Honorary President Giorgio Federici Università di ... - Sib Chieti 2012

Honorary President 

Giorgio Giorgio Federici Federici 

Università di Roma di Roma “Tor “Tor Vergata” Vergata” 

President 

Carmine Di Ilio Di Ilio 

Università di Chieti di Chieti “G. “G. d’Annunzio” “G. d’Annunzio” 

Scientific Committee 

Mario Mario Alberghina - Catania - Catania 

Cesare Cesare Balduini - Pavia - Pavia 

Francesco Bonomi Bonomi - Milano - Milano 

Federico Bussolino - Torino - Torino 

Filiberto Cimino Cimino - Napoli - Napoli 

Chiara Chiara Cini Cini - Roma - Roma 

Carmine Di Ilio Di Ilio - Ilio Chieti - Chieti 

Patrizia Patrizia Galletti Galletti - Napoli - Napoli 

Renato Renato Gennaro - Trieste - Trieste 

Giuseppe Paradies - Bari - Bari 

Fabiola Fabiola Sinigaglia - Novara - Novara 

Giancarlo Solaini Solaini - Bologna - Bologna 

1 

Organizing Committee 

Antonio Antonio Aceto Aceto - Chieti - Chieti 

Stefania Angelucci - Chieti - Chieti 

Tonino Tonino Bucciarelli - Chieti - Chieti 

Vincenzo De Laurenzi De Laurenzi - Chieti - Chieti 

Piero Piero Del Del Boccio Boccio - Chieti - Chieti 

Antonio Antonio Di Giulio Di Giulio - L’Aquila - L’Aquila 

Carmine Di Ilio Di Ilio - Ilio Chieti - Chieti 

Luca Luca Federici Federici - Chieti - Chieti 

Mauro Mauro Maccarrone - Teramo - Teramo 

Filippo Filippo Martini Martini - Chieti - Chieti 

Alfonso Alfonso Pennelli Pennelli - Chieti - Chieti 

Raffaele Petruzzelli - Chieti - Chieti 

Paolo Paolo Sacchetta - Chieti - Chieti

Wednesday 26 th September 

12:30 Registration 

15:00 Welcome 

15:30-16:30 Antonini Lecture 

Chairman: F. Cimino (Napoli) 

M. Brunori (Roma) 

Form and substance: morphogenesis of a protein 

16:30-19:00 Session 1 

Metabolism and regulatory functions of NAD+ 

Chairmen: A. De Flora (Genova) - G. Magni (Camerino) 

N. Raffaelli (Ancona) 

Novel components of bacterial NAD(P)+ metabolism as revealed through an 

integrated genomic approach 

M. Rizzi (Novara) 

Structural enzymology of NAD(P)+ homeostasis 

M.P. Coleman (Cambridge, UK) 

NAD+ metabolism and axon survival 

S. Bruzzone (Genova) 

NAD+ and NAD+ -converting enzymes in immune cell responses and inflammation 

D. Corda (Napoli) 

Cellular functions of the PARP-dependent Mono-ADP-ribosylation 

19:00 Welcome cocktail 

Thursday 27 th September 

9:00-11:00 Session 2 

Host Defense Peptides of Innate Immunity: from Physiology to Therapy 

Chairmen: R. Gennaro (Trieste) - D. Barra (Roma) 

J-M. Reichhart (Strasbourg, Fr) 

Antimicrobial Defense, the Drosophila paradigm.

M. Mangoni (Roma) 

A lesson from amphibian skin for the development of new anti-infective agents. 

A. Tossi (Trieste) 

Pro-rich antimicrobial peptides and their derivatives as anti-infective and 

bacterial cell penetrating agents. 

A. Pini (Siena) 

Preclinical development of a novel therapeutic peptide for bacterial infections 

11:00-11:30 Coffee Break 

11:30-13:30 Session 3 

Natural toxins: powerful tools for biochemical studies 

Chairmen: G.P. Rossini (Modena) - C. Montecucco (Padova) 

13:30-14:30 Lunch 

J-D. Troadec (Marseille-FR) 

Deciphering the Deoxynivalenol toxicity: a brain study 

C. Montecucco (Padova) 

Tetanus and Botulinum Neurotoxins and the Neuroexocytosis Nanomachine 

A. Cortelazzo (Siena) 

Contrasting aspects of snake venom toxins: deadly to humans 

and useful for the development of diagnostic tests 

G.P. Rossini (Modena) 

Microalgal toxins: potent biological agents and useful probes for 

the study of basic bio-molecular processes 

14:30-16:30 SIB groups meetings 

16:30-18:30 Poster session 1 

Friday 28 th September 

9:00-11:00 Session 4 

Role of Oxygen pressure in the control of cellular metabolism 

in physiology and pathology 

Chairmen: G. Solaini (Bologna) - P. Chiarugi (Firenze) 

M. Mazzone (Leuven-Be) 

Vessels, Oxygen & Oxidative Homeostasis

P. Chiarugi (Firenze) 

TUMOR-STROMA INTERPLAY: oxygen sensitive reciprocal METABOLIC 

REPROGRAMMING through LACTATE SHUTTLE 

G. Solaini (Bologna) 

Fuel substrates control the involvement of mitochondria in cell adaptation to hypoxia 

C. Gelfi (Milano) 

Hypoxia adaptation in humans and animal models: contribution of proteomics 


11:30-13:30 Session 5 

Biochemical mechanisms of cellular senescence 

Chairmen: C. Di Ilio (Chieti) - P. Galletti ( Napoli) 

13:30-14:30 Lunch 

F. D’Adda di Fagagna (Milano) 

Molecular mechanisms of cellular senescence 

R. Faraonio (Napoli) 

Cellular senescence and microRNAs 

P. Salomoni (London-UK) 

Role of the promyelocytic leukaemia protein in regulation 

of normal and neoplastic neural stem cells 

F. Cecconi (Roma) 

Autophagy in the control of cell survival and proliferation 

14:30-16:30 Session 6 

Cell Cycle engine and targeted-oriented therapy 

Chairmen: F. Della Ragione (Napoli) - G. Melino (Roma) 

G. Melino (Roma) 

Involvement of p73, a p53-family member, in metabolism and senescence 

M. Malumbres (Madrid-ES) 

Targeting mitosis for cancer therapy 

S. Campaner (Milano) 

Assessing therapeutic targets in a pre-clinical model of Myc-induced lymphoma 

A. Borriello (Napoli) 

Targeting cell cycle: novel insights into p27Kip1 and p57Kip2 metabolism

16:30-18:30 Poster session 2 

18:00-19:30 SIB assembly 

21:00 Social Dinner 

Saturday 29 th September 

9:00-11:00 Session 7 

Steroid receptors, more out than in 

Chairmen: F. Sinigaglia (Novara) - A. Migliaccio (Napoli) 

M. Marino (Roma) 

The diversity of estrogen receptor signaling influences estrogen effects 

on skeletal muscle cells 

A. Migliaccio (Napoli) 

Nuclear export of Estradiol Receptor α 

A. Bertoni (Novara) 

The platelet as a model system for the non-genomic actions of steroid receptors 

M. Maggiolini (Cosenza) 

G protein-coupled estrogen receptor (GPER/GPR30): a new player in estrogen signaling 


11:30-13:30 Session 8 

New insights in the hemostatic processes 

Chairmen: F. Bernardi (Ferrara) - F. Bussolino (Torino) 

A. Balduini (Pavia) 

Bone marrow environment and regulation of platelet production 

G. Serini (Torino) 

Regulation of integrin function by semaphorin receptor signaling 

R. De Cristofaro (Roma) 

Proteolytic regulation of adhesive multimeric proteins: 

ADAMTS13 and von Willebrand factor 

M. Pinotti (Ferrara) 

Aberrant mRNA splicing in coagulation factor deficiencies: 

from molecular mechanisms to RNA-based therapeutic approach 

13:30 Concluding remarks

Wednesday 26 th 

September

Antonini Lecture 

FORM AND SUBSTANCE: 

MORPHOGENESIS OF A PROTEIN 

Maurizio Brunori 

Dipartimento di Scienze Biochimiche, Sapienza-Università di Roma, Rome, Italy 

The epiphany of the form from the substance is the essence of the folding of a protein 

whose shape is a spontaneous manifestation of its sequence, in water. The protein 

folding problem is quite unique in molecular Biochemistry being complex enough to 

be challenging yet sufficiently defined to ask meaningful questions. 

Over the past three decades enormous progress has been made in spite of the huge 

diversity in sequences and structures being an obstacle in drawing general rules just 

by comparing the folding pathway of different proteins. Progress in understanding the 

folding mechanism came from the opportunity to characterize the transition state in 

going from denatured to native; and the relationships between sequence information 

and protein fold could be takled using small globular proteins. 

A combination of protein engineering, transient kinetics and MD simulations allows 

to decipher the structure of the transition state, which is unique in providing a clue 

to the minimal interactions necessary to trace a productive pathway. Working with 

protein families characterized by the same fold but subjected to extensive mutations, 

one can infer the role of specific residues and/or overall topology in dictating folding 

pathways; this will be illustrated by experiments on the PDZ domain family and their 

circularly permuted variants (1-3). On the other hand, the design of proteins with 

different topologies but nearly identical sequences allows one to unveil the role of the 

minimal number of amino acids necessary to dictate the native structure; experiments 

on the G A /G B heteromorphic pair will be presented to illustrate this approach (4-6). 

I guess that Professor Antonini would have been thrilled to be able to successfully 

exploit the effective power of his scientific pet, the stopped-flow, in assessing the 

crucial molecular interactions dictating the protein fold: from substance to form(s) via 

kinetics. 

1. Ivarsson, Y., Travaglini-Allocatelli, C., Brunori, M., Gianni, S. (2008) J. Biol. Chem. 283, 8954 

2. Ivarsson, Y., Travaglini-Allocatelli, C., Brunori, M., Gianni, S. (2009) J. Am. Chem. Soc. 131, 11727 

3. Gianni, S., Ivarsson, Y., De Simone, A., Travaglini-Allocatelli, C., Brunori, M., Vendruscolo, M., (2010) 

Nature Struct. Mol. Biol. 17, 1431 

4. Morrone, A., McCully, M.E., Bryan, P.N., Brunori, M., Daggett, V., Gianni, S., Travaglini-Allocatelli, C. (2011) 

J. Biol. Chem. 286, 3863 

5. Brunori, M., Gianni, S., Giri, R., Morrone, A., Travaglini-Allocatelli, C. (2012) Biochem. Soc. Trans. 40, 429. 

6. Giri, R., Morrone, A., Travaglini-Allocatelli, C., Jemth, P., Brunori, M., Gianni, S., (2012) Proc. Natl. Acad. 

Sci. USA. Published early edition. 

9

Wednesday 26 th 

September 

Session 1

Wednesday 26 th September Session 1 

Novel components of bacterial NAD(P) metabolism as 

revealed through an integrated genomic approach 

nadia raffaelli 

Dipartimento di Scienze Agrarie, Alimentari e Ambientali, 

Università Politecnica delle Marche, Ancona 

NAD(P) is an ancient and ubiquitous metabolite which besides acting as a redox 

cofactor for several dehydrogenases, is utilized in many metabolic and regulatory 

processes as a consumable co-substrate for the modification of proteins and nucleic 

acid. Maintaining the coenzyme’s homeostasis is therefore of paramount importance 

for the cell and it is pursued through a strict regulation of its biosynthesis. A peculiar 

feature of NAD biosynthesis is the existence of several different de novo, salvage, 

and recycling pathways, which can occur in different combinations depending on the 

organism and cell-type. In bacteria, this metabolic versatility reflects a substantial 

diversity of the NAD biosynthetic machinery in the various bacterial species, which 

parallels the variety of bacterial habitats and lifestyles; in mammals it correlates 

with a marked diversification of the coenzyme synthesis at tissue, cellular, and even 

subcellular level. In recent years the complexity of the NAD biosynthetic machinery 

has begun to be unraveled in the majority of eukaryotic and prokaryotic species 

using approaches that combine comparative genomic analysis, metabolic pathways 

reconstruction, and experimental characterization. Bacteria, in particular, offer a 

good opportunity for this kind of analysis, given their highly diversified metabolic 

capabilities, availability of large amounts of genome sequence data, and the presence 

of conserved operons that reveal functional interactions between genes involved in 

the same metabolic pathways. By using genome context analysis techniques, in 

particular clustering of functionally coupled genes on the prokaryotic chromosome, 

domain fusions, phylogenetic profiling, and analysis of conserved regulatory sites, we 

contributed to uncover new players in bacterial NAD biosynthesis. In particular, we 

were able to identify a novel transcriptional regulator family (NrtR), controlling various 

aspects of NAD biosynthesis in a broad range of bacterial species, as well as the longsought 

gene coding for NMN deamidase (PncC), a key enzyme in NAD salvaging, 

widely conserved among bacteria. 

The NrtR regulator was identified through an in-depth analysis of conserved NAD 

biosynthetic operons in hundreds bacterial species. The structural analysis of 

the predicted regulator revealed that it is composed of an N-terminal domain 

homologous to ADP ribose pyrophosphatase of the Nudix family and a C-terminal 

HTH-like domain likely involved in DNA binding. NrtR-binding sites were predicted 

12


in a variety of bacterial genomes, allowing in silico reconstruction of NrtR regulons 

that include genes involved in various aspects of NAD synthesis, like de novo and 

salvage pathways, niacin uptake, and PRPP synthesis. Experimental characterization 

of different members of the NrtR family revealed that NrtR acts as a transcriptional 

repressor and that binding of the NAD catabolite ADP ribose to the Nudix domain 

prevents DNA binding. In the proposed mechanism of transcriptional regulation, ADP 

ribose acts as the NrtR antirepressor: high ADP-ribose levels in the cell, which might 

be indicative of active NAD turnover, would signal the activation of NAD biosynthetic 

gene expression via inhibiting the repressor function of NrtR. 

NMN deamidase was originally described in Enterobacteria, but the corresponding 

gene eluded identification for over 30 years. A genomics-based reconstruction of 

NAD metabolism across hundreds bacterial species suggested that NMN deamidase 

reaction is the only possible way of nicotinamide salvage in some species. This 

prediction was verified via purification of native NMN deamidase from Shewanella 

oneidensis followed by the identification of the respective gene pncC. Enzymatic 

characterization of the PncC protein, as well as phenotype analysis of deletion 

mutants, confirmed its proposed physiological role in NAD salvaging, also allowing 

the identification of a novel amidohydrolase fold. 

In conclusion, the characterization of NrtR and PncC are examples of how comparative 

genomics has been exploited to assign functional roles to “unknown genes” (nrtR) 

and to identify “missing genes” (pncC) in the NAD biosynthetic pathway. 

This work was partly supported by the Italian Minister of Foreign Affairs, “Direzione Generale per la 

Promozione del Sistema Paese” 

13


Structural enzymology of NAD(P) homeostasis 

GaravaGlia Silvia, roSSi franca, rizzi Menico 

Dipartimento di Scienze del Farmaco, 

Università del Piemonte Orientale “A Avogadro”, 28100 Novara 

The structural analysis of all enzymes in a metabolic pathway is a prerequisite for 

answering fascinating questions such as those relating to the evolutionary relationships 

between enzymes within the same and related pathways. In this framework we are 

working on enzymes involved in NAD(P) homeostasis in human, mosquitoes and 

bacteria. NAD(P) biosynthesis and recycling show striking differences amongst 

living organisms. We carried out extensive structural characterization of enzymes 

in this pathway in different organisms including bacterial and human Nicotinic acid 

adenylyltransferase, bacterial NAD synthetase and bacterial NAD kinase. We also 

determined the crystal structure of Haemophilus influenzae NAD nucleotidase (NadN) 

and exploited the structural data to uncover a possible novel function of CD73, the 

human ortholog of bacterial NadN, in extracellular NMN processing. We are currently 

targeting human nicotinic acid phosphoribosyltransferase, a potential novel target in 

cancer, bacterial NMN deamidases and the recently identified enzymes involved in the 

repair of hydroxylated NAD in both prokaryotes and eukayotes. Along the kynurenine 

pathway of tryptophan degradation, i.e. the de novo NAD synthesis in eukaryotes, 

we solved the structures of human kynurenine aminotransferases I and II, A. gambiae 

3-hydroxykynurenine aminotransferase and human picolinic carboxylase. Overall, our 

investigations are used for the design of small molecules of potential medical interest 

and revealed fascinating evolutionary relationships. 

14


NAD metabolism and axon survival 

Michael coleMan 

Recent studies show that NAD metabolism is particularly important for axon survival. 

As axons are lost early in many neurodegenerative disorders it is important to 

understand the underlying mechanism. We use axon transection, which induces 

Wallerian degeneration of distal axons, as a model system to understand this 

mechanism. 

The slow Wallerian degeneration protein (WldS) is an aberrant protein that delays 

Wallerian degeneration by tenfold. Structure-function studies of this protein and 

related Nmnat isoforms indicate that Nmnat activity within axons is critical for axon 

survival. Of the three mammalian isoforms, only Nmnat2 has been confirmed to be an 

endogenous axonal protein. As it is a labile protein, axons are vulnerable to interruptions 

in anterograde delivery of Nmnat2, whether through injury, disease or reduced 

expression. Thus, axons in primary culture undergo Wallerian-like degeneration when 

Nmnat2 is knocked down, and peripheral nerve growth fails without Nmnat2. 

While Nmnat2 is delivered to axons on membrane-bound transport vesicles, we find 

that this is not its main site of action. Its limited protective capacity for injured axons 

is greatly enhanced by dissociation from these vesicles to a level similar to WldS, 

suggesting a cytosolic site of action. Intriguingly, Nampt, another protein that we 

find to be a critical determinant of axon survival, is also cytosolic. This proximity may 

be important for Nmnat2 to sequester the Nampt product, NMN, which accumulates 

in injured and explanted nerves and induces axon degeneration unless an enzyme 

activity is present to remove it. Thus, Nampt inhibition phenocopies WldS. The 

pathway relationship with other emerging regulators of axon survival such as Sarm1 

will be the key to identifying the most promising therapeutic targets for disorders of 

anterograde axonal transport. 

15


NAD + and NAD + - converting enzymes in immune cell 

responses and inflammation 

Bruzzone Santina 1 , Grozio aleSSia 1 , MaGnone Mirko 1 , Bauer inGa 2 , 

uccelli antonio 3 , Parenti Marco daniele 4 , del rio alBerto 4 , 

zocchi elena 1 , de flora antonio 1 and nencioni aleSSio 2 

1 Department of Experimental Medicine, Section of Biochemistry, 2 Department of 

Internal Medicine, and 3 Department of Neuroscience, Ophthalmology and Genetics, 

University of Genova, Italy; 4 Laboratory of Biochemoinformatics, Department of 

Experimental Pathology, Alma Mater Studiorum, University of Bologna, Italy. 

Intracellular NAD + levels ([NAD + ]i) regulate human T lymphocyte survival, cytokine 

secretion and response to antigenic stimuli, as NAD + depletion or repletion affect T cell 

function in opposite directions. 

Activated, but not resting, T lymphocytes undergo NAD + depletion upon FK866mediated 

Nicotinamide phosphoribosyltransferase inhibition, causing impaired 

proliferation, reduced cytokine production, and eventually cell death. In NAD + -depleted 

cells, impairment of Sirtuin 6 (SIRT6), a NAD + -dependent deacetylase, is responsible for 

defective cytokine release. 

NAD+-derived Ca 2+ -mobilizing second messengers, produced by CD38, play a pivotal 

role in T cell activation. Accordingly, FK866 decreases the mitogen-induced [Ca 2+ ]i 

rise in activated T lymphocytes and reduces the Ca 2+ content of Thapsigargin (TG)sensitive 

Ca 2+ stores. As a result of its NAD + -depleting action, FK866 strikingly reduces 

the neurological damage and the clinical manifestations of murine experimental 

autoimmune encephalomyelitis, a model of T-cell mediated autoimmune disease. 

Conversely, when NAD + levels are increased by supplementing T lymphocytes with 

NAD + precursors, the Ca 2+ content of TG-sensitive Ca 2+ stores and cell response to 

mitogens in terms of [Ca 2+ ]i elevation and functional activities are up-regulated. 

Also in pancreatic cancer cells, SIRT6 activity enhances the expression of inflammatory 

mediators by sequentially increasing intracellular levels of ADP-ribose, activating the 

TRPM2 Ca 2+ channel and increasing the [Ca 2+ ]i. Thus, SIRT6 inhibitors could reduce 

inflammation associated with pancreatic cancer. Using computational approaches, a 

database of compounds was screened to identify new SIRT6 inihibitors. Candidate 

molecules were tested using both in vitro fluorescence-based assay with the 

recombinant protein and cell-based assays. Some compounds showed an inhibition 

profile in the micromolar range concentration and might be used as a starting point for 

lead optimization purposes. 

SIRT6 inhibitors and NAD + -lowering molecules might prove effective therapeutic 

strategies against inflammation and T cell-mediated autoimmune diseases; conversely, 

SIRT6 activators and NAD + -replenishing pharmacologic interventions could potentiate 

T cell-mediated immune responses. 

16


Cellular functions of the PARP 

dependent Mono-ADP-ribosylation 

GriMaldi Giovanna 1 , Giuliana 1 , valente carMen 1 , 

turacchio GaBriele 1 , Pucci Piero 2,3 and corda daniela 1 

1 Institute of Protein Biochemistry, 

National Research Council, Via Pietro Castellino 111, 80131 Naples, Italy; 

2 Department of Chemical Sciences and CEINGE-Biotecnologie Avanzate, 

3 ‘Federico II’ University, Naples, Italy. 

Mono-ADP-ribosylation (mono-ADPR) is a reversible post-translational modification 

of proteins catalyzed by ADP-ribosyltransferases (ARTs). The physiological role of 

the mono-ADPR is now recognised in processes such as membrane traffic, immune 

response and signalling. While the bacterial ARTs (such as pertussis, diphtheria, 

clostridium toxins) have been known for long time, the list of eukaryotic enzymes 

and relative substrates is still incomplete. Some members of the poly-ADP-ribosylpolymerase 

(PARPs) family are predicted to possess ART activity based on the 

observed substitutions in the H-Y-E triad of the catalytic domain. Indeed, PARP10 and 

PARP16 have been reported to be mART and, in particular, PARP16 has been shown 

to ADP-ribosylate KaryopherinB1. 

In in vitroADP-ribosylation assays, we have observed that PARP12 has a pronounced 

activity, consistent with its predicted ART nature. We have now characterised the 

subcellular localisation, function and activity of PARP12 and shown that indeed it is 

a mono-ART. Immunofluorescence experiments in HeLa cells have indicated that this 

enzyme is localised at the Golgicomplex. Here PARP12 plays important roles, since 

it is involved in both the regulation of the early steps of the secretory pathway and 

in the maintenance of the Golgi complex structure. Based on this evidence, and with 

the aim of investigating the molecular mechanism involved, we used a proteomic 

approach and identified 25 intracellular substrates and interactors of PARP12. Among 

these, Rab1a, a Golgi-localised small GTPase, was further analysed since, similarly to 

PARP12, it controls transport from the endoplasmic reticulum to the Golgi complex, 

as well as the Golgi complex structure. Hence, PARP12 could play a role in membrane 

traffic through the ADP-ribosylation and regulation of Rab1a. In addition, clustering of 

the numerous PARP12 substrates suggests that PARP12 is involved in diverse cellular 

functions, including the regulation of small-GTPases, RNA control and membrane 

traffic. 

17

Thursday 27 th 

September 

Session 2

Thursday 27 th September Session 2 

Antimicrobial Defense, the Drosophila paradigm 

JM reichhart 

We have been working since 1985 on the innate immune system. The adaptative 

immune system with its antibodies, B and T cells arose only once during evolution, 

around 500 million years ago probably in the first vertebrates. This specific immune 

system acts in concert with the innate immune system in roughly 45000 vertebrate 

species as a defence against invading microorganisms. In all invertebrates, the 

defence mechanisms are purely innate. 

In Drosophila, an infection provokes the rapid synthesis of powerful antibiotic peptides 

by the fat body. As an example, the basal level of expression of the antifungal peptide 

DROSOMYCIN, is increased a thousand fold within 30 minutes of septic injury in 

larvae or adults. The control of this expression involves at least two pathways that, 

for sake of simplicity, I refer to as the TOLL and the IMD pathways. The paramount 

role of the TOLL and IMD pathways in the host defence of Drosophila is illustrated 

by experiments in which mutant flies are challenged with fungi or bacteria. In 

TOLL-deficient mutants, survival to fungal, but not to bacterial infection, is severely 

compromised. By contrast, IMD mutants are markedly affected by bacterial infections 

but resist fungi with a survival pattern similar to that of wild-type flies. 

In Vertebrates, recognition of microbes by the innate immune system takes place at 

the cellular level by a family of transmembrane receptors homolog to Drosophila Toll, 

namely the Toll like receptors (TLRs). In Drosophila however, these recognition events 

take place in the open circulatory system via soluble excreted recognition proteins 

like the peptidoglycan recognition proteins (PGRPs) and the glucan binding proteins 

(GNBPs). In turn, these recognition steps must be conveyed onto Toll by extracellular 

proteolytic signalling pathways. Upstream of the IMD pathway, other PGRPs are 

recognizing Gram-negative microbial determinants. We are now interested in how 

these two pathways are activated and regulated. 

20


A lesson from amphibian skin for the development 

of new anti-infective agents 

Maria luiSa ManGoni 

Department of Biochemical Sciences, University of Rome “La Sapienza” 

Piazzale Aldo Moro, 5-00185-Rome Italy 

Ribosomally-made antimicrobial peptides (AMPs) are key components of the innate 

immune response of all living organisms (1). Amphibian skin secretions represent 

one of the richest sources for such molecules, which are synthesized and stored 

within granules of holocrine-type glands and released upon stimulation (2). Recently, 

a particular attention has been devoted to the temporin family because of their unique 

properties: (i) they are among the smallest AMPs (10-16 residues) with the lowest 

number of cationic amino acids; ; (ii) some isoforms display a fast membranolytic 

effect against bacteria, yeasts and protozoa of Leishmania genus (2); (iii) some 

others can neutralize the toxic effect of the lipopolysaccharide (LPS), by inhibiting 

TNF-α release from LPS-activated macrophages; and (iv) they display among them 

a synergistic effect against Gram-negative bacteria to overcome microbial resistance 

imposed by the LPS-outer membrane (3,4); (v) the same temporin combinations can 

also synergize in the LPS-detoxification. 

Another interesting AMP from frog skin is the 1-18 fragment of esculentin-1b from 

Rana esculenta. It displays a rapid bactericidal effect against important nosocomial 

pathogens, such as Pseudomonas aeruginosa. Its in vivo antimicrobial activity and 

mode of action has been studied using the mini-host model of Caenorhabditis elegans 

(5). It can promote survival of Pseudomonas-infected nematodes and permeate the 

membrane of Pseudomonas cells inside the worm’s gut. Furthermore, it has been 

used to treat dairy cows affected by clinical mastitis giving promising results. 

Overall, these studies suggest frog-skin AMPs as attractive molecules to assist in the 

future design and manufacturing of new peptide-based drugs for treatment of lifethreatening 

microbial infections. 

1. Boman H.G. Annu. Rev. Immunol. 1995; 13:61-92 

2. Mangoni, ML et al. FASEB J. 2001; 15:1431-1432 

3. Mangoni ML et al. J. Biol Chem. 2008; 283:22907-22917 

4. Bhunia A et al. J. Biol Chem. 2011; 286:24394-24406 

5. Uccelletti D. et al. Antimicrob Agents Chemother. 2010; 54:3853-3860. 

21


Pro-rich antimicrobial peptides and their derivatives 

as anti-infective and bacterial cell penetrating agents. 

aleSSandro toSSi, Marco Scocchi, renato Gennaro 

Department of Life Sciences, Building Q - Lab 109, 

University of Trieste, Via Giorgieri 1, I-34127 Trieste 

The Pro-rich cathelicidin Bac7 is selectively active against some Gram-negative 

bacterial species. It acts without initial damage to bacterial membranes, and like 

other Pro-rich AMPs (PR-AMPs) from mammals and insects, its action requires outer 

membrane penetration, cytoplasmic membrane transport by a specific machinery 

(involving the protein SbmA in E.coli) and interaction with internal targets leading 

to bacterial inactivation. One or more of these processes requires stereoselective 

interactions as all-D Bac7 is much less active. This and other PR-AMPs are examples 

of cell penetrating peptides (CPP) for susceptible Gram-negative bacteria, with 

both intrinsic antimicrobial activity and potential capacity to internalize impermeant 

antibiotic cargo. The mechanism of action was studied using genetic and biochemical 

approaches, as well as specific bacterial deletion mutants, to characterize the 

transport machinery, intracellular targets, and role of the outer membrane barrier. 

Fluorescently labelled Bac7 fragments of different lengths allowed identifying the 

minimum sequence requirements for efficient internalization into bacteria and to 

correlate this with the minimum antimicrobial requirements. The essential role of the 

N-terminal arginine residues in mediating transport/and or cidal activity was probed 

by substituting these with natural or unnatural analogs with altered structural and 

physico-chemical features. Results indicate that Bac7 may be internalised using an 

integrated transport system involving inner and outer membrane components. A 

Bac7 fragment was capable of protecting animals against bacterial infections with 

remarkably low toxicity, although clearance was rapid. Formulations are being studied 

to extend the active lifetime. 

22


Preclinical development of a novel therapeutic peptide 

for bacterial infections 

aleSSandro Pini 

Department of Biotechnology, University of Siena, Via Fiorentina 1, 53100 Siena 

The antimicrobial peptide M33 derives from the random selection of a combinatorial 

library incubated with E. coli cells and a rational optimization phase that produced a 

peptide with typical features of antimicrobial peptides [1]. 

M33 peptide is currently synthesized in tetra-branched form, a structure that confers 

to peptides a high resistance to circulating peptidases, thus becoming particularly 

suitable for in vivo use. M33 peptide is predominantly active against Gram-negative 

bacteria with MICs comparable to many traditional antibacterial agents already used in 

the clinic. Its mechanism of action has been characterized for membrane interaction, 

pore formation, biofilm eradication, DNA binding and LPS neutralization. Neutralization 

of LPS, demonstrated as a reduction in TNF-α production by macrophages, is a 

crucial aspect because it suggests that in vivo the peptide is involved not only in the 

direct killing of bacteria but possibly also in the reduction of cytokines that generate 

inflammation. This aspect, along with the low MIC shown by M33 against clinical 

isolates of P. aeruginosa from patients with Cystic Fibrosis or sepsis, increases the 

interest of this molecule as a new drug for diseases where inflammation triggered by 

bacterial infection is a major element of pathology progression. 

The current preclinical development of peptide M33 consists of efficacy experiments 

in animal models of sepsis, pneumonia and skin infection. Here we present results 

where strong reduction of bacterial load in-vivo and high animal survival rates are 

obtained through M33 administration. These results, along with toxicity and PK 

studies, are in their final steps and M33 is running to start its clinical trials within the 

next future. 

1. Pini A, Falciani C, Mantengoli E, Bindi S, Brunetti J, Iozzi S, Rossolini GM and Bracci L (2010) A Novel 

Tetra-Branched Antimicrobial Peptide Which Neutralizes Bacterial Lipopoly-saccharide and Prevents 

Septic Shock In Vivo. FASEB J 24:1015-1022. 

23


September 

Session 3


Deciphering the Deoxynivalenol toxicity: a brain study 

Jean-deniS troadec 

Deoxynivalenol (DON), one of the most abundant trichothecenes found on cereals, 

has been implicated in mycotoxicoses in both humans and farm animals. Low doses 

toxicity is characterized by reduced weight gain, diminished nutritional efficiency 

and immunologic effects. The levels and patterns of human food commodities 

contamination justify that DON consumption constitutes a public health issue. 

Its stability during processing and cooking explains its widespread presence in 

human food. Despite the described modulation of feeding behavior induced by 

DON consumption, the data aiming to characterize this effect are limited and the 

mechanisms by which this toxin exerts its anorexigenic action remain largely unknown. 

We recently characterized DON intoxication by showing that the toxin concomitantly 

affects feeding behavior, body temperature and locomotor activity in mice after both 

per os and central administration. Using c-Fos expression mapping, we identified the 

neuronal structures activated in response to DON and observed that the pattern of 

neuronal populations activated by the toxin resembled those induced by inflammatory 

signals. By real-time PCR, we report the first evidences for a DON-induced central 

inflammation, attested by the strong up-regulation of IL-1α, IL6, TNF-α, COX-2 and 

mPGES-1 mRNA. On the other hand, we provide the first demonstration that DON 

reduced feeding behavior and modified satiation and satiety by interfering with 

central neuronal networks dedicated to food intake regulation i.e. POMC and NUCB2/ 

nesfatin-1 expressing neurons. 

Altogether, these results strongly suggest that during intoxication DON reaches the 

brain where it modifies anorexigenic balance. These results were obtained with an 

animal model using acute intoxication. Accordingly, extrapolation on human health 

should be made with caution. However, we believe these new results are sufficient to 

reconsider the impact of DON on human population chronically exposed to the toxin. 

26


Tetanus and Botulinum Neurotoxins and the 

Neuroexocytosis Nanomachine 

a. MeGiGhian 1 , M. zordan 2 , S. Pantano 3 , M. Scorzeto 1 , 

M. riGoni 1,4 , d. zannini 2 , o. roSSetto 1,4 , c. Montecucco 1,4 

1 Department of Biomedical Sciences and 2 Department of Biology, 

University of Padova, Italy, 3 Institut Pasteur de Montevideo, 

Uruguay and 4 CNR Institute for Neuroscience, Italy 

Tetanus and botulinum neurotoxins are the etiological agents of the neuroparalytic 

diseases known as tetanus and botulism, respectively. They cause a persistent 

inhibition of neurotransmitter release by displaying inside neurons a zinc-endopeptidase 

activity specific for either VAMP/synaptobrevin or SNAP-25 or syntaxin, which are 

the three proteins forming the SNARE complex, the essential component of the 

neuroexocytosis nanomachine. During my presentation I will illustrate experiments 

performed with botulinum A and E neurotoxin which indicate that multiple SNAP-25 

molecules participates in a single event of synaptic vesicle fusion with release of the 

neurotransmitter. On this basis and of molecular modelling, we have elaborated a 

radial model for the nanonomachine of neuroexocytosis which consists of 8 SNARE 

complexes interacting each other via the C-terminus of SNAP25 and the syntaxin 

segment 250-255 (Drosophila melanogaster numbering of syntaxin isoform 1). This 

model predicts that Arg206 of SNAP-25 and Asp253 of syntaxin form an ionic couple 

whose role appears to be that of registering the position of the individual SNARE 

complexes within the octameric supercomplex. 

To test this prediction we generated transgenic Drosophila lines which allowed the 

expression of the mutated SNARE along with the wild type protein and analyzed 

electrophysiologically the neuromuscular junction, which is the most tightly controlled 

synapse. The results obtained and the controls performed indicate that these two 

residues (D. melanogaster SNAP-25 Arg206 and Dm Syntaxin Asp253 are essential 

for neuroexocytosis. These data support a rosette-like radial assembly of several 

SNARE complexes which interact with each other via the ionic couple formed by 

these two residues. 

27


Contrasting aspects of snake venom toxins: 

deadly to humans 

and useful for the development of diagnostic tests 

cortelazzo aleSSio 

Department of Internal medicine, Endocrine-Metabolic Sciences and Biochemistry, 

University of Siena, Siena, Italy 

The first studies on snake venom date back to 1700 and were made by the Italian 

scientist Felice Fontana (1720-1805), who first discovered the coagulant effects of 

snake venom. He noted that following an injection of viper venom in the rabbit jugular 

vein, the blood of animal clot quickly and it followed the death. Nowadays, the snake 

bite poisoning is a medical and social problem of considerable importance, showing 

from 94.000 to 125.000 deaths per year [1]. 

Up to now the potential of snake venom has not yet been fully explored, it is also 

incomplete understanding of the proteome and its components devoid of enzymatic 

activity [2]. Snake venoms, in particular those of Viperidae and Elapidae, contain 

hundreds of important molecules, pharmacologically active, with low molecular weight, 

including histamine, polyamide, alkaloids, small peptides and other allergens. Snake 

venom is a complex mixture consisting mainly of proteins and peptides (representing 

about 60-80% of dry weight), belonging to several major families including enzymes 

(serine proteases, Zn 2+ -metalloproteases, L-amino oxidase, PLA 2 ), and proteins 

without enzymatic activity (disintegrins, lectin-type C, natriuretic peptides, ohanin, 

miotoxins, secretory proteins rich in cysteines, nerves and vascular endothelium 

growth factors), although, it should be noted that different poisons show a distinct 

profile distribution of these proteins [3]. Proteases affect platelet aggregation, blood 

coagulation, fibrinolysis, the complement system, blood pressure and nervous 

system. Mainly, the proteases involved in hemostasis are divided in metalloproteases 

and serine proteases. Both can serve as tools for studying the molecular details of 

the activation of specific factors involved in coagulation, the fibrinolytic process and 

treatment of various pathological conditions of hemostasis and thrombotic [4]. 

In addition to proteolytic enzymes, snake venom is rich in phosphatase, 

phosphodiesterase, acetylcholinesterase and oxidase, which are enzymes able 

to degrade nucleic acids and 5’ -nucleotidases [5]. Many snake venoms contain 

secretory PLA 2 , involved in digestion of prey, in the toxicity and danger of poisoning. 

The venom PLA 2 , are capable of inducing a wide variety of pathological effects, 

including neurotoxicity, myotoxicity, cardiotoxicity, cytotoxicity and necrosis [6]. In 

most cases, poisoning requires medical emergency and treatment of victims with 

28

antivenom that are often effective but, in some cases, needs to be supplemented with 

support for respiratory or renal failure. Animal-derived antivenoms have been used to 

treat snake envenomation for more than 100 years. Major technological advantages 

in the past 30 years have produced antivenoms that are highly purified and chemically 

modified to reduce the risk of acute hypersensitivity reactions. 

In many cases the problem is addressed with the use of therapies with traditional 

medicinal plants. Previous articles have reported that plants belonging to the 

Asteraceae, Flucourticaceae, Boraginaceae, Apocymacaea, Fabaceae and Musaceae 

families are able to counteract the lethal effect of snake venom. The components 

that can minimize or totally inhibit the effect of snake venom are steroids such as 

cholesterol, diterpenes, triterpenes and phenolic compounds [7]. Among the plants 

with medicinal antivenom properties we studied Mucuna pruriens (Fabaceae ), which 

is used in traditional Nigerian medicine. Our previous work demonstrated that the 

aqueous extract of Mucuna pruriens seeds is able to protect mice from the effects of 

Echis carinatus venom [8]. The research on snake venom can lead to give structure 

to folk medicine and also to enrich with new and important information the medical 

Biochemistry, Proteomics and in particular the Venomics. The search for possible 

solutions to the toxic effects of snake venom is a field of study and research is still 

evolving. It may be concluded that information is now available to establish that 

snake venom toxin may serve as a starting material for drug design to combat several 

pathophysiological problems. 

References 

1. Guerranti R., Cortelazzo A., Hope-Onyekwere N. S., Furlani E., Cerutti H., Puglia M., Bini L., Leoncini R. In 

vitro effects of Echis carinatus venom on the human plasma protome. Proteomics 2010; 10: 3712-3722. 

2. Georgieva D., Arni R. K., Betzel C. Proteome analysis of snake venom toxins: pharmacological insights. 

Expert Rev. Proteomics 2008; 5: 787-797. 

3. Calvete J. J., Sanz L., Angulo Y., Lomonte B., Gutiérrez J. M. Venoms, venomics, antivenomics. FEBS 

Lett. 2009; 583: 1736-1743. 

4. Cortelazzo A., Guerranti R., Bini L., Hope-Onyekwere N., Muzzi C., Leoncini R., Pagani R. Effects of 

snake venom proteases on human fibrinogen chains. Blood Transfus. 2010; Suppl. 3: 120-125. 

5. Russel F. E., Dart R. C. Toxic effect of animal toxin. Toxic agents. 1984; 22: 753-765. 

6. Valentin E., Lambeau G. Increasing molecular diversity of secreted phospholipase A2 and their receptors 

and binding proteins. Biochimica and Biophysica Acta 2000; 1488: 59-70. 

7. Soares A. M., Ticli F. K., Marcussi S., Lourenço M. V., Januário A. H., Sampaio S. V., Giglio J. R., Lomonte 

B., Pereira P. S. Medicinal plants with inhibitory properties against snake venoms. Curr. Med. Chem. 

2005;12 (22): 2625-2641. 

8. Guerranti R., Ogueli I. G., Bertocci E., Muzzi C., Aguiyi J. C., Cianti R., Armini A., Bini L., Leoncini R., 

Marinello E., Pagani R. Proteomic analysis of the pathophysiological process involved in the antisnake 

venom effect of Mucuna pruriens extract. Proteomics. 2008 Jan;8 (2): 402-412. 

29


Microalgal toxins: potent biological agents and useful 

probes for the study of basic bio-molecular processes 

roSSini Gian Paolo 

Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, 

Via G. Campi 287 

Some species of marine microalgae produce toxic secondary metabolites which can 

be classified in distinct chemical groups and represent sources of risks for human 

and animal health. Humans can become exposed to microalgal toxins primarily 

by consumption of contaminated seafood, resulting from feeding habits of marine 

organisms. The filter-feeding behavior of bivalve mollusks, in particular, can lead to 

accumulation of high levels of toxins in shellfish flesh, which may be eventually eaten 

by humans and lead to cases of poisoning. The toxicity of these components in human 

and animal systems has originally driven most investigations on their chemical and 

functional properties. Over the years, however, some features of these substances 

have attracted the attention of the scientific community. On the one hand, microalgal 

toxins are very potent compounds, showing effective concentrations in the 10 -11 -10 -9 

M range in cellular systems. On the other hand, the molecular targets of microalgal 

toxins characterized so far include key components of the machinery responsible for 

cellular functioning, such as ion pumps and channels, neurotransmitter receptors, as 

well as proteins involved in signal transduction and the control of cellular processes. 

The mechanistic features of microalgal toxins can explain most of their adverse 

effects in biological systems, and justify their notable potential as tools to study basic 

biological functions at a molecular level. In fact, microalgal toxins are being used in 

ongoing investigations whose scope may not be limited to toxicological issues posed 

by individual toxin groups, but rather refer to the clarification of mechanistic features 

of basic bio-molecular processes selectively targeted by individual toxins. 

This presentation is dedicated to the late Romano Viviani and Ernesto Fattorusso, 

whose studies gave pioneering contributions in the field of microalgal toxins. 

30

Friday 28 th 

September 

Session 4

Friday 28 th September Session 4 

Vessels, Oxygen & Oxidative Homeostasis 

M. Mazzone 

Despite the panoply of therapeutic options currently available in oncology, their 

success is limited by the abnormal tumor vasculature that impairs drug delivery. In 

addition, the efficacy of anti-cancer treatment is challenged by the severity of side 

effects. PHD2 is an oxygen and redox sensitive enzyme that regulates the stability 

of the hypoxia-inducible transcription factors (HIFs) and thereby induces cellular 

adaptations to stress conditions. Here I will show that reduced activity of PHD2 in 

endothelial cells normalizes the tumor vasculature and thus optimizes perfusion. 

Tumor vessel normalization in Phd2 haplodeficient (Phd2 +/- ) mice increases the 

delivery of chemotherapeutic drugs to the tumor and hence their anti-tumor and 

anti-metastatic effects. Similar results were also elicited by acute deletion of one or 

twoPhd2 alleles in the tumor stroma, regardless of simultaneous Phd2 targeting in 

tumor cells. In response to chemotherapy-induced oxidative burst, the deletion of 

one or two Phd2 alleles in healthy organs reinforces a HIF-mediated detoxification 

program that is independent of hypoxia. This ultimately prevents oxidative damage, 

organ failure, and tissue demise. Altogether, our study discloses novel perspectives 

for the optimization of chemotherapy. 

32


TUMOR-STROMA INTERPLAY: OxYGEN SENSITIVE 

RECIPROCAL METABOLIC REPROGRAMMING THROUGH 

LACTATE SHUTTLE 

Paola chiaruGi 

Department of Biochemical Sciences, University of Florence, 

viale Morgagni 50, 50134 Firenze, Italy 

Tumor cells form an heterogeneous microenvironment through their narrow relationship 

with stromal cells, including cancer associated fibroblasts or macrophages (CAFs 

and CAMs), other inflammatory cells, as well as endothelial cells. Several emerging 

data indicate CAFs as active participants in tumor progression and cancer cell 

dissemination. We recently reported that in aggressive prostate cancers, carcinoma 

cells (PCa) engage with their CAFs a biunivocal diabolic circuitry driving PCa cells to 

activate epithelial mesenchymal transition (EMT) thereby achieving stem cells traits 

and increasing spontaneous metastatic dissemination. We now speculate that the 

reciprocal interplay between CAFs and PCa cells goes beyond the engagement of 

EMT and embraces a real metabolic reprogramming of both cells types. Co-cultures 

of CAFs and PCa cells, as well as reciprocal treatment with conditioned media (CM), 

actually reveals a profound alteration in metabolism of both CAFs and PCa cells. Upon 

PCa contact CAFs undergo a typical Warburg effect, driven by the oxygen sensitive 

hypoxia inducible transcription factor-1 (HIF-1), as indicated by increase in GLUT1 

glucose transporter, increased lactate production and extrusion through a de novo 

expressed monocarboxylate transporter-4 (MCT4). Conversely PCa cells, upon CAF 

contact, undergo a “reverse Warburg effect”, showing a decrease in GLUT1 expression 

and an increase in the MCT1 lactate transporter, which regulate upload of lactate. The 

metabolic reprogramming of both stromal and cancer cells is under strict regulation 

of hypoxia inducible factor-1 (HIF1), which drives a redox- and SIRT3-dependent 

stabilization of HIF1 in normoxic conditions. PCa cells gradually become independent 

from glucose consumption, although they develop a dependence on lactate upload 

to drive ATP production, anabolic pathways, and thereby cell growth. In agreement, 

pharmacological inhibition of MCT1-mediated lactate upload dramatically affects PCa 

cells survival. Hence, CAFs are able to mimic an hypoxia-like transcriptional response 

in PCa cells leading stromal cells to synergize with cancer cells by supporting their 

survival and growth in hostile environment lacking nutrients. 

33


FUEL SUBSTRATES CONTROL THE INVOLVEMENT OF 

MITOCHONDRIA IN CELLS ADAPTATION TO HYPOxIA 

Solaini Giancarlo, SGarBi Gianluca, 

Padula anna, Baracca aleSSandra 

Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna, Italia 

Mitochondria are the predominant organelles for oxygen utilization and disposal, 

their regulatory response to the decrement in oxygen saturation is proposed to 

be an important mediator of the overall functional adaptation of cells to hypoxia. 

It is well established that the effects of hypoxia on cells are both time dependent: 

acute, intermittent or chronic, and degree dependent: the absolute reduction in the 

partial pressure of oxygen. These hypoxia-induced changes include modulation in 

mitochondrial respiratory capacity, activation of the mitochondrial biogenesis/removal 

regulatory program, and induction of mitochondrial antioxidant defence systems. 

These parameters seem to be affected also by the energy substrates available to 

the cells, but the issue has been scarcely addressed. We set the present study to 

establish how normal cells adapt to different oxygen pressure and oxidative fuel 

during a prolonged exposure. 

Human skin fibroblasts were exposed to 0.5 to 21 % oxygen tension, grown in glucose 

deficient media (galactose substituted glucose) and compared with those grown under 

the currently used conditions. At variance to fibroblasts grown in glucose, fibroblasts 

grown in glucose deficiency adapted to hypoxia by reducing their mass only slightly, 

preserving a well structured mitochondrial network, and producing low ROS. 

Interestingly, the oxidative phosphorylation rate of the mitochondria grown in glucosefree 

medium was enhanced. The pivotal transcription factor induced by hypoxia, HIF- 

1α as well as BNIP3, a factor activating mitophagy that has been associated to HIF, 

was found expressed both in presence and in absence of glucose, but at significantly 

different levels in presence or absence of glucose. Therefore, our data show that the 

HIF/BNIP3 pathway can induce mitochondrial network fragmentation and mitophagy 

in presence of glucose only, and that the effect of HIF on the mitochondrial OXPHOS 

is modulated by glucose. Overall, the data concur with the idea that the plasticity 

in mitochondrial regulation and function facilitates adaptations to a limited oxygen 

supply. 

Acknowledgements: This work was supported by a Grants from MIUR, Italy [PRIN 2008], and from 

Fondazione Del Monte di Bologna e Ravenna, Italy [2010]. 

34


HYPOxIA ADAPTATION IN HUMANS AND ANIMAL 

MODELS: CONTRIBUTION OF PROTEOMICS 

cecilia Gelfi 

Dipartimento di Scienze Biomediche per la Salute, 

Via Fratelli Cervi 93, Segrate , Milano 

The exploration of cellular mechanisms underlying beneficial adaptive processes and 

detrimental responses to hypoxia represents the object of several current research 

studies leading to improve the comprehension on molecular aspects related to 

hypoxia adaptation and possibly to develop new treatments for human diseases. 

Proteome analysis on skeletal muscles represents a tool for profiling molecular 

changes leading to adaptation to hypoxia under physiological conditions.Previous 

studies indicated that prolonged exposure to altitudes above 5000 meters (m) as in 

the course of Himalayan expeditions induces an average 10-15% lower limbs fat-free 

mass loss compared with control conditions whereas maximum aerobic power is 

only slightly affected. Muscle mass changes are accompanied by a 20-25% reduction 

of mitochondrial volume density and by a proportional decrease of oxidative enzyme 

as appears from photometric assessments on muscle biopsies made upon return. 

Also the anaerobic maximum exercise performance undergoes a significant reduction 

despite only moderate changes in the muscle glycolytic enzyme pattern. Thanks to 

the development of advanced proteomic techniques, the use of differential proteomics 

and the adoption of stringent statistical methods have made it possible to identify in 

humans a large number of quantitative and qualitative protein changes induced by 

physiologic, pathologic as well as paraphysiologic variables such hypoxia. Muscles 

from animal models exposed to acute and prolonged hypoxia, human muscles 

biopsies from individuals at sea level and at high altitude after nine days, two weeks 

and after fifty days of exposure were utilized to investigate the alteration occurring 

in skeletal muscle proteome identifying some of the cellular processes involved in 

hypoxia adaptation. Proteome analysis was performed utilizing 2D-DIGE and MALDI- 

ToF /ToF mass spectrometry. 2D-DIGE analysis revealed that the majority of changed 

proteins is represented by metabolic enzymes belonging to glycolysis, tricarboxylic 

acid cycle, fatty acid beta oxidation and oxidative phosphorylation, and by proteins 

involved in the regulation of protein translation. All these proteins were considerably 

less abundant under hyoxia versus normoxia. Among proteins involved in stress 

response a down-regulation in the early fase and an up-regulation of some antioxidant 

enzymes in the long term exposure were identified. A marked modulation was present 

also in cytoskeletal and contractile proteins. In addition markers of autophagy, 

35


apoptosis, energy production and mitochondrial biogenesis appeared significantly 

changed during adaptation. Proteomic analysis provided a broad picture of events 

occurring in muscle tissue under short and long term hypoxia exposure. Particularly, 

it allows to underline the more relevant changes at the metabolic, functional and 

structural level involved in the tissue adaptation at the limit of humans tolerance of O 2 

deprivation. 

Aknowledgements. Suppoted by MIUR (grant: FIRBRBRNO7BMCT) 

36

Friday 28 th 

September 

Session 5


MOLECULAR MECHANISMS OF CELLULAR SENESCENCE 

faBrizio d’adda di faGaGna 

Cellular senescence is a powerful mechanism of tumor suppression that also limits 

the proliferation of healthy cells and thus is associated with organismal ageing. I will 

propose a unifying mechanism for different types of cellular senescence based on 

DNA damage generation and persistent DNA damage response (DDR) activation. I will 

also discuss our most recent results on RNA and DDR modulation. 

38


Cellular senescence and microRNAs 

faraonio r. 1 

1 Dipartimento di Biochimica e Biotecnologie Mediche, 

Università di Napoli Federico II, Italy 

Cellular senescence is a permanent cell cycle arrest caused by various stimuli (i.e. short 

telomeres, activated oncogenes, oxidative stress) that result in DNA damage which, in 

turn, triggers a DNA damage response (DDR) that if persistent induces the senescence 

program. Senescence is considered a potent tumor suppressive mechanism and 

increasing evidences point to a link between cellular senescence and aging/agerelated 

diseases. Senescent cells display many characteristics, some of which reflect 

mechanisms (such as DDR) that contribute to the senescent phenotype, others (like 

senescence-associated b-galactosidase [SA-b-Gal] activity) that accompany the 

execution of the senescence program. Changes in gene expression patterns play 

an integral role in senescence. MicroRNAs (miRNAs) are short non-coding RNAs 

that regulate gene expression at post-transcriptional level. They decrease protein 

synthesis through translational repression or degradation of target mRNAs. We have 

recently reported the identification of 24 miRNAs (SAmiRs) that were either up-or 

down-regulated in replicative senescent IMR90 fibroblasts (1). The expression of 

most of these SAmiRs was also deregulated in senescence induced by DNA damage 

(etoposide) or oxidative stress (DEM). We focused on up-regulated SAmiRs and we 

showed that 5 of them (miR-210, miR-376a*, miR-486-5p, miR-494 and miR-542-5p) 

are causally related to the induction of senescence. In fact, their overexpression in 

young IMR90 cells is able to foster a senescent phenotype with induction of specific 

senescence-associated markers. We have also shown that these SAmiRs promote 

double-strand DNA breaks, DDR and reactive oxygen species accumulation. These 

data prompt us to study the molecular pathways in which SAmiRs are involved by 

identification of their targets. To this aim, we used Two Dimensional Differential-In- 

Gel Electrophoresis (2D-DIGE) to compare protein expression patterns of young 

IMR90 cells with senescent (replicative or oxidative stress induced) cells and with 

miR-494-transfected young cells. 243 protein spots out of 1449 analyzed resulted 

differentially expressed. Mass-spectrometry analysis of a subset of 78 spots allowed 

the identification of various proteins including cytoskeletal, heat shock, metabolic and 

redox proteins, as well as proteins involved in DDR and others of unknown function. 

1. R. Faraonio, P. Salerno, F. Passaro, C. Sedia, A. Iaccio, R. Bellelli, T.C. Nappi, M. Comegna, S. Romano, 

G. Salvatore, M. Santoro and F. Cimino. “A set of miRNA participates in the cellular senescence program 

in human diploid fibroblasts” Cell Death Differ. 19, 713-21 (2012) 

39


Role of the promyelocytic leukaemia protein in regulation 

of normal and neoplastic neural stem cells 

Paolo SaloMoni 

Samantha Dickson Brain Cancer Unit, UCL Cancer Institute 

The control of cell fate in neural stem/progenitor cells (NPCs) is critical for central 

nervous system (CNS) development as well as adult neurogenesis. Disruption of 

this process can lead to development of brain neoplasms, including glioblastoma 

multiforme (GBM). Within established GBM, a subpopulation of stem-like tumourinitiating 

cells is believed to underlie disease progression. Revealing the mechanisms 

underlying cell fate control within normal and neoplastic neural stem cells is therefore 

key to understanding tumour initiation and progression in the CNS. The promyelocytic 

leukaemia protein (PML) gene is involved in the t(15;17) chromosomal translocation 

of acute promyelocytic leukaemia (APL). The PML protein localises to PML nuclear 

bodies and functions as a growth/tumour suppressor. Our work has implicated PML 

in the regulation of NPCs in the developing neocortex. As a result, PML-deficient 

mice display alterations of corticogenesis and smaller brains. PML function is not 

limited to the embryonic brain, as PML loss affects expansion and differentiation of 

NPCs also in the postnatal brain. Unexpectedly, PML is highly expressed in highgrade 

GBM tumours and GBM-initiating neural stem cells, suggesting that it may 

play a non-tumour suppressive role within established CNS tumours. Overall, our 

findings implicate PML in regulation of both normal and neoplastic NPCs, and may 

have important implications for the understanding of brain cancer pathogenesis. 

40


Autophagy in the control of cell survival and proliferation 

franceSco cecconi 1,2 

1 Dulbecco Telethon Institue at the Department of Biology, 

University of Tor Vergata, Rome Italy 

2 IRCCS Santa Lucia Foundation, Rome Italy 

Autophagy is a cellular mechanism to degrade bulk cytosol, damaged organels and 

long-lived proteins. Besides its clear role in cleaning up the cells from potentially 

dangerous agents, ranging from pathogens to protein aggregates, a role is emerging for 

autophagy in finely regulating a cell’s life. Due the fast kinetics of autophagy activation 

and progression, to the easy mobilization of its main effectors (the autophagosomes) 

within the cytosol, and to the high metabolic advantages resulting from its function, 

autophagy is the best candidate to help cells removing entire sets of structural or 

functional factors, resetting the cell cytosol (and the nucleus) to respond to new 

environmental cues. Developmental signals, for example, can activate autophagy 

and a rapid demise and recycling of transcription factors, signallers, cytoskeletal 

components and modified organels may accompany or even trigger the differentiation 

of neuroepithelial precursors or, more in general epithelial cells. Since autophagy is 

continuously cross-talking with the cell death machinery, the key choice of a cell 

between survival and death is also regulated by autophagy. Last, we are now aware 

that autophagy induction may also control cell proliferation and protein ubiquitylation, 

this implying a role for this signalling cascade in the global coordination of cell fate. 

I will discuss here the implication of these evidence in human diseases, and the 

potential of manipulating this process while fighting neurodegeneration, muscle 

dystrophies, lysosomal storage disorders and cancer. 

41

Friday 28 th 

September 

Session 6


Involvement of p73, a p53-family member, 

in metabolism and senescence 

Melino G. 

University Tor Vergata, Rome, Italy; Medical Research Council, 

Toxicology Unit, Leicester, UK 

p63 and p73 have been identified as the ancestral members of the p53 family. 

Despite the high sequence and structural similarity, the mouse knockouts revealed 

a crucial role in neural development for p73 and in epidermal formation for p63. We 

identified several transcriptional targets, the mechanisms of regulation of cell death, 

and the p63 isoform involved in epithelial development. Both genes are involved in 

female infertility and maternal reproduction as well as in cancer formation, although 

with distinct mechanisms. TAp73 knockout mice (TW Mak G&D 2008) show high 

tumor incidence with hippocampal dysegensis. Conversely, ΔNp73 knockout mice 

(TW Mak G&D 2010) show a very low incidence of cancer, with sign of moderate 

neurodegeneration with a significant loss of cellularity in the cortex. This indicate a 

tumor suppressor role for TAp73 and an oncogenic role for ΔNp73. 

p73 transcriptional activity requires a Tyr-99 phosphorylation by c-Abl. p73 steady state 

protein levels are kept low under normal physiological conditions through degradation 

by the 26S proteasome, mediated by the HECT-containing E3 ubiquitin ligase ITCH. We 

developed an ELISA high throughput screening for ITCH auto-ubiquitylation, resulting 

in several positive compounds that are able to modulate chemosensitivity at 10 µM 

concentration. These compounds could be effective in cancer treatment. In addition 

to this major degradation pathway, we have also described two novel mechanisms of 

degradation. Firstly, we identified that the orphan F-box protein FBXO45, can target 

both TAp73 and ΔNp73 isoforms to degradation by poly-ubiquitylation. FBXO45 is 

the human ortholog of the C.elegans F-box protein FSN-1, therefore this novel finding 

elucidates a conserved pathway evolved from nematode to human, by which the 

p53 family members are regulated by an SCF-dependent mechanism. Secondly, 

we identified and characterized a novel transcriptional target of TAp73, the ring 

finger domain ubiquitin ligase PIR (p73-induced Ring Finger). PIR seems to be the 

first ubiquitin ligase able to differentiate between the TAp73 and ΔNp73 isoforms. 

Indeed, in response to DNA damage TAp73 is activated to induce cell cycle arrest 

or apoptosis, while ΔNp73 is rapidly degraded, highlighting the significance of the 

relative ratio of each isoform. In conclusion, we describe novel ITCH inhibitors and 

two novel mechanisms of p73 degradation, by FBXO45 and by PIR. 

44


Here, we describe the involvement of p73 in senescence and metabolism. TAp73null 

mice show a significant premature spontaneous aging phenotype at 12 months 

of age: alopecia, epidermal thinning, reduced subucutaneous fat, increased visceral 

fat TAp73, osteoporosis with scoliosis. This indicate a significant phenotype related 

to obesity and ageing. Both in vivo and in vivo TAp73-null mice show unbalanced 

redox defences. TAp73 is able to drive the expression of glutaminase type 2 (GLS2), 

acting on specific binding sites present on its promoter. In agreement with these in 

vitro data, TAp73-null cells show clear metabolic defects in the glutamine pathway 

affecting GSH and redox balance. In keeping, we show a role for TAp73 in the 

regulation of metabolic pathways. 

