />
increased α-MSH immunoreactivity as determined by RIA. Mass spectroscopy (MALDI-MS<br />
and MS/MS) revealed generation <strong>of</strong> stable peptides from ACTH1-39 by HMEC-1, such as<br />
ACTH18-28 and ACTH5-12 , whereas degradation in the presence <strong>of</strong> NEP inhibitors resulted<br />
in ACTH5-14 and a dehydrated ACTH1-14 , which involves additional enzyme activity.<br />
Likewise, HDMEC membranes (NEPlow /ACEhigh ) fragmented ACTH similar to HMEC-1<br />
membranes (NEPhigh /ACElow ) in the presence <strong>of</strong> NEP inhibitors. In addition, proteolytic<br />
processing <strong>of</strong> ACTH and α-MSH by recombinant ACE or NEP generated peptide fragments<br />
that partially overlapped with that produced by EC membranes. Thus, NEP and ACE are<br />
involved in, but not exclusively responsible for the EC processing <strong>of</strong> stress hormones<br />
(ACTH) and anti-inflammatory peptides (α-MSH). Since NEP and ACE are regulated by<br />
inflammatory mediators and UV light, this may be important for ACTH/MSH-modulated<br />
skin inflammation.<br />
Literature<br />
1. Scholzen, T.E., et al. (2000). J. Invest. Dermatol. 115:1021-1028.<br />
2. Scholzen, T.E., et al. (2003). Endocrinology 144:360-370.<br />
3. Scholzen, T. et al., (1998). Exp. Dermatol. 7:81-96.<br />
contact:<br />
Ph.D. Thomas E Scholzen<br />
University <strong>of</strong> Muenster<br />
Ludwig-Boltzmann-Institute & Dept. <strong>of</strong> Dermatology<br />
thoscho@uni-muenster.de<br />
Von-Esmarch-Strasse 58<br />
48167 Münster (Germany)
Christina Schreier, Werner Kremer, Bernd Fakler, Hans Robert Kalbitzer<br />
Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type<br />
voltage gated potassium channels<br />
The N-terminal, cytoplasmic tetramerization domain (T1) has been shown to be essential<br />
in the formation <strong>of</strong> functional tetramers <strong>of</strong> voltage-gated K+ channels (Kv) in vivo.<br />
Deletion <strong>of</strong> T1 impairs tetramerization and thus the assembly <strong>of</strong> the functional K+<br />
channel. In most Kv channels the T1 domain (or an intracellular β-subunit which docks<br />
to the T1 N-terminus) carries an inactivation domain which is also known as the ball<br />
peptide as a reference to the classic ‘Ball and Chain’ concept. The lacking <strong>of</strong> the ball<br />
peptide leads to significant changes in the inactivation characteristics <strong>of</strong> the ion channel.<br />
It is currently being dicussed, whether inactivation results from the interaction or binding<br />
<strong>of</strong> the ball peptide to the transmembrane pore region or to the T1 domain.<br />
To study the interaction <strong>of</strong> Kv tetramerization domains with ball peptides by solution<br />
NMR-spectroscopy Shaker and Shaw type T1-domains were expressed in E.coli. The<br />
proteins could be purified in quantities necessary for NMR-spectroscopy. As shown by 1D<br />
¹H NMR experiments they are well-folded. Diffusion NMR experiments and gel filtration<br />
show that the proteins form stable tetramers under quasiphysiological conditions in<br />
solution. Monomers <strong>of</strong> T1 domain could only be observed at harsh denaturing conditions<br />
that enforce at least partial unfolding <strong>of</strong> the protein.<br />
contact:<br />
Christina Schreier<br />
Universität Regensburg<br />
Lehrstuhl für Biophysik (Pr<strong>of</strong>. Dr. Dr. Kalbitzer)<br />
christina.schreier@biologie.uni-regensburg.de<br />
Universiätsstrasse 31<br />
93040 Regensburg (Deutschland)
Katrin Stolle, Michael Schnoor, Jürgen Rauterberg, Paul Cullen, Stefan Lorkowski<br />
TGF-β1 inducible genes in coronary smooth muscle cells<br />
According to the ‘response-to-injury’ hypothesis, the initial step in atherogenesis is the<br />
accumulation <strong>of</strong> leukocytes as a consequence <strong>of</strong> injury or dysfunction <strong>of</strong> the<br />
endothelium. These leukocytes release various growth factors and chemokines such as<br />
platelet-derived growth factor or transforming growth factor-β1 (TGF-β1). TGF-β1 is a<br />
cytokine involved in atherosclerosis that is produced by macrophages and smooth<br />
muscle cells <strong>of</strong> the arterial wall. In vitro studies have shown that TGF-β1 inhibits smooth<br />
muscle cell proliferation and supports pathologic accumulation <strong>of</strong> excessive extracellular<br />
matrix. To better understand the role <strong>of</strong> TGF-β1 in atherogenesis, knowledge <strong>of</strong> TGF-β1<br />
inducible genes is <strong>of</strong> great interest. To investigate such genes, we monitored in vitro<br />
changes <strong>of</strong> mRNA expression in coronary smooth muscle cells under the influence <strong>of</strong> TGFβ1<br />
using microarrays. More than 6.600 (30%) <strong>of</strong> the 22.000 monitored genes were<br />
expressed in coronary smooth muscle cells. After incubation with 10 ng/ml TGF-β1 the<br />
expression <strong>of</strong> 91 genes was increased more than two-fold. 18 genes were identified that<br />
were more than two-fold downregulated. Genes with increased expression such as<br />
interleukin-11, connective tissue growth factor (CTGF) or vascular endothelial growth<br />
factor (VEGF), which are known to be sensitive to TGF-β1 prove the integrity <strong>of</strong> our<br />
experiment. In addition we identified several genes which are not known to be regulated<br />
by TGF-β1. For an interesting panel <strong>of</strong> genes we confirmed differential expression using<br />
real-time RT-PCR. We identified two new proteins – a Rab GTPase interacting factor and<br />
a RAS like small GTPase – which are differentially expressed during TGF-β1 treatment,<br />
identified their genomic structure and performed further characterization <strong>of</strong> these<br />
proteins including analysis <strong>of</strong> tissue-specific expression and cellular localization.<br />
contact:<br />
Dr. Stefan Lorkowski<br />
Universität Münster<br />
Institut für Arterioskleroseforschung<br />
stefan.lorkowski@uni-muenster.de<br />
Domagkstraße 3<br />
48149 Münster (Deutschland)
Brian Anderton<br />
The ageing neuronal cytoskeleton and neurodegenerative<br />
disease<br />
Abnormalities <strong>of</strong> the two principal structural constituents <strong>of</strong> the neuronal cytoskeleton,<br />
microtubules and neur<strong>of</strong>ilaments, underlie at least two different types <strong>of</strong> neuronal<br />
inclusion bodies that characterise different neurodegenerative diseases. In the case <strong>of</strong><br />
Alzheimer’s disease (AD), there is a total loss <strong>of</strong> microtubules in neurons that contain<br />
paired helical filaments which are composed <strong>of</strong> the microtubule-associated protein tau.<br />
In amyotrophic lateral sclerosis (ALS), neur<strong>of</strong>ilaments accumulate in cell bodies and<br />
proximal regions <strong>of</strong> the axons <strong>of</strong> motor neurons. In both cases, aberrant phosphorylation<br />
<strong>of</strong> tau and neur<strong>of</strong>ilament proteins may be the underlying cause <strong>of</strong> their pathogenic<br />
aggregation and formation <strong>of</strong> the inclusion bodies may lead directly to neuronal cell<br />
death. The risk <strong>of</strong> developing these neurodegenerative conditions increases with age and<br />
hence aberrant phosphorylation may be a feature <strong>of</strong> an ageing cytoskeleton in<br />
susceptible individuals. In our group, we have therefore been attempting to identify<br />
phosphorylation sites, investigate mechanisms <strong>of</strong> phosphorylation <strong>of</strong> tau and<br />
neur<strong>of</strong>ilaments and study the consequences <strong>of</strong> phosphorylation on their properties. We<br />
have found several new sites in tau isolated from Alzheimer brain and investigated the<br />
effects on axonal transport <strong>of</strong> both tau and neur<strong>of</strong>ilaments <strong>of</strong> interfering with the<br />
phosphorylation states <strong>of</strong> these proteins and these data will be presented.<br />
contact:<br />
Pr<strong>of</strong>essor Brian Anderton<br />
King's College London<br />
Institute <strong>of</strong> Psychiatry<br />
b.anderton@iop.kcl.ac.uk<br />
De Crespigny Park<br />
SE5 8AF London (UK)
Claus Kerkh<strong>of</strong>f, Wolfgang Nacken, Malgorzata Czarny, Marie Claire Dagher, Claudia<br />
Sopalla, Jacques Doussiere<br />
The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
The Ca 2+ - and arachidonic acid-binding S100A8/A9 protein complex was recently<br />
identified by in vitro-studies as a novel partner <strong>of</strong> the phagocyte NADPH oxidase. The<br />
present study demonstrated its functional relevance by the impaired oxidase activity in<br />
both neutrophil-like NB4 cells, after specific blockage <strong>of</strong> S100A9 expression, and<br />
polymorphonuclear neutrophils from S100A9 -/- mice. The impaired oxidase activation<br />
could also be mimicked in a cell-free system by pre-treatment <strong>of</strong> neutrophil cytosol with<br />
a S100A9-specific antibody, pointing to the identity <strong>of</strong> S100A8/A9 with the cytosolic<br />
factor enhancing NADPH oxidase. Our finding that mutant S100A8/A9 complexes unable<br />
to bind arachidonic acid did not promote NADPH oxidase activity is indicative for the<br />
S100A8/A9 facilitated transfer <strong>of</strong> the c<strong>of</strong>actor arachidonic acid to NADPH oxidase. In<br />
vitro-analysis <strong>of</strong> oxidase activation as well as protein-protein interaction studies revealed<br />
that S100A8/A9 acts by binding to cytosolic factors <strong>of</strong> oxidase activation and by<br />
delivering AA to the membrane-bound flavocytochrome b.<br />
Beside the role <strong>of</strong> S100A8/A9 in the activation <strong>of</strong> the phagocyte NADPH oxidase, our<br />
finding has further interesting implications. In addition to their specific expression in<br />
myeloid cells, the two S100 proteins are present in psoriasis keratinocytes. In<br />
preliminary experiments epithelial HeLa cells showed a drastic increase <strong>of</strong> oxidase<br />
activity after S100 gene transfection. Due to growing evidence that reactive oxygen<br />
species (ROS) are involved in the pathogenesis <strong>of</strong> psoriasis, it is tempting to speculate<br />
that overexpression <strong>of</strong> S100A8/A9 might result to hyperproliferation <strong>of</strong> psoriais<br />
keratinocytes.<br />
Literature<br />
Doussiere, J., Bouzidi, F., Vignais, P.V. (2001) Biochem. Biophys. Res. Commun. 285,<br />
1317-1320<br />
Kerkh<strong>of</strong>f, C., Klempt, M., Sorg, C. (1998) Biochim. Biophys. Acta 1448, 200-211<br />
Kerkh<strong>of</strong>f, C., Klempt, M., Kaever, V., Sorg, C. (1999) J. Biol. Chem. 274, 32672-32679<br />
Kerkh<strong>of</strong>f, C., Vogl, T., Nacken, W. Sopalla, C., Sorg, C. (1999) FEBS Lett. 460, 134-138<br />
Sopalla, C., Leukert, N., Sorg, C., Kerkh<strong>of</strong>f, C. (2002) Biol. Chem. 383, 1895-1905<br />
Kerkh<strong>of</strong>f, C., H<strong>of</strong>mann, H.A., Vormoor, J., Melkonyan, H., Roth, J., Sorg, C., Klempt, M.<br />
(2002) J. Biol. Chem. 274, 41879-41887<br />
Kerkh<strong>of</strong>f, C., Nacken, W., Czarny, M., Dagher, M.C., Sopalla, C., Doussiere, J. (2004)<br />
(submitted).<br />
contact:<br />
Dr. Claus Kerkh<strong>of</strong>f<br />
Münster<br />
Institut <strong>of</strong> Experimental Dermatology<br />
kerkhoc@uni-muenster.de<br />
Röntgenstr. 21<br />
48149 Münster (Germany)
Niklas Feldhahn, Florian Klein, Markus Müschen<br />
The BCR-ABL1 kinase bypasses selection for the expression <strong>of</strong><br />
a pre-B cell receptor in pre-B acute lymphoblastic leukemia<br />
cells<br />
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant<br />
transformation <strong>of</strong> human pre-B cells. To identify novel target genes <strong>of</strong> BCR-ABL1mediated<br />
transformation and to elucidate how the oncogenic BCR-ABL1 kinase may<br />
interfere with normal pre-B cell receptor signaling, we compared genome-wide gene<br />
expression <strong>of</strong> BCR-ABL1+ ALL cells with their normal counterpart.<br />
While normal pre-B cells are selected for the expression <strong>of</strong> a functional pre-B cell<br />
receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH<br />
allele and silence the expression <strong>of</strong> genes involved in pre-B cell receptor signaling. In the<br />
cases that do not harbor a productively rearranged IGH allele, we identified traces <strong>of</strong><br />
secondary VH gene rearrangements, which may have rendered an initially productive VH<br />
region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region<br />
gene are unresponsive to pre-B cell receptor engagement and exhibit autonomous<br />
oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition <strong>of</strong><br />
BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and<br />
differentiate into immature B cells expressing Ig light chains. BCR-ABL1 kinase activity is<br />
linked to defective pre-B cell receptor signaling and the expression <strong>of</strong> a truncated<br />
is<strong>of</strong>orm <strong>of</strong> the pre-B cell receptor-associated linker molecule BLNK. Also in primary<br />
leukemia cells, truncated BLNK is expressed before but not after treatment <strong>of</strong> the<br />
patients with STI571.<br />
We conclude that inhibition <strong>of</strong> BCR-ABL1 reconstitutes selection for a functional (pre-) B<br />
cell receptor signaling in pre-B ALLs. Hence, a functional (pre-) B cell receptor signaltransduction<br />
cascade provides these cells with a potential mechanism to escape<br />
apoptotic cell death induced by STI571.<br />
contact:<br />
Niklas Feldhahn<br />
Universität Düsseldorf<br />
Lab. for Molecular Stem Cell Biology<br />
feldhahn@itz.uni-duesseldorf.de<br />
Moorenstr. 5<br />
40225 Düsseldorf (D)
David Denis S<strong>of</strong>eu Feugaing, Hans Kresse, Martin Götte<br />
The dermatan sulfate proteoglycans Decorin and Biglycan<br />
follow partially diverging endocytic routes<br />
The dermatan sulfate proteoglycans decorin and biglycan are efficiently internalized by<br />
receptor-mediated endocytosis, resulting in lysosomal degradation(1-3).We studied<br />
uptake <strong>of</strong> 35Sulfate-labelled decorin and biglycan in human skin fibroblasts in the<br />
presence <strong>of</strong> inhibitors <strong>of</strong> specific endocytic routes.Tyrphostin AG1478, an inhibitor <strong>of</strong><br />
EGFR phosphorylation & chlorpromazine, an inhibitor <strong>of</strong> clathrin-dependent endocytosis<br />
inhibited decorin endocytosis by 50% without additive effects. In the presence <strong>of</strong> filipin,<br />
which inhibits caveolae formation, decorin uptake was reduced by 30%.Chlorpromazine<br />
and filipin showed additive effects.Chlorpromazine treatment reduced biglycan<br />
endocytosis by 40%, whereas filipin & Tyrphostin AG1478 did nor alter biglycan uptake.<br />
Confocal microscopy revealed a partial colocalization <strong>of</strong> decorin with the transferrin<br />
receptor after 10 min uptake, which disappeared after 10 min <strong>of</strong> chase. CD44 or caveolin-<br />
1 did not colocalize with decorin after 10 min, and no colocalization <strong>of</strong> decorin and<br />
internalized hyaluronan was observed after 30 min. High expression <strong>of</strong> the clathrinbinding<br />
Hrs protein leads to EGFR trapping in early endosomes(4). NIH3T3 cells<br />
transfected with Hrs displayed markedly decreased decorin degradation, while<br />
internalization was not significantly inhibited. Decorin was trapped in clustered vesicular<br />
structures. Our results suggest that decorin follows different uptake routes which are<br />
influenced by EGFR and uses components whose intracellular trafficking are influenced<br />
by chlorpromazine, filipin & Hrs.Based on the differential susceptibility to<br />
pharmacological inhibition, biglycan endocytosis differs at least in part mechanistically<br />
from decorin uptake.<br />
Literature<br />
(1)Götte, M. et al. (1995) Eur J Cell Biol 66:226-33<br />
(2)Schaefer, L. et al. (1998) Kidney Int 54:1529-41<br />
(3)Götte, M. et al. (2004) Cellular & Molecular Biology Letters 9: in press<br />
(4)Raiborg, C. et al. (2001) EMBO J. 20:5008-21<br />
contact:<br />
Dipl. Biochem. David Denis S<strong>of</strong>eu Feugaing<br />
Münster University Hospital<br />
Deptartment <strong>of</strong> Obstetrics and Gynecology<br />
ds<strong>of</strong>eu@yahoo.com<br />
Domagkstr.11<br />
D-48149 Münster (Germany)<br />
additional information<br />
H.K., M.G.: Dept. <strong>of</strong> Physiological Chemistry and Pathobiochemistry, Münster University Hospital<br />
H.K.: deceased<br />
corresponding author: M. Götte
Jörg Andrä, Sylvie E. Blondelle, Roman Jerala, Guillermo Martinez de Tejada, Michel H.<br />
J. Koch, Karl Lohner, Klaus Brandenburg<br />
The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Lact<strong>of</strong>errin is an antibacterial protein <strong>of</strong> mucosal epithelia contributing to the first line <strong>of</strong><br />
defence against invading pathogens. Its antibacterial activity is located in the cationic Nterminal<br />
domain which, in vivo, is released as an active peptide, termed lact<strong>of</strong>erricin. In<br />
addition to their antibacterial action, epithelial peptides may be involved in the<br />
neutralization <strong>of</strong> lipopolysacharide (LPS, endotoxin) which is released from the envelope<br />
<strong>of</strong> Gram-negative bacteria. LPS is a very potent activator <strong>of</strong> immune cells leading to a<br />
massive production <strong>of</strong> cytokines, systemic inflammation, and septic shock. Till now, no<br />
effective therapy for the treatment <strong>of</strong> septic shock is available and thus the mortality<br />
rate is very high. To address this problem, we are designing and developing compounds<br />
which combine endotoxin neutralization and antibacterial activity (1,2). For this, we have<br />
synthesized the LPS-binding domain <strong>of</strong> lact<strong>of</strong>erricin, and derivatives there<strong>of</strong>, and<br />
modified it further by linkage to alkyl chains. We determined the biological activities (LPSneutralizing,<br />
antibacterial, and cytotoxic activity) <strong>of</strong> the peptides and investigated the<br />
molecular basis <strong>of</strong> the peptide/LPS interaction by biophysical methods. Our data allowed<br />
us to deduce a molecular model <strong>of</strong> the peptide/LPS interaction and the structural<br />
requirements <strong>of</strong> the peptides for an effective neutralization <strong>of</strong> LPS: high affinity binding<br />
that can be achieved by a combination <strong>of</strong> good-fitting electrostatic interaction and<br />
penetration <strong>of</strong> a hydrophobic anchor <strong>of</strong> the peptide into the acyl chain moiety <strong>of</strong> the LPS<br />
aggregates.<br />
Supported by grant ANEPID (QLK2-CT-2002-01001) from the EU.<br />
Literature<br />
(1) J. Andrä, M. H. J. Koch, R. Bartels und K. Brandenburg. Antimicrob. Agents<br />
Chemother. 48, 1593-1599 (2004)<br />
(2) J. Andrä, M. Lamata, G. Martinez de Tejada, R. Bartels, M. H. J. Koch und K.<br />
Brandenburg. Biochem. Pharmacol. in press (2004)<br />
contact:<br />
Dr. Jörg Andrä<br />
Research Center Borstel, Leibniz-Center for Medicine and Biosciences<br />
Division <strong>of</strong> Biophysics<br />
jandrae@fz-borstel.de<br />
Parkallee 10<br />
23845 Borstel (Germany)
Frank Dietz, Friedericke Lehmann, Heiko Gäthje, Maija Slaidina, Daniel Daraban, Sörge<br />
Kelm<br />
The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
The Myelin-associated Glycoprotein (MAG) is a nervous system cell adhesion molecule,<br />
belonging to the family <strong>of</strong> sialic acid binding proteins called Siglecs (sialic acid binding<br />
immunoglobulin like lectin) exclusively expressed on oligodendrocytes and Schwann cells<br />
[1]. So far, the function <strong>of</strong> MAG in the maintenance <strong>of</strong> myelin and stabilisation <strong>of</strong> myelinaxon<br />
contacts has only been described for mammalian species (e.g. rodents and<br />
humans) except for its possible orthologue from birds SMP [2]. Within these species<br />
MAG occurs in two splice variants called L- and S-MAG (for long- and short MAG) which<br />
differ only in a short stretch <strong>of</strong> the intracellular C-termini. This unique portion <strong>of</strong> the Cterminus<br />
seems to play an important role in different signal transduction events.<br />
To get more information about the evolution <strong>of</strong> MAG we used the almost complete gene<br />
assemblies <strong>of</strong> two genomes belonging to the fish phylum (Takifugu rubripes and Danio<br />
rerio) to screen them for MAG encoding genes [3]. Based on the information available<br />
from the gene banks, cDNAs for MAG orthologues from Takifugu rubripes and Danio rerio<br />
have been cloned, sequenced and expressed as recombinant proteins which show all<br />
expected properties <strong>of</strong> functional Siglecs including sialic acid binding. With its<br />
mammalian orthologues fish MAG shares an expression pattern restricted to the nervous<br />
system. Interestingly, we found three instead <strong>of</strong> two alternatively spliced C-termini in<br />
three fish species (including Cyprinus carpio). Two <strong>of</strong> these splice variants correlate with<br />
L- and S-MAG known from mammalian MAG (s.a.). However, on the amino acid level<br />
these variants differ significantly between fish and mammals. The third splice variant<br />
contains an extra exon leading to an extension <strong>of</strong> the C-Terminus <strong>of</strong> L-MAG and have<br />
been termed XL-MAG. Furthermore, we found so called ITIMs (Immunoreceptor based<br />
tyrosine inhibitory motif) usually in mammals only found in Siglecs belonging to the<br />
immune system [4]. These findings suggest that the signalling pathways mediated by<br />
MAG are not identical in fish and mammals and has to be investigated in further studies.<br />
Literature<br />
1. Crocker P.R. et al. (1998): Glycobiology 8, v<br />
2. Angata T, Varki A. (2002): Chem Rev. 102 (2): 439-69<br />
3.Venkatesh B., Gilligen P., Brenner S. (2000): FEBS Lett. 476 (1-2): 3-7<br />
4.Crocker P.R. and Varki A. (2001): Immunology 103: 137-145<br />
contact:<br />
Dr Frank Dietz<br />
Universität Bremen<br />
Centre <strong>of</strong> Biomolecular Interactions Bremen<br />
fdietz@uni-bremen.de<br />
Leobener Str. im NW2<br />
28359 Bremen (Deutschland)
Daniel Braas, Holger Gundelach, Karl-Heinz Klempnauer<br />
The glioma amplified sequence 41 gene (GAS41) is a direct<br />
Myb target gene<br />
The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which transforms<br />
myelomonocytic cells in vivo and in vitro. It is thought that v-Myb exerts its biological<br />
effects by deregulating the expression <strong>of</strong> specific target genes, most <strong>of</strong> which are still<br />
unknown. The chicken glioma amplified sequence 41 gene (GAS41) is located<br />
immediately downstream <strong>of</strong> the lysozyme gene, a known Myb-regulated gene. The<br />
GAS41 promoter colocalizes with a CpG island which also functions as an origin <strong>of</strong><br />
replication. Since the GAS41 promoter contains several potential Myb-binding sites<br />
(MBS) we have investigated whether GAS41 is a v-Myb target gene. Our results show<br />
that the GAS41 gene is directly activated by a v-Myb/estrogen receptor fusion protein.<br />
Furthermore, our studies reveal that the GAS41 promoter is stimulated by v-Myb in cotransfection<br />
experiments and that the DNA-binding activity <strong>of</strong> v-Myb is crucial for<br />
transactivation <strong>of</strong> the promoter. Electrophoretic mobility shift assays(EMSA) indicate that<br />
four <strong>of</strong> several other potential Myb-binding sites are bound in vitro by Myb and seem to<br />
be essential for Myb inducibility <strong>of</strong> the GAS41 promoter. Finally, chromatin<br />
immunoprecipitation assays demonstrate that v-Myb is bound to the GAS41 promoter in<br />
vivo. Taken together this work identifies the GAS41 gene to be a novel v-Myb target<br />
gene.<br />
contact:<br />
Daniel Braas<br />
Universität Münster<br />
Inst. f. Biochemie<br />
braas@uni-muenster.de<br />
Wilhelm-Klemm Str.2<br />
48149 Münster (Deutschland)
Martin Götte, Laura Rossi, Robert Greb, Dorothe Spillmann, Antonia Joussen, Merton<br />
Bernfield, Ludwig Kiesel<br />
The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Leukocyte adhesion to endothelial cells (EC) during inflammation is a multistep process<br />
that involves leukocyte rolling, adhesion and extravasation mediated by selectins, CAM's,<br />
integrins and chemokines. Heparan sulfate (HS) modifies the function <strong>of</strong> many <strong>of</strong> these<br />
molecules under physiological conditions(1,2). We used mice deficient in the cell surface<br />
HS proteoglycan syndecan-1 (Sdc1 KO)(3) to study its role in leukocyte-endothelial<br />
interactions in vivo, using a perfusion technique with FITC-coupled ConA and intravital<br />
microscopy. Sdc1 KO mice showed dramatically enhanced leukocyte-EC adhesion in the<br />
perfusion model (5-fold, p < 0.005) and in intravital microscopy (80-fold, TNF-alphastimulation,<br />
p < 0.001). This effect was leukocyte-dependent, as shown in hematopoietic<br />
chimaeras and by in vitro assays measuring adhesion <strong>of</strong> Sdc1 KO monocytes and PMN’s<br />
to HUVEC cells. Addition <strong>of</strong> heparin (10 µg/ml) reverted increased adhesion <strong>of</strong> Sdc1 KO<br />
cells to WT levels, suggesting an involvement <strong>of</strong> the Sdc1 HS chains. However, a HS<br />
fingerprint analysis revealed no qualitative differences in the organ-specific disaccharide<br />
patterns <strong>of</strong> HS isolated from Sdc1 KO and wild-type mice. A dramatic increase in<br />
leukocyte extravasation was observed both by intravital microscopy following TNF-alphastimulation<br />
and in a skin inflammation model (contact hypersensitivity). Furthermore,<br />
increased angiogenesis was observed in a cornea assay. Our data demonstrate a role for<br />
syndecan-1 as a negative regulator <strong>of</strong> inflammatory responses, which acts at the stage<br />
<strong>of</strong> leukocyte-endothelial interactions. Selectins, integrins, CAM‘s and chemokines interact<br />
with heparan sulfate and are potential targets for syndecan-1 in this process(4-6).<br />
Literature<br />
(1) Götte, M. (2003) FASEB J. 17:575-91<br />
(2) Bernfield, M., Götte, M. et al. (1999) Annu Rev Biochem 68:729-77<br />
(3) Götte, M. et al. (2002) Invest Ophthalmol Vis Sci 43:1135-41<br />
(4) Li, M. et al. (2002) Cell 111:635-46<br />
(5) Götte, M., Echtermeyer, F. (2003) Sci World J 3:1327-31<br />
(6) Elenius, V., Götte, M. et al. (2004) J Biol Chem 279: in press<br />
contact:<br />
Dr. Martin Götte<br />
Universitätsklinikum Münster<br />
Frauenheilkunde und Geburtshilfe (Forschungslabor)<br />
mgotte@uni-muenster.de<br />
Domagkstr. 11<br />
48149 Münster (Deutschland)<br />
additional information<br />
D.S.:Department <strong>of</strong> Medical Biochemistry and Microbiology,Uppsala University, Sweden<br />
A.J.,M.B.: Harvard Medical School, Boston, USA
Christoph Becker-Pauly, Markus-N. Kruse, Daniel Lottaz*, Irene Yiallouros, Hans-Willi<br />
Krel**, Erwin E. Sterchi*, Walter Stöcker<br />
The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Meprin is a zinc-endopeptidase <strong>of</strong> the astacin family, which has been known to be<br />
expressed as membrane bound or secreted protein on mammalian brush-border<br />
membranes, on intestinal leukocytes and on certain cancer cells. We now show that both<br />
meprin-subunits are expressed in human epidermal skin. However, they are not colocalized.<br />
Meprin α has been detected in the basal cell layer <strong>of</strong> the epidermis, whereas<br />
meprin Β is expressed in keratinocytes <strong>of</strong> the Stratum granulosum immediately beneath<br />
the Stratum corneum. Thus, one can assume that this protease might be involved in<br />
epithelial differentiation. The two types <strong>of</strong> meprin subunits are translated as proenzymes,<br />
forming disulfide bonded homo- and hetero-oligomers. They have to be<br />
activated by removal <strong>of</strong> an N-terminal pro-peptide. While both human α and Β are<br />
activated by trypsin in the gut, only meprin α but not Β is processed to its mature form<br />
by plasmin outside the intestinal tract. Despite their completely different cleavage<br />
specificities, both human meprin α and Β are able to hydrolyse basement membrane<br />
components like collagen IV, nidogen-1 and fibronectin, but do not cleave fibrillar<br />
collagen I, thus they display a gelatinase-like activity. Human meprin is inhibited by<br />
hydroxamic acid derivatives like batimastat, galardin, Pro-Leu-Gly-hydroxamate, tumour<br />
necrosis factor α protease inhibitors (TAPI-0, TAPI-2), and by thiol based compounds like<br />
captopril. The most effective inhibitor <strong>of</strong> human meprin α is the naturally occurring<br />
hydroxamate actinonin (K i = 20 nM). The marked variance in the cleavage specificities<br />
<strong>of</strong> human meprin α and Β is reflected by their interaction with the TACE inhibitor Ro 32-<br />
7315, whose affinity to the Β subunit is by three orders <strong>of</strong> magnitude weaker than to α.<br />
contact:<br />
Dipl. Biologe Christoph Becker-Pauly<br />
Westfälische Wilhelms-Universität<br />
Institut für Zoophysiologie<br />
christoph.becker@uni-muenster.de<br />
Hindenburgplatz 55<br />
48143 Münster (Deutschland)<br />
additional information<br />
*) Institute <strong>of</strong> Biochemistry and Molecular Biology, University <strong>of</strong> Bern, Switzerland<br />
**) Roche Diagnostics GmbH, Pharma Research, Penzberg, Germany<br />
Supported by a start up grant <strong>of</strong> the University <strong>of</strong> Münster and by a research grant <strong>of</strong> the Swiss<br />
National Foundation to E.S. (Nr. 3200-052736.97).
María Jose Gómez-Lechón, Alfonso Serralta, Teresa Donato, Enrique O´Connor, Jose<br />
Vicente Castell, Jose Mir<br />
The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
FK506) and CsA are potent immunosuppressive drugs used for the prevention <strong>of</strong> graft<br />
rejection in organ transplantation. Experimental and clinical studies have shown<br />
correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell<br />
death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very<br />
likely involved in allograft rejection and experimental evidences have shown a decrease<br />
<strong>of</strong> FasR expression in mouse hepatocytes produced by the drugs. On the basis <strong>of</strong> these<br />
findings, we have investigated the protective effect <strong>of</strong> FK506 in comparison with CsA on<br />
Fas-induced apoptosis, by analysing the activation <strong>of</strong> downstream effector caspases in<br />
human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies<br />
against FasR, which resulted in a significant activation <strong>of</strong> caspase 3 after 12 hours. A<br />
significant reduction (ca. 30-40%) <strong>of</strong> caspase 3 activation by 5 mM FK506 and CsA was<br />
observed. Along with less activation <strong>of</strong> caspase-3 a decrease <strong>of</strong> apoptotic DNA<br />
fragmentation was found. In addition, FK506 significantly reduced caspase 8 but also<br />
caspase 9 activation, in a similar extend than CsA thus suggesting a strong protective<br />
effect at the mitochondrial level <strong>of</strong> this drug, as it has been already reported for CsA.<br />
Prevention <strong>of</strong> the downstream activation <strong>of</strong> the caspase cascade and apoptosis was<br />
observed when hepatocytes were pretreated for 3 hours with the drugs. These effects <strong>of</strong><br />
FK506 help to explain its strong anti-rejection properties <strong>of</strong> the drug and suggests<br />
promising benefits <strong>of</strong> pharmacological preconditioning on ischemia-reperfusion injury<br />
following liver transplantation.<br />
contact:<br />
PhD María Jose Gómez-Lechón<br />
Hospital La Fe<br />
Centro de Investigación-Unidad de Hepatología Experimental<br />
gomez_mjo@gva.es<br />
Avda Campanar 21<br />
46009 Valencia (Spain)
Lutz Hilterhaus, Stefan Malcharek, Hans-Joachim Galla<br />
The Influence <strong>of</strong> Cholesterol and POPE on mono- and<br />
multilayers conatining surfactant protein C<br />
Film balance, fluorescence microscopy (FM) and scanning force microscopy (SFM) results<br />
show, that the compression <strong>of</strong> a model system containing DPPC/DPPG/SP-C leads to a<br />
formation <strong>of</strong> multilayer structures at high surface pressure (1). The structure <strong>of</strong> these<br />
protrusions can be influenced by adding non-bilayer lipids, such as POPC, POPE or DPPE,<br />
to this model system: The topography <strong>of</strong> these lipid-protein mixtures show smaller<br />
protrusions. This can be interpreted as a stabilisation <strong>of</strong> the edges <strong>of</strong> squeezed out<br />
multilayers by the cone shaped non-bilayer lipids. Following this interpretation an<br />
addition <strong>of</strong> bilayer stabilizing lipids, e.g. cholesterol, should lead to the formation <strong>of</strong><br />
broad domains. Our approach was, to verify this by means <strong>of</strong> film balance, FM, SFM and<br />
Time-<strong>of</strong>-Flight Secondary Ion Mass Spectrometry (ToF-SIMS).<br />
Experiments were performed on a DPPC/DPPG/SP-C (80:20:0.4 mol%) system and<br />
compared to the protein free DPPC/DPPG (80:20 mol%). To both systems different<br />
amounts <strong>of</strong> Cholesterol or POPE were added.<br />
The results <strong>of</strong> the film balance experiments for these systems show a shift to smaller<br />
molecular area with increasing amounts <strong>of</strong> cholesterol, which is interpreted as a<br />
decrease <strong>of</strong> rigidity <strong>of</strong> the films. With increasing amount <strong>of</strong> POPE, a higher<br />
compressibility could be observed. This means that the fluidity is increased by POPE.<br />
For characterisation <strong>of</strong> the phase behaviour the fluorescence microscopy at the air-water<br />
interface was used. We compared the domain structure (2) at different pressures and<br />
ratios <strong>of</strong> cholesterol or POPE: at a given surface pressure the fluid domain sizes increase<br />
with increasing cholesterol content. At 30 mol% Cholesterol and a pressure <strong>of</strong> p = 30<br />
mN/m we observed a domain free surface for the protein free film. The same effectwas<br />
observed at p = 45 mN/m for the protein containing film. By increasing the content <strong>of</strong><br />
POPE a phase separation at high pressure for both types <strong>of</strong> film could be shown.<br />
Additionaly the observed protrusions get smaller.<br />
SFM was used to visualize the topography <strong>of</strong> the SP-C containing samples. For all<br />
containing systems protrusions with steps <strong>of</strong> 6 nm in height could be observed. These<br />
protrusions get broader upon increasing cholesterol ratio, contrary to the POPE<br />
containing systems, which show protrusion structure which is gets more narrow upon<br />
increasing amount <strong>of</strong> POPE.<br />
This results presented here are in line with our expectations for cholesterol as a bilayer<br />
stabilizing and POPE as a bilayer destabilizing compound.<br />
Literature<br />
[1] Galla H.-J; Malcharek S; Bourdos N; In: Nag K, editor. Lung Surfactant<br />
(Dys)function, New York: Marcel Dekker, INC.;2003<br />
[2] Veldhuizen R; Nag K; Oreig S; Possmayer F; Biochim. Biophys. Acta 1998; 1408: 90-<br />
108<br />
contact:<br />
Dipl. Chem. Lutz Hilterhaus<br />
WWU Münster<br />
Institut für Biochemie<br />
hilterha@uni-muenster.de<br />
Wilhelm-Klemm-Str. 2<br />
48149 Münster (Deutschland)
Ute Klenz, Hans-Joachim Galla<br />
The Influence <strong>of</strong> Membrane Fluidity on Vesicle Insertion in a<br />
SP-B or SP-C containing Monolayer<br />
One <strong>of</strong> the main functional unit in lung is the lung surfactant, a complex protein lipid<br />
mixture [1]. This monolayer which lines the interface between the liquid hypophase and<br />
the gase phase reduces the surface tension at the air-water interface and forms<br />
reversible membrane protrusions during breathing cycle [2]. These protrusions consist <strong>of</strong><br />
lipid multilayer which are connected and stabilized by surfactant specific proteins A, B<br />
and C. Thereby SP-C acts with its alpha helix like an anchor between two monolayers<br />
whereas SP-B is capable <strong>of</strong> forming discoidal structures for material exchange [1]. While<br />
saturated lipid components (especially Dipalmitoylphosphatidylcholine - DPPC) are<br />
responsible for the reduction <strong>of</strong> the surface tension unsaturated lipids appear to enable<br />
the necessary dynamic <strong>of</strong> the system [3].<br />
To get further information about the influence and the interaction <strong>of</strong> unsaturated lipid<br />
components with saturated differently charged lipids and the surfactant proteins B and C<br />
we did kinetic and fluorescence measurements on a Langmuir film balance. In a<br />
DPPC/DPPG 4:1 monolayer with or without either SP-B (0,2 mol%) or SP-C (0,4 mol%)<br />
saturated lipids were systematically replaced with unsaturated POPC or POPG. After<br />
preparing the monolayer on 25 mM HEPES + 3 mM Ca2+ buffer at 20 °C DPPC/DPPG<br />
4:1 vesicles were injected into the subphase. The increase <strong>of</strong> surface pressure was<br />
determined as a function <strong>of</strong> time. The resulting rearrangement <strong>of</strong> the monolayer was<br />
also documented by scanning force microscopy.<br />
Model systems with unsaturated lipids should lead to a decrease <strong>of</strong> the multiplayer<br />
structures in comparison to the standard system DPPC/DPPG/SP-C or SP-C what seems<br />
to be enhanced by the combination <strong>of</strong> an unsaturated acyl chain with a negatively<br />
charged headgroupin a single molecule. Furthermore unsaturated lipids are likely to<br />
support the formation <strong>of</strong> a SP-B or SP-C rich network in the liquid-expanded phase<br />
around condensed-phase domains.<br />
Literature<br />
[1] von Nahmen, A., A. Post, H.-J. Galla, M. Sieber (1997) The phase behavior <strong>of</strong> lipid<br />
monolayers containing pulmonary surfactant protein C studied by fluorescence light<br />
microscopy<br />
[2] Ross, M., S. Kroll, A. Jansh<strong>of</strong>f, H.-J. Galla (2002) Kinetics <strong>of</strong> phospholipid insertion<br />
into monolayers containing the lung surfactant proteins SP-B or SP-C<br />
[3] Walters, R. W., R. R. Jenq, S. B. Hall (2000) Distinct steps in adsorption <strong>of</strong><br />
pulmonary surfactant to an air-liquid interface<br />
contact:<br />
Ute Klenz<br />
Universität Münster<br />
Biochemie<br />
klenz@uni-muenster.de<br />
Wilhelm-Klemm Str. 2<br />
41849 Münster (Germany)
Pariya Na Nakorn, Carol Flach, Rich Mendelsohn, Hans-Joachim Galla<br />
The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C 17±palm )<br />
on the surface properties <strong>of</strong> lipid monolayers at the air/waterinterface<br />
The pulmonary surfactant is a mixture <strong>of</strong> surface active substances, which are presented<br />
at the air/water-interface <strong>of</strong> the mammalian lung. The important surfactant proteins, SP-<br />
B and SP-C, are important for the dynamic adaptation <strong>of</strong> the surface tension <strong>of</strong> the<br />
alveoli during the breathing cycle. SP-C facilitates the respreading <strong>of</strong> films from<br />
multilayers that have been formed during the compression [1, 2]. It has been reported<br />
that insertion <strong>of</strong> the α-helical (C-terminal) part <strong>of</strong> SP-C into a lipid bilayers occurs when<br />
protrusions are formed at high surface pressures [3]. We are interested in the role <strong>of</strong> Spalmitoylation<br />
for the formation <strong>of</strong> lipid-protein protrusions. In consideration <strong>of</strong> these<br />
fatty acid chains may act as an anchor between two bilayers, identified as protrusions.<br />
Our experimental model systems are composed <strong>of</strong> the major surfactant phospholipids<br />
(DPPC/DPPG 80:20 mol %) with SP-C or truncated SP-C (SP-C 17±palm ). Truncated SP-<br />
C 17±palm lacks the C-terminal segment <strong>of</strong> native SP-C and is either used in palmitoylated<br />
(SP-C 17+palm ) or non-palmitoylated (SP-C 17-palm ) to investigate the role <strong>of</strong> Spalmitoylation.<br />
Film balance technique, fluorescence light microscopy (FLM) and<br />
scanning force microscopy (SFM) have been used to investigate biophysical properties<br />
and the topography <strong>of</strong> lipid-peptide monolayers.<br />
We could show that the N-terminal part <strong>of</strong> SP-C is not able to build up protrusions and to<br />
stabilize bilayer formation. These observations are supported by an IRRAS study<br />
performed by Bi et al. [4], which shows that the protein fragment becomes more<br />
exposed to the water subphase at high surface pressure suggesting a squeeze out <strong>of</strong> the<br />
peptide from the hydrophobic region <strong>of</strong> monolayers.<br />
These results support the idea that the C-terminal part <strong>of</strong> SP-C plays a major role in<br />
multilayer formation during the breathing cycle in pulmonary surfactant.<br />
Literature<br />
[1] Qanbar, R., Cheng, S., Possmayer, F. and Schürch, S. (1999) Am J Physiol 271,<br />
L572-L580<br />
[2] Nag, K., Munro, J.G., Inchley, K., Schürch, S., Petersen, N.O. and Possmayer, F.<br />
(1999) Am J Physiol 277, L1179-L1189<br />
[3] Galla, H.-J., Bourdos, N., von Nahmen, A., Amrein, M. and Sieber, M. (1998) Thin<br />
Solid Films 327-329, 632-635<br />
[4] Bi, X., Flach, C.R., Perez-Gil, J., Plasencia, J., Andreu, D., Oliveira, E. and<br />
Mendelsohn, R. (2002) Biochemistry 41, 8385-8395<br />
contact:<br />
Pariya Na Nakorn<br />
University <strong>of</strong> Muenster<br />
Institute <strong>of</strong> Biochemistry<br />
pariya@uni-muenster.de<br />
Wilhelm Klemm Str.2<br />
48149 Muenster (Germany)
S<strong>of</strong>ia Depner, Wibke Schwarzer, Nicole Daum, Jean Kadathukalam, Norbert E. Fusenig,<br />
Dirk Breitkreutz<br />
The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
Besides its crucial metabolic role insulin regulates various cell functions like growth, in<br />
part shared by insulin like growth factor (IGF). Herein we have searched for possible<br />
physical/functional links <strong>of</strong> insulin-receptor (IR), IGF-1R and membrane as well as<br />
signalling components in human epidermal cells, which may elucidate the proneness to<br />
certain skin lesions and cancer in diabetics. Using human HaCaT cells, immunocoprecipitation<br />
(anti-IR, western blots) revealed constitutive IR linkage to integrin<br />
alpha6beta4, while insulin enhanced caveolin-1, protein kinase C delta (PKCd), but<br />
decreased PKCa interaction with IR. Accordingly, insulin induced tyrosine<br />
phosphorylation <strong>of</strong> IR, caveolin-1, PKCd, and tetraspanin CD151, while decreasing that<br />
in alpha6beta4. To reduce IR or IGF-1R levels respective siRNAs were developed,<br />
showing in transient transfections reduction down to about 10 and 20% (western). This<br />
also lowered growth and additionally induced apoptosis in case <strong>of</strong> IGF-1R, seen by vast<br />
cell detachment. By stable transfection with IR siRNA mostly cell clones with moderate<br />
reduction <strong>of</strong> IR (about 50%) evolved, only one showing a drop to about 20%. This<br />
implies that many repressed cells were not able to form larger colonies, which should be<br />
even more severe for IGF-1R suppression. Similarly, introducing single alpha6 chains<br />
(not beta4) had a deleterious effect on cell survival. A functional link between IR or IGF-<br />
1R signalling, integrin status and adhesion/apoptosis, presumably involving also PKC,<br />
shall be explored by tetracycline-inducible expression using established HaCaT-tet<br />
repressor cells. Finally, analysis <strong>of</strong> those components in lipid rafts indicates such<br />
functional units within cell membranes.<br />
contact:<br />
Frau S<strong>of</strong>ia Depner<br />
Deutsches Krebsforschungszentrum Heidelberg<br />
s.depner@dkfz.de<br />
Im Neuenheimer Feld 280<br />
69120 Heidelberg (Deutschland)
Regine Willumeit, Mont Kumpugdee, Sebastian Linser, Thomas Hauss, Sergio S.<br />
Funari, Jörg Andrä<br />
The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Neutron diffraction and X-ray scattering can useful tools to study the interaction <strong>of</strong><br />
peptide antibiotics with model membranes. These systems are <strong>of</strong> increasing interest<br />
since it is well known that infectious diseases are one <strong>of</strong> the leading causes <strong>of</strong> death<br />
world wide. Together with a broader resistance <strong>of</strong> bacterial pathogens against classical<br />
antibiotics an alternative for the development <strong>of</strong> antibacterial drugs is needed. One<br />
possibility could be antimicrobial peptides.<br />
In this study we present results <strong>of</strong> the interaction <strong>of</strong> the potent peptide antibiotic NK-2<br />
(1) with model membranes composed <strong>of</strong> POPE or POPG, two phospholipids most<br />
abundant in bacterial membranes. Neutron diffraction was used among other techniques<br />
(for example SAXS, fluorescence spectroscopy (2) and Fourier-transform infrared<br />
spectroscopy FTIR) to evaluate the position <strong>of</strong> the peptide in the membrane. A<br />
significant difference in the position <strong>of</strong> the peptide was detected for both membranes<br />
types: While for POPE a surface attachment <strong>of</strong> the peptide is likely, in the case <strong>of</strong> POPG<br />
the peptide inserts parallel to the membrane surface by approximately 1 nm into the<br />
bilayer. In both cases a pore formation like in the barrel stave model was not detected.<br />
Literature<br />
(1) Andrä, J. & Leippe, M. (1999). Candidacidal activity <strong>of</strong> shortened synthetic analogs <strong>of</strong><br />
amoebapores and NK-lysin. Med Microbiol Immunol. 420, 1-8.<br />
(2) Schröder-Borm, H., Willumeit, R., Brandenburg, K. & Andä, J. (2003). Molecular<br />
basis for membrane selectivity <strong>of</strong> NK-2, a potent peptide antibiotic derived from NKlysin.<br />
Biochem. Biophys. Acta 1612, 164-171.<br />
contact:<br />
PD Dr. Regine Willumeit<br />
GKSS Forschungszentrum Geesthacht<br />
Institut für Werkst<strong>of</strong>fforschungs<br />
willumeit@gkss.de<br />
Max-Planck-Str. 1<br />
21502 Geesthacht (Deutschland)
Raiko Stephan, C. Klämbt, S. Bogdan<br />
The Kette/Abi/Sra-1 complex regulates actin dynamics by<br />
inhibting wave and activating wasp function in Drosophila<br />
The F-actin cytoskeleton is a fundamental component <strong>of</strong> all eukaryotic cells mediating<br />
the dynamics <strong>of</strong> many cellular processes. Actin nucleation is catalyzed by the Arp2/3<br />
complex, which is in turn activated by WASP and WAVE. WASP proteins are autoinhibited<br />
whereas WAVE proteins appear to be trans-inhibited. In Drosophila WASP and<br />
WAVE are both regulated by the same protein complex comprising Kette, Abi and Sra-1.<br />
We have previously shown that this complex suppresses the activity <strong>of</strong> WAVE but is also<br />
able to activate WASP function at the membrane. The Kette protein is linked to WASP via<br />
Abi, which can directly bind to Kette and WASP.<br />
Here were present a further characterization <strong>of</strong> the Abi protein. As Kette and Sra-1, Abi<br />
is highly expressed in the central nervous system during development. Using gain and<br />
loss <strong>of</strong> function experiments, we show that Abi is essential for WASP-dependent<br />
processes such as bristle development in the peripheral nervous system <strong>of</strong> Drosophila.<br />
Since the Kette/Abi/Sra-1 complex can also regulate WAVE-dependent F-actin formation,<br />
it must exert an important role in cytoskeletal organization.<br />
Literature<br />
Bogdan, S., Grewe, O., Strunk, M., Mertens, A., Klämbt, C. (2004). Sra-1 interacts with<br />
Kette and Wasp and is required for neuronal and bristle development in Drosophila.<br />
Development, in press<br />
Bogdan, S. and Klämbt, C. (2003). Kette regulates actin dynamics and genetically<br />
interacts with Wasp and Wave. Development 130, 4427-4437<br />
contact:<br />
Raiko Stephan<br />
Westfälische Wilhelms-Universität Münster<br />
Institut für Neurobiologie<br />
raiko.stephan@uni-muenster.de<br />
Badestrasse 9<br />
48149 Münster (Deutschland)
Jürgen Meyer zu Tittingdorf, Sascha Rexroth, Eva Schäfer, Ralf Schlichting, Christoph<br />
Giersch, Nobert A. Dencher, Holger Seelert<br />
The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
The chloroplast H + -ATP synthase is a key component for the energy supply <strong>of</strong> higher<br />
plants and green algae. An oligomer <strong>of</strong> identical protein subunits III is responsible for<br />
the conversion <strong>of</strong> an electrochemical gradient into rotational motion. To date it is highly<br />
controversal if the oligomer III stoichiometry is affected by the metabolic state <strong>of</strong> the<br />
organism or not. Here, the intact oligomer III <strong>of</strong> the ATP synthase from Chlamydomonas<br />
reinhardtii has been isolated for the first time. Due to the importance <strong>of</strong> the subunit III<br />
stoichiometry for energy conversion, a gradient gel system was established to<br />
distinguish oligomers with different stoichiometries, solely by their migration behavior.<br />
With this methodology, a possible alterability <strong>of</strong> this stoichiometry in respect to the<br />
metabolic state <strong>of</strong> the cells was examined. Several growth parameters, i.e., light<br />
intensity, pH-value, carbon source, and CO 2 concentration, were varied to determine<br />
their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the<br />
oligomer III <strong>of</strong> the chloroplast H + -ATP synthase always consists <strong>of</strong> a constant number <strong>of</strong><br />
monomers over a wide range <strong>of</strong> metabolic states. Furthermore, mass spectrometry<br />
indicates that subunit III from both spinach and C. reinhardtii is not modified posttranslationally.<br />
Data suggest a subunit III stoichiometry <strong>of</strong> the algae ATP synthase<br />
divergent from higher plants.<br />
contact:<br />
Dipl. Biol. Jürgen Meyer zu Tittingdorf<br />
Technische Universität Darmstadt<br />
Institut für Physikalische Biochemie<br />
jmwmzt@hrzpub.tu-darmstadt.de<br />
Petersenstrasse 22<br />
64287 Darmstadt (Deutschland)
Hans Schöler, Luca Gentile, James Kehler, Karin Hübner, Michele Boiani<br />
The Oocyte – Embryonic Stem Cell Cycle<br />
Mouse embryonic stem cells in culture can develop into oogonia that enter meiosis and<br />
recruit adjacent cells to form follicle-like structures and later develop into blastocysts<br />
(Hübner et al. 2003). Oogenesis in culture should contribute to various areas including<br />
somatic cell nuclear transfer. Reprogramming, in the context <strong>of</strong> the transfer <strong>of</strong> a<br />
differentiated nucleus into an oocyte, can be defined as the transformation <strong>of</strong> a somatic<br />
cell nucleus into a functional embryonic nucleus capable <strong>of</strong> forming a viable organism.<br />
Correct expression <strong>of</strong> embryonic genes is a prerequisite for development and is<br />
indicative <strong>of</strong> nuclear reprogramming. Oct4 is a gene essential for peri-implantation<br />
development and embryonic pluripotency. It is expressed during cleavage stages and<br />
essential for the differentiation <strong>of</strong> the blastocyst. Oct4 expression becomes restricted to<br />
the inner cell mass and epiblast. After gastrulation Oct4 is active only in germ cells and<br />
silent in somatic cells. Oct4 was used as markers for which gene reprogramming could<br />
be directly related to developmental potential <strong>of</strong> somatic cell clones (Boiani et al. 2002).<br />
Cumulus cell clones initiate Oct4 expression at the correct stage but exhibit an incorrect<br />
spatial expression in the majority <strong>of</strong> blastocysts. The quality <strong>of</strong> an Oct4 GFP transgenic<br />
signal in blastocysts correlates with the ability to generate outgrowths maintaining GFP<br />
expression, and frequency <strong>of</strong> ES cell derivation. Mouse clone blastocysts have less than<br />
half the normal cell number, and <strong>of</strong>ten a faulty expression <strong>of</strong> Oct4. Clone-clone<br />
aggregates <strong>of</strong> genetically identical, but epigenetically different embryos do not form<br />
more blastocysts, but the majority expresses Oct4 normally and has higher rates <strong>of</strong> fetal<br />
and postnatal development (Boiani et al. 2003). Although cloning efficiency is<br />
augmented 8-fold, ES cell derivation is not significantly improved. Our results indicate<br />
that the derivation <strong>of</strong> ES cell lines from clone blastocysts and reproductive cloning are<br />
distinct in their requirements.<br />
Literature<br />
Boiani M, et al. Genes Dev16: 1209, 2002.<br />
Boiani M, et al. EMBO J. 22: 5304, 2003.<br />
Hübner, K et al. Science: 300, 1251, 2003.<br />
contact:<br />
Pr<strong>of</strong>. Dr. Hans-Robert Schöler<br />
MPI für Molekulare Biomedizin<br />
schoeler@mpi-muenster.mpg.de<br />
Mendelstrasse 7<br />
48149 Münster (Deutschland)<br />
additional information<br />
Second Address: Center for Animal Transgenesis and Germ Cell Research, The School <strong>of</strong> Veterinary<br />
Medicine, University <strong>of</strong> Pennsylvania, Kennett Square, USA
Christina Zechel, Ingrid Koziollek-Drechsler, Ulrike Sattler, Marek Samochocki<br />
The orphan receptor GCNF in neuronal differentiation<br />
The germ cell nuclear factor GCNF is a repressor <strong>of</strong> transcription which is required for<br />
embryonic survival and development, and the regulation <strong>of</strong> fertility. We used a<br />
transgenic approach to investigate its role in RA-induced neural differentiation. As model<br />
we chose the embryonal carcinoma cell line PCC7-Mz1, which reproducibly differentiates<br />
into a tissue-like pattern <strong>of</strong> neuronal and non-neuronal cells after exposure to RA.<br />
The phenotype <strong>of</strong> both, gcnf sense and antisense clones consistently indicated that the<br />
expression level <strong>of</strong> GCNF determined the ratio <strong>of</strong> the neuronal to non-neuronal fates that<br />
developed after exposure to RA. Moreover, GCNF expression was required for efficient<br />
induction <strong>of</strong> a subset <strong>of</strong> pro-neuronal genes, as well as up-regulation <strong>of</strong> the neuronal<br />
proteins MAP2 and synaptophysin. Differentiation and maturation <strong>of</strong> neuronal precursor<br />
cells was largely delayed in gcnf antisense cultures. This involved the down-regulation <strong>of</strong><br />
nestin, a marker for undifferentiated neural stem cells, and the acquirement <strong>of</strong><br />
conductance properties <strong>of</strong> mature neurons. Our data suggest that GCNF promotes the<br />
neuronal fate and subsequent neuronal differentiation and neuron maturation.<br />
contact:<br />
Dr. Christina Zechel<br />
Johannes Gutenberg University Mainz<br />
Institute <strong>of</strong> Physiological Chemistry and Pathobiochemistry<br />
zechel@uni-mainz.de<br />
Duesberg Weg 6<br />
55099 Mainz (Germany)
Sven Huelsmann, Christina Hepper, Rolf Reuter<br />
The PDZ-GEF Dizzy regulates cell form and cell adhesion via<br />
rap1 and integrins in macrophages <strong>of</strong> the Drosophila embryo.<br />
In the Drosophila embryo macrophages originate from the cephalic mesoderm and<br />
perform a complex but stereotype migration through the entire embryo. The cellular and<br />
molecular mechanisms regulating this migration remain largely unknown.<br />
We identified the PDZ G-nucleotide exchange factor (PDZ-GEF) Dizzy as one <strong>of</strong> the<br />
components being essential for normal macrophage migration. In mutants lacking Dizzy,<br />
macrophages have smaller cellular protrusions, and migration is slowed down<br />
significantly. In contrast, macrophages overexpressing Dizzy are vastly enlarged and<br />
form long, multiple protrusions. These cell shape changes depend on the function <strong>of</strong> the<br />
small GTPase Rap1: In rap1 mutants Dizzy is unable to induce the large protrusions.<br />
Furthermore, forced expression <strong>of</strong> a constitutively active form <strong>of</strong> Rap1, but not <strong>of</strong> the<br />
wild-type form, induces cell shape changes identical to those induced by Dizzy. These<br />
findings suggest that Dizzy acts through Rap1. Previously, we have shown that integrins<br />
are essential for macrophage migration in the Drosophila embryo. We propose that<br />
integrins are a Rap1-mediated target <strong>of</strong> Dizzy activity: In integrin mutants Dizzy cannot<br />
induce shape changes <strong>of</strong> macrophages. These data provide the first link between a PDZ-<br />
GEF, the corresponding small GTPase and cell adhesion for cell migration in vivo.<br />
We have isolated full-length cDNAs for dizzy and observed alternative mRNA splicing<br />
events <strong>of</strong> unknown relevance. Currently, we generate transgenic flies which allow the<br />
conditional expression <strong>of</strong> the splice is<strong>of</strong>orms and <strong>of</strong> various mutant forms <strong>of</strong> dizzy. The<br />
effect <strong>of</strong> these forms on macrophage morphology and migration in vivo will allow to<br />
elucidate the structure-function-relationship <strong>of</strong> the Dizzy protein in regulating cell<br />
motility.<br />
contact:<br />
Dipl. Biol. Christina Hepper<br />
Universität Tübingen<br />
Interfakultäres Institut f. Zellbiologie; Abt. Genetik d. Tiere<br />
christina.hepper@uni-tuebingen.de<br />
Auf der Morgenstelle 28<br />
72076 Tübingen (Deutschland)
Heike Bruhn, Matthias Leippe<br />
The pore-forming mechanism <strong>of</strong> amoebapore A characterized<br />
by mutational analysis<br />
Killing <strong>of</strong> foreign cells by pore-forming proteins for the purpose <strong>of</strong> defence or attack is a<br />
common principle used by many organisms. Interestingly, some members <strong>of</strong> the diverse<br />
family <strong>of</strong> saposin-like proteins (SAPLIPs) possess the capability <strong>of</strong> fulfilling this function.<br />
SAPLIPs are widespread in eukaryotic organisms and are characterized by a common<br />
fold and, in spite <strong>of</strong> their distinct and different functions, by the ability to interact with<br />
lipids. From an evolutionary view, the archetype <strong>of</strong> the pore-forming members <strong>of</strong> this<br />
family are the amoebapores from the protozoan parasite Entamoeba histolytica. The<br />
molecular features <strong>of</strong> the recently solved NMR structure <strong>of</strong> amoebapore A, namely a<br />
defined exposed hydrophobic surface region as well as specific electrostatic interactions,<br />
allowed us to postulate a hitherto unique mechanism <strong>of</strong> action by which a member <strong>of</strong><br />
this protein family forms stable pores into a cellular membrane. Here, we elucidate the<br />
mechanistic details by the analysis <strong>of</strong> recombinantly expressed mutants.<br />
contact:<br />
Dr Heike Bruhn<br />
Universität Würzburg<br />
Zentrum für Infektionsforschung<br />
heike.bruhn@mail.uni-wuerzburg.de<br />
Röntgenring 11<br />
97070 Würzburg (Deutschland)
A. Markert, S. Kucht, J. Gross, M. Ahimsa-Mueller, Y. Hussein, U.* Steiner, E. Leistner<br />
The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Ergoline alkaloids are constituents <strong>of</strong> Clavicipitaceous fungi living on Poaceae plants.<br />
Ergoline alkaloids are also present in Ipomoea asarifolia Roem. & Schult<br />
(Convolvulaceae). Elimination <strong>of</strong> epibiotic and endophytic fungi by fungicides (1) results in<br />
loss <strong>of</strong> fungi and ergoline alkaloids from the Ipomoea plant indicating that accumulation<br />
<strong>of</strong> alkaloids in the plant depends on the presence <strong>of</strong> plant-associated fungus.<br />
Results <strong>of</strong> experiments are discussed in which fungi isolated from the plants were<br />
examined for the presence <strong>of</strong> alkaloids.<br />
Literature<br />
(1) Kucht et al., Planta. 2004 Apr 15<br />
contact:<br />
Anne Markert<br />
Rheinische Friedrich-Wilhelms-Universität Bonn<br />
Insitut für Pharmazeutische Biologie<br />
A.Markert@uni-bonn.de<br />
Nußallee 6<br />
53115 Bonn (Deutschland)<br />
additional information<br />
*)Institut für Pflanzenkrankheiten der Universität Bonn
Yanfeng Li, Jan Dudek, Bernard Guiard, Nikolaus Pfanner, Peter Rehling, Wolfgang Voos<br />
The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner<br />
to Pam18<br />
Most <strong>of</strong> mitochondrial proteins are imported post-translationally into the organelle.<br />
Preproteins destined for the matrix are directed by N-terminal presequences across both<br />
mitochondrial membranes. The preproteins cross the membranes via two channels<br />
formed by the translocase <strong>of</strong> the outer membrane (TOM) and <strong>of</strong> the inner membrane<br />
(TIM23).<br />
The translocation <strong>of</strong> entire precursor polypeptides into the matrix requires the function <strong>of</strong><br />
the mitochondrial Hsp70 (mtHsp70) that forms the core <strong>of</strong> presequence translocaseassociated<br />
motor (PAM). In the PAM complex, mtHsp70 cooperates with four<br />
cochaperones: the nucleotide exchange-factor Mge1, the translocase-associated fulcrum<br />
Tim44, the J-protein Pam18, and Pam16. Pam18 and Pam16 show a significant<br />
homology to proteins <strong>of</strong> the DnaJ family. Pam18 and Pam16 form a stable complex that<br />
associates with the TIM23 translocase. Pam18 contains a conserved J-domain while<br />
Pam16 only contains a J-like domain where the signature motif HPD is missing. We could<br />
show that Pam18 stimulates the ATPase activity <strong>of</strong> mtHsp70. In contrast, Pam16 inhibits<br />
the Pam18-mediated stimulation <strong>of</strong> the ATPase activity <strong>of</strong> mtHsp70. Moreover, Pam 16<br />
cannot be converted to a J-type cochaperone by inclusion <strong>of</strong> the HPD motif. In Pam16<br />
mutants, the interaction <strong>of</strong> mtHsp70 with incoming preproteins is defective. We propose<br />
that Pam16 thus does not represent a typical Hsp70-cochaperone <strong>of</strong> the J-protein family,<br />
but rather serves to control the activity <strong>of</strong> Pam18.<br />
Literature<br />
1. Truscott, K. N. et al. (2003) J. Cell Biol. 163, 707-713.<br />
2. Frazier, A. et al. (2004) Nat. Struct. Mol. Biol. 11, 226-233<br />
3. Li ,Y. et al. (2004) J. Biol. Chem. in press<br />
contact:<br />
Yanfeng Li<br />
University <strong>of</strong> Freiburg<br />
Institute <strong>of</strong> Biochemistry and Molecular Biology<br />
yanfeng.li@biochemie.uni-freiburg.de<br />
Hermann-Herder-Str.7<br />
79104 Freiburg (Germany)
Florian Garczarek, Klaus Gerwert<br />
The Proton Release Mechanism <strong>of</strong> Bacteriorhodopsin’s WT,<br />
E204D and E194D revealed with Time-Resolved Step-Scan and<br />
H/D-Exchange FTIR Measurements<br />
It was proposed that the proton release group <strong>of</strong> bR constitutes not <strong>of</strong> a single proton<br />
binding site at one amino acid, but that the released proton is delocalised in a hydrogenbonded<br />
network (1). In the L to M transition the pK <strong>of</strong> the network decreases and the<br />
proton is released to the protein surface. This was shown by a negative continuum<br />
absorbance change in the spectral region from 1900 cm -1 to 1800 cm -1 . Here, the<br />
influence <strong>of</strong> several mutants on the proton release network is investigated. Especially<br />
the continuum absorbance change is analyzed. These studies yield the information about<br />
the location <strong>of</strong> the network, and reveal the participating residues necessary for its<br />
stabilization. Together with MD-simulations (2) we identifed a protonated water cluster,<br />
consisting <strong>of</strong> one proton and five water molecules surrounded by six residues and two<br />
backbone groups. Hence it is clear why so many different mutated residues are able to<br />
perturb the proton release. This is due to their influence on the protonated hydrogenbonded<br />
network by their electrostatic properties, the spatial rearrangement and the<br />
difference in their H-bond formation capability. Besides the investigation <strong>of</strong> the temporal<br />
behavior <strong>of</strong> the continuum absorption we take a closer look on its spectral band shape<br />
by expanding the range to 3050 cm -1 (3,4), and find it in a good agreement with<br />
quantum chemical calculations <strong>of</strong> excess protons in water. Furthermore we introduce a<br />
new method to resolve the protonation state <strong>of</strong> carboxylic acids in proteins, where we<br />
compare the 2 nd derivatives <strong>of</strong> direkt H/D exchange FTIR absorbance spectra. Using this<br />
technique we specifie the carbonyl stretch <strong>of</strong> Glu204 and Asp204 within the bR ground<br />
state <strong>of</strong> the mutants E194D and E204D, but we do not find Glu204 or Glu194 protonated<br />
in the ground state <strong>of</strong> bR-WT (4).<br />
Literature<br />
(1)Rammelsberg R., Huhn G., Lubben M., Gerwert K. 1998. Biochemistry 37:5001-5009.<br />
(2)Kandt C., Schlitter J., Gerwert K. 2004. Biophys. J. 86:705-717.<br />
(3)Garczarek F., Wang J., El-Sayed Mostafa A., Gerwert K. (submitted)<br />
(4)Garczarek F. and Gerwert K. (submitted)<br />
contact:<br />
Florian Garczarek<br />
Ruhr-Universität-Bochum<br />
Lehrstuhl für Biophysik<br />
garflo@bph.rub.de<br />
Universitätsstr. 150<br />
44780 Bochum (Germany)
Beat A. Imh<strong>of</strong>, Michel Aurrand-Lions, Chrystelle Lamagna<br />
The role <strong>of</strong> junctional adhesion molecule-C (JAM-C) in<br />
angiogenesis and inflammation<br />
Junctional adhesion molecules are members <strong>of</strong> an immunoglobulin superfamily subset.<br />
They possess two extracellular immunoglobulin domains and a cytoplasmic PDZ binding<br />
sequence. We recently cloned JAM-C and found that it is part <strong>of</strong> a cell polarity complex.<br />
JAM-C is located at vascular junctions and controls vascular permeability and leukocyte<br />
transendothelial migration. More recently we found JAM-C expression upregulated in<br />
tumor blood vessels. Antibodies against JAM-C blocked in vitro vessel formation and<br />
prevented angiogenic growth under hypoxy in vivo. More importantly, the antibodies<br />
blocked tumor angiogenesis and as a consequence tumor growth in a mouse model.<br />
In conclusion, JAM-C is a versatile vascular adhesion molecule and a novel target to<br />
block angiogenesis.<br />
contact:<br />
PhD, Pr<strong>of</strong>. Beat A. Imh<strong>of</strong><br />
Centre Médical Universitaire<br />
Dept <strong>of</strong> Pathology & Immunology<br />
Beat.Imh<strong>of</strong>@medecine.unige.ch<br />
1, rue Michel Servet<br />
CH1211 GENEVA 4 (Switzerland)
O. Bergner, C. Brendel, Claudia Koch-Brandt<br />
The role <strong>of</strong> the lipoprotein receptor LRP1 in connective tissue<br />
factor (CTGF) induced signal transduction: Implications for<br />
the pathogenesis <strong>of</strong> atherosclerosis<br />
The elevated production <strong>of</strong> CTGF in atherosclerotic lesions suggests an important role for<br />
this growth factor in atherogenesis. Expression <strong>of</strong> CTGF is subject to complex regulation<br />
and is induced by growth factors such as TGFβ and PDGF-BB. Recently J. Herz and<br />
coworkers have shown that by controlling PDGF receptor activity LRP1 plays a pivotal<br />
role in the protection <strong>of</strong> the vessel wall and the prevention <strong>of</strong> atherosclerosis. In addition<br />
LRP1 directly binds to CTGF and might not only promote its internalization and<br />
degradation, but also induce cellular signalling cascades. In cooperation with the Herz<br />
group we here propose to characterize the role <strong>of</strong> LRP1 in CTGF induced cellular<br />
signaling in fibroblasts as well as in a vascular smooth muscle cell line and primary<br />
smooth muscle cells. Comparative analysis will be performed in the presence or absence<br />
<strong>of</strong> CTGF and/or PDGF in cells that express wt LRP1, or mutant forms <strong>of</strong> this receptor, or<br />
are defective in the gene or are depleted <strong>of</strong> this receptor by siRNA expression. After<br />
growth factor stimulation the activation <strong>of</strong> known and putative components <strong>of</strong> LRP1 and<br />
CTGF signalling pathways will be examined. These experiments will reveal the role <strong>of</strong><br />
LRP1 and the transduction pathways involved in CTGF-dependent signaling and will yield<br />
new insights into the pathogenesis <strong>of</strong> atherosclerosis.<br />
All work will be done in collaboration with J. Herz, UTSW Dallas, USA<br />
contact:<br />
Pr<strong>of</strong>. Dr. Claudia Koch-Brandt<br />
Universität Mainz<br />
Institut für Biochemie<br />
koch@mail.uni-mainz.de<br />
Becherweg 30<br />
55099 Mainz (D)
Jan Barnikow, Ron Tynes, Maik Kindermann, Stefan Steurer, Andreas Brecht<br />
The SNAP-tag TM - a unique tool for covalent labeling and<br />
immobilization <strong>of</strong> proteins<br />
We present a novel protein tag and its applications which range from protein labeling<br />
over immobilization to purification. The SNAP-tag is based on the DNA-repair enzyme<br />
alkylguanine-DNA-alkyltransferase. 1 It reacts with O 6 –alkylguanine derivatives forming a<br />
thioether bond between the protein and the alkylgroup <strong>of</strong> its substrate. 2<br />
The selectivity <strong>of</strong> the reaction in combination with the formation <strong>of</strong> a stable linkage<br />
between the protein-tag and its substrate <strong>of</strong>fers attractive possibilities for handling and<br />
labeling <strong>of</strong> recombinant fusion proteins even in complex samples. 3 The in vivo labeling <strong>of</strong><br />
SNAP-tag fusion proteins has been tested with a range <strong>of</strong> fluorescent dye substrates in<br />
several mammalian cell lines. The nontoxic compounds allow live cell imaging. Cellular<br />