45


Targeting Mitosis for Cancer Therapy 

MarcoS MaluMBreS 

Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO) 

Madrid 

Mitosis is the process by which cells segregate the previously duplicated genome into 

two daughter cells. This process is mainly controlled by protein phosphorylation and 

degradation. Several mitotic kinesins and kinases, including members of the Aurora 

or Polo families, are required for mitotic entry and progression and are currently 

considered as attractive cancer targets. On the other hand, cell cycle-dependent 

proteolysis is partially regulated by the Anaphase-Promoting Complex/Cyclosome 

(APC/C), an E3-ubiquitin ligase that mediates the ubiquitination of a wide variety of cell 

cycle regulators during mitotic exit and before S-phase. The APC/C is modulated by 

two cofactors, Cdc20 and Cdh1, which select and target the proper substrates such 

as kinases and kinase regulators for degradation. We have recently addressed the 

relevance of some of these mitotic regulators using conditional gene-targeted alleles 

in the mouse. Mitotic kinases are essential for life and their complete ablation results 

in lethality in embryos and also at the cellular level. Interestingly, partial deficiency 

in some of these kinases reveals new tissue-specific activities for some of the core 

cell-cycle kinases, such as Plk1, that may have critical implications in cancer therapy. 

The APC/C cofactor is also essential since proliferating cells, including progenitor or 

very aggressive tumor cells, arrest in metaphase upon deletion of the APC/C cofactor 

Cdc20 in vivo. Strikingly, prolonged metaphase arrest is not tolerated by cells and 

Cdc20-null cells die in a few hours with no exception. We are currently investigating 

the mitotic cell death pathways that mediate this phenotype in Cdc20-null cells or 

cells treated with mitotic poisons currently used in the Clinic such as taxol. 

46


Assessing therapeutic targets in a pre-clinical model 

of Myc-induced lymphoma 

caMPaner Stefano 

Team Leader Center for Genomic Science of IIT@SEMM Fondazione Istituto Italiano 

di Tecnologia (IIT) at the IFOM-IEO Campus Via Adamello 16, 20139 Milan - Italy 

The plethora of molecular studies focusing on c-Myc has enhanced our understanding 

of the physiological and pathological role of this transcription factor frequently 

deregulated in cancer cells, thus inspiring novel insights fuelling the exploration of new 

opportunities for targeted cancer therapy. We will present recent data pointing at some 

relevant pathways involved in progression and maintenance of Myc dependent tumors. 

In a first line of research we focused on the role the ATR/CHK1 pathway in licensing 

Myc induced hyper-proliferation. Using a combination of genetic and pharmacological 

approaches, we found that bypassing the replicative checkpoint through ATR 

or CHK1 inhibition resulted in a robust DNA damage response, leading to potent 

cytotoxicity in cancer cells. Long-term treatments with CHK1 inhibitors effectively 

reduced tumor burden and dissemination in mice. Thus, despite the Myc induced in 

S-phase increase may predispose cells to trigger a replicative stress response, the 

concomitant Myc dependent engagement of the ATR/CHK1 pathway, prevents intra 

S-phase arrest, allowing Myc dependent hyper-proliferation. We therefore propose 

that CHK1 inhibition may represent an effective way of treating tumors with elevated 

Myc activity and suggest that targeting the ATR/CHK1 pathway may represent a 

valuable therapeutic option in tumors with an activated replicative stress checkpoint. 

In a second line of research we analyzed the role of Pin1, a proposed regulator of 

Myc stability and activity, during Myc induced lymphomagenesis. We show here that 

genetic ablation of Pin1 in Eµ-myc transgenic mice starkly reduced lymphoma onset 

and penetrance. In pre-malignant Pin1-deficient B-cells, the proliferative response 

to Myc was selectively impaired, in the absence of changes in either steady-state 

Myc levels or Ser 62 phosphorylation. Thus, while Pin1 is not essential for normal 

cell growth and mouse development, it is required to support the oncogenic activity 

of Myc in B-cells. Indeed, Eµ-myc lymphoma cells were sensitive to Pin1 inhibition, 

making Pin1 an attractive therapeutic target in Myc-driven tumors. 

47


Targeting cell cycle: novel insights into p27 Kip1 

and p57 Kip2 metabolism 

adriana Borriello 

Department of Biochemistry, Biophysics and General Pathology, 

Second University of Naples, Via De Crecchio 7, 80138, Napoli 

Deregulation of cell cycle is a frequent feature of malignant transformation and many 

therapeutic strategies have been suggested in the last few years to inhibit the altered 

cycling of cancer cells. Cyclin/cyclin-dependent kinase (cyclin/CDK) complexes are 

the major players of cell proliferation, as they drive progression of cells through the 

different phases of the division cycle by acquiring catalytic activity only at specific 

points. These serine/threonine kinases phosphorylate several substrates that, in turn, 

promote the phase transitions; therefore, CDKs activity must be tightly regulated to 

ensure orderly cell cycle progression. 

A number of mechanisms play an important role in modulating CDK activity. These 

include the binding to specific cyclin partner(s), the phosphorylation at peculiar 

residues and the interaction with CDK inhibitors (CKI). 

Two major families of CKI have been identified, the INK4 and the CIP/Kip. The CIP/ 

Kip family includes three members, namely p21 Cip1 , p27 Kip1 and p57 Kip2 . In human 

cancers, CIP/Kip proteins show frequent alterations that mostly result in a decrease 

of their level and cell mislocalization. It is also to underscore that CKIs target, 

beside CDK/cyclin complexes, also proteins capable of regulating cell motility (and 

metastatization), apoptosis, autophagy, gene transcription and genome duplication. 

Although the relevance of CKIs in cancer is undisputable, the knowledge of their 

metabolism is still incomplete, thus reducing the possibility of their targeting. 

A major mechanism of the CIP/Kip proteins control is their post-synthetic modifications. 

Here, our recent findings on p27 Kip1 and p57 Kip2 phosphorylation will be reported. 

In particular, the role of specific p27 Kip1 phosphorylations in correlation with the CKI 

functions, localization and metabolism will be addressed. These findings might give 

novel opportunities in the context of a CKI handling for therapeutic goals. Preliminary 

data on post-synthetic modifications of p57 Kip2 and their role in the activity of the 

CKI will also be discussed. 

48

Saturday 29 th 

September 

Session 7

Saturday 29 th September Session 7 

The diversity of estrogen receptor signaling influences 

estrogen effects on skeletal muscle cells 

Maria Marino and Marco PelleGrini 

Department of Biology, University Roma Tre, 

viale G. Marconi, 446 I-00146, Rome, Italy 

The estrogen decrease in post-menopausal women has been associated with a 

number of negative outcomes, including a greater incidence of injury as well as a delay 

in recovery from these injuries. Over the past two decades, our understanding of the 

protective effects of estrogen against various types of injury and disease states has 

grown immensely. In skeletal muscle, studies with animals have demonstrated that 

sex and estrogen, in particular 17β-estradiol (E2), could influence muscle contractile 

properties and stimulate muscle repair and regenerative processes. E2 could exert 

its protective effects upon skeletal muscle damage, inflammation and repair by: (i) 

acting as an antioxidant, thus limiting oxidative damage and/or (ii) governing the 

regulation of a number of downstream genes and molecular targets. Until a decade 

ago, all E2 signaling was thought to occur by E2 binding to nuclear estrogen receptor 

(ERα) which, in turn, bind to DNA and function as ligand-activated transcription factor. 

Estrogen binding to the receptor alters gene expression, thereby altering cell function. 

The discovery of a second receptor subtype (i.e., ERβ) together with the considerable 

recognition that E2 also binds to ERs tethered to the plasma membrane, resulting 

in acute activation of signaling kinases, was a breakthrough in endocrinology. Thus, 

E2 can alter skeletal cell function by binding to different ERs which trigger distinct 

signal transduction pathways. Here, the different contribution of ER subtypes and 

their signaling mechanisms to the E2 effects in skeletal muscle cell will be discussed. 

50


“Nuclear export of Estradiol Receptor α” 

GaBriella caStoria, Pia Giovannelli, Marzia di donato, 

ferdinando auricchio and antiMo MiGliaccio. 

Department of Biochemistry, Biophysics and General Pathology. 

2 nd University of Naples, Via L De Crecchio, 7, 80138 Naples 

In breast cancer cells, cytoplasmic localization of the estradiol receptor-α (ERα) 

regulates estradiol-dependent S phase entry. We identified a nuclear export 

sequence (NES) in ERα and show that its export is dependent on both estradiolmediated 

phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome 

region maintenance 1 (CRM1). Interestingly, NES-ERα mutants unable to exit 

the nucleus inhibit estradiol-induced S phase entry, without impairing hormonedependent 

transcription. ERα is associated with “ForKHead_in_Rhabdomyosarcoma” 

(FKHR) protein in the nucleus, and estradiol stimulates nuclear exit of both proteins. 

ERαknockdown or ERv NES mutations prevent nuclear export of both ERα and FKHR. 

Conversely, the mutant of FKHR (FKHR-AAA), which cannot be phosphorylated AKT, 

is trapped in the nucleus with ERα, inhibiting S phase entry. Thus, estradiol-induced 

AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby 

triggering complex export from the nucleus necessary for initiation of DNA synthesis 

and S phase entry. The intracellular localization of ERα and DNA synthesis is also 

regulated by ERα tyrosine phosphorylation at position 537 by Src. In fact, inhibition 

of Src or use of a peptide mimicking the ERα p-Tyr537 sequence inhibiting tyrosine 

phosphorylation traps the receptor in nuclei of estradiol-treated MCF-7 cells. The 

ERα mutant carrying Tyr537�Phe mutation (ER537F) persistently localizes in nuclei of 

various cell types. In contrast with ERα wt, ER537F does not associate with Ran and 

its interaction with Crm1 is insensitive to estradiol. Thus, independently of estradiol, 

ER537F is retained in nuclei, where it entangles FKHR, driving cell cycle arrest. ChIP 

analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR 

interaction with Cyclin D1 promoter down regulating Cyclin expression. Remarkably, 

this mutant also counteracts cell transformation by the activated forms of Src or PI3-K. 

In conclusion, ERα localization together with its phosphorylation by Src is required for 

hormone responsiveness of DNA synthesis in breast cancer cells. 

51


The platelet as a model system for the non-genomic 

actions of steroid receptors 

Bertoni aleSSandra 1,2 , reineri Stefania 1 , raStoldo aleSSandro 1 , 

SiniGaGlia faBiola 1,2 

1 Department of Translational Medicine Novara, University of Piemonte Orientale 

A. Avogadro, Novara, IT 

2 BRMA, University of Piemonte Orientale A. Avogadro, Novara 

Steroid hormones are known to act through nuclear receptors and regulate gene 

transcription. Recently it is been shown that they can also elicit rapid non-genomic 

effects on target cells. 

The recent findings that nuclear receptors are expressed in anucleated human platelets 

renders these cells an excellent model to study the non-genomic effects of these 

hormones. 

Platelets express a number of nuclear receptors, including receptors for sex steroids, 

glucocorticoids, peroxisome proliferator-activated receptors and retinoid X receptors. 

The impact of sex steroids on the cardiovascular system and their ability to regulate 

platelet function is still an open and debated question. We investigated the effect of 

estrogens and dehydroepiandrosterone on platelets function. 

In human platelets 17β-estradiol causes the rapid phosphorylation of the tyrosine kinases 

Src and Pyk2, and the formation of a signaling complex, which includes Src, Pyk2, 

and PI3-K. These events are dependent on membrane-associated estrogen receptor 

(ER) β. Lipid rafts are critical, cholesterol-enriched membrane domains which play a 

major role in blood platelet activation processes. ERβ translocated to the raft fractions 

upon stimulation with 17β-estradiol in a time-dependent fashion. In 17β-estradiolstimulated 

platelets a rapid and transient translocation of the active forma Src and Pyk2 

to membrane raft domains occurred. These results suggest that in human platelets 

the estrogen signaling might be mediated through some reorganization of the lipid raft 

domains. 

Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, are the most abundant 

sex steroids circulating in human blood. DHEA-S, but not DHEA, inhibited in vitro 

thrombin-dependent platelet aggregation in a dose-dependent manner. DHEA-S exerted 

this effect by decreasing thrombin-dependent dense granule secretion, and so impairing 

the positive feed-back loop provided by ADP. Furthermore, DHEA-S inhibited thrombindependent 

activation of Akt, ERK1/2, and p38 MAP kinase. Although both DHEA-S 

and DHEA directly activated in platelets the inhibitory cGMP/PGK/VASP pathway, 

these events were not responsible for the inhibitory action of DHEA-S in platelets. In 

addition DHEA-S acted in synergism with nitric oxide in inhibiting platelet aggregation. 

In conclusion DHEA-S inhibited platelet activation caused by a mild stimulus without 

completely hampering platelet functionality and thus DHEA-S may participate in the 

physiological mechanisms that maintain circulating platelets in a resting state. 

52


G protein-coupled estrogen receptor (GPER/GPR30): 

a new player in estrogen signaling 

M. MaGGiolini 

17β-estradiol (E2) binds to and activates the estrogen receptor (ER)α and ERβ, which 

regulate the transcription of genes involved in numerous biological responses, including 

the growth of normal and neoplastic tissues. In recent years, increasing evidence have 

demonstrated that the G protein-coupled receptor named GPR30/GPER mediates 

rapid E2-dependent functions in different cell contexts. GPER signaling activated 

not only by estrogens, but also by antiestrogens and endocrine disruptors is clearly 

distinct from that dependent on ERs, although the two transduction pathways may 

interact and cooperate in certain circumstances. The expression and the physiological 

function of GPER have been assessed in diverse tissues like both male and female 

reproductive organs, bone, pancreatic islets, the cardiovascular, immune and nervous 

systems. Likewise, the role of GPER in cancer has been evidenced in numerous 

studies performed in breast, endometrial, ovarian, thyroid, prostate and testicular 

germ tumor cells. In accordance with these findings, the overexpression of GPER was 

associated with an aggressive phenotype of some estrogen-sensitive tumors. In this 

regard, the EGFR-dependent signaling was shown to up-regulate GPER expression 

in both ERα-negative and positive cancer cells, providing additional evidence that 

estrogens and growth factor transduction pathways may cooperate in amplifying 

mitogenic stimuli in these cells. Considering that EGFR-mediated signals contributes 

to tamoxifen resistance in patients with breast cancer and 4-hydroxytamoxifen binds 

to and activates GPER, the EGFR-dependent regulation of GPER could contribute to 

the mechanisms involved in the tamoxifen failure in breast tumors. The IGF-I system 

also regulates GPER expression and function leading to the activation of a signaling 

network that stimulates the migration and proliferation of cancer cells. In particular, 

the up-regulation of GPER expression by IGF-I was demonstrated to occurs through 

the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway in breast and endometrial 

cancer cells. In addition, an important hallmark of cancer growth and resistance to 

chemotherapy such as hypoxia, was shown to induce GPER expression in breast 

tumor cells and even in cardiomyocytes. On the basis of these observations, GPER 

regulation may be included among the mechanisms involved in the adaptation to 

hypoxia in cancer and the cardiovascular system. 

Since its identification to date, the transduction signaling and gene expression profile 

triggered by GPER have been widely characterized. A whole sequence of biological 

events has been shown to follow GPER activation such as the EGFR transactivation, the 

rapid phosphorylation of ERK1/2, the activation of PI3K and PLC, the cAMP increase 

53

and the intracellular calcium mobilization. As it concerns the GPER-activated gene 

signature, diverse studies have demonstrated that the main GPER target gene, CTGF, 

is involved in relevant biological effects stimulated by estrogens like the proliferation 

and migration in diverse cancer cells as well as in breast cancer associated fibroblasts 

(CAFs), which strongly contribute to cancer progression. 

The subcellular localization of GPER is a further interesting area of investigation. 

GPER was localized to the endoplasmic reticulum and the plasma membrane as 

well. Recent studies have intricated this issue because the nuclear localization and 

function of GPER was demonstrated in CAFs, standing for an intriguing role exerted 

by GPER as a transcription factor in these cells. 

GPER exhibits many of the expected characteristics of an estrogen receptor, 

including the capability to bind to estrogens, phyto- and xenoestrogens and even 

the ER antagonists 4-hydroxytamoxifen (OHT) and fulvestrant (ICI 182 780). Unlike 

the antagonistic properties displayed by OHT and ICI with respect to the classical 

ERs, both compounds act as GPER agonists. Conversely, the well known ER agonist 

estriol exerts inhibitory effects on GPER-mediated signaling, confirming the potential 

opposite functions elicited by estrogenic/anti-estrogenic agents through each 

estrogen receptor type. Recently, a novel compound (referred to as MIBE) was shown 

to exhibit the unique property to bind to and inhibit both GPER- and ERα-mediated 

signaling in breast cancer cells. This compound (and further derivatives) may be 

used in innovative therapeutic approaches targeting both estrogen receptors types 

in cancer cells. 

As it concerns the GPER-mediated action in the different tissues, accumulating 

evidence regarding in particular the cardiovascular system suggest that GPER 

is responsible for a variety of beneficial effects of estrogens, hence this receptor 

may represent a novel target to develop effective strategies for the treatment of 

cardiovascular diseases by tissue-specific, selective activation of estrogen-dependent 

molecular pathways devoid of side effects seen with conventional hormone therapy. 

Data available may allow to consider GPER as a new player in estrogen-mediated 

signaling. However, the role elicited by GPER as an ER is not universally accepted. 

Moreover, various GPER-deficient mouse strains have been generated but with 

somewhat divergent phenotypes. Overall, diverse questions concerning the molecular 

and cell biology of GPER remain as open issues that require further efforts to be fully 

understood. 

54

Saturday 29 th 

September 

Session 8


Bone Marrow Environment and Regulation 

of Platelet Production 

Balduini aleSSandra 

Department of Molecular Medicine, Laboratory of Biotechnology, 

University of Pavia, IRCCS San Matteo Foundation, Pavia, Italy and 

Department of Biomedical Engineering, Tufts University, Medford, MA, USA. 

Platelets production occurs in the bone marrow after megakaryocyte (Mk) migration 

from an osteoblastic niche to a vascular niche, where mature Mks elongate proplatelets 

through the vascular endothelium and release platelets in the blood stream. These 

events occur in a complex microenvironment where extracellular matrices (ECMs) 

are fundamental regulatory components. However, mechanisms underlying Mk 

development remain unsolved. Our results demonstrated that different ECMs differently 

regulate Mk function and platelet production. Specifically, we showed that type IV can 

support proplatelet formation, while type I collagen, located in the osteoblastic niche, 

is the only ECM component known to inhibit this process. Further, we demonstrated 

that human Mks express and synthesize cellular fibronectin (cFN), with a predominance 

of the EDA isoform, and transglutaminase FXIII-A and we proposed that these 

two molecules are involved in a new regulatory mechanism of Mk-type I collagen 

interaction. This mechanism seemed to be mediated by the exposure of cFN to the 

cell membrane and maintained by FN polymerization catalyzed by FXIII-A. Moreover, 

by Atomic Force Microscopy, we documented that the tensile strength of fibrils in 

type I collagen structure regulates cytoskeleton contractility of human Mks through 

activation of the Rho-ROCK pathway and MLC-2 phosphorylation. Importantly, only 

the ECM receptors integrin alpha2beta1 and GPVI were extensively studied in Mks, 

whereas the expression and function of other receptors on human Mks are unknown. 

Our results also demonstrated, for the first time, that human Mks express the collagen 

receptor Discoidin Domain Receptor 1 (DDR1) that regulates megakaryocyte Sykmediated 

migration through activation of the tyrosine phosphatase SHP1. Finally, 

we recapitulated some of the bone marrow niche function in a silk based 3D model 

resulting in functional platelet production from human derived Mks. Thus, modeling 

the bone marrow environment is fundamental to understand the elaborate regulation 

of megakaryocyte function and platelet production. 

56


Regulation of integrin function by 

semaphorin receptor signaling 

Guido Serini 

Laboratory of Cell Adhesion Dynamics - IRCC, Institute for Cancer Research and 

Treatment and Department of Oncological Sciences - University of Torino School 

of Medicine Strada Provinciale 142, Km 3.95, 10060 Candiolo, Torino, Italy. 

In multicellular organisms, extracellular matrix (ECM) proteins and their cognate 

integrin adhesive receptors play key roles in several physiological processes such as 

embryonic development, hemostasis, immunity, and wound healing, and in pathologic 

conditions such as thrombosis, bleeding disorders, cancer metastasis, cardiovascular 

and inflammatory disease. Integrins can exist in different conformational states with 

respect to their affinity for ECM proteins and regulation of conformational integrin 

activation is crucial for their biological functions. 

We have previously shown that in the embryo the development of a properly patterned 

network of blood vessels relies upon the fine modulation of integrin conformational 

activation by chemoattractant and chemorepulsive cues, such as vascular endothelial 

growth factor (VEGF)-A and semaphorin 3A (SEMA3A). Indeed, during physiological 

angiogenesis endothelial cells (ECs) dynamically remodel their integrin-mediated 

interactions with the ECM in response to the guidance cues that regulate their 

motility. SEMA3A signals through a holoreceptor complex formed by neuropilin (Nrp)1 

and type A/D plexin, which respectively constitute the ligand binding and signal 

transducing subunit. Plexins function through their GTPase activating protein (GAP) 

cytodomain which inactivates integrin regulating small GTPases, namely Rap1 and 

R-Ras. While it is well known that Rap1-GTP via RIAM activates talin, which in turn 

binds the cytoplasmic domain of integrins to elicit their conformational activation, the 

mechanisms through which R-Ras promotes integrin-mediated cell adhesion are still 

unknown. 

We identified the Ras and Rab interactor 2 (RIN2) protein as a key R-Ras mediator that, 

by physically and functionally coupling R-Ras and Rab5 GTPases at ECM adhesion 

sites and on early endosomes, elicits EC-to-ECM adhesion, migration, and vascular 

morphogenesis. Upon cell binding to the ECM, the association of RIN2 to R-Ras-GTP 

lessens its Rab5 guanosine exchange factor (GEF) activity and maximizes its docking 

function. As adaptor protein, on the one hand RIN2 concentrates a pool of Rab5 at 

ECM adhesions, while on the other hand it promotes the Rab5-dependent topological 

relocation of active R-Ras to Rac1-containing early endosomes. Accordingly, R-Ras- 

GTP via RIN2/Rab5 specifically elicits the endocytosis of ECM-bound/active integrins 

57


from the plasma membrane while the concurrent association of RIN2 with active R-Ras 

and Rab5, which at steady state mainly occurs on EEA1-positive early endosomes, 

is instead required to stimulate the activation of Rac1 and the ensuing adhesion of 

ECs to ECM proteins. Furthermore, stimulation of cell adhesion by RIN2 depends 

on TIAM1, a Rac GEF that can be activated on early endosomes by the coincident 

detection of two Rab5-triggered phenomena: the VPS34-mediated production of 

PIns(3)P and the RIN2-dependent translocation of R-Ras-GTP. Along the same line, 

we also found that Nrp1, independently from its function of co-receptor for VEGF-A 

and SEMA3A, supports EC adhesion to fibronectin by promoting the fast vesicular 

traffic of the active conformation of α5β1 integrin back and forth from ECM adhesion 

sites. Therefore, during vascular morphogenesis modulation of cell adhesion to the 

ECM results from the regulation of integrin conformation, traffic, and endosomal 

signaling. 

Acknowledgements 

These studies have been supported by AIRC, Telethon Italy, Compagnia di San Paolo, Ministero dell 

Salute, Fondazione Piemontese per la Ricerca sul Cancro, Associazione Augusto per la vita, and Regione 

Piemonte. 

Selected publications 

1. G. Serini 1 , V. Valdembri, S. Zanivan, G. Morterra, C. Burkhardt, F. Caccavari, L. Zammataro, L. Primo, 

L. Tamagnone, M. Logan, M. Tessier-Lavigne, M. Taniguchi, A.W. Püschel, and F. Bussolino 1 . Class 3 

semaphorins control vascular morphogenesis by inhibiting integrin function. Nature 2003, 424: 391-397. 

1 

Corresponding authors 

2. D. Valdembri, P.T. Caswell, K.I. Anderson, J.P. Schwarz, I. König, E. Astanina, F. Caccavari, J.C. Norman, 

M.J. Humphries, F. Bussolino, and G. Serini. Neuropilin−1/GIPC1 Signaling Regulates α5β1 Integrin 

Traffic and Function in Endothelial Cells. PLoS Biology 2009, 7:e1000025. 

3. C. Sandri, F. Caccavari, D. Valdembri, C. Camillo, S. Veltel, M. Santambrogio, L. Lanzetti, F. Bussolino, 

J. Ivaska, and G. Serini. The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and 

morphogenesis via active integrin endocytosis and Rac signaling. Cell Research 2012, Jul 24. doi: 

10.1038/cr.2012.110. 

4. D. Valdembri and G. Serini. Regulation of adhesion site dynamics by integrin traffic. Current Opinion in 

Cell Biology 2012, in press. 

58


PROTEOLYTIC REGULATION OF ADHESIVE MULTIMERIC 

PROTEINS: 

ADAMTS13 AND VON WILLEBRAND FACTOR 

raiMondo de criStofaro 

Dipartimento di Scienze Mediche – Servizio Malattie Emorragichee Trombotiche 

Facoltà di Medicina e Chirurga “A. Gemelli” 

Università Cattolica S. Cuore – ROMA 

The zinc-protease a disintegrin-like and metalloprotease with thrombospondin type I 

repeats (ADAMTS13) cleaves the Tyr1605-Met1606 peptide bond of von Willebrand 

factor (VWF), avoiding the accumulation of ultra large VWF multimers. The proteolysis 

takes place only under high stress conditions, where VWF unfolds and exposes the 

scissile bond in the A2 domain of the protein. The larger VWF multimers in plasma are 

the most haemostatically reactive not only because they contain more ligand binding 

sites, but also because they are more conformationally responsive to vascular shear 

forces. The catalysis is influenced by two ionizable groups, whose pKa values were 

equal to 6.41±0.08 (ionization enthalpy = 32.6 ±1.7 kJ/mol) and 4±0.1 (ionization 

enthalpy 3.8 ± 0.4 kJ/mol), whereas these values were equal to 6±0.1 and 4.1±0.1, 

respectively, in Co 2+ -substituted ADAMTS13. The catalytic process showed negative 

activation entropy (-144 kJ/mol), suggesting that the transition state becomes more 

ordered than the ground state of the reactants. The k cat /K m values were not linearly 

correlated with temperature, as expression of change of the kinetic ‘‘stickiness’’ of 

the substrate. The Met1606-Arg1668 peptide product acted as hyperbolic mixedtype 

inhibitor of FRETS-VWF73 hydrolysis. Asp1653, Glu1655, Glu1660, Asp1663, 

together with the hydrophilic side chain of Thr1656 were shown to form a ‘‘hot spot’’ in 

the VWF A2 sequence, which drives the molecular recognition and allosteric regulation 

of binding to ADAMTS13. The interaction of the Met1606-Arg1668 region of VWF with 

ADAMTS13 involves basic residues of the protease and is thus progressively inhibited 

at pH values .8.50. The functional interactions between ADAMTS13 and VWF that 

have been characterized have been shown to involve VWF residues that are C terminal 

to the scissile bond. However, besides these C-terminal residues. the VWF P3 position 

in VWF, Leu1603, was demonstarted to be critical for VWF proteolysis. Extensive sitespecific 

mutagenesis studies of the metalloprotease domain of ADAMTS13 identified 

two spatially separated clusters centered on Leu198 or Val195 as possible subsites 

interacting with VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 

Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The 

recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported 

59


remote exosites C terminal of the cleavage site, suggests a mechanism whereby the 

VWF P1-P1scissile bond is brought into position over the active site for cleavage. A 

molecular model of the VWF A2 region containing the scissile bond in complex with 

the ADAMTS-13 active site showed that the substrate can fit into the active site only 

if ADAMTS13 assumes a C-like shape and, interacting with the acidic 1653–1668 

region of VWF, properly orients the Tyr1605-Met1606 peptide bond for the cleavage 

by the zinc-aquo complex in the active site. 

60


ABERRANT mRNA SPLICING IN COAGULATION FACTOR 

DEFICIENCIES: 

FROM MOLECULAR MECHANISMS TO RNA-BASED 

THERAPEUTIC APPROACHES 

Mirko Pinotti 

Department of Biochemistry and Molecular Biology, University of Ferrara, Italy 

Pre-mRNA splicing, in which exons are precisely identified and joined together to form 

the mature mRNA, reveals a large complexity. Natural mutations inducing aberrant 

splicing, a frequent cause of human disease (>15%), represent peculiar models to 

elucidate molecular mechanisms underlying exon definition. 

As models, we chose a panel of mutations occurring at either donor (5’ss) or acceptor 

(3’ss) splice sites of coagulation F7 exon 7 or F9 exon 5, which were found in patients 

with severe factor VII (FVII) or IX (FIX) deficiency. Expression studies with FVII/FIX 

minigenes demonstrated that these mutations, altering sequences relevant for 

spliceosome assembly, caused exon skipping and/or activation of cryptic 5’ss. 

In the earliest splicing step, the exon is defined by binding of the U1 small nuclear 

ribonucleoprotein (U1snRNP) to the 5’ss through its U1snRNA component. Therefore, 

5’ss mutations reducing complementarity with the U1snRNA, or combination of 3’ss 

mutations with weak natural 5’ss inefficiently recognized by the U1snRNA, might 

explain our experimental observations. 

To test this hypothesis, we assessed the ability of engineered U1snRNA, with improved 

complementarity to the 5’ss of F7 exon 7 or F9 exon 5, to restore mRNA processing. In 

the cellular models of FVII/FIX deficiency, loading of U1snRNA to the normal or mutant 

5’ss re-directed usage of the correct splice sites in the presence of different mutations 

types, and significantly rescued synthesis of the properly spliced mRNA (from barely 

detectable to >20% of transcripts). In the F9 model, we also evaluated a series of 

U1snRNA variants targeting non-conserved intronic sequences downstream of the 

5’ss (Exon Specific U1-ExSpeU1), which should ensure gene specificity and reduce 

potential off-target effects. Intriguingly, we identified a unique ExSpeU1 that was able 

to virtually restore splicing impaired by different mutations at the 5’ss or 3’ss. 

Moreover, through the expression of splicing-competent FVII/FIX minigenes, we 

demonstrated that the U1snRNA-mediated rescue resulted in appreciable levels 

(10-20 ng/ml; up to 100% as compared to wild-type FVII/FIX for some mutants) of 

secreted FVII/FIX molecules with normal enzymatic (procoagulant) activity. These 

results, if achieved in coagulation factor deficient patients in whom even tiny amounts 

of circulating protein ameliorate the clinical phenotype, would have a therapeutic 

impact. 

61


In conclusion, we elucidated the molecular mechanisms underlying aberrant splicing in 

coagulation factor genes, which result from the interplay between the strength of 5’ss 

and 3’ss. Depending on the specific exon features and sequence context, restoration 

of complementarity with the U1snRNA might strongly improve exon definition, thus 

suggesting an innovative RNA-based correction strategy for coagulation diseases 

caused by aberrant splicing. Studies in animal models, which are required to evaluate 

the ability of U1snRNA to modulate splicing in vivo, are in progress. 

62

Poster 

Session 1 


September 

16:30 - 18:30

Ammine 

Biogene 

AMB

Ammine Biogene AMB 1 

Spermine and spermidine exert a synergistic effect 

on DNA aggregation 

SePPi claudio and Balduini ceSare 

Dept of Biology and Biotecnology “L: Spallanzani” 

Division of Biochemistry - University of Pavia 

The most relevant function of polyamines (spermine, spermidine and putrescine) 

is their ability to bind nucleic acids in particular DNA. The binding of spermine 

and spermidine induces the condensation, the aggregation and in some cases, a 

conformational change of DNA. Most studies concerning DNA aggregation have 

investigated the effect of one polyamine at a time. We have analyzed DNA aggregation 

induced by mixtures containing different amounts of spermine and spermidine and 

found that these polyamines cooperate in inducing DNA aggregation. In order to test if 

this effect is synergistic or merely additive, we undertook an isobolographic analysis. 

An isobologram is a plot of pairs of doses that in combination, yield a specified 

level of effect and is often utilized in the analysis of the combined effects of simple 

chemical mixtures. Spermine and spermidine show the same maximal effect but their 

log dose-effect curves are not parallel. In these cases the use of dose equivalence 

leads to two possible isoboles of additivity. We have calculated these isoboles by 

the method described by Tallarida (Tallarida RJ J Pharmacol Exp Ther. 2006 319:1- 

7) for the aggregating effect ranging from 10% to 90% of the maximum. We have 

then evaluated the dose-response curves for mixture of spermine and spermidine 

with ratios ranging from 1:10 to 1:50. Our results show that at low doses the two 

polyamines act synergistically, whereas for doses causing an aggregation higher than 

50% the effect is additive. These observations are in agreement with the hypothesis 

that spermine could induce DNA aggregation also by forming a crosslink between two 

double strands. 

I Poster Session 

66


TRANSGLUTAMINASE 2 IS INVOLVED IN 

HOMOCYSTEINE-INDUCED INFLAMMATORY RESPONSE 

OF THP-1 MONOCYTES 

riSitano roBerto, currò Monica, GanGeMi chiara, ferlazzo nadia, 

ientile riccardo, caccaMo daniela 

Dept. of Biomedical Sciences and Morphological and Functional Imaging, 

University of Messina 

Clinical studies indicate that hyperhomocysteinemia is associated with vascular 

disease including cerebrovascular, peripheral arterial occlusive diseases and 

neuroinflammation [1]. 

High levels of homocysteine (Hcy) increase cell adhesion and induce a proinflammatory 

state in the vessel wall by promoting the expression of adhesion molecules and 

recruitment of monocytes. In THP-1 cells Hcy promotes NF-kB activation and 

stimulates intercellular adhesion molecules (ICAM-1) [2]. High levels of Hcy also 

induce the expression and secretion of the chemoattractant protein of monocyte 

(MCP-1) and interleukin 8 (IL-8) in endothelial cells, smooth muscle cells and human 

monocytes [3]. 

It is also known that transglutaminase 2 (TG2), a calcium-dependent enzyme that 

catalyzes transamidation reaction producing either polyaminated or deamidated or 

polymeric proteins, is involved in the inflammatory process through the activation of 

NF-kB. 

We aimed to evaluate the involvement of TG2 in cell response to pro-inflammatory 

stimuli using human THP-1 monocytes subjected to a chronic treatment (2-5 days) 

with mild Hcy concentrations (50 µM). 

The results show that Hcy is able to induce TG2, especially in response to a 

prolonged stimulus (5 days), and increase TNF-α, IL-6, and RANKL mRNA levels, 

as well as NF-κB activation and HIF-1α induction. Upon specific inhibition of TG2 

activity by R283, we also provided evidence that TG2 was involved in these Hcy 

effects. 

To summarize, Hcy was able to activate inflammatory response in THP-1 cells, and 

the increased expression of TG2 is part of these events. Furthermore, TG2 activation 

could be associated to changes involving HIF-1α pathway. 

1. Finch & Joseph Cardiovasc Hematol Disord Drug Targets 10:241-5, 2010 

2. Postea et al. Arterioscler Thromb Vasc Biol 26: 508–513, 2006. 

3. Wang et al. Arterioscler Thromb Vasc Biol 22: 1777–1783, 2002. 


67


Differential mRNA transcription of transglutaminase genes 

in human periodontal disease: reduction of both TGM1 

and TGM3 expression in affected gingival tissues 

currò Monica 1 , MatareSe Giovanni 2 , iSola Gaetano 2 , 

caccaMo daniela 1 , ventura valeria 1 , 

cordaSco Giancarlo 2 , ientile riccardo 1 

1 Department of Biomedical Sciences and Morphofunctional Imaging 

2 Department of Specialized Surgery, University of Messina 

Transglutaminases are involved in the stabilisation of matrix molecules by protein 

cross-linking also leading to formation of keratin envelopes formed during epidermal 

cell differentiation. However different isoforms of tranglutaminases can be involved in 

different functions using different protein substrates. 

Transglutaminase 1 (TG1) isoform is primarily expressed in stratified squamous 

epithelia [1]. Additionally, transglutaminase 3 (TG3) has also been suggested to 

play important roles in epidermal keratinization and the formation of the cornified 

envelope [2]. Indeed, TG3 is expressed predominantly in differentiating keratinocytes, 

corneocytes and hair follicles [3], and it is also found extracellularly [4]. 

Following periodontal disease, gingival epithelial tissue is characterized by cell 

proliferation and migration of the cells over the tooth and altered connective tissue 

substratum. 

The aim of this study was to evaluate the mRNA transcripts of different transglutaminase 

isoforms in the human gingival tissues from patients with periodontal disease and 

relative controls. Gene expression for TG1, TG2, TG3 was evaluated by Real-time 

PCR, whereas the expression of FactorXIIIa was evaluated by RT-PCR. The TGM1 and 

TGM3 mRNA were the most abundantly expressed in gingival tissues. In comparison 

to normal tissues, mRNAs of TGM1 and TGM3 show a significant decrease in 

periodontal disease. TG2 mRNA was significantly less abundant in comparison to 

mRNAs of other transglutaminases, slight and unsignificant increase in the expression 

of TGM2 gene was obseved in samples from patients in comparison to control 

subjects. We suggest that TGs gene expression may be modified in the damaged 

gingival tissue and emphasize the key role of these enzymes in gingival remodelling/ 

healing and adaptive processes. 

1. Yamada K et al. (1997) Hum Mol Genet. 6:2223-31. 

2. Candi E et al. (2005) Nat Rev Mol Cell Biol. 6:328-40. 

3. Hitomi K et al. (2001) Int J Biochem Cell Biol. 33:491-8 

4. Jans R et al. (2008) J Invest Dermatol.128:517-29. 


68


Biogenic Amines Counteract Transglutaminase Catalyzed 

Crosslinking of a Recombinant Ovine Prion Protein 

Sorrentino anGela 1 , GioSafatto concetta valeria l. 1 , 

SiranGelo ivana 2 , de SiMone carMela 1 di Pierro ProSPero 1 , 

Porta raffaele 1 and Mariniello loredana 1 

1 Department of Food Science, University of Naples “Federico II”, 

Via Università, 100 – 80055 Portici, Napoli, Italy 

2 Department of Biochemistry and Biophysics “F. Cedrangolo”, 

Second University of Naples, Via L. De Crecchio, 7 - 80138 - Napoli Italy 

Prion proteins are known as the main agents of transmissible spongiform 

encephalopathies affecting humans as well as animals. A recombinant ovine prion 

protein was found to be able to act in vitro as effective substrate for a microbial 

isoform of transglutaminase, an enzyme catalyzing the formation of isopeptide 

bonds inside the proteins (1). We proved that several primary amines and polyamines 

(hydroxylamine, mono dansylcadaverine, spermidine and spermine) effectively 

counteract transglutaminase-mediated prion protein crosslinking. Mass spectrometry 

experiments demonstrated the formation of three intramolecular crosslinks that give 

to the protein the property to be more competent in amyloid formation compared to 

the unmodified one. In addition, the intramolecularly crosslinked prion protein was 

also shown to be more resistant to proteinase K digestion. Our findings suggest a 

possible use of both transglutaminase and biogenic primary amines in investigating 

the molecular basis of the conversion of the prion protein into its pathological form. 

References 

1. Sorrentino A. et al., Biochim. Biophys. Acta, in press. 


69


IN VITRO EFFECTS OF FERMENTED PAPAYA 

(Carica Papaya, L.) 

SUPPLEMENTATION IN TYPE 2 DIABETIC PATIENTS 

raffaelli f 1 , nanetti l 1 , alidori a 1 , Montecchiani G 1 , Borroni f 1 , 

Giulietti a 1 , Sforza G 1 , faloia e 2 , Mazzanti l 1 , viGnini a 1 

1 Department of Clinical Sciences,Faculty of Medicine, Marche 

Polytechnic University – Ancona, Italy 

2 Clinic of Endocrinology, Faculty of Medicine, Marche 

Polytechnic University – Ancona, Italy 

ABSTRACT 

Aims: To evaluate the fermented papaya (FPP) in vitro effect supplementation in DM2 

patients and healthy controls in order to use it as adjuvant of daily therapy. 

Results: The study was performed on 30 patients and 15 healthy subjects, matched 

for age and sex. Platelets were isolated from peripheral venous blood. The study was 

divided into 2 phases: patients and controls platelets were analyzed at recruitment 

(T ) and after in vitro supplementation with FPP at 50 g/ml for 3 hours at 37°C (T ). 

0 1 

On all samples were determined: Na + /K + ATPase activity, membrane fluidity tested 

by anisotropy of fluorescent probes TMA-DPH and DPH, Total Antioxidant Capacity 

(TAC), Superoxide Dismutase (SOD) activity and conjugated dienes levels. 

In vitro FPP supplementation counteracts oxidative damage associated with diabetes 

and its complications; on one hand improves platelet function through increased 

membrane fluidity (tested by decreased anisotropy TMA-DPH and DPH anisotropy) 

and Na + /K + ATPase activity, on the other, enhances antioxidant systems functionality 

by increasing SOD activity and TAC, as well as by decreasing conjugated dienes 

levels significantly in patients versus controls. This platelet functionality enhancement, 

which results in reduction of hyperactivity and hyperaggregability, characteristics of 

diabetes mellitus, suggests a new method of secondary prevention of complications 

associated with platelet dysfunction. 

Innovation and Conclusions: It can be assumed that FPP utilization in the diet of 

diabetics may have, at first, a preventive role and then protective in the progression of 

oxidative damage associated with diabetes and its complications, although the exact 

protective mechanism is still under study. 


70

Biochimica 

Marina e 

dell’Ambiente 

BMA

Biochimica Marina e dell’Ambiente BMA 1 

In vivo E. foetida coelocytes response and ex-vivo 

coelomocytes sub-population sensitivity to DNA damaging 

agents : soil contamination biomarker 

Mincarelli laura 1 , viSchetti coStantino 1 , Monaci elGa 1 , 

tiano luca 2 

1 Università Politecnica delle Marche, 

Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Ancona. 

2 Università Politecnica delle Marche, Dipartimento di Scienze Cliniche Specialistiche 

ed Odontostomatologiche, Ancona. 

Among soil organism earthworms play an essential role in regulating soil processes 

and providing soil fertility. Thanks to their capacity to ingest soil particles and to 

transfer inorganic and organic toxicants trough-out the food chain. Moreover they 

represent relevant soil contamination bio-indicators. Several studies reported the 

use of earthworm coelomoytes to assess pesticides and metals genotoxicity in soil 

organisms using comet assay. coelomocytes are immunocompetent cells, circulating 

in the coelomic fluid, particularly exposed to soil pollutants and they can be extruded 

by exposure of the organism to a saline solution containing ethanol 5%. Subsequently 

celomocyte can be separated by density centrifugation in less etherogeneous 

subpopulations. Data obtained from a mixed population, such as the total extruded 

celomocytes, could be influenced by sub-population specific sensitivity 

In the present study we performed in vivo genotoxicity assay using Cu 2 SO 4 at 75 mg/ 

Kg, 150 mg/kg, 300mg/kg for 3,9, 27 days of exposure; and ex-vivo stress exposure 

of different celomocytes subpopulation using H 2 O 2 in the range of concentration 15- 

120 µM in order to define cell specific dose response and to identify an homogeneous 

cell population to be used as a relevant biomarker. In vivo data confirmed a dose 

response in DNA damage, not affected by exposure time. 

In ex-vivo experiments 3 enriched population were obtaineid at 15%, 25% and 35% 

percoll gradients. All density separated sub-population and the total unseparated 

population show a dose response in DNA damage levels in the tested range. However 

15% percoll sub-population appears more susceptible to the ex-vivo stress, while 

the other two sub-population seem to be more resistant in comparison to the total 

celomocyte population and 15% percoll sub-population. Reported data suggest 

that 15% percoll celomocyte subfraction might represent a sensitive biomarker of 

environmental genotoxic agents; further studies on ceolomocytes sub-populations 

obtained from in vivo exposure should be performed to integrate and confirm these 

results. 


72

Biochimica Marina e dell’Ambiente BMA 2 

Expression of tryptophan hydroxylase (TPH-1) in Carassius 

auratus of different size 

ferlazzo a.M., zanGhì G., Miano M., BruSchetta G., d’aScola a. 1 , 

G. naStaSi 1 , a. avenoSo 1 

Department of Morphology, Biochemistry, Physiology and Animal Production, 

Biochemistry Unit; 1 Department of Biochemical, Physiological and Nutritional 

Sciences, Chemistry Unit, University of Messina 

Tryptophan hydroxylase 1 (TPH-1) is the key enzyme of peripheral serotonin (5-HT) 

synthesis. 5-HT tissue distribution was investigated in Conger conger, Scyliorhinus 

stellaris and Oncorhynchus mykiss (1). TPH-1 mRNA expression was found in tissues 

of small size Carassius auratus (2). Aim of this study was to compare body weight 

influence in TPH-1 expression. Two groups of n 5 fish each were transferred into 2 

tanks of 150 l of fresh water for 2 weeks (Group 1, bw 10g; Group 2, bw 30g; fish 

density 7,5 Kg/m 3 ). Both groups were fed for 3 days with freeze-dried foods. Once 

fish were sacrificed, total RNA extracted from each tissue was quantified. One µg was 

retrotranscribed into copy DNA for PCR Real Time reactions (ABI 7500). Two Assays 

on Demand (TPH-1 and beta-actin) were used. Results were expressed according 

to ΔΔCt calculation and by a relative quantization software. PCR Real Time results 

showed a significant TPH-1 mRNA expression in Group 2 intestine vs Group 1, which 

could be explained with 5-HT inhibitory effect on pituitary growth hormone release 

(3). Whereas Group 1 showed higher expression in gills, probably related to small size 

fish osmoregulation demand. No variation was found in liver of both groups due to 

the same diet. 

1. Caamaño-Tubío R.I. et al. (2007) Comp. Biochem. Physiol., Part C 145, 245–255 

2. Zanghì G. et al. (2012) BMA Messina, 8 giugno 2012, Atti p.20 

3. Somoza G.M. and Peter R.E. (1991) Gen. Comp. Endocrinol., 82,103-110 


73

Biochimica 

Cellulare 

BCe

Biochimica Cellulare BCE 1 

Guanosine-Induced Antiproliferative Effects Are Modulated 

by GPCR Expression in Human Glioma 

and Melanoma Cell Lines 

roBerta Garozzo 1 , valentina di liBerto 2 , Patricia Giuliani 3 , 

franceSco caciaGli 3 , daniele filiPPo condorelli 1 , 

natale Belluardo 2 

1 Department of Chemical Sciences, Sect. of Biochemistry, 

University of Catania, Catania, Italy. 

2 Department of Experimental Biomedicine and Clinical Neuroscience, 

University of Palermo, Palermo 

3 Department of Biomedical Sciences, University of Chieti-Pescara, Chieti, Italy 

Guanine, Guanosine and GMP exerted a marked growth inhibition in the U87 

glioma cell line that was not seen with other tested nucleotides, nucleosides and 

nucleobases and such effect was replicated in several different human tumoral cell 

lines. The loss of guanine effect in a cell line bearing a mutated inactive Hypoxhantine- 

Guanine Phosphorybosil Transferase (HGPRT) and the decreased potency of GUA 

in U87 cells, silenced for HGPRT transcripts, demonstrated the central role of the 

intracellular metabolism of GUA in growth-inhibitory effects. These results do not 

exclude that additional mechanisms, involving activation of membrane receptors, 

might be responsible for cell type-specific biological responses. In the present study, 

the expression of different G protein-coupled receptors was down-regulated by RNA 

interference in the U87 cell line. The silencing of one of this GPCR reduced significantly 

the antiproliferative effects of both guanosine and guanine. Different stably transfected 

cell clones over-expressing this GPCR showed an increased sensitivity to guaninebased 

purines, while their sensitivity to other antiproliferative compounds was not 

modified. Finally, the expression of the GPCR in the HGPRT-mutated melanoma cell 

line re-established a growth-inhibitory response to these substances. These data 

suggest an involvement of GPCRs in modulating cell responses to guanine and 

guanosine and stimulate further research aimed at verifying the hypothesis that a 

membrane protein may act as a functional receptor for guanine-based purines. 

However, the existence of a HGPRT-independent intracellular mechanism potentiated 

by the GPCR expression is an alternative explanation that cannot be excluded on the 

basis of this results. 


76


Protective effects of PrxI over-expressed in neuronal 

model on the toxicity induced by beta-amyloid 

annaMaria ciMini, roBerta Gentile, eliSaBetta Benedetti, 

franceSco anGelucci, GiuSePPina Pitari, rodolfo iPPoliti 

Dept. of Health, Life and Environmental Sciences, 

University of L’Aquila, I-67100 Coppito. 

Peroxiredoxins are ubiquitous proteins that recently attracted major interests in view 

of the strict correlation observed in several cell lines and/or tissues between different 

levels of their expression and the increased capacity of cells to survive in different 

pathophysiological conditions. They are recently considered as the most important 

enzymes regulating the concentration of hydroperoxides inside the cells. In CNS 

all the Prx isoforms are present though with different expression pattern depending 

on cell phenotype. PrxI and IV are constitutively expressed in glial cells but not in 

neurons, whereas PrxII, III, IV and V are expressed in neurons. Interestingly, neurons 

treated with amyloid beta peptide (Abeta), the main component of amyloid plaques 

found in the brain of patients of Alzheimer disease known to alter redox hoemostatsis 

inside cells, showed an overexpression of PrxI. In these conditions, PrxI is only 

moderately hyperxodized while PrxII is mainly found in this form. This finding supports 

the observed protective role of PrxI when it is transfected in PC12 cells or primary 

hippocampal neurons subjected to Abeta exposure. This treatment recovers neurons 

from injury and likely triggers stress oxidative signaling which maintain cell viability. 

In this work taking advantage on cells trasfected by a construct where human PrxI is 

fused with a Green fluorescent protein (GFP) at the C-terminus, we characterized and 

shed light on some events at the basis of cell survival after Abeta injury suggesting 

possible new signal cascades dealing with the antiapoptotic effect of PrxI. The results 

obtained indicated protective role of Prx1 in counteracting Aß damage by increasing 

cell viability, preserving neurites and lowering cell death. 


77


Abscisic acid enhances adipocyte diffentiation and glucose 

uptake in murine 3T3-L1 and in human ATMSC. 

1 Sturla l , 2 Scarfì S, Mannino e, 1 Bruzzone S, 3 aMeri P, 

1 de flora a, 1 zocchi e. 

1 DIMES, Section of Biochemistry, CEBR, University of Genova; 

2 DISTAV, University of Genova; 3 DiSEM, University of Genova. 

The plant hormone abscisic acid (ABA) has been demonstrated to behave as an 

endogenous pro-inflammatory hormone in humans. ABA is released by activated 

inflammatory cells and stimulates several functional activities both autocrinally and 

paracrinally. Interestingly, ABA is also produced and released by human and murine 

pancreatic beta cells and adipocytes in response to glucose, moreover nanomolar ABA 

stimulates glucose-dependent and potentiates glucose-independent insulin release. 

release. Finally, ABA concentration in human plasma increases in healthy subjects 

after oral glucose load. To evaluate the effect of ABA on adipocyte differentiation, 

murine 3T3-L1 and human ATMSC were cultured in differentiation medium in the 

presence of nanomolar ABA concentration; ABA enhances adipogenesis inducing 

lipid accumulation, detected by Oil Red analysis, and triglyceride deposition. As a 

result of the ABA induced adipocyte differentiation, the gene expression of PPARγ 

and its target genes, such adiponectin, AP2 and glucose transporter (GLUT) 4 was 

increased. Moreover, in mature 3T3-L1 adypocytes ABA significantly stimulated the 

basal glucose uptake mediating by GLUT-4 traslocation to the plasmamenbrane, 

evaluated with FACS analysis, confocal imaging, and subcellular fractionation 

experiments. ABA stimulates Akt phosphorylation, similarly to insulin, and the effects 

on glucose uptake are decreased by cotreatment with a PI3K inhibitor, suggesting 

that ABA activates Akt signaling pathway. 

Furthermore, we demonstrated that human adipose tissue incubated in the presence 

of glucose and others inflammatory stimuli releases ABA in the culture supernatant. 

As ABA stimulates insulin release, the endogenous hormone ABA produced by the 

adipose tissue could be involved in regulation of glucose metabolism in mammals. 


78


Targeting ErbB-3 impairs NRG-1-driven mammosphere 

formation of ErbB-2/ErbB-3 expressing 

breast cancer stem cells. 

Graziano vincenzo 1 , Barcaroli daniela 1 , GhaSeMi reza 1 , 

StaSSi GiorGio 2 , 

de laurenzi vincenzo 1 , iacoBelli Stefano 1-3 and Sala Gianluca 1-3 

1 Dipartimento di Scienze Biomediche, Universita’ “G. d’Annunzio” Chieti-Pescara, 

Fondazione “G. D’Annunzio, Centro Studi sull’Invecchiamento, 

Ce.S.I., Via Colle dell’Ara 1, 66100, Chieti, ITALY 

2 Department of Surgical and Oncological Sciences, Cellular and Molecular 

Pathophysiology Laboratory, University of Palermo, 90127 Palermo, Italy 

3 MediaPharma s.r.l. Chieti, Italy 

Cancer stem cells (CSCs) are a small proportion of heterogeneous cells within a tumor 

which show a greater capacity to maintain tumorigenesis than the rest of the cells, and 

are more resistant to traditional cancer therapies. It has been proposed that targeting 

cancer stem cells is possibly a way to obtain durable cancer treatment responses. 

Here we identified ErbB-3 receptor as a molecular target of breast cancer stem cells 

(BCSCs). We found that the ErbB-3-ligand NRG-1 strongly induces clonogenicity 

of BCSCs possibly by activation of the Akt and Erk pathways. Abrogation of ErbB- 

3 expression by stable RNAi completely inhibits the NRG-1-driven mammosphere 

formation and AKT and Erk activation. Moreover, treatment with EV20, a novel 

humanized antibody targeting ErbB-3 inhibits NRG-1-induced ErbB-3 phosphorylation 

and promotes receptor down-regulation. All together these data strongly suggest that 

the NRG-1/ErbB-3/AKT axis may be a relevant target in ErbB-2/ErbB-3 expressing 

BCSCs and make EV20 a promising molecules to be developed for the treatment of 

tumors allowing elimination of cancer stem cells with activated ErbB-3. 


79


Alkaptonuria is a novel human 

secondary amyloidogenic disease 

Millucci l. 1 , SPreafico a. 2 , tinti l. 2 , Braconi d. 1 , Ghezzi l. 1 , 

PaccaGnini e. 3 , Bernardini G. 1 , aMato l. 1 , laSchi M. 1 , luPetti P. 3 , 

orlandini M. 1 , Santucci a 1 . 

1 Dipartimento di Biotecnologie, Chimica e Farmacia, 

2 Dipartimento di Medicina Clinica e Scienze Immunologiche, 

3 Dipartimento di Biologia Evolutiva, Università degli Studi di Siena. 

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic 

acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces 

a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy 

for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, 

Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU 

specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated 

human chondrocytes and cartilage. SAA and SAP deposition was examined using 

immunofluorescence and their levels were evaluated in the patients’ plasma by ELISA. 

2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins 

involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 

7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular 

tissues. SAA and SAP composition of the deposits assessed secondary type 

of amyloidosis. High levels of SAA and SAP were found in AKU patients’ plasma. 

Systemic amyloidosis was assessed by Congo Red staining of patients’ abdominal 

fat and salivary gland. AKU is the second pathology after Parkinson’s disease where 

amyloid is associated with a form of melanin. Aberrant expression of proteins involved 

in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a 

novel type II AA amyloidosis open new important perspectives for its therapy, since 

methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid 

aggregates. 

Grants FMPS 2008-2009-2010, TLSF-Orphan 0108, Telethon GGP10058, FP7-304985. 

1 Millucci L., et al. Biochim Biophys Acta. 2012. In Press. 


80


Fibronectin, laminin and type IV collagen are key 

components of the sinusoids-associated megakaryocyte 

“niche” within bone marrow 

currao Manuela 1 , Malara aleSSandro 1 , GruPPi criStian 1,2 , 

tira M. enrica 2 , Balduini aleSSandra 1,3 . 

1 Department of Molecular Medicine, Laboratory of Biotechnology, 

University of Pavia, IRCCS San Matteo Foundation, Pavia, Italy, 

2 Department of Biology and Biotechnology, University of Pavia, Pavia, Italy 

3 Department of Biomedical Engineering, Tufts University, Medford, MA, USA. 

Megakaryocytes (Mks) develop within the bone marrow environment by interacting 

with different extracellular matrices (ECMs) located at bone and vascular structures 

level. Recent evidences demonstrated that Mks contribute to the establishment of 

stem cell niches by regulating matrix deposition from environmental cells, releasing 

pro- or anti-angiogenic factors and ECM cross-linking enzymes, and supporting 

fibronectin fibrillogenesis. In this work we have analyzed the spatial distribution of 

Mks and ECMs by immunofluorescence in murine femur sections. We found that Mks 

were predominantly located in the femur diaphysis with only 20% of Mks within 50µm 

from the endosteal surface and more than 80% of Mks located less than 50µm from a 

sinusoid. Correlation between Mk distance from sinusoids and dimension suggested 

a gradient of maturing Mks towards the vascular niche. Next, we deciphered bone 

marrow ECM composition by western blotting and mapped the location in situ of 

different collagens (I, III, IV, VI) and glycoproteins (fibronectin, thrombospondin, laminin, 

von Willebrand factor). We found that all these proteins were differently located in the 

endosteal, arteriolar and sinusoidal districts supporting the concept that regulation 

of hemopoiesis, in the bone marrow, may also depend from matrix distribution. 

Further, we showed, for the first time, that Mks were surrounded by a pericellular 

matrix mainly composed of fibronectin, laminin and type IV collagen. Interestingly, 

these three proteins were also demonstrated to promote thrombopoietin-dependent 

Mk differentiation in in vitro cultures of bone marrow hemopoietic progenitor cells 

(3,53±0,86, 1,19±0,11, 1,40±0,23 fold increase, respectively). Finally, fibronectin, 

laminin and type IV collagen were also demonstrated to be expressed and synthesized 

by differentiated Mks in vitro as demonstrated by PCR and western blotting analysis. 

All together these results suggested that Mks are important ECM-producing bone 

marrow cells and that released ECMs support megakaryopoyesis and concur to the 

generation of bone marrow niches. 


81


VEGF receptor-1 involvement in the pericyte loss induced 

by E. coli in an in vitro model of blood brain barrier 

Motta c. a , SalMeri M. B , anfuSo c.d. a , aModeo a. B , Scalia M. c , 

toScano M.a. B , alBerGhina M. a and luPo G. a 

a Dept. Biomedicina Clinica e Molecolare, University of Catania, Italy; 

b Dept. Scienze Bio-Mediche, University of Catania, Italy; 

c Dept. Anatomia, Biologia e Genetica, University of Catania, Italy 

The key aspect of neonatal meningitis is related to the ability of pathogens to invade 

the blood-brain barrier (BBB) and to penetrate the central nervous system [1]. 

In an in vitro model of BBB, on the basis of co-culturing primary bovine brain 

endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), VEGF release 

was higher in comparison with BRPC or BBEC mono-cultures. The growth factor may 

act as a stabilizing agent for co-cultures. E. coli K1, adhere and enter BBEC, but not 

BRPC, by a vesicle-mediated mechanism. In our model of BBB infection, a significant 

loss of pericytes (PC) was observed. Following VEGFR-1, but not VEGFR-2, blockade 

by the specific antibody, elevated TEER values, reduction of E. coli induced-BBB 

permeability and reversion of the PC loss were found. These data suggest that 

functional inhibition of pericytal VEGFR-1 increases their survival. E. coli could exert 

their biological effects on BBB by BRPC, VEGFR-1-mediated, coverage ablation. 

Since VEGF amplified production in co-cultures may be related to the increase in 

prostaglandin biosynthesis, we measured PGE 2 concentration and cytosolic and 

Ca 2+ -independent phospholipase A 2 (cPLA 2 and iPLA 2 ) enzyme activities related to 

arachidonate release [2]. In BBEC in co-culture, as well as in mono-culture [3], bacteria 

are able to stimulate cPLA 2 and iPLA 2 activities, whereas in BRPC no stimulation of 

both activities was found. 