localization experiments with various fusion proteins targeting the label to cytosceleton,<br />
plasmamembrane, nucleus or cytoplasm resulted in the respective staining in<br />
combination with very low background signals.<br />
In vitro, the labeling proceeds with more than 90% labeling achieved within 10 min (RT,<br />
10 µM substrate, up to 5 µM fusion protein). Fluorescence polarization allows to follow<br />
the reaction in realtime with dyes that cover a broad range <strong>of</strong> the spectrum. Labeled<br />
SNAP-tag fusion proteins can be used in fluorescent interaction assays. Another<br />
attractive application is the detection and quantitation <strong>of</strong> labeled fusion proteins on SDSgels<br />
without further staining or blotting and immunodetection steps.<br />
The technology is currently tested and further applications are developed in a number <strong>of</strong><br />
joint research projects with academical and industrial partners.<br />
Literature<br />
1) A. Juillerat, T. Gronemeyer, A. Keppler, S. Gendreizig, H. Pick, H. Vogel, K. Johnsson,<br />
2003, Chemistry&Biology 10: 313-7.<br />
2) A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K. Johnsson, 2003,<br />
Nature Biotechnology 21: 86 - 89.<br />
3) A. Keppler, K. Kindermann, S. Gendreizig, H. Pick, H. Vogel, K. Johnsson, 2004,<br />
Methods 32: 437-44.<br />
contact:<br />
Dr. Jan Barnikow<br />
Covalys Biosciences AG<br />
jan.barnikow@covalys.com<br />
Benkenstr. 254<br />
4108 Witterswil (Switzerland)
Stefan Harjes, Peter Bayer, Axel Scheidig<br />
The structure <strong>of</strong> human 3'-phosphoadenosine-5'phosphosulfate<br />
synthetase 1 - A molecular pendulum?<br />
The high energy sulfate donor PAPS made by human PAPS synthetase 1 is involved in<br />
sulfate conjugation <strong>of</strong> extracellular matrix, hormones and drugs. PAPS is synthesized by<br />
two subsequent reactions from ATP and sulfate. ATP sulfurylase forms APS, then APS<br />
kinase phosphorylates the APS intermediate to PAPS. Up to now the interaction between<br />
the two enzymatic activities remained elusive, mainly because <strong>of</strong> missing structural<br />
information. Here we present the crystal structure <strong>of</strong> human PAPSS1 at 1.8 Å resolution.<br />
The structure reveals a homodimeric, asymmetric complex with the shape <strong>of</strong> a chair. The<br />
two monomers have different structures and only one <strong>of</strong> them binds ADP. ATP<br />
sulfurylase and APS kinase span an angle which differs between the two monomers by<br />
40 degrees, strongly indicating conformational changes. We further verified the<br />
asymmetry <strong>of</strong> hPAPSS1 by an enzymatic assay in solution.<br />
contact:<br />
Dr. Stefan Harjes<br />
Max-Planck Institut fuer Moleculare Physiologie<br />
Molekulare und Strukturelle Biophysik<br />
stefan.harjes@mpi-dortmund.mpg.de<br />
Otto-Hahn-Str. 11<br />
44227 Dortmund (D)
Kristina Beck, Karl-Heinz Klempnauer<br />
The transcription factor C/EBP&beta triggers phosphorylation<br />
<strong>of</strong> the coactivator p300: A new mechanism <strong>of</strong> cross-talk<br />
between transcription factors and coactivators?<br />
Coactivators play key roles in the regulation <strong>of</strong> eukaryotic gene expression by acting as<br />
bridging proteins between transcription factors and the basal transcriptional machinery<br />
as well as by modulating chromatin structure. Coactivators are targets <strong>of</strong> posttranslational<br />
modifications, e.g. phosphorylation. We have previously shown that binding<br />
<strong>of</strong> the coactivator p300 to the transcription factor CCAAT-enhancer-binding protein beta<br />
(C/EBP&beta) or certain other transcription factors triggers its phosphorylation by an as<br />
yet unknown protein kinase (Schwartz et al. 2003).<br />
To elucidate the molecular mechanism by which C/EBP&beta triggers p300<br />
phosphorylation we have already mapped the regions <strong>of</strong> p300 and C/EBP&beta that are<br />
necessary for C/EBP&beta dependent phosphorylation <strong>of</strong> p300. Furthermore, we have<br />
identified several sites <strong>of</strong> p300 which are phosphorylated. In the present work, we were<br />
trying to identify the protein kinase responsible for C/EBP&beta-dependent<br />
phosphorylation <strong>of</strong> p300.<br />
To investigate whether a protein kinase is bound to C/EBP&beta or p300 we<br />
immunoprecipitated C/EBP&beta, p300 or a complex <strong>of</strong> both from transfected cells<br />
followed by in vitro protein kinase assays. Our results suggest that protein kinase<br />
activity is recruited to a complex <strong>of</strong> p300 and C/EBP&beta more efficiently than to either<br />
<strong>of</strong> these proteins alone. This protein kinase phosphorylates both p300 and C/EBP&beta.<br />
Literature<br />
C. Schwartz, K. Beck, S. Mink, M. Schmolke, B. Budde, D. Wenning and K.H.<br />
Klempnauer. (2003) Embo J 22, 882-892.<br />
contact:<br />
Kristina Beck<br />
Universität Münster<br />
Biochemie<br />
beckkr@uni-muenster.de<br />
Wilhelm-Klemm-Str. 2<br />
48149 Münster (Deutschland)
Maret Böhm, Nadja Bitomsky, Karl-Heinz Klempnauer<br />
The transformation supressor Protein Pdcd4 binds to RNA and<br />
is involved in the regulation <strong>of</strong> c-Jun activity.<br />
The pdcd4 gene, a direct target gene <strong>of</strong> the transcription factor c-Myb, was originally<br />
identified as a gene whose expression is strongly induced during apoptosis in various<br />
murine cells. Recent work has implicated pdcd4 as a tumor suppressor gene in the<br />
development <strong>of</strong> lung cancer. Analyses <strong>of</strong> the molecular function <strong>of</strong> Pdcd4 suggest that<br />
the protein may play multiple roles. Pdcd4 interacts with eukaryotic translation initiation<br />
factors eIF4A and eIF4G and appears to have a regulatory role during protein<br />
translation. We were interested to see whether the protein is also an RNA-binding<br />
protein. To address this issue, we determined the ability <strong>of</strong> Pdcd4 to bind to agarose<br />
carrying covalently bound poly-adenylic acid and tested the RNA-binding activity <strong>of</strong><br />
Pdcd4 in a nitrocellulose filter-binding assay, using bacterially expressed and purified<br />
Pdcd4 and radiolabeled RNA. Our data show that Pdcd4 binds to RNA and map the RNA<br />
binding domain to the aminoterminal region <strong>of</strong> the protein. Presently we are trying to<br />
determine if Pdcd4 binds to specific RNA species. Another interesting feature <strong>of</strong> Pdcd4 is<br />
its ability to suppress TPA-induced transformation <strong>of</strong> mouse keratinocytes and to inhibit<br />
the activation <strong>of</strong> AP-1 dependent reporter genes by c-Jun, suggesting that the Pdcd4<br />
protein might inhibit neoplastic transformation by blocking the function <strong>of</strong> c-Jun. We<br />
have addressed the mechanism by which Pdcd4 affects c-Jun activity. Our data show<br />
that Pdcd4 interferes with the phosphorylation <strong>of</strong> c-Jun by Jun-N terminal-kinase and the<br />
recruitment <strong>of</strong> the coactivator p300 by c-Jun.<br />
contact:<br />
Maret Böhm<br />
Westfälische Wilhelms-Universität<br />
Institut für Biochemie<br />
maboehm@uni-muenster.de<br />
Wilhelm-Klemm-Str. 2<br />
48149 Münster (Germany)
Christoph Hutter, Ingo Neumann, Stefan Burdach, Martin S. Staege<br />
The tumor specific chimeric transcription factor EWS-FLI1<br />
down-regulates gene expression<br />
Ewing family tumors (EFT) are defined by expression <strong>of</strong> EWS-ETS fusion proteins which<br />
are involved in the transformation process. In the huge majority <strong>of</strong> EFT, reciprocal<br />
translocations <strong>of</strong> the EWS gene on 22q12 with FLI1 on 11q24 are detectable. The<br />
product <strong>of</strong> the EWS-FLI1 translocation is considered to be an aberrant transcription<br />
factor that is capable to transform normal cells in vitro. It was shown that EWS-FLI1<br />
expression is necessary for tumor cell growth. Yet, the mechanisms <strong>of</strong> transformation by<br />
EWS-FLI1 are unclear and only a few target genes <strong>of</strong> EWS-FLI1 have been identified. We<br />
transiently and stably transfected HEK293 cells with a vector allowing simultaneous<br />
expression <strong>of</strong> EWS-FLI1 and EGFP as reporter gene. Expression <strong>of</strong> EWS-FLI1 in<br />
transiently and stably transfected cells was confirmed by RT-PCR and western blotting.<br />
Using high-density DNA-microarrays (Affymetrix HG-U133A) we analyzed the gene<br />
expression program <strong>of</strong> these cells in comparison to wild type and mock-transfected cells.<br />
Additionally, EWS-FLLI1 positive (A673, SK-N-MC) and negative (SiMa, CHP-126, SH-<br />
SY5Y) tumor cell lines and native tumor samples from Ewing tumors (EWS-FLI1 positive)<br />
and neuroblastomas (EWS-FLI1 negative) were analyzed on the same microarray<br />
platform. EWS-FLI1 induced expression <strong>of</strong> several genes in transiently as well as stably<br />
transfected HEK293 cells. Surprisingly, EWS-FLI1 also suppressed expression <strong>of</strong> a large<br />
number <strong>of</strong> genes, especially in stably EWS-FLI1 expressing cells. In these cells<br />
approximately four times more genes were down-regulated then induced. A significant<br />
proportion <strong>of</strong> these down-regulated genes was also down-regulated in EWS-FLI1-positive<br />
tumor cell lines and patient samples in comparison to negative samples.<br />
From our data we conclude that EWS-FLI1, in addition to switching on expression <strong>of</strong><br />
target genes, acts also as a direct or indirect suppressor <strong>of</strong> gene expression. This may<br />
also explain the maintenance <strong>of</strong> the very low differentiated phenotype <strong>of</strong> Ewing tumor<br />
cells.<br />
contact:<br />
Christoph Hutter<br />
Universitätsklinik und Poliklinik für Kinder- und Jugendmedizin<br />
Forschungslabor für Krebskranke Kinder/BioZentrum<br />
christoph.hutter@medizin.uni-halle.de<br />
Weinbergweg 22<br />
06120 Halle (Saale) (Deutschland)
Andrea Schulze, Sybille Standera, Elke Bürger, Marjolein Kikkert, Emmanuel Wiertz,<br />
Peter-Michael Kloetzel, Michael Seeger<br />
The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate<br />
Proteasomes are the main protein degrading machineries <strong>of</strong> the cell. The proteins that<br />
are destined for degradation by the proteasome are covalently conjugated to the small<br />
protein ubiquitin by an enzymatic cascade involving the three enzymes E1, E2 and E3.<br />
During the past years many proteins were characterised that contain a domain with<br />
strong homology to ubiquitin. These proteins are called ubiquitin-domain proteins<br />
(UDPs). UDPs appear to be involved in different cellular functions. But they all share the<br />
ability to interact with the proteasome via their ubiquitin-like domain.<br />
We are analysing a human UDP which contains a ubiquitin-like domain at the very Nterminus<br />
and transmembrane helices at the C-terminus. This protein, called HERP,<br />
localises to the endoplasmatic reticulum (ER) and its expression is induced upon ER<br />
stress. Co-immunoprecipitations demonstrate the interaction <strong>of</strong> HERP with components<br />
<strong>of</strong> the ubiquitin-proteasome system. A siRNA mediated knock-down <strong>of</strong> HERP resulted in<br />
an early onset <strong>of</strong> ER-stress induced apoptosis. Therefore we hypothesised that HERP has<br />
a role in ERAD. This idea is supported by experiments showing that HERP is stabilised<br />
upon overexpression <strong>of</strong> one <strong>of</strong> its interaction partners. In addition overexpression <strong>of</strong><br />
HERP results in an accelerated degradation <strong>of</strong> an ERAD substrate.<br />
contact:<br />
Andrea Schulze<br />
Charite<br />
Institut für Biochemie<br />
andrea.schulze@charite.de<br />
Monbijoustr. 2<br />
10117 Berlin (Germany)
Nicole Reifschneider, S. Goto, Norbert A. Dencher, Frank Krause<br />
Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong><br />
Rattus norvegicus studied by peptide mass fingerprinting<br />
Understanding the role <strong>of</strong> mitochondria not only in normal cell physiology but also in<br />
cellular dysfunction and cellular death is one <strong>of</strong> the main topics in current proteomic<br />
research. Especially blue-native polyacrylamide gel electrophoresis (BN-PAGE) combined<br />
with subsequent 2D SDS-PAGE is an ideal tool for this purpose as it is a simple method<br />
providing high throughput <strong>of</strong> various soluble and/or membrane proteins in their native<br />
state including protein-protein interactions.<br />
Mitochondria <strong>of</strong> different tissues (liver, kidney, brain, muscle) isolated from Rattus<br />
norvegicus were solubilized with the mild detergent digitonin under conditions<br />
maintaining respiratory supercomplexes and ATP synthase oligomers. The digitoninextracts<br />
were analysed using BN-PAGE and the more gentle CN (colourless-native)-<br />
PAGE. Many mitochondrial integral membrane proteins, besides oxidative<br />
phosphorylation complexes, as well as matrix proteins were identified by trypsinization<br />
and subsequent matrix assisted laser desorption/ionisation mass spectrometry (MALDI-<br />
MS) in one gel. Using this technique a partial mitochondrial proteome map could be<br />
assembled providing physiological significant information about protein-protein<br />
interactions. Also tissue specific proteins could be observed and will be discussed.<br />
contact:<br />
Nicole Reifschneider<br />
Tu-Darmstadt<br />
Physikalische Biochemie<br />
nicoler@pop.tu-darmstadt.de<br />
Petersenstr. 22<br />
64287 Darmstadt (Germany)
Peter Hinterdorfer<br />
Topography and Recognition Imaging using AFM<br />
We have developed a method for the localization specific binding sites and epitopes with<br />
nm positional accuracy by combining dynamic force microscopy with single molecule<br />
recognition force spectroscopy. A magnetically driven AFM tip containing a ligand<br />
covalently bound via a tether molecule was oscillated at 5 nm amplitude while scanning<br />
along the surface. Since the tether had a length <strong>of</strong> 8 nm, the ligand on the tip was<br />
always kept in close proximity to the surface and showed a high probabilty <strong>of</strong> binding<br />
when a receptor site was passed. We showed that for cantilevers with low Q-factor<br />
driven at frequencies below resonance the surface contact only influenced the downward<br />
deflections <strong>of</strong> the oscillations, while binding <strong>of</strong> the ligand on the tip to the receptors on<br />
the surface reduced only the upwards deflections <strong>of</strong> the oscillations. The recognition<br />
signals were well separated from the topographic signals arising from the surface. In this<br />
way, topography and recognition image were gained simultaneously and independently<br />
with nm lateral resolution.<br />
This work was supported by the Austrian Science Foundation Project P-14549 and the<br />
GEN-AU initiative.<br />
contact:<br />
Ass. Pr<strong>of</strong>. Dr. Peter Hinterdorfer<br />
Johannes Kepler University <strong>of</strong> Linz<br />
Institute for Biophysics<br />
peter.hinterdorfer@jku.at<br />
Altenbergerstr. 69<br />
A-4040 Linz (Austria)
Henriette Brüggemann, Antje Berken, Alfred Wittingh<strong>of</strong>er<br />
Towards the function <strong>of</strong> plant Rop GTPases in actin<br />
reorganisation<br />
Growing evidence reveals that Rop proteins, the Rho GTPase homologues <strong>of</strong> plants, play<br />
a significant role in regulating actin dependent processes in higher plants (1). However,<br />
the molecular mechanisms <strong>of</strong> regulation are largely unknown. Like their mammalian and<br />
yeast counterparts Rho, Rac and Cdc42, Rops act as molecular switches that cycle from<br />
a GDP-bound “Off” to a GTP-bound “On” state. In this cycle, GTPase inactivation occurs<br />
via GTPase activating proteins (GAPs) while GTPase activation requires guanine<br />
nucleotide exchange factors (GEFs). Unlike mammalian RhoGAPs, RopGAPs feature a<br />
Cdc42/Rac interactive binding (CRIB) motif that is believed to regulate RopGAP activity<br />
(2). We analyzed bacterially expressed and purified Rop4 and RopGAP2 from Arabidopsis<br />
thaliana. RopGAP2 specifically interacted with activated Rop4. Moreover, Yeast Two<br />
Hybrid studies showed that there are two binding sites for the Rop GTPase in RopGAP2,<br />
the CRIB and the GAP domain. Rop4 nucleotide <strong>of</strong>f rates were found to be rather low<br />
implying that RopGEFs exist in plants although no RhoGEF homologues <strong>of</strong> the Dbl-type<br />
have yet been identified throughout the sequenced genomes. GFP-Rop4 overexpression<br />
in NIH3T fibroblasts resulted in the formation <strong>of</strong> lamellipodia and membrane ruffles<br />
indicating that Rops are involved in the yet unknown signalling pathways towards actin.<br />
We demonstrate that definite Rop-interactive CRIB-motif containing proteins (RICs) (3)<br />
differentially interact with activated Rops, which may mediate particular Rop<br />
downstream effects.<br />
Literature<br />
(1) Zheng, Z. L., and Yang, Z. (2000). The Rop GTPase: An emerging signalling switch in<br />
plants. Plant Mol. Biol. 44, 1-9.<br />
(2) Wu, G., Li, H., and Yang, Z. (2000). Arabidopsis RopGAPs are a novel family <strong>of</strong> rho<br />
GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for ropspecific<br />
GTPase stimulation. Plant Physiol 124, 1625-1636.<br />
(3) Wu, G., Gu, Y., Li, S., and Yang, Z. (2001). A genome-wide analysis <strong>of</strong> Arabidopsis<br />
Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets. Plant Cell<br />
13, 2841-2856.<br />
contact:<br />
Henriette Brüggemann<br />
Max-Planck-Institut für molekulare Physiologie<br />
Strukturelle Biologie<br />
henriette.brueggemann@mpi-dortmund.mpg.de<br />
Otto-Hahn-Str. 11<br />
44227 Dortmund (Deutschland)
Jana Mooster, Florian Klein, Niklas Feldhahn, Mieke Sprangers, Markus Müschen<br />
Tracing the pre-B to immature B cell transition in human<br />
leukemia cells reveals a coordinated sequence <strong>of</strong> primary and<br />
secondary IGK gene rearrangement, IGK deletion and IGL<br />
gene rearrangement<br />
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant<br />
transformation <strong>of</strong> pre-B cells in humans and prevents further development. We studied<br />
whether inhibition <strong>of</strong> BCR-ABL1 kinase activity by the anti-leukemic drug STI571 can<br />
relieve this differentiation block: STI571 treatment <strong>of</strong> leukemia patients induced<br />
expression <strong>of</strong> the Ig light chain-associated transcription factors IRF4 and SPIB. In<br />
parallel, upregulation <strong>of</strong> RAG1 and 2, increased light chain germline transcription, and<br />
rearrangement <strong>of</strong> IGK and IGL genes were observed in cell culture experiments.<br />
However, STI571-treated pre-B ALL cells expressed l- but almost no k-light chains. This<br />
was consistent with STI571-induced rearrangement <strong>of</strong> the k-deleting element (KDE),<br />
which can delete productively rearranged Vk-Jk joints. Amplifying short-lived DNA<br />
intermediates from double-strand breaks at recombination signal sequences within the<br />
IGK, KDE and IGL loci revealed a coordinated sequence <strong>of</strong> rearrangement events<br />
induced by STI571: Recombination <strong>of</strong> IGK gene segments is already initiated within one<br />
hour after STI571-treatment, followed by KDE-mediated deletion <strong>of</strong> Vk-Jk-joints six<br />
hours later and, ultimately, IGL gene rearrangement after 12 hours. Continued activity<br />
<strong>of</strong> the recombination machinery induced secondary IGK gene rearrangements, which<br />
shifted preferential usage <strong>of</strong> upstream located Jk- to downstream Jk- gene segments.<br />
Thus, inhibition <strong>of</strong> BCR-ABL1 in pre-B ALL cells i.) recapitulates early B cell development,<br />
ii.) directly shows that IGK, KDE and IGL genes are rearranged in sequential order and<br />
iii.) provides a model for Ig light chain gene regulation in the human.<br />
contact:<br />
Jana Mooster<br />
Universität Düsseldorf<br />
Lab. for Molecular Stem Cell Biology<br />
mooster@itz.uni-duesseldorf.de<br />
Moorenstr. 5<br />
40225 Düsseldorf (D)
Krzyszt<strong>of</strong> Wandzik, Katrin Dassler, Matthias Kaup, Hendrik Fuchs<br />
Transferrin receptor expression and shedding during<br />
megakaryopoiesis<br />
Transferrin receptor (TfR) is a transmembrane, homodimeric glycoprotein that mediates<br />
cellular uptake <strong>of</strong> iron via endocytosis <strong>of</strong> ferri-transferrin. Little is known <strong>of</strong> the<br />
expression and shedding <strong>of</strong> the transferrin receptor leading to soluble transferrin<br />
receptor (sTfR) during megakaryopoiesis. The erythroleukemic cell line K562 was used<br />
as a model system for studying early steps <strong>of</strong> megakaryopoiesis. Phorbol-12-N-myristate<br />
13-acetate (PMA) stimulates these cells to differentiate into megakaryocytic-like cells.<br />
This development is characterized by acquisition <strong>of</strong> megakaryocytic markers like CD 41 /<br />
61, polyploidisation, increase in cellular size and appearance <strong>of</strong> eosinophilic areas in the<br />
cytoplasm with a concomitant loss <strong>of</strong> erythroic characteristics. High levels <strong>of</strong> ploidy can<br />
be induced by staurosporine, a potent inhibitor <strong>of</strong> protein kinase C. We investigated the<br />
expression and shedding <strong>of</strong> TfR during megakaryocytic maturation in K562 cells. We<br />
found that differentiation <strong>of</strong> K562 cells for 24 h with PMA (100 nM) or staurosporine (40<br />
nM) in the megakaryocytic pathway is accompanied by a down regulation <strong>of</strong> TfR-protein<br />
expression (western blot analysis). After 48- and 72-h-treatment <strong>of</strong> K562 cells with PMA<br />
the TfR-protein expression decreased strongly. Release <strong>of</strong> sTfR after treatment with PMA<br />
for 24, 48 and 72 h or with staurosporine for 24 h correlates with TfR-protein expression<br />
(sTfR-ELISA and ferri-transferrin-sepharose affinity chromatography). To examine the<br />
morphological changes in the K562 cells during differentiation into megakaryocytes, cells<br />
were fixed and stained with May-Grünwald-Giemsa. Upon PMA-induced differentiation<br />
the megakaryocytic-like morphology was characterized by a lower nuclear-tocytoplasmic<br />
ratio, a lobulated nucleus and more acidophilic cytoplasm (15%, 25% and<br />
40% <strong>of</strong> the cells after treatment with PMA for 24 h, 48 h and 72 h respectively).<br />
contact:<br />
Krzyszt<strong>of</strong> Wandzik<br />
Charité-Universitätsmedizin Campus Benjamin Franklin<br />
Institute <strong>of</strong> Clinical Chemistry and Pathobiochemistry<br />
krzyszt<strong>of</strong>.wandzik@medizin.fu-berlin.de<br />
Hindenburgdamm 30<br />
12200 Berlin (Germany)
Peter Rehling, Agnieszka Chacinska, Maria Lind, Ann E. Frazier, Jan Dudek, Nikolaus<br />
Pfanner<br />
Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
More than 99% <strong>of</strong> the mitochondrial proteins are encoded by nuclear genes and need to<br />
be imported into the organelle from the cytosol. Multiprotein machineries in the outer<br />
and inner membranes <strong>of</strong> mitochondria mediate posttranslational protein transport into<br />
the various subcompartments <strong>of</strong> the organelle; the outer membrane, the intermembrane<br />
space, the inner membrane, and the matrix (1). The TOM complex transports proteins<br />
across the outer membrane. After passage through TOM two distinct TIM complexes in<br />
the inner membrane mediate selective transport <strong>of</strong> proteins into or across the inner<br />
membrane. The twin-pore translocase (TIM22 complex) specifically inserts<br />
multispanning membrane proteins with internal targeting signals, such as the metabolite<br />
carriers, into the inner membrane (2). Recent isolation <strong>of</strong> the translocase, its<br />
reconstitution and in organello analysis shows that protein insertion into the membrane<br />
is a multistep process that uses the membrane potential as an external force. In<br />
contrast, proteins with N-terminal targeting signals utilize the presequence translocase<br />
(TIM23 complex) for transport across or into the inner membrane. This membrane<br />
embedded translocase consists <strong>of</strong> a channel forming module to which an import motor<br />
(PAM) is associated (3,4). PAM is required to drive protein translocation across the inner<br />
membrane into the matrix. We have isolated this translocase and identified novel<br />
constituents (3,4,5). Detailed analysis <strong>of</strong> the novel Tim and Pam proteins reveals now a<br />
highly dynamic organization <strong>of</strong> the translocase. The modular organization <strong>of</strong> the TIM23<br />
complex allows the translocase to adapt to the various classes <strong>of</strong> precursor proteins that<br />
are transported along this pathway.<br />
Literature<br />
1. Rehling, P. et al. (2004). Nature Rev. Mol. Cell Biol. In press<br />
2. Rehling, P., et al. (2003). Science 299: 1747-1751<br />
3. Truscott, K.N., et al (2003). J. Cell Biol. 163: 707-713<br />
4. Frazier, A.E., et al. (2004). Nature Struct. Mol. Biol.11: 226-233<br />
5. Geissler, A. et al. Cell 111: 507-518<br />
contact:<br />
PD Dr. Peter Rehling<br />
Albert-Ludwigs Universität Freiburg<br />
Inst. für Biochemie & Molekularbiologie<br />
peter.rehling@biochemie.uni-freiburg.de<br />
Hermann-Herder-Str. 7<br />
79104 Freiburg (Germany)
Andrea Scrima, Ingrid Vetter, Alfred Wittingh<strong>of</strong>er<br />
TrmE, a GNBP involved in tRNA-modification<br />
TrmE is a member <strong>of</strong> the guaninenucleotide-binding proteins (GNBP), which bind and<br />
hydrolyse GTP. This class <strong>of</strong> proteins cycles between a GTP bound state, which<br />
represents the active state <strong>of</strong> the protein, and an inactive, GDP bound state.<br />
In contrast to the family <strong>of</strong> small GTPases, that regulate many crucial cellular processes<br />
like differentiation, cell-cell-adhesion and nuclear and vesicular transport by “switching”<br />
signalling pathways on and <strong>of</strong>f, TrmE is directly involved in an enzymatic reaction, the<br />
modification <strong>of</strong> tRNA at the wobble position uridine 34 in tRNA. U34 is modified in<br />
bacteria at the 2 and 5 position <strong>of</strong> the uridine-base. The modification at the 5 position<br />
requires many different proteins: TrmE is involved in the first step <strong>of</strong> the base<br />
modification, the formation <strong>of</strong> 5-carboxymethylaminomethyl-2-thiouridine(cmnm5s2U),<br />
which also requires the protein GidA. In a following modification step TrmC catalyses the<br />
formation <strong>of</strong> 5-methylaminomethyl-2-thiouridine (mnm5s2U). It has been shown that<br />
the modification at position U34 in the tRNA affects the anticodon-codon-interaction and<br />
influences frameshifting during the translation process. Mutant alleles <strong>of</strong> the GidA and<br />
TrmE homologues Mto1 and MSS1 in S.cerevisiae reveal a respiratory-deficient<br />
phenotype. Those proteins may also be involved in several human diseases based on<br />
mutations in mitochondrial genes like the non-syndromic-deafness or my<strong>of</strong>ibrillar<br />
myopathy (MERRF/MELAS).<br />
The main target <strong>of</strong> our work is to elucidate the role <strong>of</strong> TrmE in the modification reaction<br />
and wheter GTP-hydrolysis has a regulatory effect or provides the energy for the<br />
reaction. In this work the biochemical properties <strong>of</strong> TrmE have been investigated and we<br />
will present the structure <strong>of</strong> TrmE from Thermotoga maritima solved by x-ray<br />
crystallography.<br />
Literature<br />
[1] Yim L, Martinez-Vicente M, Villarroya M, Aguado C, Knecht E, Armengod ME. J Biol<br />
Chem. 2003 Aug 1;278(31):28378-87.<br />
[2] Cabedo H, Macian F, Villarroya M, Escudero JC, Martinez-Vicente M, Knecht E,<br />
Armengod ME. EMBO J. 1999 Dec 15;18(24):7063-76.<br />
contact:<br />
Andrea Scrima<br />
Max-Planck-Institut für molekulare Physiologie<br />
andrea.scrima@mpi-dortmund.mpg.de<br />
Otto-Hahn-Strasse 11<br />
44227 Dortmund (Germany)
Martin W. Volmer, Yvonne Radacz, Stephan A. Hahn, Susanne Klein-Scory, Kai<br />
Stühler, Marc Zapatka, Wolff Schmiegel, Helmut E. Meyer, Irmgard Schwarte-Waldh<strong>of</strong>f<br />
Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the<br />
anti-adhesive invasion-promoting matricellular protein SPARC<br />
Smad4 is a central mediator in the TGF-beta signal cascade and also a pivotal point in<br />
the cross-talk with other signal cascades. It functions both as a signal transmitter and as<br />
a transcriptional comodulator which influences the expression <strong>of</strong> a variety <strong>of</strong> genes by<br />
interaction with other transcription factors. The functional inactivation <strong>of</strong> Smad4 is a<br />
common event in the process <strong>of</strong> carcinogenesis <strong>of</strong> certain tumors. At a significant rate<br />
this event occurs in one half <strong>of</strong> pancreatic and one third <strong>of</strong> metastatic colon carcinomas.<br />
The reexpression <strong>of</strong> the tumor suppressor Smad4 in Smad4-deficient SW480 human<br />
colon carcinoma cells inhibits tumor growth and modulates cell-cell and cell-matrix<br />
adhesion. To decipher molecules involved in altered interactions <strong>of</strong> the tumor cells with<br />
their environment proteins contained in conditioned media from Smad4-deficient and<br />
Smad4-reconstituted SW480 cell clones were separated on large 2D-gels and visualized<br />
by silver staining. Differential protein spots were excised, trypsin digested and identified<br />
using MALDI-TOF mass-spectrometry. Northern and Western blot analyses were<br />
performed to validate the results.<br />
This led to the identification <strong>of</strong> novel Smad4 targets, among them SPARC (Secreted<br />
Protein Acidic and Rich in Cysteins). SPARC is exclusively expressed by Smad4-deficient<br />
SW480 clones and is a very prominent protein in their supernatants. Northern blot<br />
analysis revealed that Smad4 suppresses SPARC expression at the level <strong>of</strong> transcription.<br />
SPARC has previously been characterized as an anti-adhesive protein and is known to<br />
play an important role in tissue remodelling in development and repair. In invading<br />
tumors strong SPARC expression is characteristically detected at the tumor-host tissue<br />
border. A role in invasion has also been functionally proven using in vitro invasion<br />
assays. Thus, a role <strong>of</strong> Smad4 as a negative regulator <strong>of</strong> SPARC expression is consistent<br />
with its tumor suppressor function.<br />
contact:<br />
Martin Volmer<br />
Ruhr-Universität Bochum<br />
Medizinisches Proteom-Center<br />
Martin.Volmer@ruhr-uni-bochum.de<br />
Universitätsstr. 150<br />
44780 Bochum (Deutschland)
Heidrun Rhode, M. Schulze, E. Mitre, G.A. Cumme, T. Rausch, D. Philipp, A. Horn<br />
Turbo-mixing in microplates<br />
Currently microplate technology has occupied all fields <strong>of</strong> rapid analysis and<br />
combinatorial chemistry, including high-throughput schedules with rapid reactions.<br />
However, sufficiently rapid mixing in microplates still represents an unsolved and<br />
underestimated or neglected problem. E.g., a microplate shaker on its highest speed<br />
takes about 50 min to produce homogeneous mixtures <strong>of</strong> samples and reagent solutions<br />
in 384-well plates.<br />
Here we introduce a mixing method using the phenomenon <strong>of</strong> Marangoni convection.<br />
After deliverance <strong>of</strong> the watery solutions to be mixed into microplate wells, complete<br />
mixing is brought about simply by spotting liquid droplets to the liquid surfaces. These<br />
drops contain water miscible organic solvents or detergents which may be selected for<br />
compatibility with sample components. In order to check method performance, the<br />
microplate wells were filled successively with layers <strong>of</strong> liquids differing markedly in pH<br />
and density, the upmost layer containing an indicator with pH dependent optical<br />
absorbance or fluorescence. Upon spotting liquid droplets to the surfaces, mixing was<br />
followed-up by optical signal change. Within wells <strong>of</strong> 96- and 384-well microplates,<br />
mixing by Marangoni convection took about one second. The amount <strong>of</strong> organic solvents<br />
necessary for instantaneous production <strong>of</strong> an ideally homogeneous mixture is small<br />
enough not to alter bioactive molecules, neither by heat <strong>of</strong> solution nor by the resulting<br />
concentration <strong>of</strong> solvent spotted. Moreover, solvents may rapidly evaporate from the<br />
mixture. If necessary, mixing may be achieved also by spotting solvent repeatedly into<br />
the same well.<br />
Applications to enzyme-activity determination and evaluation <strong>of</strong> liquid handling devices<br />
are presented.<br />
contact:<br />
PD Dr. med. habil Heidrun Rhode<br />
Friedrich-Schiller-Universität<br />
Institut für Biochemie I<br />
Heidrun.Rhode@mti.uni-jena.de<br />
Klinikum<br />
07740 Jena (Deutschland)
Sven Gathmann, Dorothée Walter, Dirk Schneider<br />
Uncovering the mechanisms for protein sorting in<br />
cyanobacteria<br />
Signal sequences mediate protein translocation across many biological membranes. In<br />
chloroplastst and bacteria the majority <strong>of</strong> proteins get translocated via the well<br />
characterized Sec-pathway. Gram-negative cyanobacteria display characteristics <strong>of</strong> both<br />
chloroplasts as well as bacteria and cyanobacteria contain two internal membrane<br />
systems: the thylakoid and the cytoplasmic membrane. These two membranes separate<br />
different compartments from the cytoplasm with each compartment having a distinct<br />
protein composition. All translocated proteins are synthesized in the cyanobacterial<br />
cytoplasm as precursors, containing an N-terminal cleavable signal sequence and this<br />
signal gets cleaved <strong>of</strong>f by a signal peptidase after translocation.<br />
Interestingly, in the genome <strong>of</strong> cyanobacteria two lepB genes encoding for type I signal<br />
peptidases can be found. But wherefore do cyanobacteria need two signal peptidases if<br />
one is sufficient for other bacteria? To study the activity and specificity <strong>of</strong> the two<br />
proteins from Synechocystis PCC 6803, a method for overexpression and refolding <strong>of</strong> the<br />
proteins in vitro was established. A characterization <strong>of</strong> the refolded proteins suggests<br />
that they have distinct specificities and different activities. These in vitro results were<br />
further supported by in vivo analysis.<br />
Furthermore, we are interested in the role <strong>of</strong> individual signal sequences for protein<br />
sorting in Synechocystis. Fusion <strong>of</strong> various signal sequences to a reporter results in<br />
different efficiencies <strong>of</strong> protein translocation in vivo indicating that the signal sequences<br />
can play an important role for protein sorting in cyanobacteria.<br />
contact:<br />
Sven Gathmann<br />
Albert-Ludwigs- Universität Freiburg<br />
Institut für Biochemie und Molekularbiologie<br />
Sven.Gathmann@biochemie.uni-freiburg.de<br />
Hermann-Herder-Strasse 7<br />
79104 Freiburg i. Br. (Deutschland)
Antje Burse, Sabrina Discher, Jürgen Kuhn, Wilhelm Boland<br />
Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in<br />
Leaf Beetle Larvae<br />
The defensive chemistry <strong>of</strong> leaf beetle larvae <strong>of</strong> the subtribus Chrysomelina constitutes<br />
an excellent model system to evaluate the co-evolutionary processes leading to<br />
adaptation <strong>of</strong> insect and plant. Most <strong>of</strong> the Chrysomelina species are highly specialized in<br />
their feeding habit, and qualitative analyses <strong>of</strong> their defensive secretion revealed three<br />
levels <strong>of</strong> host plant dependence: full, partial, or lack <strong>of</strong> dependence. Sequestration <strong>of</strong><br />
plant derived compounds was analyzed by feeding experiments with thioglucosides<br />
which combine a unique structural similarity to natural substrates with stability against<br />
hydrolysis. These experiments provided strong evidence that uptake <strong>of</strong> plant glucosides<br />
into the gut tissue followed by entering the defensive system is mediated by transport<br />
proteins. In order to characterize the transport processes, in vitro-uptake experiments<br />
were performed on dissected gut tissue <strong>of</strong> Phaedon cochleariae using different inhibitors.<br />
The inhibition <strong>of</strong> glucose transport prevented also the import <strong>of</strong> thioglucosides. Based on<br />
these results, a GLUT-homologous transporter was identified and further characterized.<br />
The transport proteins responsible for uptake <strong>of</strong> plant glucosides may serve as bottle<br />
neck during host plant shifts <strong>of</strong> Chrysomelina larvae and thus, play a crucial role for<br />
changes <strong>of</strong> leaf beetle’s chemical defense.<br />
contact:<br />
Dr. Antje Burse<br />
MPI for Chemical Ecology<br />
Department <strong>of</strong> Bioorganic Chemistry<br />
aburse@ice.mpg.de<br />
Hans-Knöll-Str. 8<br />
07745 Jena (Germany)
Volker Kroehne, Ingo Heschel*, Frank Schügner*, Jörg W. Bartsch, Harald Jockusch<br />
Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure<br />
for Muscle Cell Differentiation in Culture and in Grafts<br />
The aim <strong>of</strong> the present work was to evaluate macromolecular scaffolds and culture<br />
conditions for myogenic cells in order to transplant muscle precursor cells or immature<br />
muscle fibres (myotubes) into recipient mice and to achieve the formation <strong>of</strong> skeletal<br />
muscle tissue in situ. A structure providing matrix is necessary, since injection <strong>of</strong><br />
suspensions <strong>of</strong> myogenic cells usually leads only to minor contribution to regenerating<br />
muscle. Scaffold cultures were analyzed directly, or after grafting into a muscle bed <strong>of</strong><br />
GFP labelled nude host mice; the recipient GFP label served to distinguish host from<br />
donor cells [1]. For pilot experiments, the permanent mouse myogenic cell line C 2 C 12<br />
was used. Its potential to yield contractile myotubes upon change to differentiation<br />
medium was verified by culturing in Matrigel TM (BD Biosciences), a reconstituted<br />
basement membrane matrix. It is long known that a collagen (coated) substrate is<br />
beneficial for growth and differentiation <strong>of</strong> myogenic cells (cf. [2]). Hence collagen<br />
sponges with icecrystal-induced channels <strong>of</strong> 50 •m diameter, running through their<br />
whole thickness ([3], Matricel GmbH) were used as novel scaffolds. C 2 C 12 cells were<br />
seeded onto pieces <strong>of</strong> collagen sponge and, after a phase <strong>of</strong> proliferation, cultured in<br />
fusion medium to induce differentiation. The sponges provided an architecture suitable<br />
for differentiation into long multinucleated myotubes oriented in parallel, with sufficient<br />
mechanical stability for transplantation. For this purpose sponges were used that were<br />
similar in size and shape to the muscle to be replaced (M. tibialis anterior, TA). The fate<br />
<strong>of</strong> these scaffold cultures in the environment <strong>of</strong> a host muscle bed will be described.<br />
Supported by the Stem Cell Network North Rhine Westphalia, Ministry <strong>of</strong> Science and<br />
Research NRW and by Fonds der Chemischen Industrie<br />
Literature<br />
1. Jockusch & Voigt (2003) J. Cell Sci. 116, 1611-1616<br />
2. Bardouille, C., Lehmann, J., Heimann, P., Jockusch, H. (2001) Appl. Microbiol.<br />
Biotechnol. 55, 556-562<br />
3. Scho<strong>of</strong>, H., Apel, J., Heschel, I., Rau, G. (2001) J. Biomed. Mater. Res. 58, 352-357<br />
contact:<br />
Volker Kroehne<br />
Bielefeld University<br />
Developmental Biology & Molecular Pathology<br />
vkroehne@uni-bielefeld.de<br />
33501 Bielefeld (Germany)<br />
additional information<br />
*) Matricel GmbH, D-52134 Herzogenrath
Masamitsu Futai, Ge-Hong Sun-Wada, Yoh Wada<br />
Vacuolar-type proton pump ATPases (V-ATPases) and inside<br />
acidic organelles/compartments: rotational mechanism and<br />
diverse physiological roles.<br />
The vacuolar-type H+-ATPases (V-type or V- ATPase) are ubiquitous eukaryotic proton<br />
pumps. They are present in endomembrane organelles such as vacuoles, lysosomes,<br />
endosomes, Golgi apparatus, synaptic vesicles, coated vesicles, and also in the plasma<br />
membranes <strong>of</strong> specialized cells including osteoclasts and kidney epithelial cells. They<br />
pump protons into corresponding organelles/compartments.<br />
V-ATPase consists <strong>of</strong> a membrane periphereal V1 catalytic domain and an integral Vo<br />
proton pathway, and has similarities with F-ATPase (ATP synthase) including rotational<br />
mechanism for catalysis and proton translocation, but different in composition <strong>of</strong><br />
subunits and their diverse is<strong>of</strong>orms. The Vo with “subunit a” is<strong>of</strong>orms, a1, a2, and a3<br />
show different subcellular localization, whereas that with a4 is specifically expressed in<br />
kidney intercalated cell plasma membrane. The endosomes/lysosomes having V-ATPase<br />
with a3 are transported to the cell periphery and assembled into the plasma membrane<br />
<strong>of</strong> osteoclasts during osteoclast differentiation.<br />
Subunit is<strong>of</strong>orms are also found in the V1 sector: kidney (d2, G3, and C2-b), testis (E1),<br />
brain (G2), lung lamellar bodies (C2-a). The different is<strong>of</strong>orms affect energy coupling<br />
and ATPase catalysis. Thus, unique V-ATPases with different is<strong>of</strong>orms are involved in<br />
bone resorption, renal ion homeostasis, fertilization, transmitter uptake and surfactant<br />
excretion. These results indicate that diverse physiological roles <strong>of</strong> V-ATPase are<br />
established through utilization <strong>of</strong> specific is<strong>of</strong>orms, the basic subunit structure and<br />
enzyme mechanism being maintained.<br />
contact:<br />
pr<strong>of</strong>essor Masamitsu Futai<br />
Osaka University<br />
Division <strong>of</strong> biological Sciences and CREST, Japan Science and Technology Corporation<br />
m-futai@sanken.osaka-u.ac.jp<br />
Ibaraki<br />
567-0047 Osaka (Japan)
Klaus-Peter Vogel, Monique Karl, Heinz-Jürgen Steinh<strong>of</strong>f, Wolfgang H. Ziegler<br />
Vinculin phospholipid interaction studied by EPR utilizing sitedirected<br />
spin labeling<br />
X-band electron paramagnetic resonance (EPR) spectroscopy has been used to study the<br />
interaction <strong>of</strong> the cytoskeletal protein vinculin with phospholipid vesicles.<br />
Vinculin links filamentuous actin to cell-matrix adhesions and plays an important role in<br />
cell adhesion and migration [1,2]. Vinculin acts as a sensor in lipid regulation <strong>of</strong><br />
adhesion site turnover [3]. To study the interaction <strong>of</strong> the two proposed lipid binding<br />
sites <strong>of</strong> the protein tail domain with acidic phospholipids by EPR, site-directed spin<br />
labeling (SDSL) was applied [4]. Functionality <strong>of</strong> all mutants was confirmed by protein<br />
ligand binding.<br />
- EPR spectra at room temperature were taken to obtain the mobility <strong>of</strong> the spin label<br />
side chain (SLSC), giving information on the proximate secondary structure[4].<br />
- Different paramagnetic quenchers were used in power saturation measurements to<br />
determine their accessibility to the SLSC, providing information about the orientation <strong>of</strong><br />
the SLSC with respect to the water and the lipid phases [4].<br />
- Spectra taken at 160K allowed to determine the hyperfine tensor, as a measure <strong>of</strong> the<br />
polarity <strong>of</strong> the environment [4].<br />
Results: Measurements in absence and in presence <strong>of</strong> phospholipids show the interaction<br />
<strong>of</strong> the vinculin tail domain with lipid membranes and effects on the secondary structure<br />
<strong>of</strong> the protein. Effects <strong>of</strong> lipid binding sites are discriminated using selective binding site<br />
mutants.<br />
Literature<br />
[1] Critchley, D.R., Current Opinion in Cell Biology 12, 133-139 (2000)<br />
[2] Xu, W. et al., Development 125, 327-337 (1998)<br />
[3] Chandrasekhar, I. et al., submitted<br />
[4] Steinh<strong>of</strong>f, H.-J., Frontiers in Bioscience 7, c97-110 (2002)<br />
contact:<br />
Klaus-Peter Vogel<br />
Universität Osnabrück<br />
FB Physik, AG Makromolekülstruktur<br />
kpvogel@uos.de<br />
Barbarastraße 7<br />
49069 Osnabrück (Deutschland)
Maria Heiser, Birgit Hutter-Paier, Lidia Jerkovic, Michael Becker-Andre, Hans<br />
Dieplinger, Roswitha Pfragner, Manfred Windisch<br />
Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Alzheimer´s disease (AD) is a progressive neurodegenerative disorder affecting millions<br />
<strong>of</strong> people worldwide demanding pr<strong>of</strong>ound medical and social care. Increasing evidence<br />
points out that oxidative stress is a putative factor in the pathogenesis <strong>of</strong> AD. Therefore,<br />
antioxidants such as vitamin E have become attractive as therapeutic agents in the<br />
treatment <strong>of</strong> AD. This lipophilic free radical scavenger is known to be transported in<br />
plasma by lipoproteins, yet no specific transport protein for α-tocopherol, especially for<br />
extravascular fluids like cerebrospinal fluid (CSF), has been reported. However, Afamin,<br />
an 87 kDa human plasma glycoprotein with specific binding properties for vitamin E (αtocopherol)<br />
was recently characterized. In the present study the in vitro effects <strong>of</strong> native<br />
human Afamin, <strong>of</strong> Afamin pre-loaded with vitamin E, and <strong>of</strong> vitamin E on neuronal cells<br />
were investigated. Isolated cortical chicken neurons were maintained either under<br />
apoptosis-inducing low serum conditions or exposed to oxidative stress by the addition<br />
<strong>of</strong> H 2 O 2 or beta-amyloid peptide Aß 25-35 . Afamin and vitamin E synergistically enhance<br />
the survival <strong>of</strong> cortical neurons under apoptotic conditions. Furthermore, Afamin alone<br />
protects cortical neurons from cell death in both experimental settings. Therefore, the<br />
plasma glycoprotein Afamin apparently displays a neuroprotective activity not only by<br />
virtue <strong>of</strong> binding and transporting vitamin E but also on its own.<br />
Literature<br />
Heiser M, Hutter-Paier B, Jerkovic L, Pfragner R, Windisch M, Becker-Andre M, Dieplinger<br />
H (2002)Vitamin E binding protein Afamin protects neuronal cells in vitro. J Neural<br />
Transm, Suppl.62:337-345<br />
contact:<br />
Maria Heiser<br />
Medizinische Universität Graz<br />
Institut für Pathophysiologie<br />
heiser@gmx.at<br />
Heinrichstr. 31<br />
8010 Graz (Österreich)
Marten Wikstrom<br />
Warburg's Atmungsferment: A molecular energy transducer<br />
The respiratory enzyme accounts for >90% <strong>of</strong> the oxygen consumption in biology. This<br />
membrane-bound metalloprotein is member <strong>of</strong> a large family <strong>of</strong> enzymes, the haemcopper<br />
oxidases, which are found in all aerobic organisms. In all cases the catalysed<br />
reaction is the reduction <strong>of</strong> O2 to water at a structurally conserved bimetallic haemcopper<br />
catalytic site, the immediate electron donor being another haem group in the<br />
close vicinity.<br />
What is remarkable about this key system <strong>of</strong> aerobic life is, on the one hand, the safe<br />
O2 reduction mechanism that occurs without release <strong>of</strong> toxic reactive oxygen species,<br />
and on the other, conservation <strong>of</strong> much <strong>of</strong> the free energy released during exergonic O2<br />
reduction by generation <strong>of</strong> an electrochemical proton gradient across the membrane. It<br />
is this gradient that is subsequently used as an energy source for production <strong>of</strong> ATP.<br />
Both these aspects <strong>of</strong> the function will be discussed, as well as their linkage, which is<br />
fundamental to our understanding the energy-transducing function <strong>of</strong> the respiratory<br />
enzyme.<br />
contact:<br />
Pr<strong>of</strong>essor Marten Wikstrom<br />
Univ. <strong>of</strong> Helsinki<br />
Inst. <strong>of</strong> Biotechnology<br />
marten.wikstrom@helsinki.fi<br />
Viikinkaari 1, PB 65<br />
00014 Helsinki (Finland)
Karen Strenge, Paul Crocker, Nicolai Bovin<br />
What are the functions <strong>of</strong> siglecs on human monocytes and<br />
macrophages?<br />
Siglecs are sialic acid (Sia)-specific lectins which are mainly expressed on cells <strong>of</strong> the<br />
hematopoietic system. Although the distinct functions <strong>of</strong> the siglecs are still unknown,<br />
most siglecs are thought to be involved in the regulation <strong>of</strong> cell activation since they<br />
contain cytoplasmic immuno tyrosine-based inhibitory signaling motifs.<br />
To analyse the role <strong>of</strong> siglecs for monocyte functions it was first necessary to investigate<br />
their expression patterns. We observed specific changes in siglec expression upon in<br />
vitro differentiation and activation <strong>of</strong> monocytes. Although monocytes express high<br />
levels <strong>of</strong> Siglec-3, -5, -7 and-9, no sialoside probe binding could be observed. This<br />
indicates that on monocytes all siglec Sia binding sites are occupied by binding to<br />
sialylated cis-ligands on the same cell surface. However, upon differentiation some<br />
unmasking <strong>of</strong> binding sites for sialylated glycoconjugates occurs. This suggests that on<br />
monocytes siglecs may be kept in a "masked" state in order to act on the same cell<br />
surface as a negative regulator or to prevent undisirable interactions with inappropriate<br />
trans-targets until they are ready to be used in specific situations.<br />
Interestingly, macrophages can recognise Sia on tumor cells, leading to a Sia-dependent<br />
TNF-a production. Since Fc-chimera <strong>of</strong> Siglec-1, -7, -9 and -10 can bind with high affinity<br />
to different human tumor cell lines it is imaginable that a direct interaction between<br />
siglecs and so far unknown glycoconjugates expressed on tumor cells occurs. Further<br />
functional co-culture studies <strong>of</strong> monocytes or macrophages with the different tumor cells<br />
in the presence <strong>of</strong> blocking anti siglec antibodies will show, whether the Sia-dependent<br />
TNF-a production is mediated by one <strong>of</strong> the siglecs.<br />
contact:<br />
Dr. Karen Stenge<br />
Universität Bremen<br />
Institut für Biochemie<br />
kstrenge@uni-bremen.de<br />
Leobenerstr.<br />
28359 Bremen (Deutschland)
Tatjana M. E. Schwabe, Daniela Schlüsener, Jochen Kruip<br />
Ycf3 from Synechocystis sp. PCC 6803: Localisation,<br />
Oligomerisation and Role in Photosystem I Biogenesis<br />
Cyanobacterial photosystem I (PS I) is our model system to elucidate the biogenesis <strong>of</strong> a<br />
membrane protein complex on the molecular level [1]. PS I is comprised <strong>of</strong> 11 -12<br />
protein subunits and a large number <strong>of</strong> c<strong>of</strong>actors, embedded in the thylakoid membranes<br />
<strong>of</strong> cyanobacteria and chloroplasts [2]. It functions as one <strong>of</strong> two light-driven electron<br />
pumps in photosynthetic electron transport.<br />
Several potential biogenesis-proteins have been identified, based on knockout mutants,<br />
but their molecular function is largely unknown. Here, we focus on Ycf3, a soluble TPRmotif<br />
containing protein. Overexpression in E. coli yielded recombinant protein, for which<br />
we have developed purification protocols [3]. Size exclusion chromatography shows<br />
homo-oligomerisation, with Ycf3 existing in oligomeric complexes <strong>of</strong> 260 kDa. These<br />
data are supported by further experiments using yeast-2-hybrid system and ligandoverlay<br />
assays. Ycf3 is loosely associated with thylakoid membranes <strong>of</strong> Synechocystis,<br />
as revealed by immunoelectronmicroscopy and immunoblot analysis. Deletion mutants in<br />
Synechocystis sp. PCC 6803 show characteristic phenotypes: cells lacking ycf3 posses no<br />
photosystem I and are unable to grow photoautotrophically. Their membrane proteome<br />
is similar to a PSI-deletion strain, as evidenced by 2D-blue native/SDS-PAGE. We are<br />
currently employing affinity chromatography using his-tagged recombinant Ycf3 to<br />
search for possible interaction partners in Synechocystis. We are also conducting<br />
proteomic analyses <strong>of</strong> wildtype in comparison with knock-out mutants using 2D IEF/SDSelectrophoresis<br />
and MALDI-ToF-PMF.<br />
Literature<br />
1. Schwabe, T.M.E. and J. Kruip; Indian J Biochem Biophys, 2000. 37(6): p. 351-359.<br />
2. Jordan, P., et al.; Nature, 2001. 411(6840): p. 909-17.<br />
3. Schwabe, T.M., et al.; J Chromatogr B, 2003. 786(1-2): p. 45-59.<br />
contact:<br />
Dr. Tatjana M. E. Schwabe<br />
Ruhr-Universität Bochum<br />
Biochemie der Pflanzen<br />
tatjana.schwabe@rub.de<br />
Universitätsstr. 150<br />
44801 Bochum (Deutschland)<br />
additional information<br />
J.K. present address: Aventis Pharma Germany, 65926 Frankfurt, Germany
Symposium:<br />
Cytoskeleton dynamics<br />
Mohammad Reza Ahmadian<br />
New insights into structure-function relationships in GTPase-effector<br />
interaction<br />
Judith Austermann, Max Koltzscher, Volker Gerke<br />
Characterization <strong>of</strong> the calcium-regulated S100P-ezrin interaction<br />
Buzz Baum<br />
Dissecting actin cytoskeletal organization in Drosophila using RNAi<br />
Henriette Brüggemann, Antje Berken, Alfred Wittingh<strong>of</strong>er<br />
Towards the function <strong>of</strong> plant Rop GTPases in actin reorganisation<br />
Marian Farkasovsky, Peter Herter, Beate Voß, Alfred Wittingh<strong>of</strong>er<br />
Nucleotide binding and filament assembly <strong>of</strong> recombinant yeast septin<br />
complexes.<br />
Dennis Fiegen, Lars-Christian Haeusler, Lars Blumenstein, Ulrike Herbrand, Radovan Dvorsky,<br />
Ingrid R. Vetter, Mohammad R. Ahmadian<br />
Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating GTPase<br />
M. Tharwat Ghoneim*, A.M. Baraka*, M.M . Matta**, H. Kamal**<br />
New insights into the modulatory role <strong>of</strong> serotonin reuptake inhibitors in<br />
myocardial ischemia reperfusion injury in the rabbit<br />
Verena Goebeler, Ursula Rescher, Volker Gerke<br />
Biochemical Characterization <strong>of</strong> Annexin A9 and Identification <strong>of</strong> Interacting<br />
Proteins<br />
Lars Hemsath, Patricia Stege, Mohammad Reza Ahmadian<br />
Molecular basis <strong>of</strong> Cdc42 recognition by Wiskott-Aldrich Syndrome Proteins<br />
Markus Knop, Elin Aareskjold, Volker Gerke<br />
Rab3d and annexin A2 play a role in regulated secretion <strong>of</strong> vWF but not tPA<br />
from human endothelial cells<br />
Bettina Krüger, Susanne Mühlich, Thomas Huff, Angela Graness, Margarete Goppelt-Strübe<br />
Alterations in the actin and microtubular network influence CTGF expression in<br />
endothelial cells<br />
Eva-Maria Mandelkow, J. Biernat, T. Timm, E. Thies, X.-Y. Li, E. Mandelkow<br />
Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite growth and<br />
degeneration<br />
Georg Reiser, Mohan Tulapurkar, Theo Hanck<br />
Sequestration and recycling pathway <strong>of</strong> P2Y2 nucleotide receptor: Clathrinand<br />
actin cytoskeleton-dependent receptor endocytosis, live cell visualization<br />
<strong>of</strong> green fluorescent protein tagged receptor<br />
Markus Rüffer, Randy Kurz, Winnie Weigel, Andrée Rothermel, Jürgen Trzewik, Gerhard<br />
Artmann, Andrea A. Robitzki<br />
Active cardiomyocytes on collagen gels for biohybrid systems<br />
Manfred Schliwa<br />
Portrait <strong>of</strong> a molecular motor<br />
Raiko Stephan, C. Klämbt, S. Bogdan<br />
The Kette/Abi/Sra-1 complex regulates actin dynamics by inhibting wave and<br />
activating wasp function in Drosophila<br />
Hameeda Sultana, Angelika.A Noegel
Cyclase associated protein CAP in the regulation <strong>of</strong> the actin cytoskeleton and<br />
cell polarity in Dictyostelium<br />
Tadaomi Takenawa, Shiro Suetsugu, Daisuke Yamazaki<br />
Differential functions <strong>of</strong> WAVE1 and 2 in cell migration and spreading.<br />
Anke Vahrmann, Anna Szkodowska, Christoph Linke, Monika C. M Mueller, Tilly Bakker-<br />
Grunwald, Henning Scholze<br />
Structural characterization and localization <strong>of</strong> annexin E1 in trophozoites <strong>of</strong><br />
Giardia lamblia
Symposium:<br />
Development and Aging <strong>of</strong> the Neuronal Cytoskeleton<br />
Brian Anderton<br />
The ageing neuronal cytoskeleton and neurodegenerative disease<br />
Carlos Dotti<br />
Neuronal membrane cholesterol loss results in low plasmin activity yet higher<br />
beta-secretase cleavage <strong>of</strong> APP<br />
Lubov Rochlina, Thomas Linn, Reinhard Bretzel, Eugen Kolossov, Juergen Hescheler, Irina<br />
Drobinskaya<br />
Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the Murine<br />
Embryonic Stem Cell System<br />
Johann Gross, Astrid Machulik, Amarjagal Nyamaa, Birgit Mazurek<br />
Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat cochlea<br />
Stefan Helling, Petra Lutter, Petra Weingarten, Christoph Hüls, Helmut Jonuleit, Edgar Schmitt,<br />
Helmut E. Meyer, Katrin Marcus<br />
Differential analysis <strong>of</strong> T cell membrane proteins<br />
Claudia Hoemme, Jens Schwamborn, Alexander Antipenko, Dimitar B. Nikolov, Andreas W.<br />
Püschel<br />
Structural and functional analysis <strong>of</strong> the axon guidance molecule Sema3A and<br />
its receptor<br />
Thorsten Hoppe, Giuseppe Cassata, José M. Barral, Wolfdieter Springer, Alex H. Hutagalung,<br />
Henry F. Epstein, Ralf Baumeister<br />
Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a Novel E3/E4-<br />
Multiubiquitylation Complex<br />
Andrée Rothermel, Randy Kurz, Thomas Biedermann, Markus Rüffer, Winnie Weigel, Andrea<br />
Robitzki<br />
Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Oliver Schmidt, Helmut E. Meyer, Katrin Marcus<br />
Multi-dimensional chromatography <strong>of</strong> proteins<br />
Gert Schwach, M. Tschemmernegg, E. Schreiner, M. Windisch, R. Pfragner, E. Masliah, E.<br />
Rockenstein, E. Ingolic<br />
Stably transfected rat neuronal cell lines expressing alpha-Synuclein GFP<br />
Fusion Proteins as an in vitro model <strong>of</strong> Parkinson's Disease
Symposium:<br />
ECTS Short contributions<br />
Roland C. Grafström, Claudia A Staab, Jan Anders Nilsson<br />
Quantitative Genomics <strong>of</strong> Cultured Normal and transformed Human Oral<br />
Keratinocytes<br />
Marion Jech, Birgit Hutter-Paier, Elisabeth Ingolic, Harald Höger, Elisabeth Grygar, Sepp Porta,<br />
Brigitte Poncza, Manfred Windisch, Roswitha Pfragner<br />
In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines - New Models<br />
for Neuroendocrine Research<br />
John R Masters<br />
Cell line misrepresentation<br />
Dietrich Möbest, Stephan Göttig, Bithiah Grace, Reinhard Henschler<br />
A role for Rho A is indicated in migration and hematopoietic regeneration after<br />
bone marrow transplantation demonstrated by inactivation <strong>of</strong> small GTPases<br />
with clostridial toxins<br />
Jan Anders Nilsson, Claudia A. Staab, Jan-Olov Höög, Roland C. Grafström<br />
Formaldehyde-related toxicity and gene expression in cultured human oral<br />
keratinocytes<br />
Roswitha Pfragner, A. Stift*, J. Friedl*, B. Niederle*, M. Gnant*<br />
Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent Advances in<br />
Immunotherapy<br />
Ester Martín-Villar, Maria Marta Yurrita, Carlos Gamallo, Miguel Quintanilla<br />
PA2.26 antigen (T1alpha, podoplanin), a membrane mucin associated with<br />
cancer which is involved in epithelial-mesenchymal transitions<br />
Wolfgang D. Schmitt, Stephan Ruhla, Steffen Hauptmann<br />
Detection <strong>of</strong> Retrotransposition in Cancer<br />
Claudia Alma Staab, Jan-Anders Nilsson, Zsolt Sarang, Jan-Olov Höög, Roland Grafström<br />
Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in human normal and<br />
transfromed oral keratinocytes<br />
Alexander Teplyashin, Natalia Tchupikova, Svetlana Sharifullina, Mariya Rostovskaya, Svetlana<br />
Korjikova, Igor Abdrakhmanov, Alexander Dunaev, Sergey Petrin, Valeriy Eremenko, Irina<br />
Savchenkova<br />
Characterization <strong>of</strong> human MSC-like cells isolated from bone marrow, adipose<br />
tissue, skin and placenta.
Symposium:<br />
ECTS/GZG: Stem Cells<br />
Jonas Frisen<br />
Generation <strong>of</strong> neurons from stem cells in the adult brain<br />
Jürgen Hescheler<br />
Embryonic stem cells<br />
Timm Schroeder, Hella Kohlh<strong>of</strong>, Nikolaus Rieber, Satomi Nishikawa, Georg Bornkamm, Shin-Ichi<br />
Nishikawa, Tasuku Honjo, Ursula Just<br />
Notch signalling in embryonic and adult hematopoietic stem cells<br />
Volker Kroehne, Ingo Heschel*, Frank Schügner*, Jörg W. Bartsch, Harald Jockusch<br />
Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for Muscle Cell<br />
Differentiation in Culture and in Grafts<br />
Dyakonov Lev, Galnbek Tatjana, Simonova Alla, Savenko Natalia, Klenovitsky Petr, Ernst Lev,<br />
Zinovieva Natalia, Shevcova Natalia<br />
Caryological characteristics <strong>of</strong> some interspecific hybryd anymal cell cultures<br />
Monika Lichtenauer, Wolfgang E Thasler, Anja Gräbe, Birgit Jahn, Karl-Walter Jauch, Hans-<br />
Jürgen Schlitt, Thomas S Weiss<br />
Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic progenitor<br />
cells from surgical resected human liver tissue<br />
Matthias F. Melzig, Philipp Hebestreit<br />
Synergistic action <strong>of</strong> saponins with type I Ribosime-inactivating proteins<br />
agrostin and saporin<br />
Jana Mooster, Florian Klein, Niklas Feldhahn, Mieke Sprangers, Markus Müschen<br />
Tracing the pre-B to immature B cell transition in human leukemia cells<br />
reveals a coordinated sequence <strong>of</strong> primary and secondary IGK gene<br />
rearrangement, IGK deletion and IGL gene rearrangement<br />
Roswitha Pfragner, Werner Emberger, Sigrun Sodia, Olaf Reich, Sigrid Regauer<br />
Human mucosa-associated malignant melanomas: cytogenetic and molecular<br />
genetic characterization<br />
Alexandra Rolletschek, Cornelia Wiese, Gabriela Kania, Przemyslaw Blyszczuk, Barbara<br />
Steinfarz*, Ihor Zahanich+, Thomas Braun#, Oliver Brüstle*, Kenneth R. Boheler~, Anna M.<br />
Wobus<br />
Nestin-positive progenitor cells generated from adult mouse intestinal<br />
epithelium differentiate into cells expressing neural, hepatic and pancreatic<br />
properties<br />
Hans Schöler, Luca Gentile, James Kehler, Karin Hübner, Michele Boiani<br />
The Oocyte – Embryonic Stem Cell Cycle<br />
Mieke Sprangers, Hui Wang, Niklas Feldhahn, Florian Klein, Markus Müschen<br />
Lineage infidelity in pre-B acute lymphoblastic leukemia cells carrying an MLL-<br />
AF4 gene rearrangement<br />
Cornelia Wiese, Alexandra Rolletschek, Ihor Zahanich#, Jürgen F. Heubach#, Jaroslaw Czyz,<br />
Anne Navarrete-Santos+, Olaf Horstmann*, Kenneth R. Boheler§, Anna M. Wobus<br />
Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell type from adult<br />
mouse and human intestinal epithelium
Symposium:<br />
Endothelial and epithelial barriers<br />
Rüdiger Adam, Tobias Tenenbaum, Hans-Joachim Galla, Horst Schroten<br />
Bacterial Meningitis: Interactions at the Blood-CSF-Barrier<br />
Tatiana Afanasieva, Victoria Jensen, Jochen Seebach, Ute Stroeher, Heinz Feldmann, Hans-J.<br />
Schnittler<br />
Endothelial activation and change <strong>of</strong> barrier function by soluble Ebola virus<br />
glycoproteins<br />
Jörg Andrä, Sylvie E. Blondelle, Roman Jerala, Guillermo Martinez de Tejada, Michel H. J. Koch,<br />
Karl Lohner, Klaus Brandenburg<br />
The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and endotoxin<br />
neutralization<br />
Gabriele Bixel, Björn Petri, Stephan Kloep, Stefan Butz, Engelhardt Britta, Vestweber Dietmar<br />
Mouse CD99 participates in T cell recruitment into inflamed skin<br />
Angelika Bondzio, Erwin Reinwald<br />
Adhesion <strong>of</strong> Trypanosoma congolense activates signalling pathways in<br />
endothelial cells<br />
Vania Braga<br />
Rho GTPases and cell-cell adhesion<br />
S<strong>of</strong>ia Depner, Wibke Schwarzer, Nicole Daum, Jean Kadathukalam, Norbert E. Fusenig, Dirk<br />
Breitkreutz<br />
The insulin-receptor signaling complex in epidermal keratinocytes<br />
Anja Drees, Tanja Eisenblätter, Hans-Joachim Galla<br />
ABC-Transporters at the blood-brain-barrier<br />
Sabine Fuchs, Ron Unger, Maria Iris Hermanns, Kirsten Peters, Charles James Kirpatrick<br />
Different populations <strong>of</strong> endothelial progenitor cells for applications in tissue<br />
engineering<br />
Sabine Fuchs, Michael Laue, Maria Iris Hermanns, Charles James Kirkpatrick<br />
Endocytosis in an in vitro model <strong>of</strong> the human alveolar epithelial barrier:<br />
Uptake <strong>of</strong> GM-1 by human alveolar epithelial cells in primary culture<br />
Erik Hauzman, Annette Staebler, Martin Götte, Kathrin Tausch, Igor B. Buchwalow, Olaf<br />
Buchweitz, Ludwig Kiesel, Robert R. Greb<br />
Molecular analysis <strong>of</strong> the angiogenic status in endometriotic lesions and<br />
eutopic endometrium<br />
Pia Wülfing, Martin Götte, Christian Kersting, Joke Tio, Christopher Poremba, Raihanatou Diallo,<br />
Werner Böcker, Ludwig Kiesel<br />
Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Martin Götte, Laura Rossi, Robert Greb, Dorothe Spillmann, Antonia Joussen, Merton Bernfield,<br />
Ludwig Kiesel<br />
The heparan sulfate proteoglycan syndecan-1 is a novel regulator <strong>of</strong> leukocyteendothelial<br />
cell adhesion and leukocyte transmigration<br />
Maria Iris Hermanns, Sabine Fuchs, Ron Unger, Charles James Kirkpatrick<br />
Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-Well Screening<br />
<strong>of</strong> Barrier Properties<br />
Evelyn Hollnack, Tanja Eisenblätter, Hans-Joachim Galla<br />
Substrate and inhibitor specificity <strong>of</strong> the brain multidrug resistance protein<br />
(BMDP) at he blood-brain barrier<br />
Beat A. Imh<strong>of</strong>, Michel Aurrand-Lions, Chrystelle Lamagna
The role <strong>of</strong> junctional adhesion molecule-C (JAM-C) in angiogenesis and<br />
inflammation<br />
Kenji Irie, T. Sakisaka, W. Ikeda, H. Ogita, Yoshimi Takai<br />
Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and polarity<br />
Claus Kerkh<strong>of</strong>f, Wolfgang Nacken, Malgorzata Czarny, Marie Claire Dagher, Claudia Sopalla,<br />
Jacques Doussiere<br />
The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase<br />
activation in intact cells<br />
Gabriele Bixel, Stephan Kloep, Stefan Butz, Björn Petri, Britta Engelhardt, Dietmar Vestweber<br />
Mouse CD99 is required for transendothelial migration <strong>of</strong> T cells<br />
Romy Kronstein, Jochen Seebach, Hans Schnittler<br />
Association <strong>of</strong> Eps15 with beta-Catenin in endothelial cells: a novel mechanism<br />
how endothelial cells might regulate barrier function<br />
Markus-N. Kruse, Karim Sabrane, Larissa Fabritz, Ines Veltrup, Melanie Voß, Boris Skryabin,<br />
Michaela Kuhn<br />
Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong> atrial<br />
natriuretic peptide<br />
Angela Magin, Thomas Noll<br />
Feasibility <strong>of</strong> the Use <strong>of</strong> HUVEC for Autologous Co-Culture Expansion <strong>of</strong> HSC<br />
from Umbilical Cord Blood<br />
Angela Magin, Heike Partenheimer, Claudia Lange, Michael Lietz, Thomas Noll<br />
Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
Karl Matter<br />
Signalling at tight junctions in the regulation <strong>of</strong> epithelial proliferation and<br />
differentiation<br />
Luitpold Miller, Astrid Tiefenbacher, Laurenz Wurzinger, Manfred Gratzl<br />
Antibody to E-selectin inhibits SCLC cell adhesion to endothelial cells<br />
Daniela Rehder, Dietmar Vestweber, Klaus Ebnet<br />
Junctional Adhesion Molecule-A (JAM-A) participates in tight junction<br />
formation and the establishment <strong>of</strong> cell polarity in epithelial cells<br />
Christoph Riethmüller, Joachim Wegener*, Marianne Wilhelmi, Pia Jungmann, Hans Oberleithner<br />
Bradykinin enhances vesicular transport <strong>of</strong> solutes across endothelial cell<br />
layers<br />
Christina Rommel, Jan Endell, Ralf Wehrspohn, Petra Göring, Claudia Steinem, Andreas Jansh<strong>of</strong>f,<br />
Hans-Joachim Galla, Joachim Wegener<br />
Probing transepithelial substrate permeation with sub-cellular lateral<br />
resolution.<br />
Bernd Schmeck, Ralph Gross, Phillipe Dje N´Guessan, Andreas C. Hocke, Sven Sven<br />
Hammerschmidt, Tim J. Mitchell, Simone Rosseau, Norbert Suttorp, Stefan Hippenstiel<br />
Streptococcus pneumoniae induced caspase 6-deptendent apoptosis in lung<br />
epithelium<br />
Thomas E Scholzen<br />
Terminating the stress: proteolytic processing <strong>of</strong> proopiomelanocortin-derived<br />
stress and anti-inflammatory hormones by dermal microvascular endothelial<br />
cell (EC) extracellular peptidases<br />
Sebastian Schrot, Hans-Joachim Galla<br />
Neutrophile Granulocytes Transendothelial Migration in vitro<br />
Jochen Seebach, Beata Wojciak-Stothard, Hans-Joachim Schnittler<br />
Rac mediates shear stress induced increase in endothelial barrier-function<br />
S. Ramponi, A. Grotti, S. Vultaggio, A. Morisetti, G. Alfieri, V. Lorusso<br />
Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial permeability and<br />
morphology: an in vitro model
Frank Wegmann, Klaus Ebnet, Louis Du Pasquier, Dietmar Vestweber, Stefan Butz<br />
Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor<br />
MAGI-1 and recruits it to cell contacts<br />
Josef H. Wissler<br />
Physiologic Roles for Receptors for Advanced Glycosylation End Products<br />
[RAGE] <strong>of</strong> Endothelial Cells: RNA-, Redox- and Metalloregulated Non-Mitogenic<br />
Angio-Morphogenesis in Health and Disease.<br />
Ulf Schulze Topph<strong>of</strong>f, Patrick Zeni, Hans-Joachim Galla<br />
Regulation <strong>of</strong> matrix metalloproteases and their endogenous inhibitors at the<br />
choroid plexus epithelium in vitro
Symposium:<br />
Engineering and application <strong>of</strong> aut<strong>of</strong>luorescent proteins<br />
Nediljko Budisa<br />
Designing novel classes <strong>of</strong> fluorescent proteins with an expanded genetic code<br />
Nediljko Budisa, Prajna Paramita Pal, Sandra Lepthien, Barbara Mulinacchi, Luis Moroder<br />
Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold” class <strong>of</strong> green<br />
fluorescent proteins<br />
Martin Kahms, Corinna Schroth, Julia Fiebach, Jürgen Kuhlmann<br />
Aut<strong>of</strong>luorescent Proteins in Interaction Analysis<br />
Konstantin Lukyanov, Dmitry Chudakov, Vladislav Verkhusha, Mikhail Matz, Dmitry Shagin, Yurii<br />
Yanushevich, Ekaterina Barsova, Sergey Lukyanov<br />
Novel fluorescent proteins: diversity, mutagenesis and applications<br />
Tanja Meyer, Christian Schwöppe, Antje von Schaewen<br />
Molecular properties <strong>of</strong> a new G6PD-like is<strong>of</strong>orm from Arabidopsis
Symposium:<br />
Extracellular matrices and cell migration<br />
Christoph Becker-Pauly, Markus-N. Kruse, Daniel Lottaz*, Irene Yiallouros, Hans-Willi Krel**,<br />
Erwin E. Sterchi*, Walter Stöcker<br />
The human Metalloprotease Meprin - A Candidate for epithelial differentiation<br />
Till Biskup, Uta Joos, Jana Kriegsmann, Christian Heise, Ines Westphal, Götz Pilarczyk, Claus<br />
Duschl<br />
Chemically modified glass surfaces for growth <strong>of</strong> culture cells examined by<br />
light microscopy.<br />
Cord Brakebusch, Aleksandra Czuchra, Xunwei Wu, Tanja Bosse, Klemens Rottner<br />
Cdc42 is not required for the formation <strong>of</strong> filopodia and lamellipodia, but<br />
crucial for the uptake <strong>of</strong> Listeria<br />
Kerstin Brands, Ferda Cevikbas, Frank Echtermeyer<br />
My<strong>of</strong>ibroblast differentiation and TGF-b1 signalling is affected in syndecan-4<br />
deficient fibroblasts<br />
Ferda Cevikbas, Kerstin Brands, Frank Echtermeyer<br />
Syndecan-4 Deficiency leads to an upregulation <strong>of</strong> syndecan-1 in skin<br />
fibroblasts<br />
Jaros•aw Czyz, Katarzyna Miekus, Marta Czernik, Jolanta Sroka, Zbigniew Madeja<br />
Interrelation between rat prostate carcinoma cell coupling and motility in the<br />
determination <strong>of</strong> their invasiveness and metastatic activity<br />
Johannes A. Eble, Ulrike Mayer, Jörg Haier<br />
Snake venom inhibitors <strong>of</strong> collagen- and laminin-binding integrins suppress<br />
tumor cell migration and invasion<br />
Alexandre Fedotov, Ivan Ovcharuk<br />
Research <strong>of</strong> the mechanism <strong>of</strong> self-assembly fibronectins in modelling system.<br />
Peter Friedl<br />
Dynamic imaging <strong>of</strong> adhesion receptor and protease function in tumor cell<br />
invasion<br />
Sven Huelsmann, Christina Hepper, Rolf Reuter<br />
The PDZ-GEF Dizzy regulates cell form and cell adhesion via rap1 and integrins<br />
in macrophages <strong>of</strong> the Drosophila embryo.<br />
Vera Hintze, Markus Höwel, Carsten Wermter, Irene Yiallouros, Walter Stöcker<br />
Procollagen C-proteinase: Substrate recognition by C-terminal domains<br />
Caroline E. Chwieralski, Ingo Schnurra, Lars Thim, Werner H<strong>of</strong>fmann<br />
Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-2B bronchial<br />
epithelial cells depend on different signaling cascades<br />
Michael Schnoor, Katrin Stolle, Jürgen Rauterberg, Paul Cullen, Stefan Lorkowski<br />
Expression <strong>of</strong> collagens by human monocyte-derived macrophages<br />
Wittaya Pimtong, Raimund Wagener, Mats Paulsson, Peter Bruckner, Uwe Hansen<br />
Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Anke Rattenholl, Stephan Seeliger, Jörg Buddenkotte, Astrid Grevelhörster, Martin Steinh<strong>of</strong>f<br />
Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor in Skin<br />
Carcinogenesis<br />
Björn Reiss, Andreas Jansh<strong>of</strong>f, Claudia Steinem, Joachim Wegener<br />
Functionalized vesicles as a model for cell adhesion studies<br />
Markus Rüegg
Neuromuscular diseases: Concepts and new approaches for therapies<br />
David Denis S<strong>of</strong>eu Feugaing, Hans Kresse, Martin Götte<br />
The dermatan sulfate proteoglycans Decorin and Biglycan follow partially<br />
diverging endocytic routes<br />
Joerg Stetefeld<br />
Alternative mRNA-splicing in agrin- A tool for structural and functional<br />
diversity<br />
Daniela Villone, Leena Bruckner-Tuderman, Peter Bruckner, Uwe Hansen<br />
Human dermal basement membrane zone: Characterization <strong>of</strong> functional<br />
suprastructures<br />
Martin W. Volmer, Yvonne Radacz, Stephan A. Hahn, Susanne Klein-Scory, Kai Stühler, Marc<br />
Zapatka, Wolff Schmiegel, Helmut E. Meyer, Irmgard Schwarte-Waldh<strong>of</strong>f<br />
Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the anti-adhesive<br />
invasion-promoting matricellular protein SPARC<br />
Joachim Wegener, Charles Keese*, Ivar Giaever*<br />
Kinetics <strong>of</strong> cell spreading monitored by electric cell-substrate impedance<br />
sensing<br />
Carsten Wermter, Markus Höwel, Vera Hintze, Irene Yiallouros, Walter Stöcker<br />
Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition and Specificity<br />
Eva C. Woenne, Cathrine Schmidt, Nick Mirancea, Roswitha Nischt, Neil Smyth, U. Werner, Hans-<br />
Jürgen Stark, Norbert E. Fusenig, M. Gerl, Dirk Breitkreutz<br />
Nidogen-1 or -2 Promote Basement Membrane Formation in Skin -Type 3D-<br />
Cocultures<br />
Frank Möhrlen, Sebastian Gornik, Melanie Mertens, Ina Rattmann, Irene Yiallouros<br />
Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong> metalloproteinases
Symposium:<br />
Ion channels and transporters<br />
Poonam Balani, Christian Weidenfeller, Carsten Mim*, Christ<strong>of</strong> Grewer*, Thomas Rauen<br />
Glutamate transporter function in synaptic transmission<br />
Ernst Bamberg<br />
Conformational dynamics <strong>of</strong> P-type ATPases: Intraprotein signalling and<br />
subunit interaction probed by voltage fluorometry<br />
Anja Bruehl, Arnd Baumann<br />
Molecular characterization <strong>of</strong> rat calcium-activated chloride channels<br />
Antje Burse, Sabrina Discher, Jürgen Kuhn, Wilhelm Boland<br />
Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in Leaf Beetle<br />
Larvae<br />
Gaby-Fleur Böl, Regina Brigelius-Flohé<br />
Intracellular trafficking <strong>of</strong> the Interleukin-1 receptor (IL-1RI) associated<br />
kinase IRAK depends on its kinase-activity<br />
Florian Garczarek, Klaus Gerwert<br />
The Proton Release Mechanism <strong>of</strong> Bacteriorhodopsin’s WT, E204D and E194D<br />
revealed with Time-Resolved Step-Scan and H/D-Exchange FTIR<br />
Measurements<br />
Günter Gisselmann, Christian Wetzel, Hanns Hatt<br />
Functional characterization <strong>of</strong> Ih-channel splice variants in insects<br />
Günter Gisselmann, Hermann Pusch, Hanns Hatt<br />
Novel ligand gated channels in D. melanogaster: GABA-gated cation channels<br />
and gating by multiple neurotransmitters<br />
Melina Haupt, Murray Coles, Horst Kessler, Marc Bramkamp, Karlheinz Altendorf<br />
Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC complex, a P-type<br />
ATPase, are not driven by ATP-induced conformational changes<br />
Daniel Hilger, Gunnar Jeschke, Christoph Wegener, Heinz-Jürgen Steinh<strong>of</strong>f, Etana Padan,<br />
Heinrich Jung<br />
Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli probed by EPR<br />
spectroscopy<br />
Daniel Hilger, Jeschke Gunnar, Christoph Wegener, Heinz-Jürgen Steinh<strong>of</strong>f, Heinrich Jung<br />
Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP <strong>of</strong> E. coli: a<br />
protein chemical and EPR spectroscopic analysis<br />
Ana Kilic, Ana Velic, Sevdalina Yurukova, Larissa Fabritz, Eberhard Schlatter, Michaela Kuhn<br />
Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in Guanylyl cyclase-A<br />
deficient mice<br />
Viktoria Kukhtina, Denise Kottwitz, Chris Weise, Ferdinand Hucho<br />
Intracellular domain - terra incognita <strong>of</strong> the nicotinic acetylcholine receptor<br />
Martin Kühn, Lakshmi Pulagam, Denis Duché, Anton Savitsky, Klaus Möbius, Heinz-Juergen<br />
Steinh<strong>of</strong>f<br />
Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Jürgen Meyer zu Tittingdorf, Sascha Rexroth, Eva Schäfer, Ralf Schlichting, Christoph Giersch,<br />
Nobert A. Dencher, Holger Seelert<br />
The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not affect the<br />
stoichiometry <strong>of</strong> its chloroplast ATP synthase oligomer III<br />
Dessy Nikova, Volodymyr Nechyporuk-Zloy, Christian Stock, Albrecht Schwab, Hans<br />
Oberleithner, Hermann Schillers<br />
Cell membrane isolation for high resolution AFM imaging
Peter Pohl<br />
Fluid transport through epithelial barriers: the importance <strong>of</strong> ion channels and<br />
transporters<br />
Hermann Schillers, Hans-Georg Koch, Astrid Stumpf, Kerstin Wenners-Epping, Mike Waelte,<br />
Dessy Nikova, Hans Oberleithner<br />
Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Andreas Krieger, Marcel Kamp, Margit Henry, Alexey Pereverzev, Marco Weiergräber, Jürgen<br />
Hescheler, Toni Schneider<br />
Signalling through the E-type voltage-gated calcium channel. Identification <strong>of</strong><br />
interaciton partners.<br />
Christina Schreier, Werner Kremer, Bernd Fakler, Hans Robert Kalbitzer<br />
Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type voltage gated<br />
potassium channels<br />
Albrecht Schwab, Volodymyr Nechyporuk-Zloy, Christian Stock<br />
Endocytic internalization <strong>of</strong> potassium channels in migrating cells<br />
Helena Schwaßmann, Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger<br />
Seelert, Norbert A. Dencher<br />
H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas reinhardtii<br />
Heike Schäfer, Jean-Pierre Chervet, Christian Bunse, Cornelia Joppich, Helmut E. Meyer, Katrin<br />
Marcus<br />
A peptide pre-concentration approach for nano-HPLC to diminish memory<br />
effects<br />
Holger Seelert, Ansgar Poetsch, Sascha Rexroth, Norbert A. Dencher, Daniel J. Müller, Jinshu Yu,<br />
Werner Kühlbrandt, Janet Vonck<br />
Properties <strong>of</strong> the proton translocating oligomer in chloroplast FOF1 ATP<br />
synthase<br />
Uwe Schulte, Jörg Oliver Thumfart, Wolfgang Bildl, Hans-Günther Knaus, Claudia Sailer,<br />
Matthias Mann, Jens Andersen, Martin Biniossek, Volker Rohde, Bernd Fakler<br />
Identification and functional characterization <strong>of</strong> protein supercomplexes<br />
associated with Kv1.1 potassium channels<br />
Christoph Wegener, Anton Savitsky, Mathias Pfeiffer, Igor Grigorev, Klaus Möbius, Heinz-Jürgen<br />
Steinh<strong>of</strong>f<br />
High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled bacteriorhodopsin: A<br />
study <strong>of</strong> the polarity and the local pH in the Bacteriorhodopsins proton<br />
channel.