Our results show the role played by the BBEC as well as BRPC during a bacterial 

attack to BBB. A better understanding of the mechanisms by which E. coli enter the 

nervous system and how bacteria alter the communication between endothelial cells 

and PC may provide exciting new insight for clinical intervention. 

References 

1. Zhu, L. et al. (2010) Infect Immun 78: 3554-9. 

2. Alberghina, M. (2010) Microvasc Res 80: 280-285. 

3. Salmeri, M., et al. (2012) Neurosci Lett 511: 33-7. 


82


p57 Kip2 is an early effector of imatinib activity in chronic 

myelogenous leukemia 

caldarelli i, traMontano a, BencivenGa d, BorGia a, Pirozzi a, 

oliva a, della raGione f and Borriello a. 


Second University of Naples, Via De Crecchio 7, 80138, Naples - Italy 

Chronic myelogenous leukemia (CML) is characterized by the expression of BCR–ABL 

tyrosine kinase, which causes increased cell proliferation and inhibition of apoptosis. 

The introduction in therapy of imatinib mesylate (STI571), a highly potent and selective 

inhibitor of BCR–ABL tyrosine kinase activity, led to a tremendous positive impact 

in the treatment of CML and to the development of new target oriented therapies 

in cancer treatments. However, BCR–ABL transcripts are still detectable in patients 

receiving imatinib and the responses in accelerated or blastic phases of the disease, 

when they occur, are generally short-lived. Moreover, a number of imatinib-resistant 

CML cases were evidenced. These findings have fueled the interest in developing 

novel agents to override these limitations and to shed new light on the molecular basis 

of Philadelphia-positive CML. 

In this study, we investigated the effect of imatinib on the cell cycle engine of K562 cells, 

a BCR–ABL-positive cell line. We observed that imatinib causes the accumulation of 

p57 Kip2 in K562 cells. Interestingly, p57 Kip2 increase precedes the reported STI571dependent 

up-regulation of p27 Kip1 and is observed at low concentration of STI571. 

The imatinib-dependent p57 Kip2 increase occurs only in STI571-responsive cells, 

while the CKI accumulation was not evidenced in an imatinib-resistant clone. We 

also demonstrated that p57 Kip2 build-up is due to the transcriptional activation of 

CDKN1C, the p57 Kip2 -encoding gene; while neither p57 Kip2 half-life elongation nor its 

cell relocalization was observed. We demonstrated that p57 Kip2 increase plays an 

important role in the imatinib effects on cell division cycle engine. Finally, nilotinib and 

dasatinib, two second-generation BCR–ABL inhibitors, were also found to upregulate 

p57 Kip2 , acting at very low concentrations. Our findings identify a functional link 

between BCR–ABL and p57 Kip2 , suggesting that this CKI might be a critical effector of 

the anti-proliferative activity of BCR–ABL targeting drugs. 


83


NAD + levels control Ca 2+ stores replenishment and 

mitogen-induced increase of cytosolic Ca 2+ 

by ADPR-dependent TRPM2 gating in human T 

lymphocytes 

MaGnone Mirko 1 , Bauer inGa 2 , PoGGi aleSSandro 3 , 

Mannino elena 1 , Sturla laura 1 , Brini MariSa 4 , zocchi elena 1,5 , 

de flora antonio 1 , nencioni aleSSio 2 and Bruzzone Santina 1,5 

1 Dept of Experimental Medicine, Section of Biochemistry and Center of Excellence 

for Biomedical Research (CEBR), University of Genova, Italy; 

2 Dept of Internal Medicine, University of Genova, Italy; 

3 Molecular Oncology and Angiogenesis Unit, IRCCS AOU San Martino IST-National 

Institute for Cancer Research, Genova, Italy; 

4 Dept of Comparative Biomedicine and Food Science, University of Padova, Italy; 

5 Advanced Biotechnology Center (ABC), Genova, Italy. 

Intracellular NAD + levels ([NAD + ] i ) are important in regulating human T lymphocytes 

survival, cytokine secretion and the capacity to respond to antigenic stimuli. NAD + - 

derived Ca 2+ -mobilizing second messengers, produced by CD38, play a pivotal role 

in T cell activation. 

Here we demonstrate that [NAD + ] i modifications in T lymphocytes affect intracellular 

Ca 2+ homeostasis, both in terms of mitogen-induced [Ca 2+ ] i increase and of 

endoplasmic reticulum Ca 2+ store replenishment. Lowering [NAD + ] i by FK866-mediated 

Nicotinamide phosphoribosyltransferase inhibition, decreased the mitogen-induced 

[Ca 2+ ] i rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca 2+ content 

of Thapsigargin-sensitive Ca 2+ stores was greatly reduced in these cells in the presence 

of FK866. When NAD + levels were increased by supplementing peripheral blood 

lymphocytes with the NAD + precursors nicotinamide, nicotinic acid or nicotinamide 

mononucleotide, the Ca 2+ content of Thapsigargin-sensitive Ca 2+ stores, as well as 

cell responsiveness to mitogens in terms of [Ca 2+ ] i elevation, were up-regulated. 

The use of specific siRNA showed that the changes of Ca 2+ homeostasis induced 

by NAD + precursors are mediated by CD38 and the consequent ADPR-mediated 

TRPM2 gating. Finally, the presence of NAD + precursors up-regulated important T cell 

functions, such as proliferation and IL-2 release in response to mitogens. 


84


Regulation of IP6K1 gene expression: role of the prossimal 

promoter GC-rich Sp1 binding region 

de luca antonella a , Sanna faBio a , Sacchetta Paolo a , 

di ilio carMine a , Saiardi adolfo B , favaloro Bartolo a 

a Department of Sperimetal and Clinical Sciences, 

University of Chieti “G. D’Annunzio” School of Medicine, 

b Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, 

Department of Cell and Developmental Biology, University College London. 

Inositol serves as a module for the generation of a high level of molecular 

diversity through the combinatorial attachment and removal of phosphate groups. 

Inositolphosphates can be further phosphorylated by three inositol hexakisphosphate 

kinase isoforms (IP6K1, 2 and 3) to form inositol pyrophosphates (IP7). Inositol 

pyrophosphates are organic molecules with the highest proportion of phosphate 

groups, they have been linked to phosphate homeostasis and they are capable of 

regulating many biological processes, possibly by controlling energetic metabolism 

and adenosine triphosphate (ATP) production 1 . 

The function of IP6K and inositol pyrophosphates is still unclear. There is some 

evidence that IP6Ks are involved in vesicle trafficking and cell proliferation, as well as 

in tumorigenesis 2 . 

There are three different IP6K isoforms in humans, suggesting that IP6Ks are involved 

in more than one signaling pathway and might be regulated in a spatial and temporal 

manner. In this work 1815 bp of sequence located upstream of the first exon of ip6k1 

gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector 

and transfected into different cell lines. Sequentially smaller fragments of the ip6k1 

promoter region were generated by PCR, and expression levels were monitored from 

these constructs within HEK-293 and MCF7 human cell lines. A pivotal role for Sp1 

in the constitutive expression of the ip6k1 gene was demonstrated through transient 

expression of mutant IP6K1 promoter-reporter gene constructs and chromatin 

immunoprecipitation experiments. These studies offer additional insights into 

mechanisms regulating IP6K expression that can ultimately impact carcinogenesis. 

References. 

1. Szijgyarto, Z., Garedew, A., Azevedo, C. & Saiardi, A. Influence of inositol pyrophosphates on cellular 

energy dynamics. Science 334, 802-805 (2011). 

2. Morrison, B.H., et al. Gene deletion of inositol hexakisphosphate kinase 2 predisposes to aerodigestive 

tract carcinoma. Oncogene 28, 2383-2392 (2009). 


85


Redox regulation of the tumor suppressor Phosphatase 

and tensin homolog deleted on chromosome ten (PTEN) 

by methionine sulfoxide reductase A 

de luca antonella, Sanna faBio, Sacchetta Paolo, di ilio carMine, 

favaloro Bartolo 

Fondazione “G. D’Annunzio”, Centro Studi sull’Invecchiamento, 

Department of Experimental and Clinical Sciences, 

University of Chieti “G. D’Annunzio” Chieti, Italy 

Breast cancer is one of the most frequent malignancies, and it is therefore 

fundamental to identify the underlying molecular mechanisms leading to cancer 

transformation, progression and metastatization. Among other causative agents in 

the development of breast cancers, an important role for reactive oxygen species 

(ROS) has emerged. Very low levels of H2O2 can oxidize methionine (Met) residues in 

proteins to methionine sulfoxide (MetO). Methionine sulfoxide reductase A (MsrA) is 

an antioxidant repair enzyme that reduces MetO to Met. This cyclic interconversion of 

Met and MetO residues of proteins is involved in several different biological processes 

such as scavenging of ROS, regulation of enzyme activities, and cell signaling. We 

have previously shown that MsrA is down-regulated in a number of human breast 

cancers leading to the development of two important hallmarks of tumorigenesis, 

cell proliferation and extracellular matrix degradation 1 . The underlying molecular 

mechanisms involve increased H2O2 levels, and activation of the phosphoinositide 

3-kinase (PI3K) pathway that is up-regulated in several human cancer. Our hypothesis 

is based on the fact that MsrA down-regulation in cancer can potentiate the H2O2 

signaling pathway by disactivating the tumor suppressor PTEN. In an effort to expand 

our previous work, here we show that silencing of MsrA in MDA-MB231 cells induces 

post-translational downregulation of PTEN both in vitro as well as in vivo models. 

References 

1 De Luca A., Sanna F., Sallese M., Ruggiero C., Grossi M., Sacchetta P., Rossi C., De Laurenzi V., Di Ilio 

C., Favaloro B. Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a 

more aggressive phenotype. Proc Natl Acad Sci U S A 107, 18628-18633 (2010). 


86


Fzd6 a Novel Prognostic Marker in Breast Cancer 

lattanzio roSSano 1,2,6 , MottoleSe Marcella 3 , natali Pier GiorGio 4,6 , 

Graziano vincenzo 1 , de laurenzi vincenzo 1 , Sala Gianluca 1,6 , 

iacoBelli Stefano 1,6 , Sala arturo 5 and Piantelli Mauro 1,2,6 

1 Departement of Biomedical Sciences, University “G. D’Annunzio” Chieti, Italy 

2 Center of Excellence for Research on Aging, Foundation 

University “G. D’Annunzio” Chieti, Italy 

3 Department of Pathology, Regina Elena Cancer Institute, Rome, Italy 

4 CINBO Laboratories, University “G. D’Annunzio” Chieti, Italy 

5 Brunel Institute of Cancer Genetics and Pharmacogenomics, 

Brunel University, London, UK 

6 MediaPharma s.r.l. Chieti, Italy 

Wnt signaling regulates cell fate decisions throughout embryonic development and is 

involved in human cancer 1-3 . The seven trans-membrane protein frizzled (Fzd) transduce 

the Wnt signal and 10 receptors have been so far identified.We have previously 

reported that the expression of one of such receptors, Fzd6, predicts poor survival 

in neuroblastoma patients 4 . We have recently analyzed Fzd6 expression in a panel of 

breast cancers comparing it with the clinical outcome of patients with node negative, 

T1/T2 breast cancers. The study group consisted of 252 patients presenting with 

primary unilateral breast carcinoma (T1-2), with no evidence of nodal involvement and 

distant metastases. Fzd6 protein expression was assessed by immunohistochemistry 

on tissue microarrays. We also studied Fzd6 distribution in breast cancer molecular 

subtypes 5 . The results were correlated with clinical data using Kaplan–Meier curves 

and multivariate Cox regression analysis. In tumor cells the expression of Fzd6 was 

membranous, cytoplasmic and nuclear. Only the overexpression of cytoplasmic 

Fzd6 (c-Fzd6) was a significant and independent negative prognostic factor for 

distant metastases. This was observed in the whole population and in patients with 

Luminal-B/HER2 negative cancers. Thus, c-Fzd6 could be a new prognostic marker 

and potential target of therapy in breast cancer. 

References 

1) Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005;434:843-50. 

2) Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469-80. 

3) MacDonald BT, et al. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 

2009;17:9-26. 

4) Cantilena S, et al. Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and 

human neuroblastomas. Oncotarget 2011;2:976-83. 

5) Goldhirsch A, et al. Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the 

St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann 

Oncol;22:1736-47. 


87


Ceramide flow from the Endoplasmic Reticulum to the 

Golgi apparatus is impaired in Glucolipotoxic conditions in 

INS-1 β cells 

GiuSSani Paola 1 , GJoni enida 1 , cinque aleSSandra 1 , 

le Stunff hervé 2 , riBoni laura 1 , viani Paola 1 

1 Department of Medical Biotechnology and Translational Medicine, 

University of Milano, LITA Segrate, Via Fratelli Cervi, 93, 20090 Segrate (MI) Italy 

2 Unité Biologie Fonctionnelle et Adaptative - EAC CNRS 4413, 

Laboratoire de Biologie et Pathologie du Pancréas Endocrine, 

Université PARIS- DIDEROT (7),4, rue Marie-Andrée Lagroua Weill-Halle, 

75205 PARIS Cedex 13 France 

In type 2 diabetes the detrimental effects of chronic exposure to NEFA, in particular 

palmitate, on β-cell function and viability have been associated to hyperglycaemia. 

ER stress is among the molecular pathways and regulators involved in these negative 

effects. Moreover, it is known that accumulation of ceramide due to glucolipotoxicity 

can be associated to the induction of ER stress. The increased ER ceramide levels can 

be due to an enhanced ceramide biosynthesis and a decrease in ceramide utilization 

as well. In the control of ER ceramide levels it is crucial ceramide trafficking between 

ER and Golgi, which represents the site of complex sphingolipid biosynthesis. In order 

to gain insight into the molecular mechanism(s) of glucolipotoxicity we studied the 

effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells. 

Metabolic studies show that treatment with palmitate results in the inhibition of 

ceramide utilization for SM and glycosphingolipid biosynthesis. Fluorescence 

microscopy studies suggest a reduced ER to Golgi flow of ceramide. To evaluate 

if ceramide accumulation could be due to a defect in the vesicular and/or CERTmediated 

transport of ceramide we studied CERT expression and ceramide 

metabolism in INS-1 cells silenced for CERT incubated with glucose in the presence 

or not of palmitate. Glucolipotoxicity decreased the total amount of CERT and, by 

inducing CERT phosphorylation, prevented its localization to the Golgi of pancreatic 

β-cells. Moreover in CERT-downregulated cells palmitate still inhibited ceramide 

utilization for SM biosynthesis. These results demonstrate that both the vesicularmediated 

and CERT-mediated transport are involved in the reduced ER to Golgi flow 

of ceramide in glucolipotoxic conditions. 

Altogether these data suggest that the accumulation of ceramide can be due to a 

decrease in utilization of newly synthesized ceramide for SM biosynthesis thus 

supporting a role of ER-associated ceramide in the regulation of β-cell death induced 

by gluco-lipotoxicity. 


88


Chemotherapy associated oxidative stress: role of 

methionine sulfoxide reductase A 

Sanna faBio a , de luca antonella a , ciavardelli doMenico a , 

lanuti Paola B , roSSi claudia a , Sacchetta Paolo a , di ilio carMine a , 

favaloro Bartolo a 

a Department of Sperimetal and Clinical Sciences, 

University of Chieti “G. D’Annunzio” School of Medicine, 

b Department of Medicine and Aging Science (D.M.S.I.), University G. d’Annunzio 

of Chieti-Pescara, Via dei Vestini 31, 66013, Chieti Scalo (CH), Italy. 

Doxorubicin is currently considered to be one of the most effective agents in treatment 

of breast cancer, unfortunately, resistance to this agent is common, representing 

a major obstacle to successful treatment 1 . The anthracyclines generate by far the 

highest level of reactive oxygen species (ROS) of all anti-neoplastic agents, in contrast 

to the above noted families of antineoplastic agents, such as paclitaxel a member of 

taxanes family 2 . 

MsrA is a member of methionine sulfoxide reductases system, known to protect 

proteins from oxidation and act as a ROS scavenger. Msr system is considered an 

important component of the defence mechanism against oxidative damage. In a 

previously paper we shown that down regulation of methionine sulfoxide reductase 

A in breast cancer results in a more aggressive phenotype 3 . Here we show, that 

loss of MsrA sensitizes MDA-MB231 cells to the cytotoxic effect of doxorubicin but 

not paclitaxel. Our hypothesis is based on the fact that MDA-MB231 silenced for 

the MsrA are likely to be more vulnerable to damage by further ROS insults induced 

by exogenous agents. To investigate whether MsrA silenced MDA-MB231 (shMSrA) 

and control MDA-MB231 (mock) differed in sensitivity after doxorubicin or paclitaxel 

treatment, we evaluate cell growth by using MTT assay on classic monolayer (2D) as 

well as in Matrigel cultured cells (3D). Here we also demonstrate that the increase of 

intracellular ROS in shMsrA MDA-MB231 correlate with the reduced MsrA enzyme 

activity detected in these cells. Leading to the hypothesis that, cancer cells with 

increased oxidative stress are likely to be more vulnerable to damage by further ROS 

insults induced by exogenous agents. 

References 

1. Smith L, Watson MB, O’Kane SL, Drew PJ, Lind MJ, Cawkwell L. The analysis of doxorubicin resistance 

in human breast cancer cells using antibody microarrays. Mol Cancer Ther 5, 2115-2120 (2006). 

2. Conklin, K.A. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. 

Integr Cancer Ther 3, 294-300 (2004). 

3. De Luca A., Sanna F., Sallese M., Ruggiero C., Grossi M., Sacchetta P., Rossi C., De Laurenzi V., Di Ilio 

C., Favaloro B. Methionine sulfoxide reductase A down-regulation in human breast cancer cells results 

in a more aggressive phenotype. Proc Natl Acad Sci U S A 107, 18628-18633 (2010). 


89


New perspective in the assessment 

of total intracellular magnesium 

1 SarGenti azzurra, 2 Merolle lucia, 3 andreani Giulia, 

1 caPPadone concettina, 1,4 farruGGia Giovanna, 2,4 iotti Stefano, 

5 Marraccini chiara. 

1 Dipartimento di Biochimica “G. Moruzzi”, Alma Mater Studiorum, 

Università di Bologna; 2 Dipartimento di Medicina Interna dell’Invecchiamento e 

delle Malattie Nefrologiche, Alma Mater Studiorum Università di Bologna; 

3 Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum, 

Università di Bologna; 4 Istituto Nazionale Biostrutture e Biosistemi; 

5 Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia 

Aim of this study is to perform a quantitative assessment of total intracellular 

magnesium by a new hydrossiquinoline derivative chemosensor. 

Although magnesium is essential for numerous biological processes, its homeostasis 

have not been thoroughly elucidated. However some peculiarities in its regulation 

are newly emerging. Free and total magnesium undergo to independent regulatory 

mechanisms therefore discriminating between the two pools is crucial. Atomic 

absorption spectroscopy (AAS) is the reference technique to measure total magnesium 

content but it requires volatilization of the sample and a high number of cells (several 

millions). Recently, growing interest has been raised by a new family of fluorescent 

probes DCHQ (Diaza-18-Crown-6-8-HydroxyQuinolines.), as it shows high affinity 

and specificity for magnesium. In particular, the mother probe DCHQ1 is capable to 

image intracellular Mg in living cells and to assess total cellular magnesium in small 

samples, providing overlapping results with AAS [1]. However, DCHQ1 showed some 

limitations, such as poor intracellular retention and a certain degree of fluorescence 

instability, invalidating its use, for instance, in time-based measures of magnesium 

fluxes. A new phenyl-derivative (DCHQ5) showed peculiarities that exceed these 

limitations, being highly retained within cells and remaining stable up to 30 minutes 

of incubation [2]. DCHQ5 displayed higher fluorescence enhancement upon Mg 

binding than DCHQ1, being excitable also in the visible range. In this work we tested 

DCHQ5 analytical capability: this probe can quantitatively assess the intracellular total 

magnesium, allowing to detect variations due different proliferative states, in samples 

even smaller than those required for DCHQ1. 

In conclusion DCHQ5 can be considered a reference molecular probe for the study 

of intracellular magnesium homeostasis, broadening the field of application of DCHQ 

chemosensors. 

1 Farruggia G et al. (2008) J Fluoresc, 19:11-19. 

2 Marraccini C et al. (2012) Chem Sci, 3:727-734. 


90


Imaging flow cytometry: the prospective cellular analysis 

Gallorini Marialucia 1 , Sancilio Silvia 1 , de colli Marianna 1 , 

di valerio valentina 2 , di GiacoMo viviana 1 

1 Pharmacy Department, University “G. d’Annunzio”, Chieti-Pescara, Italy. 

2 Medicine and Ageing Sciences Department, 

University “G. d’Annunzio”, Chieti-Pescara, Italy. 

The image flow cytometry provides a compromise between the statistically data sets 

and the multiparametric, high throughput of a conventional flow cytometer and the 

spatial information of a traditional fluorescence microscope 1 . 

Here we focused on membrane β1-integrin expression in human gingival fibroblasts 

treated with HEMA (2-hydroxyethilmethacrilate), a monomer released by materials 

commonly used in restorative dentistry and co-coltured with Streptococcus mitis, a 

microorganism usually present into the oral cavity. 

Even if in HEMA and saliva treated sample the β1-integrin positive cell percentage 

is reduced with respect to the control sample, the fluorescence intensity in cell 

membrane is augmented. Such subtle data analysis is obtained by means of the new 

IDEAS 5.0 software, which allows operators to visualize each cell and to leave out 

nonspecific fluorescent ones. In addition to conventional flow cytometry parameters, 

the software is equipped with a variety of new tools, such as different kind of masks 

(sets of pixels which contain a region of interest), which is possible to combine each 

others in order to specify the results obtained. 

Moreover IDEAS, finding the best discriminating feature among two different 

populations, gives the most insightful cell analysis possible and enables a broad 

range of applications that would be impossible using either technique alone 2 . 

1. Filby A, Davies D. Cytometry A. 2012 Jul 3. doi: 10.1002/cyto.a.22091. 

2. Basiji DA, Ortyn WE et al. Clin Lab Med. 2007, 27:653-70. 


91


Monitoring magnesium efflux cAMP-induced in HL60 cells 

by using a new hydroxyquinoline fluorescent chemosensor 

1 Marraccini chiara, 2 SarGenti azzurra, 3 Merolle lucia, 

2 caPPadone concettina, 2,4 farruGGia Giovanna, 2,4 iotti Stefano. 

1 Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia; 


Università di Bologna; 

3 Dipartimento di Medicina Interna dell’Invecchiamento e delle Malattie Nefrologiche, 

Alma Mater Studiorum Università di Bologna; 

4 Istituto Nazionale Biostrutture e Biosistemi. 

This work aimed at monitoring the intracellular magnesium movements cAMPinduced 

by using DCHQ5, the phenyl-derivative of hydroxyquinolines fluorescent 

probe family, which is suitable for the determination of total magnesium [1]. Cellular 

magnesium homeostasis is still unclear, although this cation is essential for numerous 

cell functions. Several studies documented the occurrence of fluxes of magnesium 

across the plasmamembrane within minutes from the application of metabolic or 

hormonal stimuli. These fluxes result in limited variation of free intracellular Mg 2+ 

concentration and large changes in total magnesium content. 

DCHQ5 probe, which is stable up to 30 minutes of incubation and highly retained within 

loaded cells, results to be the proper tool for monitoring the magnesium fluxes [1]. 

It has been reported that a stimulation with cAMP caused a movement of total 

magnesium within 10 minute after treatment in cardiomyocytes, suggesting that this 

stimuli caused a magnesium efflux in excitable cells [2]. In this study we tested this 

hypothesis in HL60 leukemic cells, not excitable but highly proliferating cell model. We 

evaluated Mg flux by DCHQ5 using spectrofluorimetric and cytofluorimetric assays: a 

marked decrease of intracellular total magnesium was observed in the first 3 minutes. 

We also verified that at least 10% of the total intracellular amount of magnesium 

moved in the supernatant of stimulated cells, confirming the extracellular Mg efflux 

induced by cAMP. The evaluation of free intracellular magnesium by Mag-Fluo-4 

probe revealed no variation of this fraction. 

These results highlight the capability of DCHQ5 for the quantification of total 

intracellular magnesium, as well as for monitoring magnesium cellular fluxes, hence 

representing a suitable tool to deepen our knowledge on magnesium homeostasis. 

1 Marraccini C et al. (2012) Chem Sci, 3:727-734. 

2 Romani A et al. (1991) J Biol Chem, 266:24376-24384. 


92


Intracellular magnesium content changes during 

mitochondria mediated apoptosis: in depth study of early 

events on Δψ 

1 Merolle lucia, 2 caPPadone concettina, 2,3 farruGGia Giovanna, 

4 Marraccini chiara, 2 SarGenti azzurra, 5 colanardi aleSSia, 

1,3 iotti Stefano. 

1 Dipartimento di Medicina Interna dell’Invecchiamento e delle Malattie Nefrologiche, 

Alma Mater Studiorum, Università di Bologna, 


Università di Bologna; 3 Istituto Nazionale Biostrutture e Biosistemi, 

4 Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia; 

5 Istituto di Farmacologia Traslazionale- CNR L’Aquila 

A recent study showed that an indole-derivative induces mitochondria-mediated 

apoptosis and a variation of intracellular magnesium concentration in HT29 colon 

cancer cells [1]. After 48 hours of treatment several effects were observed: cell 

cycle arrest in G2/M phase, depolarization of mitochondria membrane, release of 

cytochrome c from mitochondrial compartment and caspase activation [2]. These 

events were accompanied by a decrease of intracellular magnesium, evaluated by 

flowcytometry and atomic absorption spectroscopy techniques. 

Mitochondria are known to be the main intracellular Mg 2+ store and there are many 

experimental evidences showing that mitochondrial depolarization triggers Mg 2+ 

release [3]. Aim of this work was to monitor magnesium fluctuations throughout 

apoptosis, from the early to the final stage of the process. It is known that bound and 

free magnesium fractions undergo to different regulatory mechanisms, therefore we 

measured both free and total intracellular concentrations of the cation. In addition 

the current literature on the relationship between magnesium and apoptosis focuses 

on the free fraction, which represents only a 10% of the total amount. We exploited 

the characteristics of a new chemosensor of the DCHQ family, which allows the 

quantitative assessment of total intracellular magnesium. 

The early events characterizing the apoptosis induction showed a correlation between 

ΔΨ and free intracellular magnesium content while no significant variations in the total 

pool were detected. It is crucial to clarify whether these different behaviour that occurs 

following the apoptotic stimulus was a coincidental event or a causative determinant 

of the apoptotic cascade. In conclusion the role of magnesium in apoptosis is far 

from being defined: further experimental data are needed to elucidate the involvement 

of this cation in the apoptotic process. 

1 Cappadone C., et al (in press) Mag. Res. 

2 Andreani A, et al., (2012) Med Chem; 55:2078-88. 

3 Kubota T. et al., (2005) Bioch. et Biophys. Acta 1744 19–28. 


93


NF-κB mediated down regulation of collagen synthesis 

upon HEMA treatment of hgf/S.mutans co-cultured cells 

Pacella StePhanie 1 , zara SuSi 2 , raPino Monica 3 , Marconi Guya 2 , 

cataldi aMelia 2 , Grande roSSella 2 

1 Medicine and Ageing Sciences Department, University “G. d’Annunzio”, 

Chieti-Pescara, Italy. 

2 Pharmacy Department, University “G. d’Annunzio”, Chieti-Pescara, Italy 

3 Institute of Molecular Genetics CNR Unit of Chieti 

2-hydroxyethyl methacrylate (HEMA) is a key component of resin-based compounds, 

frequently found in aqueous eluates from polymerized dental biomaterials whose 

effects on eukaryotic and prokaryotic cells have been separately investigated (1-3). 

Since in our lab a human HGF/ S. mutans co-culture model has been set up, the effect 

of 3mM HEMA on the intracellular signalling pathway, regulating the adhesion of such 

cells, has been evaluated. 

Even though no significant difference is detected in the expression of collagen I gene, 

an HEMA dependent decrease in collagen protein expression is observed even in 

the presence of S. mutans. Because a direct correlation has been previously shown 

between collagen suppression and nuclear transcription factor NFκB activation, 

attention has been focused on IκBα, the most important regulator of NF-κb which 

,once phosphorylated, allows the translocation of NFκB. So an increase of IκB 

phosphorylation in HEMA treated HGF, which persists also in co-culture, in the 

presence or not of HEMA, is found. 

These data suggest that HEMA-dependent collagen decrease biosynthesis in HGF 

results from activation of NF-κB even though it seems to be modulated by S. mutans 

presence. 

1. J.T. Samuelsen et al. Dent Mater. 2008; 24: 134-40. 

2. G. Teti et al.J Biomed Mater Res A. 2009; 90: 256-62. 

3.D’Ercole et al. Lett Appl Microbiol. 2011; 5: 193-200. 


94


TNF-α modulates HEMA-dependent inflammatory response 

in human gingival fibroblasts: an in vitro study 

di niSio chiara 1 , di GiacoMo viviana 1 , zara SuSi 1 , 

Pacella StePhanie 2 , cataldi aMelia 1 

1 Pharmacy Department , G. d’Annunzio University, Chieti, Italy; 

2 Medicine and Ageing Sciences Department, G. d’ Annunzio University, Chieti, Italy 

2-Hydroxyethyl methacrylate (HEMA) is a component of dentin-bonding systems. 

Our study aimed to investigate the inflammatory response in human gingival fibroblasts 

(HGFs) exposed to a relatively low HEMA concentration (3mM for 24 and 96 h) and 

the molecular mechanisms underlying this effect, evaluating gene expression of tumor 

necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), nuclear factor kappa B1 

(NFkB1 or p50), RelA or p65 and inhibitor of kappa light polypeptide gene enhancer 

in B-cells, kinase beta (IKBKB) by real-time reverse transcription-polymerase chain 

reaction. 

Cultured HGFs were exposed to 3 mM HEMA for 0, 24 or 96 h. 

24-h treatment enhances TNF-α mRNA levels of about 66% compared to control. 

TNF-α gene expression is about fivefold higher in 96-h-treated cells compared to 

control. 

COX-2 mRNA levels are about twofold higher in 24-h-treated cells and fourfold higher 

in 96-h-treated HGFs compared to control. 

NFkB1 gene expression is about twofold higher in 24-h-treated cells compared to 

control; 96-h incubation decreases NFkB1 mRNA levels of about 30% compared to 

control. 

24-h treatment enhances RelA gene expression of about 32%, while 96-h exposure 

decreases it of about 31% compared to control. 

24-h incubation increases IKBKB mRNA levels of about 59%, while 96-h treatment 

decreases them of about 38% compared to control. 

TNF-α – stimulated HGFs activate COX-2 gene transcription via NFkB [1]: in our 

experimental model, 24-h HEMA exposure could therefore induce an inflammatory 

response modulated by the increase in TNF-α gene expression, the consequent 

increase of NFkB1 and RelA mRNA levels and the activation of COX-2 gene 

transcription. Decreased NFkB1 and RelA mRNA levels after 96-h HEMA incubation 

could derive from severe cytotoxic effects, as shown by trypan blue dye exclusion 

test, even though COX-2 response persists. 3 mM HEMA exposure can therefore 

induce in HGFs both inflammatory and cytotoxic effects. 

[1] Nakao S et al. (2002). Mol Cell Biochem 238:11-18. 


95


Increased iNOS activity in Vascular Smooth Muscle Cells 

from diabetic rats: potential role of Ca 2+ /Calmodulindependent 

Protein Kinase IIδ 2 (CaMKIIδ 2 ). 

di Pietro natalia 1,4,6 , di toMo PaMela 1,4,6 , di SilveStre Sara 1,4 , 

Giardinelli annaliSa 1,4 , PiPino caterina 1,4 , MoraBito caterina 2,4 , 

forMoSo Gloria 3,4 , MariGGiò Maria addolorata 2,4 , 

Pandolfi aSSunta 1,4 

1 Department of Experimental and Clinical Sciences, 

2 Department of Neuroscience and Imaging, 

3 Department of Medicine and Aging Science, University “G. d’Annunzio”; 

4 Aging Research Center, Ce.S.I., “G. d’Annunzio” 

University Foundation, Chieti-Pescara, Italy. 

Objective. 

Inducible nitric oxide synthase (iNOS) expression may be increased by cytokine 

plasma levels contributing to vascular damage in diabetes. Besides transcriptional 

regulation, Ca 2+ /CaMKIIδ 2 may play a role in post-translationally controlled iNOS 

activity. We accordingly investigated the involvement of the Ca 2+ /CaMKIIδ 2 signaling 

pathway in regulating lipopolysaccharide (LPS)-induced iNOS activity in cultured 

aortic vascular smooth muscle cells (VSMCs) from diabetic rats. 

Methods and Results. 

VSMCs obtained from 10 diabetic rats (DR) and 10 control rats (CR) were stimulated 

with 20 µg/ml LPS. After 24 hours, iNOS protein levels were 1.37 fold increased in DR- 

vs CR-VSMCs (p < 0.05; Western Blot), while iNOS activity (conversion L-(3H)-arginine 

into L-(3H)-citrulline) and intracellular nitrotyrosine levels (immunofluorescence) 

were about 2.7 fold greater in DR- than in CR-VSMCs. Interestingly, LPS increased 

intracellular Ca 2+ levels (Fluorescence video imaging) more markedly in DR- than 

in CR-VSMCs. This was associated with CaMKII activation by phosphorylation, a 

decreased amount of co-immunoprecipitating iNOS/CaMKIIδ 2 (Western Blot) and 

increased iNOS activity. The CaMKII-inhibitor KN-93 abolished all the LPS-effects. 

Conclusion. 

These results indicate that the Ca 2+ /CaMKIIδ 2 signaling pathway may be an important 

regulator of iNOS activity in diabetes, and hence contribute to the potential development 

of innovative therapeutic strategies for vascular complications in diabetes. 


96


β-Carotene and lycopene affect endothelial response to 

TNF-α reducing nitro-oxidative stress and interaction 

with monocytes 

di toMo PaMela 1,2 , canali raffaella 3 , ciavardelli doMenico 2,4 , 

di SilveStre Sara 1,2 , de Marco aleSSandro 2,5 , 

Giardinelli annaliSa 1,2 , PiPino caterina 1,2 , di Pietro natalia 1,2 , 

virGili faBio 3 and Pandolfi aSSunta 1,2 . 

1 Department of Experimental and Clinical Sciences, 

‘G. d’Annunzio’’ University, Chieti-Pescara, Italy. 

2 Aging Research Center, Ce.S.I., 

‘Gabriele d’Annunzio’’ University Foundation, Chieti-Pescara, Italy. 

3 National Research Institute for Food and Nutrition, (INRAN), Roma, Italy. 

4 Faculty of Motor and Health Science, Kore University, Enna, Italy. 

5 Department of Medicine and Aging Science, 

‘‘G. d’Annunzio’’ University, Chieti-Pescara, Italy. 

Scope: 

Cardiovascular disease (CVD) is associated with vascular oxidative imbalance and 

inflammation. Increased reactive oxygen species (ROS) generation is associated with 

a functional inactivation of nitric oxide (NO) due to the reaction with O2 - , leading to 

peroxynitrite (ONOO - ) formation and subsequent reduction in the beneficial effect 

of vascular NO bioavailability. Carotenoids’-rich diets have been associated with 

decreased risk of CVD, but the underlying mechanism is still unknown. 

Methods and Results: 

In human umbilical vein endothelial cells (HUVECs), both β-carotene (BC) or 

lycopene (Lyc) significantly affected Tumor Necrosis Factor-α (TNF-α)-induced 

inflammation, being associated with a significant decrease in the generation of ROS 

(spectrofluorometry) and nitrotyrosine (an index of ONOO- formation, cytofluorimetry), 

an increased NO/cGMP (cyclic guanosine monophosphate) levels (EIA), and a downregulation 

of NF-kB-dependent adhesion molecule expression (Western blot and 

EMSA) and monocyte–HUVEC interaction (adhesion assay). Our results indicate that 

BC or Lyc treatment reduce the inflammatory response in TNF-α-treated HUVECs. This 

is due to the redox balance protection and to the maintenance of NO bioavailability. 

Conclusion: 

Our observations provide background for a novel mechanism for carotenoids’antiinflammatory 

activity in the vasculature and may contribute to a better understanding 

of the protective effects of carotenoid-rich diets against CVD risk. 


97


Characterization of Nitric Oxide enzymatic production in 

red blood cells from End Stage Renal Disease patients 

di Pietro natalia 1,3 , Giardinelli annaliSa 1,3 , Sirolli vittorio 2 , 

di toMo PaMela 1,3 , PiPino caterina 1,3 , di SilveStre Sara 1,3 , 

de Marco aleSSandro 3 , BonoMini Mario 2 , Pandolfi aSSunta 1,4 . 

1 Department of Experimental and Clinical Sciences; 

2 Department of Medicine University “G. d’Annunzio”; 3 Aging Research Center, 

Ce.S.I., “G. d’Annunzio” University Foundation, Chieti-Pescara, Italy. 

Aims 

Red blood cells (RBCs) play a fundamental role in Nitric oxide (NO) bioavailability, 

providing the binding for NO by haemoglobin contained in the RBCs. Although, to 

date NO vascular synthesis has been ascribed exclusively to endothelium through 

endothelial Nitric Oxide Synthase (eNOS) activity, recently eNOS-like expression 

and activity in human RBCs has been demonstrated. Since end-stage-renal-disease 

(ESRD) patients suffer from endothelial dysfunction associated to reduced NO 

bioavailability, the aim of this study is to characterize enzymatic NO production in 

ESRD-RBCs . 

Methods and Results 

RBCs from control subjects (C, N=12) or ESRD patients (N=12) on chronic 

maintenance hemodialysis were collected and processed to determine: eNOS 

expression and phosphorylation levels (Real Time PCR, Immunoprecipitation, 

Immunofluorescence Confocal Microscopy, Flow Cytometry); Calmodulin (CaM), Heat 

shock protein 90 (HSP90) and Caveolin-1 (cav-1) levels and their eNOS interaction 

by Immunoprecipitation. Erythrocytes NOS activity (DAF-2DA measurement by Flow 

Cytometry) and NO bioavailability (intracellular cGMP levels by Enzymeimmunoassay) 

were evaluated following insulin (100 nM) stimulation in the presence or absence of 

selective eNOS inhibitor (L-NAME, 1 mM). 

Results show that both C- and ESRD-RBCs express a functional eNOS. Of note, 

basal eNOS expression/activation and cGMP levels were significantly higher in ESRD- 

than C-RBCs. On the other hand, insulin significantly increased both NO release and 

cGMP levels only in C-RBCs. 

Conclusions 

Our results showed for the first time an increased basal enzymatic NO production and 

cGMP accumulating levels into the RBC from uremic patients. This may contribute 

to define the erythrocyte’s role in the reduced plasma bioavailability of NO in uremia. 


98


Class I histone deacetylases as molecular switches in the 

regulation of lipid and energy metabolism 

in adipose tissues 

a. GalMozzi 1 , n. Mitro 1 , a. ferrari 1 , e. GerS 1 , e. fiorino 1 , 

f. Gilardi 1 , c. Godio 1 , G. cerMenati 1 , a. Gualerzi 2 , e. donetti 2 , 

u. Guerrini 1 , d. caruSo 1 , a. Mai 3 , e. Saez 4 , 

e. de faBiani 1 and M. creStani 1 

1 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano 

2 Dipartimento di Morfologia Umana e Scienze Biomediche, Università degli Studi di Milano 

3 Dipartimento di Chimica e Tecnologie del Farmaco, 

Università degli Studi di Roma “La Sapienza” 

4 Department of Chemical Physiology, The Scripps Research Institute, La Jolla CA 

Transcriptional and epigenetic mechanisms regulate lipid metabolism. Histone 

deacetylases (HDACs) and nuclear receptors play a pivotal role in the regulation of 

lipid metabolism in adipose tissues, liver and skeletal muscles in normal and disease 

states. Here, we aimed to investigate the role of HDACs in the regulation of energy 

metabolism under diabetic and obese conditions, focusing on metabolic regulation in 

adipose tissues. The class I selective HDAC inhibitor MS275 improved the diabetic 

phenotype of db/db mice by stimulating energy expenditure in multiple tissues. MS275 

increased mitochondrial biogenesis and oxidative metabolism in C2C12 myotubes 

via upregulation of PGC-1α, the master regulator of mitochondrial biogenesis. Knock 

down of HDAC3 by RNAi increased the expression of PGC-1α and recapitulated the 

effects of MS275. In diabetic and obese db/db mice MS275 reduced brown adipocyte 

size and increased interscapular brown adipose tissue (BAT) area as assessed by 

MRI analysis. The improved functionality of BAT in mice on MS275 was confirmed 

during a cold challenge showing that these mice retained body temperature better 

than control mice. Surprisingly, visceral WAT of db/db mice treated with MS275 

underwent Prdm16-independent reprogramming towards brown-like phenotype. 

MS275 upregulated the expression of Ucp1, Adrb3 and other BAT specific mRNAs in 

WAT. Bioinformatic analysis of Adrb3 gene promoter identified putative PPARγ binding 

sites, suggesting a role of this nuclear receptor in transcriptional regulation of Adrb3. 

We conclude that biochemical inhibition of class I HDACs revealed a mitochondrial 

signature mediated by Pgc-1α in skeletal muscle and by the Pgc-1α/Pparγ axis in 

adipose tissue, ameliorating the “diabesogenic” phenotype of db/db mice. 

(Funded by EU FP6 LSHM-CT2006-037498, Cariplo Foundation 2008.2511, The Armenise-Harvard 

Foundation and PRIN 2008 ZTN724) 


99


Zoledronic acid encapsulated in self-assembly pegylated 

nanoparticles as a new delivery system 

against brain tumors 

Silvia zaPPaviGna 1 , aMalia luce 1 , alida Panico 1 , Manuela Porru 2 , GiuSePPina 

Salzano 3 , Sara luSa 3 , GiuSePPe de roSa 3 , carlo leonetti 2 and Michele caraGlia 1 

1 Department of Biochemistry and Biophysics, Second University of Naples; 2 Experimental Chemotherapy 

Laboratory, Regina Elena Cancer Institute, Rome; 3 Department of Chemistry and Pharmaceutical 

Chemistry Toxicology, University “Federico II” of Naples. 

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of 

new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse 

in all cases. The blood–brain barrier (BBB) is the most important limiting factor for the development of new 

drugs and drug delivery for the central nervous system (CNS). Different methods have been used to facilitate 

the transporting of drugs into the brain (1, 2). Recently, small peptide-vectors have been used to enhance the 

uptake of several therapeutic drugs into the brain, and most of them can facilitate transportation safely and 

efficiently. Zoledronic acic (ZOL) is a drug used for the treatment of bone metastases and recent data report 

that beneficial effect of ZOL may result from a direct anti-tumour activity. One of the most important limits of 

ZOL is its limited delivery in tumour tissues and excessive accumulation in the bone. On these bases, there is 

a need to develop new ZOL formulations with a lower affinity for bone and a longer half-life in the circulation 

that would result in increased probability to affect peripheral tumours. Moreover, the functionalization of these 

nanoparticles with transferrin (TRF) could allow their crossing through BBB. 

The delivery system consists in self-assembly PEGylated nanoparticles (NPs), based on calcium/phosphate 

NPs and cationic liposomes. The preparation conditions were optimized in order to achieve NPs easily 

prepared before use, with colloidal dimensions and high ZOL loading. Moreover, to improve the targeting of 

ZOL in the brain we designed ZOL-containing NPs (NPs-ZOL) functionalized with transferrin (TRF) able to 

bind specific receptors on endothelial cells of BBB. We have evaluated the effects of NPs-ZOL functionalized 

or not with transferrin on growth inhibition of Ln229, U373MG and U87MG glioblastoma cells by MTT 

assay. The encapsulation in NPs functionalized with TRF resulted in higher in vitro cytotoxic activity than 

free ZOL on all three glioblastoma cell lines. However, the potentiation of anti-proliferative activity of TRFconjugated 

NPs-ZOL was equal (Ln229) or less (U373MG and U87MG) than that one induced by NPs-ZOL 

not functionalized with TRF and correlated with TRF receptor expression on tumour cells. On the other hand, 

TRF-NPs-ZOL showed a higher antitumor efficacy if compared with that one caused by naked NPs-ZOL in 

mice intramuscularly bearing glioblastoma tumors, inducing a significant tumor weight inhibition (TWI) of 41%, 

a tumor growth delay (T-C) of 10 days and an increase of ILS (increase of mice survival) of 23%. Moreover, 

we have performed interaction experiments with temozolomide (TMZ), a gold standard for the treatment of 

glioblastomas, nanoZOL functionalized with TRF or not, and free ZOL alone and in combination on growth 

inhibition using, as evaluation method of the synergism, the dedicated software Calcusyn. The sequences 

TMZ at the day 1 followed by TRF-NPs-ZOL or NPs-ZOL or free ZOL at the day 2 were in all cases strongly 

synergistic in inducing growth inhibition on glioblastoma cells while the reverse sequence TRF-NPs-ZOL 

followed by TMZ was synergistic only in LN229, which expressed TRF receptor at higher levels than U373MG 

and U87MG. All other combinations were additive or antagonistic. 

Conclusion. These preliminary results showed that the delivery of ZOL by NPs increases the antitumor efficacy 

of this drug in glioblastoma cells also in combination with standard cytotoxic agents. These data strongly 

warrant further investigations in intracranially injected glioblastoma cells preclinical models. 

1. Pardrige WM. New approaches to drug delivery through the blood–brain barrier. Trends Biotechnol. (1994) 12 239– 245. 

2. Jolliet-Riant P, Tillement JP. Drug transfer across the blood– brain barrier and improvement of brain delivery. (1999) 

Fundam. Clin. Pharmacol. 13 16– 26. 


100


Proteomic profile of cancer stem cells differentiation 

shows alteration of glycolytic pathway 

ciavardelli doMenico 1, 2* , roSSi claudia 1, 3* , forlì federica 1, 3 , 

Barcaroli daniela 1, 3 , zocchi loredana 1, 3 , de cola antonella 1, 3 , 

zucchelli Mirco 1, 3 , conSalvo ada 1, 3 , d’aGuanno SiMona 4, 5 , 

di ilio carMine 3 , Sacchetta Paolo 3 , todaro Matilde 6 , 

StaSSi GiorGio 6 , de laurenzi vincenzo 1, 3 4, 5 

, urBani andrea 

1 Center of Excellence on Aging (Ce.S.I.), University Foundation, Chieti, Italy; 

2 Faculty of Engineering, Architecture, and Motor Science, Kore University, Enna, Italy; 

3 Experimental and Clinical Science Department, 

“G. d’Annunzio” University, Chieti-Pescara, Italy; 

4 Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy; 

5 IRCCS S. Lucia Foundation, Rome, Italy. 

6 Department of Surgical and Oncological Sciences,University of Palermo, Italy. 

Cancer stem cells (CSCs) have a key role in solid tumours development, recurrence, 

and resistance to pharmacological treatment. In vitro, CSCs grow as three-dimensional 

cellular aggregates, called spheroids. Breast CSCs have been found to be resistant 

to conventional chemotherapy in vitro and in animal models and are thought to be 

able to regenerate the tumor after the bulk of more differentiated cells have been 

killed by therapy. To further characterize CSCs features we proposed a label free 

shotgun proteomics analysis to compare the protein expression profiles in CSCs 

grown as spheres or allowed to differentiate for 3.5 and 7 days. Our results show 

that the majority of changes occur already after 3.5 days of differentiation, indeed we 

identified 44 proteins down-regulated at this time point as compared to spheres. 19 of 

these were still down-regulated after 7 days of differentiation and only the expression 

of 4 additional proteins was reduced only after 7 days. Interestingly only 10 proteins 

increased in differentiated cells as compared to spheres. Functional analysis of the 

differentially expressed proteins reveals that CSCs have higher expression of a number 

of proteins related to carbohydrate metabolism and glycolysis, and of proteins related 

to free radical scavenging. Our findings suggest that breast CSCs exhibit a phenotype 

characterized by a higher glucose metabolism and increased defence from oxidative 

stress. We functionally confirmed our proteomic data by evaluating LDH activity and 

the effect of 2-deoxyglucose and rotenone, inhibitors of glycolysis and oxidative 

phosphorylation, respectively. Anyway, further studies are required but it is intriguing 

to speculate that breast CSCs preferentially perform anaerobic glucose metabolism 

on the basis of the significant increase of LDHA and PKM2 expression levels. 

*These authors equally contributed to the study. 


101


Treatment of AML cells with a G-quadruplex selective 

ligand displaces nucleophosmin from nucleoli 

antonella de cola, erMinia carletti, daniela Barcaroli, 

vincenzo Graziano, carMine di ilio, 

vincenzo de laurenzi and luca federici 

Ce.S.I. Centro Scienze dell’Invecchiamento and Dipartimento di Scienze 

Sperimentali e Cliniche, Università di Chieti “G. D’Annunzio”, 66013 Chieti – Italy. 

Nucleophosmin (NPM1 also known as B23) is a shuttling, multifunctional 

phosphoprotein mainly localized in the nucleolus. NPM1 is involved in cellular activities 

related to both proliferation and growth-suppression pathways. It partecipates in 

ribosome biogenesis, genomic stability and in the response of stress and oncogenic 

stimuli. NPM1 is implicated in human tumorigenesis and is the most frequently 

mutated gene in acute myeloid leukemia (AML). The AML mutations lead to the 

destabilization and the unfolding of the C-terminal domain of NPM1 and promote the 

aberrant cytoplasmic localization of the protein. 

We have previously shown that NPM1 C-terminal domain binds with high affinity 

G-quadruplex structures at DNA. Here show, by chromatin immunoprecipitation 

expriments, that NPM1 interacts in vivo with several G-quadruplex regions found at 

rDNA in the nucleoli of OCI-AML2 cells, that bear wild-type NPM1 on both alleles. 

We further show that the G-quadruplex selective ligand TmPyP4 is able to displace 

NPM1 from nucleoli to the nucleoplasm in OCI-AML2 cells and the loss of NPM1 

nucleolar localization leads to cell death. Conversely, OCI-AML3 cells, bearing 

heterozygous NPM1 mutation, are more resistant to TmPyP4 treatment, in line with 

recent evidence suggesting that mutated NPM1 confers protection from cell death 

through cytosolic gain of function properties. 


102


BIOCHEMICAL CHARACTERIZATION OF FOLLICULAR 

FLUIDS IN PATIENTS UNDERGOING IVF-ET: 

OxIDATIVE STRESS MARKERS, 

NO PRODUCTION AND EMBRYO GRADING 

tiano luca 1 , Mazzanti laura 1 , GiannuBilo Stefano r. 2 , 

BruGè franceSca 1 , Sforza Giulia, turi anGelo 2 , 

littarru Gian Paolo 1 , tranquilli andrea l. 2 , viGnini. arianna 1 

1 Department of Clinical Science, Section of Biochemistry, and 2 Section of Woman 

Health Science, Università Politecnica delle Marche, Ancona, Italy 

Cultural and social changes in the modern society produced a significant delay in 

reproductive age and a decrease in pregnancy rates (PR). Oocyte quality is one of the 

most important factors associated with successful pregnancy, especially in in vitro 

fertilization and embryo transfer (IVF–ET). Accumulation of oxidative damage have 

been indicated as a major cause of age-associated infertility as well as other causes 

of reproductive impairment such as obesity and diabetes. Reactive oxygen species 

(ROS) were shown to be involved in human reproduction acting as physiological 

mediators. However when ROS outnumber antioxidant defences, they might impair 

reproductive processes. In particular, at follicular level oxidative stress might interfere 

with this complex microenvironment altering maturation. Among biochemical 

mediators involved in folliculogenesis nitric oxide (NO) plays a role in the regulation 

of ovarian blood flow. The vasodilatory effect of estrogen in humans may be partially 

mediated by NO, a potent vasodilator synthesized in the arterial wall and human 

uterine artery blood flow. 

In this study we evaluated NO, lypophilic antioxidant CoQ10 and total antioxidant 

capacity in follicular fluids from 30 patients undergoing IVF-ET and correlated these 

parameters with embryo grading. Data have shown an increase in total antioxidant 

capacity during the follicular maturation process with significant differences between 

immature/dysmorphic and mature oocyte. This was associated with a significant 

decrease in CoQ10 content in FF associated with grading III–IV embryos. Moreover a 

direct correlation between follicular NO and embryo grading (r = 0.61; P < 0.001) was 

also observed. Our data highlight the relevance of the oxidative state of the follicular 

environment in promoting maturation and the role of NO as a mediator in this process, 

whose concentrations need to be tightly controlled since an excess might produce 

detrimental effects, probably due to its inactivation to the highly reactive peroxynitrite 

(ONOO - ). 


103


Extracellular sphingosine-1-phosphate contributes to 

survival properties of glioblastoma stem cells 

riccitelli e., GiuSSani P., di vito c., condoMitti G., trinGali c., 

Galli r., viani P., riBoni l. 

Department of Medical Biotechnology and Translational Medicine, 

University of Milan, LITA Segrate, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy 

Sphingosine-1-phosphate (S1P) is a potent bioactive lipid formed from sphingosine by 

sphingosine kinases (SKs) action. S1P is considered as an onco-promoter molecule, 

favouring growth, invasion, and therapy-resistance of different tumours, including 

glioblastomas, the most frequent and aggressive human intracranial cancers. Despite 

the introduction of the alkylating agent temozolomide in glioblastoma therapy has 

improved patient survival, drug resistance limits its benefits. Accumulating literature 

indicates that glioblastoma stem-like cells (GSCs), a subpopulation of cells with 

the exclusive ability to self-renew and maintain the tumor, might contribute to 

glioblastoma aggressiveness and resistance to therapy. This study investigated S1P 

secretion by GSCs, and its possible role in cell survival. To this purpose we used 

GSCs isolated from the human U87 glioblastoma cell line (U-SC) and GSCs derived 

from a primary culture of human glioblastoma (L0627). We found that both GSC 

models efficiently form neurospheres in mitogen-defined medium, and express high 

levels of recognized neural-stem cell markers. Moreover, GSCs exhibited resistance 

to temozolomide, despite not expressing the DNA repair protein MGMT, a major 

contributor to temozolomide-resistance. Further analyses revealed the presence of 

S1P not only inside the cells, but also in the culture medium from both GSCs and 

U87. Notably the extracellular S1P level was found much higher in GSC models than 

in U87 cells, and the ratio between extracellular and intracellular S1P was 1:10 and 

1:1 in U87 and GSCs, respectively. Enzyme activity assays excluded SKs presence 

in GSC medium, implicating an efficient secretion of S1P in GSCs. Intriguingly, 

concomitant treatment with temozolomide and a SKs inhibitor made GSCs sensitive 

to drug toxicity. Furthermore, S1P administration promoted cell survival after this cotreatment. 

Altogether our data implicate GSCs as an important source of extracellular 

S1P, which might act as an autocrine signal contributing to the survival properties of 

GSCs. 


104


Electrospun poly(L-lactic acid)-calcium-deficient 

hydroxyapatite nanocomposite mats drive multipotent and 

pluripotent stem cell osteogenic differentiation 

SaBata Martino 1 , arMentano ilaria 2 , franceSco d’anGelo 1 , ilaria cacciotti 3 , 

roBerto tiriBuzi 1 , Mattia quattrocelli 4 , coStantino del Gaudio 3 , elena fortunati 2 , 

Marcello iMBriani 5 , auro caraffa 6 , Giuliano GiorGiocerulli 6 , livia viSai 5,7 , 

JoSè Maria 2 kenny 8 , Maurilio SaMPaoleSi 4 , aleSSandra Bianco 3 , aldo orlacchio 1,9 . 

1 Department of Experimental Medicine and Biochemical Science, Section of Biochemistry and Molecular Biology, 

University of Perugia, Perugia, Italy; 2 Materials Engineering Centre, UdR INSTM, NIPLAB, University of Perugia, 

Terni, Italy; 3 Department of Industrial Engineering, UdR INSTM Roma Tor Vergata, University of Roma “Tor Vergata”, 

Roma, Italy; 4 Translational Cardiomyology Lab, SCIL, Catholic University of Leuven, Leuven, Belgium; 5 Department 

of Molecular Medicine and Center for Tissue Engineering (C.I.T), University of Pavia, Pavia, Italy; 6 Department of 

Orthopedics and Traumatology, University of Perugia, Perugia, Italy; 7 Salvatore Maugeri Foundation IRCCS, Pavia, Italy 

and International Centre For Studies And Research In Biomedicine (I.C.B.), L-2015, Luxembourg; 8 Institute of Polymer 

Science and Technology, CSIC, Madrid, Spain; 9 GeBiSa Research Foundation, Perugia. 

In this study we investigated whether multipotent (human bone marrow-derived mesenchymal stem 

cells [hBM-MSCs]) and pluripotent stem cells (murine induced pluripotent stem cells [iPSCs] and murine 

embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded 

with 1%wt or 8%wt calcium deficient nanohydroxyapatite (d-HAp). 

Bianco et al. (1) produced a –based microfibrous nanocomposite with different amount (i.e. 1 %wt to 8 

%wt) of synthesized deficient nanohydroxyapatite(d-Hap) means of electrospinning technique. Notable the 

addition of different amounts of d-HAp to PLLA produceda set of materials with similar architecture and 

tunable mechanical properties (1-2). 

We selected adult human bone marrow-mesenchymal stem cells (hBM-MSCs) as representative 

multipotential stem cells and on the basis of their capability to generate differentiated cells even if they are 

cultured on biomaterials. As pluripotent stem cells we chosen induced pluripotent stem cells (iPSCs). These 

stem cells may be generated in vitro from somatic differentiated cells and offer the advantage to produce 

specific donor cells for cell replacement and/or tissue engineering applications. Finally we adopted murine 

embryonic stem cells (ESCs), as natural stem cell control of iPSCs and based on their pluripotency capability. 

Our results showed that PLLA/d-HAp nanocomposite mats have an active role in inducing human multipotent 

(hBM-MSCs) and murine pluripotent (iPSCs and ESCs) stem cell differentiation towards the osteogenic 

lineage in the absence of exogeneous soluble differentiating agents. 

The lack of osteogenic differentiation of both murine pluripotent and human multipotent stem cells cultured 

on neat PLLA under the above experimental conditions addresses this result to the new properties acquired 

by the PLLA/d-HAp nanocomposite mats. 

Altogether these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp 

nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cellpolymeric 

nanocomposite and the new mechanical properties acquired by the PLLA/d-HAp nanocomposites 

key steps for the differentiation process. 

REFERENCES 

1) Bianco A. et al. J. Bioactive and Compatible Polymers. 2011, 26, 225-241. 

2) D’angelo F. et al. Biomacromolecule.2012 april 12 

ACKNOWLEDGMENTS 

The authors would like to thank the Italian Fondazione Cassa di Risparmio di Perugia (grant no. 2010.011.0445 to A.O.), the 

Italian Ministero dell’Istruzione, dell’Università e della Ricerca (grant: PRIN no.20084XRSBS_001 to A.O.). 


105


Calpain system is involved in mediating the extremely low 

frequency (ELF) electromagnetic field (EMF) stimulatory 

effect on myoblast migration 

Bennato franceSca 1 , BriSdelli faBrizia 1 , toGnolatti Piero 2 , 

Mancini faBrizio 2 , cardiGno roSella 1 , and iorio roBerto 1 

1 Department of Biotechnological and Applied Clinical Sciences, University of 

L’Aquila, via Vetoio, 67100 L’Aquila, tel +39-0862433443, Fax +39-086243343. 

2 Department of Electrical and Information Engineering, University of L’Aquila, 

via Gronchi, 67100 L’Aquila, tel +39-0862434402, Fax +39-0862434403. 

Many studies have demonstrated that exposure to extremely low frequency (ELF) 

electromagnetic fields (EMFs) affects various cell functions from regulation of 

cell proliferation and differentiation to the modulation of the membrane receptors 

functionality. In this study our working hypothesis was that ELF-EMF (sinusoidal 

waveform; 1 mT; 50 Hz) may influence myogenesis process due to its ability to 

modify the cellular redox state and level of intracellular Ca 2+ in C2C12 cells [1]. The 

results presented here demonstrate that ELF-EMF is able to positively modulate 

myoblast migration, an important step of myogenesis, and provides evidence for 

the involvement of calpain system in mediating this effect. This statement is based 

on the following observations: myoblast exposure to ELF-EMF for 6 h resulted in a 

transient but significant increase in cell motility; this stimulatory effect on myoblast 

migration was associated with a marked increase of µ- and m-calpain activity and 

the concomitant shift in their subcellular localization; the increase in calpain activity 

was not associated with significant changes in intracellular distribution and protein 

levels of the endogenous calpain inhibitor, calpastatin; the peak of calpain activity 

resulted in a progressive and significant decrease of myristoylated alanine-rich 

C-kinase substrate (MARCKS) expression, an actin-binding protein and substrate of 

calpain; this progressive decrease of MARCKS protein levels was associated with 

modifications in actin dynamics; and inhibiting calpains through the pharmacological 

inhibitors calpeptin prevented the ELF-EMF stimulatory effect on myoblast migration 

in a dose-dependent fashion. In conclusion, our findings clearly demonstrate the 

involvement of ubiquitous calpains in ELF-EMF-mediated myoblast migration 

probably through the cleavage of MARCKS actin-binding protein. 

Reference 

[1] C. Morabito, F. Rovetta, M. Bizzarri, G. Mazzoleni, G. Fanò, and M.A. Mariggiò. Modulation of redox 

status and calcium handling by extremely low frequency electromagnetic field in C2C12 cells: A realtime, 

single-cell approach. Free Radic. Biol. Med., 48:579-589. 


106


Extremely low frequency (ELF) electromagnetic field 

(EMF) reduces quercetin-induced apoptosis in K562 cells: 

possible role of HSP-70 

BriSdelli faBrizia 1 , Bennato franceSca 1 , Sellitri doriana 1 , 

Mancini faBrizio 2 , toGnolatti Piero 2 , Bozzi arGante 1 , 

aMicoSante Gianfranco 1 and iorio roBerto 1 

1 Department of Biotechnological and Applied Clinical Science; 

2 Department of Electrical and Information Engineering, University of L’Aquila 

The extremely low frequency electromagnetic fields (ELF-EMFs) have been found 

to produce a variety of biological effects. In particular, according to the specific 

characteristics of the EMF applied and/or to the type of cell used, the electromagnetic 

stimuli can cause cytotoxicity or cytoprotection. This study was aimed to investigate 

whether the ELF-EMF can induce a synergistic or antagonistic effect on the apoptosisinducing 

activities of quercetin in human chronic myeloid leukemia (K562) cells. Cell 

growth and viability, cell cycle distribution, apoptosis rate, caspase 3 activity, ROS 

levels, and HSP70 expression were analyzed up to 72 h upon incubation with 25 µM 

quercetin alone or in the presence of ELF-EMF (sinusoidal waveform; 1 mT, 50 Hz). 

Our results showed that the ELF-EMF reduced cytotoxic effects of quercetin in K562 

cells. In particular, the ELF-EMF increased cell growth and viability and decreased 

(about 50%) the number of apoptotic cells and caspase-3 activity in quercetin-treated 

cells after 48-72 h of incubation. 

In addition, the quercetin-induced cell cycle arrest in G1 phase was suppressed by 

ELF-EMF treatment. Focusing on the stress-related pathways, the ELF-EMF exposure 

did not induced any significant change in reactive oxygen species (ROS) levels, but 

increased the expression of the chaperone heat shock protein HSP70. In conclusion, 

our findings support a cytoprotective action of the electromagnetic stimulation able 

to antagonize the quercetin-induced apoptosis in K562 cells; in particular, this major 

resistance associated to ELF-EMF exposure may be attributed to higher expression 

of HSP70. 


107


ALK1/ACVRL1 overexpression in Human Ascending 

Thoracic Aortic Aneurysm and atherosclerotic lesions 

lenato Gennaro M 1 , Buttari BriGitta 2 , neri MarGherita 3 , 

di GiaMMarco GaBriele 4 , ProfuMo eliSaBetta 2 , fineSchi vittorio 3 , 

Botella luiSa-Marìa 5 , BernaBéu carMelo 5 , reSta franceSco 1 , 

SaBBà carlo 1 , riGanò rachele 2 

1 Sovra-Unit Center for Rare Diseases and Geriatric Unit, University Hospital of Bari, Bari, 

2 Department of Infectious, Parasitic and Immune-mediated Diseases Istituto Superiore di Sanità, Roma, 

3 Department of Surgery, Unit of Legal Medicine, University of Foggia, Foggia, 4 Department of 

Neuroscience and Imaging, Unit of Cardio-Thoracic Surgery, University of Chieti-Pescara, Chieti, Italy, 

5 Centro Investigaciones Biologicas, CSIC, Madrid, Spain 

Ascending Thoracic Aortic Aneurysm (ATAA) is defined as a dilatation of the ascending 

trait of thoracic aorta, ultimately leading to aortic wall dissection. Unlike aneurysms 

of abdominal aorta, ATAA usually has a non-atheromasic aetiology. ATAA entails a 

maladaptive remodelling of the aortic wall extracellular matrix, which is abnormally 

shifted towards an enhanced degradation. Several reports outline that aberrant 

extracellular matrix turnover is associated to dysregulated responsiveness of aortic 

wall cells to Transforming-Growth-Factor-Beta/Bone-Morphogenic-Protein (TGFBeta/ 

BMP). Furthermore, mutations in genes encoding receptors/effectors of TGFBeta/BMP 

signalling, such as ACVRL5, TGFBR2, MADH3, are responsible for genetic syndromes 

including ATAA within their clinical spectrum. Endoglin and ALK1/ACVRL1, the genes 

mutated in the Mendelian disorder Hereditary Haemorrhagic Telangiectasia, are involved 

in TGFBeta/BMP-mediated vascular development/remodelling, but their role in ATAA 

has never been investigated. In the present study, we aimed to elucidate the expression 

pattern of different TGFBeta/BMP receptors in human specimen from ATAA patients. 

Samples of ascending aorta were obtained from ATAA patients and compared to 

samples of control aorta from healthy individuals. The expression of different TGFBeta/ 

BMP receptors was studied by immunohistochemcal analysis. 

Healthy aortas showed a very low expression of Endoglin. A modest expression of 

ALK1/ACVRL1 and a strong expression of ALK5/TGFBR1 and TGFBR2 were evident. 

Aneurysmal samples showed a marked overexpression of ALK1/ACVRL1, as well as 

TGFBeta1, ALK5/TGFBR1 and TGFBR2. Overexpression was even more pronounced 

in areas corresponding to small atherosclerotic plaques. By contrast, no Endoglin 

upregulation was observed in aneurysmal samples compared with control aortas. 

However, atherosclerotic areas showed a strong upregulation of Endoglin, mainly 

paralleling that of ALK1/ACVRL1, TGFBeta1 and TGFBR2. 

Our results report for the first time an ALK1/ACVRL1 overexpression in ATAA. The lack of 

Endoglin overexpression in aneurysmal lesions and the parallel upregulation of Endoglin 

and ALK1/ACVRL1 in atherosclerotic plaques indicate that the two overexpression 

mechanisms are differently regulated. 


108


Tubulin polymerization and mitochondrial potential: 

a link under exploration 

da Pozzo eleonora 1 ; coSta BarBara 2 ; la reGina GiuSePPe 3 ; 

SilveStri roMano 3 ; novellino ettore 4 ; Martini claudia 1 . 

1 Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, 

Università di Pisa; 

2 Dipartimento di Morfologia Umana e Biologia Applicata, Università di Pisa; 

3 Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie 

del Farmaco, Sapienza Università di Roma; 

4 Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli Federico II. 

In mitochondria, transport of respiratory substrates (ATP, ADP and phosphate) across 

the mitochondrial inner membrane occurs through a variety of specific transporters. In 

contrast, metabolite exchange across the outer membrane occurs primarily through the 

voltage-dependent anion channel (VDAC). Thus the maintenance of the mitochondrial 

membrane potential (ΔΨ) depends on flux of respiratory substrates through the 

VDAC. Tubulin, the heterodimeric subunit of microtubules, binds to mitochondria 

specifically at VDAC [1], promoting a single-channel closure of VDAC. Recently free 

tubulin has been proposed as dynamic regulator of ΔΨ in human hepatoma cells, 

lung carcinoma and head and neck cancer cells [2]. Classical inhibitors of microtubule 

assembly, such as colchicine, vincristine and vinblastine (all of which prevent tubulin 

polymerization), provoke microtubule destabilization, arrest of mitotic progression 

and cell death [3]. Interfering with these cellular processes is therefore a strategy to 

inhibit the proliferation of cancer cells. 

In the present study, we used the arylthioindole anti-mitotic agents, potent inhibitors 

of tubulin polymerization [4], to investigate the effect of tubulin polymerization arrest 

on U87MG cells, a cell culture model of human glioblastoma multiforme (GBM). We 

assessed U87MG cell survival and ΔΨ in the presence of arylthioindoles. We evidenced 

a dose- and time-dependent inhibition of U87MG cell growth and a collapse of the 

mitochondrial membrane potential. We also measured generation of reactive oxygen 

species (ROS) immediately after the anti-mitotic agent treatment (1’, 5’, 15’ and 60’); 

the level of ROS were increased in respect to control after 1 hour. 

The present findings may suggest that free tubulin dynamically regulates mitochondrial 

metabolism in GBM cancer cells. 

References 

[1] J Biol Chem 2002; 277: 33664–9 

[2] Cancer Res 2010; 70(24): 10192–201 

[3] Nature 2004; 428: 198−202 

[4] J Med Chem 2011; 54: 8394−406 


109


Therapeutic potential of a pyridoxal-based vanadium(IV) 

complex showing selective cytotoxicity 

for cancer vs. healthy cells 

anna BaSile, a Maria StrianeSe, B antonio Mazzone, B 

Silvana Morello, a Maria caterina turco, a* claudio Pellecchia B* 

a) Department of Pharmaceutical and Biomedical Sciences and BioUniverSA SRL, 

University of Salerno, via Ponte Don Melillo, Fisciano (SA), Italy 

b) Department of Chemical and Biology, 

University of Salerno, via Ponte Don Melillo, Fisciano (SA), Italy 

Abstract 

Vanadium compounds can exert anticancer effects, partly due to inhibition of tyrosine 

phosphatases. Here we report the effect of N,N’-ethylenebis (pyridoxylideneiminato) 

vanadium (IV) complex [Pyr 2 enV(IV)], that induced 93% and 57% of cell mortality 

in A375 (human melanoma) and A549 (human lung carcinoma) cells, respectively; 

the mortality was


Pancreatic adenocarcinomas release 

BAG3 protein in serum 

antonia falco 1,2 , aleSSandra roSati 1,2 , Michelina feSta 1,2 , 

anna BaSile 1,2 , MarGot de Marco 1,2 Morena d’avenia 1,2 , 

Maria PaScale 1,2 , rita BiSoGni 2 , faBrizio dal Piaz 1 , Michael hahne 3 , 

PaSquale BartoloMeo Berloco 4 , franceSco nudo 4 , claudio tiBerti 5 , 

claudio arra 6 , antonio BarBieri 6 , Marianna Gala 6 , 

doMenica rea 6 , franceSca tavano 7 , faBio franceSco di Mola 7 , 

PierluiGi di SeBaStiano 7 , Michele caraGlia 8 , antonio feBBraro 9 , 

Gaetano di coStanzo 10 , daniela Barcaroli 11 , aldo ScarPa 12 , 

vincenzo de laurenzi 2,11 , raffaele Pezzilli 13 , 

Maria caterina turco 1,2 . 

1. Department of Pharmaceutical and Biomedical Sciences, 

University of Salerno, Fisciano (SA), Italy; 

2. BIOUNIVERSA srl, University of Salerno, Fisciano (SA), Italy; 

3. Institut de Génétique Moléculaire de Montpellier, CNRS UMR5535, Montpellier, France; 

4. Chirurgia Generale e Trapianti d’Organo, 

La Sapienza Università di Roma, Umberto I Policlinico di Roma, Rome, Italy; 

5 . Department of Clinical Sciences, University of Rome Sapienza, Rome, Italy; 

6. Struttura Semplice Dipartimentale Sperimentazione Animale, Istituto Nazionale Tumori, 

IRCCS “Fondazione G. Pascale”, Naples, Italy; 

7. Department of Surgery, Casa “Sollievo della Sofferenza” Hospital, 

IRCCS, San Giovanni Rotondo, Foggia, Italy; 

8. Department of Biochemistry and Biophysics, Second University of Naples; 

9. Department of Oncology, “Fatebenefratelli” Hospital, Benevento, Italy; 

10 . Servizio di Medicina Trasfusionale, Istituto Nazionale Tumori, 

IRCCS “Fondazione G. Pascale”, Naples, Italy; 

11 Department of Biomedical Sciences, University “G. D’Annunzio” Chieti-Pescara and 

Fondazione “G. D’Annunzio, Centro Studi sull’Invecchiamento, Ce.S.I., Chieti, Italy; 

12. ARC-NET Centre for Applied Research on Cancer and Department of Pathology and 

Diagnostics, University and Hospital Trust of Verona, Verona, Italy; 

13. Pancreas Unit, Department of Digestive Diseases and Internal Medicine, 

Sant’Orsola-Malpighi Hospital, Bologna, Italy. 

Abstract 

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers and 

novel markers are required for noninvasive diagnosis and follow up as well as for 

prognostic assessment. We have recently shown that BAG3 is over-expressed in 

all PDACs we analyzed (346 cases) as compared to normal pancreatic tissue and 

expression levels correlated with patient survival. Here we show that PDACs also 

release BAG3 both in vivo and in vitro and BAG3 can be detected in sera of patients 

thus representing a potential marker for this disease. 


111


Nitric Oxide Depletion Alter The Hematopoietic Stem Cell 

Commitment Toward Immunogenic Dendritic Cells 

tiriBuzi roBerto 1 , criSPoltoni lucia 1 , tartacca franceSco 2 , 

Martino SaBata 1 , PalMerini carlo alBerto 2 and orlacchio aldo 1,3 . 

1 Dipartimento di Medicina Sperimentale e Scienze Biochimiche, 

Sezione di Biochimica e Biologia Molecolare, Università di Perugia, Via del Giochetto, 06100 Perugia, Italy. 

2 Dipartimento di Medicina Interna, Università di Perugia, Via del Giochetto, 06100 Perugia, Italy. 

3 GeBiSa Research Foundation, Perugia. 

Nitric oxide (NO• ) is a free radical that operates as an endogenous messenger in most 

cells in both autocrine and paracrine signaling modes. It’s involved in the proliferation and 

differentiation balance along several developmental and differentiation settings. In this work 

we investigated the contribution of NO• during the differentiation of human CD34 + cells 

toward i-DCs 

Methods Isolation of human HSCs: Human peripheral blood mononuclear cells (PBMCs) 

were isolated by density gradient centrifugation, after which CD34 + cells were purified 

through immunomagnetic selection using the mini-magnetic-activated cell sorter (MACS) 

system. 

NO• inhibitor treatment: CD34-enriched cells were cultured for 14 days in BC to generate 

i-DCs cells. 5mM NG-Monomethyl-L-arginine, Monoacetate (L-NMMA) was added as NOS 

inhibitor and 10 µM oxy-haemoglobin (HbO ) as NO 2 • scavenger as well as both L-NMMA 

plus HbO . To increase the NO 2 • concentration sodium nitroprusside (SNP) was used as 

sources. 

Analysis of treated cells: We have evaluated the proliferating rate by BrdU incorporation, 

immunophenotypic markers by FACScan flow cytometer and clonogenic ability by 

MethoCultTM assay of cells treated with L-NMMA, HbO and L-NMMA plus HbO respect 

2 2 

to BC induced cells. 

Results Compared to standard BC-cultures, the inhibition of NOS with L-NMMA or the 

depletion of paracrine NO• with HbO as scavenger, hamper the differentiation process of 

2 

i-DCs and correlated with (I) an active proliferation state at day 14th (II) a significant reduction 

in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high 

expression of the CD34 marker; (III) a retrieved clonogenic ability on MethoCultTM assay. 

Conclusion Globally, our data indicate that the depletion of NO • during the commitment 

stage block the CD34 + HSCs differentiation into i-DCs and maintains a undifferentiated cell 

population underlying the prevalence of the autocrine to paracrine effect of NO • . 

Grant: Italian Ministero dell’Istruzione, dell’Università e della Ricerca (PRIN 2008 –No. 20084XRSBS_001) to Aldo Orlacchio. 


112


TRAP1-DEPENDENT QUALITY CONTROL: COUPLING 

PROTEIN SYNTHESIS TO DEGRADATION 

MataSSa Swann d 1 , aMoroSo Mr 1 , crudele v 2 , landriScina M 3 , 

eSPoSito f 1 . 

Some co-translationally acting chaperones, the so-called ‘chaperones linked to 

protein synthesis’ (CLIPS), have been recently identified in Saccharomyces cerevisiae. 

Despite the extensive efforts, little is known at present about analogous factors in 

higher eukaryotes. We previously demonstrated that TRAP1, the only mitochondrial 

member of the HSP90 family is involved, through the interaction with the regulatory 

proteasome protein particle TBP7 in the endoplasmic reticulum, in the quality control 

of two nuclear-encoded mitochondrial proteins i.e. the β subunit of F1ATPase and the 

p18 Sorcin isoform: the regulation of TRAP1 client protein’s expression relies on their 

ubiquitination control and does not involve changes in the overall protein’s stability. 

Two observations allowed us to hypothesize a control of protein translation by TRAP1: 

i) TRAP1-interacting proteins are mitochondrial, therefore they must enter unfolded 

into mitochondria; ii) it has been estimated that up to 30% of newly synthesized 

proteins are co-translationally degraded by the ubiquitin-proteasome system, 

implying efficient quality control mechanisms that couple protein translation, protein 

folding and proteasomal degradation. Here we demonstrate that TRAP1 is associated 

to ribosomes and to several translation initiation and elongation factors, and that 

TRAP1 control on its client proteins may involve the coupling of protein synthesis and 

degradation. Moreover, we show that TRAP1 regulates the rate of protein synthesis 

through the GCN2-eIF2α pathway, by favouring the activation of the kinase GCN2 

with the consequent phosphorylation of eIF2α and attenuation of cap-dependent 

translation. As a result TRAP1-interfered cells contain reduced levels of downstream 

effectors, such as the transcription factor ATF4, the ER chaperone BiP, the cystine 

antiporter system light chain xCT, which sensitizes these cells to ER stress, nutrient 

deprivation and oxidative damage. 

1 Department of Biochemistry and Medical Biotechnologies, University of Naples “Federico II”, Via Pansini 

5, Naples 80131, Italy. 

2 Department of General Pathology, Second University of Naples, Via de Crecchio 7, Naples 80138, Italy. 

3 Clinical Oncology Unit, Department of Medical Sciences, University of Foggia, Foggia, Italy. 


113


The endocannabinoid system in MEG-01 cells 

GaSPeri valeria 1 , evanGeliSta daniela 1 , aviGliano luciana 1 , 

Maccarrone Mauro 2,3 , catani Maria valeria 1 

1 Department of Experimental Medicine & Surgery, 

University of Rome Tor Vergata, Rome, Italy. 

2 European Center for Brain Research (CERC)/IRCCS 

S. Lucia Foundation, Rome, Italy. 

3 Department of Biomedical Sciences, University of Teramo, Teramo, Italy. 

Anandamide (AEA) and 2-arachidonoylglycerol (2-AG), the most biologically active 

endocannabinoids (ECs), are ubiquitous lipid mediators known to exert different 

biological effects. In blood, AEA and 2-AG endowed with distinct functions: 2-AG acts 

as a differentiating agent for megakaryocytes and as a trigger of platelet activation; 

instead, AEA regulates platelet choice between death and survival. Recently, we 

demonstrated that 2-AG triggered commitment to the megakaryoblastic lineage, in 

the pluripotent HEL cells; here, we investigated the role of ECs in the megakaryocytic 

MEG-01 cell line, able to fully differentiate and to produce platelet-like fragments, thus 

representing a more advanced stage of differentiation respect to HEL cells. 

First, we characterized the expression of proteins which bind and metabolize ECs in 

proliferating cells. As demonstrated by Western blot, MEG-01 cells express cannabinoid 

type-1 (CB1) and 2 (CB2) receptors, as well as type 1 vanilloid receptor (TRPV1). 

Cells also express ECs-generating enzymes: N-acyl-phosphatidylethanolamineshydrolyzing 

phospholipase D (NAPE-PLD), for AEA, and sn-1-specific diacylglycerol 

lipase (DAGL) for 2-AG. Next, we looked for the presence of specific hydrolases [fatty 

acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)] terminating the 

biological activity of AEA and 2-AG, respectively; as assessed by Western blot and 

enzymatic assay, MEG-01 cells express both enzymes. 

Then, we investigated whether ECs regulate lineage determination and platelet 

generation. MEG-01 cells were treated for up to 4 days with the phorbol ester TPA 

(a classical inducer of thrombopoiesis), as well as with increasing concentrations of 

2-AG or AEA; by phase-contrast microscopy we found that, like TPA, ECs induce the 

appearance of ruffled edges on MEG-01 cells, commonly observed during membrane 

blebbing of platelets, with 2-AG being more efficient. 

Our preliminary results showed that ECs are able to induce megakaryopoiesis and 

thrombopoiesis, thus triggering platelet generation from mature megakaryocytes. 


114


2-Arachidonoylglycerol modulates the initial steps of the 

leukocyte adhesion cascade 

evanGeliSta daniela 1 , GaSPeri valeria 1 , aviGliano luciana 1 , 

Maccarrone Mauro 2,3* , catani Maria valeria 1* 

1 Department of Experimental Medicine & Surgery, 

University of Rome Tor Vergata, Rome, Italy. 

2 European Center for Brain Research (CERC)/IRCCS S. Lucia Foundation, 

Rome, Italy. 

3 Department of Biomedical Sciences, University of Teramo, Teramo, Italy. 

*senior co-authors 

Accumulating evidence points to an immuno-modulatory role for endocannabinoids 

(ECs), although the exact mechanism of action remains not fully elucidated. Several 

data support an anti-inflammatory effect through modulation of chemokines and 

cytokines, but a pro-inflammatory role can be sustained as well. Furthermore, the 

knowledge gained to date presents some shortcomings, since literature data concern 

single cell types or analysis of late stages of the inflammatory process. Therefore, 

we aimed at characterizing the role of ECs in early events of endothelium/leukocyte 

interactions. We found that 2-arachidonoylglycerol (2-AG) was able to initiate and 

complete the leukocyte adhesion cascade, by modulating the expression of selectins 

and their ligands. A short exposure of endothelial ECV304 cells to 2-AG was 

sufficient to prime them towards a pro-inflammatory state: within 1 hour of treatment, 

endothelial cells showed time-dependent plasma membrane expression of P- and 

E-selectins, which trigger the initial steps (namely capture and rolling) of inflammation. 

Commitment to inflammation was permanent, since activated endothelial cells released 

the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) for up to 24 hours, 

despite the removal of 2-AG after 1 hour of incubation. TNF-α-containing medium is, 

then, able to promote leukocyte recruitment; Jurkat cells grown in the conditioned 

medium derived from 2-AG-treated endothelial cells showed enhanced L-selectin and 

P-selectin glycoprotein ligand-1 (PSGL1, the specific ligand for selectins) expression, 

as well as increased efficiency of adhesion and trans-migration. In conclusion, 2-AG 

indirectly promotes leukocyte recruitment into inflamed sites by acting on endothelial 

cells, thus representing a potential therapeutic target for treatment of inflammatory 

diseases. 


115


IN VITRO EFFECTS OF FERMENTED PAPAYA 

(Carica Papaya, L.) SUPPLEMENTATION IN TYPE 2 

DIABETIC PATIENTS 

raffaelli f 1 , nanetti l 1 , alidori a 1 , Montecchiani G 1 , Borroni f 1 , 

Giulietti a 1 , Sforza G 1 , faloia e 2 , Mazzanti l 1 , viGnini a 1 

1 Department of Clinical Sciences,Faculty of Medicine, 

Marche Polytechnic University – Ancona, Italy 

2 Clinic of Endocrinology, Faculty of Medicine, 

Marche Polytechnic University – Ancona, Italy 

ABSTRACT 

Aims: 

To evaluate the fermented papaya (FPP) in vitro effect supplementation in DM2 

patients and healthy controls in order to use it as adjuvant of daily therapy. 

Results: 

The study was performed on 30 patients and 15 healthy subjects, matched for age and 

sex. Platelets were isolated from peripheral venous blood. The study was divided into 

2 phases: patients and controls platelets were analyzed at recruitment (T 0 ) and after 

in vitro supplementation with FPP at 50 g/ml for 3 hours at 37°C (T 1 ). On all samples 

were determined: Na + /K + ATPase activity, membrane fluidity tested by anisotropy of 

fluorescent probes TMA-DPH and DPH, Total Antioxidant Capacity (TAC), Superoxide 

Dismutase (SOD) activity and conjugated dienes levels. 

In vitro FPP supplementation counteracts oxidative damage associated with diabetes 

and its complications; on one hand improves platelet function through increased 

membrane fluidity (tested by decreased anisotropy TMA-DPH and DPH anisotropy) 

and Na + /K + ATPase activity, on the other, enhances antioxidant systems functionality 

by increasing SOD activity and TAC, as well as by decreasing conjugated dienes 

levels significantly in patients versus controls. This platelet functionality enhancement, 

which results in reduction of hyperactivity and hyperaggregability, characteristics of 

diabetes mellitus, suggests a new method of secondary prevention of complications 

associated with platelet dysfunction. 

Innovation and Conclusions: 

It can be assumed that FPP utilization in the diet of diabetics may have, at first, a 

preventive role and then protective in the progression of oxidative damage associated 

with diabetes and its complications, although the exact protective mechanism is still 

under study. 


116


Bioenergetic consequences of PARK2 mutations in 

hereditary early-onset Parkinson’s Disease 

f. aMati 1 , n.a. Martino 2 , a. Gnoni 1 , r. trentadue 1 , c. criScuolo 3 , 

G. de Michele 3 , M.e. dell’aquila 2 , S.PaPa 1,4 , a.M. Sardanelli 1 

1 Department of Medical Basic Sciences, University of Bari “Aldo Moro”, Bari, Italy 

2 Department of Animal Production, University of Bari “Aldo Moro”, Bari, Italy 

3 Department of Neurological Sciences, Federico II University, Naples, Italy 

4 Institute of Biomembranes and Bioenergetics, CNR, Bari, Italy 

There is substantial evidence pointing to a critical role of mitochondrial dysfunction 

in the pathogenesis of sporadic and early onset PD [1, 2]. The PARK2 gene encodes 

Parkin, a protein associated with the outer mitochondrial membrane that functions 

as an E3 ubiquitin ligase. Recent observations suggest that Parkin participates in 

the quality control of mitochondrial proteins, autophagy and mitochondrial structural 

dynamics. Pathogenic Parkin mutations associated with autosomal recessive juvenile 

parkinsonism (AR-JP) are distributed throughout the Parkin gene and include missense, 

nonsense and frameshift mutations, as well as exon deletions and multiplications [3]. 

We investigated on the impact of different Parkin mutations on mitochondrial function, 

analyzing human primary dermal fibroblasts from four Italian patients affected by early 

onset Parkinson’s disease carrying the following mutations: ex3-4del; Cys253Tyr/ 

ex5del; ex2del/ex2-4del. We found a significant decrease in patients’ fibroblasts of 

complex I, IV and V specific activity and a significant reduction of the uncoupled 

respiration rates independent of the substrate used as compared to controls. 

Oligomycin-sensitive ATP hydrolase and ATP synthase activity of CV measured in the 

mitoplast fraction revealed a decrease in the patients’ cells as compared with controls. 

Cellular total ATP content, measured under basal conditions or under strict glycolytic 

conditions, indicates that the defective ATP production by OXPHOS is compensated 

by glycolytic supply. Mitochondrial dysfunction in PD patients is associated with a 

significant decrease in ΔΨ and increase in ROS production. The pattern and severity 

of mitochondrial dysfunction varying between different mutations, correlated with the 

clinical features, providing evidence that this dysfunction in Parkin mutant fibroblasts, 

is involved in the pathogenesis of Parkinson. 

[1] Papa S et al. (2009) J Bioenerg Biomembr, 41, 509-16. 

[2] Sai, Y., et al. (2012) Neurosci. Biobehav. Rev. Epub ahead of print 

[3] Sriram S.R. (2005) Hum. Mol. Genet. 14 (17): 2571-2586. 


117


Fibroblasts transfer lipids and proteins to tumor cells 

through cargo vesicles supporting their growth 

Santi alice 1 , caSelli anna 1 , ranaldi franceSco 1 , Paoli Paolo 1 , 

d’aMico MaSSiMo 2 , Stinziani Stefano 1 , cirri Paolo 1 

1 Dipartimento di Scienze Biochimiche and 

2 Dipartimento di Patologia e Oncologia Sperimentali; 

Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy 

Until a few years ago it was thought that intercellular communication was mediated 

by electrical or chemical signals. In the last decades an additional mechanism of 

cellular communication, which involves the traffic of small membrane vesicles among 

neighboring cells, has been proposed [1]. These vesicles are secreted by many cells 

(including cells of hematopoietic origin [2, 3], intestinal epithelial cells [4], tumor cells 

[5] and neuronal cells [6]) and can participate in physiological as well as pathological 

processes. 

In the context of tumor microenvironment study we have shown that cancer cells 

increase their growth rate of 30-40% when co-cultured with primary human fibroblasts 

and that only a minor part of this effect is due to soluble factors-mediated cellular 

crosstalk. 

Cytofluorimetry, confocal microscopy and radiolabelled protein experiments revealed 

that fibroblasts transfer both proteins and lipids to cancer cells. On the contrary, 

various lines of cancer cells are not able to perform this kind of effect neither on 

themselves nor on fibroblasts. 

Our hypothesis is that the capability of fibroblasts to mediate the transfer of their 

own proteins and lipids to other cell types represents one of their major physiologic 

functions, i.e. to be the responsible of tissue trophism. In the simplest hypothesis, in 

the context of tumor microenvironment, these phenomena could enhance cancer cells 

growth rate by increasing the rate of mass accumulation to the lower limit necessary 

for cell division. 

1. Bastida E et al., (1984), Blood 64, 177-184; 

2. Raposo G et al., (1996), J. Exp. Med. 183, 1161-1172; 

2. Zitvogel L et al., (1998), Nat. Med. 4, 594-600; 

4. Van Niel G et al., (2001), Gastroenterology 121, 337-349; 

5. Wolfers J et al., (2001), Nat. Med. 7, 297-303; 

6. Faure J et al., (2006), Mol. Cell. Neurosci. 31, 642-648. 


118


Identification and characterization of cancer stem 

cells from Head and Neck squamous cell carcinoma 

cell line Hep-2. 

Pozzi valentina 1 , rocchetti roMina 2 , Santarelli andrea 1 , 

ruBini corrado 2 , MorGanti Stefano 1 , Giuliante rachela 1 , 

Sartini davide 1 , lo Muzio lorenzo 3,4 , eManuelli Monica 1 . 

1 Dipartimento di Scienze Cliniche Specialistiche e Odontostomatologiche, 

2 Dipartimento di Scienze Biomediche e Sanità Pubblica, 

Università Politecnica delle Marche, Ancona, ITALY; 

3 Dipartimento di Scienze Chirurgiche, Università di Foggia, Foggia, ITALY; 

4 I.R.C.C.S. - C.R.O.B., Rionero in Vulture, Potenza, ITALY. 

Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumourrelated 

mortality. Despite the recent improvements, the increase in overall survival 

has been nominal and cancer recurrence and treatment failures continue to occur in a 

significant percentage of patients. In recent years, several studies have demonstrated 

that only a distinct subpopulation of tumor cells, termed cancer stem cells (CSCs), 

contains the capability to undergo self-renewal and differentiation and hence has the 

ability to initiate tumorigenesis and support ongoing cancer growth. Cancer stem 

cell theory has important implications both for early detection of cancer and for the 

identification of new therapeutic targets. In this regard, CSC-enriched populations 

could be a more reliable tool to study the biology of tumours, rather than profiling the 

entire tumour, which is composed of heterogeneous cell populations. The purpose of 

the present study was to identify and characterize CSCs obtained from the HNSCC 

cell line Hep-2. CSCs were enriched through sphere formation, by cultivating HNSCC 

cell line in defined serum-free medium. Real-time PCR and immunocytochemical 

analysis revealed that a number of stem cell markers, including CD-133, CD-44, 

Oct-4, Sox2, Nanog and Nestin, were upregulated in CSC-enriched populations. 

In addition, tumorigenic potential of CSC-enriched populations was evaluated in 

vivo. Hep-2 cells and CSC-enriched populations were injected subcutaneously 

into immunocompromised mice. Interestingly, CSC-enriched populations showed a 

stronger capability to form tumours in vivo compared to the parental cell line. Our 

findings give new insights on the molecular features of cancer stem cells. A more 

detailed functional analysis of CSCs may provide a novel tool for investigating the 

tumorigenic process of HNSCC. 

Acknowledgments: 

This work has been supported by MIUR through PRIN 2008 (20089E83YR_004). 


119


Effect of zidovudine on the PKC of erythroleukemic K562 

and acute lymphoblastic leukemia HSB-2 cells 

carnicelli veronica, lizzi anna rita, Gualtieri Giancaterino, 

franceSchini nicola, Bozzi arGante and di Giulio antonio 

Dept. of Biotechnological and Applied Clinical Sciences University of L’Aquila 

This work is dedicated to the memory of Prof. Arduino Oratore who left us prematurely 

on May 5 th 2010. He was actively involved in the guidance of the Department of 

Biomedical Sciences and Technologies for many years. 

AZT is a drug used for the treatment of HIV-infected patients. In addition to this 

clinical use, “in vitro” studies have demonstrated that this drug alters some enzymatic 

activities [1], slows the rate of transferrin receptor endocytosis [2], decreases cell 

proliferation. Since it is known that PKC is involved in both normal and leukemic 

hematopoietic cell proliferation and differentiation, we investigated the possible 

relationship between the PKC activity and its isoforms expression with AZT treatment 

of human erythroleukemic undifferentiated K562 and acute lymphoblastic leukemia 

HSB-2 cells. 

The cell proliferation was already reduced and AZT caused a significant inhibition of 

activity at short times in both cells, whereas, a longer exposure (> 24 h) increased the 

PKC activity. A possible alteration by AZT in the membrane fluidity, was investigated 

by EPR technique. The zidovudine did not alter the membrane structure in K562 cells, 

while in the case of HSB-2 caused an increase in the fluidity membrane; this cell line 

appeared more sensitive to the AZT action on the membrane lipids. 

The PKC α and PKC ß II expression in K562 cells enhanced upon 8 h incubation, but the 

effect on PKC α was weaker than that measured on PKC ß II . Therefore AZT modifies 

the PKC activity and expression in both cell lines, with a different responsiveness. 

References 

1. Carnicelli V., Di Giulio A., Bozzi A., Strom R. and Oratore A.: Zidovudine inhibits protein kinase C activity 

in human chronic myeloid (K562) cells. Basic and Clinical Pharmacology and Toxicology 2006, 99, 317– 

322. 

2. D‘Alessandro AM, D‘Andrea G, Di Ciccio L, Brisdelli F, Rinaldi AC, Bozzi A, Oratore A. 3‘-Azido-3‘deoxythymidine 

reduces the rate of transferrin receptor endocytosis in K562 cells. 

Biochim Biophys Acta. 1999; 1450(3):232-41. 


120


FLAVIN ADENINE DINUCLEOTIDE METABOLISM 

IN THE NUCLEUS 

PaneBianco concetta, GiancaSPero tereSa anna, 

MiccoliS anGelica, BuSco Giovanni, carMone claudia, 

colella Matilde, Barile Maria. 

Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche, 

Università degli Studi di Bari “Aldo Moro”, via Orabona 4, 70125, Bari. 

Following its uptake from outside, in the cell riboflavin undergoes an ATP-dependent 

phosphorylation catalyzed by riboflavin kinase (RFK, E.C. 2.7.1.26) to form FMN, most 

of which is further converted by FAD synthase (FADS, E.C. 2.7.7.2) into FAD, the 

cofactor for many flavoenzymes involved in several biological processes in different 

cellular compartments. 

We previously demonstrated that beside a cytosolic enzyme (i.e. isoform 2 of 

human FLAD1 gene product) a mitochondrial isoform of FADS exists (isoform 1) 

(1, 2), presumably responsible for the biogenesis of mitochondrial flavoenzymes. 

Interestingly, the possibility of unexpected localizations for FADS has arisen (3) and, 

meanwhile, at least two new isoforms of FADS have been deposited in databases. 

Among FAD-dependent enzymes, lysine-specific demethylase-1 (E.C. 1.14.11.B1) is 

a nuclear protein recently shown to regulate cellular energy balance depending on 

FAD availability (4). 

These observation prompted us to investigate on the presence of flavin coenzymes in 

the nucleus and on a possible metabolism of FAD in this organelle. 

To this aim HPLC analysis of acid-precipitable flavins in nuclei isolated from rat liver 

were performed. The observation of a time-dependent decrease of FAD suggested 

us the existence of a hydrolytic activity, as confirmed by spectrofluorimetric 

measurements revealing an increase in fluorescence due to conversion of FAD into its 

more fluorescent precursor FMN/riboflavin (5). 

Immunoblotting experiments performed on rat liver nuclei using a home-made 

antibody against FADS revealed the enrichment of an immunoreactive band in respect 

to the other sub-cellular fractions. 

Confocal microscopy on several mammalian cells confirmed the existence of FADS 

co-localizing with nuclear markers. 

1. Barile et al., Eur J Biochem, 2000, 15, 4888-900. 

2. Torchetti et al., Mitochondrion, 2010, 10, 263-73. 

3. Lin et al., J Neurol, 2009, 256, 774-82. 

4. Hino et al., Nat Commun, 2012, 3, 758. 

5. Brizio et al., Eur J Biochem, 1997, 3, 777-85. 


121


Identification of nuclear proteins which interact 

with H1° mRNA 

di lieGro carlo Maria 1 , Saladino Patrizia 1 , Proia Patrizia 2 , 

Schiera GaBriella 1 , and di lieGro italia 3 

Departments of 1 Molecular and Biomolecular Technological Sciences (STEMBIO), 

2 Sport Sciences (DISMOT), and 3 Experimental Biomedicine and Clinical 

Neurosciences (BIONEC), University of Palermo, Palermo, Italy 

In developing rat brain the synthesis of H1° histone is mainly regulated at posttranscriptional 

level and probably depends on RNA-binding proteins (RBPs) (1). We 

previously identified RBPs apparently specific for this messenger (2) and cloned two 

novel proteins by screening an expression cDNA library by binding to radiolabeled 

RNA (3-10). Here we report the use of biotinylated H1° RNA as bait to isolate by 

chromatography nuclear proteins which interact with H1° mRNA. We identified by 

mass spectrometry abundant RBPs and molecular chaperones. By western blot we 

also evidenced, among the RNA-bound proteins, the cold shock domain-containing 

protein 2 (CSD-C2, also know as PIPPin), a brain-enriched RNA-binding protein 

cloned in our laboratory. Co-immunoprecipitation assays were performed to confirm 

interactions. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 

whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 

interacts with hnRNP A1 and with the Y box-binding protein 1 (YB1). We also got 

evidences that CSD-C2 interacts with Hsc70. In conclusion, we characterized a set 

of interactions which suggest the existence of a ribonucleoprotein particle, that might 

control H1º histone expression in maturing brain. 

REFERENCES: 

1.Derrigo M et al (2000) Int J Mol Med 5:111 

2.Scaturro M et al (1998) J Biol Chem 273:22788 

3.Castiglia D et al (1996) Biochem Biophys Res Commun 218:390 

4.Nastasi T et al (1999) J Biol Chem 274:24087 

5.Nastasi T et al (2000) Neuroreport 11:2233 

6.Raimondi L et al (2003) J Cell Mol Med 7:35 

7.Scaturro M et al (2003) Int J Mol Med 11:509 

8.Sala A et al (2007) Int J Mol Med 19:501 

9.Bono E et al (2007) Endocrinology 148:252 

10.Saladino P et al. (2012) Int J Mol Med. 29:141 


122


L-Carnosine inhibits KRAS-mediated HCT116 proliferation, 

by affecting ROS production and Hypoxia inducible factor 

(HIF-1α expression. 

iovine B., nocella f., Garofalo M., Pricolo M.r., orefice M., 

Bevilacqua M.a. 

Dipartimento di Biochimica e Biotecnologie Mediche. 

Università degli Studi di Napoli Federico II 

Cancer cells with respect to normal cells adapt themselves to hypoxia by using 

this physiological system for tumor growth, invasion, and metastasis. The hypoxiainducible 

factor (HIF-1) plays a central role in O 2 homeostasis and is considered a 

central regulator of the adaptation responses of cancer cells to hypoxia (1). HIF-1 

is a heterodimeric transcription factor consisting in an O 2 -regulated HIF-1α and a 

constitutively expressed HIF-1α subunit. Considerable effort has been directed to the 

discovery the chemical or natural molecules that target HIF-1α protein and regulate 

its_signaling pathway. In a proteomic approach, some authors support the concept 

that carnosine (α-Ala-His), a naturally occurring dipeptide, affects tumor cell growth in 

human glioma cell line, by an interference with protein folding/ processing and HIF-1α 

signalling (2). Recently, it has been shown that carnosine inhibits the proliferation of 

human HCT116 colon cancer cells (3). In this cell line, the activating KRAS mutation 

contributes to oncogenic transformation and to activation of HIF-1α promoting the 

ATP production by glycolysis and_the generation of mitochondrial reactive oxygen 

species (ROS). We observed that pre-treatment with 50-100mM carnosine decreases 

ATP and ROS concentrations as well as Erk1/2 phosphorylation and inhibits the cell 

cycle progression in the G1 phase. We assume that the anti-proliferative effect of 

carnosine in HCT116 cells is related to its anti-oxidant action and its ability to affect 

glycolysis (3). Next, we investigated the effects of carnosine on the expression of 

HIF-1α and evaluated how the glucose metabolism was affected. Pre-treatment of 

HCT116 cells with 50-100mM carnosine reduced HIF-1α protein level and also the 

hypoxia-inducible genes recognized by the hypoxia responsive element (HRE) such 

as aldolase and pyruvate dehydrogenase kinase 1 (PDK-1). 

(1) Semenza G.L. Nat.Rev.Cancer 2003 3(10):721-32 

(2) Asperger, A., et al., Cancer Invest.2011 29(4):272-81 

(3) Iovine B., et al., Cancer Letters 2012 28:315(2): 122-8 


123


c-Met transactivation by FPRL1 requires NADPH oxidasedependent 

superoxide generation in human prostate 

epithelial cell line PNT1A 

cattaneo f., PariSi M., caSo v. M., aMMendola r. 

Dipartimento di Biochimica e Biotecnologie Mediche, 

Università di Napoli Federico II, Via S. Pansini 5, 80131 Naples, Italy. 

Cross-communication between G-protein-coupled receptors (GPCR) and tyrosine 

kinase receptors (TKR) provides a signalling mechanism essential to amplify the 

network of information exchange throughout the cell. We investigated, in human 

prostate epithelial PNT1A cells, the ability of the formyl peptide receptor-like 1 (FPRL1), 

a member of the GPCR superfamily, to trans-phosphorylate c-Met, a TKR expressed 

in several cell types and involved in many physiological processes. We observed that 

these cells express FPRL1 and that stimulation with its agonist WKYMVm induces 

NADPH oxidase-dependent superoxide generation and the phosphorylation of 

tyrosine 1313/1349/1356 residues of c-Met. As a result of c-Met transactivation, the 

phosphotyrosine located in the multifunctional docking site (Y 1349 VHVNATY 1356 VNV) 

provide binding sites for a variety of Src homology-2 (SH2)-containing signal 

transducers and effectors. We observed the trigger of STAT3 pathway, as well as 

PLC-γ1 and PI3K(p85) phosphorylation, which catalyze PKCα and Akt activation, 

respectively. WKYMVm-induced c-Met transactivation and the intracellular signalling 

triggered by FPRL1 are prevented by the FPRL1-selective antagonist WRWWWW, 

by pertussis toxin and by SU11274, a specific inhibitor of c-Met. The critical role 

of NADPH oxidase-dependent superoxide generation in this cross-talk mechanism 

is supported by the finding that apocynin, a specific NADPH oxidase inhibitor, or a 

siRNA against p22 phox , prevent c-Met transactivation, STAT3 pathway activation, as 

well as PI3K(p85) and Akt phosphorylation. These results highlight the function of 

FPRL1 to activate a highly interconnected signalling network by crosstalk with c-Met, 

and suggest novel possibilities for rational therapeutic interventions. 

1. Pavone L.M., Cattaneo F., Rea S., De Pasquale V., Spina A., Sauchelli E., Mastellone V., Ammendola R. 

(2011). Cell. Signal., 23: 1961-1971. 

2. F. Cattaneo, A. Iaccio, G. Guerra, S. Montagnani, R. Ammendola (2011). Free Rad. Biol. & Med. 

51:1126-1136. 


124


Nox4 inhibitory activity of new synthesized oxindole 

derivatives 

franceSco vieceli dalla SeGa, laura zaMBonin, diana fiorentini, 

Benedetta rizzo, Silvana hrelia and cecilia Prata 

Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna – Italy 

Recently, the Nox4 inhibitory activity shown by some oxindole derivatives has been 

described [Borbély G et al. J. Med. Chem., 2010]. In this study, a panel of new 

synthesized derivatives active against different leukaemia cell lines in the NCI assays 

were tested for the ability to inhibit Nox4 in B1647 cells, a human acute myeloid 

leukaemia cell line. Nox4 is known to be constitutively active and to play the major 

role in the generation of ROS required for sustaining cellular growth and proliferation 

of B1647 cells. MTT assay confirmed the anti-proliferative activity of all the tested 

molecules in a dose-dependent manner at a concentration of 5 and 10µM. ROS 

production was measured in B1647 cells after an acute treatment (30 min) with the 

selected compounds. Results indicate that two compounds (C5 and C7) are able to 

inhibit ROS generation in a dose-dependent manner. As B1647 cells express Nox2 

and Nox4, in order to rule out the role of Nox2-derived ROS, the acute inhibition of 

ROS production was measured following RNA silencing of Nox2. Data show that the 

two selected compounds were able to inhibit ROS production at the same extent 

either in Nox2 silenced cells or in the control. Finally, Western blotting analysis was 

performed to investigate Nox4 expression in B1647 cells after 24 h-incubation with the 

compounds. Data show that treatments with C5 and C7 were able to downregulate 

Nox4. In conclusion, results here reported indicate that Nox4 could be a target for 

two of the new synthesized oxindole derivatives and that Nox4 inhibition seems to 

contribute to the anti-proliferative effect of the compounds in acute leukaemia B1647 

cells and to be a promising strategy for a novel antileukemic therapy. 

This work was supported by Fondazione del Monte di Bologna e Ravenna (Italy). 


125


Bag3 polymorphisms may be associated to heart failure 

due to stress cardiomyopathy 

d’avenia Morena 1,2 , de Marco MarGot 1,2 , citro rodolfo 3 , 

roSati aleSSandra 1,2 , falco antonia 1,2 , PaScale Maria 1,2 , 

de laurenzi vincenzo 2,4 , turco Maria caterina 1,2 . 

1.Department of Pharmaceutical and Biomedical Sciences, 

University of Salerno, Fisciano (SA), Italy; 

2. BIOUNIVERSA srl, University of Salerno, Fisciano (SA), Italy; 

3. Department of Cardiology, University Hospital 

“San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy; 

4. Department of Biomedical Sciences, University “G. d’Annunzio” Chieti-Pescara 

and Fondazione “G. D’Annunzio, Centro Studi sull’Invecchiamento, 

Ce.S.I.”, Chieti, Italy; 

BAG3 (BCL2-associated athanogene 3) is a cytoplasmic protein expressed at high 

levels in the cardiac muscle tissue. It supports the survival and the contractile 

activity of muscle. It was reported that BAG3 knock-out mice die 4 weeks after 

birth because of fulminant myopathy with heart apoptosis (Homma et al., 2006). A 

mutated form of BAG3, heterozygous Pro209Leu, (Selcen et al. 2009) was reported 

in three patients suffering of childhood cardiomyopathy and a severe and progressive 

muscle weakness. Moreover, increased levels of BAG3 were detected in rat hearts 

after temporary occlusion of descending coronary artery, suggesting BAG3 role in the 

regulation of cardiomyocytes survival under oxidative stress. 

We evaluated whether BAG3 could be involved in the Tako Tsubo cardiomyopathy 

(or stress cardiomyopathy) pathogenesis that is characterized by left ventricular 

dysfunction, with symptoms that can mimic an acute coronary syndrome. Stress 

cardiomyopathy arises predominantly in postmenopausal women, suggesting that 

myocardial stunning might be provoked by estrogen deficiency (Brenner R et al. 2012), 

and it arises also after a stressful event, suggesting “the catecholamine hypothesis”. 

CAs are among the first compounds released during stress (Dewachter P et al 2011), 

and in those patients something collapses the physiological responses. 

In particular, we found that epinephrine in vitro stimuli increase bag3 expression 

(mRNA and protein) in wild type human PBMC and adult human cardiomyocytes. 

The absence of significant cardiovascular risk factors in patients affected by stress 

cardiomyopathy suggested that it might be associated with a possible genetic etiology. 

Therefore, we sequenced bag3 gene to check for polymorphisms in 50 patients and 

65 healthy donors. Four polymorphism were high represented among patients (R71Q, 

C151R, P407L and one in the non-coding region) and patients’ PBMC (carrying some 

of those polymorphisms) stimulated in vitro with epinephrine showed mRNA bag3 

levels lower than healthy donors. 


126


Epigenetic modulation of anandamide hydrolase by 

estrogen 

Maccarrone Mauro 1,2 , Pucci MarianGela 1 , di Siena Sara 3 , 

di GiacoMo daniele 3 , Pirazzi valentina 1 , GereMia raffaele 3 and 

GriMaldi Paola 3 

1 Department of Biomedical Sciences, University of Teramo, 64100 Teramo, Italy 

2 European Center for Brain Research (CERC)/Santa Lucia Foundation, 

00179 Rome, Italy 

3 Department of Public Health and Cellular Biology, 

University of Rome ”Tor Vergata”, 

00133 Rome, Italy 

Estrogen (E 2 ) regulates spermatogenesis, yet its direct target genes have not yet been 

identified. Here, we cloned the proximal 5’ flanking region of the mouse anandamide 

hydrolase (fatty acid amide hydrolase, faah) gene upstream of the luciferase reporter 

gene, and demonstrated its promoter activity and E 2 inducibility in primary mouse Sertoli 

cells. Specific mutations in the E 2 response elements (ERE) of the faah gene showed 

that two proximal ERE sequences (ERE2/3) are essential for E 2 -induced transcription, 

and chromatin immunoprecipitation experiments showed that E 2 induced estrogen 

receptor β binding at ERE2/3 sites in the faah promoter in vivo. Moreover, the histone 

demethylase LSD1 was found to be associated with ERE2/3 sites and to play a role 

in mediating E 2 induction of FAAH expression. E 2 induced epigenetic modifications at 

the faah proximal promoter compatible with transcriptional activation, by remarkably 

decreasing methylation of both DNA at CpG site and histone H3 at lysine 9. Finally, 

FAAH silencing abolished E 2 protection against apoptosis induced by the FAAH 

substrate anandamide. 

Conclusions. 

Taken together, our results identify FAAH as the first direct target of E 2 . 

Acknowledgements. 

This work was supported by grants from Agenzia Spaziale Italiana to RG and MM, and Fondazione TERCAS 

(2009-2012 project) to MM. 


127


Transcriptional and epigenetic regulation of Anandamide- 

Hydrolase (FAAH) in patients with late-onset Alzheimer disease 

di franceSco andrea 1 , d’addario claudio 1 , aroSio Beatrice 2 , 

GuSSaGo criStina 2 , dell’oSSo Bernardo 3 , GaliMBerti daniela 4 , 

ScarPini elio 4 , altaMura carlo a 3 , Mari daniela 2 1, 5 , Mauro 

1 Dept. of Biomedical Sciences, University of Teramo, 64100 Teramo 

2 Geriatric Unit, University of Milan, Fondazione Cà Granda, 

IRCCS Ospedale Maggiore Policlinico, 20122 Milan, Italy 

3 Dept. of Psychiatry, University of Milan, Fondazione Cà Granda, 

IRCCS Ospedale Maggiore Policlinico, 20122 Milan, Italy 

4 Dept. of Neurological Sciences, “Dino Ferrari” Center, University of Milan, 

Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico, 20122 Milan, Italy 

5 European Center for Brain Research (CERC)/ IRCCS Santa Lucia Foundation, 00142 Rome, Italy 

Abstract 

Alzheimer disease (AD) is the most frequent form of dementia in the elderly, 

affecting more than 25 million people worldwide; current treatments for AD provide 

only palliative approaches and in the search of new targets for its therapy, the 

“endocannabinoid system” (ECS) recently emerged as a promising candidate due to 

its role in neuroinflammatory and neurodegenerative diseases. 

We have studied the gene expression status and the epigenetic regulation of ECS 

components in peripheral blood mononuclear cells (PBMCs) of subjects with lateonset 

AD (LOAD) and age-matched controls (CT). 

Gene expression of all major ECS elements in LOAD subjects and healthy controls was 

quantified and only FAAH mRNA was found to be altered by the disease. A significant 

increase of faah gene expression was observed in LOAD subject compared to controls 

(LOAD: 2.30 ± 0.48; CONT: 1.00 ± 0.14;* p < 0.05). Consistently, we also observed 

in LOAD subjects an increase in FAAH protein levels (CT: 0.75 ± 0.04; LOAD: 1.11 ± 

0.15;* p < 0.05), as well as a reduction in DNA methylation at faah gene promoter (CT: 

55.90 ± 4.60 %; LOAD: 41.20 ± 4.90 %;* p < 0.05). 

In conclusion, this study adds new information on ECS alterations in normal or 

pathological aging, highlighting the importance of epigenetic mechanisms in faah 

gene regulation. On the basis of the relationship between the brain and the periphery 

in AD, it also suggests a possible role for FAAH not only as a peripheral biomarker but 

also as new possible therapeutic target for AD. 

References 

D’Addario C, Di Francesco A, Arosio B, et al. Epigenetic regulation of Fatty Acid amide hydrolase in 

Alzheimer disease. PLoS One.2012;7:e39186. 

Aknowledgment 

This investigation was partially supported by Fondazione TERCAS (grant 2009–2012 to MM). ADF was 

supported by a “Fondazione TERCAS-Progetto Speciale Assegni di Ricerca 2011-2013”fellowship. 


128


ExPRESSION AND PURIFICATION OF RECONBINANT 

HISTONE DEACETYLASE 1 

PreSta enrica, naSo federica, Parolin carola, calonGhi natalia 

and Sartor GiorGio 

Alma Mater Studiorum, Università di Bologna, 

Dipartimento di Biochimica “G.Moruzzi. 

9-hydroxystearic acid (9-HSA) is an endogenous product of lipid peroxidation 

identified in several human and murine cell lines (1). 9-HSA exogenous administration 

to a human colon adenocarcinoma cell line (HT29) arrests the cells in G0/G1, the block 

of the cell cycle progression being characterized by an increase in the expression of 

p21 WAF1 in an immediate-early, p53 independent fashion (2). 

In HT29 cells, such agents induce effects over imposable to those induced by 9-HSA, 

whose activity as a histone deacetylase 1 (HDAC1) (EC 3.5.1.98) inhibitor has been 

reported (3). The reconstruction of the 3D model of the human enzyme, not yet 

crystallized, and docking studies have evidenced that the hydroxyacid can interact 

with the catalytic site and inhibit its activity (3). 

Several studies are currently under way: first we try to express and isolate the protein 

HDAC1 for crystallographic and interaction studies with the 9-HSA cloning the gene in 

plasmid vector pcDNA3.1 to be expressed in HeLa S3, this method does not gave us 

appreciable results. We then utilize an expression system that uses Baculovirus that 

allow us to optimize the amount of protein obtained. 

In order to purify the HDAC1 recombinant protein we use two chromatographic steps 

in inverse phase HPLC and affinity chromatography techniques. 

References 

1. Masotti et al., Ann. NY Acad. Sci. 1988; 551:47-57. 

1. Calonghi et al., Biochem. Biophys. Res. Commun. 2004 30; 314(1):138-142. 

1. Calonghi et al., J. Lipid Res. 2005; 46(8):1596-1603 


129


Identification of specific proteins affected by microRNAs in 

cellular senescence 

coMeGna M. 1 , naPolitano M. 2 , Succoio M. 1 , vitale M. 1,3 , 

d’aMBroSio c. 4 , Scaloni a. 4 , zaMBrano n. 1,3 , faraonio r. 1 , 

PaSSaro f. 1 and ciMino f. 1,2 

1 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy 

2 Fondazione SDN – Istituto di Ricerca Diagnostica e Nucleare, Napoli, Italy 

3 CEINGE-Biotecnologie Avanzate s.c. a r.l, Napoli, Italy 

4 Proteomics and Mass Spectrometry Laboratory, National Research Council, Napoli, Italy 

Cellular senescence can be induced by various endogenous or exogenous stresses 

triggering DNA damage and DNA Damage Response (DDR). Cells undergoing 

senescence have characteristic biochemical features sustained by a reprogrammed 

gene expression. MicroRNAs (miRNAs) are small non-coding RNAs that act as potent 

negative regulators at post-transcriptional level. We recently identified 24 miRNAs 

(SAmiRs) that were either up-or down-regulated in senescent cells (1). SAmiRs 

expression was also deregulated in senescence induced by DNA damage (etoposide) 

or oxidative stress (DEM). Upon adoptive overexpression, 7 up-regulated SAmiRs 

induced senescence-associated heterochromatin foci (SAHFs) and senescenceassociated 

beta-gal staining (SAbeta-gal) that were accompanied, in the case 

of miR-210, miR-376a*, miR-486-5p, miR-494 and miR-542-5p, by reduced cell 

proliferation, induction of double strand DNA breaks, DDR and reactive oxygen 

species accumulation. Based on these results, our aim is to study regulatory networks 

induced by SAmiRs in senescence through the identification of their direct targets. 

To address this point, we used two different approaches. In the first case, we used 

target-prediction algorithms to generate a list of mRNAs downregulated in replicative 

senescent fibroblasts, harboring potentially SAmiRs recognition elements within their 

3’UTR. Preliminary results indicate that ID4 and CDCA2 are targets of miR-486-5p and 

miR-494, respectively. In the second case, we performed Two Dimensional Differential- 

In-Gel Electrophoresis (2D-DIGE) and mass-spectrometry analysis to identify proteins 

differentially expressed between senescent (replicative or DEM induced) IMR90 

fibroblasts and young cells, or after miR-494 overexpression compared to young 

cells. Among all the protein spots that were differentially detected and identified, we 

found cytoskeletal, heat shock, metabolic and redox proteins, as well as polypeptide 

components involved in DDR and others of unknown function. Validation of these 

results is in progress by integrating in silico computational analysis, luciferase-based 

and western blot assays. 

1. R. Faraonio et al. Cell Death Differ., 19, 713-21 (2012) 


130


Serotonin role in development, function and disease of 

thyroid gland 

1,2 Silviana rea, 2 SiMona tafuri, 2 anna coStaGliola, 

1,3 valeria de PaSquale, 2 vincenzo MaStellone, 

1,3 luiGi Michele Pavone 

1 Department of Biochemistry and Medical Biotechnologies, 

2 Department of Biological Structures, Functions and Technologies, 

University of Naples Federico II, Naples, Italy; 

3 CEINGE, Advanced Biotechnology, Via Gaetano Salvatore, 486, 80145 Naples, Italy. 

Serotonin is an ubiquitous monoamine which exerts both a morphogenetic and 

neurotransmitter activity in different tissues. It acts through seven families of receptors, 

however, its activity is strictly regulated by transporter SERT which modulates 

serotonin concentration in the extracellular fluid. Alterations of serotonergic system 

are associated with a variety of disorders ranging from depression, schizophrenia 

and anxiety to hypertension, eating disorders, heart valve disease, irritable bowel 

syndrome and others. 

We focused our interest on the role of serotonin in thyroid development, function and 

disease. A Cre/loxP-based fate mapping approach was used to follow the regions of 

the mouse thyroid labeled by serotonin transporter SERT. Reporter gene expression 

(lacZ) is activated by Cre expression from the SERT locus in SERT(Cre/+);ROSA26R 

compound mouse embryos. Cell labeling, first detected in thyroid primordium at E10.5 

prenatal stage, was followed until postnatal day P30. Co-localization of lacZ staining 

in the same cells that express the transcription factors Nkx2.1 and Pax8 at E12.5 

stage confirmed their identity as thyroid cell precursors. SERT immunohistochemistry 

on thyroid sections of E18.5 embryos showed SERT expression in thyroid follicular 

cells. Western blotting analysis confirmed the expression of the protein in adult thyroid 

tissue and cultured FRTL-5 cells. Then, we investigated the molecular signalling 

activated by serotonin interaction with 5HT2A receptor in cultured rat epithelial 

cells (FRT). RT-PCR demonstrated the expression of SERT and 5-HT2A mRNAs 

and immunoblotting confirmed the presence of the two proteins in FRT cell system. 