Symposium:<br />
Key players in RNA-silencing<br />
Witold Filipowicz, Caroline Artus, Lukasz Jaskiewicz, Fabrice Kolb, Ramesh Pillai, Haidi Zhang<br />
RNAi and microRNA machineries in mammalian cells<br />
Blaga Popova, Markus Kuhlmann, Markus Kaller, Wolfgang Nellen<br />
Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene silencing<br />
Ulrich Sentner, Sebastian Fettig<br />
P1/HcPro: viral suppressors <strong>of</strong> gene silencing in wheat
Symposium:<br />
Mechanisms <strong>of</strong> Cellular Compartimentalization<br />
Lutz Hilterhaus, Stefan Malcharek, Hans-Joachim Galla<br />
The Influence <strong>of</strong> Cholesterol and POPE on mono- and multilayers conatining<br />
surfactant protein C<br />
Ute Klenz, Hans-Joachim Galla<br />
The Influence <strong>of</strong> Membrane Fluidity on Vesicle Insertion in a SP-B or SP-C<br />
containing Monolayer<br />
Stefan Malcharek, Christian Schachtrup, Friedrich Spener, Hans-Joachim Galla<br />
Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong> H- and E- FABP<br />
double knock-out mice<br />
Günter Müller, Susanne Wied, Christian Jung, Andrea Schulz, Norbert Tennagels, Wendelin Frick<br />
Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-Talk to the<br />
Insulin Signaling Cascade by the Antidiabetic Drug Glimepiride<br />
Pariya Na Nakorn, Carol Flach, Rich Mendelsohn, Hans-Joachim Galla<br />
The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C17±palm ) on the surface<br />
properties <strong>of</strong> lipid monolayers at the air/water-interface<br />
Peter Rehling, Agnieszka Chacinska, Maria Lind, Ann E. Frazier, Jan Dudek, Nikolaus Pfanner<br />
Transport <strong>of</strong> nuclear encoded proteins across and into the inner mitochondrial<br />
membrane<br />
Gerhard Schütz, Manuel Mörtelmaier, Mario Brameshuber, Karel Drbal, Hannes Stockinger<br />
Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
Silke Steffens, Jens Oldendorf, Lifeng Chi, Günter Haufe, Hans-Joachim Galla<br />
Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters<br />
Robert Tampe<br />
INs and OUTs <strong>of</strong> Antigens - The Transport Machinery TAP in the Cellular<br />
Immune System<br />
Regine Willumeit, Mont Kumpugdee, Sebastian Linser, Thomas Hauss, Sergio S. Funari, Jörg<br />
Andrä<br />
The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes: Insights by<br />
Neutron Diffraction and X-ray Scattering<br />
Olaf Zschörnig, Christ<strong>of</strong> Gutsche, Matthias Müller, Klaus Arnold<br />
Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing vesicles
Symposium:<br />
Molecular recognition mechanism in plant-microbe interactions<br />
Steffen Amme, Twan Rutten, Bernhard Schlesier, Michael Melzer, Hans-Peter Mock<br />
Proteome analysis <strong>of</strong> tobacco trichomes<br />
Ton Bisseling, Rene Geurts, Caroline Franken, Erik Limpens, Patrick Smit<br />
Nod factor perception and transduction<br />
Susanne Berger, Martina Papadopoulos, Katharina Bonfig, Ulrich Schreiber, Thomas Roitsch<br />
Complex regulation <strong>of</strong> gene expression, photosynthesis and sugar levels by<br />
pathogen infection<br />
Nour Eddine El Gueddari, Bruno Moerschbacher<br />
Chitosan Activates Resistance Against Pathogens After eXposure (CARAPAX)<br />
Rüdiger Hampp, Mika Tarkka, Uwe Nehls<br />
Symbiotic interactions at the root level <strong>of</strong> forest trees: receive, deliver and get<br />
protected<br />
Tina Jordan, Annett Meyer, Kristin Peter, Patrick Römer, Sebastian Schornack, Ulla Bonas,<br />
Thomas Lahaye<br />
Pepper Bs3 and tomato Bs4 - distinct molecular principles for perception <strong>of</strong><br />
highly-related Xanthomonas AvrBs3 and AvrBs4 proteins<br />
A. Markert, S. Kucht, J. Gross, M. Ahimsa-Mueller, Y. Hussein, U.* Steiner, E. Leistner<br />
The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline alkaloids in Ipomoea<br />
asarifolia (Convolvulaceae)<br />
Jan Scheffer, Yvonne Rolke, Paul Tudzynski<br />
Signalling in early stages <strong>of</strong> pathogenic development <strong>of</strong> Claviceps purpurea.<br />
Nicholas J. Talbot, Martin J. Gilbert, Darren M. Soanes, Madhumita Barooah, Zheng Yi Wang,<br />
Sarah Ahmad, Claire Veneault-Fourrey, Joanna M. Jenkinson, Lucy J. Holcombe, Gurpreet<br />
Bhambra<br />
Functional genomics <strong>of</strong> plant infection by the rice blast fungus Magnaporthe<br />
grisea<br />
Claus Wasternack, Irene Stenzel, Bettina Hause, Gerd Hause, Otto Miersch<br />
Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong> tomato<br />
S. Wings, C. Pullen, T. Boettcher, E. Leistner<br />
Observations on the erratic occurrence <strong>of</strong> maytansinoids in plants and<br />
microorganisms
Symposium:<br />
New bioanalytical techniques and developments<br />
Heiko Andresen, Kim Zarse, Carsten Grötzinger, Oliver J. Kreuzer, Marc Birringer, Eva<br />
Ehrentreich-Förster, Frank F. Bier<br />
Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Jan Barnikow, Ron Tynes, Maik Kindermann, Stefan Steurer, Andreas Brecht<br />
The SNAP-tagTM - a unique tool for covalent labeling and immobilization <strong>of</strong><br />
proteins<br />
Nadja Bilko, Denys Bilko<br />
Ex vivo proliferation and differentiation <strong>of</strong> cord blood cells in gel culture<br />
system<br />
Matthias Böcker, Pia Heidenreich, Harald Fuchs, Tilman Schäffer<br />
Scanning ion conductance microscope with shear-force control<br />
Andreas Böhm, Albert Sickmann<br />
A Clustering Solution for Distributed Data Analysis in Mass Spectrometry<br />
(paOla)<br />
Andreas Böhm, Albert Sickmann<br />
Automatic Synchronizing and self-Updating Protein Sequence Database<br />
Management System<br />
Andreas Böhm, Florian Grosse-Coosmann, Albert Sickmann<br />
Calculating theoretical ms spectra for given sequences<br />
Gregor Witte, Claus Urbanke, Ute Curth<br />
Analytical Ultracentrifugation pinpoints the interaction <strong>of</strong> DNA polymerase III<br />
and primase with EcoSSB to its C-terminal region<br />
Andreas Gorschlüter, L.H. Mak *, M. Knoll *, N. Dankbar, C. Sundermeier<br />
Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding Forces between<br />
Single Molecules<br />
Sergey V. Tokalov, Herwig O. Gutzeit, Jutta Ludwig-Müller, Barbara Kind, Alexander Franz,<br />
Yvonne Henker<br />
Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism in human cells<br />
Pia M. Heidenreich, Sebastian Schrot, Hans-Joachim Galla, Harald Fuchs, Tilman E. Schäffer<br />
Scanning Ion Conductance Microscopy - Topographical non-contact imaging <strong>of</strong><br />
s<strong>of</strong>t surfaces<br />
Robert Henderson, David Dryden, Michael Edwardson, Michael Waring<br />
DNA, Drugs and Proteins: Insights from the Atomic Force Microscope<br />
Alejandro Heredia, C. C. Bui, Ueli Suter, Peter Young, Tilman Schäffer<br />
Mechanical properties <strong>of</strong> myelinated and de-myelinated peripheral axons with<br />
the atomic force microscope<br />
Sonja Hess, Xiaoli Chen, Samuel W. Cushman, Lewis K. Pannell<br />
Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong> adipocytes<br />
Peter Hinterdorfer<br />
Topography and Recognition Imaging using AFM<br />
Heinrich Hörber<br />
Single molecule mechanics<br />
Bernhard Andreas Krenig<br />
A new developed biochemical process in purifikation
Stefan Kreusch, Heidrun Rhode, Horst Hoppe, Thomas Moore, Renate Bublitz, Joachim<br />
Misselwitz, Margarete Schulze, Gerd Cumme<br />
Separation and analysis <strong>of</strong> native human proteomes using parallel<br />
chromatography with microplates: Alport syndrome versus healthy serum<br />
Ziv Reich<br />
Exploring Protein-Binding Mechanisms and Energy Landscapes by Single-<br />
Molecule Mechanical Unbinding Experiments<br />
Nicole Reifschneider, S. Goto, Norbert A. Dencher, Frank Krause<br />
Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong> Rattus<br />
norvegicus studied by peptide mass fingerprinting<br />
Sascha Rexroth, Jürgen Meyer zu Tittingdorf, Frank Krause, Holger Seelert, Norbert Dencher<br />
Identification <strong>of</strong> integral thylakoid membrane proteins from Chlamydomonas<br />
reinhardtii by peptide mass fingerprinting and MALDI-MS<br />
Heidrun Rhode, M. Schulze, E. Mitre, G.A. Cumme, T. Rausch, D. Philipp, A. Horn<br />
Turbo-mixing in microplates<br />
Thomas Schulenborg, Heike Schäfer, Christian Bunse, Helmut E. Meyer, Katrin Marcus<br />
Detection <strong>of</strong> phosphorylation sites using a hybrid triple quadrupole/linear ion<br />
trap mass spectrometer<br />
Barbara Sitek, Ognjan Apostolov, Kathy Pfeiffer, Kai Stühler, Helmut E. Meyer, Angelika Eggert,<br />
Alexander Schramm<br />
Identification <strong>of</strong> proteins differentially expressed upon neurotrophin receptor<br />
activation using DIGE and MALDI-MS<br />
Manfred Wuhrer, Carolien A. M. Koeleman, André M. Deelder, Cornelis H. Hokke<br />
Analysis <strong>of</strong> protein glycosylation using normal-phase nano-scale liquid<br />
chromatography-mass spectrometry <strong>of</strong> glycopeptides and oligosaccharides
Symposium:<br />
Nuclear structure and gene expression<br />
Kristina Beck, Karl-Heinz Klempnauer<br />
The transcription factor C/EBP&beta triggers phosphorylation <strong>of</strong> the<br />
coactivator p300: A new mechanism <strong>of</strong> cross-talk between transcription<br />
factors and coactivators?<br />
Peter Becker<br />
Lifting a chromosome - Dosage Compensation in Drosophila<br />
Angelika Bondzio, Christoph Gabler, Dagmar Beier, Siegfried Risse, Ralf Einspanier<br />
Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia Virus (BLV)<br />
expression through Toll-like receptor 4<br />
Daniel Braas, Holger Gundelach, Karl-Heinz Klempnauer<br />
The glioma amplified sequence 41 gene (GAS41) is a direct Myb target gene<br />
Maret Böhm, Nadja Bitomsky, Karl-Heinz Klempnauer<br />
The transformation supressor Protein Pdcd4 binds to RNA and is involved in<br />
the regulation <strong>of</strong> c-Jun activity.<br />
Olesya Chayka, Jörg Kintscher, Karl-Heinz Klempnauer<br />
Identification <strong>of</strong> MYB and C/EBP responsive enhancer <strong>of</strong> the chicken mim-1<br />
gene<br />
Ralph Goethe, Katrin Hübner, Loc Phi-van<br />
Possible involvement <strong>of</strong> Ca2+/calmodulin dependent protein kinase II<br />
(CaMKII) in lysozyme pre-mRNA splicing<br />
Christoph Hutter, Ingo Neumann, Stefan Burdach, Martin S. Staege<br />
The tumor specific chimeric transcription factor EWS-FLI1 down-regulates<br />
gene expression<br />
Ingo Neumann, Christoph Hutter, Angelika B. Reske-Kunz, Stefan Burdach, Martin S. Staege<br />
Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene expression pr<strong>of</strong>ile<br />
<strong>of</strong> neuroblastoma cells identifies new options for immunotherapy<br />
Teodora Nikolova, Jaroslaw Czyz, Alexandra Rolletschek, Przemyslaw Blyszczuk, Jürgen<br />
Schuderer, Niels Kuster, Anna M Wobus<br />
Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and apoptosisrelated<br />
genes in p53-deficient embryonic stem (ES) cells and ES-derived<br />
neural progenitors<br />
Bas van Steensel, Maartje Vogel, Martin Loden, Elzo de Wit, Frauke Greil, Ben Abbas<br />
Regulatory networks revealed by genomic maps <strong>of</strong> heterochromatin protein<br />
binding in flies and humans<br />
Christina Zechel, Ingrid Koziollek-Drechsler, Ulrike Sattler, Marek Samochocki<br />
The orphan receptor GCNF in neuronal differentiation
Symposium:<br />
Plenary Lectures<br />
Wolfgang Baumeister, Martin Beck, Ariane Briegel, Marek Cyrklaff, Thomas Keil, Julia Kuerner,<br />
Vladan Lucic, Ohad Medalia, Stephan Nickell, Juergen Plitzko<br />
Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Peter Fromherz<br />
Cells 'n Chips or Ionics meets Electronics<br />
Masamitsu Futai, Ge-Hong Sun-Wada, Yoh Wada<br />
Vacuolar-type proton pump ATPases (V-ATPases) and inside acidic<br />
organelles/compartments: rotational mechanism and diverse physiological<br />
roles.<br />
Frank R. Neumann, M.R. Gartenberg, A. Taddei, T. Laroche, M. B•aszczyk, Susan Gasser<br />
Imaging functional chromosomes: tethers and dynamics in the yeast nucleus<br />
Hermann Gaub<br />
Single Molecule Force Spectroscopy- A Bettter Understanding <strong>of</strong> Biomolecules<br />
with Newton?<br />
Oliver Hantschel, Bhushan Nagar, John Kuriyan, Giulio Superti-Furga<br />
Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases<br />
Franz Hillenkamp<br />
MALDI Mass Spectrometry <strong>of</strong> Nucleic Acids<br />
Keiichi Namba<br />
Self-assembly and switching <strong>of</strong> the bacterial flagellum<br />
Tom Rapoport<br />
Structure and function <strong>of</strong> a protein-conducting channel<br />
Christian Ungermann<br />
Protein palmitoylation during membrane fusion<br />
Anna M. Wobus<br />
Stammzellforschung: Potenzial, Perspektiven und Probleme
Symposium:<br />
Small non coding RNAs<br />
Jerome Cavaille, Hervé Seitz, Hélène Royo, Shau-Ping Lin *, Neil Youngson *, Anne C. Ferguson-<br />
Smith *<br />
Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
Tamas Kiss<br />
Nuclear organization <strong>of</strong> human modification guide RNA machinery<br />
Jost Seibler, Birgit Küter-Luks, Heidrun Kern, Frieder Schwenk<br />
Rapid method for reproducible, shRNA-mediated gene silencing in vivo
Symposium:<br />
Structural Basis <strong>of</strong> Electron Transport<br />
Daniele Penzo, Valeria Petronilli, Alessia Angelin, Claudia Cusan, Raffaele Colonna, Luca<br />
Scorrano, Francesco Pagano, Maurizio Prato, Fabio Di Lisa, Paolo Bernardi<br />
Arachidonic Acid Mediates Calcium-dependent Apoptosis through the<br />
Mitochondrial Pathway<br />
Oliver Bremm, Andre Remy, Klaus Gerwert<br />
Coupling <strong>of</strong> light-induced electron transfer to proton uptake in photosynthesis<br />
Thomas Carell, Alexandra Mees, Tobias Klar, Andre P. M. Eker, Lars-Oliver Essen<br />
Crystal structure <strong>of</strong> DNA Photolyase complexed to double stranded DNA<br />
Prasad Gajula, Igor Borovykh*, Christian Beier, Peter Gast*, Heinz-Juergen Steinh<strong>of</strong>f<br />
Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled photosynthetic reaction<br />
centers from Rhodobacter sphaeroides based on molecular dynamics<br />
simulations and EPR spectroscopy.<br />
Stefan Kerscher, Ljuban Grgic, Aurelio Gar<strong>of</strong>ano, Noushin Kashani-Poor, Ulrich Brandt<br />
A yeast genetics approach to study mitochondrial complex I<br />
Oliver Einsle, Peter MH Kroneck<br />
Structural Basis <strong>of</strong> Denitrification<br />
Tatjana M. E. Schwabe, Daniela Schlüsener, Jochen Kruip<br />
Ycf3 from Synechocystis sp. PCC 6803: Localisation, Oligomerisation and Role<br />
in Photosystem I Biogenesis<br />
B. van den Heuvel<br />
Gene hunting and gene cloning in human OXPHOS deficiency<br />
Marten Wikstrom<br />
Warburg's Atmungsferment: A molecular energy transducer
Symposium:<br />
Structural organization and dynamics <strong>of</strong> protein<br />
Dirk Bald, Toru Hisabori, Georg Groth, Holger Lill<br />
Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and activation <strong>of</strong> F1-<br />
ATPase by tentoxin<br />
Heike Betat, Christiane Rammelt, Georges Martin, Mario Mörl<br />
A Twin Study: Chimeras between Poly(A) Polymerase and CCA enzyme show<br />
unexpected activities<br />
Marc Bramkamp, Jeff Errington<br />
Membrane bound proteins <strong>of</strong> the division machinery in bacteria<br />
Henrik Brutlach, Sadasivam Jeganathan², Martin von Bergen², Eckhard Mandelkow², Heinz-<br />
Juergen Steinh<strong>of</strong>f<br />
EPR investigation on site-directed spin labeled Tau protein<br />
Partha Chakrabarti, Yan Suveyzdis, Alfred Wittingh<strong>of</strong>er, Klaus Gerwert<br />
FTIR on the Rap-RapGAP reaction: GTPase activation without an arginine<br />
finger<br />
Vincent Lemaitre, Anthony Watts, Wolfgang Fischer<br />
Ion channels with minimalist design – from viruses.<br />
Peter Geyer, Rolf Döker, Xiaodong Zhao, Werner Kremer, Jürgen Kuhlmann, Alfred Wittingh<strong>of</strong>er,<br />
Hans Robert Kalbitzer<br />
Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2 and its<br />
interaction with Ran<br />
Matthias Gralle, Michelle Botelho, Cristiano Oliveira, Iris Torriani, Sergio Ferreira<br />
Folding and structure <strong>of</strong> the amyloid precursor protein investigated by solution<br />
scattering and spectroscopy<br />
Rico Czaja, Marc Struhalla, Katja Höschler, Wolfram Saenger, Norbert Sträter, Uli Hahn<br />
New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Udo Heinemann, Konrad Büssow, Uwe Mueller, Patrick Umbach<br />
Insight into the structure <strong>of</strong> human proteins from a structural genomics<br />
approach<br />
Roland H<strong>of</strong>weber, Gudrun Horn, Hans Robert Kalbitzer<br />
cell free studies on the function <strong>of</strong> cold shock proteins<br />
Norman Kachel, Werner Kremer, Ralph Zahn, Hans Robert Kalbitzer<br />
Intermediate states and implications for the species barrier <strong>of</strong> the human<br />
prion protein revealed by high pressure NMR<br />
Carsten Kötting, Katrin Beckmann, Marco Blessenohl, Sven Brucker, Partha Chakrabarti, Carolin<br />
Eichholz, Angela Kallenbach, Yan Suveyzdis, Klaus Gerwert<br />
Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved FTIR-<br />
Spectroscopy<br />
Michael Lammers, Rolf Wagner, Atsushi Shimada, Alfred Wittingh<strong>of</strong>er<br />
Structural and biochemical characterisation <strong>of</strong> the Rho effector mDia<br />
Yanfeng Li, Jan Dudek, Bernard Guiard, Nikolaus Pfanner, Peter Rehling, Wolfgang Voos<br />
The presequence translocase-associated protein import motor <strong>of</strong><br />
mitochondria: Pam16 functions in an antagonistic manner to Pam18<br />
Julian Ollesch, Eva Zobeley, Rudi Glockshuber, Klaus Gerwert<br />
α-β-Transition Studies In Proteins And Peptides With Timeresolved FTIR<br />
Alexander Prodöhl, Thomas Volkmer, Dirk Schneider
C<strong>of</strong>actor Binding to Membrane Proteins - From the Two-Stage to a Three-Stage<br />
Model?<br />
Andrea Scrima, Ingrid Vetter, Alfred Wittingh<strong>of</strong>er<br />
TrmE, a GNBP involved in tRNA-modification<br />
Michael Spörner, Thosten Graf, Burkhard König, Hans Robert Kalbitzer<br />
Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour therapy<br />
Enrica Brodignon, Johann Klare, Martin Engelhard, Heinz-Jürgen Steinh<strong>of</strong>f<br />
EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the photo-transducer<br />
NpHtrII<br />
Heinz-Jürgen Steinh<strong>of</strong>f<br />
Multi-frequency EPR reveals the conformational dynamics <strong>of</strong> membrane<br />
proteins: colcin A and the sensory rhodopsin-transducer complex<br />
Robin Vetter, Melanie Wagner, Jürgen Kuhlmann<br />
Effects <strong>of</strong> an inhibitor <strong>of</strong> Acyl Protein Thioesterase 1 on Ras-mediated signal<br />
transduction<br />
Klaus-Peter Vogel, Monique Karl, Heinz-Jürgen Steinh<strong>of</strong>f, Wolfgang H. Ziegler<br />
Vinculin phospholipid interaction studied by EPR utilizing site-directed spin<br />
labeling<br />
Birgit von Janowsky, Karin Röttgers, Martin Krayl, Wolfgang Voos<br />
Structural properties <strong>of</strong> the substrate protein determine degradation by the<br />
mitochondrial AAA+ protease PIM1<br />
Christiane Wiegand, Marco Hagedorn, Harald Jockusch<br />
Misfolded TMV Coat Proteins: New Mutants and Pathogenicity in Plant and<br />
Animal Cells
Symposium:<br />
_Other (free) topic_<br />
Mekky Mohammed Abouzied, Heba Mahmoud El-tahir, Volkmar Gieselmann, Sebastian Franken<br />
Evidence for a cell surface receptor <strong>of</strong> HDGF<br />
Iris G. Altug, Harro J. Bouwmeester, Wilfried A. König<br />
Isolation and functional expression <strong>of</strong> cDNAs encoding sesquiterpene<br />
synthases, including the enantiomeric (+)- and (-)-Germacrene D-Synthases<br />
from Solidago canadensis L.<br />
Jürgen Alves, Wolfgang Küster, Imke Peters<br />
Mutants <strong>of</strong> the EcoRI Restriction Endonuclease Exhibit Changes <strong>of</strong> Specificity<br />
Imre Berger, Daniel Fitzgerald, Timothy Richmond<br />
New Baculovirus Expression Tools for Multiprotein Applications<br />
Astrid Blume, Andrew J. Benie, Stephan Hinderlich2, Werner Reutter2, Richard R. Schmidt3,<br />
Thomas Peters<br />
New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic acid biosynthesis,<br />
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, with<br />
different ligands<br />
Heike Bruhn, Matthias Leippe<br />
The pore-forming mechanism <strong>of</strong> amoebapore A characterized by mutational<br />
analysis<br />
Alexander Bubikat, Bernd Zetsche, Larissa Fabritz, Axel Gödecke, Michaela Kuhn<br />
Local ANP prevents cardiac remodeling in hypertensive eNOS-deficient mice<br />
Gerald Bäuml, Gudrun Horn, Widmar Tanner, Hans Robert Kalbitzer<br />
Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the Saccharomyces<br />
cerevisiae cell adhesion molecule a-agglutinin<br />
Frank Dietz, Friedericke Lehmann, Heiko Gäthje, Maija Slaidina, Daniel Daraban, Sörge Kelm<br />
The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new splice variant<br />
Radovan Dvorsky, Lars Blumenstein, Patricia Stege, Mohhamad Reza Ahmadian<br />
RhoA-p160Rock communication<br />
Niklas Feldhahn, Florian Klein, Markus Müschen<br />
The BCR-ABL1 kinase bypasses selection for the expression <strong>of</strong> a pre-B cell<br />
receptor in pre-B acute lymphoblastic leukemia cells<br />
Sven Gathmann, Dorothée Walter, Dirk Schneider<br />
Uncovering the mechanisms for protein sorting in cyanobacteria<br />
R. Gerke, M. Schmeer, T. Seipp, U. Pliquett, E. Neumann<br />
Spatial Resolution <strong>of</strong> Gene Expression by Membrane Electroporation <strong>of</strong> CHO<br />
Cell Layers<br />
María Jose Gómez-Lechón, Alfonso Serralta, Teresa Donato, Enrique O´Connor, Jose Vicente<br />
Castell, Jose Mir<br />
The immunusupresive drug FK506 prevents Fas-induced apoptosis and<br />
mitochondrial dysfunction in human hepatocytes.<br />
Stefan Harjes, Peter Bayer, Axel Scheidig<br />
The structure <strong>of</strong> human 3'-phosphoadenosine-5'-phosphosulfate synthetase 1 -<br />
A molecular pendulum?<br />
Romano Hebeler, P. P. Dijkwel, H. E. Meyer, B. Warscheid<br />
Quantitative proteomics for the investigation <strong>of</strong> leaf senescence in Arabidopsis<br />
thaliana
Maria Heiser, Birgit Hutter-Paier, Lidia Jerkovic, Michael Becker-Andre, Hans Dieplinger,<br />
Roswitha Pfragner, Manfred Windisch<br />
Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical chicken<br />
neurons in vitro<br />
Angela Hirtreiter, Luis Figueiredo, Daniel Klunker, Bernd Haas, Debbie Ang*, Dean Naylor,<br />
Michael Kerner, Costa Georgopoulos*, Ulrich Hartl, Manajit Hayer-Hartl<br />
Functional Characterization <strong>of</strong> group I and II chaperonins in the archaeon<br />
Methanosarcina mazei<br />
Anna Kantola, Jorma Keski-Oja, Katri Koli<br />
Promoter characterization <strong>of</strong> the latent transforming growth factor (TGF)-β<br />
binding protein 3 (LTBP-3)<br />
O. Bergner, C. Brendel, Claudia Koch-Brandt<br />
The role <strong>of</strong> the lipoprotein receptor LRP1 in connective tissue factor (CTGF)<br />
induced signal transduction: Implications for the pathogenesis <strong>of</strong><br />
atherosclerosis<br />
Monika Kogler-Voigt, Roswitha Pfragner, Sabine Supanz, Christa Nöhammer, Konrad<br />
Schauenstein, Karl-Heinz Preisegger<br />
Detection <strong>of</strong> rare tumor cells in peripheral blood for early identification <strong>of</strong><br />
recurrent disease<br />
Nils Kurlemann, Andreas Liese<br />
Application <strong>of</strong> immobilized benzaldehyde lyase as catalyst in the continuous<br />
synthesis <strong>of</strong> a chiral 2-hydroxy ketone<br />
Bernd Lepenies, Bernhard Fleischer, Thomas Jacobs<br />
Regulation <strong>of</strong> T cell responses by BTLA: Functional analysis using a soluble<br />
BTLAIg fusion protein<br />
Katrin Stolle, Michael Schnoor, Jürgen Rauterberg, Paul Cullen, Stefan Lorkowski<br />
TGF-β1 inducible genes in coronary smooth muscle cells<br />
Sabine Riekenberg, Birte Witjes, Gundula Key, Henning Scholze<br />
Amoebasin, a chagasin-like cysteine proteinase inhibitor in trophozoites<br />
Entamoeba histolytica<br />
Beate Rinner, Harald Greger, Brigitte Brem, Peter Pürstner, Veronika Siegl, Thomas Efferth,<br />
Roswitha Pfragner<br />
Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary thyroid<br />
carcinoma?<br />
Randy Kurz, Markus Rüffer, Christin Urban, Andrée Rothermel, Winnie Weigel, Andrea A.<br />
Robitzki<br />
Living cells on a chip – the use <strong>of</strong> electric properties for the development <strong>of</strong> a<br />
cardiomyocyte based biosensor<br />
Christiane Schaffitzel, Imre Berger, Jan Postberg, Jozef Hanes, Hans J. Lipps, Andreas Plückthun<br />
Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Birgit Scharf, Christine Rotter, Rüdiger Schmitt, Maria de Soto<br />
Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for host association<br />
are coordinated in S. meliloti<br />
Oliver Schilling, Andreas Vogel*, Brenda Kostelecky, Wolfram Meyer-Klaucke<br />
Functional diversity and metal selectivity <strong>of</strong> proteins sharing the metallo-βlactamase<br />
fold<br />
Marco Schmeer, Thomas Seipp, Sergej Kakorin, Eberhard Neumann<br />
Mechanism for the conductivity changes caused by membrane electroporation<br />
<strong>of</strong> CHO cell - pellets<br />
Frank Schnütgen, Silke De-Zolt, Petra Van Sloun, Patricia Ruiz, Thomas Floss, Wolfgang Wurst,<br />
Harald von Melchner<br />
Genomewide targeting <strong>of</strong> secreted and transmembrane proteins using the<br />
secretory gene trap U3Ceo<br />
Antje Schulte, André Schönichen, Nadine Czudnochowski, Matthias Geyer
Biochemical Characterization <strong>of</strong> the Interaction between Cyclin T1 and Hexim1<br />
and its Competition with the HIV-1 Tat protein<br />
Andrea Schulze, Sybille Standera, Elke Bürger, Marjolein Kikkert, Emmanuel Wiertz, Peter-<br />
Michael Kloetzel, Michael Seeger<br />
The ubiquitin domain protein HERP is stabilised upon interaction with a<br />
component <strong>of</strong> the ubiquitin-proteasome system and promotes degradation <strong>of</strong><br />
an ERAD substrate<br />
Dorothea Stahl, Walter Sibrowski<br />
A regulatory role <strong>of</strong> IgG2 in IgM-IgG immune complexes <strong>of</strong> normal human<br />
plasma<br />
Karen Strenge, Paul Crocker, Nicolai Bovin<br />
What are the functions <strong>of</strong> siglecs on human monocytes and macrophages?<br />
Lambrini Tontsidou, Konstantinos Bilbilis, Petra Pennekamp, Boris Skryabin, Hans-Joachim Galla,<br />
Jürgen Horst, Bernd Dworniczak<br />
GGT-Cre mice for tissue-specific gene ablation in proximal kidney tubules<br />
Markus von Nickisch-Rosenegk, Eva Ehrentreich-Förster, Berit Umann, Frank F. Bier<br />
Synthetic and recombinant zinc fingers as a nano-addressable probe to<br />
doublestranded DNA structures<br />
Marcia von Zeska Kress Fagundes, Marcela Savoldi, Joel Fernandes, Roy Larson, Maria Helana<br />
Goldman, Gustavo Goldman<br />
NpkA, a cdc2-related kinase from Aspergillus nidulans, interacts with the<br />
UvsBATR kinase<br />
Krzyszt<strong>of</strong> Wandzik, Katrin Dassler, Matthias Kaup, Hendrik Fuchs<br />
Transferrin receptor expression and shedding during megakaryopoiesis
<strong>Authors</strong> (total 1274)<br />
Name Abstract<br />
Aareskjold, Elin Rab3d and annexin A2 play a role in regulated secretion <strong>of</strong><br />
vWF but not tPA from human endothelial cells<br />
Abbas, Ben Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
Abdrakhmanov, Igor Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Abouzied, Mekky Evidence for a cell surface receptor <strong>of</strong> HDGF<br />
Mohammed<br />
Adam, Rüdiger Bacterial Meningitis: Interactions at the Blood-CSF-Barrier<br />
Afanasieva, Tatiana Endothelial activation and change <strong>of</strong> barrier function by soluble<br />
Ebola virus glycoproteins<br />
Ahimsa-Mueller, M. The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Ahmad, Sarah Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Ahmadian, Mohammad<br />
R.<br />
Ahmadian,<br />
Mohammad Reza<br />
Ahmadian, Mohammad<br />
Reza<br />
Ahmadian, Mohhamad<br />
Reza<br />
Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
New insights into structure-function relationships in GTPaseeffector<br />
interaction<br />
Molecular basis <strong>of</strong> Cdc42 recognition by Wiskott-Aldrich<br />
Syndrome Proteins<br />
RhoA-p160Rock communication<br />
Alfieri, G. Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
Alla, Simonova Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Altendorf, Karlheinz Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC<br />
complex, a P-type ATPase, are not driven by ATP-induced<br />
conformational changes<br />
Altug, Iris G. Isolation and functional expression <strong>of</strong> cDNAs encoding<br />
sesquiterpene synthases, including the enantiomeric (+)- and (-<br />
)-Germacrene D-Synthases from Solidago canadensis L.<br />
Alves, Jürgen Mutants <strong>of</strong> the EcoRI Restriction Endonuclease Exhibit Changes<br />
<strong>of</strong> Specificity<br />
Amme, Steffen Proteome analysis <strong>of</strong> tobacco trichomes<br />
Andersen, Jens Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Anderton, Brian The ageing neuronal cytoskeleton and neurodegenerative<br />
disease<br />
Andresen, Heiko Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Andrä, Jörg The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Andrä, Jörg The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Ang*, Debbie Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Angelin, Alessia Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Antipenko, Alexander Structural and functional analysis <strong>of</strong> the axon guidance<br />
molecule Sema3A and its receptor<br />
Apostolov, Ognjan Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Arnold, Klaus Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing<br />
vesicles<br />
Artmann, Gerhard Active cardiomyocytes on collagen gels for biohybrid systems
Artus, Caroline RNAi and microRNA machineries in mammalian cells<br />
Aurrand-Lions, Michel The role <strong>of</strong> junctional adhesion molecule-C (JAM-C) in<br />
angiogenesis and inflammation<br />
Austermann, Judith Characterization <strong>of</strong> the calcium-regulated S100P-ezrin<br />
interaction<br />
B•aszczyk, M. Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
Bakker-Grunwald, Tilly Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia<br />
Balani, Poonam Glutamate transporter function in synaptic transmission<br />
Bald, Dirk Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and<br />
activation <strong>of</strong> F1-ATPase by tentoxin<br />
Bamberg, Ernst Conformational dynamics <strong>of</strong> P-type ATPases: Intraprotein<br />
signalling and subunit interaction probed by voltage<br />
fluorometry<br />
Baraka*, A.M. New insights into the modulatory role <strong>of</strong> serotonin reuptake<br />
inhibitors in myocardial ischemia reperfusion injury in the<br />
rabbit<br />
Barnikow, Jan The SNAP-tagTM - a unique tool for covalent labeling and<br />
immobilization <strong>of</strong> proteins<br />
Barooah, Madhumita Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Barral, José M. Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Barsova, Ekaterina Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Bartsch, Jörg W. Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for<br />
Muscle Cell Differentiation in Culture and in Grafts<br />
Baum, Buzz Dissecting actin cytoskeletal organization in Drosophila using<br />
RNAi<br />
Baumann, Arnd Molecular characterization <strong>of</strong> rat calcium-activated chloride<br />
channels<br />
Baumeister, Ralf Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Baumeister, Wolfgang Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Bayer, Peter The structure <strong>of</strong> human 3'-phosphoadenosine-5'phosphosulfate<br />
synthetase 1 - A molecular pendulum?<br />
Beck, Kristina The transcription factor C/EBP&beta triggers phosphorylation<br />
<strong>of</strong> the coactivator p300: A new mechanism <strong>of</strong> cross-talk<br />
between transcription factors and coactivators?<br />
Beck, Martin Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Becker, Peter Lifting a chromosome - Dosage Compensation in Drosophila<br />
Becker-Andre, Michael Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Becker-Pauly,<br />
Christoph<br />
The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Beckmann, Katrin Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Beier, Christian Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled<br />
photosynthetic reaction centers from Rhodobacter sphaeroides<br />
based on molecular dynamics simulations and EPR<br />
spectroscopy.<br />
Beier, Dagmar Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia<br />
Virus (BLV) expression through Toll-like receptor 4<br />
Benie, Andrew J. New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Berger, Imre Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Berger, Imre New Baculovirus Expression Tools for Multiprotein Applications<br />
Berger, Susanne Complex regulation <strong>of</strong> gene expression, photosynthesis and<br />
sugar levels by pathogen infection
Bergner, O. The role <strong>of</strong> the lipoprotein receptor LRP1 in connective tissue<br />
factor (CTGF) induced signal transduction: Implications for the<br />
pathogenesis <strong>of</strong> atherosclerosis<br />
Berken, Antje Towards the function <strong>of</strong> plant Rop GTPases in actin<br />
reorganisation<br />
Bernardi, Paolo Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Bernfield, Merton The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Betat, Heike A Twin Study: Chimeras between Poly(A) Polymerase and CCA<br />
enzyme show unexpected activities<br />
Bhambra, Gurpreet Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Biedermann, Thomas Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Bier, Frank F. Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Bier, Frank F. Synthetic and recombinant zinc fingers as a nano-addressable<br />
probe to doublestranded DNA structures<br />
Biernat, J. Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth and degeneration<br />
Bilbilis, Konstantinos GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Bildl, Wolfgang Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Bilko, Denys Ex vivo proliferation and differentiation <strong>of</strong> cord blood cells in<br />
gel culture system<br />
Bilko, Nadja Ex vivo proliferation and differentiation <strong>of</strong> cord blood cells in<br />
gel culture system<br />
Biniossek, Martin Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Birringer, Marc Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Biskup, Till Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Bisseling, Ton Nod factor perception and transduction<br />
Bitomsky, Nadja The transformation supressor Protein Pdcd4 binds to RNA and<br />
is involved in the regulation <strong>of</strong> c-Jun activity.<br />
Bixel, Gabriele Mouse CD99 participates in T cell recruitment into inflamed<br />
skin<br />
Bixel, Gabriele Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Blessenohl, Marco Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Blondelle, Sylvie E. The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Blume, Astrid New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Blumenstein, Lars Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
Blumenstein, Lars RhoA-p160Rock communication<br />
Blyszczuk, Przemyslaw Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors<br />
Blyszczuk, Przemyslaw Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Boettcher, T. Observations on the erratic occurrence <strong>of</strong> maytansinoids in<br />
plants and microorganisms<br />
Bogdan, S. The Kette/Abi/Sra-1 complex regulates actin dynamics by<br />
inhibting wave and activating wasp function in Drosophila<br />
Boheler~, Kenneth R. Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties
Boheler§, Kenneth R. Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Boiani, Michele The Oocyte – Embryonic Stem Cell Cycle<br />
Boland, Wilhelm Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in<br />
Leaf Beetle Larvae<br />
Bonas, Ulla Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Bondzio, Angelika Adhesion <strong>of</strong> Trypanosoma congolense activates signalling<br />
pathways in endothelial cells<br />
Bondzio, Angelika Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia<br />
Virus (BLV) expression through Toll-like receptor 4<br />
Bonfig, Katharina Complex regulation <strong>of</strong> gene expression, photosynthesis and<br />
sugar levels by pathogen infection<br />
Bornkamm, Georg Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Borovykh*, Igor Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled<br />
photosynthetic reaction centers from Rhodobacter sphaeroides<br />
based on molecular dynamics simulations and EPR<br />
spectroscopy.<br />
Bosse, Tanja Cdc42 is not required for the formation <strong>of</strong> filopodia and<br />
lamellipodia, but crucial for the uptake <strong>of</strong> Listeria<br />
Botelho, Michelle Folding and structure <strong>of</strong> the amyloid precursor protein<br />
investigated by solution scattering and spectroscopy<br />
Bouwmeester, Harro J. Isolation and functional expression <strong>of</strong> cDNAs encoding<br />
sesquiterpene synthases, including the enantiomeric (+)- and (-<br />
)-Germacrene D-Synthases from Solidago canadensis L.<br />
Bovin, Nicolai What are the functions <strong>of</strong> siglecs on human monocytes and<br />
macrophages?<br />
Braas, Daniel The glioma amplified sequence 41 gene (GAS41) is a direct<br />
Myb target gene<br />
Braga, Vania Rho GTPases and cell-cell adhesion<br />
Brakebusch, Cord Cdc42 is not required for the formation <strong>of</strong> filopodia and<br />
lamellipodia, but crucial for the uptake <strong>of</strong> Listeria<br />
Brameshuber, Mario Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
Bramkamp, Marc Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC<br />
complex, a P-type ATPase, are not driven by ATP-induced<br />
conformational changes<br />
Bramkamp, Marc Membrane bound proteins <strong>of</strong> the division machinery in bacteria<br />
Brandenburg, Klaus The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Brands, Kerstin My<strong>of</strong>ibroblast differentiation and TGF-b1 signalling is affected<br />
in syndecan-4 deficient fibroblasts<br />
Brands, Kerstin Syndecan-4 Deficiency leads to an upregulation <strong>of</strong> syndecan-1<br />
in skin fibroblasts<br />
Brandt, Ulrich A yeast genetics approach to study mitochondrial complex I<br />
Braun#, Thomas Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Brecht, Andreas The SNAP-tagTM - a unique tool for covalent labeling and<br />
immobilization <strong>of</strong> proteins<br />
Breitkreutz, Dirk Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Breitkreutz, Dirk The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
Brem, Brigitte Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Bremm, Oliver Coupling <strong>of</strong> light-induced electron transfer to proton uptake in<br />
photosynthesis<br />
Brendel, C. The role <strong>of</strong> the lipoprotein receptor LRP1 in connective tissue<br />
factor (CTGF) induced signal transduction: Implications for the<br />
pathogenesis <strong>of</strong> atherosclerosis
Bretzel, Reinhard Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the<br />
Murine Embryonic Stem Cell System<br />
Briegel, Ariane Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Brigelius-Flohé, Regina Intracellular trafficking <strong>of</strong> the Interleukin-1 receptor (IL-1RI)<br />
associated kinase IRAK depends on its kinase-activity<br />
Britta, Engelhardt Mouse CD99 participates in T cell recruitment into inflamed<br />
skin<br />
Brodignon, Enrica EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the phototransducer<br />
NpHtrII<br />
Brucker, Sven Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Bruckner, Peter Human dermal basement membrane zone: Characterization <strong>of</strong><br />
functional suprastructures<br />
Bruckner, Peter Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Bruckner-Tuderman,<br />
Leena<br />
Human dermal basement membrane zone: Characterization <strong>of</strong><br />
functional suprastructures<br />
Bruehl, Anja Molecular characterization <strong>of</strong> rat calcium-activated chloride<br />
channels<br />
Bruhn, Heike The pore-forming mechanism <strong>of</strong> amoebapore A characterized<br />
by mutational analysis<br />
Brutlach, Henrik EPR investigation on site-directed spin labeled Tau protein<br />
Brüggemann,<br />
Henriette<br />
Towards the function <strong>of</strong> plant Rop GTPases in actin<br />
reorganisation<br />
Brüstle*, Oliver Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Bubikat, Alexander Local ANP prevents cardiac remodeling in hypertensive eNOSdeficient<br />
mice<br />
Bublitz, Renate Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Buchwalow, Igor B. Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Buchweitz, Olaf Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Buddenkotte, Jörg Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor<br />
in Skin Carcinogenesis<br />
Budisa, Nediljko Designing novel classes <strong>of</strong> fluorescent proteins with an<br />
expanded genetic code<br />
Budisa, Nediljko Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold”<br />
class <strong>of</strong> green fluorescent proteins<br />
Bui, C. C. Mechanical properties <strong>of</strong> myelinated and de-myelinated<br />
peripheral axons with the atomic force microscope<br />
Bunse, Christian A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Bunse, Christian Detection <strong>of</strong> phosphorylation sites using a hybrid triple<br />
quadrupole/linear ion trap mass spectrometer<br />
Burdach, Stefan The tumor specific chimeric transcription factor EWS-FLI1<br />
down-regulates gene expression<br />
Burdach, Stefan Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene<br />
expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma cells identifies new options<br />
for immunotherapy<br />
Burse, Antje Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in<br />
Leaf Beetle Larvae<br />
Butz, Stefan Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Butz, Stefan Mouse CD99 participates in T cell recruitment into inflamed<br />
skin<br />
Butz, Stefan Endothelial adhesion molecule ESAM binds directly to the<br />
multidomain adaptor MAGI-1 and recruits it to cell contacts<br />
Bürger, Elke The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate
Büssow, Konrad Insight into the structure <strong>of</strong> human proteins from a structural<br />
genomics approach<br />
Bäuml, Gerald Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the<br />
Saccharomyces cerevisiae cell adhesion molecule a-agglutinin<br />
Böcker, Matthias Scanning ion conductance microscope with shear-force control<br />
Böcker, Werner Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Böhm, Andreas Calculating theoretical ms spectra for given sequences<br />
Böhm, Andreas Automatic Synchronizing and self-Updating Protein Sequence<br />
Database Management System<br />
Böhm, Andreas A Clustering Solution for Distributed Data Analysis in Mass<br />
Spectrometry (paOla)<br />
Böhm, Maret The transformation supressor Protein Pdcd4 binds to RNA and<br />
is involved in the regulation <strong>of</strong> c-Jun activity.<br />
Böl, Gaby-Fleur Intracellular trafficking <strong>of</strong> the Interleukin-1 receptor (IL-1RI)<br />
associated kinase IRAK depends on its kinase-activity<br />
Carell, Thomas Crystal structure <strong>of</strong> DNA Photolyase complexed to double<br />
stranded DNA<br />
Cassata, Giuseppe Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Castell, Jose Vicente The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
Cavaille, Jerome Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
Cevikbas, Ferda My<strong>of</strong>ibroblast differentiation and TGF-b1 signalling is affected<br />
in syndecan-4 deficient fibroblasts<br />
Cevikbas, Ferda Syndecan-4 Deficiency leads to an upregulation <strong>of</strong> syndecan-1<br />
in skin fibroblasts<br />
Chacinska, Agnieszka Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
Chakrabarti, Partha Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Chakrabarti, Partha FTIR on the Rap-RapGAP reaction: GTPase activation without<br />
an arginine finger<br />
Chayka, Olesya Identification <strong>of</strong> MYB and C/EBP responsive enhancer <strong>of</strong> the<br />
chicken mim-1 gene<br />
Chen, Xiaoli Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong><br />
adipocytes<br />
Chervet, Jean-Pierre A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Chi, Lifeng Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters<br />
Chudakov, Dmitry Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Chwieralski, Caroline E. Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-<br />
2B bronchial epithelial cells depend on different signaling<br />
cascades<br />
Coles, Murray Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC<br />
complex, a P-type ATPase, are not driven by ATP-induced<br />
conformational changes<br />
Colonna, Raffaele Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Crocker, Paul What are the functions <strong>of</strong> siglecs on human monocytes and<br />
macrophages?<br />
Cullen, Paul Expression <strong>of</strong> collagens by human monocyte-derived<br />
macrophages<br />
Cullen, Paul TGF-β1 inducible genes in coronary smooth muscle cells<br />
Cumme, G.A. Turbo-mixing in microplates<br />
Cumme, Gerd Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Curth, Ute Analytical Ultracentrifugation pinpoints the interaction <strong>of</strong> DNA<br />
polymerase III and primase with EcoSSB to its C-terminal<br />
region
Cusan, Claudia Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Cushman, Samuel W. Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong><br />
adipocytes<br />
Cyrklaff, Marek Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Czaja, Rico New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Czarny, Malgorzata The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
Czernik, Marta Interrelation between rat prostate carcinoma cell coupling and<br />
motility in the determination <strong>of</strong> their invasiveness and<br />
metastatic activity<br />
Czuchra, Aleksandra Cdc42 is not required for the formation <strong>of</strong> filopodia and<br />
lamellipodia, but crucial for the uptake <strong>of</strong> Listeria<br />
Czudnochowski, Nadine Biochemical Characterization <strong>of</strong> the Interaction between Cyclin<br />
T1 and Hexim1 and its Competition with the HIV-1 Tat protein<br />
Czyz, Jaros•aw Interrelation between rat prostate carcinoma cell coupling and<br />
motility in the determination <strong>of</strong> their invasiveness and<br />
metastatic activity<br />
Czyz, Jaroslaw Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors<br />
Czyz, Jaroslaw Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Dagher, Marie Claire The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
Dankbar, N. Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding<br />
Forces between Single Molecules<br />
Daraban, Daniel The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
Dassler, Katrin Transferrin receptor expression and shedding during<br />
megakaryopoiesis<br />
Daum, Nicole The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
de Soto, Maria Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for<br />
host association are coordinated in S. meliloti<br />
de Wit, Elzo Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
De-Zolt, Silke Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
Deelder, André M. Analysis <strong>of</strong> protein glycosylation using normal-phase nanoscale<br />
liquid chromatography-mass spectrometry <strong>of</strong><br />
glycopeptides and oligosaccharides<br />
Dencher, Nobert A. The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Dencher, Norbert Identification <strong>of</strong> integral thylakoid membrane proteins from<br />
Chlamydomonas reinhardtii by peptide mass fingerprinting and<br />
MALDI-MS<br />
Dencher, Norbert A. H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
Dencher, Norbert A. Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Dencher, Norbert A. Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong><br />
Rattus norvegicus studied by peptide mass fingerprinting<br />
Depner, S<strong>of</strong>ia The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
Di Lisa, Fabio Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Diallo, Raihanatou Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Dieplinger, Hans Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Dietmar, Vestweber Mouse CD99 participates in T cell recruitment into inflamed<br />
skin
Dietz, Frank The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
Dijkwel, P. P. Quantitative proteomics for the investigation <strong>of</strong> leaf<br />
senescence in Arabidopsis thaliana<br />
Discher, Sabrina Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in<br />
Leaf Beetle Larvae<br />
Donato, Teresa The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
Dotti, Carlos Neuronal membrane cholesterol loss results in low plasmin<br />
activity yet higher beta-secretase cleavage <strong>of</strong> APP<br />
Doussiere, Jacques The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
Drbal, Karel Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
Drees, Anja ABC-Transporters at the blood-brain-barrier<br />
Drobinskaya, Irina Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the<br />
Murine Embryonic Stem Cell System<br />
Dryden, David DNA, Drugs and Proteins: Insights from the Atomic Force<br />
Microscope<br />
Du Pasquier, Louis Endothelial adhesion molecule ESAM binds directly to the<br />
multidomain adaptor MAGI-1 and recruits it to cell contacts<br />
Duché, Denis Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Dudek, Jan Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
Dudek, Jan The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner to<br />
Pam18<br />
Dunaev, Alexander Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Duschl, Claus Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Dvorsky, Radovan Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
Dvorsky, Radovan RhoA-p160Rock communication<br />
Dworniczak, Bernd GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Döker, Rolf Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Eble, Johannes A. Snake venom inhibitors <strong>of</strong> collagen- and laminin-binding<br />
integrins suppress tumor cell migration and invasion<br />
Ebnet, Klaus Endothelial adhesion molecule ESAM binds directly to the<br />
multidomain adaptor MAGI-1 and recruits it to cell contacts<br />
Ebnet, Klaus Junctional Adhesion Molecule-A (JAM-A) participates in tight<br />
junction formation and the establishment <strong>of</strong> cell polarity in<br />
epithelial cells<br />
Echtermeyer, Frank My<strong>of</strong>ibroblast differentiation and TGF-b1 signalling is affected<br />
in syndecan-4 deficient fibroblasts<br />
Echtermeyer, Frank Syndecan-4 Deficiency leads to an upregulation <strong>of</strong> syndecan-1<br />
in skin fibroblasts<br />
Edwardson, Michael DNA, Drugs and Proteins: Insights from the Atomic Force<br />
Microscope<br />
Efferth, Thomas Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Eggert, Angelika Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Ehrentreich-Förster, Eva Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Ehrentreich-Förster, Eva Synthetic and recombinant zinc fingers as a nano-addressable<br />
probe to doublestranded DNA structures<br />
Eichholz, Carolin Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Einsle, Oliver Structural Basis <strong>of</strong> Denitrification
Einspanier, Ralf Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia<br />
Virus (BLV) expression through Toll-like receptor 4<br />
Eisenblätter, Tanja Substrate and inhibitor specificity <strong>of</strong> the brain multidrug<br />
resistance protein (BMDP) at he blood-brain barrier<br />
Eisenblätter, Tanja ABC-Transporters at the blood-brain-barrier<br />
Eker, Andre P. M. Crystal structure <strong>of</strong> DNA Photolyase complexed to double<br />
stranded DNA<br />
El Gueddari, Nour<br />
Eddine<br />
Chitosan Activates Resistance Against Pathogens After<br />
eXposure (CARAPAX)<br />
Emberger, Werner Human mucosa-associated malignant melanomas: cytogenetic<br />
and molecular genetic characterization<br />
Endell, Jan Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Engelhard, Martin EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the phototransducer<br />
NpHtrII<br />
Engelhardt, Britta Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Epstein, Henry F. Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Eremenko, Valeriy Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Errington, Jeff Membrane bound proteins <strong>of</strong> the division machinery in bacteria<br />
Essen, Lars-Oliver Crystal structure <strong>of</strong> DNA Photolyase complexed to double<br />
stranded DNA<br />
Fabritz, Larissa Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Fabritz, Larissa Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide<br />
Fabritz, Larissa Local ANP prevents cardiac remodeling in hypertensive eNOSdeficient<br />
mice<br />
Fakler, Bernd Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type<br />
voltage gated potassium channels<br />
Fakler, Bernd Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Farkasovsky, Marian Nucleotide binding and filament assembly <strong>of</strong> recombinant<br />
yeast septin complexes.<br />
Fedotov, Alexandre Research <strong>of</strong> the mechanism <strong>of</strong> self-assembly fibronectins in<br />
modelling system.<br />
Feldhahn, Niklas The BCR-ABL1 kinase bypasses selection for the expression <strong>of</strong><br />
a pre-B cell receptor in pre-B acute lymphoblastic leukemia<br />
cells<br />
Feldhahn, Niklas Tracing the pre-B to immature B cell transition in human<br />
leukemia cells reveals a coordinated sequence <strong>of</strong> primary and<br />
secondary IGK gene rearrangement, IGK deletion and IGL<br />
gene rearrangement<br />
Feldhahn, Niklas Lineage infidelity in pre-B acute lymphoblastic leukemia cells<br />
carrying an MLL-AF4 gene rearrangement<br />
Feldmann, Heinz Endothelial activation and change <strong>of</strong> barrier function by soluble<br />
Ebola virus glycoproteins<br />
Ferguson-Smith *, Anne Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
C.<br />
Fernandes, Joel NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
interacts with the UvsBATR kinase<br />
Ferreira, Sergio Folding and structure <strong>of</strong> the amyloid precursor protein<br />
investigated by solution scattering and spectroscopy<br />
Fettig, Sebastian P1/HcPro: viral suppressors <strong>of</strong> gene silencing in wheat<br />
Fiebach, Julia Aut<strong>of</strong>luorescent Proteins in Interaction Analysis<br />
Fiegen, Dennis Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
Figueiredo, Luis Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Filipowicz, Witold RNAi and microRNA machineries in mammalian cells<br />
Fischer, Wolfgang Ion channels with minimalist design – from viruses.
Fitzgerald, Daniel New Baculovirus Expression Tools for Multiprotein Applications<br />
Flach, Carol The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C 17±palm )<br />
on the surface properties <strong>of</strong> lipid monolayers at the air/waterinterface<br />
Fleischer, Bernhard Regulation <strong>of</strong> T cell responses by BTLA: Functional analysis<br />
using a soluble BTLAIg fusion protein<br />
Floss, Thomas Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
Franken, Caroline Nod factor perception and transduction<br />
Franken, Sebastian Evidence for a cell surface receptor <strong>of</strong> HDGF<br />
Franz, Alexander Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Frazier, Ann E. Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
Frick, Wendelin Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-<br />
Talk to the Insulin Signaling Cascade by the Antidiabetic Drug<br />
Glimepiride<br />
Friedl, Peter Dynamic imaging <strong>of</strong> adhesion receptor and protease function in<br />
tumor cell invasion<br />
Friedl*, J. Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent<br />
Advances in Immunotherapy<br />
Frisen, Jonas Generation <strong>of</strong> neurons from stem cells in the adult brain<br />
Fromherz, Peter Cells 'n Chips or Ionics meets Electronics<br />
Fuchs, Harald Scanning Ion Conductance Microscopy - Topographical noncontact<br />
imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
Fuchs, Harald Scanning ion conductance microscope with shear-force control<br />
Fuchs, Hendrik Transferrin receptor expression and shedding during<br />
megakaryopoiesis<br />
Fuchs, Sabine Different populations <strong>of</strong> endothelial progenitor cells for<br />
applications in tissue engineering<br />
Fuchs, Sabine Endocytosis in an in vitro model <strong>of</strong> the human alveolar<br />
epithelial barrier: Uptake <strong>of</strong> GM-1 by human alveolar epithelial<br />
cells in primary culture<br />
Fuchs, Sabine Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-<br />
Well Screening <strong>of</strong> Barrier Properties<br />
Funari, Sergio S. The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Fusenig, Norbert E. Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Fusenig, Norbert E. The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
Futai, Masamitsu Vacuolar-type proton pump ATPases (V-ATPases) and inside<br />
acidic organelles/compartments: rotational mechanism and<br />
diverse physiological roles.<br />
Gabler, Christoph Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia<br />
Virus (BLV) expression through Toll-like receptor 4<br />
Gajula, Prasad Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled<br />
photosynthetic reaction centers from Rhodobacter sphaeroides<br />
based on molecular dynamics simulations and EPR<br />
spectroscopy.<br />
Galla, Hans-Joachim The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C17±palm )<br />
on the surface properties <strong>of</strong> lipid monolayers at the air/waterinterface<br />
Galla, Hans-Joachim Regulation <strong>of</strong> matrix metalloproteases and their endogenous<br />
inhibitors at the choroid plexus epithelium in vitro<br />
Galla, Hans-Joachim Substrate and inhibitor specificity <strong>of</strong> the brain multidrug<br />
resistance protein (BMDP) at he blood-brain barrier<br />
Galla, Hans-Joachim Neutrophile Granulocytes Transendothelial Migration in vitro<br />
Galla, Hans-Joachim GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Galla, Hans-Joachim Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters
Galla, Hans-Joachim The Influence <strong>of</strong> Membrane Fluidity on Vesicle Insertion in a SP-<br />
B or SP-C containing Monolayer<br />
Galla, Hans-Joachim Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong><br />
H- and E- FABP double knock-out mice<br />
Galla, Hans-Joachim The Influence <strong>of</strong> Cholesterol and POPE on mono- and<br />
multilayers conatining surfactant protein C<br />
Galla, Hans-Joachim ABC-Transporters at the blood-brain-barrier<br />
Galla, Hans-Joachim Bacterial Meningitis: Interactions at the Blood-CSF-Barrier<br />
Galla, Hans-Joachim Scanning Ion Conductance Microscopy - Topographical noncontact<br />
imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
Galla, Hans-Joachim Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Gamallo, Carlos PA2.26 antigen (T1alpha, podoplanin), a membrane mucin<br />
associated with cancer which is involved in epithelialmesenchymal<br />
transitions<br />
Garczarek, Florian The Proton Release Mechanism <strong>of</strong> Bacteriorhodopsin’s WT,<br />
E204D and E194D revealed with Time-Resolved Step-Scan and<br />
H/D-Exchange FTIR Measurements<br />
Gar<strong>of</strong>ano, Aurelio A yeast genetics approach to study mitochondrial complex I<br />
Gartenberg, M.R. Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
Gasser, Susan Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
Gast*, Peter Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled<br />
photosynthetic reaction centers from Rhodobacter sphaeroides<br />
based on molecular dynamics simulations and EPR<br />
spectroscopy.<br />
Gathmann, Sven Uncovering the mechanisms for protein sorting in<br />
cyanobacteria<br />
Gaub, Hermann Single Molecule Force Spectroscopy- A Bettter Understanding<br />
<strong>of</strong> Biomolecules with Newton?<br />
Gentile, Luca The Oocyte – Embryonic Stem Cell Cycle<br />
Georgopoulos*, Costa Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Gerke, R. Spatial Resolution <strong>of</strong> Gene Expression by Membrane<br />
Electroporation <strong>of</strong> CHO Cell Layers<br />
Gerke, Volker Rab3d and annexin A2 play a role in regulated secretion <strong>of</strong><br />
vWF but not tPA from human endothelial cells<br />
Gerke, Volker Biochemical Characterization <strong>of</strong> Annexin A9 and Identification<br />
<strong>of</strong> Interacting Proteins<br />
Gerke, Volker Characterization <strong>of</strong> the calcium-regulated S100P-ezrin<br />
interaction<br />
Gerl, M. Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Gerwert, Klaus Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Gerwert, Klaus The Proton Release Mechanism <strong>of</strong> Bacteriorhodopsin’s WT,<br />
E204D and E194D revealed with Time-Resolved Step-Scan and<br />
H/D-Exchange FTIR Measurements<br />
Gerwert, Klaus Coupling <strong>of</strong> light-induced electron transfer to proton uptake in<br />
photosynthesis<br />
Gerwert, Klaus α-β-Transition Studies In Proteins And Peptides With<br />
Timeresolved FTIR<br />
Gerwert, Klaus FTIR on the Rap-RapGAP reaction: GTPase activation without<br />
an arginine finger<br />
Geurts, Rene Nod factor perception and transduction<br />
Geyer, Matthias Biochemical Characterization <strong>of</strong> the Interaction between Cyclin<br />
T1 and Hexim1 and its Competition with the HIV-1 Tat protein<br />
Geyer, Peter Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Ghoneim*, M. Tharwat New insights into the modulatory role <strong>of</strong> serotonin reuptake<br />
inhibitors in myocardial ischemia reperfusion injury in the<br />
rabbit
Giaever*, Ivar Kinetics <strong>of</strong> cell spreading monitored by electric cell-substrate<br />
impedance sensing<br />
Giersch, Christoph The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Gieselmann, Volkmar Evidence for a cell surface receptor <strong>of</strong> HDGF<br />
Gilbert, Martin J. Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Gisselmann, Günter Novel ligand gated channels in D. melanogaster: GABA-gated<br />
cation channels and gating by multiple neurotransmitters<br />
Gisselmann, Günter Functional characterization <strong>of</strong> Ih-channel splice variants in<br />
insects<br />
Glockshuber, Rudi α-β-Transition Studies In Proteins And Peptides With<br />
Timeresolved FTIR<br />
Gnant*, M. Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent<br />
Advances in Immunotherapy<br />
Goebeler, Verena Biochemical Characterization <strong>of</strong> Annexin A9 and Identification<br />
<strong>of</strong> Interacting Proteins<br />
Goethe, Ralph Possible involvement <strong>of</strong> Ca2+/calmodulin dependent protein<br />
kinase II (CaMKII) in lysozyme pre-mRNA splicing<br />
Goldman, Gustavo NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
interacts with the UvsBATR kinase<br />
Goldman, Maria Helana NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
interacts with the UvsBATR kinase<br />
Gómez-Lechón, María<br />
Jose<br />
Goppelt-Strübe,<br />
Margarete<br />
The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
Alterations in the actin and microtubular network influence<br />
CTGF expression in endothelial cells<br />
Gornik, Sebastian Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong><br />
metalloproteinases<br />
Gorschlüter, Andreas Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding<br />
Forces between Single Molecules<br />
Goto, S. Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong><br />
Rattus norvegicus studied by peptide mass fingerprinting<br />
Grace, Bithiah A role for Rho A is indicated in migration and hematopoietic<br />
regeneration after bone marrow transplantation demonstrated<br />
by inactivation <strong>of</strong> small GTPases with clostridial toxins<br />
Graf, Thosten Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour<br />
therapy<br />
Grafström, Roland Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in<br />
human normal and transfromed oral keratinocytes<br />
Grafström, Roland C. Quantitative Genomics <strong>of</strong> Cultured Normal and transformed<br />
Human Oral Keratinocytes<br />
Grafström, Roland C. Formaldehyde-related toxicity and gene expression in cultured<br />
human oral keratinocytes<br />
Gralle, Matthias Folding and structure <strong>of</strong> the amyloid precursor protein<br />
investigated by solution scattering and spectroscopy<br />
Graness, Angela Alterations in the actin and microtubular network influence<br />
CTGF expression in endothelial cells<br />
Gratzl, Manfred Antibody to E-selectin inhibits SCLC cell adhesion to<br />
endothelial cells<br />
Greb, Robert The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Greb, Robert R. Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Greger, Harald Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Greil, Frauke Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
Grevelhörster, Astrid Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor<br />
in Skin Carcinogenesis
Grewer*, Christ<strong>of</strong> Glutamate transporter function in synaptic transmission<br />
Grgic, Ljuban A yeast genetics approach to study mitochondrial complex I<br />
Grigorev, Igor High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Gross, J. The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Gross, Johann Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat<br />
cochlea<br />
Gross, Ralph Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Grosse-Coosmann, Calculating theoretical ms spectra for given sequences<br />
Florian<br />
Groth, Georg Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and<br />
activation <strong>of</strong> F1-ATPase by tentoxin<br />
Grotti, A. Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
Grygar, Elisabeth In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Gräbe, Anja Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Grötzinger, Carsten Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Guiard, Bernard The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner to<br />
Pam18<br />
Gundelach, Holger The glioma amplified sequence 41 gene (GAS41) is a direct<br />
Myb target gene<br />
Gunnar, Jeschke Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP<br />
<strong>of</strong> E. coli: a protein chemical and EPR spectroscopic analysis<br />
Gutsche, Christ<strong>of</strong> Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing<br />
vesicles<br />
Gutzeit, Herwig O. Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Gäthje, Heiko The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
Gödecke, Axel Local ANP prevents cardiac remodeling in hypertensive eNOSdeficient<br />
mice<br />
Göring, Petra Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Götte, Martin The dermatan sulfate proteoglycans Decorin and Biglycan<br />
follow partially diverging endocytic routes<br />
Götte, Martin Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Götte, Martin Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Götte, Martin The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Göttig, Stephan A role for Rho A is indicated in migration and hematopoietic<br />
regeneration after bone marrow transplantation demonstrated<br />
by inactivation <strong>of</strong> small GTPases with clostridial toxins<br />
Haas, Bernd Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Haeusler, Lars-Christian Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
Hagedorn, Marco Misfolded TMV Coat Proteins: New Mutants and Pathogenicity<br />
in Plant and Animal Cells<br />
Hahn, Stephan A. Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Hahn, Uli New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Haier, Jörg Snake venom inhibitors <strong>of</strong> collagen- and laminin-binding<br />
integrins suppress tumor cell migration and invasion<br />
Hampp, Rüdiger Symbiotic interactions at the root level <strong>of</strong> forest trees: receive,<br />
deliver and get protected
Hanck, Theo Sequestration and recycling pathway <strong>of</strong> P2Y2 nucleotide<br />
receptor: Clathrin-and actin cytoskeleton-dependent receptor<br />
endocytosis, live cell visualization <strong>of</strong> green fluorescent protein<br />
tagged receptor<br />
Hanes, Jozef Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Hansen, Uwe Human dermal basement membrane zone: Characterization <strong>of</strong><br />
functional suprastructures<br />
Hansen, Uwe Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Hantschel, Oliver Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases<br />
Harjes, Stefan The structure <strong>of</strong> human 3'-phosphoadenosine-5'phosphosulfate<br />
synthetase 1 - A molecular pendulum?<br />
Hartl, Ulrich Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Hatt, Hanns Novel ligand gated channels in D. melanogaster: GABA-gated<br />
cation channels and gating by multiple neurotransmitters<br />
Hatt, Hanns Functional characterization <strong>of</strong> Ih-channel splice variants in<br />
insects<br />
Haufe, Günter Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters<br />
Haupt, Melina Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC<br />
complex, a P-type ATPase, are not driven by ATP-induced<br />
conformational changes<br />
Hauptmann, Steffen Detection <strong>of</strong> Retrotransposition in Cancer<br />
Hause, Bettina Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong><br />
tomato<br />
Hause, Gerd Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong><br />
tomato<br />
Hauss, Thomas The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Hauzman, Erik Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Hayer-Hartl, Manajit Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Hebeler, Romano Quantitative proteomics for the investigation <strong>of</strong> leaf<br />
senescence in Arabidopsis thaliana<br />
Hebestreit, Philipp Synergistic action <strong>of</strong> saponins with type I Ribosime-inactivating<br />
proteins agrostin and saporin<br />
Heidenreich, Pia Scanning ion conductance microscope with shear-force control<br />
Heidenreich, Pia M. Scanning Ion Conductance Microscopy - Topographical noncontact<br />
imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
Heinemann, Udo Insight into the structure <strong>of</strong> human proteins from a structural<br />
genomics approach<br />
Heise, Christian Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Heiser, Maria Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Helling, Stefan Differential analysis <strong>of</strong> T cell membrane proteins<br />
Hemsath, Lars Molecular basis <strong>of</strong> Cdc42 recognition by Wiskott-Aldrich<br />
Syndrome Proteins<br />
Henderson, Robert DNA, Drugs and Proteins: Insights from the Atomic Force<br />
Microscope<br />
Henker, Yvonne Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Henry, Margit Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Henschler, Reinhard A role for Rho A is indicated in migration and hematopoietic<br />
regeneration after bone marrow transplantation demonstrated<br />
by inactivation <strong>of</strong> small GTPases with clostridial toxins<br />
Hepper, Christina The PDZ-GEF Dizzy regulates cell form and cell adhesion via<br />
rap1 and integrins in macrophages <strong>of</strong> the Drosophila embryo.<br />
Herbrand, Ulrike Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase
Heredia, Alejandro Mechanical properties <strong>of</strong> myelinated and de-myelinated<br />
peripheral axons with the atomic force microscope<br />
Hermanns, Maria Iris Different populations <strong>of</strong> endothelial progenitor cells for<br />
applications in tissue engineering<br />
Hermanns, Maria Iris Endocytosis in an in vitro model <strong>of</strong> the human alveolar<br />
epithelial barrier: Uptake <strong>of</strong> GM-1 by human alveolar epithelial<br />
cells in primary culture<br />
Hermanns, Maria Iris Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-<br />
Well Screening <strong>of</strong> Barrier Properties<br />
Herter, Peter Nucleotide binding and filament assembly <strong>of</strong> recombinant<br />
yeast septin complexes.<br />
Heschel*, Ingo Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for<br />
Muscle Cell Differentiation in Culture and in Grafts<br />
Hescheler, Juergen Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the<br />
Murine Embryonic Stem Cell System<br />
Hescheler, Jürgen Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Hescheler, Jürgen Embryonic stem cells<br />
Hess, Sonja Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong><br />
adipocytes<br />
Heubach#, Jürgen F. Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Hilger, Daniel Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP<br />
<strong>of</strong> E. coli: a protein chemical and EPR spectroscopic analysis<br />
Hilger, Daniel Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Hillenkamp, Franz MALDI Mass Spectrometry <strong>of</strong> Nucleic Acids<br />
Hilterhaus, Lutz The Influence <strong>of</strong> Cholesterol and POPE on mono- and<br />
multilayers conatining surfactant protein C<br />
Hinderlich2, Stephan New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Hinterdorfer, Peter Topography and Recognition Imaging using AFM<br />
Hintze, Vera Procollagen C-proteinase: Substrate recognition by C-terminal<br />
domains<br />
Hintze, Vera Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition<br />
and Specificity<br />
Hippenstiel, Stefan Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Hirtreiter, Angela Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Hisabori, Toru Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and<br />
activation <strong>of</strong> F1-ATPase by tentoxin<br />
Hocke, Andreas C. Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Hoemme, Claudia Structural and functional analysis <strong>of</strong> the axon guidance<br />
molecule Sema3A and its receptor<br />
H<strong>of</strong>fmann, Werner Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-<br />
2B bronchial epithelial cells depend on different signaling<br />
cascades<br />
H<strong>of</strong>weber, Roland cell free studies on the function <strong>of</strong> cold shock proteins<br />
Hokke, Cornelis H. Analysis <strong>of</strong> protein glycosylation using normal-phase nanoscale<br />
liquid chromatography-mass spectrometry <strong>of</strong><br />
glycopeptides and oligosaccharides<br />
Holcombe, Lucy J. Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Hollnack, Evelyn Substrate and inhibitor specificity <strong>of</strong> the brain multidrug<br />
resistance protein (BMDP) at he blood-brain barrier<br />
Honjo, Tasuku Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Hoppe, Horst Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum
Hoppe, Thorsten Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Horn, A. Turbo-mixing in microplates<br />
Horn, Gudrun cell free studies on the function <strong>of</strong> cold shock proteins<br />
Horn, Gudrun Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the<br />
Saccharomyces cerevisiae cell adhesion molecule a-agglutinin<br />
Horst, Jürgen GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Horstmann*, Olaf Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Hucho, Ferdinand Intracellular domain - terra incognita <strong>of</strong> the nicotinic<br />
acetylcholine receptor<br />
Huelsmann, Sven The PDZ-GEF Dizzy regulates cell form and cell adhesion via<br />
rap1 and integrins in macrophages <strong>of</strong> the Drosophila embryo.<br />
Huff, Thomas Alterations in the actin and microtubular network influence<br />
CTGF expression in endothelial cells<br />
Hussein, Y. The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Hutagalung, Alex H. Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Hutter, Christoph The tumor specific chimeric transcription factor EWS-FLI1<br />
down-regulates gene expression<br />
Hutter, Christoph Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene<br />
expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma cells identifies new options<br />
for immunotherapy<br />
Hutter-Paier, Birgit In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Hutter-Paier, Birgit Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Hübner, Karin The Oocyte – Embryonic Stem Cell Cycle<br />
Hübner, Katrin Possible involvement <strong>of</strong> Ca2+/calmodulin dependent protein<br />
kinase II (CaMKII) in lysozyme pre-mRNA splicing<br />
Hüls, Christoph Differential analysis <strong>of</strong> T cell membrane proteins<br />
Höger, Harald In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Hörber, Heinrich Single molecule mechanics<br />
Höschler, Katja New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Höwel, Markus Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition<br />
and Specificity<br />
Höwel, Markus Procollagen C-proteinase: Substrate recognition by C-terminal<br />
domains<br />
Höög, Jan-Olov Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in<br />
human normal and transfromed oral keratinocytes<br />
Höög, Jan-Olov Formaldehyde-related toxicity and gene expression in cultured<br />
human oral keratinocytes<br />
Ikeda, W. Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and<br />
polarity<br />
Imh<strong>of</strong>, Beat A. The role <strong>of</strong> junctional adhesion molecule-C (JAM-C) in<br />
angiogenesis and inflammation<br />
Ingolic, E. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Ingolic, Elisabeth In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Irie, Kenji Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and<br />
polarity<br />
Jacobs, Thomas Regulation <strong>of</strong> T cell responses by BTLA: Functional analysis<br />
using a soluble BTLAIg fusion protein<br />
Jahn, Birgit Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Jansh<strong>of</strong>f, Andreas Functionalized vesicles as a model for cell adhesion studies
Jansh<strong>of</strong>f, Andreas Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Jaskiewicz, Lukasz RNAi and microRNA machineries in mammalian cells<br />
Jauch, Karl-Walter Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Jech, Marion In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Jeganathan², Sadasivam EPR investigation on site-directed spin labeled Tau protein<br />
Jenkinson, Joanna M. Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Jensen, Victoria Endothelial activation and change <strong>of</strong> barrier function by soluble<br />
Ebola virus glycoproteins<br />
Jerala, Roman The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Jerkovic, Lidia Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Jeschke, Gunnar Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Jockusch, Harald Misfolded TMV Coat Proteins: New Mutants and Pathogenicity<br />
in Plant and Animal Cells<br />
Jockusch, Harald Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for<br />
Muscle Cell Differentiation in Culture and in Grafts<br />
Jonuleit, Helmut Differential analysis <strong>of</strong> T cell membrane proteins<br />
Joos, Uta Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Joppich, Cornelia A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Jordan, Tina Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Joussen, Antonia The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Jung, Christian Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-<br />
Talk to the Insulin Signaling Cascade by the Antidiabetic Drug<br />
Glimepiride<br />
Jung, Heinrich Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP<br />
<strong>of</strong> E. coli: a protein chemical and EPR spectroscopic analysis<br />
Jung, Heinrich Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Jungmann, Pia Bradykinin enhances vesicular transport <strong>of</strong> solutes across<br />
endothelial cell layers<br />
Just, Ursula Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Kachel, Norman Intermediate states and implications for the species barrier <strong>of</strong><br />
the human prion protein revealed by high pressure NMR<br />
Kadathukalam, Jean The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
Kahms, Martin Aut<strong>of</strong>luorescent Proteins in Interaction Analysis<br />
Kakorin, Sergej Mechanism for the conductivity changes caused by membrane<br />