Cell exposure to different amounts of serotonin resulted in a proliferation response 

at a dose of 5 µM, whereas 50 µM serotonin caused cell death. Cell proliferation 

was induced by serotonin through the activation of ERK/STAT3 pathway. Our 

results suggest a previously unappreciated role of serotonin in physiological thyroid 

embryonic development as well as in pathological thyroid dysfunctions. 


131


MOLECULAR MECHANISM OF ACTION OF POLYPHENON 

E, A GREEN TEA ExTRACT, IN BENIGN AND 

TUMORIGENIC PROSTATE EPITHELIAL CELLS 

rizzi federica 1,2,3 , Silva aleSSandro 1 , naPonelli valeria 1,2 , 

tardito Saverio 1 , raMazzina ileana 1,2 , Bonacini Martina 1 and 

Bettuzzi Saverio 1,2,3 

1 Department of Biomedicine, Biotechnology and Translational Research and 2 Centre 

for Molecular and Translational Oncology (COMT), University of Parma, Parma, Italy; 

3 National Institute of Biostructure and Biosystems (INBB), Rome, Italy. 

Green Tea Extracts (GTE) show potent chemoprevention activity for prostate cancer 

(PCa), but the mechanism of action is still obscure. Increasing doses of Polyphenon 

E (PolyE), a standardized GTE, were given to immortalized PNT1a, mimicking initial 

stages of PCa, or metastatic, androgen-independent PC3, mimicking advanced 

stages. Growth arrest, decreased viability and cell death occurred in both cell lines, 

but PNT1a were more sensible to PolyE (IC 50 = 35 µg/mL) than PC3 (IC 50 = 145 µg/ 

mL). Cell cycle arrest occurred at G0/G1 phases for PNT1a with up-regulation of p21 

and down regulation of cyclin D1, while at G 2/ M phases for PC3 cells, in which only 

Cyclin D1 was down-regulated. Activation of caspase-3, -7, -9 and cleavage of PARP- 

1 occurred in PNT1a, while the BH3-only family (PUMA and Bax) and AIF increased 

(and caspases were not activated) in PC3. Autophagy was transiently activated only 

in PNT1a cells. 

We found that the target of GTE is ER: induction of ER core stress proteins peIF2a 

and ATF4 and ER stress protein markers GRP78/BiP, CHOP/GADD153 and GADD34 

occurred in both cell lines, but with different time-course. PNT1A overcome ER 

stress, then rapidly died by cell-detachment, caspase-dependent apoptosis (anoikis). 

At difference, in PC3 cells prolonged ER stress caused progressive loss of ER 

morphology, extensive intracellular vacuolization, persistent activation of p-EIF2a and 

ATF-4, activation of XBP1 mRNA splicing and dramatic up-regulation of CHOP and 

GADD34 (key proteins of ERS-mediated cell death). Under these conditions, PC3 died 

very slowly: cells shrink, but remain attached to the surface, with formation of calceinnegative 

extensive vacuolization due to enlarged ER. Cell membranes remain intact 

also in presence of chromatin margination and detachment of DNA-free cell bodies. 

The combination of molecular markers and morphological analysis is compatible with 

necroptosis, a highly regulated kind of caspase-independent cell death, specifically 

occurring in metastatic PC3 cells. 


132


CLUSTERIN IS EPIGENETICALLY REPRESSED IN HUMAN 

PROSTATE CANCER CELLS 

rizzi federica 123 , coletta MarianGela 1 , raMazzina ileana 1,2 , 

Bonacini Martina 1 , naPonelli valeria 1,2 , Silva aleSSandro 1 and 

Bettuzzi Saverio 1,2,3 

1 Department of Biomedicine, Biotechnology and Translational Research and 

2 Centre for Molecular and Translational Oncology (COMT), University of Parma, 

Parma, Italy; 3 National Institute of Biostructure and Biosystems (INBB), Rome, Italy. 

Clusterin (CLU) is a conserved protein involved in apoptosis and cell survival downregulated 

in many tumours (including prostate cancer and neuroblastoma), where it 

acts as a tumour suppressor. Several CLU transcripts, different at the 5’ end, are 

produced from the single copy human CLU gene, but very little is known about 

their functions and regulation. We studied the expression of two CLU mRNAs in 

immortalized or cancer human cells in comparison with benign controls, investigating 

how expression of CLU is affected by epigenetic drugs such as 5-Aza-2’-deoxycytidine 

(AZA) or trichostatin A (TSA). 

Total RNA was extracted from normal embryonic lung fibroblasts (WI-38), immortalized 

BEAS2B, HT125, PNT1a and cancer epithelial A549, MCF7, PC3, DU145 cells 

under standard condition. Relative expression of CLU mRNAs was evaluated by RT- 

PCR. Prostate cancer PC3 and DU145 cells have been treated with AZA, TSA or a 

combination of the two drugs. Methylation of the CLU promoter was assessed by 

Methylation Specific PCR. Histone H3 tail modification by acetylation/methylation at 

the CLU promoter was assessed by Chromatin Immunoprecipitation (ChIP) assay and 

quantitative RT-PCR (RT-qPCR). 

One of the two CLU mRNAs studied is constitutively expressed across all cell lines 

tested, while the other is virtually absent in cancer cells, being only detectable in 

normal or immortalized cells. A CpG island on CLU promoter is only methylated in 

cancer cells. The CLU mRNA silenced by cell transformation is re-expressed by 

AZA and TSA. Re-expression of silenced CLU mRNA was associated to nuclear 

localization of CLU and induction of apoptotic cell death. TSA alone, or with AZA, 

increases H3 post-translational modification (H3-Ace, H3Lys9Ace, H3Lys9Met3) in 

the CLU promoter, causing active transcription. In conclusion, a specific CLU mRNA 

transcript is epigenetically down-regulated in prostate cancer cells through chromatin 

remodelling. Re-expression of CLU by epigenetic drugs unravels its tumoursuppressor 

activity. 


133


Protective role of 17-β-estradiol toward IL-6 leukocyte 

expression triggered by intense training in elite young 

female athletes 

trinGali criStina 1 , Scala loredana 1 , SilveStri ilaria 1 , 

Scurati raffaele 2 , Michielon Giovanni 2 alBerti GiaMPietro 2 and 

venerando Bruno 1 


University of Milan, School of Exercise Sciences, LITA, 

via F.lli Cervi 93, Segrate, Milan, 20090, Italy 

2 Department of Biomedical Sciences for Health, University of Milan, 

School of Exercise Sciences 

Exercise performed at a competitive level could deeply modify the immune system 

and the cytokine response of athletes. In this report, we demonstrated that young elite 

female athletes, engaged in synchronized swimming and artistic gymnastics, showed 

an increase of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) mRNAs 

expression of blood mononuclear cells, in comparison to young girls performing sport 

at a recreational level. The increase of IL-6 and TNF-α mRNAs directly depended on 

the intensity of the training. IL-6 gene expression appeared to be modulated by the 

levels of circulating oestrogens and pre-pubertal girls revealed the higher increases of 

IL-6. In addition, BMI (body mass index) percentile was inversely correlated with both 

the increases of IL-6 and TNF-α in pre-pubertal athletes. The consequence of these 

events was the shift of the cytokine profile toward a pro-inflammatory status. IL-6 and 

TNF-α are able to impair the growth hormone (GH)- insulin growth factor -1 (IGF-1) 

axis. Therefore, these modifications induced by training performed at an elite level risk 

to negatively affect the growth of female children athletes. 


134


Activation by 2-arachidonoylglycerol of platelet p38MAPK/ 

cPLA 2 pathway 

SiGnorello Maria Grazia, GiacoBBe enrica and leoncini Giuliana 

Department of Experimental Medicine, Biochemistry section, 

Genoa University, Genoa, Italy 

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a 

platelet agonist able to induce aggregation and to increase intracellular calcium. In 

the present report we have confirmed these data and demonstrated that the inhibitor 

of p38MAPK SB203580 and the inhibitor of cPLA 2 metabolism ETYA affect both these 

parameters. Thus we aimed to define the role of p38MAPK/cytosolic phospholipase 

A 2 (cPLA 2 ) pathway in 2-AG-induced human platelet activation. p38MAPK activation 

was assayed by phosphorylation. cPLA 2 activation was assayed by phosphorylation 

and as arachidonic acid release and thromboxane B 2 formation. It was shown that 

2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 µM 

after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, 

indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific 

inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of 

type-2 cannabinoid receptor SR144528. Moreover the incubation of platelets with 

2-AG led to the phosphorylation of cPLA 2 and its activation. Platelet pretreatment 

with SB203580, inhibitor of p38MAPK abolished both cPLA 2 phosphorylation 

and activation. In addition SR141716 strongly impaired cPLA 2 phosphorylation, 

arachidonic acid release and thromboxane B 2 formation, whereas SR144528 did not 

change these parameters. Finally platelet stimulation with 2-AG led to an increase in 

free oxygen radical species. In conclusion data provide insight into the mechanisms 

involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA 2 pathway 

could play a relevant role in this complicated process. 


135


Expression of the antiapoptotic protein BAG3 is a feature 

of pancreatic adenocarcinoma and overexpression is 

associated with poorer survival 

roSati aleSSandra 1,2 , BerSani SaMantha 3 , tavano franceSca 4 , dalla 

Pozza eliSa 5 , MarGot de Marco 1,2 , PalMieri Marta 5 , 

de laurenzi vincenzo 2,6 , franco renato 7 , ScoGnaMiGlio GioSuè 7 , 

Palaia raffaele 8 , fontana andrea 9 , di SeBaStiano PierluiGi 4 , 

donadelli MaSSiMo 5 , dando ilaria 5 , MedeMa Jan Paul 10 , 

diJk frederike 11 , wellinG lieke 12 , di Mola faBio franceSco 4 , 

Pezzilli raffaele 13 , turco Maria caterina 1,2 *, ScarPa aldo 3 

1.Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano (SA), Italy; 2. BIOUNIVERSA srl, 

University of Salerno, Fisciano (SA), Italy; 3. ARC-NET Center for Applied Research on Cancer and Department of Pathology 

and Diagnostics, Hospital and University Trust , Verona, Italy; 4. Department of Surgery Casa Sollievo della Sofferenza” Hospital, 

IRCCS, San Giovanni Rotondo, Italy; 5. Department of Life and Reproduction Sciences, Biochemistry Section, University of 

Verona, Verona, Italy; 6. Department of Biomedical Sciences, University “G. d’Annunzio” Chieti-Pescara and Fondazione “G. 

D’Annunzio, Centro Studi sull’Invecchiamento, Ce.S.I., Chieti, Italy; 7. U.O.C. Anatomia Patologica, Istituto Nazionale Tumori 

Fondazione G. Pascale, Naples, Italy; 8. Abdominal Surgery Unit, Istituto Nazionale Tumori Fondazione G. Pascale, Naples, Italy; 

9. Unit of Biostatistics “Casa Sollievo della Sofferenza” Hospital, IRCCS, San Giovanni Rotondo, Italy; 10. LEXOR, Center for 

Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands; 11. Department of 

Pathology and 12. Department of Surgery, Academic Medical Center, University of Amsterdam, The Netherlands; 

13. Pancreas Unit, Department of Digestive Diseases and Internal Medicine, Sant’Orsola-Malpighi Hospital, Bologna, Italy. 

Abstract 

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers being 

the fourth cause of cancer- related deaths. Less than 5% of patients have a long term 

survival reaching 15% in those that undergo surgery while in inoperable cases the 

median survival is only 6 months. A deeper understanding of PDAC biology as well 

as novel prognostic markers are therefore required to improve the outcome. Here we 

report that BAG3, a protein with recognized antiapoptotic activity, was expressed in 

all 346 PDACs analyzed while it was not expressed by the surrounding non neoplastic 

tissue. Interestingly, in a cohort of 66 patients who underwent R0 resection, patients 

with high BAG3 expression had a significantly shorter survival (median survival = 12 

months) than those with low BAG3 expression (median survival = 23 months) (p = 

0.0013). Furthermore, we show that BAG3 expression in PDAC-derived cell lines 

protects from apoptosis and confers resistance to gemcitabine offering a partial 

explanation to the survival data. Our results indicate that BAG3 protein plays a relevant 

role in PDAC biology and suggest that BAG3 expression levels might be a potential 

marker to predict outcome of patients. 


136


Failing cardiomyocytes release BAG3 that activates 

macrophages 

de Marco MarGot 1, 2 , falco antonia 1, 2 , BaSile anna 1, 2 , roSati aleSSandra 1, 

2 1, 2 1, 2 1 

, feSta Michelina , d’avenia Morena , franceSchelli Silvia 

dal Piaz faBrizio 1 , BiSoGni rita 2 , Barcaroli daniela 3 , Marzocco Stefania 1 , 

Pinto aldo 1 , coPPola GiuSePPe 4 , PiScione federico 5 , GiGantino alBerto 5 , 

de roSa roBerta 6 , vitulano Gennaro 5 , virtuoSo nicola 5 , ManGanelli 

fiore 7 , PalerMo erneSta 7 , roSato GiuSePPe 7 , 

hahne Michael 8 , tiBerti claudio 9 , de laurenzi vincenzo 2, 3 , 

1, 2 

turco Maria caterina 

1.Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano (SA), Italy; 

2. BIOUNIVERSA srl, University of Salerno, Fisciano (SA), Italy; 3. Department of Biomedical 

Sciences, University “G. d’Annunzio” Chieti-Pescara and Fondazione “G. D’Annunzio, Centro Studi 

sull’Invecchiamento, Ce.S.I.”, Chieti, Italy; 4. Unit of Transfusion Medicine, University Hospital 

“San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy; 5. Department of Cardiology, University Hospital 

“San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy; 6. Department of Clinical Medicine, Cardiovascular 

Sciences and Immunology, Federico II University, Naples, Italy; 7. Department of Cardiology, “A.O.S.G. 

Moscati” Hospital, Avellino, Italy; 8. Institut de Génétique Moléculaire de Montpellier, CNRS UMR5535, 

Montpellier, France; 9. Department of Clinical Sciences, University of Rome Sapienza, Rome, Italy. 

Rationale: BAG3 is constitutively expressed in very few cell types including cardiomyocytes 

where it is known to stabilize myofibril structure and integrity during mechanical stress. In 

general its function seems to be sustaining cell survival through regulation of signaling 

proteins. Since cardiomyocytes are known to release protective factors in response to 

stress, we analyzed whether they could also release BAG3. 

Objective: Study of the release of BAG3 by cardiomyocytes in response to stress and 

definition of its potential function. 

Methods and Results: Western blot analysis of media from cultured cardiomyocytes shows 

that BAG3 is released upon serum starvation. Proteomic analysis confirms identity. By 

subcellular fractionation we show that extracellular BAG3 is associated to exocytic vesicles. 

We also show that auto-antibodies against BAG3 can be detected in sera of patients with 

chronic heart failure. Furthermore we developed a specific ELISA test for these antibodies 

and analyzed sera from 98 congestive heart failure patients and 89 healthy donors showing 

that patients have significantly higher levels of circulating auto-antibodies. 

Finally we show that FITC-conjugated rBAG3 specifically binds to macrophages. BAG3 

activates macrophages as demonstrated by increased iNOS and COX-2 levels as well as 

production of nitrites and IL-6. 

Conclusions: We showed that BAG3 is actively released in the extracellular space in 

response to stress and activates macrophages. Moreover, patients with chronic heart 

failure develop auto-antibodies against BAG3 that can potentially be used as markers of 

disease progression or response to therapy. 


137


Activation of Platelet-Activating Factor Receptor and SIRT1 

signaling in endothelial progenitor cells 

BaleStrieri Maria luiSa, eSPoSito aleSSandra, d’onofrio nunzia, 

caSale roSario, Giovane alfonSo, Servillo luiGi. 

Department of Biochemistry and Biophysics, 

Second University of Naples, 80138 Naples, Italy. 

Endothelial progenitor cells (EPC) are a subset of bone marrow-derived cells 

committed to the maintenance and preservation of vascular turnover, remodeling, and 

homeostasis. The therapeutic application of this progenitor cell population affects the 

recovery of blood flow after ischemia and atherosclerosis, suggesting that EPC are 

active players in maintaining a healthy cardiovascular system. However, hyperglycemia, 

cardiovascular risk factors, and the release of pro-inflammatory mediators, such 

as Platelet-Activating Factor (PAF), may compromise the EPC therapeutic efficacy 

by affecting their number and functional activities (1). Moreover, the reduction of 

EPC number following treatment with high-glucose concentration occurs through 

a mechanism that involves downregulation of SIRT1 (2). Here, we investigated the 

possible relationship between PAF-receptor activation and SIRT1 signaling in early 

EPC isolated from total peripheral blood mononuclear cells of healthy human donors. 

Results indicated that treatment with PAF (50 ng/ml) for 6 hours in serum free media 

before 3-day culture determined a reduction of EPC number with a concomitant 

decrease of SIRT1 activity and expression levels. Notably, both the reduction of EPC 

number and the decreased SIRT1 activity induced by PAF were abolished by CV3988, 

a PAF receptor antagonist. These findings unveil an interplay between PAF and SIRT1 

pathways in EPC and provide further insight into the mechanisms by which EPC 

exhibit impaired functionality. 

(1) Balestrieri ML, Giovane A, Milone L, Servillo L. Endothelial progenitor cells express PAF receptor and 

respond to PAF via Ca(2+)- dependent signaling. Biochim Biophys Acta. 2010, 1801:1123-32. 

(2) Balestrieri ML, Rienzo M, Felice F, Rossiello R, Grimaldi V, Milone L, Casamassimi A, Servillo L, Farzati 

B, Giovane A, Napoli C. High glucose downregulates endothelial progenitor cell number via SIRT1. 

Biochim Biophys Acta. 2008, 1784:936-45. 


138


Proteomic analysis of molecular responses to algal toxins 

azaspiracid-1 and yessotoxin shows a shared protein 

effector of their toxicity pathways in human cells 

Sala Gian luca, roSSini Gian Paolo 

Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia 

Via Campi 287, I-41125 Modena, Italy 

The toxic responses of biological systems can be viewed as signalling pathways 

whose organization and molecular effects lead to functional impairment of the system 

when sufficiently perturbed by noxious stimuli. A full account of different toxicity 

pathways triggered by toxicants is a prerequisite for a complete understanding of 

adverse effects they exert in living system. To approach this level of complexity, we 

studied the molecular changes triggered by two marine biotoxins, azaspiracid-1 

(AZA-1) and yessotoxin (YTX) at the system level. These toxins belong to two different 

chemical classes of toxins, for which previous investigations indicated a convergence 

between their toxicity pathways, by a blockade of endocytic processes. Based on this 

evidence, we matched the molecular perturbations induced by AZA-1 and YTX, by the 

analysis of changes they could induce in the proteome of our major model system, 

represented by a human epithelial cell line, the MCF-7 breast cancer cells. After a 

24 h exposure of MCF-7 cells to 1 nM final concentration of AZA-1 and YTX, whole 

cells lysates were prepared and subjected to bidimensional electrophoresis and gel 

staining. Digital elaboration of images showed 7 components significantly affected by 

AZA-1 treatment as compared to controls. Two of these proteins were also changed by 

YTX treatment, with the same trend (underexpression). Mass spectrometric analysis 

of these components led to their identification, and showed that cellular levels of 

cathepsin D had been lowered as a consequence of exposure to either AZA-1 or 

YTX. The decreased levels of the lysosomal protease induced by two distinct toxins, 

provide a preliminary indication that altered protein turnover at the level of lysosomes 

participates to the toxicity pathways they induce in sensitive cells. 


139


The mitochondrial citrate carrier (CIC) regulates insulin 

secretion by human male gamete. 

caPPello anna r. 1 , Guido carMela 1,3 , Santoro antonella 1 , 

Santoro Marta 3 , caPoBianco loredana 4 , Montanaro daniela 3 , 

Madeo Marianna 1 , andò SeBaStiano 2,3 , aquila Saveria 1,3 

and dolce vincenza 1 

Departments of Pharmaco-Biology 1 and Cellular Biology 2 and Centro Sanitario 3 , 

University of Calabria, Italy; Department of Biological and Environmental Sciences 

and Technologies 4 , University of Salento, Italy 

A lack of knowledge exists on how mammalian spermatozoa manage their energy 

status, a vital point for the understanding the unique physiology of sperm and the 

mechanisms implied in mammalian sperm acquisition of capacitation, a prerequisite of 

egg fertilization. The present study provides biochemical and morphological evidence 

that mitochondrial citrate carrier (CIC) is present in ejaculated human sperm and is 

restricted to the midpiece. The inhibition of CIC with the specific substrate analog 

1,2,3-benzenetricarboxylate resulted in the reduction of cholesterol efflux, protein 

tyrosine phosphorylation, phospho-AKT, phospho-p60src, hyperactivated motility 

and acrosome reaction, suggesting a role for this mitochondrial carrier in sperm 

physiology. Furthermore, inhibition of CIC by 1,2,3-benzenetricarboxylate resulted in 

a reduction of glucose-stimulated insulin secretion and autocrine insulin secretion by 

sperm. Remarkably, blocking CIC also reduced glucose-6-phosphate dehydrogenase 

activity, probably in accordance with its regulation on insulin secretion. Capacitation 

and glucose metabolism were stimulated by glucose as well as citrate, the specific 

substrate of CIC, implying a similar action because glucose and citrate both induced 

insulin secretion by sperm. In the present finding, we discovered a new site of action 

for CIC in the regulation of metabolism, and it may be assumed that CIC works with 

other factors in the regulation of sperm energy metabolism to sustain capacitation 

process and acrosome reaction. It may be taken into account as molecular target in 

the therapies designed for male infertility disorders. 


140


The Endocannabinoid System in Stem Cells from the 

Amniotic Fluid 

di toMMaSo Monia 1 , Bari Monica 2 , antonucci ivana 3 , 

BattiSta natalia 1,4 , StuPPia liBorio 3,* and Maccarrone Mauro 1,4,* 

1 Department of Biomedical Sciences and StemTeCh Group, 

University of Teramo, Teramo, Italy. 

2 Department of Experimental Medicine and Surgery, 

University of Rome “Tor Vergata”, Rome, Italy. 

3 Department of Oral Sciences, Nano and Biotechnologies and StemTeCh Group, 

“G. d’Annunzio” University of Chieti, Chieti, Italy. 

4 European Center for Brain Research (CERC)/Santa Lucia Foundation I.R.C.C.S., 

Rome, Italy. * Equally senior authors. 

Abstract 

Stem cells can differentiate into different lineages, and harbor the potential for selfrenewal 

that can be used to replace dysfunctional cells within a tissue. Amniotic Fluid 

Stem Cells (AFSCs) express Oct-4 mRNA, a specific marker of pluripotent stem cells, 

and are able to differentiate into cells of the three germ layers. Moreover, AFSCs are 

not tumorigenic, retain long telomerase and maintain normal karyotype for over 250 

population doublings. Additionally, they do not raise the ethical concerns associated 

with embryonic stem cells. 

Endocannabinoids are mainly arachidonic acid derivates that sustain manifold signalling 

pathways. Along with their G protein-coupled cannabinoid receptors (especially CB1 and CB ) and the enzymes involved in their biosynthesis and degradation, these lipids 

2 

form the so-called endocannabinoid system (ESC). Recently, ECS expression has 

been reported in the bone marrow niche, and functional CB and CB were found 

1 2 

in human and murine hematopoietic stem and progenitor cells, as well as in mouse 

embryonic stem cells [1]. 

In this study, we aimed at determining the mRNA and protein expression of metabolic 

enzymes and receptors of endocannabinoids in AFSCs, and their modulation during 

the differentiation process. 

Quantitative real time-PCR assays and Western blot analysis showed that AFSCs 

express the mRNA and the proteins of the enzymes responsible for the metabolism 

of AEA (N-arachidonoylethanolamine) and 2-AG (2-arachidonoylglycerol), and of their 

binding receptors. Remarkably, the expression of ECS elements was found to be 

modulated upon differentiation of AFSCs into cardiomyocytes. 

Overall, our study suggests that the ECS might have a key-role in stem cell biology. 

References 

Bari, M., Tedesco, M., Battista, N., Pasquariello, N., Pucci, M., Gasperi, V., Scaldaferri, M.L., Farini, D., De 

Felici, M. and Maccarrone, M. (2011) Characterization of the endocannabinoid system in mouse embryonic 

stem cells. Stem Cells Dev. 20: 139-147. 


141


Transcriptional regulation of the human FPR2/ALx gene: 

evidence of a heritable genetic variant that impairs 

promoter activity 

cianci eleonora, 2 SiMiele felice, 1 recchiuti antonio, 1 

MattoScio doMenico, 1 de luca antonella, 1 franchi Sara, 3 

Gatta valentina, 3 Parolari aleSSandro, 4,5 ,werBa JoSè PaBlo, 4 

caMera Marina, 4,5 favaloro Bartolo 1 and roMano Mario 1 . 

1 Dipartimento di Scienze Biomediche, Chieti. 

2 Dipartimento di Medicina e Scienze dell’Invecchiamento, Chieti. 

3 Dipartimento di Scienze Orali Nano e Biotecnologia, 

Centro di Eccellenza Scienze dell’Invecchiamento, Chieti. 

4 Centro Cardiologico Monzino Istituto Di Ricovero e Cura a Carattere Scientifico 

(IRCCS), Milano 

5 Dipartimento Scienze Farmacologiche Università di Milano. 

FPR2/ALX, a G-protein-coupled receptor, expressed in leukocytes, epithelial and 

endothelial cells, is recognized by mediators that promote inflammation resolution 

such as lipoxin (LX)A 4 , resolvin D1 and annexin A1. Although FPR2/ALX expression is 

pivotal for inflammation resolution, the FPR2/ALX promoter has not yet been localized. 

Here, we mapped a 346 bp region with strong promoter activity 307 bp upstream the 

transcription start site (TSS) of the FPR2/ALX gene. Chromatin immunoprecipitation 

and site direct mutagenesis revealed that specific protein 1 binds to the FPR2/ALX 

promoter and is crucial for its full activity, which is also tightly regulated by methylation. 

FPR2/ALX promoter activity as well as mRNA and protein expression was significantly 

enhanced by LXA 4 , the ligand with the highest affinity for this receptor, uncovering 

a positive feedback loop of resolution during inflammation. We also identified a 

single nucleotide polymorphism, (A/G) in the FPR2/ALX core promoter at -220 

bp from the TSS in one subject with history of cardiovascular disease and in his 

two daughters. This mutation reduced by 35-90% the basal promoter activity and 

made the core promoter unresponsive to LXA 4 in vitro. Moreover, neutrophils from 

individuals carrying the A/G variant displayed ~ 10- and 3-fold reduction in FPR2/ALX 

mRNA and protein, respectively, compared with cells from their relatives or healthy 

volunteers expressing the wild-type allele. Together, these results unravel the FPR2/ 

ALX transcription machinery and provide the first evidence of genetic variants within 

the FPR2/ALX promoter, which impair gene expression at the transcriptional level. In 

light of the evidence, in vivo, that FPR2/ALX expression levels represent per se a key 

pro-resolution mechanism, our data may open the way for the discovery of a novel 

class of anti-inflammatory drugs, based on the modulation of FPR2/ALX expression. 


142

Biochimica 

della 

Nutrizione 

BNu

Biochimica della Nutrizione BNu 1 

SAFFRON: A POTENTIAL CANDIDATE FOR A NOVEL 

ANTICANCER DRUG AGAINST HUMAN PROSTATE 

CANCER 

1 Mancini, a., 2 lizzi, a.r., 3 de SiMone a., 3 Marroccella c.e., 

2 Gravina G.l., 1 tatone c., 2 feStuccia c., 1 d’aleSSandro, a.M. 

1 Life, Health and Environmental Sciences, University of L’Aquila; 

2 Clinical and Biotechnology Sciences, University of L’Aquila; 

3 Chamber of Commerce, Agenzia per lo Sviluppo; 

Cancer of prostate is the most commonly diagnosed solid-malignancy in men living in 

developed countries. Saffron consists of dried stigmas of Crocus sativus L. flower and 

is known for anti-cancer properties. In this study, we investigated the antitumor effects 

and underlying mechanisms of Saffron extract (SE) and its major constituent crocin 

against prostate cancer in vivo. Aqueous and ethanolic Saffron extract (SE) (1g/20ml) 

were purified and characterized by HPLC . In order to asses the activity of Saffron 

extract and crocin in vivo randomized groups of 22rv1-bearing mice were treated 

orally with SE and crocin at 400mg/Kg and 200mg/Kg body weight, respectively, for 

28 days. In previous in vitro studies we demonstrated that SE and crocin altered cell 

cycle progression and induced apoptosis in a time- and concentration-dependent 

manner. Results from this in vivo study showed that SE and crocin inhibited xenograft 

growth of established 22rv1 tumors: a significant reduction in solid tumor volume 

was found in crocin-treated mice when compared with control animals (56.8% vs 

control, 621 +/- 87 vs 1438 +/- 167 mg, p


Role of α-Tocopheryl-phosphate on the expression of 

Sestrin in pre-adipocytes NIH-3T3L1. 

duGo laura 1 , liranGi Melania 1 , dachà Marina 1 , zinGG Jean Marc 2 

and azzi anGelo 2 

1 Laboratorio di Biochimica, Chimica e Nutrizione, Centro di Ricerca Integrato (CIR) 

Università Campus Bio-Medico di Roma, Roma, Italy 

2 Vascular Biology Laboratory, JM USDA-HNRCA at Tufts University, Boston, USA 

Sestrin is a family of proteins whose main role is the suppression of lipids accumulation, 

the prevention of cardiac malfunctions and the protection of muscle from age-related 

degeneration. 

Sestrin accumulates in cells in response to stress factors, potentiate the activity of 

AMPK and inhibit the activation of target gene of rapamycin (mTOR). Expression of 

Sestrin is increased by the activity of p53 and decreased by the insulin-AKT signalling 

pathway. 

α-Tocopheryl-phosphate (α-TP) is the phosphorylated derivate of α-tocopherol. α-TP 

has been reported to act as a regulator of gene expression and cell signal transduction 

(1). Preliminary experiments showed that the addition of α−TP (but not α−tocopherol) 

to NIH3T3-L1 pre-adipocytes, transcriptionally activates a set of genes: TRB3 (Tribbles 

Homolog 3), C8FW (anti-G-Protein-Coupled Receptor Induced Protein), Sestrin-2 

and Insulin Induced Gene 1 (INSIG), potentially preventing fat accumulation in these 

cells. α-TP could therefore have a role in the prevention of lipids accumulation during 

adipocytes differentiation. 

We report here the effect of α-tocopherol and α−TP on the expression of Sestrin in 

NIH3T3-L1 pre-adipocytes. Cells were treated with α-tocopherol and α−TP at the 

concentrations of 10, 20 and 40 µM for 8 and 24 h. 

Sestrin gene and protein expression was investigated by RT-PCR and western blot. 

Our results show that, after 24 h treatment of cells with α−TP (40 µM), the expression 

of both Sestrin mRNA and Sestrin protein were significantly increased compared to 

the cells treated with α-tocopherol in the same conditions. 

The regulation of Sestrin expression could indicate a role for α-TP in the modulation 

of adipose tissue metabolism and energetic homeostasis. 

Further investigation will be necessary to clarify the role of α-TP on the regulation of 

gene expression in adipocytes before and during differentiation. 

1. Azzi, A. (2007) Molecular mechanism of alpha-tocopherol action. Free radical biology & medicine 43,16- 21. 


145


Redox imbalance activates PGC-1α-mediated 

mitochondrial antioxidant response during metabolic stress 

aquilano katia 1 , Baldelli Sara 1 , PaGliei Beatrice 1 , 

ciriolo Maria roSa 1,2 

1 Dept. of Biology, University of Rome “Tor Vergata”, 

Via della Ricerca Scientifica, 00133 Rome Italy 

2 IRCCS San Raffaele La Pisana, Via di Val Cannuta 249, 00166 Rome, Italy 

Glutathione (GSH) is the most abundant low molecular weight thiol in mammalian 

cells and acts as the major cellular non-enzymatic antioxidant. GSH also has a pivotal 

function in buffering the intracellular production of nitric oxide (NO), particularly in 

brain wherein NO acts as an important neuromodulator and neurotransmitter. We have 

found that nutrient limitation, a dietary regimen that is generally linked to longevity, 

is associated with a decline of GSH level in mouse brain. This event is the primum 

movens of NO-mediated induction of the transcriptional co-activator peroxisome 

proliferator-activated receptor γ coactivator 1 α (PPARGC1A or PGC-1α) via a novel 

pathway involving p53. We have demonstrated that such pathway culminates in the 

enhancement of the mitochondrial antioxidant response (i.e. SOD2 increase) through 

the nuclear factor (erythroid-derived 2)-like2 (NFE2L2). Moreover, the pharmachological 

depletion of GSH results in the activation of such antioxidant pathway in the brain. 

Overall our results suggest that the decline of GSH level occurring with normal ageing 

and upon nutrient limitation could be a beneficial rather than a detrimental event and 

may serve as a molecular signal for adaptation to environmental/pathological stress 

and possibly in the extension of lifespan. 

*This work was partially supported by Fondazione Roma (Grant 2008) and by MIUR 


146


DHA MODIFIES LIPID RAFT STRUCTURE AND FUNCTION 

IN MDA-MB-231 BREAST CANCER CELLS 

corSetto Paola a. 1 , aMi diletta 2 , Montorfano GiGliola 1 , 

creMona andrea 1 , doGlia Silvia M. 2 , rizzo anGela M. 1 

1 Dipartimento di Scienze Farmacologiche e Biomolecolari, 

Università degli Studi di Milano 

2 Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca 

In vivo and in vitro studies suggest that omega-3 poliunsaturated fatty acids (n-3 PUFA) 

can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer 

therapy (1). N-3 PUFA, like docosaexaenoic acid (DHA), could alter tumor growth by 

influencing cell replication, interfering with cell cycle components or increasing cell 

death, either by inducing necrosis or apoptosis. 

We have observed that DHA induces in MDA-MB-231 cells apoptosis and reduction 

of cell viability. Moreover, DHA significantly reduces the EGFR level and completely 

inhibits EGFR activation. 

However, the mechanism by which n-3 PUFA inhibit breast cancer growth, is not yet 

well understood. It was suggested that these fatty acids might change cell membrane 

fluidity and structure, especially of lipid rafts. 

Our data obtained by HPLC-GC analysis demonstrate that DHA is incorporated and 

metabolized in MDA-MB-231 membrane. In particular, DHA is incorporated in breast 

cancer lipid rafts with different specificity for the phospholipid moiety and induces a 

reduction of cholesterol and sphingomyelin content, indicating a possible change in 

raft organization. Changes in the lipid raft structure were also analyzed with atomic 

force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). The AFM 

analysis of breast cancer lipid rafts indicates after DHA incorporation a reduction of 

microdomains number. Moreover PUFA treated rafts are dimensionally different from 

control rafts (2). 

Then, when DHA is incorporated into cell membrane, it may induce the formation of 

“declustered rafts” with reorganization of lipids and proteins that may greatly influence 

signal transduction. 

1 Calviello G et al., Nutr Cancer. 2009. 

2 Corsetto PA et al., Cell Biochem Biophys. 2012. 


147


Brutieridin and Melitidin: natural products with potential 

hypocholesterolemic activity 

Martello eManuela 1 , di donna leonardo 2 , iacoPetta doMenico 1 , 

caPPello anna r. 2 , Gallucci GiSelda 2 , Sindona Giovanni 1 and 

dolce vincenza 2 

1 Department of Chemistry, and 2 Department of Pharmaco-Biology, 

University of Calabria, Arcavacata di Rende (CS) Italy. 

Background: Statins are the pharmacological inhibitors of cholesterol biosynthesis, 

acting on key enzyme 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR). They are 

the most effective and practical class of drugs for reduce low-density lipoproteins 

(LDL) concentrations, although cardiovascular disease remains the main cause of 

mortality in westernized countries. The pursuit of novel therapies to target the residual 

risk is focused on raising the levels of high-density lipoproteins (HDL). 

Methods: The serum and hepatic lipids content, in a model of hypercholesterolemic 

induced rats treated with brutieridin and melitidin enriched fraction (BMF) extract from 

bergamot fruit or with simvastatin, has been measured by HPLC or direct enzymatic 

assays. Additionally, the expression levels of HMGR, low-density lipoprotein receptor 

and fatty acid synthase were determined by RT-PCR and Western blot analysis. 

Results: Simvastatin and the BMF are found able to reduce the levels of total 

cholesterol (TC), tryglicerides (TG) and LDL, whereas an increase of HDL content is 

observed exclusively in the BMF treated rats. The results of RT-PCR and Western blot 

analyses evidenced an appreciable increased of all genes and proteins analyzed in 

treated rats respect to the untreated hypercholesterolemic rats. 

Conclusions: Our study revealed that the daily supplementation with BMF is able to 

significantly reduce the TC, TG and LDL in a similar extent to simvastatin treatment 

and more important, contemporaneously is able to increase the HDL blood levels. 

General significance: These data show that intake of BMF extract from bergamot fruit 

is a promising therapeutic tool for the treatment of hypercholesterolemia. 


148


Hesperetin and vitamin C anti-inflammatory properties in 

endothelial ECV304 cells 

Savini iSaBella, aviGliano luciana, evanGeliSta daniela, GaSPeri 

valeria, catani Mariavaleria 

Department of Experimental Medicine & Surgery, 

University of Rome Tor Vergata, Rome, Italy 

Prospective studies have reported an inverse relationship between citrus fruits 

consumption and risk of cardiovascular diseases 1 . Clinical and preclinical studies with 

orange juice demonstrated its beneficial effect on cardiovascular risk factors, such as 

blood pressure, colesterolemia, endothelial and platelet functions. Among the naturally 

occurring citrus compounds, vitamin C (50 mg/100 g) and the flavanone hesperidin 

(35-150 mg/100 g; 95% of the total flavonoids) may contribute to the preventive 

effects of orange fruits. Hesperidin (hesperetin-7-O-rutinoside) is hydrolyzed in the 

distal part of the intestine and colon by the enteric microflora, thus producing the 

active aglycone hesperetin; it has been reported that hesperetin improves endothelial 

function and reduces of circulating inflammatory biomarkers such as high-sensitivity 

C-reactive protein, and soluble E-selectin in patients with metabolic syndrome 2 . 

Here, we attempted to characterize the anti-inflammatory effects of vitamin C 

and hesperetin on endothelial cells. To this end, endothelial cell vein (ECV304) 

cells were pre-loaded with L-ascorbic acid (AA) then incubated with 100 ng/mL 

lipopolysaccharide (LPS), a classical inducer of inflammation; release of the proinflammatory 

cytokine tumour necrosis factor-α (TNF-α) was assessed by ELISA 

assay. We found that both compounds abrogated LPS effects: hesperetin (10 µM, 

for 30 minutes) and AA (50 µM, for 30 minutes) suppressed the LPS-induced TNF-α 

release; the anti-inflammatory action was seen with the two compounds used alone 

(79 and 75% inhibition, respectively), as well as when hesperetin and AA were 

administered together (100% inhibition). 

Our results suggest that both hesperetin and AA antagonize the LPS-induced secretion 

of pro-inflammatory cytokines and that they act synergistically. Therefore, prevention 

of vascular inflammatory diseases may be more efficaciously achieved by consuming 

hesperetin- and vitamin C-containing foods instead of single supplements. 

1. Chanet et al., J Agric Food Chem. 2012, DOI: 10.1021/jf300669s 

2. Rizza et al., J Clin Endocrinol Metab. 2011, 96:E782-92. 


149


Correlation between polyphenolic profile and antioxidant, 

antiproliferative and cytoprotective activities of old and 

modern common wheat varieties 

leoncini eManuela 1 , Prata cecilia 1 , MalaGuti Marco 1 

Marotti ilaria 2 , SeGura carretero antonio 3 , catizone Pietro 2 , 

dinelli Giovanni 2 , hrelia Silvana 1 

1 Department of Biochemistry “G. Moruzzi” 

University of Bologna, via Irnerio, 48, 40126 Bologna, Italy 

2 Department of Agroenvironmental Science and Technology, 

University of Bologna, viale Fanin, 44, 40127 Bologna, Italy 

3 Department of Analytical Chemistry, University of Granada, 

c/Fuentenueva s/n, 18003 Granada, Spain 

Epidemiological studies correlated whole grains intake to a reduced incidence of 

chronic disease such as cardiovascular disease and cancer. This potentially protective 

effect could be attributed in part to phenolic compounds present in whole grains and 

to their strong antioxidant properties (1). 

Aim of this study was to characterize the phenolic profile of 6 common wheat 

genotypes (1 modern and 5 old cultivars) and to evaluate their potential antiproliferative 

and cytoprotective effect in different cell culture systems. A leukemic cell line, HL60, 

and cultured rat cardiomyocytes, were supplemented with different phenolic extracts 

for 24 h and oxidative stress was induced by H 2 O 2 -treatment. Antiproliferative and 

cytoprotective effects were measured in terms of intracellular ROS level decrease and 

influence on cell viability. 

Old varieties displayed higher polyphenol and flavonoid content which positively 

correlated (P


Short-term effects of olive oil polyphenols on lipid 

synthesis in cultured rat hepatocytes 

Priore Paola 1 , Siculella luiSa 1 , cavallo aleSSandro 1 , 

Gnoni GaBriele vincenzo 1 

1 Laboratory of Biochemistry, DiSTeBA, University of Salento, 

via Monteroni,73100 Lecce (Italy) 

Olive oil is the principale source of fat in the Mediterranean diet, which has been 

associated with a lower incidence of coronary heart disease and certain cancers. 

Oleuropein (Ole), hydroxytyrosol (H-Tyr) and tyrosol (Tyr), which are antioxidant 

molecules found in olive leaves and oil, have been reported to exert several biochemical 

and pharmacological effects. 

In the present study, we investigated the short-term effect of these compounds on 

hepatic lipogenesis in cultured rat liver cells obtained as in (1). Ole, H-Tyr and Tyr 

dose-dependently (in the range 2.5-100 µM) inhibited both de novo fatty acid and 

cholesterol syntheses without effect on cell viability. The inhibitory effect of individual 

compounds, Ole > H-Tyr > Tyr, was already evident within 1 h of polyphenol addition 

to the hepatocytes, and increased with time up to 4 h, only after Ole treatment. A 

noticeable polyphenols-induced reduction of the de novo triacylglycerol synthesis 

was also detected. 

To clarify the anti-lipogenic mechanism of these compounds, their influence on the 

activity of key enzymes of cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA 

reductase, HMGCR) and fatty acid biosynthesis (acetyl-CoA carboxylase, ACC; 

fatty acid synthase, FAS) was investigated in situ by using digitonin-permeabilized 

hepatocytes. 

Within 1 h of treatment, the three tested compounds were able to inhibit both ACC 

and HMGCR activities, while no change in FAS activity was observed. These findings 

suggest a hypolipidemic action of minor components of olive oil in rat cultured 

hepatocytes, and add further support to the idea that olive oil, with its antioxidants, 

may play a role in the reduced incidence of diseases related to alteration of lipid 

metabolism in the Mediterranean area. 

This work was supported by a grant from Apulia Region PS101 

1. Giudetti AM, Leo M, Geelen MJ, Gnoni GV. Short-term stimulation of lipogenesis by 3,5-L-diiodothyronine 

in cultured rat hepatocytes. Endocrinology. 2005 146(9):3959-66. 


151


Pomegranate ellagitannins inhibit in vitro carbohydrate 

digestion 

davide taGliazucchi, elena verzelloni, andrea BelleSia, 

anGela conte 

Department of Life Sciences, University of Modena and Reggio Emilia, 

Via Amendola, 2-Pad. Besta, 42100 Reggio Emilia, Italy 

Pomegranate is an ancient fruit studied for its anti-diabetic properties. The aim of 

the present work was to identify the α-glucosidase inhibitors in a pomegranate juice 

polyphenol-rich extract (PPRE). The PPRE, obtained using C18 solid-phase extraction 

contained 5.87 ± 0.26 mmol of ellagic acid equivalent (EAE)/L and showed an inhibitor 

activity on rat intestinal α-glucosidase with an IC 50 value of 922.2 ± 7.1 µmol of EAE/L. 

The PPRE was fractioned in three different fractions using C18 columns and 

glucosidase inhibitory activity was recovered in an ellagitannins-enriched fraction. 

After HPLC separations punicalagin, punicalin and ellagic acid were identified as 

α-glucosidase inhibitors with IC 50 values of 140.2 ± 1.1, 191.4 ± 1.3 and 380.9 ± 3.5 

µmol/L, respectively. 

Kinetic analysis showed that the inhibition is of mixed type with a Ki of 770.04 ± 

233.54 µmol/L. 

Pomegranate ellagitannins interact directly with the α-glucosidase and the nonspecific 

binding of ellagitannins may alters the protein structure reducing the velocity 

of the catalysis and altering the accessibility to the active site of the substrate. 

The inhibitory effect on carbohydrate hydrolysis of PPRE was tested against a real 

food system (potatoes) using an in vitro digestion protocol. The presence of PPRE 

produced a decrease in the amount of released glucose. During digestion punicalin 

and punicalagin concentration decreased as a consequence of their binding to salivary 

or potatoes proteins and of their hydrolysis. Despite this loss, the PPRE retained high 

inhibitory activity. 

In conclusion, our data are consistent with the hypothesis that pomegranate juice can 

be considered a very useful food supplement to ameliorate postprandial hyperglycemia 

linked to type 2 diabetes and hyperglycemia-induced vascular complications. 


152


Antioxidant activity of in vitro digested bovine milk 

elena verzelloni, ahMed helal, davide taGliazucchi, anGela conte 

Department of Life Sciences, University of Modena and Reggio Emilia, 

Via Amendola, 2-Pad. Besta, 42100 Reggio Emilia, Italy 

The objective of the present study was to analyze the antioxidant properties of different 

types of bovine milk subjected to simulated gastro-pancreatic digestion. Whole, semiskimmed, 

and skimmed milk were digested in sequence with pepsin (300 U/ml; pH 

2.5; 2h) then with pancreatin (0.8 g/l; pH 7.5; 2h) and bile salts (5 mg/ml). At the end 

of digestion, samples were subjected to ultrafiltration (3 KDa) and the high (HMW) 

and low (LMW) molecular weight fractions analyzed with ABTS assay. All milk types 

contain antioxidants with whole milk having the highest amount (622.3 ± 44.5 mg 

vitamin C/L). About 90% of antioxidant activity was in the HMW fraction. The degree 

of protein hydrolysis (DH) after digestion was different considering the different milk 

types (20.8 ± 0.4, 24.3 ± 0.3 and 30.7 ± 0.8 in whole, semi-skimmed and skimmed 

milk, respectively). Pancreatic digestion caused an increase in the antioxidant activity 

for all the analyzed milk (post-pancreatic ABTS value of 3374.3 ± 104.6, 2657.1 ± 

39.6 and 2751.2 ± 46.9, in whole, semi-skimmed and skimmed milk, respectively). 

The distribution of the antioxidant activity between the LMW and HMW fractions was 

different considering the diverse milk types. The percentage of antioxidant activity 

in the LMW fraction increased to 38%, 79% and 90% in whole, semi-skimmed and 

skimmed milk, respectively, according to the increase in the DH. 

HPLC analysis of LMW fractions revealed that all the digested milk had the same 

peptides pattern but the peptides concentrations in skimmed milk was higher than 

in semi-skimmed and whole milk. On-line HPLC-ABTS analysis of LMW fractions 

showed that two peptides are the major contributor to the antioxidant activity of this 

fraction. The two peptides were identified by MS/MS. Our data suggest that digestion 

of milk increased the antioxidant activity, potentially reducing the oxidative stress in 

the gastro-intestinal tract. 


153


Bone mass density is independently associated with 

oxidative stress in post- but not in pre-menopausal women 

eleonora creMonini 1 , arianna roMani 1 , Gloria BonaccorSi 2 , 

carlo M. BerGaMini 1 , alfredo Patella 2 , criStina caStaldini 3 , 

Stefania ferrazzini 3 , aleSSandra caPatti 3 , enrica fila 2 , 

caSali f 5 , fantini Gf 5 , vincenzo lanzara 1 ,leo MaSSari 3,4 

and carlo cervellati 1 

1 Department of Biochemistry and Molecular Biology, 

University of Ferrara, Ferrara, Italy. 

2 Gynecologic Obstetrics Clinic University of Ferrara, Ferrara, Italy. 

3 Menopause and Osteoporosis Centre, University of Ferrara, Ferrara, Italy. 

4 Department of Biomedical Sciences and Advanced Therapies, 

Section of Orthopedic Clinic, Hospital “S. Anna”, University of Ferrara, Ferrara, Italy 

5 Laboratory Medicine Dept., San Marino State Hospital, San Marino 

Background: Postmenopausal osteoporosis (PO) is a widespread disease with a 

spectrum ranging from asymptomatic bone loss to disabling fractures especially in 

hip, femur and vertebra. The decline in 17β-estradiol (E2) occurring during menopausal 

transition represents, along with aging, is crucial for PO development in women. 

Experimental studies in animal models and cell cultures have suggested that the fall 

in E2 might contribute to developing oxidative stress (OS) which in turn is believed 

to play an important role in PO pathogenic mechanism. The lack of human studies 

on this issue, led us to explore the effects of the pre- and post-menopausal status 

on OS and bone health. Methods: Serum parameters of oxidative challenge (lipid 

hydroperoxides) and antioxidant defense (total serum antioxidants) were assessed in 

a sample of 161 women (78 pre- and 83 post-menopausal, of whom 30 osteoporotic). 

Parameters of bone mineral density (BMD) at femoral neck and lumbar spine were 

also assessed. Results: Pearson’s correlation analysis unveiled that spinal BMD was 

negatively correlated with lipid hydroperoxides in overall postmenopausal subsample 

(r=-0.234, p=0.008), while no significant link between these two variables was detected 

in women in reproductive age (r=-0.045, p=0.854). Noteworthy, multivariate analysis 

showed that the association found in postmenopausal women retained significance 

after taking into consideration potential confounding factors (p=0.001). Conclusion: 

our data showed that markers of oxidative damage are linked with bone loss in women 

in postmenopausal status. We interpret these results as a proof that menopauserelated 

oestrogen withdrawal might trigger oxidative injury to bone thereby increasing 

bone resorption and the risk of PO development. 


154


Mechanisms behind sulforaphane cardioprotective effect 

against methylglyoxal induced citotoxicity 

faBBri daniele, anGeloni criStina, hrelia Silvana 

Department of Biochemistry “G. Moruzzi” – University of Bologna, Italy 

Advanced glycation end products (AGEs) are a group of molecules that are generated 

through non-enzymatic glycation and accumulate under diabetic condition 1 . The most 

investigated AGEs precursor is methylglyoxal, a metabolic compound formed mainly 

from the glycolytic intermediate glyceraldehyde-3-phosphate. Methylglyoxal is more 

reactive than glucose and shows a stronger ability to cross-link with protein amino 

groups to form AGEs. Methylglyoxal -induced cytotoxicity may be at least partially 

responsible for diabetes-related impairments of cardiovascular function. methylglyoxal 

omeostasis is controlled by the glyoxalase system that consists of two enzymes, 

glyoxalase 1 (GLO1) and glyoxalase 2. In a recent study, Xue et al. 2 demonstrated that 

the transcriptional levels of GLO1 are controlled by NF-E2-related factor 2 (Nrf2). The 

isothiocyanate sulforaphane, derived from the hydrolysis of glucoraphanin abundantly 

present in broccoli, represents one of the most potent inducers of phase II enzymes 

through the Keap1–Nrf2 pathway 3 . 

Aim of this study was to investigate, in primary cultures of rat cardiomyocytes, the 

biological effects of SF on methylglyoxal-induced cardiotoxicity focusing on the 

signaling pathways of apoptosis and the modulation of the glyoxalase system. 

Cell viability was evaluated by MTT assay, caspase 3 activity by a fluorimetric 

method, ERK1/2, p38 MAPK, JNK and Akt phosphorylation and GLO1 expression by 

immunoblotting, GLO1 activity spectrophotometrically. 

We observed that sulforaphane treatment significantly counteracted cell death and 

apoptosis induced by methylglyoxal, inhibited methylglyoxal induced activation of the 

pro-apoptotic kinases JNK and p38. Moreover, for the first time, we demonstrated that 

sulforaphane is able to significantly increase GLO1 protein expression and activity. 

These findings suggest that targeting the glyoxalase system might be a promising 

therapeutic strategy to counteract cardiovascular diseases of diabetic patients. 

Supported by Fondazione del Monte di Bologna e Ravenna 

1Brownlee, Diabetes 1993, 43:836-841. 

2Xue, Biochem J 2012 443:213-222. 

3Lee, Cancer Lett 2005 224:171-184. 


155


Effect of polyunsaturated fatty acids on lipogenic factors 

caPuto Mariella, de roSa Maria caterina, reSciGno tania, 

zirPoli hylde, vaSSallo antonio, de toMMaSi nunziatina, 

torino Gaetano, tecce Mario felice 

Department of Biomedical and Pharmaceutical Science, 

University of Salerno, Via Ponte Don Melillo, Fisciano (Sa), Italy 

Polyunsaturated fatty acids (PUFA) are nutrients with relevant biological activities 

and preventive effects of cardiovascular and neoplastic diseases and therefore it is 

important to characterize the molecular processes involved in their action. On this 

purpose, we evaluated the effect of PUFA on Stearoyl-CoA Desaturase 1 (SCD1), the 

rate limiting enzyme in PUFA biosynthesis and on Sterol Regulatory Element Binding 

Protein (SREBP-1c), a transcription factor which directly modulate its expression. 

SCD1, introducing a double bond in fatty acyl-CoA substrates, is of particular interest 

since alterations of its activity seem to be involved in several dyslipidemic conditions. 

Considering that Liver X Receptor (LXR) activation induces lipogenic pathways 

also trough SREBP-1c regulation, we analyzed the effect of a specific LXR agonist 

molecule, T0901317, on a human hepatoma cell line (HepG2). Our data show a 

significant induction of SREBP-1c and SCD1 by T0901317. In addition, we analyzed 

some putative transcription factor binding sites within SCD1 gene promoter sequence. 

Using chromatin immunoprecipitation (ChIP) assay, we identified a functional binding 

site (-789/-659 SRE1 region). Data show an increased SREBP-1c binding after LXR 

activation. We analyzed the effects of individual PUFA on this mechanism. Results 

show that docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) 

and arachidonic acid (AA, C20:4) are able to reduce SREBP-1c binding on SRE1 

region, while a saturated stearic acid (SA) did not give any effect. To further understand 

if PUFA produce these effects through a direct interaction with LXR, we have also 

carried out a surface plasmon resonance (SPR) analysis which showed a direct 

binding of DHA, EPA and AA on LXR transcription factor, while no interaction was 

found with saturated SA, indicating that these effects are specific. We conclude that 

analyzed PUFA selectively down-regulate lipogenic pathways through LXR inhibition 

and consequent SREBP-1c and SCD1 expression down-regulation. 


156


Effect of steviol glycosides on glucose transport activity in 

different cell types 

B. rizzo, l. zaMBonin, e. leoncini, f. vieceli dalla SeGa, c. Prata, 

d. fiorentini and S. hrelia 

Dipartimento di Biochimica “G. Moruzzi”, Alma Mater Studiorum 

Università di Bologna, Italy 

Overweight or obese people is continuously rising in industrialized nations. This 

people is subjected to an increased risk for a number of deleterious health conditions 

including type 2 diabetes, heart diseases and cancer. Type 2 diabetes is associated 

to impaired insulin secretion and insulin resistance in peripheral tissues, leading to 

hypoinsulinemia and hyperglycemia. Therefore, non toxic, weight controlling sugar 

substitutes as well as newer and safer agents for treatment of type 2 diabetes are 

needed. Steviol glycosides, non caloric, natural sweeteners extracted from Stevia 

rebaudiana, have been used as antihyperglycemic agents (1), but little is known about 

their mechanism of action at molecular level, especially on their potential interaction 

with cellular glucose transport system. 

We studied glucose transport activity in different cell types expressing GLUT1, the 

basal glucose transporter, and GLUT4, the insulin sensitive one (2). It has been known 

that GLUT4, largely confined in intracellular storage site, becomes recruited to the 

cell surface under the influence of insulin and other stimuli, but also the isoform 

GLUT1 translocates to the plasma membrane in response to several stimuli (3). Thus, 

we investigated whether rebaudioside A and stevioside could be able to modulate 

glucose uptake of these cells and possibly to influence the intracellular distribution of 

GLUT isoforms. 

Supported by Ministry of Economic Development (Project Made in Italy-over 50) 

1. Narissara L et al., 2004, Metabolism 53, pp 101-107 

2. Hrelia S et al., 2002, Biochimica et Biophysica Acta 1567, pp 150-156 

3. Maraldi T et al., 2007, Antioxidants & Redox Signaling, 9, pp 271-279 


157


Modifications of lipoxygenase-1 from genetically modified 

soybean 

anGelucci clotilde B. 1 , SaBatucci annalaura 1 , 

di franceSco andrea 1 , ciaBatti ilaria 2 , daineSe enrico 1,3* , 

Maccarrone Mauro 1,3* 

1 Departimento di Scienze Biomediche, Università di Teramo, Teramo, Italia, 

2 Centro di Referenza Nazionale per la Ricerca di OGM (CROGM), Roma, Italia, 

3 Centro Europeo di Ricerca sul Cervello/Fondazione “Santa Lucia”, Roma. 

*Equally senior authors. 

In U.S.A., Canada, and several developing countries (e.g., China, Brazil, Argentina) 

the use of genetically modified plants (GMPs) in agriculture is rapidly growing and 

expanding. In this countries, GM soybeans (MON-04032-6), modified for resistance to 

the herbicide glyphosate, is one of the most cultivated GMPs (data from ISAAA 2011). 

The European Food Safety Agency (EFSA) in its guidelines emphasizes the importance 

of identifying in GMPs the presence of functional changes in proteins involved in plant 

defense mechanisms and alterations in lipid profiles. Aim of this work was to verify 

possible alterations of an important enzyme involved in plant defense mechanisms: 

lipoxygenase-1 (LOX-1). We show major structural and functional changes of LOX-1 

in GM soybeans with respect to wild-type counterparts. In particular, we demonstrate 

a strongly reduced expression and catalytic efficiency of LOX-1 in MON-04032-6 

soybean, and a 70% reduction of iron in its active site. These LOX-1 alterations were 

not paralleled by significant changes in iron bioavailability between the GM soybeans 

and conventional counterparts, and led to a significant decrease of peroxidized lipids 

in seeds. Interestingly, we recently demonstrated that LOX-1 activity and iron removal 

can be modulated upon exposure to bacteria of the Streptomyces species (1). Overall, 

the here reported modifications of LOX-1 could be related to alterations of hostpathogen 

relationships in GM soybeans with respect to the conventional counterparts. 

1. Dainese E., Angelucci C.B., Sabatucci A., De Filippis V., Mei G. and Maccarrone M. 

(2010) A novel role for iron in modulating the activity and membrane-binding ability of 

a trimmed soybean lipoxygenase-1, The FASEB Journal 24, 1725-1736. 


158


Caspase 3 mediated PARP cleavage in lymphocytes from 

newborn babies of diabetic mothers 

l. criSPoltoni 1 , r. tiriBuzi 1,# , f. tarquini 2,# , S. Porcellati 1 , 

a. del Pino 3 , S. Martino 1 , G. coata 2 , S. arnone 4 , e. torlone 4 , 

B. caPPuccini 5 , G.c. di renzo 2, and a. orlacchio 1,6 . 

1 :Department of Experimental Medicine and Biochemical Sciences, University of Perugia; 

2 :Department of Obstetrics and Gynaecology, University Hospital, Perugia; 3 Department of 

Agronomy and Environmental Sciences, University of Perugia; 4 :Department of Internal Medicine, 

Endocrinology and Metabolism, University of Perugia; 5 :Department of Neonatology, University 

Hospital, Perugia. 6 : GeBiSa Research Foundation, Perugia. 

Several epidemiological studies showed that maternal diabetes during pregnancy is a 

recognized cause of a series of congenital defects (neural, cardiac, renal) and of long-term 

alterations in cardiovascular, metabolic and renal function in offspring due to increased 

apoptosis, altered fetal programming and epigenetic damages. Many factors, including 

the cysteine proteases caspase, have been shown to be involved in these events. 

The aims of the study was to investigate the presence and processing of caspase 3 and 

PARP (Poly ADP Ribose Polymerase) in cord blood cells in relation to glycaemic control 

during intrauterine life. 

Methods 

Patients: Normotolerant pregnant women to glucose (n=20) and pregnant women with 

GDM (Gestational Diabetes Mellitus, n=18) attending to the Perinatal and Reproductive 

Centre, at the University of Perugia (Italy), have been enrolled and all delivered at term. 