electroporation <strong>of</strong> CHO cell - pellets<br />
Kalbitzer, Hans Robert Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour<br />
therapy<br />
Kalbitzer, Hans Robert Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type<br />
voltage gated potassium channels<br />
Kalbitzer, Hans Robert cell free studies on the function <strong>of</strong> cold shock proteins<br />
Kalbitzer, Hans Robert Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the<br />
Saccharomyces cerevisiae cell adhesion molecule a-agglutinin<br />
Kalbitzer, Hans Robert Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Kalbitzer, Hans Robert Intermediate states and implications for the species barrier <strong>of</strong><br />
the human prion protein revealed by high pressure NMR
Kallenbach, Angela Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Kaller, Markus Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene<br />
silencing<br />
Kamal**, H. New insights into the modulatory role <strong>of</strong> serotonin reuptake<br />
inhibitors in myocardial ischemia reperfusion injury in the<br />
rabbit<br />
Kamp, Marcel Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Kania, Gabriela Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Kantola, Anna Promoter characterization <strong>of</strong> the latent transforming growth<br />
factor (TGF)-β binding protein 3 (LTBP-3)<br />
Karl, Monique Vinculin phospholipid interaction studied by EPR utilizing sitedirected<br />
spin labeling<br />
Kashani-Poor, Noushin A yeast genetics approach to study mitochondrial complex I<br />
Kaup, Matthias Transferrin receptor expression and shedding during<br />
megakaryopoiesis<br />
Keese*, Charles Kinetics <strong>of</strong> cell spreading monitored by electric cell-substrate<br />
impedance sensing<br />
Kehler, James The Oocyte – Embryonic Stem Cell Cycle<br />
Keil, Thomas Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Kelm, Sörge The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
Kerkh<strong>of</strong>f, Claus The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
Kern, Heidrun Rapid method for reproducible, shRNA-mediated gene silencing<br />
in vivo<br />
Kerner, Michael Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Kerscher, Stefan A yeast genetics approach to study mitochondrial complex I<br />
Kersting, Christian Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Keski-Oja, Jorma Promoter characterization <strong>of</strong> the latent transforming growth<br />
factor (TGF)-β binding protein 3 (LTBP-3)<br />
Kessler, Horst Inter-domain motions <strong>of</strong> the N-domain <strong>of</strong> the KdpFABC<br />
complex, a P-type ATPase, are not driven by ATP-induced<br />
conformational changes<br />
Key, Gundula Amoebasin, a chagasin-like cysteine proteinase inhibitor in<br />
trophozoites Entamoeba histolytica<br />
Kiesel, Ludwig Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Kiesel, Ludwig Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Kiesel, Ludwig The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Kikkert, Marjolein The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate<br />
Kilic, Ana Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Kind, Barbara Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Kindermann, Maik The SNAP-tagTM - a unique tool for covalent labeling and<br />
immobilization <strong>of</strong> proteins<br />
Kintscher, Jörg Identification <strong>of</strong> MYB and C/EBP responsive enhancer <strong>of</strong> the<br />
chicken mim-1 gene<br />
Kirkpatrick, Charles<br />
James<br />
Kirkpatrick, Charles<br />
James<br />
Endocytosis in an in vitro model <strong>of</strong> the human alveolar<br />
epithelial barrier: Uptake <strong>of</strong> GM-1 by human alveolar epithelial<br />
cells in primary culture<br />
Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-<br />
Well Screening <strong>of</strong> Barrier Properties
Kirpatrick, Charles James Different populations <strong>of</strong> endothelial progenitor cells for<br />
applications in tissue engineering<br />
Kiss, Tamas Nuclear organization <strong>of</strong> human modification guide RNA<br />
machinery<br />
Klar, Tobias Crystal structure <strong>of</strong> DNA Photolyase complexed to double<br />
stranded DNA<br />
Klare, Johann EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the phototransducer<br />
NpHtrII<br />
Klein, Florian The BCR-ABL1 kinase bypasses selection for the expression <strong>of</strong><br />
a pre-B cell receptor in pre-B acute lymphoblastic leukemia<br />
cells<br />
Klein, Florian Tracing the pre-B to immature B cell transition in human<br />
leukemia cells reveals a coordinated sequence <strong>of</strong> primary and<br />
secondary IGK gene rearrangement, IGK deletion and IGL<br />
gene rearrangement<br />
Klein, Florian Lineage infidelity in pre-B acute lymphoblastic leukemia cells<br />
carrying an MLL-AF4 gene rearrangement<br />
Klein-Scory, Susanne Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Klempnauer, Karl-Heinz The transformation supressor Protein Pdcd4 binds to RNA and<br />
is involved in the regulation <strong>of</strong> c-Jun activity.<br />
Klempnauer, Karl-Heinz The glioma amplified sequence 41 gene (GAS41) is a direct<br />
Myb target gene<br />
Klempnauer, Karl-Heinz The transcription factor C/EBP&beta triggers phosphorylation<br />
<strong>of</strong> the coactivator p300: A new mechanism <strong>of</strong> cross-talk<br />
between transcription factors and coactivators?<br />
Klempnauer, Karl-Heinz Identification <strong>of</strong> MYB and C/EBP responsive enhancer <strong>of</strong> the<br />
chicken mim-1 gene<br />
Klenz, Ute The Influence <strong>of</strong> Membrane Fluidity on Vesicle Insertion in a SP-<br />
B or SP-C containing Monolayer<br />
Kloep, Stephan Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Kloep, Stephan Mouse CD99 participates in T cell recruitment into inflamed<br />
skin<br />
Kloetzel, Peter-Michael The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate<br />
Klunker, Daniel Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Klämbt, C. The Kette/Abi/Sra-1 complex regulates actin dynamics by<br />
inhibting wave and activating wasp function in Drosophila<br />
Knaus, Hans-Günther Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Knoll *, M. Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding<br />
Forces between Single Molecules<br />
Knop, Markus Rab3d and annexin A2 play a role in regulated secretion <strong>of</strong><br />
vWF but not tPA from human endothelial cells<br />
Koch, Hans-Georg Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Koch, Michel H. J. The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Koch-Brandt, Claudia The role <strong>of</strong> the lipoprotein receptor LRP1 in connective tissue<br />
factor (CTGF) induced signal transduction: Implications for the<br />
pathogenesis <strong>of</strong> atherosclerosis<br />
Koeleman, Carolien A. M. Analysis <strong>of</strong> protein glycosylation using normal-phase nanoscale<br />
liquid chromatography-mass spectrometry <strong>of</strong><br />
glycopeptides and oligosaccharides<br />
Kogler-Voigt, Monika Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease<br />
Kohlh<strong>of</strong>, Hella Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Kolb, Fabrice RNAi and microRNA machineries in mammalian cells<br />
Koli, Katri Promoter characterization <strong>of</strong> the latent transforming growth<br />
factor (TGF)-β binding protein 3 (LTBP-3)
Kolossov, Eugen Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the<br />
Murine Embryonic Stem Cell System<br />
Koltzscher, Max Characterization <strong>of</strong> the calcium-regulated S100P-ezrin<br />
interaction<br />
Korjikova, Svetlana Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Kostelecky, Brenda Functional diversity and metal selectivity <strong>of</strong> proteins sharing<br />
the metallo-β-lactamase fold<br />
Kottwitz, Denise Intracellular domain - terra incognita <strong>of</strong> the nicotinic<br />
acetylcholine receptor<br />
Koziollek-Drechsler, The orphan receptor GCNF in neuronal differentiation<br />
Ingrid<br />
Krause, Frank Identification <strong>of</strong> integral thylakoid membrane proteins from<br />
Chlamydomonas reinhardtii by peptide mass fingerprinting and<br />
MALDI-MS<br />
Krause, Frank Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong><br />
Rattus norvegicus studied by peptide mass fingerprinting<br />
Krause, Frank H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
Krayl, Martin Structural properties <strong>of</strong> the substrate protein determine<br />
degradation by the mitochondrial AAA+ protease PIM1<br />
Krel**, Hans-Willi The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Kremer, Werner Intermediate states and implications for the species barrier <strong>of</strong><br />
the human prion protein revealed by high pressure NMR<br />
Kremer, Werner Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Kremer, Werner Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type<br />
voltage gated potassium channels<br />
Krenig, Bernhard A new developed biochemical process in purifikation<br />
Andreas<br />
Kresse, Hans The dermatan sulfate proteoglycans Decorin and Biglycan<br />
follow partially diverging endocytic routes<br />
Kreusch, Stefan Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Kreuzer, Oliver J. Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Krieger, Andreas Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Kriegsmann, Jana Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Kroehne, Volker Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for<br />
Muscle Cell Differentiation in Culture and in Grafts<br />
Kroneck, Peter MH Structural Basis <strong>of</strong> Denitrification<br />
Kronstein, Romy Association <strong>of</strong> Eps15 with beta-Catenin in endothelial cells: a<br />
novel mechanism how endothelial cells might regulate barrier<br />
function<br />
Kruip, Jochen Ycf3 from Synechocystis sp. PCC 6803: Localisation,<br />
Oligomerisation and Role in Photosystem I Biogenesis<br />
Kruse, Markus-N. Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide<br />
Kruse, Markus-N. The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Krüger, Bettina Alterations in the actin and microtubular network influence<br />
CTGF expression in endothelial cells<br />
Kucht, S. The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Kuerner, Julia Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Kuhlmann, Jürgen Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Kuhlmann, Jürgen Aut<strong>of</strong>luorescent Proteins in Interaction Analysis<br />
Kuhlmann, Jürgen Effects <strong>of</strong> an inhibitor <strong>of</strong> Acyl Protein Thioesterase 1 on Rasmediated<br />
signal transduction
Kuhlmann, Markus Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene<br />
silencing<br />
Kuhn, Jürgen Uptake <strong>of</strong> Plant Glycosides by Intestinal Membrane Proteins in<br />
Leaf Beetle Larvae<br />
Kuhn, Michaela Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Kuhn, Michaela Local ANP prevents cardiac remodeling in hypertensive eNOSdeficient<br />
mice<br />
Kuhn, Michaela Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide<br />
Kukhtina, Viktoria Intracellular domain - terra incognita <strong>of</strong> the nicotinic<br />
acetylcholine receptor<br />
Kumpugdee, Mont The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Kuriyan, John Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases<br />
Kurlemann, Nils Application <strong>of</strong> immobilized benzaldehyde lyase as catalyst in<br />
the continuous synthesis <strong>of</strong> a chiral 2-hydroxy ketone<br />
Kurz, Randy Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Kurz, Randy Active cardiomyocytes on collagen gels for biohybrid systems<br />
Kurz, Randy Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
Kuster, Niels Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors<br />
Kühlbrandt, Werner Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Kühn, Martin Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Küster, Wolfgang Mutants <strong>of</strong> the EcoRI Restriction Endonuclease Exhibit Changes<br />
<strong>of</strong> Specificity<br />
Küter-Luks, Birgit Rapid method for reproducible, shRNA-mediated gene silencing<br />
in vivo<br />
König, Burkhard Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour<br />
therapy<br />
König, Wilfried A. Isolation and functional expression <strong>of</strong> cDNAs encoding<br />
sesquiterpene synthases, including the enantiomeric (+)- and (-<br />
)-Germacrene D-Synthases from Solidago canadensis L.<br />
Kötting, Carsten Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Lahaye, Thomas Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Lamagna, Chrystelle The role <strong>of</strong> junctional adhesion molecule-C (JAM-C) in<br />
angiogenesis and inflammation<br />
Lammers, Michael Structural and biochemical characterisation <strong>of</strong> the Rho effector<br />
mDia<br />
Lange, Claudia Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
Laroche, T. Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
Larson, Roy NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
interacts with the UvsBATR kinase<br />
Laue, Michael Endocytosis in an in vitro model <strong>of</strong> the human alveolar<br />
epithelial barrier: Uptake <strong>of</strong> GM-1 by human alveolar epithelial<br />
cells in primary culture<br />
Lehmann, Friedericke The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
Leippe, Matthias The pore-forming mechanism <strong>of</strong> amoebapore A characterized<br />
by mutational analysis<br />
Leistner, E. The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Leistner, E. Observations on the erratic occurrence <strong>of</strong> maytansinoids in<br />
plants and microorganisms<br />
Lemaitre, Vincent Ion channels with minimalist design – from viruses.
Lepenies, Bernd Regulation <strong>of</strong> T cell responses by BTLA: Functional analysis<br />
using a soluble BTLAIg fusion protein<br />
Lepthien, Sandra Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold”<br />
class <strong>of</strong> green fluorescent proteins<br />
Lev, Dyakonov Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Lev, Ernst Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Li, X.-Y. Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth and degeneration<br />
Li, Yanfeng The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner to<br />
Pam18<br />
Lichtenauer, Monika Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Liese, Andreas Application <strong>of</strong> immobilized benzaldehyde lyase as catalyst in<br />
the continuous synthesis <strong>of</strong> a chiral 2-hydroxy ketone<br />
Lietz, Michael Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
Lill, Holger Regulation <strong>of</strong> single Motor Protein molecules: inhibtion and<br />
activation <strong>of</strong> F1-ATPase by tentoxin<br />
Limpens, Erik Nod factor perception and transduction<br />
Lin *, Shau-Ping Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
Lind, Maria Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
Linke, Christoph Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia<br />
Linn, Thomas Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the<br />
Murine Embryonic Stem Cell System<br />
Linser, Sebastian The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Lipps, Hans J. Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Loden, Martin Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
Lohner, Karl The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Lorkowski, Stefan Expression <strong>of</strong> collagens by human monocyte-derived<br />
macrophages<br />
Lorkowski, Stefan TGF-β1 inducible genes in coronary smooth muscle cells<br />
Lorusso, V. Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
Lottaz*, Daniel The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Lucic, Vladan Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Ludwig-Müller, Jutta Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Lukyanov, Konstantin Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Lukyanov, Sergey Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Lutter, Petra Differential analysis <strong>of</strong> T cell membrane proteins<br />
Machulik, Astrid Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat<br />
cochlea<br />
Madeja, Zbigniew Interrelation between rat prostate carcinoma cell coupling and<br />
motility in the determination <strong>of</strong> their invasiveness and<br />
metastatic activity<br />
Magin, Angela Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
Magin, Angela Feasibility <strong>of</strong> the Use <strong>of</strong> HUVEC for Autologous Co-Culture<br />
Expansion <strong>of</strong> HSC from Umbilical Cord Blood<br />
Mahmoud El-tahir, Heba Evidence for a cell surface receptor <strong>of</strong> HDGF<br />
Mak *, L.H. Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding<br />
Forces between Single Molecules
Malcharek, Stefan Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong><br />
H- and E- FABP double knock-out mice<br />
Malcharek, Stefan The Influence <strong>of</strong> Cholesterol and POPE on mono- and<br />
multilayers conatining surfactant protein C<br />
Mandelkow, E. Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth and degeneration<br />
Mandelkow, Eva-Maria Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth and degeneration<br />
Mandelkow², Eckhard EPR investigation on site-directed spin labeled Tau protein<br />
Mann, Matthias Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Marcus, Katrin Multi-dimensional chromatography <strong>of</strong> proteins<br />
Marcus, Katrin Detection <strong>of</strong> phosphorylation sites using a hybrid triple<br />
quadrupole/linear ion trap mass spectrometer<br />
Marcus, Katrin A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Marcus, Katrin Differential analysis <strong>of</strong> T cell membrane proteins<br />
Markert, A. The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Martin, Georges A Twin Study: Chimeras between Poly(A) Polymerase and CCA<br />
enzyme show unexpected activities<br />
Martín-Villar, Ester PA2.26 antigen (T1alpha, podoplanin), a membrane mucin<br />
associated with cancer which is involved in epithelialmesenchymal<br />
transitions<br />
Martinez de Tejada,<br />
Guillermo<br />
The dual role <strong>of</strong> antimicrobial peptides - antibiotic activity and<br />
endotoxin neutralization<br />
Masliah, E. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Masters, John R Cell line misrepresentation<br />
Matta**, M.M . New insights into the modulatory role <strong>of</strong> serotonin reuptake<br />
inhibitors in myocardial ischemia reperfusion injury in the<br />
rabbit<br />
Matter, Karl Signalling at tight junctions in the regulation <strong>of</strong> epithelial<br />
proliferation and differentiation<br />
Matz, Mikhail Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Mayer, Ulrike Snake venom inhibitors <strong>of</strong> collagen- and laminin-binding<br />
integrins suppress tumor cell migration and invasion<br />
Mazurek, Birgit Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat<br />
cochlea<br />
Medalia, Ohad Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Mees, Alexandra Crystal structure <strong>of</strong> DNA Photolyase complexed to double<br />
stranded DNA<br />
Melzer, Michael Proteome analysis <strong>of</strong> tobacco trichomes<br />
Melzig, Matthias F. Synergistic action <strong>of</strong> saponins with type I Ribosime-inactivating<br />
proteins agrostin and saporin<br />
Mendelsohn, Rich The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C17±palm )<br />
on the surface properties <strong>of</strong> lipid monolayers at the air/waterinterface<br />
Mertens, Melanie Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong><br />
metalloproteinases<br />
Meyer, Annett Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Meyer, H. E. Quantitative proteomics for the investigation <strong>of</strong> leaf<br />
senescence in Arabidopsis thaliana<br />
Meyer, Helmut E. Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Meyer, Helmut E. Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Meyer, Helmut E. Multi-dimensional chromatography <strong>of</strong> proteins
Meyer, Helmut E. A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Meyer, Helmut E. Detection <strong>of</strong> phosphorylation sites using a hybrid triple<br />
quadrupole/linear ion trap mass spectrometer<br />
Meyer, Helmut E. Differential analysis <strong>of</strong> T cell membrane proteins<br />
Meyer, Tanja Molecular properties <strong>of</strong> a new G6PD-like is<strong>of</strong>orm from<br />
Arabidopsis<br />
Meyer zu Tittingdorf,<br />
Jürgen<br />
Meyer zu Tittingdorf,<br />
Jürgen<br />
Meyer zu Tittingdorf,<br />
Jürgen<br />
Identification <strong>of</strong> integral thylakoid membrane proteins from<br />
Chlamydomonas reinhardtii by peptide mass fingerprinting and<br />
MALDI-MS<br />
H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Meyer-Klaucke, Wolfram Functional diversity and metal selectivity <strong>of</strong> proteins sharing<br />
the metallo-β-lactamase fold<br />
Miekus, Katarzyna Interrelation between rat prostate carcinoma cell coupling and<br />
motility in the determination <strong>of</strong> their invasiveness and<br />
metastatic activity<br />
Miersch, Otto Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong><br />
tomato<br />
Miller, Luitpold Antibody to E-selectin inhibits SCLC cell adhesion to<br />
endothelial cells<br />
Mim*, Carsten Glutamate transporter function in synaptic transmission<br />
Mir, Jose The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
Mirancea, Nick Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Misselwitz, Joachim Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Mitchell, Tim J. Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Mitre, E. Turbo-mixing in microplates<br />
Mock, Hans-Peter Proteome analysis <strong>of</strong> tobacco trichomes<br />
Moerschbacher, Bruno Chitosan Activates Resistance Against Pathogens After<br />
eXposure (CARAPAX)<br />
Moore, Thomas Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Mooster, Jana Tracing the pre-B to immature B cell transition in human<br />
leukemia cells reveals a coordinated sequence <strong>of</strong> primary and<br />
secondary IGK gene rearrangement, IGK deletion and IGL<br />
gene rearrangement<br />
Morisetti, A. Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
Moroder, Luis Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold”<br />
class <strong>of</strong> green fluorescent proteins<br />
Mueller, Monika C. M Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia<br />
Mueller, Uwe Insight into the structure <strong>of</strong> human proteins from a structural<br />
genomics approach<br />
Mulinacchi, Barbara Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold”<br />
class <strong>of</strong> green fluorescent proteins<br />
Mühlich, Susanne Alterations in the actin and microtubular network influence<br />
CTGF expression in endothelial cells<br />
Müller, Daniel J. Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Müller, Günter Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-<br />
Talk to the Insulin Signaling Cascade by the Antidiabetic Drug<br />
Glimepiride
Müller, Matthias Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing<br />
vesicles<br />
Müschen, Markus The BCR-ABL1 kinase bypasses selection for the expression <strong>of</strong><br />
a pre-B cell receptor in pre-B acute lymphoblastic leukemia<br />
cells<br />
Müschen, Markus Tracing the pre-B to immature B cell transition in human<br />
leukemia cells reveals a coordinated sequence <strong>of</strong> primary and<br />
secondary IGK gene rearrangement, IGK deletion and IGL<br />
gene rearrangement<br />
Müschen, Markus Lineage infidelity in pre-B acute lymphoblastic leukemia cells<br />
carrying an MLL-AF4 gene rearrangement<br />
Möbest, Dietrich A role for Rho A is indicated in migration and hematopoietic<br />
regeneration after bone marrow transplantation demonstrated<br />
by inactivation <strong>of</strong> small GTPases with clostridial toxins<br />
Möbius, Klaus High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Möbius, Klaus Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Möhrlen, Frank Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong><br />
metalloproteinases<br />
Mörl, Mario A Twin Study: Chimeras between Poly(A) Polymerase and CCA<br />
enzyme show unexpected activities<br />
Mörtelmaier, Manuel Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
Na Nakorn, Pariya The influence <strong>of</strong> truncated Surfactant-Proteins C (SP-C17±palm )<br />
on the surface properties <strong>of</strong> lipid monolayers at the air/waterinterface<br />
Nacken, Wolfgang The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
Nagar, Bhushan Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases<br />
Namba, Keiichi Self-assembly and switching <strong>of</strong> the bacterial flagellum<br />
Natalia, Savenko Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Natalia, Shevcova Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Natalia, Zinovieva Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Navarrete-Santos+,<br />
Anne<br />
Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Naylor, Dean Functional Characterization <strong>of</strong> group I and II chaperonins in<br />
the archaeon Methanosarcina mazei<br />
Nechyporuk-Zloy,<br />
Volodymyr<br />
Nechyporuk-Zloy,<br />
Volodymyr<br />
Endocytic internalization <strong>of</strong> potassium channels in migrating<br />
cells<br />
Cell membrane isolation for high resolution AFM imaging<br />
Nehls, Uwe Symbiotic interactions at the root level <strong>of</strong> forest trees: receive,<br />
deliver and get protected<br />
Nellen, Wolfgang Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene<br />
silencing<br />
Neumann, E. Spatial Resolution <strong>of</strong> Gene Expression by Membrane<br />
Electroporation <strong>of</strong> CHO Cell Layers<br />
Neumann, Eberhard Mechanism for the conductivity changes caused by membrane<br />
electroporation <strong>of</strong> CHO cell - pellets<br />
Neumann, Frank R. Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
Neumann, Ingo The tumor specific chimeric transcription factor EWS-FLI1<br />
down-regulates gene expression<br />
Neumann, Ingo Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene<br />
expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma cells identifies new options<br />
for immunotherapy<br />
Nickell, Stephan Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Niederle*, B. Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent<br />
Advances in Immunotherapy
Nikolov, Dimitar B. Structural and functional analysis <strong>of</strong> the axon guidance<br />
molecule Sema3A and its receptor<br />
Nikolova, Teodora Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors<br />
Nikova, Dessy Cell membrane isolation for high resolution AFM imaging<br />
Nikova, Dessy Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Nilsson, Jan Anders Quantitative Genomics <strong>of</strong> Cultured Normal and transformed<br />
Human Oral Keratinocytes<br />
Nilsson, Jan Anders Formaldehyde-related toxicity and gene expression in cultured<br />
human oral keratinocytes<br />
Nilsson, Jan-Anders Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in<br />
human normal and transfromed oral keratinocytes<br />
Nischt, Roswitha Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Nishikawa, Satomi Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Nishikawa, Shin-Ichi Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Noegel, Angelika.A Cyclase associated protein CAP in the regulation <strong>of</strong> the actin<br />
cytoskeleton and cell polarity in Dictyostelium<br />
Noll, Thomas Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
Noll, Thomas Feasibility <strong>of</strong> the Use <strong>of</strong> HUVEC for Autologous Co-Culture<br />
Expansion <strong>of</strong> HSC from Umbilical Cord Blood<br />
Nyamaa, Amarjagal Expression <strong>of</strong> prestin mRNA in the organotypic culture <strong>of</strong> rat<br />
cochlea<br />
Nöhammer, Christa Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease<br />
N´Guessan, Phillipe Dje Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Oberleithner, Hans Bradykinin enhances vesicular transport <strong>of</strong> solutes across<br />
endothelial cell layers<br />
Oberleithner, Hans Cell membrane isolation for high resolution AFM imaging<br />
Oberleithner, Hans Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Ogita, H. Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and<br />
polarity<br />
Oldendorf, Jens Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters<br />
Oliveira, Cristiano Folding and structure <strong>of</strong> the amyloid precursor protein<br />
investigated by solution scattering and spectroscopy<br />
Ollesch, Julian α-β-Transition Studies In Proteins And Peptides With<br />
Timeresolved FTIR<br />
Ovcharuk, Ivan Research <strong>of</strong> the mechanism <strong>of</strong> self-assembly fibronectins in<br />
modelling system.<br />
O´Connor, Enrique The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
Padan, Etana Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Pagano, Francesco Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Pal, Prajna Paramita Expansion <strong>of</strong> the genetic code enables design <strong>of</strong> a novel “gold”<br />
class <strong>of</strong> green fluorescent proteins<br />
Pannell, Lewis K. Quantitative proteomics <strong>of</strong> the secretory proteome <strong>of</strong><br />
adipocytes<br />
Papadopoulos, Martina Complex regulation <strong>of</strong> gene expression, photosynthesis and<br />
sugar levels by pathogen infection<br />
Partenheimer, Heike Surface Marker Pr<strong>of</strong>iling <strong>of</strong> WJC, MSC, SMC and HUVEC<br />
Paulsson, Mats Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Pennekamp, Petra GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules
Penzo, Daniele Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Pereverzev, Alexey Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Peter, Kristin Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Peters, Imke Mutants <strong>of</strong> the EcoRI Restriction Endonuclease Exhibit Changes<br />
<strong>of</strong> Specificity<br />
Peters, Kirsten Different populations <strong>of</strong> endothelial progenitor cells for<br />
applications in tissue engineering<br />
Peters, Thomas New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Petr, Klenovitsky Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Petri, Björn Mouse CD99 participates in T cell recruitment into inflamed<br />
skin<br />
Petri, Björn Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Petrin, Sergey Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Petronilli, Valeria Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Pfanner, Nikolaus Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
Pfanner, Nikolaus The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner to<br />
Pam18<br />
Pfeiffer, Kathy Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Pfeiffer, Mathias High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Pfragner, R. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Pfragner, Roswitha Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Pfragner, Roswitha Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease<br />
Pfragner, Roswitha Human mucosa-associated malignant melanomas: cytogenetic<br />
and molecular genetic characterization<br />
Pfragner, Roswitha In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Pfragner, Roswitha Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Pfragner, Roswitha Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent<br />
Advances in Immunotherapy<br />
Phi-van, Loc Possible involvement <strong>of</strong> Ca2+/calmodulin dependent protein<br />
kinase II (CaMKII) in lysozyme pre-mRNA splicing<br />
Philipp, D. Turbo-mixing in microplates<br />
Pilarczyk, Götz Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Pillai, Ramesh RNAi and microRNA machineries in mammalian cells<br />
Pimtong, Wittaya Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Pliquett, U. Spatial Resolution <strong>of</strong> Gene Expression by Membrane<br />
Electroporation <strong>of</strong> CHO Cell Layers<br />
Plitzko, Juergen Exploring the inner space <strong>of</strong> cells by cryoelectron tomography<br />
Plückthun, Andreas Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Poetsch, Ansgar Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase
Pohl, Peter Fluid transport through epithelial barriers: the importance <strong>of</strong><br />
ion channels and transporters<br />
Poncza, Brigitte In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Popova, Blaga Mechanisms <strong>of</strong> antisense RNA and RNAi mediated gene<br />
silencing<br />
Poremba, Christopher Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Porta, Sepp In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Postberg, Jan Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Prato, Maurizio Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Preisegger, Karl-Heinz Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease<br />
Prodöhl, Alexander C<strong>of</strong>actor Binding to Membrane Proteins - From the Two-Stage<br />
to a Three-Stage Model?<br />
Pulagam, Lakshmi Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Pullen, C. Observations on the erratic occurrence <strong>of</strong> maytansinoids in<br />
plants and microorganisms<br />
Pusch, Hermann Novel ligand gated channels in D. melanogaster: GABA-gated<br />
cation channels and gating by multiple neurotransmitters<br />
Pürstner, Peter Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Püschel, Andreas W. Structural and functional analysis <strong>of</strong> the axon guidance<br />
molecule Sema3A and its receptor<br />
Quintanilla, Miguel PA2.26 antigen (T1alpha, podoplanin), a membrane mucin<br />
associated with cancer which is involved in epithelialmesenchymal<br />
transitions<br />
Radacz, Yvonne Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Rammelt, Christiane A Twin Study: Chimeras between Poly(A) Polymerase and CCA<br />
enzyme show unexpected activities<br />
Ramponi, S. Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
Rapoport, Tom Structure and function <strong>of</strong> a protein-conducting channel<br />
Rattenholl, Anke Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor<br />
in Skin Carcinogenesis<br />
Rattmann, Ina Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong><br />
metalloproteinases<br />
Rauen, Thomas Glutamate transporter function in synaptic transmission<br />
Rausch, T. Turbo-mixing in microplates<br />
Rauterberg, Jürgen Expression <strong>of</strong> collagens by human monocyte-derived<br />
macrophages<br />
Rauterberg, Jürgen TGF-β1 inducible genes in coronary smooth muscle cells<br />
Regauer, Sigrid Human mucosa-associated malignant melanomas: cytogenetic<br />
and molecular genetic characterization<br />
Rehder, Daniela Junctional Adhesion Molecule-A (JAM-A) participates in tight<br />
junction formation and the establishment <strong>of</strong> cell polarity in<br />
epithelial cells<br />
Rehling, Peter Transport <strong>of</strong> nuclear encoded proteins across and into the<br />
inner mitochondrial membrane<br />
Rehling, Peter The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner to<br />
Pam18<br />
Reich, Olaf Human mucosa-associated malignant melanomas: cytogenetic<br />
and molecular genetic characterization<br />
Reich, Ziv Exploring Protein-Binding Mechanisms and Energy Landscapes<br />
by Single-Molecule Mechanical Unbinding Experiments<br />
Reifschneider, Nicole Tissue diversity <strong>of</strong> the mitochondrial membrane proteome <strong>of</strong><br />
Rattus norvegicus studied by peptide mass fingerprinting<br />
Reinwald, Erwin Adhesion <strong>of</strong> Trypanosoma congolense activates signalling<br />
pathways in endothelial cells
Reiser, Georg Sequestration and recycling pathway <strong>of</strong> P2Y2 nucleotide<br />
receptor: Clathrin-and actin cytoskeleton-dependent receptor<br />
endocytosis, live cell visualization <strong>of</strong> green fluorescent protein<br />
tagged receptor<br />
Reiss, Björn Functionalized vesicles as a model for cell adhesion studies<br />
Remy, Andre Coupling <strong>of</strong> light-induced electron transfer to proton uptake in<br />
photosynthesis<br />
Rescher, Ursula Biochemical Characterization <strong>of</strong> Annexin A9 and Identification<br />
<strong>of</strong> Interacting Proteins<br />
Reske-Kunz, Angelika B. Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene<br />
expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma cells identifies new options<br />
for immunotherapy<br />
Reuter, Rolf The PDZ-GEF Dizzy regulates cell form and cell adhesion via<br />
rap1 and integrins in macrophages <strong>of</strong> the Drosophila embryo.<br />
Reutter2, Werner New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Rexroth, Sascha Identification <strong>of</strong> integral thylakoid membrane proteins from<br />
Chlamydomonas reinhardtii by peptide mass fingerprinting and<br />
MALDI-MS<br />
Rexroth, Sascha Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Rexroth, Sascha H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
Rexroth, Sascha The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Rhode, Heidrun Turbo-mixing in microplates<br />
Rhode, Heidrun Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Richmond, Timothy New Baculovirus Expression Tools for Multiprotein Applications<br />
Rieber, Nikolaus Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Riekenberg, Sabine Amoebasin, a chagasin-like cysteine proteinase inhibitor in<br />
trophozoites Entamoeba histolytica<br />
Riethmüller, Christoph Bradykinin enhances vesicular transport <strong>of</strong> solutes across<br />
endothelial cell layers<br />
Rinner, Beate Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Risse, Siegfried Bacterial Lipopolysaccharide (LPS) activates Bovine Leukemia<br />
Virus (BLV) expression through Toll-like receptor 4<br />
Robitzki, Andrea Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Robitzki, Andrea A. Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
Robitzki, Andrea A. Active cardiomyocytes on collagen gels for biohybrid systems<br />
Rochlina, Lubov Live Monitoring <strong>of</strong> the Endoderm–like Cell Differentiation in the<br />
Murine Embryonic Stem Cell System<br />
Rockenstein, E. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Rohde, Volker Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Roitsch, Thomas Complex regulation <strong>of</strong> gene expression, photosynthesis and<br />
sugar levels by pathogen infection<br />
Rolke, Yvonne Signalling in early stages <strong>of</strong> pathogenic development <strong>of</strong><br />
Claviceps purpurea.<br />
Rolletschek,<br />
Alexandra<br />
Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Rolletschek, Alexandra Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors
Rolletschek, Alexandra Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Rommel, Christina Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Rosseau, Simone Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Rossi, Laura The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Rostovskaya, Mariya Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Rothermel, Andrée Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Rothermel, Andrée Active cardiomyocytes on collagen gels for biohybrid systems<br />
Rothermel, Andrée Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
Rotter, Christine Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for<br />
host association are coordinated in S. meliloti<br />
Rottner, Klemens Cdc42 is not required for the formation <strong>of</strong> filopodia and<br />
lamellipodia, but crucial for the uptake <strong>of</strong> Listeria<br />
Royo, Hélène Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
Ruhla, Stephan Detection <strong>of</strong> Retrotransposition in Cancer<br />
Ruiz, Patricia Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
Rutten, Twan Proteome analysis <strong>of</strong> tobacco trichomes<br />
Rüegg, Markus Neuromuscular diseases: Concepts and new approaches for<br />
therapies<br />
Rüffer, Markus Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Rüffer, Markus Active cardiomyocytes on collagen gels for biohybrid systems<br />