Lymphocytes isolation: Diluted cord blood was layered on a density gradient and 

centrifuged (800xg, 20min) to isolate peripheral blood mononuclear cells (PBMCs). 

Lymphocytes were collected as cell population remaining from PBMCs after depletion 

of CD14 + monocytes. 

Analysis of caspase 3 and PARP: Lymphocyte extract was analyzed in order to evaluate 

the presence and processing of caspase 3 and PARP by immunoblotting. 

Results: Our results showed the activation of caspase 3 only in cells from newborn 

babies of GDM mother’s that do not react an optimal glycemic control during pregnancy. 

Moreover, in the same samples we detect the presence of inactivated PARP enzymes, 

with a MW=89kDa, indicating a specific cleavage mediated by active Caspase 3. 

Conclusion: This study reports for the first time a direct relation between the rate of 

glycaemic control and Caspase 3 and PARP expression and processing, contributing to 

clarify the mechanism by witch the failure in achieving early glycemic control in maternal 

metabolism contributes to excessive fetal and neonatal morbidity attributable to diabetes 

in pregnancy. 

Grant: Fondazione CRPg, n: 2012.0126.021 to AO 


159


Modification of caseins as functional biomarkers of 

polycyclic aromatic hydrocarbons contamination of bovine 

milk 

di Michele antonio 1,2 , PieraGoStino daMiana 2,3 , 

d’aleSSandro nicola 1 Paolo 2,3 , urBani andrea 3,4 , 

del Boccio Piero 2,3 . 

1 Dept of Sciences, University “G. d’Annunzio” of Chieti-Pescara Italy 

2 Dept. of Experimental and Clinical Sciences, 

University “G. d’Annunzio” of Chieti-Pescara Italy 

3 Research Centre on Aging (Ce.S.I), 

University “G. d’Annunzio” of Chieti-Pescara Italy 

4 Dept. of Internal Medicine, University of Rome Tor Vergata, Rome, Italy 

Polycyclic aromatic hydrocarbons (PAHs) have been included as toxic compounds 

by Agency for Toxic Substances and Disease Registry because of their mutagenic 

and carcinogenic properties. After internalization in biological pathways, PAHs are 

metabolized and generate complex mixtures of several compounds such as hydroxylmetabolites. 

1-OH pyrene and 3-OH phenanthrene were found in bovine milk, thus 

metabolites of PAHs should be taken into consideration when evaluating the food 

safety1. However, the detection of PAH levels in biological matrices could be not 

sufficient to give a right qualitative estimation of the final food products. The purpose 

of this study was to highlight the interaction of caseins-PAHs, after photo-irradiation 

in order to identify functional biomarkers of contamination. Alpha-casein (protein 

that constitutes a 32% of the caseins pool in bovine milk2) was used as a model 

of contamination in vitro added with PAHs and their hydroxyl-metabolites. Samples 

were subjected to photo-irradiation to emphasize the reactivity of contaminants. 

After enzymatic digestion of the incubated protein, peptides were extracted and 

analyzed by mass spectrometry. Differential peptides were highlighted, indicating 

the occurrence of protein modifications incubated with hydroxyl-metabolites. 

Tandem mass spectrometry was employed to characterize and identify the modified 

peptides in vitro. Successively the potential biomarker highlighted were searched to 

contaminated bovine milk. These preliminary results allowed us to highlight protein- 

PAHs adducts as markers of contamination. If confirmed, these evidences could be 

exploited to develop new marker indicators of quality agri-food industry. 

References 

1. Lutz S. et al. Journal of Agricultural and Food Chemistry.2006.54, 263-268 

2. Philippe A. Guy, Francois Fenaille Wiley Periodicals, Inc., Mass Spec Rev.2006.25:290-326 


160


Identification of phosphorylated components in milk of 

different species 

BruSchetta G., fazio e. 1 , fraGalà S. 1 , PariSi M. 2 , notti a. 2 , 

ferlazzo a.M. 

Department of Morphology, Biochemistry, Physiology and Animal Production, 

Biochemistry Unit, 

1 Physiology Unit; 

2 Department of Organic and Biological Chemistry, University of Messina 

Milk is a biological fluid well suited for nutritional studies (1) and diagnostic 

purposes (2), as it can be collected routinely and noninvasively. 31 P high-resolution 

NMR spectroscopy does not require an extensive chemical manipulation of 

samples and can easily highlight differences and/or similarities between samples 

as complex as milk. 31 P NMR analysis using detergents, a method already used 

for the analysis of plasma and erythrocyte membrane phospholipid composition 

(3), showed the presence, in all milk samples analyzed, of hydrolysis products of 

phosphatidylcholine and phosphatidylethanolamine: glycerophosphorylcholine 

(GPC), known for its neuroprotection effect on age-related oxidative damage (4), 

glycerophosphorylethanolamine (GPE) and phosphorylcholine (PC). Phosphocreatine 

is also present except in human and cow milk. In addition, minor phosphorylated sugars, 

that probably are metabolic intermediates of biochemical pathways, were identified 

(galactose-1-phosphate, N-acetylglucosamine-1-phosphate) in greater amounts in 

donkey milk than in human, cow, goat and camel milk. These data confirm the natural 

variability of milk samples among different species. Moreover, this simplified method 

could be proposed to carry on a comparative study of phosphorylated components, 

used as membrane stabilizers in the antioxidant therapy (5). 

1. Andreotti G. et al. (2006) Journal of Food Composition and Analysis 19, 843-849 

2. Klein M.S. et al. (2012) J. Proteome Res. 11, 1373-1381 

3. Ferlazzo A.M. et al. (2011) Vet. Res. Commun. 35, 521-530 

4. Suchy J. et al. (2009) Nutr. Res. 29, 70-74 

5. Savastano M. et al. (2007) Arch. Med. Res. 38, 456-459 


161


HUMAN PROSTATE CANCER PREVENTION BY GREEN TEA 

CATECHINS: 

THE CLINICAL TRIAL AND PUTATIVE MOLECULAR 

MECHANISMS OF ACTION 

rizzi federica 1,2 and Bettuzzi Saverio 1,2 

1 Department of Biomedicine, Biotechnology and Translational Research and 

Centre for Molecular and Translational Oncology (COMT), 

2 University of Parma, Parma, Italy; National Institute of Biostructure and Biosystems 

(INBB), Rome, Italy. 

Prostate cancer (PCa) is one of the major cancer killer in aged men living in Western 

Countries. Little is known about the molecular mechanisms leading to progression. 

Advanced PCa is almost incurable, because therapeutic options are largely 

unsatisfactory. Therefore it is urgent to search for effective therapies when disease is 

still initial and potentially curable. The long latency makes PCa an ideal candidate for 

chemoprevention intervention before invasion and metastatic spread occurs. 

Catechins extracted for Green Tea (GTE) posses potent anti-cancer activity in vitro. 

We showed that GTE administration was effective at inhibiting progression of PCa 

to clinical evidence in patients bearing isolated pre-neoplastic lesion (HGPIN). 

Improvement was stable 2 years after suspension of treatment. We searched for genes 

whose expression was specifically changed by GTE administration in vivo by DNA 

microarray in the prostate of GTE-treated TRAMP mice (a transgenic model for PCa) 

versus controls, looking for biomarkers to monitor GTE activity and tissue response. 

Interestingly, we found that the number of genes whose expression is significantly 

changed by GTE are relatively few. Among these, one of the most interesting gene 

was Clusterin (CLU). CLU is involved in cell survival and cell death and considered to 

drive the fate of the cell. During PCa progression in TRAMP mice, CLU expression is 

lost at early stages. When animals are treated with 0.3% GTE in drinking water and 

respond to chemoprevention, CLU expression gets back to high levels, and sensitivity 

to apoptotic death is restored. At difference, when chemoprevention fails in GTEresistant 

PCa, CLU expression is still absent and cells are resistant to apoptosis. Longterm 

administration of GTE was found safe in humans. Therefore, chemoprevention 

with GTE may be the best option for a better clinical managing of PCa, but more 

molecular data are necessary to discriminate responsive from resistant PCa. 


162


Pro-apoptotic activity of the phytochemical Indicaxanthin 

on colorectal carcinoma cells (Caco-2) and epigenetic 

CpG demethylation of the promoter and reactivation of the 

expression of p16 

naSelli f, Modica M, attanzio a, caradonna f, Gentile c, 

teSoriere l, livrea Ma 

Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, 

Università di Palermo 

Phytochemicals play prominent roles in human diet and nutrition as protective 

redo-active substances in prevention of several disorders and chronic diseases in 

humans. Today, their function as potent modulators of the mammalian epigenomeregulated 

gene expression is rapidly emerging. In the present study antiproliferative 

effects of Indicaxanthin (Ind) from the fruits of Opuntia ficus-indica (1), and potential 

influence on DNA methylation has been investigated on Caco-2 cells, a human cell 

line of colorectal carcinoma. Ind caused a clear dose- and time-dependent decrease 

of the cell proliferation (IC (50) 50 µM) associated to apoptosis as demonstrated by 

phosphatidylserine externalization and depolarization of mithocondrial membrane. 

Ind decreased the Go-G1phase whereas increased S and G2-M phases of the cell 

cycle. The phytochemical did not altered the intracellular ROS levels but decreased 

the [Ca 2+ ] i . Investigation on DNA methylation using MESAP-PCR (Methylation- 

Sensitive Arbitrarily-Primed Polymerase Chain Reaction) (2), showed that 100 µM Ind 

induced a slight global demethylation after a 48 h treatment. Analysis of epigenetic 

changes in the DNA methylation pattern at CpG promoter of p16 (INK4a), using 

MSRE (Methylation-Sensitive Restriction Endonucleases Multiplex-Polymerase Chain 

Reaction), showed that Ind caused CpG demethylation. Western blotting analysis 

carried out with p16 monoclonal antibody, confirmed the reactivation of the protein 

expression. Present data, suggesting that a long-term exposure to indicaxanthin in 

diet might potentially affect epigenetic machines of the intestinal cells, preventing 

or repairing initial derangements/disorders, encourage studies on the mechanism 

involved. 

1. Livrea M.A and Tesoriere L. in Herbal and Traditional Medicine, 537-556, (eds. Parker L., Ong C, Halliwell 

B) Marcel Dekker 

2. Caradonna F, Barbata G, Sciandrello G (2000). Nova Science Publishers, Inc. New York USA 4th quarter, 

ISBN.1-60021-875-1. 


163


The diet phytochemical Indicaxanthin suppresses 

7-ketocholesterol-induced THP-1 cell apoptosis via 

inhibition of redox unbalance, cytosolic Ca 2+ increase and 

NF-kB activation 

attanzio a, Pizzuto e, teSoriere l, alleGra M, livrea Ma 

Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, 

Università di Palermo 

Indicaxanthin (Ind) is the betalain pigment characterizing the edible fruits of Opuntia 

ficus indica. Antioxidant activity of Ind in humans (1) and its bioactivity have been 

reported in a number of studies in cell cultures as well as in vivo (2). This study 

investigated the activity of ind in THP-1 cells, a human monocyte/macrophage cell 

line, submitted to injury by 7-ketocholesterol (7-KC), a component of oxidized LDL 

which plays a central role in atherosclerosis (3). In our model a pathophysiological 

environment has been simulated co-incubating 2.5 µM Ind with 16 µM 7-KC, i.e. 

concentrations comparable with those measured in human plasma after cactus pear 

fruit intake (2), and known to be present at the atheroschlerotic plaque (3), respectively. 

Ind counteracted the 7-KC–induced cell growth inhibition, preventing the arrest of 

the cell cycle at the G0-G1 phase. A number of pro-apoptotic hallmarks have been 

considered, including nuclear morphological changes, externalization of PS in 

plasma-membrane, depolarization of mitochondrial membrane, PARP cleavage and 

BAD de-phosphorylation. Ind totally prevented all the 7-KC induced pro-apoptotic 

alterations. Ind inhibited the increase of key second messangers regulating cell death, 

such as ROS and Ca 2+ ions. Finally, Ind prevented the 7-KC-dependent thiol depletion 

and the activation of the cellular effector of inflammation NF-kB. Taking into account 

localization of Ind at the lipid bilayers (4), redox activity of Ind with interception of 

ROS generated by the macrophage NADPH-oxidase NOX-4, and/or interference with 

mechanisms controlling Ca 2+ influx, may be hypothesized. 

1. Livrea M.A and Tesoriere L. in Herbal and Traditional Medicine, 537-556, (eds. Parker L., Ong C, Halliwell 

B) Marcel Dekker 

2. Tesoriere L. et al., Am J Clin Nutr, 2004, 80, 941-945. 

3. Lemaire-Ewing S et al., Cell Biol Toxicol, 2005, 21, 97-114. 

4. Turco Liveri ML et al., J Agric Food Chem 2009, 57, 10959–10963 


164


Nutraceutical and antioxidant potential of LAB: biochemical 

and proteomic methods to elucidate the Se-fixing pathway 

1 Giunta carlo, 1 ManGiaPane erika, 2 Galano euGenio, 

2 PalMeSe anGelo, 1 PeSSione aleSSandro, 2 aMoreSano anGela, 

1 PeSSione enrica 

1 Life Sciences and Systems Biology Department, University of Torino, Italy 

2 Department of Organic Chemistry and Biochemistry, University of Napoli, Italy 

Probiotic lactic acid bacteria have fundamental importance for a correct functioning 

of the human gut. They are able to produce several metabolites such as short 

chain fatty acids, conjugated linoleic acids, bacteriocins, bio-active molecules and 

exopolysaccharides that, in different ways, can bring a benefit to the host. Among 

these various possibilities, the capability to internalize and fix selenium has recently 

received a great attention. 

Selenium (Se) is an essential dietary trace element for humans and microorganisms. 

Se deficiency is associated with various chronic diseases such as cardiovascular 

diseases, some kinds of cancer and oxidative stress, since Se is present in the 

active site of glutathione peroxidase. The use of Se-enriched lactobacilli is an 

interesting approach to solve selenium-deficiency especially during ageing: these 

microorganisms, grown in presence of inorganic forms of selenium, are able to convert 

it into more bio-available organic forms, introducing it into proteins. The addition of 

these microorganisms to a particular food generates a functional food. 

In order to better understand the behavior of Lactobacillus reuteri Lb2 BM DSM 16341 

when grown on selenium, several strategies were applied: a comparative proteomic 

approach was used to highlight changes in the protein pattern in several cellular districts 

and pI ranges. These analyses revealed the up-regulation of the energetic metabolism 

(glycolysis and pentose phosphate pathway), a high expression of adhesion-involved 

proteins and, lust but not least, the up-regulation of a selenocysteine lyase, responsible 

for the insertion of selenocysteine into proteins. To shed more light on the ability of the 

strain to fix selenium into proteins, SEC and HPLC coupled with ICP-MS experiments 

were carried out. The obtained results represent a further confirmation of the ability 

of L. reuteri Lb2 BM to get more bio-available forms of selenium (selenized proteins). 


165


β-lactoglobulin structural rearrangement at interfaces: 

relevance to its immunogenic behavior 

Miriani Matteo 1 , Gentilini Matteo 1,2 , MarenGo Mauro 1 , 

iaMetti Stefania 1 , frøkiær hanne 2 and BonoMi franceSco 1 

1) Section of Chemistry and Biomolecular Sciences, DeFENS, University of Milan 

2) Institute of Chemistry and Biochemistry, Faculty of Life Sciences, 

University of Copenhagen 

The physiological properties of many proteins, relies on their behavior at the molecular 

scale, which often is affected by their surface interactions. A central issue for the in 

vivo action of allergens, but also of antibacterial or otherwise bioactive peptides, is the 

surface behavior in terms of interaction with other molecules and surfaces present in 

the same environment. 

β-lactoglobulin (BLG) is the major bovine whey protein. A 36 kDa dimer of identical 

subunits, BLG is considered the major milk allergen. A general relationship between 

structure and immunoreactivity of milk proteins has not been established. The aim of 

this work is to understand whether and how the BLG conformational changes derived 

from the interaction with two model hydrophobic interfaces (a liquid oil-in-water 

nanoemulsion, and solid latex nanoparticles), can modulate its immunoreactivity and 

its absorption by human cells involved in the immune response. 

Conformational changes in the absorbed protein were demonstrated through 

spectroscopic and limited proteolysis/MS approaches. ELISA experiments 

demonstrated that immunoreactivity of BLG increases upon adsorption on either liquid/ 

liquid or solid/liquid hydrophobic interfaces, likely as a consequence of recognition 

of cognate regions of the protein by the antibodies. Indeed, after hydrolysis with 

trypsin, the overall immunoreactivity of the absorbed protein remains high compared 

with that of the protein hydrolyzed in solution. Also, the kinetics of internalization by 

monocytes of BLG adsorbed on the oil nanodroplets is with respect to free BLG, 

which is absorbed more efficiently and rapidly than BLG adsorbed on an emulsion 

interface. Competition experiments, carried out by adding free unlabeled protein to 

both free and emulsion BLG systems shows how the free protein seems to acts as 

an enhancer for the internalization of the oil-bound protein. Whether these evidences 

indicate independent patterns for intracellular uptake of free and nanoparticulatebound 

BLG remains to be verified. 


166


Natural and synthetic forms of vitamin E as inhibitors 

of secretory phospholipase A 2 (sPLA 2 ) in cystic fibrosis 

epithelial cells 

Pilolli franceSca, leGnaioli Silvia, Bianconi Marcello, 

Piroddi Marta, Galli franceSco and Goracci GianfranceSco 

Dipartimento di Medicina Interna, Università degli Studi di Perugia 

Secretory phospholipase A 2 (sPLA 2 ) GIIA is a form of the heterogeneous family of 

PLA 2 enzymes highly expressed in inflammatory tissues and in a variety of human 

tumors (Graff, R.J. et al. Clin. Cancer Res., 2001). Beside the key role in arachidonate 

metabolism and thus in the control of inflammatory reactions, this isoenzyme can 

produce different biological responses when released in the extracellular medium 

such as cell killing and cell-to-cell signaling, as well as tissue degeneration and 

remodelling. According with Chandra et al. (Chandra et al J. Mol. Biol. 2002), the 

fat-soluble antioxidant vitamin E, in the form of alpha-tocopherol, could specifically 

target on sPLA 2 leading to competitive inhibition of the enzyme activity (Ki = 1.59 ± 

0.09 µM). Here we preliminarily investigated sPLA 2 GIIA expression and activity in lung 

epithelial cells expressing the cystic fibrosis (CF) mutation Δ508 and assessed enzyme 

inhibition and the cytoprotective and anti-inflammatory effects of some natural and 

synthetic forms of vitamin E before and after the challenge of CF epithelial cells with 

active Pseudomonas Aeruginosa (PAO). 

Using a novel assay procedure set up in this study to screen sPLA 2 activity in largescale 

pharmacological tests, we confirmed the inhibitory activity of vitamin E forms 

with gamma-tocopherol and alpha-TEA, an ether derivative of alpha-tocopherol, as 

two of the most effective test molecules. The inhibitory activity was present even 

after the bacterial challenge with PAO. Reactive oxygen species, IL-8 production 

and COX2 activity were also influenced by the treatments that were well tolerated at 

pharmacological concentrations of < 20 µM. The findings in this study suggest a role 

for the pharmacological inhibition of sPLA2 activity in the control of lung inflammation 

of CF patients. Further pre-clinical investigation of vitamin E-derived sPLA2 inhibitors 

in CF model systems are awaited. 


167

Immunologia 

Biochimica 

IMB

Immunologia Biochimica IMB 1 

METABOLOMICS OF MULTIPLE MYELOMA 

franceSca fontana 1,2, JoSè Manuel Garcia ManteiGa 1,2 , 

Silvia Mari 1,3 , eliSaBetta Mariani 1 , enrico caneva 4 , 

franceSco caMnaSio 1 , MaGda Marcatti 1 , edoardo Gaude 1 , 

Giovanna MuSco 1 , roBerto Sitia 1,2 , faBio ciceri 1,2 , 

and SiMone cenci 1,2 

1. San Raffaele Scientific Institute, Milano, Italy 

2. Università Vita-Salute San Raffaele, Milano, Italy 

3. R4R, Research for Rent, Italy 

4. Università degli Studi di Milano, Italy 

Multiple Myeloma (MM) is an incurable neoplastic disorder of plasma cells, which 

invade the bone marrow, secrete monoclonal immunoglobulins, and induce bone 

lesions, hypercalcemia, anemia and renal failure. MM is preceded by a frequent 

condition, monoclonal gammopathy of undetermined significance (MGUS), but is 

diagnosed only once end-organ damage ensues. 

In search for early biomarkers of MM, we deployed MS-based metabolic profiling to 

investigate the correlates of myeloma tumorigenesis and progression in peripheral and 

bone marrow plasma. As a whole, metabolic footprinting successfully discriminated 

active disease from controls, correlating with bone marrow tumor cellularity. Renal 

dysfunction was independently addressed, leading to the identification of a number 

of metabolites previously associated with chronic kidney disease, and providing 

the power to discriminate cancer growth from end-organ damage. MM-associated 

features included a significant decrease in one lipid class and the increase of a few rare 

metabolites, suggesting unexpected pathomechanisms. Furthermore, hypothesizing 

extracellular lysolipids to be consumed by MM cells, we obtained in vitro evidence 

supporting a trophic role on myeloma cells. 

MM consists of intramedullary dissemination of malignant cells causing osteolytic 

lesions at high risk of fracture. Aiming to address the local heterogeneity of myeloma 

bone disease, we also set up to investigate MM lesions by intact tissue HR-MAS 

NMR. Metabolic fingerprinting efficiently matched histological findings, clustering 

according to tissue identity. Moreover, peak assignment revealed that areas of high 

tumor cellularity were associated with high concentration of lipids. 

In all, our data suggest that metabolomics is a powerful strategy to address the 

complexity of MM pathophysiology, and develop novel diagnostic strategies. 


170


LGALS3BP induces VEFG in human breast cancer cells and 

promotes angiogenesis 

traini Sara 1,2 , Piccolo enza 1,2 , tinari nicola 1,2 , fichera iMMa 1,2 , 

d’eGidio Maurizia 1,2 , di riSio annaliSa 1,2 , natoli clara 1 , and 

iacoBelli Stefano 1,2 . 

1 MediaPharma s.r.l., Via dei Vestini, 31, 66100 Chieti, Italy; 

2 Department of Biomedical Sciences, G. D’Annunzio University and Foundation, 

66100 Chieti, Italy. 

Background: Elevated serum or tissue levels of lectin galactoside-binding soluble 

3 binding protein (LGALS3BP) have been associated with short survival and 

development of metastasis in a variety of human cancers. However the role of 

LGALS3BP, particularly in the context of tumor-host relationships, is still missing. 

Methods: MDA-MB-231 human breast cancer cells were stably silenced for human 

LGALS3BP and assayed for the content of vascular endothelial growth factor (VEGF) 

by ELISA. For adhesion assay, cells were plated onto 96-well culture dishes precoated 

with Collagen IV, Collagen V, Fibronectin, or Laminin. Adherent cells were stained 

and their absorbance measured at 550 nm. For transmigration assay, calcein-labeled 

MDA-MB-231 cells were stratified over HUVEC in transwell chambers and the number 

of transmigrating tumor cells counted using fluorescent microscopy. Tubulogenesis 

was performed by seeding HUVEC on matrigel and counting the number of branch 

point/field under 10X magnification. In vivo angiogenesis was done using matrigel 

plugs implanted subcutaneously in athymic (nu+/nu+) mice. Seven days later, plugs 

were were removed and the hemoglobin content assayed. 

Results: LGALS3BP knockdown in MDA-MB-231 cells was accompanied to a 

decreased cell adhesion to fibronectin, a reduced transendothelial migration and, 

more importantly, a reduced expression of VEGF. Production of VEGF, which was 

restored by exposure of silenced cells to recombinant LGALS3BP, required an intact 

PI3K/Akt signaling. Furthermore, LGALS3BP was able to directly stimulate in vitro 

HUVEC tubulogenesis and in vivo angiogenesis in a VEGF-independent manner. 

Immunohistochemical analysis of human breast cancer tissues showed a positive 

correlation between LGALS3BP expression, VEGF expression and blood vessel 

density. 

Conclusions: In addition to its prometastatic role, LGALS3BP secreted by human 

breast cancer cells may function as a pro-angiogenic factor through a dual mechanism, 

i.e by induction of tumor VEGF and stimulation of endothelial cells angiogenesis. 


171


EV20, a novel humanized ErbB-3 antibody, suppresses 

NRG1β/ErbB3/AKT axis in vitro and boost the antitumor 

activity of trastuzumab in xenografted BxPC-3 pancreatic 

tumor in mice 

GhaSeMi reza 1 , iacoBelli Stefano 1-2 and Sala Gianluca 1-2 

1 Dipartimento di Scienze Biomediche, Universita’ “G. d’Annunzio” Chieti-Pescara, 

Fondazione “G. D’Annunzio, Centro Studi sull’Invecchiamento, 

Ce.S.I., Via Colle dell’Ara 1, 66100, Chieti, ITALY 

2 Mediapharma s.r.l., Via dei Vestini 1, 66100 Chieti, ITALY 

Deregulated signaling through the EGFR (ErbB-1) and ErbB-2 members of the 

epidermal growth factor receptor family (ErbB) frequently occurs in solid tumors, 

including pancreatic cancer. Consequently, these two receptors have been the most 

targeted proteins in cancer. However, increasing evidence proposes that ErbB-3, 

another member of the ErbB family, is a key player in driving oncogenic signaling and 

resistance to therapy. Indeed, re-activation of the ErbB-3/PI3K/AKT axis is thought 

to be the major mechanism of acquired resistance to ErbB-1 and ErbB-2 inhibitors. 

In this study we aimed to evaluate the antitumor potentials of the concomitant inhibition 

of ErbB-2 and ErbB-3 receptors in the BxPC-3 pancreatic cancer cell model, using 

trastuzumab, the clinical approved ErbB-2 inhibitor, and EV20, a novel humanized 

anti-ErbB-3 monoclonal antibody developed in our lab. 

We first found that neuregulin 1 (NRG-1) the ErbB-3 ligand, more strongly than EGF, 

the ErbB-1 ligand, induces the activation of both AKT and ERK in BxPC-3 cells. 

We explored then that the inhibition of ErbB-2 (by trastuzumab) or ErbB-3 (by RNAi 

or EV20) strongly impairs the NRG-1-driven AKT activation and cell proliferation, 

suggesting a key role for ErbB-2/ErbB-3 receptors in this cell line. Intriguingly, the 

concomitant targeting of both ErbB-2 (by trastuzumab) and ErbB-3 (by EV20) resulted 

in the complete shutdown of the NRG-1-driven AKT activity as well as reduction of cell 

survival. This in vitro antitumor and trastuzumab-enhancing activity of EV20 was also 

confirmed in vivo in the xenografted pancreatic tumors developed by subcutaneous 

injection of BxPC-3 cells in the nude mice. 

Altogether, our data underscore the importance of NRG-1-driven activation of ErbB-2/ 

ErbB-3 dimer in a pancreatic cancer model and propose the dual targeting of ErbB-2/ 

ErbB-3 receptors as a new option for pancreatic cancer therapy. 


172


Tumor Necrosis Factor-a Neutralizing Antibodies Induced 

by a Glycolaldehyde-Modified C-terminal Polypeptide 

oStuni anGela, BavoSo alfonSo, Bracalello anGelo and 

1 traMontano alfonSo 

Dept. of Science , University of Basilicata, 85100 Potenza, Italy 

1 UC Davis – Medical School, Dept. of Pediatrics, Davis, CA 95616, USA 

TNF-a is a pro-inflammatory cytokine that is overexpressed in diverse inflammatory 

states such as rheumatoid arthritis. Anti-TNF therapies, including monoclonal 

antibodies (Abs), have demonstrated remarkable success in management of arthritis 

and other chronic inflammatory diseases. The possibility to elicit autoAbs to TNF-a 

(beneficial autoimmunity) has been proposed as an immunotherapeutic approach 

to neutralize systemic TNF-a (1). Studies using a DNA vaccination strategy in rat 

adjuvant arthritis mapped several epitopes for natural anti-TNF-a autoAbs in the 

C-terminal portion of the sequence. We previously showed that aldehyde-tagged 

peptide conjugates representing immunogenic epitopes of toxic shock syndrome 

toxin–1, could induce anti-toxin neutralizing Abs without the need of potent adjuvants 

(2). In the present study, using a C-terminal TNF-a recombinant polypeptide, we 

followed a similar approach to generate neutralizing Abs against TNF-a in Lewis rats. 

Immunized rats showed less severe symptoms in the collagen induced arthritis ( CIA) 

model. 

Dalum I. et. al. Therapeutic antibodies elicited by immunization against TNF-alpha. Nat Biotechnol 1999. 

17:666-669. 

Bavoso A, Ostuni A, De Vendel J, Pollaro F, Armentano F, Knight T, Makker S. and A. Tramontano. Aldehyde 

modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome 

toxin-1. J Pept Sci 2006. 12:843-849. 


173


Branched peptides as novel tumor-targeting agents for 

bladder cancer 

falciani c 1 , lelli B 1 , Brunetti J 1 , Pini a 1 , ravenni n 1 , dePau l 1 , 

lozzi l 1 , Minervini a 2 , carini M 2 , Bracci l 1 . 

1 University of Siena, Department of Biotechnology, Siena. 

2 University of Florence, Department of Urology, Careggi Hospital, Florence. 

Bladder cancer (BC) ranks ninth in worldwide cancer incidence. It is the seventh most 

common cancer in men and the 17 th most common cancer in women, with more than 

330,000 new cases and more than 130,000 deaths each year [1]. The main clinical 

impact of BC is related to recurrence and progression of disease. Patients with BC 

have the highest lifetime treatment costs per patient among all cancers, due to the 

numerous retreatments for recurrence. This suggests the clear need for more effective 

adjuvant treatments. In this direction, the selective targeting of tumor cells should be 

the goal of modern cancer diagnosis and therapy. Peptide receptor tumor targeting 

strategy is particularly attractive, since it provides the possible tumor target and its 

candidate peptide targeting agent at the same time. 

Peptides reproducing the sequence of the endogenous peptide neurotensin (NT) in 

its tetrameric form (NT4) proved to bind tumor cell membrane and can be coupled 

to different effector units for either imaging or therapy of cancer cells [2]. Indeed, 

NT4 can efficiently discriminate between tumor and healthy tissue in human surgical 

samples from colon or pancreas adenocarcinoma [3] and bladder cancer. Conjugation 

of cytotoxic drugs to NT4 provides an in vitro and in vivo peptide receptor mediated 

cytotoxic effect, increasing drug selectivity toward receptor-positive cells. Furthermore, 

drug-armed nanoparticles decorated with NT4 showed to be much more effective 

than nude liposomes for intracellular drug delivery in cancer cells [4]. 

1. Venturini M et al. The Numbers of Cancer in Italy 2011 Intermedia Editore 

2. Falciani C et al. Mol. Cancer Ther. 2441–2448 (2007). 

3. Falciani C et al. Curr. Cancer Drug Targets 10(7), 695–704 (2010). 

4. Falciani C et al. Chem. Med. Chem. 6(4), 678–685 (2011). 


174


Carbon nanotubes for biomedical application 

deloGu lucia GeMMa 1 , venturelli enrica 2 , Manetti roBerto 3 , 

PeScatori Mario 4 , Bianco alBerto 2 and franceSco SGarrella 1 . 

1 Dipartimento di scienze del farmaco, Universitˆ degli Studi di Sassari, 

via muroni 23 a, 07100 Sassari, Italy 

2 CNRS, Institut de Biologie MolŽculaire et Cellulaire, Laboratoire d’Immunologie 

et Chimie ThŽrapeutiques, 67000 Strasbourg, France 

3 Dipartimento di Medicina Clinica, Sperimentale e Oncologica, 

Università degli Studi di Sassari, Sassari, Italy. 

4 Department of Surgical Oncology Erasmus MC Rotterdam NL. 

Carbon nanotubes (CNTs) are intensively investigated nanomaterials for their possible 

biological applications (Menard-Moyon C Chembiol 2010, Zhuang et al. Nano Res 

2009). Most of the previous studies were performed focusing on the possible toxicity 

of CNTs and on the possibility to use them as drug carrier. Indeed, CNTs might 

behave as a possible new immunomodulator system. To explore this hypothesis 

we investigated the impact using four different functionalization of multi-walled 

CNTs (MWCNTs). To have representative cells for both types of immune response, 

adaptive and innate, we used Jurkat cell line (T lymphocytes) and THP1 (monocytes). 

We first investigated the uptake, viability and apoptosis. Our experiments showed 

a significant uptake and a lack of toxicity for all types of MWCNTs in both types 

of cells. Since the immune system is always a balance of switching genes on and 

off, we performed a whole genome expression evaluation. The Affimetrix microarray 

technique was applied to understand the immune system interaction with the different 

MWCNTs. Our data showed that MWCNTs were inert for T cells with only 74 genes 

affected. On the opposite, we found a significant distinction in the genome-wide 

pattern of gene expression between the controls and the MWCNT treated monocytes 

for a total of 328 genes affected. In particular we found, for cells treated with OX- 

MWCNT-NH3+ that the genes involved in the immune response and inflammation 

were massively induced. This work on MWCNTs genome expression modulation 

confirm our previous study conducted ex-vivo on lymphomonocytes (Delogu LG et 

al. Nanomedicine Lond 2011) and lead to the possible use of OX-MWCNTNH3+ as 

potential immunostimulatory systems. 


175


Identification, cloning and functional characterization of 

a novel splice variant of human BST1/CD157 ADP-ribosyl 

cyclase 

Parrotta roSSella, lo Buono nicola, GiacoMino alice, Morone 

SiMona, ortolan erika, ferrero enza and funaro ada 

Dept. of Medical Sciences, University of Torino Medical School, Turin, Italy 

CD157 is a GPI-anchored ectoenzyme belonging to the NADase/ADP-ribosyl 

cyclase gene family involved in the control of leukocyte polarization, migration and 

diapedesis. We recently demonstrated that CD157 is a molecular organizer of signaling 

competent membrane microdomains and forms part of a larger molecular machine 

ruled by integrins. The CD157-integrin partnership provides optimal adhesion and 

transmigration to human monocytes (1). CD157 is also expressed in epithelial ovarian 

cancer where it controls tumor cell migration and peritoneal invasion, and is a marker 

of poor prognosis (2). In this work we identified and cloned by “Overlap extension 

PCR cloning” technique a novel alternative splice variant of CD157, never described 

before, containing an additional exon encoding a β-sheet in the extracellular domain, 

without apparent modifications of the original structure, as shown by protein structure 

prediction software. We demonstrated that the CD157 splice variant is expressed 

by all cells expressing “conventional” CD157. The functional characterization of the 

new isoform has been carried on using engineered ovarian cancer cells as disease 

model. The results showed that forced expression of CD157 in ovarian cancer cells 

i) promotes motility as shown by in vitro wound healing and haptotaxis assays and 

ii) elicits intracellular signals resulting in increased activation of the MAPK-ERK1/2 

downstream signaling pathway. These preliminary data indicate that the novel splice 

variant of human CD157 may play a role similar to that of the canonical CD157, 

however, further investigations are needed to fully understand the molecular basis of 

this alternative splicing, which appeared in primate phylogeny. 

1. Lo Buono N.*, Parrotta R.*, et al. A. The CD157-integrin partnership controls transendothelial migration 

and adhesion of human monocyte. J Biol Chem. 2011 May 27;286(21):18681-91 

2. Erika Ortolan et al. Functional role and prognostic significance of CD157 in ovarian carcinoma. J Natl 

Cancer Inst. 2010 Aug 4;102(15):1160-77 


176

Sviluppo 

Differenziamento 

e Apoptosi 

SDA

Sviluppo Differenziamento e Apoptosi SDA 1 

TGFbeta-induced Rho kinase activation downstream of 

S1P4 in myoblasts: a novel signaling pathway accounting 

for the apoptotic effect exerted by the cytokine 

cencetti franceSca, Bernacchioni caterina, BleScia SaBrina, 

donati chiara and Bruni Paola 

Dipartimento di Scienze Biochimiche, Università di Firenze, Viale GB Morgagni 50, 

50134 Firenze; Istituto Interuniversitario di Miologia. 

S1P4 is one of the five G protein coupled receptors specific for sphingosine 

1-phosphate (S1P), a powerful bioactive lipid that exerts multiple key biological 

actions. According to literature data, expression of S1P4, differently from that of 

other receptor subtypes, is restricted to lymphoid tissue. Unexpectedly, during the 

investigation of the biological role played by the cross-talk between transforming 

growth factor beta (TGFb) and S1P signaling axis we found that in mouse myoblasts 

S1P4 is significantly up-regulated by TGFb, although it does not participates to the 

pro-fibrotic action exerted by the cytokine (1). 

Here we report that the apoptotic effect exerted by TGFb (5 ng/ml), measured by 

caspase-3 activity assay and PARP cleavage, was dependent on S1P4, since it 

was abrogated by its specific silencing or treatment with a selective antagonist and, 

conversely, was significantly enhanced in myoblasts overexpressing this receptor 

subtype. Moreover, TGFb-driven apoptotic response mediated by S1P4 was found 

to rely on biosynthesis of S1P, specifically catalyzed by sphingosine kinase-2 (SK2), 

given that the biological effect was abolished when SK2 but not SK1 was silenced and 

a pan-SK inhibitor was capable of preventing the apoptotic effect whereas a selective 

SK1 inhibitor was inefficacious. 

Notably, a novel signaling pathway that involves Rho kinase-2-dependent 

phosphorylation of PTEN and consequent reduced AKT activity was individuated as 

major transducer of the S1P4-directed apoptotic response. 

This study provides further evidence for a key role of S1P signaling axis in the TGFb 

action mechanism that could be exploited to counteract the detrimental effects of this 

cytokine in skeletal muscle regeneration. 

1. Cencetti et al. Mol Biol Cell 21, 1111 (2010) 


178


Role of endocannabinoid receptors in megakaryocyte 

differentiation 

Portanova Patrizia 1 , SaMPietro Sara 1 , nalin Michela 1 , BaGarotti 

aleSSandra 1 , SiniGaGlia faBiola 1,2 , Bertoni aleSSandra 1,2 

1 Department of Translational Medicine Novara, University of Piemonte 

Orientale A. Avogadro, Novara, IT 

2 BRMA, University of Piemonte Orientale A. Avogadro, Novara 

Introduction Megakaryocytes originate in the bone marrow from pluripotent stem 

cells through a differentiation process that involves stem cell commitment, nuclear 

polyploidization, and the cytoplasmatic maturation leading to the production of 

platelets. Several cytokines and growth factors synergistically promote growth and 

maturation of megakaryocytes in bone marrow. 

The endocannabinoid system consists of cannabinoid receptors (CB1, CB2, and 

TRPV1), endogenous ligands (2-arachidonoylglycerol and anandamide), ligand 

membrane transporters, and ligand-metabolizing enzymes (DAGL, NAPE-PLD, FAAH, 

MAGL, ABHD6 and ABHD12). 

Recent findings suggest a novel physiological role for endocannabinoids as signaling 

molecules responsible for the control of proliferation and differentiation, both centrally 

and peripherally. However, little is known concerning haematopoietic cell differentiation 

and endocannabinois. 

Methods Murine bone marrow-derived Lin-/cKit+ cells were induced to differentiate 

in ex-vivo in megakaryocytes. Protein expression was analyzed by immunoblotting, 

and gene expression was analyzed by RT-PCR. Ploidy, expression of differentiation 

markers, and cell dimensions were analyzed by flow cytometry. 

Results During megakaryocyte differentiation CB1 and TRPV1 expression showed a 

progressive and significant increase, whereas CB2 expression was not modulated. 

Moreover, the expression of FAAH and MAGL were strongly downregulated; ABHD12 

was already expressed in Lin-/cKit+ cells and was not further modulated, while ABHD6 

was not expressed megakaryocytes. Furthermore, addition of anandamide during 

differentiation significantly increased the number of intermediate and fully mature 

cells via CB1 receptor signalling, while signaling through CB2 receptors reduced the 

number of fully mature megakayocytes. However, none endocannabinoid was able to 

affect the differentiation degree of the cells since CD41 expression and cell ploidy was 

unaltered by endocannabinoid treatment. 

Conclusions: In light of all these findings, we can speculate an involvement of the 

endocannabinoid system in the regulation of the number of maturing megakaryocytes 

within the bone marrow. Further studies are necessary to better understand the molecular 

pathways activated downstream of CB receptors during megakaryocytopoiesis. 


179


ω-3 PUFA REDUCE CHOLESTEROL BIOSYNTHESIS AND 

DOxORUBICIN RESISTANCE IN COLON CANCER CELLS 

riGanti chiara 1 , corSetto Paola a. 2 , Montorfano GiGliola 2 , 

creMona andrea 2 , GhiGo dario 1 , BoSia aMalia 1 , rizzo anGela M. 2 

1 Dipartimento di Oncologia, Università degli Studi di Torino 

2 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano 

Multidrug resistance (MDR) indicates a decrease in sensitivity of cancer to a wide 

variety of cytotoxic compounds, as doxorubicin. In fact the most frequent drawback 

of doxorubicin treatment is the onset of drug resistance, due to its active efflux from 

cancer cells through P-glycoprotein (Pgp). Although a central role has been ascribed 

to Pgp in MRD, lipid membrane environment also appears to be important; Pgp is 

membrane transporter and is mainly localized in lipid rafts. In particular, membrane 

cholesterol is essential to maintain a high Pgp activity (1), consequently tumors with 

a MDR phenotype have higher concentrations of plasma membrane cholesterol than 

chemosensitive ones. 

Several studies have demonstrated that ω-3 polyunsaturated fatty acids (ω-3 PUFA) 

intake is correlated to low incidence of colorectal cancer (2). The mechanism(s) by 

which ω-3 PUFA might exhibit a protective effect remains unclear but one of the 

hypotheses indicates that they may also change the fluidity of cell membrane and 

thus influence signaling pathways. 

We treated human doxorubicin-sensitive (HT29) and doxorubicin-resistant (HT29-dx) 

colon cancer cells with EPA and DHA (50 µmol/L). 

Preliminary results indicate that DHA and EPA are incorporated in plasma membrane 

with a reduction of cholesterol content, in particular in HT29-dx cells. 

The cholesterol biosynthesis is higher in drug-resistant cells compared to drugsensitive 

ones, and is decreased dose-dependently by PUFA treatment in both cell 

lines. Moreover, ω-3 PUFA, especially DHA, determine a reduction of HMGCoAR 

activity and expression. 

Only in HT29 dx cells, we have verified a slightly reduction of SREBP-2 nuclear 

content. 

Finally, DHA induces doxorubicin accumulation in drug resistant HT29-dx cells and 

reduces the Pgp activity, measured as the ability to extrude rhodamine. 

The evaluation of lipid raft Pgp localization and lipid composition will give the indication 

of a direct effect of ω-3 PUFA on raft structure and function. 

1 Kopecka J, Journal of Controlled Release 2011. 

2 Giros A, Cancer Prevention Research 2009. 


180


New adenilic dinucleotides with antileukemic activity 

Scarfì Sonia 1 , Bruzzone Santina 2 , Giovine Marco 1 , 

de flora antonio 2 and zocchi elena 2 

1 Dept. Territory, Environment and Life, University of Genova,; 

2 Dept. Experimental Medicine, Section Biochemistry, University of Genova, Italy 

Recently, ADP-ribosyl cyclases from the marine sponge Axinella polypoides, and 

from human CD38, demonstrated to synthesize two diadenosine homodinucleotides 

from cADPR and adenine (designated P18 and P24), which are characterized by an 

unusual N-glycosidic bond between one of the adenines and ribose [1]. 

P18 and P24 affect the [Ca 2+ ] i when applied extracellularly to intact cells, not 

unexpectedly affecting cell proliferation [2]. The activity of P18 and P24 is dependent 

on their capacity to modulate the purinergic receptor P2X7 and P2Y11 [3]. In particular 

P24 activates the purinergic receptor/channel P2X7, eliciting influx of extracellular 

Ca 2+ , loss of mitochondrial function and induction of apoptotic cell death. Some 

observations indicate that cells from myeloid and lymphocytic leukemias should be 

sensitive to the cytotoxic effect of P2X7 agonists suggesting the use of P24 for cancer 

therapy. 

Indeed, results obtained in our laboratory on cells isolated from patients with AML 

or B-CLL, and on tumor cell lines (erythroleukemia, lung and breast cancer) indicate 

that P24 induces cell death by apoptosis at sub-micromolar concentrations, while 

P18 is effective at micromolar concentrations. In the same conditions, cells isolated 

from healthy donors were not affected by P24 and P18 indicating a specific cytotoxic 

effect on leukemic and on solid tumor cells. Furthermore, we could confirm that the 

cytotoxic activity of P24 was mainly attributable to the dissipation of the mitochondrial 

proton gradient, through the opening of the permeability transition pore, resulting in 

cell death. 

In conclusion, we suggest the possible use of P24 as a drug for cancer therapy in 

haematological malignancies while ongoing studies are trying to identify further cell 

types sensitive to this new antitumor drug. 

1. Basile G. et al (2005) Proc Natl Acad Sci USA 102, 14509-14 

2. Bruzzone S. et al (2007) J Biol Chem 282, 5045-52 

3. Bruzzone S. et al (2010) J Biol Chem 285, 21165-74 


181


Ceramide acts as a mediator of the antiproliferative and 

pro-death effects of the natural flavonoid luteolin in human 

colon cancer cells 

aBdelhadi.louBna, GiuSSani.Paola, viani.Paola, riBoni.laura 

Department of Chemistry, biochemistry and biotechnology for medicine 

The sphingolipid ceramide plays an important role as anticancer cellular mediator and 

is implicated in chemotherapy-induced apoptosis by different agents. Human colon 

cancers (the second most leading cause of cancer-related deaths in the western 

countries) have only half the ceramide content of normal tissue, and these low 

ceramide levels have been correlated with their increased resistance to apoptosis. 

As luteolin (3’,4’,5,7-tetrahydroxyflavone), a common dietary flavonoid, exhibits 

powerful pro-apoptotic and anti-cancer properties, and sensitizes colon cancer cells 

to therapeutic-induced cytotoxicity, we addressed the question if a relationship exists 

between the unknown anticancer mechanism of luteolin and ceramide in colon cancer 

cells. 

Using the human colon cancer cell line Caco-2 as cell model, we initially found that 

luteolin administration was associated to the inhibition of cell proliferation and, at higher 

doses, to cell death. In further experiments, we investigated the ceramide metabolism 

in Caco-2 cells and the possible effects of luteolin on it, using radiolabeled sphingosine 

as precursor. The results showed that Caco-2 cells efficiently and rapidly generate 

both ceramide and sphingomyelin from sphingosine, and that luteolin treatment 

induces a dose-dependent reduction of the sphingomyelin biosynthesis, associated 

to a significant increase of the cellular ceramide. This evidence let us hypothesize that 

ceramide may be involved in the anti-proliferative and toxic effect of luteolin. On these 

promises, we evaluated the effect of ceramide on cell survival. We found that either the 

administration of ceramide analogues or the inhibition of sphingomyelin biosynthesis 

were able to induce Caco-2 cell death, thus demonstrating that ceramide is able to 

mimic the cytotoxic effect of luteolin. 

Overall, these results demonstrate that luteolin can act as an antiproliferative and prodeath 

agent in Caco-2 cells by up-regulating cellular ceramide through inhibition of 

its metabolism to sphingomyelin, and suggest a potential role of luteolin/ceramide as 

anti-cancer agent in colon malignancies. 


182


Isolation and characterization of human rotator cuff tendon 

stem cells 

conforti erika 1 , Piccoli Marco 1 , creo PaSquale 1 , 

cirillo federica 1 , BerGante Sonia 1 , ScarinGi raffaella 1 , 

Ghiroldi andrea 1 , raGone vincenza 1 , MaSuzzo PaMela 1 , 

caBitza Paolo 1,2 , tettaManti Guido 2 , GaGliano nicoletta 2 , 

randelli Pietro 1,2 and anaStaSia luiGi 1,2,* 

(1) IRCCS Policlinico San Donato, San Donato Milanese, Italy; 

(2) Department of Biomedical Sciences for Health, University of Milan, Milan, Italy. 

Rotator cuff (RC) tendons are often prone to lesions and raptures, as 30 to 50% 

of the population over fifty suffers of partial- and full-thickness RC tears. Several 

approaches have been developed over the years, including the use of growth factors, 

bone morphogenetic proteins and, more recently, stem cells. Among adult stem 

cells, bone marrow mesenchymal stromal cells (BMSCs) are by far the most studied, 

although tendon-derived progenitor cells (TSPCs) have been found in several animal 

species, including humans (1,2). However, the isolation of a cell population with stem 

cells characteristics from the human rotator cuff has yet to be reported. 

In this work we report the isolation and characterization of two new adult stem 

cell populations from the supraspinatus (SS), which is part of the rotator cuff, and 

from the long head bicep (LHB) tendons. Cells could be cultured and expanded, 

and they showed adult stem cell characteristics, i.e. they are self-renewing in vitro, 

clonogenic, and multipotent, as they could be induced to differentiate in vitro into 

different cell types, including osteoblasts, adipocytes and skeletal muscle. These 

new cell populations were phenotypically characterized and compared to BMSCs 

and to human dermal fibroblasts (DFs). Moreover, as sphingolipids are emerging key 

regulatory molecules of stem cell proliferation and differentiation, the sphingolipid 

pattern of all cells has been determined. 

1. Lui, P. P., and Chan, K. M. (2011) Stem cell reviews 7, 883-897 

2. Bi, Y., Ehirchiou, D., Kilts, T. M., Inkson, C. A., Embree, M. C., Sonoyama, W., Li, L., Leet, A. I., Seo, B. 

M., Zhang, L., Shi, S., and Young, M. F. (2007) Nature medicine 13, 1219-1227 


183


A comparison between the role of SPARC in osteogenic 

differentiation of mesenchymal stem cells and in WIN/ 

TRAIL-induced apoptosis in osteosarcoma cells 

a. notaro, S. SaBella, o. Pellerito, M. Giuliano and G. calvaruSo 

Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, 

sezione di Biochimica, Università degli Studi di Palermo 

Mesenchymal stem cells are well known to possess multipotential differentiative 

ability and represent a good tool for clinical research. 

In our study, after induction of osteogenic differentiation of bone marrow mesenchymal 

stem cells (BM-MSC) by using conditioned media, we focused our attention on 

SPARC, a regulatory protein which affects cell differentiation, proliferation, and 

survival. During osteogenic differentiation SPARC levels raised in time-dependent 

manner and were functional to the increase of other osteogenic markers, such as 

osterix and osteopontin. The effects of SPARC downregulation were investigated 

through the use of a specific small-interfering RNA, demonstrating a reduction in the 

levels of osteogenic markers in siSPARC cells. 

Since SPARC, besides to its important role in osteogenic differentiation, can modulate 

cell-cell and cell-matrix interactions and act either as a tumor suppressor or a tumor 

promoter, we were interested to investigate its levels in osteosarcoma MG63 cells, a 

malignant osteoblastic-like cell line. In these cells the basal levels of SPARC resulted 

very high. 

It is well known that MG63 cells are resistant to TRAIL (tumor necrosis factor apoptosis 

inducing ligand) although they can be sensitised to TRAIL-induced apoptosis by 

different compounds. Treatment of these cells with a combination of the synthetic 

cannabinoid WIN and TRAIL induced marked cytotoxic effects. Since treatment 

with WIN/TRAIL combination further enhanced the levels of SPARC, we sought to 

evaluate the possible role of SPARC in WIN/TRAIL-mediated apoptotic pathway. To 

this purpose, we induced SPARC down-regulation by means of gene silencing and 

observed a decrease in the apoptotic features induced by the combined treatment. 

In conclusion, in osteosarcoma cells SPARC seems to behave like a tumor suppressor 

protein which is able to actively participate to apoptotic cell death induced by WIN/ 

TRAIL combined treatment. 


184


Autophagy and ER-stress participate to cannabinoidinduced 

apoptosis in colon carcinoma cells 

o. Pellerito, a. notaro, S. SaBella, G. calvaruSo and M. Giuliano 

Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, 

sezione di Biochimica, Università degli Studi di Palermo 

Autophagy is a highly conserved cellular process wherein cytoplasmic materials, 

including organelles, are sequestered into double-membrane vesicles called 

autophagosomes and delivered to lysosomes for degradation or recycling. Besides 

its role in cellular homeostasis, autophagy represents a form of programmed cell 

death, designated “type II programmed cell death”. Accordingly, autophagy has 

been proposed to play an important role in both tumor progression and cancer cell 

death. It is known that autophagy is also involved in the action mechanism induced by 

cannabinoids, a class of compounds which exert a wide variety of biological effects. 

In this study, we investigated the effects of the synthetic cannabinoid WIN55,212-2 

on the growth of HT29 colon carcinoma cell line. WIN was capable to curb cell growth 

by inducing an apoptotic pathway which was preceded by ER-stress, as confirmed 

by the increase in ER-stress sensors such as GRP78, CHOP and TRB3. Moreover, 

WIN treatment triggered an autophagic pathway with the increase in the level of 

beclin-1 in the early phase of treatment and a progressive time-dependent conversion 

of LC3-I to LC3-II. We confirmed the induction of autophagic process using monodansylcadaverine, 

a fluorescent molecule, which is employed as a selective marker 

for autophagic vacuoles and especially acidic autophagolysosomes. 

To understand the role of autophagy in WIN-induced apoptosis in HT29 cells, we 

employed 3-MA, a specific inhibitor of autophagic process. We observed that in the 

first phase of treatment autophagy inhibition counteract WIN-induced cell death as if 

autophagy was an important step of cell death induction. Differently, after prolonged 

treatment, 3-MA was no longer able to counteract WIN effects; this probably was a 

consequence of the activation of apoptotic cell death mediated by the high levels of 

CHOP. In fact, CHOP down-regulation by gene silencing induced a decrease in the 

levels of autophagy markers and delayed WIN-mediated apoptotic events. 


185


Glioblastoma multiforme: pharmacological re-activation of 

p53 by inhibition of p53-MDM2 interaction. 

BarBara coSta 1 , eleonora da Pozzo 2 , SiMona daniele 2 , 

Baratta chiara 2 , SaBrina taliani 3 , federico da SettiMo 3 , 

claudia Martini 2 . 

1 Department of Human Morphology and Applied Biology; 2 Department of Psychiatry, 

Neurobiology, Pharmacology and Biotechnology; 3 Department of Pharmaceutical 

Science, University of Pisa, Italy. 

The glioblastoma multiforme (GBM) responds poorly to conventional therapy, which 

consists in causing DNA damage in highly proliferating tumor cells. This cancer 

escapes the activity of genotoxic modalities because GBM cells frequently exhibit 

attenuated function of p53 protein [1]. If p53 could function fully, it could block cell cycle 

progression and activate the apoptotic program following DNA damage induction. In 

GBM, p53 availability is frequently reduced because it binds to the murine double 

minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor 

cells [2]. For this reason, the inhibition of MDM2-p53 interaction seems represent 

a way to activate p53 pathway and offer an important window for GBM therapeutic 

intervention. 

In the present study, we investigated the antitumor in vitro effects of new synthesised 

indole-based peptide mimetic compounds that were projected as MDM2 inhibitors by 

the means of molecular modeling analyses. The U87MG cell line was chosen as a cell 

culture model of human GBM because it maintains wild-type p53 and overexpresses 

MDM2 [3, 4]. The EB54 and EB56 were the most potent compounds able to dissociate 

MDM2/p53 complex isolated by U87MG cells. In these tumor cells, they re-activated 

p53 function, as indicated by the transcription stimulation of the main target genes of 

p53. The reactivation of p53 function was effective to reduce the number of tumor cells 

in a time-and dose-dependent manner. Following EB cell treatments we identified the 

upregulation of the PUMA gene expression, dissipation of mitochondrial membrane 

potential and presence of phosphatydilserine in outer layer of plasma membrane, all 

features consistent with apoptotic cell death. 

References 

[1]Cerami E, et al. PLoS One 2010;5 e8918. 

[2] He J, et al. Genes Chromosomes Cancer 1994;11 91-6. 

[3] Kondo S, et al. Oncogene 1995;10 2001-6. 

[4] Mayo LD, et al. J Biol Chem 2002;277 5484-9. 


186


Sirt-1 cleavage and shuttling between the nucleus and 

the cytoplasm: is it part of a regulatory network in breast 

cancer cells MDA-MB231? 

de BlaSio anna, MontalBano Mauro, di fiore riccardo, 

vento renza 

Section of Biochemical Sciences, Department of Experimental Biomedicine 

and Clinical Neurosciences, Polyclinic, University of Palermo, 

Via del Vespro 129, 90127 Palermo, Italy. 

SIRT1 is a NAD + -dependent protein deacetylase which acts as crucial regulator on 

several cellular processes, ranging from energy metabolism and stress response 

to tumorigenesis and aging. SIRT1 directly links cellular metabolic status to gene 

regulation, playing an important role in a number of pro-survival activities. However 

SIRT1 might play dual functions as either oncogene or tumor suppressor and this is 

currently under intense debate. Subcellular localization of SIRT1 varies in different cell 

types, and this is crucial for its impact on cellular functions (1). This study aimed to 

identify the SIRT1 protein status and its changes in glucose-deprived MDA-MB231 

breast cancer cells. Western blotting analysis of subcellular fractions showed a 

cytoplasmatic localization of full length SIRT1, and a nuclear localization of SIRT1cleaved 

forms. We also showed the nuclear localization of both sumoylated-caspase-8 

(nuclear import signal) (2) and its active cleaved-form. Interestingly, in MDA-MB231 

cells, incubation with caspase inhibitor Z-VAD-FMK, markedly lowered the levels of 

SIRT1-cleaved form, suggesting a mechanism of SIRT1 caspase-mediated cleavage. 

Evaluating the effects of nutritional scarcity on the subcellular localization of SIRT1, 

we observed that 96 h exposure to low glucose concentrations (1-10 mM) potently 

induced apoptosis. This was accompanied by a decrease in cytosolic level of fulllength 

SIRT1 with a simultaneous increase in its nuclear cleaved forms. We suggest 

that, in MDA-MB231 cells, sumoylation and nuclear import of caspase-8 might be an 

important part of a survival pathway; therefore stress resistance signals might target 

SIRT1 protein, thus modifying its function and cellular localization, and inhibiting its 

antitumor activity. Our future purpose is to understand the molecular mechanisms 

governing the dynamics of SIRT1 subcellular localization and the possible role for the 

proteolytically generated nuclear fragment. 

References 

1. Houtkooper RH et al. Nat Rev Mol Cell Biol. 2012 

2. Besnault-Mascard L et al. Oncogene. 2005 


187


Parthenolide induces caspase-independent cell death in 

osteosarcoma, melanoma and breast cancer cells through 

the induction of oxidative stress 

carliSi daniela, d’anneo antonella, lauricella Marianna, 

eManuele Sonia, Martinez roBerta, vento renza, 

teSoriere Giovanni 

Dipartimento di Biomedicina Sperimentale e Neuroscienze cliniche, 

Sezione di Scienze Biochimiche, Università degli Studi di Palermo 

Parthenolide, a sesquiterpene lactone found in European feverfew, is used in 

traditional medicine for its anti-inflammatory activity. In addition, parthenolide has 

been considered as a novel and effective anti-tumor agent because it induces 

cytotoxic effects in several tumor cell lines. 

Our studies demonstrated that parthenolide exerted strong cytotoxic effects in 

osteosarcoma MG63 and melanoma SK-Mel28 cells in culture. Staining with Hoechst 

33342 revealed in most cells after brief periods of treatments (3-5h) chromatin 

condensation and fragmentation, while only few cells were PI-positive. Prolonging 

the treatment (5-14h) PI-positive cells strongly augmented, denouncing the increase 

of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors 

were ineffective, thus suggesting a caspase-independent cell death. The study of 

the mechanism of action provided evidence that treatment with parthenolide rapidly 

stimulated (1-2 h) ROS generation, in particular by inducing activation of extracellular 

signal-regulated kinase1/2 and NADPH oxidase. This event caused depletion of 

thiol groups and glutathione, NF-κB inhibition, JNK activation and cell detachment 

from the matrix. ROS generation together with mitochondrial accumulation of Ca 2+ 

favoured dissipation of Δψm, which appeared primarily determined by the opening 

of the permeability transition pore (PTP), since Δψm loss was partially prevented by 

cyclosporin A, an inhibitor of PTP opening. 

Recently, we focused our attention on MDA-MB231 cells, a very aggressive and poorly 

differentiated breast cancer cell line, which is negative for estrogen receptor alpha. 

Preliminary results suggested that parthenolide induced cell death in these cells with 

a mechanism similar to that demonstrated in osteosarcoma and melanoma cells. 

Interestingly, we demonstrated that in MDA-MB231 cells the effect of parthenolide 

was potentiated by the addition of z-VAD-fmk, a general inhibitor of caspases. Studies 

are in progress to elucidate the mechanism of this interaction which could suggest 

new strategies for the treatment of ER-α negative breast cancer. 


188


Mutant p53 gain of function in human osteosarcoma 

3AB-OS cancer stem cells 

di fiore riccardo, draGo-ferrante roSa, Marcatti Michela, 

vento renza 

Section of Biochemical Sciences, Department of Experimental Biomedicine 

and Clinical Neurosciences, Polyclinic, University of Palermo, 

Via del Vespro 129, 90127 Palermo, Italy. 

Our studies in human osteosarcoma MG63 cells have shown that the inhibition of 

Poly (ADP-ribose) polymerase with 3-aminobenzamide, induces a progressive 

reprogramming of gene expression (1,2) until the irreversible selection of a novel cell 

line. This cell line (termed 3AB-OS) possesses all the required characters established 

for Cancer Stem Cells (3,4). We have also genetically and molecularly characterized 

3AB-OS cells, finding a number of abnormalities congruent with those described in 

pediatric and adult osteosarcomas (manuscript in preparation). 

Now, we have analyzed in MG63 and 3AB-OS cells, TP53 gene (RT-PCR, Methylation- 

Specific-PCR, Fluorescent-in situ-hybridization and Sequencing analyses) and p53 

protein (Western-blot, Immunofluorescence analyses). We have shown that, while in 

MG63 cells TP53 gene is rearranged and does not express mRNA or protein, instead 

both are highly expressed in 3AB-OS cells. Additionally, in 3AB-OS cells TP53 gene is 

mutated, rearranged and in multiple copies, and p53 protein has nuclear localization 

and is stabilized by post-translational modifications (phosphorylation and acetylation). 

Aimed at evaluating whether, in 3AB-OS cells, mutant p53 (mp53) has acquired gain 

of oncogenic functions, we have depleted mp53 by small-interfering-RNA. We have 

shown that p53-silencing induces a reduction of cell proliferation, cell migration and 

apoptosis resistance. Moreover, we have shown (Real-Time-PCR analysis) that p53silencing 

induces the regulation (up/down) of a number of genes i.e., p53 regulators; 

p53, Rb and BCl-2-family members; cell-cycle regulators; invasion and epithelialmesenchymal-transition-associated 

genes; stemness genes. Overall, the results agree 

with an oncogenic role of mp53 and suggest that mp53 could strongly contribute to 

the stemness and chemoresistance characteristics of human osteosarcoma. 

References 

1. De Blasio A et al. Int J Oncol. 2003; 23:1521-1528 

2. De Blasio A et al. FEBS Lett. 2005; 579:615-620 

3. Di Fiore R et al. J Cell Physiol. 2009; 219:301-313 

4. Di Fiore R et al. J Cell Biochem. 2012. doi: 10.1002/jcb.24214 


189


Effects of the injection of pharmacologically active 

microcarriers releasing HGF/IGF-1 and transporting 

adipose-derived stem cells in the infarcted heart 

Savi M. 1 , Bocchi l. 1 , Bonafe’ f. 2,4 , frati c. 3 , karaM J.P. 5 , fiuMana e. 2,4 , 

cavalli S. 3 , MorSelli P.G. 6 , caldarera c.M. 2,4 , Guarnieri c. 2,4 , 

MuScari c. 2,4 , Montero-Menei c. 5 , Stilli d. 1 , quaini f. 4,7 , MuSSo e. 1,4 

1. Dipartimento di Medicina evolutiva e funzionale - Università di Parma 2. Dipartimento di 

Biochimica - Università di Bologna 3. Dipartimento di Patologia e medicina di laboratorio - 

Università di Parma 4. Istituto Nazionale per le Ricerche Cardiovascolari (INRC) - Bologna 

5. INSERM U1066 e Università di Angers, Francia 6. Dipartimento di Scienze Chirurgiche 

Specialistiche e Anestesiologiche, Università di Bologna 

7. Dipartimento di Medicina interna e Scienze Biomediche - Università di Parma 

From large sample size clinical trials, stem cell treatment for cardiac repair appeared so 

far marginally successful. To achieve better results, attention has been paid to several 

factors such as fine tuning of procedural aspects, tailoring of cell type and optimization 

of engraftment techniques. Adipose-Derived Stem Cells (ASCs) can be relatively easily 

harvested, expanded, safely and effectively transplanted to an autologous or allogeneic 

host and have the potential to differentiate into various cell types, including cardiomyocytes. 

ASCs were isolated by human lipoaspirates and stained with DiI to allow their in vivo tracking. 

Pharmacologically active microcarriers (PAMs) are biodegradable and non-cytotoxic poly 

(lactic-co-glycolic acid) microspheres which exhibited therapeutic potential by successfully 

interacting with cells conveyed on their surface and tissue microenvironment. PAMs 

were coated with fibronectin and poly-lysine to improve ASCs adhesion and biological 

function. The Hepatocyte Growth Factor or the Insulin-Like Growth Factor-1 (GFs) were 

encapsulated during the microsphere formulation. We tested the hypothesis that stem cell 

based cardiac treatment via local delivery, mobilization or both can be improved by PAMs. 

We used 44 adult male Wistar rats with one-month old MI and treated with ASCs (n=8), 

ASCs+PAMs (n=7), GFs-releasing PAMs (n=6) ASCs+GFs-releasing PAMs (n=6) or vehicle 

(V, n=6). Eleven rats died in the peri-operative periods. Five additional rats were sham 

operated (SO). Two weeks after treatment, invasive hemodynamic measurements were 

performed and subsequently inducibility of ventricular arrhythmias was assessed during 

programmed stimulation in Langendorff-perfused hearts. Eventually, the hearts were 

subjected to anatomical, morphometric, and immunohistochemical analyses. Preliminary 

data indicate that the largest improvement in cardiac mechanics occurred in ADSC+GFreleasing 

PAM rats which in addition exhibited a significant extent of cell engraftment 

within the infarcted heart. By contrast the same animals trended to be more vulnerable to 

arrhythmias suggesting that caution should be paid on the electrophysiological impact of 

the physical interaction of microspheres with the myocardium. 


190


Role of oxidative stress in chemical hypoxia induced 

apoptosis 

creMonini eleonora 1 , roMani arianna 1 , cervellati franco 2 , 

cervellati carlo 1 , Pavan BarBara 2 , Sticozzi claudia 2 , and 

valacchi GiuSePPe 2-3 

1 Department of Biochemistry and Molecular Biology, University of Ferrara, Italy 

2 Department of Biology and Evolution, University of Ferrara, Italy 

3 Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea 

Proliferative retinopathies are the major cause of blindness in industrialized countries 

and they are characterized by neuronal depolarization, calcium influx and oxidative 

stress (OS). This condition evokes an unbalanced release of pro- and anti-angiogenic 

factors from the retinal pigment epithelium (RPE) towards the pro-angiogenic ones, with 

a consequent aberrant new vessel formation that, together with neurodegeneration, 

causes irreversible visual loss. It is well known that hypoxia and increase of reactive 

oxygen species (ROS) which commonly accompany proliferative retinopathies, alter 

the balance between pro- and anti-angiogenic factors. In the present study we have 

evaluated a possible role of OS in the modulation of hypoxia activated (HA) factors 

that induce apoptosis in human RPE-derived cell line (HRPE). 

After treatment of HRPE cells with 200mM CoCl 2 there was a strong induction of 

HIF1α, confirming that the experimental approach was an acceptable model for invitro 

hypoxia. The cells showed a significantly reduced viability. These data, together 

with the increased expression of VEGF confirmed that the HRPE cells respond to the 

lost of oxygen by increasing cell death (most likely apoptosis as shows by a dramatic 

increase in caspase-3 expression). 

These effects seem to be related to OS with an increased level of F 2 -isoprostane in the 

cellular medium and intra-cellular 4HNE protein adducts. The responses of the HRPE 

cells to the increased OS is the induction of NFkB activity and this was confirmed 

by immunohystochemistry analysis. In addiction the increase of OS in these cells is 

shown by flow cytometry. 

These results provide evidence of the possible mechanistic pathway involved in 

retinopathy, where hypoxic event, which leads to increased OS that via the activation 

of NFkB stimulates the production of VEGF in HRPE cells. Moreover the increase of 

cells mortality suggest that OS will induce apoptosis. 


191


Reversine induces endoreplication only when cell-cycle 

checkpoints are deregulated 

Piccoli Marco 1 , Palazzolo GiacoMo 1 , conforti erika 1 , 

creo PaSquale 1 , PaPini nadia 2 , venerando Bruno 2 , 

tettaManti Guido 1 and anaStaSia luiGi 1 , 2 

(1) Laboratory of Stem Cells for Tissue Engineering, IRCCS Policlinico San Donato, 

San Donato Milanese, Italy; 

(2) Department of Biomedical Sciences for Health, University of Milan, Milan, Italy 

The synthetic purine reversine has been shown to possess a dual activity as it 

promotes de-differentiation of adult cells, including fibroblasts [1], but it also induces 

cell growth-arrest and ultimately cell death of cancer cells. Previous mechanistic 

studies revealed that the molecule is an inhibitor of MEK1 and of non-muscle myosin 

heavy chain (NMMII), and possess a potent inhibitory capacity on MPS1, a crucial 

kinase of the spindle assembly checkpoint (2, 3). However, the apparent selectivity 

of reversine for cancer cells needed further elucidation, especially in the perspective 

of a possible application of the molecule as an anti-cancer drug. On these bases 

we performed a comparative analysis of the effects of reversine on several human 

tumor cells and normal human fibroblasts. Cell-cycle analysis revealed that reversine 

treatment caused the formation of mixed diploid/tetraploid populations in all treated 

cells. However, while normal fibroblasts showed the presence of two populations 

(diploid and tetraploid) in their respective G 1 phases, all cancer cells continued to 

perform aberrant nuclei divisions, without cytokinesis, leading to the irreversible 

formation of multinucleated aneuploid cells that died of mitotic catastrophe. These 

evidences seem to support the hypothesis that reversine treatment could be lethal for 

cells with deregulated cell-cycle checkpoints, as they can’t block endoreplication. In 

fact, chemical and genetical inhibition of p53 in fibroblasts renders them sensitive to 

reversine treatment, and cells died of aberrant endoreplications, analogously to what 

it was observed in cancer cells. 

[1] Anastasia L. et al. Cell Death and Differentiation, 2006, 13:2042-51 

[2] Chen S. et al. PNAS, 2007, 104:10482-7 

[3] Santaguida S. et al. JCB, 2010, 190:73-87 


192


Synergic effects of all-trans retinoic acid in 

limphomonocytes co-cultures on the growth of human 

keratinocytes and GRIM-19 expression 

1 Sardaro nicola, 1 PaPa franceSco, 2 tatullo Marco, 

1 nicaStro annarita, 1 d’oria SuSanna, 1 laiSo Giuliana, B Marrelli 

MaSSiMo, 1 loruSSo Michele, 3 liPPoliS roSa and 1 Scacco Salvatore 

1. Department of Basic Medical Sciences, University of Bari “A. Moro”; 

2. Calabrodental s.r.l., Crotone; 3. IBBE, CNR, Bari. 

Head and neck squamous cell carcinoma (HNSCC) ranks among the most common 

cancers. Cytokines and inflammatory signal pathways appear to be implicated 

in the development of HNSCC as well as other oral tissues preneoplastic lesions 

of unknown origin. Cytokines regulate immunity, inflammation and cell growth and 

include interleukins (ILs), interferons (IFNs), tumor necrosis factors (TNFs), and 

growth factors. Our studies reveal a significant inhibitory effect of all-trans retinoic 

acid on keratinocytes growth. This effect appears to be related to increased levels 

of protein GRIM-19 (Genes associated with Retinoid-IFN-induced Mortality) [Angell 

JE. et al. J. Biol. Chem. 2000, 275:33416-26; Papa F. et al. Int. J. Immun. Pharmacol. 

2007, 20:719-29]. A new experimental approach by co-culturing keratinocytes and 

peripheral blood limphomonocytes shows a singular synergic effect of ATRA treatment 

on inhibition of cell growth. The ATRA effects on the two different cell lines may trigger 

the mitochondrial pathway of apoptosis through enhancement of GRIM-19 actions. 


193


New insights into the mechanism of JNK1 inhibition by 

glutathione transferase P1-1 

caccuri anna Maria 1 , de luca anaStaSia 1 , federici luca 2 , de 

canio Michele 3 , Stella lorenzo 1 

1 Department of Chemical Sciences and Technologies, 

University of “Tor Vergata”, Rome, Italy; 

2 Department of Biomedical Sciences, University of Chieti G D’Annunzio, 

CeSI Center of Excellence on Aging, 66013 Chieti, Italy; 

3 Department of Internal Medicine, University of Rome Tor Vergata, 

Via Montpellier 1, 00133, Rome, Italy. 

The role played by glutathione transferase P1-1 (GSTP1-1) in modulating the c-Jun 

N-terminal kinase (JNK) pathway has been extensively investigated using JNK isoforms 

known to exert opposite effects in the cells. We have expressed the isoform JNK1α2, 

that has been reported to transmit a pro-apoptotic signal, and we have analyzed both 

the phosphorylation level and the activity of this kinase in the presence of GSTP1-1. 

Contrary to what previous studies suggest, we found that GSTP1-1 is able to form 

a complex with the unphosphorylated and inactive JNK1α2 isoform, even in the 

absence of the substrate. We also analyzed the consequences of this interaction on 

the activity of both enzymes. The complex strongly reduced the activation of JNK1α2 

while preserved GSTP1-1 from inactivation. Unexpectedly glutathione (GSH) exerted a 

negative effect on the affinity of GSTP1-1 for JNK1α2, suggesting that the intracellular 

levels of this thiol may allow a fine-tuning of the MAPK signaling pathway. Moreover 

we found that the adduct formed by GSH and the strong GSTP1-1 inhibitor NBDHEX 

abolishes the interaction between the GSTP1-1 and JNK1α2. These data confirm and 

extend at the molecular level previous evidence obtained in tumor cell lines. 


194

Poster 

Session 2 

Friday 28 th 

September 

16:30 - 18:30

Biotecnologie 

e Biochimica 

Industriale 

BIB

Biotecnologie e Biochimica Industriale BIB 1 

Peroxiredoxins for nanotechnology: a biochemical 

approach to produce nanoconductors 

ardini Matteo 1 , anGelucci franceSco 1 , di leandro luana 1 , 

Scotti Stefano 1 , Bellelli andrea 2 , Saccoccia fulvio 2 , 

iPPoliti rodolfo 1* 

1 Department of Life, Health and Environmental Sciences, University of L’Aquila, 

P.le Salvatore Tommasi 1, 67100 Coppito (AQ), Italy 

2 Department of Biochemical Sciences, “Sapienza” University of Rome, 

P.le Aldo Moro 5, 00185 Roma, Italy 

* Corresponding author: Prof. Rodolfo Ippoliti, Department of Life, 

Health and Environmental Sciences, University of L’Aquila, P.le Salvatore Tommasi 1, 

67100 Coppito (AQ), Italy, Phone: +39 0862433286, Fax: +39 0862433273 

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous proteins showing the so-called 

moonlighting behaviour as they are involved in many cellular activities: in normal 

conditions they act as peroxidases able to reduce H 2 0 2 to water; this function causes 

a change of structure from reduced decamer rings to oxidized dimers which in turn 

are reduced back by thioredoxin. Instead, in oxidative stress conditions they switch 

function loosing the peroxidase activity and acquiring molecular chaperone and cell 

signaling activities (Jang et al., 2004); these functions are based on a shift of their 

oligomerization state from single decamer rings to high molecular weight (HMW) 

species such as filaments of stacked rings (Kumsta and Jacob, 2009; Barranco- 

Medina et al., 2009; Moon et al., 2005; Gourlay et al., 2003; Phalen et al., 2006, 

Saccoccia et al., 2012). 

Starting from the study on the structure-function relationships of the HMW species 

of several 2-Cys Prxs, we were able to constitutively induce the formation of long 

self-assembling filaments of stacked decamer rings by protein engineering. Size 

exclusion chromatography and transmission electron microscopy showed that these 

filaments have a tubular structure with length up to 200 nm and are constituted by 

20-30 stacked rings presenting internal and external surfaces which are amenable of 

manipulation. In this framework, these protein nanotubes (PNTs) were exploited for 

assembling metal nanowires, filling their cavity with an array of gold nanoparticles. 

In this work we present the strategy for the production of potential high conducing 

nanowires, possibly suggesting a “green chemistry” approach to produce metallic 

nanoconductors that may be used in nanocircuits and nanodevices. 

II Poster Session 

198


Identification of new thermophilic enzymatic activities for 

hemicellulose conversion 

coBucci-Ponzano Beatrice 1 , Strazzulli andrea 1 , GiGlio roSa 1 , 

iacono roBerta 1 , MaSturzo GiuSePPe 1 , faGnano MaSSiMo 2 and 

Moracci Marco 1 

1 Institute of Protein Biochemistry – Consiglio Nazionale delle Ricerche, 

Via P. Castellino 111, 80131, Naples, Italy. 

2 Dipartimento Ingegneria agraria e Agronomia, Università di Napoli Federico II, 

via Università 100, 80055 Portici (Naples), Italy. 

The use of biomass for energy production is expected to increase in future years and 

the production of biochemicals and biofuels from energy crops arises from the need 

to find alternative sources to fossil fuel. Giant reed (Arundo donax L.) is a perennial 

grass being seen as one of the most promising biomasses for the production of 

lignocellulosic bioethanol of second generation. Enzyme cocktails that hydrolyze plant 

cell wall polysaccharides are a critical component of bioprocessing configurations 

designed to transform lignocellulosic biomass into biofuels. The large variety of 

potential biomass feedstocks and available pretreatments require tailored glycoside 

hydrolase preparations that function optimally under diverse conditions, including 

high temperatures and extreme pH. This suggests that thermophilic prokaryotes 

may be an important source of these enzymes. Our group is involved since long 

time in the identification and characterization of thermophilic glycoside hydrolases 1,2 . 

Here we report two different approaches aimed to discover robust and highly active 

hemicellulose degrading glycoside hydrolases. In the first approach, enrichment 

cultures on A. donax of microorganisms living in the solfataric field of Pisciarelli 

(Naples) was performed on giant reed culms, allowing to reduce the complexity of 

the microbial communities and to select microorganism able to degrade the target 

biomass. In a second approach, suitable thermophilic enzymatic activity belonging to 

glycoside hydrolase families GH10 and GH67 have been identified and characterized. 

Preliminary data will be presented aiming to the design of a chemo-enzymatic process 

for hemicellulose conversion. 

1. Cobucci-Ponzano B., Aurilia V., Riccio G., Henrissat B., Coutinho P.M., Strazzulli A., Padula A., Corsaro 

M.M., Pieretti G., Pocsfalvi G., Fiume I., Cannio R., Rossi M., and Moracci M. J Biol Chem, 2010, 20691- 

20703. 

2. Maurelli L., Giovane A., Esposito A., Moracci M., Fiume I., Rossi M., and Morana A. Extremophiles, 

2008, 689-700. 


199


Identification of novel allosteric chaperones as potential 

therapeutic agents in the treatment of Pompe disease 

ferrara Maria carMina 1 , Porto caterina 2,3 , Meli MaSSiMiliano 4 , 

acaMPora eMMa 3 , avolio valeria 3 , roSa MarGherita 3 , 

coBucci-Ponzano Beatrice 1 , coloMBo GiorGio 4 , andria GeneroSo 3 , 

Parenti Giancarlo 2,3 , Moracci Marco 1 , 

1 Institute of Protein Biochemistry-CNR, Naples; 

2 Telethon Institute of Genetics and Medicine (TIGEM), Naples; 

3 Department of Pediatrics, Federico II University, Naples; 

4 Istituto Chimica del Riconoscimento Molecolare-CNR, Milan. 

The deficiency of the lysosomal enzyme α-glucosidase (GAA) causes a genetic 

metabolic myopathy named Pompe disease (PD). This lysosomal storage disease 

had no approved treatment since 2006 when Enzyme Replacement Therapy 

(ERT), exploiting recombinant enzyme (rhGAA), has been introduced. This therapy 

improves survival, but its efficacy is impaired by several factors. In the recent years, 

Pharmacological Chaperone Therapy (PCT), either alone or in combination with ERT, 

has been proposed as an alternative therapeutic strategy also in PD. PCT, which is 

based on the use of small molecules that assists the correct folding of the defective 

enzymes, is also partially limited because these molecules, being competitive 

inhibitors of target enzymes, might affect their action. 

Here we report the identification of a novel chaperone (N-acetylcysteine, NAC) and 

two related compounds (N-acetyl serine, NAS; N-acetyl glycine, NAG) able to stabilize 

wild type GAA at neutral pH, to enhance the residual activity of mutated GAA, and 

to improve the efficacy of rhGAA used for ERT. The study on rhGAA, supported 

also by a computational biology analysis, on cells and in vivo, indicated that these 

chaperones did not interact with the catalytic domain of GAA and consequently were 

not competitive inhibitors of the enzyme. NAC represents the first allosteric chaperone 

identified so far and, more in general, may provide a new tool for the treatment of 

lysosomal disorders. 

Porto C, Ferrara MC, Meli M, Acampora E, Avolio V, Rosa M, Cobucci-Ponzano B, Colombo G, Moracci 

M, Andria G, Parenti G, Pharmacological enhancement of alpha-glucosidase by the allosteric chaperone 

N-acetylcysteine. Mol. Therapy, 2012, in press 


200


Glycosynthases: novel engineered glycosidases for the 

synthesis of oligosaccharides for biotechnological industry 

coBucci-Ponzano Beatrice 1 , Strazzulli andrea 1 , Bedini eMiliano 2 , 

corSaro Maria Michela 2 , roSSi MoSè 1 and Moracci Marco 1 

1 Institute of Protein Biochemistry – Consiglio Nazionale delle Ricerche, 

Via P. Castellino 111, 80131, Naples, Italy. 

2 Dipartimento di Scienze Chimiche, Università di Napoli Federico II, 

Complesso Universitario di Monte S. Angelo, via Cinthia 4, 80126 Naples, Italy. 

Carbohydrates synthesis is usually hampered by laborious regio- and stereochemical 

controls leading to poor yields and limiting its applicability in industrial biotechnological 

scales. This problem, and the absence of genetic codes as for proteins and nucleic 

acids, has greatly complicated the development of automatic techniques for the 

synthesis of oligo- and polysaccharides. Therefore, glycan/glycosylated compounds, 

despite their crucial role in several biological processes, have been exploited in 

biotechnology in isolated cases, leaving a huge potential market virtually unexplored. 

Carbohydrate active enzymes, namely glycoside hydrolases and glycosyltransferases 

(GH and GT, respectively) are excellent alternative to classical methods for 

carbohydrate synthesis, but suffer of poor yields and selectivity (GH) or are expensive 

and difficult to isolate (GT). Glycosynthases, a class of mutant GH engineered in their 

active site, represent an interesting alternative producing carbohydrates with almost 

quantitative yields 1,2 . 

Our group is active in this field since more than a decade and has now accumulated 

a collection of α- and β-glycosynthases. Here we will describe how glycosynthases 

demonstrated their plasticity in different processes and specific examples of β-gluco, 

α-galacto-, and α-fucosynthases syntheses of oligosaccharides of biotechnological 

interest whose production is challenging with conventional methods 3,4 will be 

presented. 

1. Cobucci-Ponzano B., Strazzulli A., Rossi M., and Moracci M. Glycosynthases in Biocatalysis. Adv. Synth. 

Catal, 2011, 353, 2284-2300 

2. Cobucci-Ponzano B, Moracci M. Glycosynthases as tools for the production of glycan analogs of natural 

products. Nat. Prod. Rep. 2012, 29, 697-709. 

3. Cobucci-Ponzano B, Conte F, Bedini E, Corsaro M.M, Parrilli M, Sulzenbacher G, Lipski A, Dal Piaz F, 

Lepore L, Rossi M, Moracci M, Chem Biol, 2009, 16, 1097-108. 

4. Cobucci-Ponzano B, Zorzetti C, Strazzulli A, Carillo S, Bedini E, Corsaro MM, Comfort DA, Kelly RM, 

Rossi M, Moracci M, Glycobiology, 2011, 21, 448-56. 


201


Transglutaminase-Reticulated Hydrocolloid Based Films as 

Effective Coatings of Baked and Fried Foods 

roSSi-Marquez Giovanna, di Pierro ProSPero, eSPoSito Marilena, 

Mariniello loredana, and Porta raffaele 

Department of Food Science, University of Naples “Federico II”, Portici (Napoli) Italy 

Whey protein/pectin edible films were prepared under experimental conditions for 

obtaining the maximum complexation between the two hydrocolloid components. 

The experiments were carried out in the absence or presence of transglutaminase, 

an enzyme able to crosslink proteins by forming ε(γ-glutamyl)lysine isopeptide bonds 

(1,2). Our findings indicated the formation at pH 5.1 (pHc) of transglutaminasecatalyzed 

crosslinks among soluble ionic whey protein/pectin complexes and that 

the obtained hydrocolloid films good mechanical as well as water vapor and oxygen 

barrier properties. Film application by dip-coating technique allowed to create a layer 

on the surface of both baked (biscuits) and fried (doughnuts) foods able to influence 

moisture transfer and oil uptake. In fact, we observed a significant reduction of water 

absorption by biscuits which prevented the loss of their crispness, keeping normal the 

hardness parameters and improving the springiness and chewiness ones. Moreover, 

since it is well known that fried-foods contain high amounts of fat and contribute 

in determining high blood cholesterol levels,. we used the whey protein/pectin films 

obtained in the presence of transglutaminase to coat fried food with the aim to reduce 

their water loss during the frying process. Our results showed that coated doughnuts 

exhibited decreased fat uptake, reduced fat content and improved oil barrier index, 

thus indicating that the applied coating effectively prevented water migration from 

the core to the crust during frying by filling the void that normally allows the fat to 

enter inside the food. In conclusion, our data demonstrate that the introduction 

of transglutaminase-catalyzed crosslinks among whey protein/pectin complexes 

obtained at pHc confers to the hydrocolloid film a very good performance in acting as 

water barrier for both bakery and fried foods. 

References 

1. Porta R. et al. Crit. Rev. Food Sci. Nutr. 51, 223 

2. Porta R. et al. J. Biotechnol. Biomat., DOI 10.4172/2155-952X.1000102e 


202


HARV Bioreactor for 3D culture 

of human mesenchymal stem cells 

Penolazzi letizia 1 , laMBertini eliSaBetta 1 , vecchiatini renata 1 , 

GaMBari roBerto 1 , naStruzzi claudio 2 and roBerta Piva 1 

1 Department of Biochemistry and Molecular Biology, and 

2 Department of Pharmaceutical Sciences University of Ferrara, 44121 Ferrara, ITALY 

The SYNTHECON Rotary Cell Culture System (RCCS) with HARV (High Aspect 

Rotary Vessels) technology is the premiere device that efficiently creates an 

environment that enables cell cultures to grow into complex, sophisticated three 

dimensional tissue models, in vitro. The RCCS is currently being used in a wide range 

of research projects where the production of high fidelity tissue models or the long 

term maintenance of tissue explants is required. 

Little is so far known about the changes in phenotype and gene expression occurring 

when cells are transferred from 2D to 3D culture systems. This kind of information 

is particularly important in tissue engineering, and regenerative medicine because 

cells are frequently placed in 3D scaffolds for therapeutic applications. As we are 

interested in the development of innovative systems for the repair of bone defects, 

we specifically asked whether a regime of dynamic fluid flow represented by the 

RCCS bioreactor affected the osteogenic potential of human mesenchymal stem cells 

(hMSCs) entrapped in static 3D system represented by alginate microbeads. 

We used hMSCs from different sources including umbilical cord Wharton’s jelly, 

bone marrow and periodontal ligament, after standard immunophenotyping. We 

demonstrated that Slug transcription factor expression is essential for osteogenesis. 

Comparing 2D vs 3D culture conditions hMSCs osteogenic potential, cell interaction, 

osteoblastic markers expression and deposition of mineralized matrix improved when 

alginate-entrapped hMSCs were cultured in RCCS bioreactor. In addition, when 

Polymeric Micelles (self-assembling colloidal systems) were chosen to deliver in 

culture medium osteogenic inducers (instead of traditional solubilization with DMSO 

which is known to be cytotoxic), an increase of osteogenic potential was observed. 

Our data highlight the interest about the combination between hMSCs and specific 

scaffolds, and are important prerequisite for an effective bone tissue engineering. 


203

Bioinformatica 

BNF

Bioinformatica BNf 1 

Novel tools for the determination of 

intrinsic disorder in proteins 

di doMénico toMàS, walSh ian, Martin alBerto J.M., 

toSatto Silvio c.e. 

Department of Biology, University of Padova, Padova, Italy 

Disordered protein regions are key to the function of numerous processes within an 

organism and to the determination of a protein’s biological role. The scant available 

experimental annotations suggest the existence of different disorder flavors. 

We have recently developed an ensemble of protein disorder predictors called ESpritz 

[1]. These are based on bidirectional recursive neural networks and trained on three 

different flavors of disorder, including a novel NMR flexibility predictor [2]. ESpritz 

can produce fast and accurate sequence-only predictions, annotating entire genomes 

in the order of hours on a single processor core. Alternatively, a slower but slightly 

more accurate ESpritz variant using sequence profiles can be used for applications 

requiring maximum performance. Two levels of prediction confidence allow either to 

maximize reasonable disorder detection or to limit expected false positives to 5%. 

ESpritz performs consistently well on the recent CASP9 data. 

We also provide a centralized source for data on different flavours of disorder in 

protein structures, MobiDB [3], expanding already existing sources. In addition to 

the DisProt and PDB x-ray structures, we have added experimental information from 

NMR structures [2] and five different disorder predictors. These are combined into 

a weighted consensus disorder used to classify disordered regions into flexible and 

constrained disorder. Users are encouraged to submit manual annotations through 

a submission form. MobiDB features experimental annotations for 17,285 proteins, 

covering the entire PDB, and predictions for the SwissProt database, with 565,200 

annotated sequences. Depending on the disorder flavour, 6-20% of the residues are 

predicted as disordered. 

References 

[1] Walsh, I., Martin, A.J., Di Domenico, T. and Tosatto, S.C. (2012) Bioinformatics 28, 503-9. 

[2] Martin, A.J., Walsh, I. and Tosatto, S.C. (2010) Bioinformatics 26, 2916-7. 

[3] Di Domenico, T., Walsh, I., Martin, A.J. and Tosatto, S.C. (2012) Bioinformatics 28, 2080-1. 


206


Ras effectors, ERK and PI3K, differentially regulate two 

distinct migration features: speed and directionality 

SePe leandra 1,2 , ferrari Maria carla 1,2 , fioretti franceSca 1 , and 

Paolella Giovanni 1,2 

1 Dipartimento di Biochimica e Biotecnologie mediche, 

Universita’ degli Studi di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy 

2 Ceinge Biotecnologie Avanzate, Via G. Salvatore 486, 80145 Napoli, Italy 

Cell migration is essential in many physiological and pathological processes, such 

as wound healing and metastasis formation. The involvement of Ras and other 

molecules in these processes has been extensively studied, but their effect on specific 

movement features may be better characterized by coupling dynamic microscopy 

with quantitative evaluation and statistical analysis. Quantitative analysis of timelapse 

movies, by circular statistics, allowed to obtain parameters descriptive of cell migration 

under different experimental conditions and to highlight the role of Ras in specifically 

promoting directional migration, which appears to be the main factor responsible 

for accelerated wound healing in-vitro. Ras promoted directional movement may 

be restricted to the ERK downstream pathway, as inhibition of its activation blocks 

directional migration, while inhibition of PI3K pathway reduces cell speed but not 

directionality, and is not sufficient to prevent accelerated wound closure. 

An in-depth analysis of fibroblast movement was attempted by fitting different 

motion models to experimental data, obtained from wound healing assays on 

parental NIH3T3 and transformed NIHRasV12 fibroblasts, under standard conditions 

as well as in presence of ERK and PI3K inhibitors. Fibroblast movement appears 

to be well described by superdiffusive models, as the squared displacements scale 

super-linearly with time. In particular, persistent random walk model fits well to most 

experimental conditions with different degrees of persistence for 3T3 and NIHRas 

cells. A method was developed to estimate speed and persistence parameters by 

using Levenberg-Marquard algorithm and used to study the effect of inhibitors on 

speed and persistence under different experimental conditions. 


207


BAR-PIG: a database of the pig proteome with structural 

and functional statistically validated annotation 

PioveSan daMiano 1,2 , Martelli Pier luiGi 1,2 , fariSelli Piero 1,3 , 

Profiti GiuSePPe 1,4 , fontaneSi luca 5 and caSadio rita 1,2,4 . 

1 Bologna Biocomputing Group, 

2 Department of Biology, 

3 Department of Computer Science, 

5 Department of Agro-Food Science and Technology, Sezione di Allevamenti 

Zootecnici, University of Bologna, 

4 Health Science and Technologies-ICIR, University of Bologna, Italy. 

Here we describe a database of pig proteins, comprising a total of 35,381 sequences 

collected by merging 19,576 and 23,118 chains retrieved respectively from 

UniProtKB, one of the major resources of protein sequences freely available, and 

from Ensembl, the genome database for eukaryotic species. Some 90% of these 

chains are poorly annotated and their existence is inferred automatically by sequence 

alignment towards the entire protein universe database. Given the relevance of the 

pig proteome in different studies, including human complex maladies, a statistical 

validation of the annotation is required for a better understanding of the role of specific 

genes and proteins in the complex networks underlying biological processes in the 

animal. BAR-PIG is a database in which some 21,793 sequences are endowed with 

a statistically validated annotation. Statistical validation is determined by adopting a 

cluster-centric annotation procedure that allows different types of annotation from 

structure to function and when possible to both structure and function [1]. Each 

sequence in the database can be associated with a set of statistically validated Gene 

Ontologies (GO) of the three main routes (Molecular Function, Biological Process, 

Cellular Component), with Pfam functional domains and when possible with a cluster 

HMM model that allows building of the three dimensional structure of the protein. A 

database search allows some statistics demonstrating the enrichment in both GO and 

Pfam terms of the pig proteins as compared to the UniProtKB annotation. Alternatively 

the search is possible on the basis of structural information, allowing retrieval of all the 

pig sequences with the same structural characteristics. The data base is available at 

http://bar.biocomp.unibo.it/pig. 

1. Piovesan D, Martelli PL, Fariselli P, Zauli A, Rossi I, Casadio R: BAR-PLUS: the Bologna Annotation 

Resource Plus for functional and structural annotation of protein sequences. Nucleic Acids Res 2011, 

39:W197-W202. 


208


Identification of Single Nucleotide Variants in 

Gastrointestinal Stromal Tumor KIT/PDGRFA Wild Type 

(WT GIST) with Massively Parallel Sequencing 

indio valentina(1,2), taSco Gianluca(1), 

Martelli Pier l(1), caSadio rita(1,2) 

Pantaleo Maria a(2), aStolfi annaliSa(2), forMica Serena(2), 

Paterini Paola(2), BiaSco Guido(2) 

(1)Biocomputing Group, Bologna Computational Biology Network and 

(2)Center for Cancer Research “G.Prodi”, University of Bologna, Italy 

Massively parallel sequencing allows to discover new potential target genes in rare 

diseases, such as WT-GIST, for which the causative genomic alterations still remain 

unknown. 

Whole transcriptome RNA-sequencing was performed using a 75 bases pairedend 

strategy on tumor samples of two young adult patients affected by WT-GIST. 

The sequences obtained were mapped on human reference genome and the single 

nucleotide variants (SNVs) were called and compared with dbSNP and 1000genomes 

databanks in order to highlight the novel variants with respect to the common human 

variability. After SNV calling and validation, the effect of the variations at the protein 

level was predicted with SNPs&GO, a tool suited to identify disease-associated residue 

substitution on the basis of the protein sequence and its function, benchmarked as 

top scoring by Thusberg J. et al. (2010). This approach highlighted in both patients 

common disease-related mutations at the level of the A subunit of the succinate 

dehydrogenase of the mitochondrial inner membrane (SDHA) as previously reported in 

Pantaleo M.A. et al.(2011). Based on these results, all SDH subunits were sequenced 

(with Sanger method) in 32 additional WT-GIST cases, confirming the presence of SDH 

variations in 23% of the analyzed patients. Moreover also private variants were found 

in the two young adults. Among the private disease-related variants we identified in 

one patient a K1775E substitution in the myosin heavy chain 9 (MYH9) and a R206H 

substitution in the triosephosphate isomerase 1 (TPI1). The second patient carried a 

V815M mutation in a oxoglutarate dehydrogenase-like protein (OGDHL). Interestingly 

all the mutations that are labeled disease-associated with SNPs&GO in agreement 

with SIFT, are also promoting protein destabilization according to a predictor suited 

to evaluate protein destabilization upon mutation starting from the protein sequence 

(I-Mutant3). The results are confirmed by protein structure computational analysis. 

Sanger sequencing performed on both tumor and peripheral blood samples highlights 

that private mutations were germline heterozygous. 


209


The prediction of organelle targeting peptides in eukaryotic 

proteins with Grammatical Restrained Hidden Conditional 

Random Fields 

Martelli Pier luiGi 1,2 , indio valentina 1,3 , SavoJardo caStrenSe 1,4 , 

fariSelli Piero 1,4 , caSadio rita 1,2,3 

1 Biocomputing Group, 2 Department of Biology, University of Bologna, 

via San Giacomo 9/2, 40126 Bologna, Italy. 

3 ”Giorgio Prodi” Interdepartmental Center for Cancer Research, 

University of Bologna, via Massarenti, 40138 Bologna, Italy. 

4 Department of Computer Science, University of Bologna, 

via Mura Anteo Zamboni 7, 40126, Bologna, Italy. 

Targeting peptides are the most important signal controlling the import of nuclear 

encoded proteins into mitochondria and plastids. In the lack of experimental 

information, their prediction is an essential step when proteomes are annotated, for 

inferring both the localization and the sequence of mature proteins. 

We developed TPpred a new predictor of organelle targeting peptides based on 

Grammatical Restrained Hidden Conditional Random Fields, a recently introduced 

machine-learning tool well suited to solve labeling problems (Fariselli et al., 2009) 

TPpred is trained on a non-redundant dataset of proteins where the presence of a 

target peptide was experimentally validated, comprising 297 sequences. When tested 

on the 297 positive and some other 8010 negative examples, TPpred outperforms 

available methods in both accuracy and Matthews correlation index (96% and 0.59, 

respectively). Given its very low false positive rate (3.0%), TPpred is therefore well 

suited for large-scale analyses at the proteome level. We predicted that about 4% to 

9% of the sequences of human, Arabidopsis thaliana and yeast proteomes contain 

targeting peptides and are therefore likely to be localized in mitochondria and plastids. 

TPpred predictions correlate to a good extent the experimental annotation of the 

subcellular localization, when available. TPpred was also trained and tested to predict 

the cleavage site of the organelle targeting peptide on this task the average error of 

TPpred on mitochondrial and plastidic proteins is 7 and 15 residues, respectively. This 

value is lower than the error reported for other methods currently available. 

Fariselli, P., Savojardo C, Martelli PL, Casadio R (2009) Grammatical-Restrained Hidden Conditional 

Random Fields for Bioinformatics applications. Algorithms Mol. Biol., 4, 13. 


210

enzimologia 

e Regolazione 

Metabolica 

eRM

Enzimiologia e Regolazione Metabolica ERM 1 

Functional characterization of an extracellular form of 

purine nucleoside phosphorylase (PNP) in brain cells 

PetraGnani n, cicchitti S, zuccarini M, natale S, lannutti a, 

Giuliani P, caciaGli f. 

Department of Experimental and Clinical Sciences, 

University G. D’Annunzio of Chieti-Pescara, Chieti 

Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme that catalyses the 

reversible phosphorolysis of purine nucleosides into their respective nucleobases, 

playing a key role in the purine salvage pathway. 

Its deficiency is associated with T-cell lymphopenia, severe immunodeficiency and 

several neurological dysfunctions. PNP expression/activity resulted to be modified 

during several pathological conditions such as inflammation, ischemia and cancer 

progression. Indeed, alterations in serum levels of PNP metabolites have been 

reported in patients affected by pancreatic adenocarcinoma. 

In the brain, PNP has been mainly localized in glial cells. So far, no membrane 

localization has been reported in contrast with most of the enzymes involved in purine 

metabolism that are also present in the external surface of plasma membranes (ectoenzyme) 

where they regulate the catabolism of extracellular purines released from 

cells. 

Therefore, in this study, we evaluated the presence and the specific activity of PNP in 

purified membrane preparations from cultures of different rat brain cells. 

PNP has been detected by western blot, confocal and electron microscope analysis 

while in order to evaluate the enzymatic activity, we have developed a new and highly 

sensitive method using an HPLC connected to an on-line radiochemical detector to 

measure the level of de novo formed radiolabeled guanine from labeled guanosine 

added to the medium. 

Our results indicated the presence of the enzyme in purified membrane preparations 

deriving from glial cells. In the same preparations, a specific activity was detected and 

the resulting curves fitted well the Michaelis-Menten equation. 

Interestingly, we found the enzyme also in the culture medium of glial cells. PNP was 

released from cells at rest in a time-dependent manner and a dose-dependent increase 

of the released enzyme was found after cells exposure to potassium, ATP and P2X7 

receptor agonist. Among glial cells, microglial cells seem to be more efficient in the 

enzyme secretion compared to astrocytes. 


212


ROLE OF SIRT1 SIGNALLING PATHWAYS IN THE ADAPTIVE 

RESPONSE TO OxIDATIVE STRESS AND AGING IN MOUSE 

OOCYTES 

di eMidio Giovanna 1 , falone Stefano 1 , vitti Maurizio 1 , 

Santonocito Manuela 2 , vento Marilena 3 , di Pietro cinzia 2 , 

d’aleSSandro anna Maria 1 , aMicarelli fernanda 1 and 

tatone carla 1 

1 Department of Life, Health and Environmental Sciences, University of L’Aquila, 

Via Vetoio, 67100 L’Aquila. 

2 Dipartimento Gian Filippo Ingrassia, Sezione di Biologia, Genetica, Genomica 

Cellulare e Molecolare Giovanni Sichel, Università degli Studi di Catania. 

3 Servizio di PMA/Azienda Ospedaliera Cannizzaro, Catania. 

SIRT1 is a class III NAD+ dependent histone deacetylase belonging to the sirtuin family, 

which plays an important role in stress responses and delays changes associated with 

aging and oxidative stress. SIRT1-dependent deacetylation of the transcription factor 

FOXO3a protects cells from reactive oxygen species by up-regulating scavenging 

enzymes, such as superoxide dismutase (MnSOD). 

In the present work we investigated SIRT1-related pathways in mouse oocytes by 

analysing changes in gene expression of SIRT1, FOXO3a and MnSOD under oxidative 

stress or aging, and by considering the possible regulatory role of miR132, a small 

non-coding RNA. 

After RT-qPCRs, we observed that immature (GV) oocytes expressed higher level 

of SIRT1 when compared to mature (MII) oocytes. In GV oocytes exposed to H 

2 

O 

2 

and processed for RT-qPCR after different recovery times, a significant 7-fold upregulation 

of SIRT1 was detected after 1.5 h recovery, along with an up-regulation of 

FOXO3a and MnSOD. Expression analysis of miR132 and SIRT1 showed a significant 

anti-correlation between the levels of these transcripts in control and stressed 

oocytes. Lower SIRT1 levels were also observed in young oocytes when compared 

to oocytes from reproductively old mice. Finally, GV oocytes matured in presence of 

SIRT1 inhibitors EX527, sirtinol and nicotinamide presented a significant inhibition of 

polar body formation. 

Our results provide the first evidence that: 1) SIRT1 is involved in the adaptive 

response to oxidative stress in mouse oocytes and is differentially regulated during 

reproductive aging; 2) miR132 might be involved in post-transcriptional regulation 

of SIRT1; 3) inhibition of SIRT1 negatively affects in vitro completion of meiosis; 4) 

SIRT1 is likely to be relevant to cellular processes occurring during oogenesis and 

folliculogenesis rather than in fertilization and early embryo development. These 

observations suggest that manipulation of SIRT1 and related microRNAs may help to 

establish new strategies to promote or rescue oocyte competence. 


213


Matrix metalloproteases (MMPs) activity in pancreatic juice 

after surgical resection 

SulPizio Sara 1 , franceSchini nicola 1 , locatelli Marcello 2 , 

carlucci GiuSePPe 2 , BaSSi claudio 3 , MaScetta GiuSePPe 3 , 

cotelleSe roBerto 4 , innocenti Paolo 4 , SelvaGGi federico 4 

1 Department of Biotechnological and Applied Clinical Sciences, 

University of L’Aquila, L’Aquila, Italy 

2 Department of Pharmaceutical Sciences, “G. d’Annunzio” 

University, Chieti-Pescara, Chieti, Italy 

3 Department of Surgery, University of Verona, Verona, Italy 

4 Unit of General and Laparoscopic Surgery, “G. d’Annunzio” 

University, Chieti-Pescara, Chieti, Italy 

Matrix metalloproteases (MMPs) are a family of endopeptidases with the ability of 

degrading extracellular matrix components. MMPs have been shown to play a critical 

role in the mechanisms of wound healing. The role of MMPs in the development 

of pancreatic fistula is still not well-defined. The incidence of pancreatic fistula 

varies between 3% after pancreatic head resection and up to 30% following distal 

pancreatectomy and this represents a severe complication. 

We designed this study to investigate the activity of MMPs in human pancreatic 

juice. Twenty-one patients that underwent pancreatic resection with postoperative 

drainage have been observed. The majority of patients received infusion of octreotide. 

Pancreatic fistula was diagnosed in 8 cases. Zimography assay is used to analyse the 

expression of MMPs in human pancreatic fluid collected after surgery. Gelatin was the 

specific substrate used to study MMPs. 

We identified the activity of MMPs in the pancreatic juice collected after surgical 

resection of patients with pancreatic adenocarcinoma. Zimography analysis showed 

gelatinolytic bands at 72 kDa (gelatinase A) at 92, 130, and 240 kDa (gelatinase B). 

MMP-9 and MMP-2 activities were slightly enhanced in a subgroup of patients with 

pancreatic fistula. 

MMP-2 and MMP-9 activities in pancreatic juice represent a useful biomarker for 

pancreatic fistula diagnosis and for its appropriate pharmacological treatment. 

• Muhs BE, Patel S, Yee H, Marcus S, Shamamian P. Increased matrix metalloproteinase expression and 

activation following experimental acute pancreatitis. J Surg Res. 2001 Nov;101(1):21-8. 

• Bassi C, Dervenis C, Butturini G, Fingerhut A, Yeo C, Izbicki J, Neoptolemos J, Sarr M, Traverso W, 

Buchler M; International Study Group on Pancreatic Fistula Definition. Postoperative pancreatic fistula: 

an international study group (ISGPF) definition. Surgery. 2005 Jul;138(1):8-13. Review. 


214


Characterization of C-4 demethylation complex of 

cholesterol biosynthesis 

Brozzu andrea 1 , oliaro-BoSSo SiMonetta 1 , zonari daniele 1 , 

viola franca 1 , Poirier donald 2 , cunninGhaM david 3 , herMan Gail 3 , 

Jokela heili 4 , Poutanen Matti 4 , Balliano Gianni 1 

1 Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino; 

2 Department of Molecular Medicine, Research Center and Laval University, 

Quebec City, Canada; 

3 Center for Human and Molecular Genetics, the Research Institute at Nationwide 

Children’s Hospital, Columbus; 

4 Department of Physiology, Institut of Biomedicine, University of Torku, Finland 

Cholesterol biosynthesis is a highly oxygen-consuming pathway characterized by 

two sharply different sections: (i) the actual assembly line that forms the non-cyclic 

triterpene oxidosqualene (pre-squalene section), and (ii) the steroid-shape conferring 

section which first cyclizes the oxidosqualene to lanosterol and then remodels it to 

cholesterol (post-squalene section). The most dramatic remodelling steps (and also 

the most oxygen consuming) of the post-squalene section consist in the removal of 

three methyl groups from lanosterol, one from position 14 and two from position 4, 

catalyzed by the C-14 demethylation and C-4 demethylation complexes, respectively. 

The latter complex is constituted by three enzymes (C4-sterol methyloxidase, C4sterol 

decarboxylase [NSDHL] and 3-ketoreductase [HSD17B7]) and a scaffold protein 

(C14ORF1), which is supposed to tether the enzymatic proteins of the complex. In 

humans, severe inborn post-squalene cholesterol disorders are caused by mutations 

of two of the four proteins of the complex, C4-sterol methyloxidase and C4-sterol 

decarboxylase [1], and deficiencies in the other two are suspected to cause other so 

far unknown cholesterol disorders [2]. 

To unravel the interactions among the proteins of the C-4 demethylation complex 

as well as the interaction of the complex with the remainder of the post-squalene 

section of the pathway, we adopted both the inhibition and the gene-deficiency based 

approach. To this aim, (i) different mammalian cell lines were treated with inhibitors 

putatively active on 3-ketoreductase, (ii) mammalian cell lines and embryos bearing, 

each, a single gene-deficiency in the demethylation complex were assayed for their 

post-squalene enzymatic activities. 

Results confirm the pivotal role of the C-4 demethylation complex within the postsqualene 

section of cholesterol biosynthesis, pointing to it as a direct or indirect 

“guilty factor” in some cholesterol disorders. 

References 

[1] F.D. Porter et al. J. Lipid Res. 2011, 52: 6-34 

[2] H. Jokela et al. Endocrinology 2010, 151: 1884-1892. 


215


A mutation in yeast oxidosqualene cyclase (Erg7) makes it 

independent of 3-ketoreductase activity (Erg27) 

layer JacoB v. 1 , BarneS Brett M. 1 , yaMaSaki yuJi 1 , BarBuch 

roBert 2 , li linGtao 3 , taraMino Silvia 4 , oliaro-BoSSo SiMonetta 4 , 

Balliano Gianni 4 , and Bard Martin 1 

1 Department of Biology, Indiana University-Purdue University Indianapolis, 

Indianapolis, IN 46202, USA; 

2 Advion Bioanalytical Labs, A Quintiles Co., 

Purdue Research Park, Indianapolis, IN 46241, USA; 

3 Department of Pathology, University of Utah School of Medicine, 

Salt Lake City, UT 84132, USA; 

4 Dipartimento di Scienza e Tecnologia del Farmaco, 

Università degli Studi di Torino, Turin, Italy 

In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-ketoreductase, 

results in a concomitant loss of upstream enzyme, Erg7p, an oxidosqualene 

cyclase [1,2]. However, this phenomenon occurs only in fungi, as mammalian Erg27 

orthologues are unable to rescue Erg7p activity. In this study, an erg27 mutant 

containing the mouse ERG27 orthologue was isolated that was capable of growing 

without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an 

accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain was 

crossed to a wild type and daughter segregants showed an accumulation of squalene 

epoxides as well as ergosterol indicating that the mutation entailed a leaky block at 

ERG7. Upon sequencing the ERG7 gene an A598S alteration was found in a conserved 

alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation 

that mimics Erg27p binding causing it to associate with a macromolecular complex 

responsible for ergosterol synthesis within the ER. This mutation, while decreasing 

Erg7 activity allows residual Erg7 activity to function in the absence of the Erg27 

3-keto reductase enzyme. 

Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized 

combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 

demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid 

and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with 

minimal accumulation of squalene epoxides. These results indicate retention of Erg7p 

in the ER increases its activity and suggests a novel method of regulation of ergosterol 

biosynthesis. 

References 

[1] C. Mo et al. BBA 2003, 1633: 68-74 

[2] S. Taramino et al. BBA 2010, 1801:1232:1237 


216


High-fat diet feeding and streptozotocin treatment of 

wild-type and TIMP3 deficient mice reveal significant 

metabolic alterations related to insulin resistance 

roSSi claudia 1, 2 , ciavardelli doMenico 1, 3 , zucchelli Mirco 1, 2 , 

conSalvo ada 1, 2 , cavalera Michele 4 , Mavilio Maria 4 , Marzano 

valeria 4, 5 , di ilio carMine 2 , Sacchetta Paolo 2 , federici MaSSiMo 4 , 

4, 5 

urBani andrea 


2 Experimental and Clinical Science Department, “G. d’Annunzio” 

University, Chieti-Pescara, Italy; 

3 Faculty of Engineering, Architecture, and Motor Science, 

Kore University, Enna, Italy; 

4 Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy; 

5 IRCCS S. Lucia Foundation, Rome, Italy. 

The tissue inhibitor of metalloproteinase TIMP3, a stromal protein that restrains the 

activity of proteases and receptors, is reduced in insulin resistance and in inflammatory 

metabolic disorders such as diabetes mellitus and atherosclerosis. In order to 

understand the role of this protein in metabolic alterations linked to insulin resistance, 

wild-type (WT) and TIMP3 knock-out (TIMP3-/-) mice were fed a high-fat diet (HFD) 

for 16 weeks to induce insulin resistance, or were treated with streptozotocin (STZ, 

50 mg/kg injected intra-peritoneally for 5 consecutive days) to induce diabetes and 

monitored for 12 weeks. Whole blood aminoacids and acylcarnitines profiles were 

investigated by direct infusion mass spectrometry (DIMS) analysis, through the 

addition of isotopically labelled internal standards for each analyte of interest prior to 

the extraction, according to the principle of isotope dilution internal standardization. 

Whole blood was collected on filter paper cards as dried blood spots, particularly 

suitable for small volume samples. Significant differences in blood levels of free 

carnitine, some acylcarnitines and some aminoacids were found between genotypes 

and in response to diet and streptozotocin treatment. Interestingly, HFD feeding and 

streptozotocin treatment result in similar yet more evident metabolic alterations in 

TIMP3-/- mice compared with control mice, indicating TIMP3 results in reduced 

ability of mice to cope with insulin resistance. Similar changes in aminoacids and 

acylcarnitines blood levels have been reported in diabetic patients. Our data therefore 

suggest that loss or reduced TIMP3 activity might contribute to the metabolic 

alterations of diabetic patients and therefore contribute to the patients prognosis. 


217


Integrated approaches for the functional validation of 

potential mitochondrial regulators 

BrioSchi e. 1 , cerMenati G. 1 , Saez e. 2 , caruSo d. 1 , creStani M. 1 , 

de faBiani e. 1 , Mitro n. 1 

1 Dipartimento di Scienze Farmacologiche e Biomolecolari, 

Università degli Studi di Milano, Milano. 

2 Department of Chemical Physiology Skaggs Institute for Chemical Biology, 

The Scripps Research Institute, La Jolla, CA. 

Mitochondria are the organelle committed to metabolic energy production required 

for all cellular functions. Because of their ubiquitous presence, their density and 

function have a significant impact on whole-body metabolism. Consequently, 

finding new mitochondrial regulators could expand our knowledge on cell biology 

and biochemistry. Two cDNA libraries, accounting for 70% of known human and 

murine genes, were screened in Hek293 cells in a high throughput fashion by using a 

reporter system where the luciferase gene was under the control of the mitochondrial 

transcription factor A (mTFA) promoter. Positive clones were then validated for their 

ability to modulate mitochondrial density and function using staining assays. 140 

positive cDNAs were confirmed to increase these parameters. Consequently, the 

purpose of this study was to further characterize these candidates in C2C12 cells, a 

skeletal muscle cell line. 

The 140 genes were classified for their biological and molecular function using 

bioinformatic tools; this analysis revealed that 40% clones are involved in metabolic 

and cellular processes. Based on these findings we generated a priority list of 22 

candidates. Skeletal muscle is one of the main tissues involved in the energy 

production and highly rich in mitochondria, for these reasons the 22 hits identified 

were characterized in C2C12. The cDNAs were tested for their ability to increase 

mitochondrial DNA content, a hallmark of mitochondrial biogenesis, and to affect 

the expression of mTFA, the main transcription factor involved in mitochondria 

development. Most cDNAs increased both parameters. To evaluate the functional 

relevance of these observations the oxygen consumption rate of cell transfected with 

the cDNAs was estimated. We found that 9 hits significantly increased both basal and 

uncoupled respiration. 

The understanding of the role of new mitochondrial regulators could help identify 

new targets for the development of future interventions for the treatment of metabolic 

diseases associated to mitochondrial dysfunction. 


218


Aldose reductase inhibition: a new strategy to control the 

enzyme activity in hyperglycemic conditions 

del-corSo 1 antonella, di BuGnoeliSa 1 , MoSchini roBerta 1 , 

caPPiello Mario 1 , BaleStri franceSco 1 , Sartini Stefania 2 , 

la-Motta concettin 2 a, da-SettiMofederico 2 , and Mura uMBerto 1 

1 Biochemistry Unit at the Department of Biology, University of Pisa. 

2 Department of Pharmaceutical Sciences, University of Pisa – Italy. 

Aldose reductase (AR) is an NADPH-dependent reductase (EC 1.1.1.21), which acts 

on a variety of hydrophilic as well as hydrophobic aldehydes. It is currently defined 

as the first enzyme in the so-called polyol pathway. This metabolic route transforms 

glucose into fructose through the subsequent action of AR and an NAD + -dependent 

sorbitol dehydrogenase. An exaggerated flux of glucose through the polyol pathway 

with a subsequent sorbitol accumulation, was originally proposed as the basic event 

in the aethiology of secondary diabetic complications. For decades this has meant 

targeting the enzyme for a specific and strong inhibition. However, the ability of AR 

to reduce toxic alkenals and alkanals, which are products of oxidative insults, poses 

the question of whether AR might be better classified as a detoxifying enzyme, thus 

raising doubts as to the unequivocal advantages of inhibiting the enzyme. 

In this study evidence is presented of an effective action on AR activity through an 

intra-site differential inhibition. A new generation of aldose reductase “differential” 

inhibitors (ARDIs) is proposed, which are able to interdict enzyme activity on 

glucose and in general on hydrophilic substrates, without affecting the reduction of 

hydrophobic toxic aldehydes 


219


Tissue and salivary Nicotinamide N-methyltransferase: 

promising results for early and non-invasive diagnosis of 

oral squamous cell carcinoma. 

Sartini d 1 , Pozzi v 1 , MorGanti S 1 , rocchetti r 1 , ruBini c 2 , 

Santarelli a 1 , lo Muzio l 3,4 , eManuelli M 1 . 

1 Department of Clinical Sciences, Polytechnic University of Marche, Ancona, Italy. 

2 Department of Biomedical Sciences and Public Health, 

Polytechnic University of Marche, Ancona, Italy. 

3 Department of Surgical Sciences, University of Foggia, Foggia, Italy. 

4 I.R.C.C.S. – C.R.O.B., Rionero in Vulture, Potenza, Italy 

Oral squamous cell carcinoma (OSCC) is the eighth most common cancer worldwide. 

Despite recent progress in the diagnosis and therapeutic modalities of OSCC, 

the 5-year survival rate is about 50% around the world and has not improved in more 

than two decades. Sensitive and specific biomarkers for OSCC, which can be used to 

screening high-risk patients and to predict clinical outcomes and response to therapy, 

are therefore urgently needed. 

In the present study, we focused on the expression of Nicotinamide N-Methyltransferase 

(NNMT), which catalyses the N-methylation of nicotinamide, pyridines and other 

structural analogs, playing an important role in the biotransformation and detoxification 

of many xenobiotics (1, 2). We analysed NNMT enzyme activity in paired tumour and 

non-tumour tissues obtained from patients affected with OSCC. In order to explore 

the potential suitability of NNMT expression levels determination for early and noninvasive 

diagnosis of OSCC, NNMT protein levels were evaluated in saliva samples 

from patients with OSCC and healthy subjects. 

Results obtained showed that the levels of NNMT activity are significantly higher in 

OSCC compared with adjacent normal oral mucosa. Interestingly, oral epithelium 

surrounding tumor of unfavourable cases (N+) seems to display higher activity levels 

compared to that of favourable OSCCs (N0). In addition, Western blot analyses 

indicated an upregulation of salivary NNMT in patients with OSCC. 

This study shows a marked increase in NNMT catalytic activity in OSCC and suggests 

that adjacent normal tissue of unfavourable cases seems to change toward cancer. 

Moreover, results obtained from analysis performed on saliva samples support the 

hypothesis that NNMT could represent a potential biomarker for early and noninvasive 

diagnosis of oral cancer. 