Rüffer, Markus Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
Römer, Patrick Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Röttgers, Karin Structural properties <strong>of</strong> the substrate protein determine<br />
degradation by the mitochondrial AAA+ protease PIM1<br />
Sabrane, Karim Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide<br />
Saenger, Wolfram New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Sailer, Claudia Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Sakisaka, T. Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and<br />
polarity<br />
Samochocki, Marek The orphan receptor GCNF in neuronal differentiation<br />
Sarang, Zsolt Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in<br />
human normal and transfromed oral keratinocytes<br />
Sattler, Ulrike The orphan receptor GCNF in neuronal differentiation<br />
Savchenkova, Irina Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Savitsky, Anton High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Savitsky, Anton Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Savoldi, Marcela NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
interacts with the UvsBATR kinase<br />
Schachtrup, Christian Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong><br />
H- and E- FABP double knock-out mice<br />
Schaffitzel, Christiane Telomeric Guanine Quadruplex DNA in Ciliate Nuclei<br />
Scharf, Birgit Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for<br />
host association are coordinated in S. meliloti<br />
Schauenstein, Konrad Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease
Scheffer, Jan Signalling in early stages <strong>of</strong> pathogenic development <strong>of</strong><br />
Claviceps purpurea.<br />
Scheidig, Axel The structure <strong>of</strong> human 3'-phosphoadenosine-5'phosphosulfate<br />
synthetase 1 - A molecular pendulum?<br />
Schillers, Hermann Cell membrane isolation for high resolution AFM imaging<br />
Schillers, Hermann Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Schilling, Oliver Functional diversity and metal selectivity <strong>of</strong> proteins sharing<br />
the metallo-β-lactamase fold<br />
Schlatter, Eberhard Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Schlesier, Bernhard Proteome analysis <strong>of</strong> tobacco trichomes<br />
Schlichting, Ralf The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Schlitt, Hans-Jürgen Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Schliwa, Manfred Portrait <strong>of</strong> a molecular motor<br />
Schlüsener, Daniela Ycf3 from Synechocystis sp. PCC 6803: Localisation,<br />
Oligomerisation and Role in Photosystem I Biogenesis<br />
Schmeck, Bernd Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Schmeer, M. Spatial Resolution <strong>of</strong> Gene Expression by Membrane<br />
Electroporation <strong>of</strong> CHO Cell Layers<br />
Schmeer, Marco Mechanism for the conductivity changes caused by membrane<br />
electroporation <strong>of</strong> CHO cell - pellets<br />
Schmidt, Cathrine Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Schmidt, Oliver Multi-dimensional chromatography <strong>of</strong> proteins<br />
Schmidt3, Richard R. New insights into the interaction <strong>of</strong> the key enzyme <strong>of</strong> sialic<br />
acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine<br />
kinase, with different ligands<br />
Schmiegel, Wolff Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Schmitt, Edgar Differential analysis <strong>of</strong> T cell membrane proteins<br />
Schmitt, Rüdiger Regulation <strong>of</strong> motility and expression <strong>of</strong> genes essential for<br />
host association are coordinated in S. meliloti<br />
Schmitt, Wolfgang D. Detection <strong>of</strong> Retrotransposition in Cancer<br />
Schneider, Dirk C<strong>of</strong>actor Binding to Membrane Proteins - From the Two-Stage<br />
to a Three-Stage Model?<br />
Schneider, Dirk Uncovering the mechanisms for protein sorting in<br />
cyanobacteria<br />
Schneider, Toni Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Schnittler, Hans Association <strong>of</strong> Eps15 with beta-Catenin in endothelial cells: a<br />
novel mechanism how endothelial cells might regulate barrier<br />
function<br />
Schnittler, Hans-J. Endothelial activation and change <strong>of</strong> barrier function by soluble<br />
Ebola virus glycoproteins<br />
Schnittler, Hans-Joachim Rac mediates shear stress induced increase in endothelial<br />
barrier-function<br />
Schnoor, Michael Expression <strong>of</strong> collagens by human monocyte-derived<br />
macrophages<br />
Schnoor, Michael TGF-β1 inducible genes in coronary smooth muscle cells<br />
Schnurra, Ingo Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-<br />
2B bronchial epithelial cells depend on different signaling<br />
cascades<br />
Schnütgen, Frank Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
Scholze, Henning Amoebasin, a chagasin-like cysteine proteinase inhibitor in<br />
trophozoites Entamoeba histolytica<br />
Scholze, Henning Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia
Scholzen, Thomas E Terminating the stress: proteolytic processing <strong>of</strong><br />
proopiomelanocortin-derived stress and anti-inflammatory<br />
hormones by dermal microvascular endothelial cell (EC)<br />
extracellular peptidases<br />
Schornack, Sebastian Pepper Bs3 and tomato Bs4 - distinct molecular principles for<br />
perception <strong>of</strong> highly-related Xanthomonas AvrBs3 and AvrBs4<br />
proteins<br />
Schramm, Alexander Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Schreiber, Ulrich Complex regulation <strong>of</strong> gene expression, photosynthesis and<br />
sugar levels by pathogen infection<br />
Schreier, Christina Tetramerization <strong>of</strong> the T1 Domain in Shaker and Shaw type<br />
voltage gated potassium channels<br />
Schreiner, E. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Schroeder, Timm Notch signalling in embryonic and adult hematopoietic stem<br />
cells<br />
Schrot, Sebastian Neutrophile Granulocytes Transendothelial Migration in vitro<br />
Schrot, Sebastian Scanning Ion Conductance Microscopy - Topographical noncontact<br />
imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
Schroten, Horst Bacterial Meningitis: Interactions at the Blood-CSF-Barrier<br />
Schroth, Corinna Aut<strong>of</strong>luorescent Proteins in Interaction Analysis<br />
Schuderer, Jürgen Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors<br />
Schulenborg, Thomas Detection <strong>of</strong> phosphorylation sites using a hybrid triple<br />
quadrupole/linear ion trap mass spectrometer<br />
Schulte, Antje Biochemical Characterization <strong>of</strong> the Interaction between Cyclin<br />
T1 and Hexim1 and its Competition with the HIV-1 Tat protein<br />
Schulte, Uwe Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Schulz, Andrea Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-<br />
Talk to the Insulin Signaling Cascade by the Antidiabetic Drug<br />
Glimepiride<br />
Schulze, Andrea The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate<br />
Schulze, M. Turbo-mixing in microplates<br />
Schulze, Margarete Separation and analysis <strong>of</strong> native human proteomes using<br />
parallel chromatography with microplates: Alport syndrome<br />
versus healthy serum<br />
Schulze Topph<strong>of</strong>f, Ulf Regulation <strong>of</strong> matrix metalloproteases and their endogenous<br />
inhibitors at the choroid plexus epithelium in vitro<br />
Schwab, Albrecht Endocytic internalization <strong>of</strong> potassium channels in migrating<br />
cells<br />
Schwab, Albrecht Cell membrane isolation for high resolution AFM imaging<br />
Schwabe, Tatjana M.<br />
E.<br />
Ycf3 from Synechocystis sp. PCC 6803: Localisation,<br />
Oligomerisation and Role in Photosystem I Biogenesis<br />
Schwach, Gert Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Schwamborn, Jens Structural and functional analysis <strong>of</strong> the axon guidance<br />
molecule Sema3A and its receptor<br />
Schwarte-Waldh<strong>of</strong>f,<br />
Irmgard<br />
Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Schwarzer, Wibke The insulin-receptor signaling complex in epidermal<br />
keratinocytes<br />
Schwaßmann, Helena H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
Schwenk, Frieder Rapid method for reproducible, shRNA-mediated gene silencing<br />
in vivo
Schwöppe, Christian Molecular properties <strong>of</strong> a new G6PD-like is<strong>of</strong>orm from<br />
Arabidopsis<br />
Schügner*, Frank Use <strong>of</strong> a Novel Collagen Matrix with Oriented Pore Structure for<br />
Muscle Cell Differentiation in Culture and in Grafts<br />
Schütz, Gerhard Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
Schäfer, Eva The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Schäfer, Heike Detection <strong>of</strong> phosphorylation sites using a hybrid triple<br />
quadrupole/linear ion trap mass spectrometer<br />
Schäfer, Heike A peptide pre-concentration approach for nano-HPLC to<br />
diminish memory effects<br />
Schäffer, Tilman Scanning ion conductance microscope with shear-force control<br />
Schäffer, Tilman Mechanical properties <strong>of</strong> myelinated and de-myelinated<br />
peripheral axons with the atomic force microscope<br />
Schäffer, Tilman E. Scanning Ion Conductance Microscopy - Topographical noncontact<br />
imaging <strong>of</strong> s<strong>of</strong>t surfaces<br />
Schöler, Hans The Oocyte – Embryonic Stem Cell Cycle<br />
Schönichen, André Biochemical Characterization <strong>of</strong> the Interaction between Cyclin<br />
T1 and Hexim1 and its Competition with the HIV-1 Tat protein<br />
Scorrano, Luca Arachidonic Acid Mediates Calcium-dependent Apoptosis<br />
through the Mitochondrial Pathway<br />
Scrima, Andrea TrmE, a GNBP involved in tRNA-modification<br />
Seebach, Jochen Endothelial activation and change <strong>of</strong> barrier function by soluble<br />
Ebola virus glycoproteins<br />
Seebach, Jochen Association <strong>of</strong> Eps15 with beta-Catenin in endothelial cells: a<br />
novel mechanism how endothelial cells might regulate barrier<br />
function<br />
Seebach, Jochen Rac mediates shear stress induced increase in endothelial<br />
barrier-function<br />
Seeger, Michael The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate<br />
Seelert, Holger Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Seelert, Holger Identification <strong>of</strong> integral thylakoid membrane proteins from<br />
Chlamydomonas reinhardtii by peptide mass fingerprinting and<br />
MALDI-MS<br />
Seelert, Holger The metabolic state <strong>of</strong> Chlamydomonas reinhardtii does not<br />
affect the stoichiometry <strong>of</strong> its chloroplast ATP synthase<br />
oligomer III<br />
Seelert, Holger H + -ATP synthase dimers in the chloroplast <strong>of</strong> Chlamydomonas<br />
reinhardtii<br />
Seeliger, Stephan Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor<br />
in Skin Carcinogenesis<br />
Seibler, Jost Rapid method for reproducible, shRNA-mediated gene silencing<br />
in vivo<br />
Seipp, T. Spatial Resolution <strong>of</strong> Gene Expression by Membrane<br />
Electroporation <strong>of</strong> CHO Cell Layers<br />
Seipp, Thomas Mechanism for the conductivity changes caused by membrane<br />
electroporation <strong>of</strong> CHO cell - pellets<br />
Seitz, Hervé Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
Sentner, Ulrich P1/HcPro: viral suppressors <strong>of</strong> gene silencing in wheat<br />
Serralta, Alfonso The immunusupresive drug FK506 prevents Fas-induced<br />
apoptosis and mitochondrial dysfunction in human<br />
hepatocytes.<br />
Shagin, Dmitry Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Sharifullina, Svetlana Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Shimada, Atsushi Structural and biochemical characterisation <strong>of</strong> the Rho effector<br />
mDia
Sibrowski, Walter A regulatory role <strong>of</strong> IgG2 in IgM-IgG immune complexes <strong>of</strong><br />
normal human plasma<br />
Sickmann, Albert Calculating theoretical ms spectra for given sequences<br />
Sickmann, Albert Automatic Synchronizing and self-Updating Protein Sequence<br />
Database Management System<br />
Sickmann, Albert A Clustering Solution for Distributed Data Analysis in Mass<br />
Spectrometry (paOla)<br />
Siegl, Veronika Could plant extracts <strong>of</strong>fer a new chemotherapy for medullary<br />
thyroid carcinoma?<br />
Sitek, Barbara Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Skryabin, Boris Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide<br />
Skryabin, Boris GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Slaidina, Maija The fish Myelin-associated Glycoprotein - occurrence <strong>of</strong> a new<br />
splice variant<br />
Smit, Patrick Nod factor perception and transduction<br />
Smyth, Neil Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Soanes, Darren M. Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Sodia, Sigrun Human mucosa-associated malignant melanomas: cytogenetic<br />
and molecular genetic characterization<br />
S<strong>of</strong>eu Feugaing, David<br />
Denis<br />
The dermatan sulfate proteoglycans Decorin and Biglycan<br />
follow partially diverging endocytic routes<br />
Sopalla, Claudia The arachidonic acid-binding protein S100A8/A9 promotes<br />
NADPH oxidase activation in intact cells<br />
Spener, Friedrich Influence <strong>of</strong> the surfactant synthesis in alveolar Typ II cells <strong>of</strong><br />
H- and E- FABP double knock-out mice<br />
Spillmann, Dorothe The heparan sulfate proteoglycan syndecan-1 is a novel<br />
regulator <strong>of</strong> leukocyte-endothelial cell adhesion and leukocyte<br />
transmigration<br />
Sprangers, Mieke Lineage infidelity in pre-B acute lymphoblastic leukemia cells<br />
carrying an MLL-AF4 gene rearrangement<br />
Sprangers, Mieke Tracing the pre-B to immature B cell transition in human<br />
leukemia cells reveals a coordinated sequence <strong>of</strong> primary and<br />
secondary IGK gene rearrangement, IGK deletion and IGL<br />
gene rearrangement<br />
Springer, Wolfdieter Regulation <strong>of</strong> the Myosin-Directed Chaperone UNC-45 by a<br />
Novel E3/E4-Multiubiquitylation Complex<br />
Spörner, Michael Conformational equilibrium <strong>of</strong> Ras as target for anti-tumour<br />
therapy<br />
Sroka, Jolanta Interrelation between rat prostate carcinoma cell coupling and<br />
motility in the determination <strong>of</strong> their invasiveness and<br />
metastatic activity<br />
Staab, Claudia A Quantitative Genomics <strong>of</strong> Cultured Normal and transformed<br />
Human Oral Keratinocytes<br />
Staab, Claudia A. Formaldehyde-related toxicity and gene expression in cultured<br />
human oral keratinocytes<br />
Staab, Claudia Alma Expression analysis <strong>of</strong> carbonyl-metabolising enzymes in<br />
human normal and transfromed oral keratinocytes<br />
Staebler, Annette Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Staege, Martin S. The tumor specific chimeric transcription factor EWS-FLI1<br />
down-regulates gene expression<br />
Staege, Martin S. Analysis <strong>of</strong> all-trans retinoic acid induced changes in gene<br />
expression pr<strong>of</strong>ile <strong>of</strong> neuroblastoma cells identifies new options<br />
for immunotherapy<br />
Stahl, Dorothea A regulatory role <strong>of</strong> IgG2 in IgM-IgG immune complexes <strong>of</strong><br />
normal human plasma
Standera, Sybille The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate<br />
Stark, Hans-Jürgen Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Steffens, Silke Biophysical investigation <strong>of</strong> fluorinated dihydroceramides and<br />
stearicacidethylesters<br />
Stege, Patricia Molecular basis <strong>of</strong> Cdc42 recognition by Wiskott-Aldrich<br />
Syndrome Proteins<br />
Stege, Patricia RhoA-p160Rock communication<br />
Steinem, Claudia Functionalized vesicles as a model for cell adhesion studies<br />
Steinem, Claudia Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Steiner, U.* The possible role <strong>of</strong> fungi in the accumulation <strong>of</strong> ergoline<br />
alkaloids in Ipomoea asarifolia (Convolvulaceae)<br />
Steinfarz*, Barbara Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Steinh<strong>of</strong>f, Heinz-Juergen Structural Aspects <strong>of</strong> Membrane Bound Colicin A<br />
Steinh<strong>of</strong>f, Heinz-Juergen Study <strong>of</strong> conformational dynamics <strong>of</strong> spin labeled<br />
photosynthetic reaction centers from Rhodobacter sphaeroides<br />
based on molecular dynamics simulations and EPR<br />
spectroscopy.<br />
Steinh<strong>of</strong>f, Heinz-Juergen EPR investigation on site-directed spin labeled Tau protein<br />
Steinh<strong>of</strong>f, Heinz-Jürgen Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP<br />
<strong>of</strong> E. coli: a protein chemical and EPR spectroscopic analysis<br />
Steinh<strong>of</strong>f, Heinz-Jürgen High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Steinh<strong>of</strong>f, Heinz-Jürgen Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Steinh<strong>of</strong>f, Heinz-Jürgen Vinculin phospholipid interaction studied by EPR utilizing sitedirected<br />
spin labeling<br />
Steinh<strong>of</strong>f, Heinz-<br />
Jürgen<br />
Steinh<strong>of</strong>f, Heinz-<br />
Jürgen<br />
Multi-frequency EPR reveals the conformational dynamics <strong>of</strong><br />
membrane proteins: colcin A and the sensory rhodopsintransducer<br />
complex<br />
EPR structure <strong>of</strong> the membrane proximal region <strong>of</strong> the phototransducer<br />
NpHtrII<br />
Steinh<strong>of</strong>f, Martin Proteinase-Activated Receptor-2 (PAR-2): A Tumor Suppressor<br />
in Skin Carcinogenesis<br />
Stenzel, Irene Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong><br />
tomato<br />
Stephan, Raiko The Kette/Abi/Sra-1 complex regulates actin dynamics by<br />
inhibting wave and activating wasp function in Drosophila<br />
Sterchi*, Erwin E. The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Stetefeld, Joerg Alternative mRNA-splicing in agrin- A tool for structural and<br />
functional diversity<br />
Steurer, Stefan The SNAP-tagTM - a unique tool for covalent labeling and<br />
immobilization <strong>of</strong> proteins<br />
Stift*, A. Culture <strong>of</strong> Human Medullary Throid Carcinoma - Recent<br />
Advances in Immunotherapy<br />
Stock, Christian Endocytic internalization <strong>of</strong> potassium channels in migrating<br />
cells<br />
Stock, Christian Cell membrane isolation for high resolution AFM imaging<br />
Stockinger, Hannes Single Molecule Microscopy for the study <strong>of</strong> Living T Cells<br />
Stolle, Katrin Expression <strong>of</strong> collagens by human monocyte-derived<br />
macrophages<br />
Stolle, Katrin TGF-β1 inducible genes in coronary smooth muscle cells<br />
Strenge, Karen What are the functions <strong>of</strong> siglecs on human monocytes and<br />
macrophages?
Stroeher, Ute Endothelial activation and change <strong>of</strong> barrier function by soluble<br />
Ebola virus glycoproteins<br />
Struhalla, Marc New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Sträter, Norbert New structural aspects for substrate specificity <strong>of</strong> RNase T1<br />
Stumpf, Astrid Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Stühler, Kai Identification <strong>of</strong> proteins differentially expressed upon<br />
neurotrophin receptor activation using DIGE and MALDI-MS<br />
Stühler, Kai Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Stöcker, Walter Procollagen C-proteinase: Substrate recognition by C-terminal<br />
domains<br />
Stöcker, Walter Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition<br />
and Specificity<br />
Stöcker, Walter The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Suetsugu, Shiro Differential functions <strong>of</strong> WAVE1 and 2 in cell migration and<br />
spreading.<br />
Sultana, Hameeda Cyclase associated protein CAP in the regulation <strong>of</strong> the actin<br />
cytoskeleton and cell polarity in Dictyostelium<br />
Sun-Wada, Ge-Hong Vacuolar-type proton pump ATPases (V-ATPases) and inside<br />
acidic organelles/compartments: rotational mechanism and<br />
diverse physiological roles.<br />
Sundermeier, C. Electro-Magnetic Biosensor for the Measurement <strong>of</strong> Binding<br />
Forces between Single Molecules<br />
Supanz, Sabine Detection <strong>of</strong> rare tumor cells in peripheral blood for early<br />
identification <strong>of</strong> recurrent disease<br />
Superti-Furga, Giulio Regulation <strong>of</strong> the c-Abl and Bcr–Abl Tyrosine Kinases<br />
Suter, Ueli Mechanical properties <strong>of</strong> myelinated and de-myelinated<br />
peripheral axons with the atomic force microscope<br />
Suttorp, Norbert Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Suveyzdis, Yan Mechanistic studies <strong>of</strong> the Ras-Superfamily by Time-Resolved<br />
FTIR-Spectroscopy<br />
Suveyzdis, Yan FTIR on the Rap-RapGAP reaction: GTPase activation without<br />
an arginine finger<br />
Sven Hammerschmidt,<br />
Sven<br />
Streptococcus pneumoniae induced caspase 6-deptendent<br />
apoptosis in lung epithelium<br />
Szkodowska, Anna Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia<br />
Taddei, A. Imaging functional chromosomes: tethers and dynamics in the<br />
yeast nucleus<br />
Takai, Yoshimi Molecular mechanisms <strong>of</strong> formation <strong>of</strong> cell junctions and<br />
polarity<br />
Takenawa, Tadaomi Differential functions <strong>of</strong> WAVE1 and 2 in cell migration and<br />
spreading.<br />
Talbot, Nicholas J. Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Tampe, Robert INs and OUTs <strong>of</strong> Antigens - The Transport Machinery TAP in<br />
the Cellular Immune System<br />
Tanner, Widmar Expression and purification <strong>of</strong> the binding subunit Aga2 <strong>of</strong> the<br />
Saccharomyces cerevisiae cell adhesion molecule a-agglutinin<br />
Tarkka, Mika Symbiotic interactions at the root level <strong>of</strong> forest trees: receive,<br />
deliver and get protected<br />
Tatjana, Galnbek Caryological characteristics <strong>of</strong> some interspecific hybryd<br />
anymal cell cultures<br />
Tausch, Kathrin Molecular analysis <strong>of</strong> the angiogenic status in endometriotic<br />
lesions and eutopic endometrium<br />
Tchupikova, Natalia Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Tenenbaum, Tobias Bacterial Meningitis: Interactions at the Blood-CSF-Barrier
Tennagels, Norbert Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-<br />
Talk to the Insulin Signaling Cascade by the Antidiabetic Drug<br />
Glimepiride<br />
Teplyashin, Alexander Characterization <strong>of</strong> human MSC-like cells isolated from bone<br />
marrow, adipose tissue, skin and placenta.<br />
Thasler, Wolfgang E Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Thies, E. Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth and degeneration<br />
Thim, Lars Synergistic motogenic effects <strong>of</strong> EGF and TFF2 on human BEAS-<br />
2B bronchial epithelial cells depend on different signaling<br />
cascades<br />
Thumfart, Jörg Oliver Identification and functional characterization <strong>of</strong> protein<br />
supercomplexes associated with Kv1.1 potassium channels<br />
Tiefenbacher, Astrid Antibody to E-selectin inhibits SCLC cell adhesion to<br />
endothelial cells<br />
Timm, T. Role <strong>of</strong> Tau, Tau kinases, and microtubule dynamics in neurite<br />
growth and degeneration<br />
Tio, Joke Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis<br />
Tokalov, Sergey V. Cellular target proteins <strong>of</strong> quercetin and flavonoid metabolism<br />
in human cells<br />
Tontsidou, Lambrini GGT-Cre mice for tissue-specific gene ablation in proximal<br />
kidney tubules<br />
Torriani, Iris Folding and structure <strong>of</strong> the amyloid precursor protein<br />
investigated by solution scattering and spectroscopy<br />
Trzewik, Jürgen Active cardiomyocytes on collagen gels for biohybrid systems<br />
Tschemmernegg, M. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Tudzynski, Paul Signalling in early stages <strong>of</strong> pathogenic development <strong>of</strong><br />
Claviceps purpurea.<br />
Tulapurkar, Mohan Sequestration and recycling pathway <strong>of</strong> P2Y2 nucleotide<br />
receptor: Clathrin-and actin cytoskeleton-dependent receptor<br />
endocytosis, live cell visualization <strong>of</strong> green fluorescent protein<br />
tagged receptor<br />
Tynes, Ron The SNAP-tagTM - a unique tool for covalent labeling and<br />
immobilization <strong>of</strong> proteins<br />
Umann, Berit Synthetic and recombinant zinc fingers as a nano-addressable<br />
probe to doublestranded DNA structures<br />
Umbach, Patrick Insight into the structure <strong>of</strong> human proteins from a structural<br />
genomics approach<br />
Unger, Ron Different populations <strong>of</strong> endothelial progenitor cells for<br />
applications in tissue engineering<br />
Unger, Ron Development <strong>of</strong> a Human Alveolo-Capillary Barrier In Vitro: 24-<br />
Well Screening <strong>of</strong> Barrier Properties<br />
Ungermann, Christian Protein palmitoylation during membrane fusion<br />
Urban, Christin Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
Urbanke, Claus Analytical Ultracentrifugation pinpoints the interaction <strong>of</strong> DNA<br />
polymerase III and primase with EcoSSB to its C-terminal<br />
region<br />
Vahrmann, Anke Structural characterization and localization <strong>of</strong> annexin E1 in<br />
trophozoites <strong>of</strong> Giardia lamblia<br />
van den Heuvel, B. Gene hunting and gene cloning in human OXPHOS deficiency<br />
Van Sloun, Petra Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
van Steensel, Bas Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
Velic, Ana Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Veltrup, Ines Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide
Veneault-Fourrey, Claire Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Verkhusha, Vladislav Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Vestweber, Dietmar Mouse CD99 is required for transendothelial migration <strong>of</strong> T<br />
cells<br />
Vestweber, Dietmar Endothelial adhesion molecule ESAM binds directly to the<br />
multidomain adaptor MAGI-1 and recruits it to cell contacts<br />
Vestweber, Dietmar Junctional Adhesion Molecule-A (JAM-A) participates in tight<br />
junction formation and the establishment <strong>of</strong> cell polarity in<br />
epithelial cells<br />
Vetter, Ingrid TrmE, a GNBP involved in tRNA-modification<br />
Vetter, Ingrid R. Alternative splicing <strong>of</strong> Rac1 generates Rac1b, a self-activating<br />
GTPase<br />
Vetter, Robin Effects <strong>of</strong> an inhibitor <strong>of</strong> Acyl Protein Thioesterase 1 on Rasmediated<br />
signal transduction<br />
Villone, Daniela Human dermal basement membrane zone: Characterization <strong>of</strong><br />
functional suprastructures<br />
Vogel, Klaus-Peter Vinculin phospholipid interaction studied by EPR utilizing sitedirected<br />
spin labeling<br />
Vogel, Maartje Regulatory networks revealed by genomic maps <strong>of</strong><br />
heterochromatin protein binding in flies and humans<br />
Vogel*, Andreas Functional diversity and metal selectivity <strong>of</strong> proteins sharing<br />
the metallo-β-lactamase fold<br />
Volkmer, Thomas C<strong>of</strong>actor Binding to Membrane Proteins - From the Two-Stage<br />
to a Three-Stage Model?<br />
Volmer, Martin W. Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
von Bergen², Martin EPR investigation on site-directed spin labeled Tau protein<br />
von Janowsky, Birgit Structural properties <strong>of</strong> the substrate protein determine<br />
degradation by the mitochondrial AAA+ protease PIM1<br />
von Melchner, Harald Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
von Nickisch-<br />
Rosenegk, Markus<br />
Synthetic and recombinant zinc fingers as a nano-addressable<br />
probe to doublestranded DNA structures<br />
von Schaewen, Antje Molecular properties <strong>of</strong> a new G6PD-like is<strong>of</strong>orm from<br />
Arabidopsis<br />
von Zeska Kress NpkA, a cdc2-related kinase from Aspergillus nidulans,<br />
Fagundes, Marcia interacts with the UvsBATR kinase<br />
Vonck, Janet Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Voos, Wolfgang Structural properties <strong>of</strong> the substrate protein determine<br />
degradation by the mitochondrial AAA+ protease PIM1<br />
Voos, Wolfgang The presequence translocase-associated protein import motor<br />
<strong>of</strong> mitochondria: Pam16 functions in an antagonistic manner to<br />
Pam18<br />
Voß, Beate Nucleotide binding and filament assembly <strong>of</strong> recombinant<br />
yeast septin complexes.<br />
Voß, Melanie Endothelium-mediated hypotensive and hypovolemic actions <strong>of</strong><br />
atrial natriuretic peptide<br />
Vultaggio, S. Influence <strong>of</strong> Paramagnetic Contrast Media on endothelial<br />
permeability and morphology: an in vitro model<br />
Wada, Yoh Vacuolar-type proton pump ATPases (V-ATPases) and inside<br />
acidic organelles/compartments: rotational mechanism and<br />
diverse physiological roles.<br />
Waelte, Mike Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Wagener, Raimund Distribution <strong>of</strong> Matrilin-1 in Articular Bovine Cartilage<br />
Wagner, Melanie Effects <strong>of</strong> an inhibitor <strong>of</strong> Acyl Protein Thioesterase 1 on Rasmediated<br />
signal transduction<br />
Wagner, Rolf Structural and biochemical characterisation <strong>of</strong> the Rho effector<br />
mDia<br />
Walter, Dorothée Uncovering the mechanisms for protein sorting in<br />
cyanobacteria
Wandzik, Krzyszt<strong>of</strong> Transferrin receptor expression and shedding during<br />
megakaryopoiesis<br />
Wang, Hui Lineage infidelity in pre-B acute lymphoblastic leukemia cells<br />
carrying an MLL-AF4 gene rearrangement<br />
Wang, Zheng Yi Functional genomics <strong>of</strong> plant infection by the rice blast fungus<br />
Magnaporthe grisea<br />
Waring, Michael DNA, Drugs and Proteins: Insights from the Atomic Force<br />
Microscope<br />
Warscheid, B. Quantitative proteomics for the investigation <strong>of</strong> leaf<br />
senescence in Arabidopsis thaliana<br />
Wasternack, Claus Jasmonate-mediated amplification <strong>of</strong> wound signalling <strong>of</strong><br />
tomato<br />
Watts, Anthony Ion channels with minimalist design – from viruses.<br />
Wegener, Christoph Structure and dynamics <strong>of</strong> the sodium/proline transporter PutP<br />
<strong>of</strong> E. coli: a protein chemical and EPR spectroscopic analysis<br />
Wegener, Christoph High-field EPR spectroscopy <strong>of</strong> site-directed soin labeled<br />
bacteriorhodopsin: A study <strong>of</strong> the polarity and the local pH in<br />
the Bacteriorhodopsins proton channel.<br />
Wegener, Christoph Functional dynamics <strong>of</strong> the Na + /H + antiporter NhaA <strong>of</strong> E. coli<br />
probed by EPR spectroscopy<br />
Wegener, Joachim Kinetics <strong>of</strong> cell spreading monitored by electric cell-substrate<br />
impedance sensing<br />
Wegener, Joachim Functionalized vesicles as a model for cell adhesion studies<br />
Wegener, Joachim Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Wegener*, Joachim Bradykinin enhances vesicular transport <strong>of</strong> solutes across<br />
endothelial cell layers<br />
Wegmann, Frank Endothelial adhesion molecule ESAM binds directly to the<br />
multidomain adaptor MAGI-1 and recruits it to cell contacts<br />
Wehrspohn, Ralf Probing transepithelial substrate permeation with sub-cellular<br />
lateral resolution.<br />
Weidenfeller, Christian Glutamate transporter function in synaptic transmission<br />
Weiergräber, Marco Signalling through the E-type voltage-gated calcium channel.<br />
Identification <strong>of</strong> interaciton partners.<br />
Weigel, Winnie Establishment <strong>of</strong> a novel histotypic mammalian 3D in vitro<br />
Weigel, Winnie Active cardiomyocytes on collagen gels for biohybrid systems<br />
Weigel, Winnie Living cells on a chip – the use <strong>of</strong> electric properties for the<br />
development <strong>of</strong> a cardiomyocyte based biosensor<br />
Weingarten, Petra Differential analysis <strong>of</strong> T cell membrane proteins<br />
Weise, Chris Intracellular domain - terra incognita <strong>of</strong> the nicotinic<br />
acetylcholine receptor<br />
Weiss, Thomas S Characterisation <strong>of</strong> non parenchymal cell fractions with hepatic<br />
progenitor cells from surgical resected human liver tissue<br />
Wenners-Epping, Kerstin Red blood cells used for diagnosis <strong>of</strong> cystic fibrosis<br />
Wermter, Carsten Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition<br />
and Specificity<br />
Wermter, Carsten Procollagen C-proteinase: Substrate recognition by C-terminal<br />
domains<br />
Werner, U. Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Westphal, Ines Chemically modified glass surfaces for growth <strong>of</strong> culture cells<br />
examined by light microscopy.<br />
Wetzel, Christian Functional characterization <strong>of</strong> Ih-channel splice variants in<br />
insects<br />
Wied, Susanne Redistribution <strong>of</strong> Signaling Proteins within Lipid Rafts and Cross-<br />
Talk to the Insulin Signaling Cascade by the Antidiabetic Drug<br />
Glimepiride<br />
Wiegand, Christiane Misfolded TMV Coat Proteins: New Mutants and Pathogenicity<br />
in Plant and Animal Cells<br />
Wiertz, Emmanuel The ubiquitin domain protein HERP is stabilised upon<br />
interaction with a component <strong>of</strong> the ubiquitin-proteasome<br />
system and promotes degradation <strong>of</strong> an ERAD substrate
Wiese, Cornelia Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Wiese, Cornelia Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Wikstrom, Marten Warburg's Atmungsferment: A molecular energy transducer<br />
Wilhelmi, Marianne Bradykinin enhances vesicular transport <strong>of</strong> solutes across<br />
endothelial cell layers<br />
Willumeit, Regine The Interaction <strong>of</strong> the Peptide NK-2 with Model Membranes:<br />
Insights by Neutron Diffraction and X-ray Scattering<br />
Windisch, M. Stably transfected rat neuronal cell lines expressing alpha-<br />
Synuclein GFP Fusion Proteins as an in vitro model <strong>of</strong><br />
Parkinson's Disease<br />
Windisch, Manfred Vitamin E-binding protein Afamin enhances viability <strong>of</strong> cortical<br />
chicken neurons in vitro<br />
Windisch, Manfred In vitro Characterization <strong>of</strong> two Pheochromocytoma Cell Lines -<br />
New Models for Neuroendocrine Research<br />
Wings, S. Observations on the erratic occurrence <strong>of</strong> maytansinoids in<br />
plants and microorganisms<br />
Wissler, Josef H. Physiologic Roles for Receptors for Advanced Glycosylation End<br />
Products [RAGE] <strong>of</strong> Endothelial Cells: RNA-, Redox- and<br />
Metalloregulated Non-Mitogenic Angio-Morphogenesis in Health<br />
and Disease.<br />
Witjes, Birte Amoebasin, a chagasin-like cysteine proteinase inhibitor in<br />
trophozoites Entamoeba histolytica<br />
Witte, Gregor Analytical Ultracentrifugation pinpoints the interaction <strong>of</strong> DNA<br />
polymerase III and primase with EcoSSB to its C-terminal<br />
region<br />
Wittingh<strong>of</strong>er, Alfred Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Wittingh<strong>of</strong>er, Alfred TrmE, a GNBP involved in tRNA-modification<br />
Wittingh<strong>of</strong>er, Alfred Towards the function <strong>of</strong> plant Rop GTPases in actin<br />
reorganisation<br />
Wittingh<strong>of</strong>er, Alfred FTIR on the Rap-RapGAP reaction: GTPase activation without<br />
an arginine finger<br />
Wittingh<strong>of</strong>er, Alfred Structural and biochemical characterisation <strong>of</strong> the Rho effector<br />
mDia<br />
Wittingh<strong>of</strong>er, Alfred Nucleotide binding and filament assembly <strong>of</strong> recombinant<br />
yeast septin complexes.<br />
Wobus, Anna M Electromagnetic fields affect transcript levels <strong>of</strong> regulatory and<br />
apoptosis-related genes in p53-deficient embryonic stem (ES)<br />
cells and ES-derived neural progenitors<br />
Wobus, Anna M. Stammzellforschung: Potenzial, Perspektiven und Probleme<br />
Wobus, Anna M. Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Wobus, Anna M. Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Woenne, Eva C. Nidogen-1 or -2 Promote Basement Membrane Formation in<br />
Skin -Type 3D-Cocultures<br />
Wojciak-Stothard, Beata Rac mediates shear stress induced increase in endothelial<br />
barrier-function<br />
Wu, Xunwei Cdc42 is not required for the formation <strong>of</strong> filopodia and<br />
lamellipodia, but crucial for the uptake <strong>of</strong> Listeria<br />
Wuhrer, Manfred Analysis <strong>of</strong> protein glycosylation using normal-phase nanoscale<br />
liquid chromatography-mass spectrometry <strong>of</strong><br />
glycopeptides and oligosaccharides<br />
Wurst, Wolfgang Genomewide targeting <strong>of</strong> secreted and transmembrane<br />
proteins using the secretory gene trap U3Ceo<br />
Wurzinger, Laurenz Antibody to E-selectin inhibits SCLC cell adhesion to<br />
endothelial cells<br />
Wülfing, Pia Role <strong>of</strong> the endothelin axis in breast cancer angiogenesis
Yamazaki, Daisuke Differential functions <strong>of</strong> WAVE1 and 2 in cell migration and<br />
spreading.<br />
Yanushevich, Yurii Novel fluorescent proteins: diversity, mutagenesis and<br />
applications<br />
Yiallouros, Irene Procollagen C-Proteinase (PCP, BMP1): Substrate Recognition<br />
and Specificity<br />
Yiallouros, Irene Ovastacin, a new member <strong>of</strong> the astacin family <strong>of</strong><br />
metalloproteinases<br />
Yiallouros, Irene Procollagen C-proteinase: Substrate recognition by C-terminal<br />
domains<br />
Yiallouros, Irene The human Metalloprotease Meprin - A Candidate for epithelial<br />
differentiation<br />
Young, Peter Mechanical properties <strong>of</strong> myelinated and de-myelinated<br />
peripheral axons with the atomic force microscope<br />
Youngson *, Neil Imprinted microRNA genes at the mouse Dlk1-GTl2 domain.<br />
Yu, Jinshu Properties <strong>of</strong> the proton translocating oligomer in chloroplast<br />
FOF1 ATP synthase<br />
Yurrita, Maria Marta PA2.26 antigen (T1alpha, podoplanin), a membrane mucin<br />
associated with cancer which is involved in epithelialmesenchymal<br />
transitions<br />
Yurukova, Sevdalina Inhibition <strong>of</strong> Na+-H+ exchange prevents hypertrophy in<br />
Guanylyl cyclase-A deficient mice<br />
Zahanich#, Ihor Generation <strong>of</strong> a multipotent nestin-expressing progenitor cell<br />
type from adult mouse and human intestinal epithelium<br />
Zahanich+, Ihor Nestin-positive progenitor cells generated from adult mouse<br />
intestinal epithelium differentiate into cells expressing neural,<br />
hepatic and pancreatic properties<br />
Zahn, Ralph Intermediate states and implications for the species barrier <strong>of</strong><br />
the human prion protein revealed by high pressure NMR<br />
Zapatka, Marc Tumor suppressor Smad4 mediates down-regulation <strong>of</strong> the antiadhesive<br />
invasion-promoting matricellular protein SPARC<br />
Zarse, Kim Development <strong>of</strong> Peptide Chips for Biomedical Applications<br />
Zechel, Christina The orphan receptor GCNF in neuronal differentiation<br />
Zeni, Patrick Regulation <strong>of</strong> matrix metalloproteases and their endogenous<br />
inhibitors at the choroid plexus epithelium in vitro<br />
Zetsche, Bernd Local ANP prevents cardiac remodeling in hypertensive eNOSdeficient<br />
mice<br />
Zhang, Haidi RNAi and microRNA machineries in mammalian cells<br />
Zhao, Xiaodong Solution structure <strong>of</strong> the Ran binding domain 2 from RanBP2<br />
and its interaction with Ran<br />
Ziegler, Wolfgang H. Vinculin phospholipid interaction studied by EPR utilizing sitedirected<br />
spin labeling<br />
Zobeley, Eva α-β-Transition Studies In Proteins And Peptides With<br />
Timeresolved FTIR<br />
Zschörnig, Olaf Annexin II induced fusion <strong>of</strong> polyphosphoinositide containing<br />
vesicles
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