1. Sartini D, Santarelli A, Rossi V, Goteri G, Rubini C, Ciavarella D, Lo Muzio L, Emanuelli M. Mol Med, 2007. 

2. Peng Y, Sartini D, Pozzi V, Wilk D, Emanuelli M, Yee VC. Biochemistry, 2011. 


220


SIMULTANEOUS SINGLE-SAMPLE DETERMINATION 

OF NMNAT ISOZYMES ACTIVITIES IN MODEL MOUSE 

TISSUES 

cialaBrini lucia 1 , Mori valerio 1 , orSoMando GiuSePPe 1 , 

aMici adolfo 1 , Mazzola franceSca 1 , aGoStinelli SaMuele 1 , 

ruGGieri Silverio 2 , conforti laura 3 , coleMan Michael P. 4 

and MaGni Giulio 5 

1 Dept. of Clinical Sciences (DISCO), Section of Biochemistry, and 

2 Dept. of Nutritional, Environmental and Agricultural Sciences (D3A), 

Polytechnic University of Marche, Ancona, Italy; 

3 School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, 

Nottingham NG7 2UH, UK; 

4 The Babraham Institute, Cambridge CB22 3AT, UK; 

5 School of Biology and Biotechnology, University of Camerino, Italy. 

Mammalian Nicotinamide Mononucleotide Adenylyltransferase (NMNAT, EC 2.7.7.1) 

isozymes catalyse a key reaction of NAD biosynthesis in different cellular compartments. 

We here developed a novel assay for simultaneous determination of their activity in 

the mouse as a model organism. In particular, the assay was tailored to the Wallerian 

degeneration slow (Wld S ) mouse, a model for several NAD-biosynthesis-related 

peripheral neuropathies, in which local NMNAT activity plays a key role in preserving 

axon integrity and function. This spontaneous mutant possesses, alongside the three 

endogenous NMNATs, an additional, catalytically-equivalent chimeric enzyme, WldS, 

that alone determines protection of injured axons through a gain-of-function, i.e. 

complementing essential NMNAT2 activity loss. Presently, the functional relationship 

between endogenous NMNATs and mutant Wld S has been inferred mainly from protein 

quantification experiments, an approach limited by the low quality of current antibodies, 

and intrinsically unable to evaluate possible post-translational events. The hereby 

described method, enabling direct activity measurements, is intended to fill this gap. 

Suitable assay conditions were initially assessed by exploiting the metal-ion 

dependence of isozyme forms recombinantly expressed in bacteria, and further tested 

after their mixing in vitro. The individual isozyme contributions to total NAD synthesis 

in the complex mixture was calculated from measured reaction rates under selected 

assay conditions, by generating a system of linear equations that was solved through a 

substitution matrix calculus. Final assay validation was achieved in a tissue extract by 

comparing the activity and expression levels of individual isozymes, considering their 

distinctive catalytic efficiency. The proposed method was next applied to both liver 

and brain extracts from wild-type and Wld S mouse, showing its reliability for evaluating 

the rate of compartmented NAD synthesis occurring via multiple endogenous NMNAT 

isozymes, as well as for the functional assessment of the NMNAT-like WldS chimera. 


221


ROLE OF PROTEOLYTIC ENZYMES IN THE DEGRADATIVE 

ACTIVITY ASSOCIATED TO PERIPROSTHETIC 

OSTEOLYSIS 

ianni andrea 1 , Marolda Giulio 2 , zoccali carMine 2 , 

carnicelli veronica 1 , franceSchini nicola 1 

1 Dept. of Biotechnological and Applied Clinical Sciences, 

University of L’Aquila, Via Vetoio, 67100 L’Aquila, Italy. 

2 Regina Elena National Cancer Institute, Orthopaedic Division, 

Via Elio Chianesi 53, Rome, Italy. 

Background: Periprosthetic osteolysis is a common complication in prosthetic 

implantations, and probably represents the most important pathological process that 

negatively influences implant survival. This event, as reported by some authors, seems to be 

caused by biological and mechanical factors and is characterised by massive loss of bone, 

probably due to imbalanced metabolism, that renders revision surgery substantially more 

complex. To underline the role of proteases in the osteolytic mechanism, we investigated 

the presence and the activity of different matrix metalloproteases (MMPs) and cathepsins 

in periprosthetic fluids. Comparison was made with synovial fluids drawn from patients 

affected by not inflammatory diseases. 

Method: Periprosthetic fluids were submitted to gelatin zimography, western blotting 

analysis and spectrofluorimetric assays using specific synthetic substrates. 

Results: Gelatin zimography showed a complex pattern of MMPs activities with high levels 

of MMP-2 and MMP-9. Spectrofluorimetric assays were performed to evaluate MMP-13 

and cathepsin K activities. Western blotting confirmed the presence of MMP-13, cathepsin 

K, and cathepsin B. Synovial fluids drawn by patients affected by not inflammatory diseases 

are characterised by lower concentrations of MMP-2,9, and -13 and cathepsins K and B. 

Discussion: 

• Fluids analysis showed a complex proteolytic pattern characterised by the presence and 

high activity of different enzymes namely MMP-2,3,9,13 other than cathepsin B and K. 

• The contemporary presence of such enzymes suggest that their activity as well as fluid 

pressure or the presence of particles is relevant to osteolytic process. 

• Protein pattern and enzymatic activities identification could be useful to identify new 

diagnostic and prognostic biomarkers to control osteolysis progression and efficacy of 

therapeutic strategy. 

References 

1. Rossi P. et al., L’osteolisi periprotesica, Giornale Italiano di Ortopedia e Traumatologia, 2011; 37(4): 

310-317. 

2. Beck R. T. et al., Review of periprosthetic osteolysis in total joint arthroplasty: an emphasis on host 

factors and future directions, Journal Orthopaedic Research, 2011; 30(4): 541-6 


222


NADPH-dependent association of the pentose phosphate 

pathway dehydrogenases in human neutrophils 

Proietti d’eMPaire lucia, ura Blendi, trentini aleSSandro, 

dallocchio franco and hanau Stefania 

Dipartimento di Biochimica e Biologia molecolare, Università di Ferrara 

It has been reported that glucose-6-phosphate dehydrogenase (G6PD) and 

6-phosphogluconate dehydrogenase (6PGD) of human neutrophils can migrate 

from cytosolic localization and interact with other cellular components [1, 2]. We 

have investigated the effect of NADPH on the apparent molecular weight of the 

two dehydrogenases by sucrose density gradient centrifugation. In the absence of 

NADPH the G6PD shows a main peak with an apparent molecular weight >> 200 

kDa, and only minor contributions corresponding to molecular weights ≈ 200 and 

100 kDa, the expected MW for the tetrameric and dimeric enzyme. The behaviour of 

6PGD is very similar, however MW of the main peak appears slightly lower than that 

observed for G6PD. The presence of NADPH in the sucrose gradient changes the 

sedimentation pattern, suppressing the high MW peak. The same experiments were 

performed with neutrophils previously stimulated by LPS. The presence of the two 

dehydrogenases in the high MW region was decreased, however there are still present 

high MW forms that disappear in the presence of NADPH. Analysis of the high MW 

fractions by 2D electrophoresis evidences the presence of at least 20 spots that are 

absent in the presence of NADPH. The amount of protein observed in the absence 

of NADPH cannot be simply related to the association with the dehydrogenases, that 

are present at very low concentration, and indicates that NADPH levels regulate the 

association between cytosolic proteins. 

[1] Baillet A., Xu R.,Grichine A., Berthier S., Morel F., Paclet M-H. The FASEB Journal 2011, 25, 2333-2343. 

[2] Kindzelskii A. L., Ueki T., Michibata H., Chaiworapongsa T., Romero R., Petty H. R. J. Immunol. 2004,172, 

6373-6381. 


223


xanthine dehydrogenase processes retinol to retinoic acid 

in human thyroid epithelial cells 

taiBi Gennaro 1 , nicotra concetta Ma 2 , Gueli Maria concetta 1 

1 Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), 

Università degli Studi di Palermo. 

2 Centro di Oncobiologia Sperimentale (C.OB.S.), Palermo. 

Retinoic acid is considered to be the active metabolite of retinol, able to control 

proliferation and differentiation of epithelia. Retinoic acid biosynthesis has been widely 

described with the implication of multiple enzymatic activities but our understanding 

of their biological function or regulation in the cell is limited. In our previous study (1) 

we have evidenced that milk xanthine oxidase (XO) is capable to oxidize all-transretinol 

bound to CRBP to all-trans-retinaldehyde and then to all-trans-retinoic acid. 

Afterwards, we have evaluated the biosynthetic pathway of retinoic acid in a human 

mammary epithelial cell line (HMEC) (2) in which xanthine dehydrogenase (XDH) is 

expressed, and we have reported the demonstration of a novel retinol oxidation 

pathway that in the HMEC cytoplasm directly leads to retinoic acid. In this work, new 

data from human thyroid epithelial cells (HTEC) supporting the biological role of XDH in 

retinoic acid biosynthesis are given. Particularly, we report that the same biosynthetic 

retinoic acid pathway is also expressed in the HTEC primary cultures. After partial 

protein purification, the enzyme was identified as XDH by immunoassay, by its ability 

to oxidize xanthine to uric acid and its sensitivity to the oxypurinol inhibitory effect. 

We have observed that in this thyroid epithelial cell line, CRBP(s) support the catalytic 

system that, as well as in human mammary epithelial cells, directly oxidizes t-ROL to 

t-RA. Furthermore, it was also observed that XDH activity was undetectable on both 

retinol and purines after polyclonal XO/AO antibody treatment. 

1. Taibi G. and Nicotra CMA. Xanthine oxidase catalyzes the oxidation of retinol. J Enz Inhib Med Chem 

2007; 22(4): 471-476. 

2. Taibi G., Di Gaudio F., Nicotra CMA. Xanthine dehydrogenase processes retinol to retinoic acid in human 

mammary epithelial cells. J Enz Inhib Med Chem 2008; 23(3): 317-327. 


224

Glicobiologia 

GLI

Glicobiologia GlI 1 

Intracellular protein O-GlcNacylation regulates cell 

microenvironment 

deleoniBuS Sara, viGetti davide, viola Manuela, karouSou evGenia, 

de luca Giancarlo and PaSSi alBerto 

Dipartimento di Scienze Chirurgiche e Morfologiche, Università dell’Insubria, 

via J.H. Dunant 5, 21100 Varese 

INTRODUCTION: Large body of evidence supports the idea that microenvironment 

plays a critical role in several pathologies including atherosclerosis and cancer. The 

amount of hyaluronan (HA) often reflects the progression of the diseases as it promotes 

neo-angiogenesis, cell migration and inflammation. In this work, based on human 

smooth muscle cell model, we studied the intracellular regulation of HA synthesis 

at molecular level. The hexosamine biosynthetic pathway (HBP) may increase the 

concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNac) leading to 

an increase of HA synthesis. In this study we addressed the issue trying to shed light 

on HA synthesis regulation. 

RESULTS: The flux through the HBP in the regulation of HA biosynthesis in human 

primary aortic smooth muscel cells (AoSMCs) was studied, as UDP-GlcNac is 

the donor of GlcNac for O-GlcNacylation by which the GlcNac attaches to ser/ 

thr residues via an O-linked glycosidic bond. We found that the inhibition of 

O-GlcNacylation strongly reduced HA production whereas treatments that induced 

protein O-GlcNacylation increased HA secretion. Gene expression studies done by 

quantitative RT-PCR revealed that Hyaluronan synthse 2 (HAS2) mRNA was the most 

sensible to O-GlcNacylation and accumulated after its induction. Although factors 

governing constitutive HAS 2 transcription are to be elucidated, we found that the 

transcription regulator YY1 activates HAS2 expression after O-GlcNacylation. Using 

IP and recombinant 6myc-HAS2 we demonstrate that HAS2 is O-GlcNacylated and 

indentify the residue involved. Finally, as cell migration and adhesiveness are critical 

factors for neointima formation and progression, we quantified AoSMCs motility 

and monocytes binding and found that O-GlcNacylation increased cell invasion and 

inflammatory cell recruitment. 


226


Lysosome-to-plasma membrane transport of cell surface 

glycohydrolases β-hexosaminidase and β-galactosidase is 

mediated by transcription factor EB 

MaGini aleSSandro 1 , Polchi alice 1 , urBanelli lorena 1 , 

tancini Brunella 1 , ercolani luiSa 2 , Polidoro Mario 1 

and eMiliani carla 1 

1 Department of Experimental Medicine and Biochemical Sciences, 

University of Perugia, Perugia, Italy. 

2 Department of Biochemistry, Biology and Genetics, Polytechnic 

University of Marche, Ancona, Italy. 

Eukaryotic cell surface is coated with glycan structure linked to glycoproteins 

and glycosphingolipids which exert important biological functions and play a key 

role as signaling molecules in both physiological andpathological processes. The 

glycoconjugates rearrangement is determined by a complex metabolic pathway 

involving biosynthesis, catabolism and intracellular trafficking. Nonetheless, it is now 

clear that a specific role is played by several cell surface-associated glycohydrolases 

which can act directly at the plasma level to modify cell glycocalyx. a recent work, 

in which the association of fully processed glycohydrolases β-hexosaminidase (Hex) 

and β-galactosidase (Gal) toplasma membrane lipid microdomains has been provided 

[1], we speculated on the existence of a lysosome-to-plasma membrane transport 

pathway mediating the translocation of lysosomal membrane-associated enzymes to 

the cell surface [1,2,3]. To test this hypothesis we transfected Hek 293 cells by using a 

transcription factor EB (TFEB) expressing vector. Recently, it has been demonstrated 

that TFEB overexpression induces autophagy, lysosome biogenesis, up-regulation 

of lysosomal genes expression, and leads to clearance of storage material in several 

lysosomal storage disorder cell models by promoting lysosomal exocytosis [4]. Stable 

TFEB overexpression in Hek 293 cells, significantly increased lysosomal Hex and Gal 

activities and triggered their recruitment on cell surface lipid microdomains. Taken 

together these observations suggest up-regulation of the lysosomal system due to 

TFEB overexpression is mirrored by a lysosomal glycohydrolases recruitment to the 

plasma membrane, where they may be involved in glycosphingolipids oligosaccharide 

modification processes define the curvature properties of specific areas of the cell 

surface. 

[1] Magini A. et al. (2012) Biochimie 94, 684-94 

[2] Mencarelli S. et al. (2005) FEBS Letters 579, 5501-06 

[3] Magini A. et al. (2009) Biosci. Rep.28, 229-37 

[4] Medina DL. et al. (2011) Dev Cell.21, 421-30 

Acknowledgments: Work supported by ELA Foundation (Agreement n. 2011-037C1B) 


227


ANTITHETIC ACTION OF HYALURONAN 6-MER IN 

UNDIFFERENTIATED AND DIFFERENTIATED SH-SY5Y 

CELLS 

Scuruchi Michele 1,2 , avenoSo anGela 2 , Santoro enza 1 , 

d’aScola anGela 2 , naStaSi Giancarlo 2 , caMPo Salvatore 2 , 

SPina edoardo 1 , calatroni alBerto 2 and caMPo GiuSePPe Maurizio 2 

1 Department of Clinical and Experimental Medicine, University of Messina, Messina, 

Italy. 

2 Department of Biomedical Sciences and Morphological and Functional Images, 

University of Messina, Messina, Italy. 

Small hyaluronan (HA) fragments are able to induce inflammation by stimulating both 

CD44 and toll-like receptor 4 (TLR-4). CD44 and TLR-4 stimulation activates NF-kB 

nuclear translocation that in turn induces pro-inflammatory intermediates production 

. Up-regulation of CD44 and TLR-4 was also reported during neuronal inflammation in 

course of Alzheimer disease as well as in Dementia with Lewy bodies. Although the 

pro- inflammatory effect of HA small oligosaccharides has been reported in several 

cell lines such as fibroblasts, chondrocytes and synoviocytes, little is known about 

such effect in neuronal cells. The aim of this study was to investigate the effects of 

small HA oligosaccharides (HA 6-mers) treatment on both undifferentiated ,human 

neuroblastoma, and differentiated, neuron-like, SH-SY5Y cells. Messenger-RNA 

levels for TLR-2, TLR-4, CD44, TNF-alpha, iNOS as well as synphilin-1 and alpha- 

synuclein, both specific markers of Parkinson’s disease, were measured by PCR Real 

Time in 6-mer HA treated and untreated SH-SY5Y cell cultures . The results obtained 

showed an antithetic function of HA 6-mer in relation to the type of cells receiving 

the treatment . HA 6-mer treatment produced a down-regulation of all investigated 

parameters in undifferentiated SH-SY5Y cells, while it produced an up-regulation of 

the same parameters in differentiated SH-SY5Y cells, which is in line with the wellknown 

pro inflammatory effect of HA 6-mer . Therefore, these data, suggest that HA 

6-mer treatment could exhibit two antithetic modulatory effects: 

a) The known stimulatory effect on TLR-2, TLR-4 and CD44 receptors followed by 

inflammatory intermediates activation via NF-kB, in differentiated SH-SY5Y cells. 

b) A singular down-regulation of all above listed parameters, suggesting a regulatory 

mechanism by the cancerous cell in attempt to reduce any onset of the inflammatory 

cascade. 

The mechanism of such anti-inflammatory response induced by HA 6-mer in 

undifferentiated SH-SY5Y cells will be further investigated. 


228


Sialidase NEU3 is activated under hypoxia and protects 

skeletal muscle cells from apoptosis through the activation 

of the EGFR signaling pathway and HIF-1α 

ScarinGi raffaella 1,2 , Piccoli Marco 2 , PaPini nadia 1,2 , cirillo 

federica 2 , conforti erika 2 , BerGante Sonia 1,2 , trinGali criStina 

1,2 , Garatti andrea 2 , venerando Bruno 1,2 , Menicanti lorenzo 2 , 

tettaManti Guido 2 and anaStaSia luiGi 1,2 

(1) Department of Biomedical Sciences for Health, University of Milan, Milan, Italy; 

(2) Laboratory of Stem Cell for Tissue Engineering, IRCCS Policlinico San Donato, 

San Donato Milanese (Milan), Italy. 

Sialidase NEU3, a key enzyme in ganglioside metabolism, is activated in cultured 

skeletal muscle cells (C2C12) under hypoxic conditions. NEU3 up-regulation 

stimulates the EGFR signaling pathway, which activates the hypoxia inducible factor 

(HIF-1α), resulting in a final increase of cell survival and proliferation. In the same 

cells, stable over-expression of sialidase NEU3 significantly enhances cell resistance 

to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible 

to apoptosis. These data support the working hypothesis of a physiological role 

of sialidase NEU3 in protecting cells from hypoxic stress, and may suggest new 

directions in the development of therapeutic strategies against ischemic diseases, 

particularly of the cerebro-cardiovascular systems. 


229


ISOLATION AND DIFFERENTIATION OF STEM CELLS: 

SEARCHING FOR NEW MARKERS 

BerGante Sonia 1 2 , PaPini nadia 2 , creo PaSquale 1 , torretta enrica 2 , 

Gelfi cecilia 2 , nadia SeSSareGo 3 , iBatici adalBerto 3 , venerando 

Bruno 2 , tettaManti Guido 1 and anaStaSia luiGi 1 2 . 

(1) Laboratory of Stem Cells for Tissue Engineering IRCCS Policlinico San Donato, 

San Donato Milanese, Italy; 

(2) Departement of Biomedical Sciences of Health, University of Milan, Italy. 

(3) Istituto Clinico Humanitas IRCCS, Rozzano, Italy. 

Stem cells are potential therapeutic agents that offer great promises in regenerative 

medicine. Mesenchymal stromal cells (MSCs) are the most studied adult stem cells, 

as they can be isolated from almost any tissue, they show a good self-renewal 

capacity in vitro and they also possess good “plasticity”1. Oddly, MSCs are identified 

and defined by a combination of markers that are not distinctive, as they are shared 

by other cells including fibroblasts. Therefore, pure populations of MSCs cannot 

be isolated, as they are always contaminated by other adult cells that often do not 

possess stem cell plasticity. Thus, it would be very desirable to discover novel cellsurface 

markers that would allow to discriminate MSCs from other cells. 

In this direction, we focused our attention on glycosphingolipids (GSLs), a family of 

lipids composed of a ceramide backbone and a sugar head-group found in the outer 

leaflet of the plasma membrane2, as possible surface markers for the characterization 

and isolation of human bone marrow MSCs (BMMSC). Moreover, we wanted to 

investigate their possible role in the differentiation process. 

We investigated the GSLs pattern of human BMMSCs from different donors by HPTLC 

and mass spectrometry. We found that they synthesize a-series gangliosides and GD3, 

while other gangliosides including GD2 could not be detected, although erroneously 

identified in a previous published paper based on poor antibody selectivity3. On the 

other hand, we could identify different subpopulations of BMMSCs characterized by 

the expression of specific gangliosides. Moreover, we observed that GSLs pattern of 

BMMSCs significantly changes during cell differentiation toward osteoblasts, and it 

could be used to identify and characterize the differentiation status of the cell. 

1. Jiang, Y. et al., Nature 2002 418: 41-49 

2. Martinez, C. et al., Blood 2007 109(10): 4245-4248 

3. Suila, H. et al., J. Mol. Cell. Biol. 2011 99: 99-107 


230


CANT1 mutations in Desbuquois dysplasia cause a defect 

in proteoglycan synthesis 

Monti luca 1 , huBer céline 2 , de leonardiS faBio 1 , tenni ruGGero 1 , 

forlino antonella 1 , cetta GiuSePPe 1 , Bertoli Marta 2 , 

fradin Mélanie 2 , le Merrer Martine 2 , le Goff carine 2 , 

corMier-daire valérie 2 and roSSi antonio 1 

1 University of Pavia, Dept. Molecular Medicine, Pavia, Italy; 

2 University Paris Descartes, Dept. Genetics and INSERM U781, 

Hôpital Necker Enfants Malades, Paris, France. 

Desbuquois dysplasia (DD) is an autosomal recessive chondrodysplasia characterized 

by antenatal and postnatal growth retardation, multiple dislocations and advanced 

carpal ossification. Two forms of DD have been described on the basis of the presence 

(type 1) or absence (type 2) of characteristic hand deformities. Studying DD type 1 

families, mutations in the Calcium-Activated Nucleotidase 1 gene (CANT1) have been 

identified (Huber C et al (2009) Am J Med Genet, 85, 706-710 and Nizon M et al (2012) 

Hum Mut Apr. 26.). 

CANT1 is a calcium activated nucleotidase that preferentially hydrolyzes UDP, 

but its function as well as its exact cellular localization is yet unknown. DD shares 

phenotypic features with other chondrodysplasias characterized by defects in 

cartilage proteoglycan sulfation. For this reason, we hypothesized that CANT1 

may also be involved in proteoglycan synthesis. To test whether CANT1 deficiency 

interfere with the availability of UDP-sugars needed for proteoglycan synthesis, 

fibroblasts from two DD patients homozygous for the p.R300H and p.P245RfsX3 

mutations respectively, and four controls were double labeled with [ 35 S]sulfate and 

[ 3 H]glucosamine. In the patient cells glycosaminoglycan (GAG) synthesis was almost 

normal under basal conditions, but significant reduced GAG synthesis was observed 

in presence of β-D-xyloside, a compound which enhances synthesis and secretion of 

chondroitin and dermatan sulfate chains acting as a chain initiator. Furthermore gel 

filtration chromatography on Superose 6 of GAGs released from newly synthesized 

proteoglycans after β-elimination demonstrated that GAG chains were shorter 

compared to the controls. Hyaluronic acid synthesis which occurs in the plasma 

membrane, a different compartment from proteoglycans, was within normal values 

supporting the involvement of CANT1 in the ER/Golgi compartment. 

These data suggest that CANT1 plays a role in proteoglycan and GAG metabolism and support its 

involvement in the endochondral ossification process. 

Work supported by Telethon- Italy (grant no. GGP11079). 


231


SOD MIMIC MnTM-2-PyP(5+) REDUCES INFLAMMATION 

INDUCED BY DEGRADED HYALURONAN IN CHONDROCYTES 

STIMULATED WITH Fe(II) PLUS ASCORBATE 

d’aScola anGela, avenoSo anGela, Scuruchi Michele, 

naStaSi Giancarlo, Micali antonio, Puzzolo doMenico, 

calatroni alBerto, caMPo Salvatore, caMPo GiuSePPe Maurizio. 

Department of Biomedical Sciences and Morphological and Functional Images, 

School of Medicine, University of Messina, 

Policlinico Universitario, 98125 – Messina, Italy 

During pathological conditions, oxidative burst generates hyaluronan (HA) 

fragmentation with consequent increase in small HA oligosaccharide levels. These 

fragments are able to stimulate inflammatory response, in different cell types, by 

activating the CD44 and both the toll-like receptors 4 (TLR-4) and 2 (TLR-2). CD44 and 

TLRs stimulation in turn activates the NF-kB that induces the production of several 

pro-inflammatory mediators that amplify and perpetuate inflammation. We aimed 

to study the effect of the antioxidant SOD mimic synthetic manganese porphyrin, 

Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP5+) in 

preventing HA degradation in mouse articular chondrocytes stimulated with iron (II) 

plus ascorbate. 

Fe (II) plus ascorbate stimulation induced oxidative burst confirmed by high levels of 

hydroxyl radical and peroxynitrite production, and increase in both lipid peroxidation 

and HA degradation. HA fragments produced high mRNA expression and the related 

protein production of CD44, TLR-4 and TLR-2, NF-kB activation and a significant 

up-regulation of the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), 

interleukin-1beta (IL-1beta), and other pro-inflammatory mediators, such as matrix 

metalloprotease 13 (MMP-13) and inducible nitric oxide synthase (iNOS). Treatment of 

cells with MnTM-2-PyP(5+) was able to attenuate the oxidative burst, HA degradation 

and NF-kB activation, markedly decreased CD44, and TLRs mRNA expression and 

the related protein synthesis, as well as the up-regulated inflammatory mediators 

production. Addition to cells of HA blocking peptide PEP-1 (HABP) significantly 

reduced all the inflammatory parameters up-regulated by Fe (II) plus ascorbate, and 

strengthens MnTM-2-PyP(5+) activity. These data suggest that HA degradation plays 

a key role in the earliest inflammatory response of cartilage and antioxidants could be 

a useful tool to prevent the amplification of the mechanism. 


232


COMBINED TREATMENT WITH HA INHIBITOR PEP-1 AND 

A SELECTIVE ADO A2 RECEPTOR AGONIST REDUCES 

INFLAMMATION IN ExPERIMENTAL ARTHRITIS 

calatroni alBerto, avenoSo anGela, d’aScola anGela, 

naStaSi Giancarlo, Micali antonio, Puzzolo doMenico, 

PreStiPino vera, Scuruchi Michele, caMPo Salvatore, caMPo GiuSePPe M. 

Department of Biomedical Sciences and Morphological and Functional Images, School 

of Medicine, University of Messina, Policlinico Universitario, 98125 – Messina, Italy 

Abstract 

Rheumatoid arthritis (RA) is a chronic and systemic disorder characterised by the 

progressive destruction of articular cartilage and bone. Previous investigations 

reported that the degradation of native highly polymerized hyaluronan (HA) into 

small oligosaccharides may play a role in the development and progression of RA. 

Inflammatory responses occur by modulating the toll-like receptor 4 (TLR4) and the 

TLR2, and CD44, a natural HA receptor. It was also shown that the adenosine A2 

receptor (A 2A R) plays role in arthritis exerting anti-inflammatory activity. The stimulation 

of TLR4, TLR2, and CD44 converge to activate NF-kB which, in turn, stimulates 

the production of pro-inflammatory cytokines and other detrimental mediators. In 

contrast, the stimulation of the A 2A R inhibits NF-kB activation. The aim of this study 

was to investigate the effect of a combined treatment using the HA inhibitor Pep-1 

and a selective A 2A R agonist (CV-1808) in collagen-induced arthritis (CIA) in mice. 

Arthritis was induced via intradermal injection of an emulsion containing bovine type II 

collagen in complete Freund’s adjuvant. Mice were treated with Pep-1 plus CV-1808 

intraperitoneally daily for 20 days. 

CIA increased TLR4, TLR2, CD44 and A 2A R mRNA expression and the related 

proteins in the joint cartilage of arthritic mice. Significantly increased levels of both 

mRNA and related protein were observed for tumor necrosis factor alpha (TNF-α), 

interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) 

and inducible nitric oxide synthase (iNOS). Pep-1 together with CV-1808 treatment 

significantly reduced CIA damage and decreased all the up-regulated biochemical 

parameters. Reduction in biochemical parameters was supported by histological 

analysis, and synovial fluid HA levels. 

These results suggest that by acting, through apposite tools, on the HA degradation 

pathways and, at the same time with other opportune specific tools, on adenosine 

pathways, it would be possible to develop anti-inflammatory strategies that ameliorate 

arthritis symptoms more effectively. 


233


ACTIVATION OF ADENOSINE 2A RECEPTOR REDUCES 

INFLAMMATORY RESPONSE VIA EPAC AND PARTLY VIA 

PKA IN MOUSE SYNOVIAL FIBROBLASTS STIMULATED 

WITH HYALURONAN OLIGOSACCHARIDES 

Giancarlo naStaSi, anGela avenoSo, anGela d’aScola, 

Michele Scuruchi, antonio Micali, doMenico Puzzolo, 

alBerto calatroni, Salvatore caMPo, GiuSePPe Maurizio caMPo. 

Department of Biomedical Sciences and Morphological and Functional Images, 

School of Medicine, University of Messina, Policlinico Universitario, 

98125 – Messina, Italy 

Abstract 

Small hyaluronan (HA) fragments induce inflammatory response in different cell types 

by primarily stimulating the toll-like receptor 4 (TLR-4). This interaction activates a 

series of intermediates that at the end activate the nuclear factor kappaB (NF-kB). NFkB 

in turn induces the transcription of inflammation cytokines such as tumor necrosis 

factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) that prime inflammation. 

On the contrary, the purine nucleoside adenosine (ADO) interacting with the highaffinity 

A 2A receptor activates a pathway leading to increased cAMP levels. The cAMP, 

in turn activates the protein kinase A (PKA) that is able to inhibit NF-kB, hence exerting 

anti-inflammatory activity. However, as a novel cAMP physiological mediator named 

exchange proteins activated by cAMP (EPAC) may also act on NF-kB inhibition, we 

aimed to investigate the involvement of EPAC in 4-mer HA oligosaccharide-induced 

inflammatory response in mouse synovial fibroblasts (NSF). 

Treatment of NSF with 4-mer HA increased TLR-4, TNF-alpha, and IL-1beta mRNA 

expression and the related protein production, as well as NF-kB activation. Addition 

to NSF, previously stimulated with 4-mer HA oligosaccharides, of ADO significantly 

reduced NF-kB activation, TNF-alpha and IL-1-beta expression. The pre-treatment 

of NSF with cAMP and/or PKA and/or EPAC specific inhibitors significantly inhibited 

the anti-inflammatory effect exerted by ADO. In particular the EPAC inhibitor reduced 

ADO effect to a major extent than PKA inhibitor. These results mean that both PKA 

and EPAC pathways are involved in ADO-induced NF-kB inhibition, although EPAC 

seems to be more involved than PKA. 


234


AGE-β 2 -glycoprotein I activates dendritic cells and induces 

adaptive immunity in patients with antiphospholipid 

antibody syndrome 

1 Buttari BriGitta, 1 ProfuMo eliSaBetta, 2 caPozzi antonella, 

3 facchiano franceSco, SaSo luciano, 2 Sorice Maurizio, 

1 riGanò rachele 

1 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore 

di Sanità, Rome, Italy; 2 Department of Experimental Medicine, Sapienza University of 

Rome, Rome, Italy; 3 Department of Hematology, Oncology and Molecular Medicine, 

Istituto Superiore di Sanità, Rome, Italy; 4 Department of Physiology and Pharmacology 

“Vittorio Erspamer”, Sapienza University of Rome, Rome, Italy. 

In chronic disorders related to endothelial cell dysfunction the plasma β 2 -glycoprotein 

I (β 2 GPI), plays a role as a target antigen of pathogenetic autoimmune responses. 

Information is still lacking to clarify why β 2 GPI triggers autoimmunity. Possible posttranslational 

modification of the protein could be implicated such as glycation. The 

aim of our study was to explore whether a glycated form of β 2 GPI is able to interact 

and activate immature monocyte-derived dendritic cells (iDCs) from healthy human 

donors and is the target antigen of humoral response in patients with antiphospholipid 

antibody syndrome (APS). SDS-PAGE and spectrofluorimetric analysis indicated 

that β 2 GPI incubated with glucose was sugar-modified, and such modification likely 

consisted of AGE formation (AGE-β 2 GPI). AGE-β 2 GPI caused phenotypical and 

functional maturation of iDCs involving the activation of mitogen-activated protein 

kinase-p38 and ERK and nuclear factor kb. It also induced on DCs a significant upregulation 

of RAGE, the receptor for AGEs. Evidence of RAGE involvement comes 

from blocking experiments with an anti-RAGE monoclonal antibody, confocal 

analysis and coimmunoprecipitation experiments. AGE-β 2 GPI-stimulated DCs had 

increased allostimulatory ability and primed naïve T lymphocytes towards a T helper 

2 polarization. To verify whether the presence of glycation affects sera reactivity with 

the protein, a dot blot assay and ELISA were performed using sera from patients 

with APS and from sex- and age-matched healthy subjects. Densitometric analysis 

showed that the reactivity was stronger for AGE-β 2 GPI than for native β 2 GPI at 

the same protein concentration. None of healthy subject sera reacted with β 2 GPI 

preparations. Our results strongly suggest the role of glycation in rendering β 2 GPI 

able to activate iDCs and induce an humoral immune response in patients with APS. 

Overall these results indicate that glycation of β 2 GPI might be an initiating event that 

triggers the “autoimmune spiral” in APS. 

Buttari et al. Blood 2005 and 2012 


235


Estrogen receptors/EGFR signaling crosstalk: effects of 

17β-estradiol on membrane lipid environment. 

Milani SiMona, zava Stefania, corSetto Paola antonia, 

GalBiati Mariarita i , coloMBo irMa 

Dipartimento di Scienze Farmacologiche e Biomolecolari - Sezione di Biochimica, 

Biofisica, Fisiologia e Immunopatologia - I Sezione di Biomedicina e Endocrinologia 

Università degli Studi - Milano 

Recent studies of estrogen receptors (ERs) biology have highlighted the role of an 

intimate crosstalk between the both ERs (ERα and ERβ) and EGFR signaling pathways 

as a fundamental contributor to the development of resistance to endocrine therapy 

against the ER pathways (1). 

The aim of our work is to investigate the role of membrane lipid environment on ERs 

and EGFR crosstalk in MDA-MB-231 cells, a human breast tumor cell line unresponsive 

to anti-estrogens, expressing ERα, ERβ and EGFR at very low, moderate and high 

levels, respectively. The cells have been treated with physiological doses (10 nM) of 

17β-estradiol (E 2 ) for 24 hours. 

The data, obtained by HP-TLC, western blot and qRT-PCR analyses, reveal that 

E 2 treatment promotes: 1. a significant increase of the ganglioside GM 3 (25%) and 

no change in cholesterol total amount, suggesting an alteration in the membrane 

organisation; 2. a decrement of ERα, ERβ and EGFR proteins (45, 35, 25 %, 

respectively) not associate to analogue variations of corresponding transcript levels. 

This suggests a non genomic action of E 2 . 

Surprisingly, we also found that E 2 increases EGFR auto-phosphorylation. Although 

this observation is in agreement with literature data (2), it contrasts with the largely 

accepted hypothesis of GM 3 as EGFR auto-phosphorylation specific inhibitor (3,4). 

To further explore this contrasting evidence, experiments are in progress to evaluate if 

E 2 treatment affects ERs and EGFR membrane distribution, as well as to ascertain an 

eventual shift of the caveolin-1, one of the membrane proteins of lipid rafts involved 

in the formation and functional regulation of a large signalsome complex that may 

include both ERs and EGFR. 

1- Arpino G. et al. Endocrine Rew. 29:217-233 (2008) 

2- Samanta S. et al. Oncogene 1-9 (2012) 

3- Coskun Ü. et al. PNAS 108:9044-9048 (2011) 

4- Milani S. et al. BBA 1801:617–624 (2010) 


236


Sialidase NEU3 associates both to DRM and non-DRM, 

regulating the glycosphingolipid pattern therein. 

dario Bonardi 1 , nadia PaPini 2 , Mario PaSini 1 , loredana dileo 2 , 

luiGi caiMi 1 , euGenio Monti 1 , Bruno venerando 2 and roBerto BreSciani 1 

1 Department of Biomedical Sciences and Biotechnology, 

University of Brescia, Viale Europa 11, Brescia 


University of Milano, Via F.lli Cervi 93, Segrate (MI) 

NEU3 is the plasma membrane-associated form of mammalian sialidases, exhibiting 

high substrate specificity toward gangliosides, glycosphingolipids (GSL) that, together 

with cholesterol, are particularly enriched in specialized membrane domains that can 

be isolated as detergent-resistant membranes (DRM). These DRM are more ordered 

and tightly packed than the surrounding bilayer (non-DRM) and compartmentalize 

cellular processes such as protein trafficking, signaling and adhesion. It has been 

shown that NEU3 is associated with DRM but its cellular dynamics and its biological 

role in relation to the GSL pattern of these specialized areas remains still unknown. 

For this purpose we took advantage of the inducible HeLa tTA2 Tet-OFF cell model 

to express NEU3. 

During its biosynthesis NEU3 resulted to be initially associated to the non-DRM 

fraction of the cellular membranes and after 48 h expression the protein reached a 

steady-state distribution of about 50:50 between DRM and non-DRM. Cells reached 

full activity only after 72 h, without changes in the distribution of the protein. 

Interestingly, during NEU3 biosynthesis a significant decrease in GM3 and GD1a 

content, with a concomitant increase in LacCer and GM1 was observed in the DRM 

and non-DRM fractions. These changes correlate with the distribution of the protein 

between DRM and non-DRM, suggesting the importance of NEU3 in modulating the 

GSL membrane composition. 

By switching-OFF the promoter, we found that the half-life of the enzyme is roughly 8 

h, and the degradation of the non-DRM pool of the protein is faster compared to the 

DRM pool. Treatment with the proteasome inhibitor MG132 preserved the enzyme 

from degradation and caused accumulation of NEU3 in intracellular aggregates. Future 

experiments will focus on a better understanding of the role played by NEU3 and its 

membrane topology in relation to the GSL pattern and the biological phenomena in 

which these peculiar membrane molecules are involved. 


237


Prognostic value of cell sphingolipid profile and plasma 

membrane sialidase NEU3 expression in melanoma. 

SilveStri ilaria 1 , BaldaSSari Paola 2 , Mortarini roBerta 2 , 

anichini andrea 2 , venerando Bruno 1 , trinGali criStina 1 

1 Dept. of Medical Biotechnology and Translational Medicine, University of Milan, 

LITA. via F.lli Cervi 93, Segrate, Milan, 20090, Italy 

2 Human Tumors Immunology Unit, Dept. of Experimental Oncology and Molecular 

Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Itlay 

Melanoma is the most aggressive and lethal form of skin cancer. Annually, it is 

responsible for at least 48,000 deaths worldwide and its incidence has risen sharply in 

recent decades. In addition to characteristic genetic lesions, melanoma cells display a 

characteristic repertoire of sphingolipids, especially gangliosides, very different from 

healthy melanocytes. In this report, we analysed the sphingolipid pattern and the 

mRNA expression of enzymes involved in sphingolipid metabolism in a large panel 

of short-term melanoma cell lines established from surgical specimens, isolated 

from VGP (vertical growth phase) primary tumours and from metastatic lesions of 

melanoma patients. 

We demonstrated that the gangliosides profile of melanoma cells varied from patient 

to patient and that an interesting correlation between ganglioside pattern and patients’ 

survival existed. In particular, the shortest survival time was recorded in patients 

carrying tumour cells marked by the presence of GM3, GD3, and sialylparagloboside 

(Hazard ratio: 7.2, according to Kaplain Meyer survival analysis). Sialylparagloboside 

is known to be the synthetic precursor of Sialyl Lewis X epitope that is related with 

increased metastatic potential of cancer cells. In addition, also the expression of 

some key enzymes involved in gangliosides synthesis and catabolism correlated with 

patients’ survival. In particular, the expression of the plasma membrane sialidase NEU3 

was increased in many melanoma cell lines established from patients, as compared to 

expression detected in normal melanocytes. This increment was inversely correlated 

to patients’ survival (Hazard ratio: 3, according to Kaplain Meyer survival analysis). 

These results suggest the employment of gangliosides pattern and of NEU3 expression 

as markers to classify melanomas in addition to histopathologic criteria. 

1. Furukawa K et al Proteomics 2008;8(16): 3312-6. 


238


Plasma membrane sialidase NEU3 up-regulation promotes 

β1 integrin recycling to the plasma membrane, driving 

Renal cell carcinoma (RCC) progression 

trinGali criStina 1 , luPo BarBara 1 , SilveStri ilaria 1 , PaPini nadia 1 , 

anaStaSia luiGi 2,3 , tettaManti Guido 3 , venerando Bruno 1 

1 Dept. of Medical Biotechnology and Translational Medicine, 

University of Milan, LITA, Segrate, Milan, Italy 

2 Dept. of Biomedical Sciences for Health, 

University of Milan, LITA, Segrate, Milan, Italy 

3 IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy 

Renal cell carcinoma (RCC) is the most common and lethal form of kidney cancer. 

One-third of patients have metastases at presentation, and approximately 40% of 

patients treated for a localized tumor develop recurrence. Treatment of advanced 

RCC with chemo/radiotherapy has been largely unsuccessful. In this report, we 

demonstrated that, in RCC, invasion and resistance to drugs are related to the upregulation 

of plasma membrane sialidase, NEU3. We stably silence NEU3 in a human 

primary RCC cell line, CA-TC, using a lentiviral approach. We demonstrated that 

NEU3 silencing: a) reduced the resistance to etoposide, increasing apoptosis and, in 

parallel, decreasing autophagy, through an increased expression of BAX and BAD and 

a decreased expression of BCL-2; b) decreased the invasion potential, reducing the 

expression of the metalloproteases, MMP7 and MMP1. These effects are mediated 

by a different trafficking of β1 integrins: in NEU3 silencing cells, β1 integrins were 

faster internalized through caveolar endocytosis and SRC kinases activation. Then, 

in contrast to mock cells that rapidly recycled internalized β1 integrins to the plasma 

membrane, in NEU3 silencing cells, β1 integrins were directed toward lysosomes. 

These modifications of β1 integrin recycling were mediated by the up-regulation of 

RAB25 and the down-regulation of CLIC3 in NEU3 silencing cells. The increased 

degradation of β1 integrins induced, in turns, the down-regulation of FAK, AKT, and 

EGFR signaling responsible for resistance to drugs and invasion potential in RCC cells. 

Changes of caveolar endocytosis and β1 integrin trafficking appeared to be related to 

the increase of GD1a and to alteration of sphingolipid pattern consequent to NEU3 

silencing. The induction of a GD1a-rich ganglioside profile in CA-TC cells by GD1a 

incorporation significantly reproduced some of the above effects. Therefore, NEU3 

seems to be crucially involved in RCC key signaling pathways and could constitute a 

new therapeutic target. 


239

Membrane 

e 

Bioenergetica 

MeB

Membrane e Bioenergetica MEB 1 

Effect of supplementation of CoQ 10 and R,S-alpha-lipoic 

acid in preventing mitochondrial dysfunction and oxidative 

damage in peripheral blood lymphocyte exposed to 

oxidative stress ex-vivo. 

Patrick orlando, Sonia SilveStri, tatiana arMeni, 

franceSca BruGè, Gian Paolo littarru, luca tiano 

Department of Clinical and Dental Sciences, Section of Biochemistry Polytechnic 

University of Marche, Ancona 

Regular exercise is able to modulate antioxidant defence system and improve 

energetic metabolism in mitochondria. On the other hand, exhaustive exercise might be 

associated with increased ROS production causing oxidative damage. Mitochondria, 

in this respect, represent a sensible target of oxidative damage and their impairment 

might lead both to decreased energy production and enhanced ROS formation. The 

aim of our study was to evaluate whether the administration of CoQ10 alone and in 

association with lipoic acid for 15 days to 16 healthy subjects was able to prevent 

mitochondrial dysfunction and oxidative damage induced ex-vivo by incubation of 

PBL with H 2 O 2 at 37°C for 4 hours to simulate exhaustive exercise. Results highlighted 

that CoQ 10 alone and in association with lipoic acid was able to improve plasma 

oxidative status. At cellular level, the improvement of CoQ 10 oxidative status was 

associated with enhanced mitochondrial functionality and decreased susceptibility 

to oxidative insult, measured in terms of mitochondrial membrane potential using 

the nernstian probe JC-1. However, mitochondrial activity, when stimulated by 

tested mitochondrial nutrients, in the strong oxidizing environment characterizing 

our experimental setup, was also associated with increased intracellular ROS levels. 

Co-treatment with lipoic acid was able to protect DNA from oxidation even in these 

remarkably oxidising conditions. Further studies are required to better characterize 

in vivo, during exhaustive exercise, the pro energetic and puzzling anti/pro-oxidative 

effect of CoQ 10 and lipoic acid. 


242


Melatonin modulates mitochondrial function in HaCaT cells 

via nitric oxide production 

areSe Marzia 1 , MaGnifico Maria chiara 1 , MaStronicola daniela 2 , 

forte elena 1 , Giuffrè aleSSandro 3 , Grillo caterina 1 , 

altieri faBio 1 , Blanck thoMaS J. J. 3 and Sarti Paolo 1,2 

1 Department of Biochemical Sciences, Sapienza University of Rome. 

2 CNR - Institute of Molecular Biology and Pathology, Rome. 

3 Department of Anesthesiology, The New York University, New York, NY, USA 

Melatonin is a hormone produced by the pineal gland following circadian rhythms, 

although many other cell types are able to produce it. This molecule is involved in 

many cell functions including regulation of specific enzymes involved in cell redox 

homeostasis, with impact on cell bioenergetics. 

Nitric oxide (NO) is a versatile biological messenger which regulates many physiological 

responses; these include smooth muscle relaxation, neurotransmission, inhibition of 

platelet aggregation, cell migration and mitochondrial respiration. In mammals NO 

is synthesized by three different gene-encoded NO synthase (NOS), the neuronal 

NOS (nNOS or NOS1), the inducible NOS (iNOS or NOS2), the endothelial NOS 

(eNOS or NOS3) and possibly a mitochondrial NOS. The endogenously produced 

NO influences cell bioenergetics, modulating mitochondrial respiration at the level 

of Complex I and Complex IV, i.e. Cytochrome c oxidase (CcOx). The interaction of 

CcOx and NO, results in the fast and reversible inhibition of the enzyme, that may 

proceed through two alternative reaction pathways leading to different degree of 

inhibition of cell respiration. The prevalence of one pathway over the other might be 

linked to different patho-physiological condition, possibly influenced by the presence 

of signalling compounds. Our work was aimed at studying the effects of melatonin 

on NO-mediated regulation of cell energetics. HaCaT cells treated with melatonin 

showed a time and concentration-dependent increase of the nNOS mRNA production 

with higher accumulation of nitrites/nitrates compared to controls. At mitochondrial 

level, the bioenergetic parameters such as lactate accumulation, ATP production and 

mitochondrial membrane potential followed synchronous changes compatible with 

No inhibition of Complex IV. The results are discussed in the light of a metabolic 

shift (from OXPHOS to glycolisis) induced by melatonin and by the NO mediated 

modulation of the phosphorilation chain activity initiated by the melatonin. 


243


Rapamycin reduces oxidative stress 

in frataxin-deficient yeast cells 

MaroBBio carlo M.t. 1, 2 , PiSano iSaBella 1 , Porcelli vito 1 , 

laSorSa franceSco M. 2 , PalMieri luiGi 1,2 

1 Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, 

Biotechnology and Pharmacological Sciences, University of Bari 

Via E. Orabona 4, 70125 Bari, Italy 

2 CNR Institute of Biomembranes and Bioenergetics, Via Orabona 4, 70125 Bari, Italy 

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression 

of the mitochondrial protein frataxin. Oxidative stress plays a pivot role in the 

pathogenesis of the disease and it has been shown that the use of the antioxidant 

drug idebenone improves neurological function in FRDA patients. We used the yeast 

frataxin knock-out model (Δyfh1) to analyze the effects of two antioxidant compounds, 

N-acetyl-L-cysteine (NAC) and a mitochondrially-targeted analog of vitamin E 

(MitoVitE), on ROS production. Furthermore, we tested the effect of the rapamycin, 

an inhibitor of TOR kinases. Our experiments showed a decrease in ROS production 

measured in Δyfh1 cells treated with NAC or MitoVitE due to the scavenger activity 

of these antioxidants. Also following treatment of Δyfh1 cells with rapamycin, we 

observed a marked decrease in ROS production and a parallel decrease in the number 

of the cells producing ROS as estimated by flow cytometry. This decrease was not 

due to a decrease in cell viability but correlates with a reduction of mitochondrial 

mass likely due to a stimulation by rapamycin of the selective removal of damaged 

mitochondria in frataxin-deficient cells. Although additional experimentation is 

needed to investigate interactions between ROS, mitochondrial dysfunction and 

autophagy following rapamycin treatment, our observations point to mitophagy as a 

new potential therapeutic target for FRDA. 


244


Mitochondrial soluble adenylyl cyclase (sAC) cooperates 

with membrane adenylyl cyclase in the regulation of 

complex I turnover and activity 

de raSMo doMenico 1 , SiGnorile anna 2 , SanteraMo arcanGela 2 , 

caPitanio GiuSePPe 2 , PaPa SerGio 1 

1 Institute of Biomembranes and Bioenergetics (IBBE), Consiglio Nazionale delle 

Ricerche, Bari, Italy. 

2 Department of Basic Medical Sciences, Section of Medical Biochemistry, 

University of Bari Aldo Moro, Bari, Italy. 

In mammalian cells there are subcellular pools of cAMP and PKA. In response to 

activation, by extracellular effectors (like β-adrenoceptor agonists), the plasma 

membrane adenylyl cyclase (tmAC) produces cAMP in the cytosol. It has been 

showed that activation of the cytosolic cAMP/PKA system rescues the activity of 

oxidatively damaged complex I, by promoting mitochondrial import of the NDUFS4 

subunit of complex I and its assembly in the complex in exchange with the 

endogenous carbonylated subunit. There is, in the mitochondrial matrix, a soluble 

bicarbonate-activable adenylyl (sAC) and all the members of the cAMP/PKA system. 

sAC-dependent cAMP production is involved in the regulation of the cytochrome c 

oxidase activity and in activation of the mitochondrial pathway of apoptosis. Here, 

using KH7, a specific inhibitor of sAC, and CAI, an inhibitor of carbonic anhydrase, we 

show that intramitochondrial cAMP, produced by sAC, cooperates in the β-adrenergic 

dependent stimulation of complex I activity. The KH7 had no effect on the activity of 

complex I when incubated with isolated complex I or with isolated mitochondria. The 

inhibitory effect was observed when KH7 was added to the fibroblast cell cultures. 

This inhibition was reversed by concomitant incubation with 8-br-cAMP, a permeant 

analogue of cAMP. Investigation on the import in isolated rat liver mitochondria of 

the in vitro synthesized, radiolabelled complex I subunits showed that KH7 caused 

a small decrease in the level of mitochondrially imported NDUFS4 subunit but 

strongly inhibited its assembly in the complex. The KH7 inhibition was reversed by 

the concomitant presence of 8-br-cAMP but not by externally added catalytic subunit 

of PKA. In conclusion the results show that the cytosolic and mitochondrial pools of 

cAMP cooperate in the regulation of complex I activity by modulating the import and 

dynamic assembly (exchange) of peripheral subunit(s). 


245


Over-expression in E. coli and reconstitution in liposomes 

of the OCTN3 (SLC22A21) transporter 

ScaliSe MariafranceSca, Galluccio Michele, Pochini lorena and 

indiveri ceSare 

Dipartimento di Biologia Cellulare Università della Calabria, 

via Bucci 4c Arcavacata di Rende, Italy 

Membrane transporters are essential for life since mediate trafficking of metabolites. 

Among transport systems, those expressed in mammals including humans are 

particularly interesting due to their link with pathology. Several mammalian transporters 

have been successfully cloned but, only recently, some of them have been overexpressed 

in heterologous organisms. The over-expression in E. coli of human 

hOCTN1 and hOCTN2 has been reported [1,2]. The transporters belong to a small 

protein subfamily (OCTN, Organic Cation Transporter Novel) together with OCTN3. On 

the basis of studies in intact cells, OCTN2 and OCTN3 should be involved in carnitine 

homeostasis, being carnitine the preferential substrate. The coding gene for OCTN3 

has not yet been annotated in human genome but only in mouse [3]. In the present 

work the mouse OCTN3 cDNA has been amplified and cloned in pET-21a(+). The 

recombinant construct has been used for transforming E. coli Rosetta(DE3)-pLysS. 

An over-expressed protein with an apparent molecular mass of 53kDa was collected 

in the insoluble fraction of the cell lysate. The mOCTN3 protein was purified with a 

yield of 2 mg/liter of cell culture and reconstituted in liposomes by detergent removal. 

The time course of [3H]carnitine uptake in proteoliposomes occurred by a uniport 

mode of transport. Carnitine uptake was stimulated by intraliposomal (intracellular) 

ATP. The transport was Na + independent as described in cells [3]. This substantiated 

a role for OCTN3 in carnitine transport in cells. Kinetic analysis of transport revealed 

a Km for carnitine of 36 uM similar to that found in cells. On a physiological point 

of view, OCTN3 could be involved, besides in carnitine homeostasis also in neuro- 

detoxification. 

[1] Galluccio M. et al, Protein Expr Purif (2009) 68, 215-220. 

[2] Galluccio M. et al, Mol. Biotechnol (2012) 50, 1-7. 

[3] Tamai I. et al, J Biol Chem (2000) 275, 40064-40072. 


246


Autonomous ultra-circadian rhythms of mitochondrial 

respiratory activity 

olGa cela 1 , valerio Pazienza 2 , roSella ScriMa 1 , 

GiorGia BeneGiaMo 2 , GianluiGi Mazzoccoli 2 , nazzareno caPitanio 1 

1 Dept. of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy; 

2 Dept. of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, 

IRCCS Scientific Institute and Regional General Hospital Casa Sollievo 

della Sofferenza, Opera di Padre Pio da Pietrelcina, San Giovanni Rotondo (FG), Italy. 

Circadian rhythms in gene expression synchronize biochemical processes and 

metabolic fluxes with the external environment, allowing the organism to function 

effectively in response to predictable physiological challenges. In mammals, this 

daily timekeeping is driven by the biological clocks of the circadian timing system, 

composed of master molecular oscillators within the suprachiasmatic nuclei of the 

hypothalamus, pacing self-sustained and cell-autonomous molecular oscillators in 

peripheral tissues through neural and humoral signals. Consistently with this notion, 

even in the absence of external stimuli, practically all the cells exhibit intrinsic 

oscillation of clock genes. 

Using a well established in vitro synchronization protocol we measured the endogenous 

respiratory activity in HepG-2 cell line by high resolution oxymetry over a period of 

30 hours with an interval time of 3 hours. The results obtained strikingly unveils that 

in intact cells the mitochondrial resting oxygen consumption rate (OCR) undergoes a 

rhythmic oscillation with a maximum peaking every 12 hours (zenith/nadir ratio ≈ 3-4, 

n=7, P < 0.01). The same oscillation rhythm was observed in the uncoupled and the 

ATP-dependent OCRs but not in the leak-dependent OCR. No significant oscillatory 

activity was observed for the citrate synthase. The expression analysis of a panel 

of clock genes resulted in an oscillation pattern of Rev-Erbα and RORα similar to 

that of the OCR. Noticeably, the transcription inhibitor actinomycin D suppressed the 

circadian rhythms of the OCR establishing a link between these and gene expression. 

Intriguingly, the OCRs measured in digitonin-permeabilized cells with either NADH- 

and FADH 2 -related substrates did not resulted in any oscillatory patterns. Moreover, 

treatment of cultured cells with the mitochondrial Ca 2+ uniporter inhibitor ruthenium 

red prevented OCR oscillation. 

In conclusion, these results indicate the occurrence in HepG-2 cells of an autonomous 

gene-expression- and intramitochondrial Ca 2+ -dependent ultra-circadian rhythm 

controlling the mitochondrial respiratory chain activity. A working model incorporating 

the presented observations will be put forward. 


247


Hydroxytyrosol prevents mitochondrial respiratory 

dysfunctions in human fibroblasts 

SiGnorile anna 1 , Micelli loriS 1 , Gattoni Giuliano 1 , 

de raSMo doMenico 2 , PaPa franceSco 1 , SanteraMo arcanGela 1 and 

PaPa SerGio 2 

1 Section of Medical Biochemistry, Department of Basic Medical Sciences, 

University of Bari, Italy 

2 Institute of Biomembrane and Bioenergetics, CNR, Bari, Italy 

ABSTRACT 

Introduction The use of olive oil in the diet appears to be associated with a lower 

incidence of cardiovascular diseases and cancer in the Mediterranean population [1]. 

Hydroxytyrosol (HT) is considered to contribute significantly to the health benefit of 

olive oil. Studies on the action of HT have been essentially focused on its antioxidant 

value [2]. Recently stimulation by HT of the expression of the PCG-1α, TFAM, NRF1and 

NRF2 has been observed [3]. We have investigate the protective effect of HT towards 

mitochondrial dysfunction in human fibroblasts. 

Methods Human dermal fibroblasts (NHDF) were cultivated in standard culture 

conditions (DMEM-10% FBS) (CTRL) or serum limited (DMEM-0.5 % FBS) for 72 

hours in the absence (SL) or in the presence of HT (SL+HT). Intracellular H 2 O 2 level 

was determined by DCFH 2 , complex I and complex IV activities were determined 

spectrophotometrically. Mitochondrial protein synthesis was analyzed by labeling [ 35 S] 

methionine-[ 35 S] cysteine. PKA activity was performed following the phosphorylation 

of H 2 B in the presence of [γ- 32 P]ATP. 

Results HT at low concentrations of ≈1µM, which lowered reactive oxygen species 

accumulation, prevented depression of the expression and activity of complex I 

caused by serum limitation of fibroblast cultures [4]. These effects were associated 

with HT-induced promotion of the activity of PKA, of the expression of PGC-1α, NRF1, 

TFAM and stimulation of mitochondrial protein synthesis. 

Conclusion The results show that HT is a positive promoting effector of the expression 

of OXPHOS complexes and in particular of the functional capacity of complex I, a 

pacemaker of the overall OHPHOS system. 

1. Sofi et al., BMJ, 11: 337- 344, 2008. 

2. Visioli et al., Biochem Biophys Res Commun, 247: 60-64, 1998. 

3. Hao et al., J Nutr Biochem, 7: 634-644, 2010. 

4. Papa et al., Biochim Biophys Acta 1797: 649-658, 2010. 


248


Evidence for different cadmium detoxification system in 

Saccharomyces Cerevisiae 

oStuni anGela, MiGlionico rocchina, Salvia antonella, 

BiSaccia fauStino. 

Dept. of Science , University of Basilicata, 85100 Potenza, Italy 

The yeast genome contains 30 ABC proteins. Of these proteins 22 are predicted 

to contain multiple membrane spans and are thus considered to be true ABC 

transporters, while the remaining 8 presumably carry out non-transport functions 

in the cell. Phylogenetic analyses established the existence of six ABC subfamilies. 

Member of ABCC subfamily also called MRP are involved in the efflux of xenobiotics 

in eukaryotic cells. Five members of the ABCC subfamily are full length (Ycf1p, Bpt1p, 

Ybt1, Nft1p, and Vmr1p), one is short (Yor1p). While Yor1p localizes to the plasma 

membrane the others localized to the vacuolar membrane (1). In many cases yeast 

ABCCs exhibit overlapping substrate specificity. There is not a critical functional 

distinction between full-length and short ABCCs. On glucose medium the main 

transporter of cadmium-GS conjugates into the vacuole is the Yeast Cadmium Factor, 

Ycf1p (2), while on ethanol/glycerol medium Vmr1p mediates cadmium detoxification 

(3). 

In this study, experiments carried out in presence of L-Buthionine-sulfoximine (BSO), 

a specific inhibitor of γglutamylcysteine synthetase, on Saccharomyces Cerevisiae 

wild type and Vmr1p- deleted strain, suggested that on ethanol/glycerol medium 

the Vmr1p mediates cadmium detoxification but not through formation of Cd[GS]2 

complexes. 

1. Paumi C, et al. (2009) Microbiol.Mol.Biol.Rev73, 

2. Li Z, Szczypka M, Lu Y, Thiele D & Rea P. J Biol Chem(1996)7: 6509–6517. 

3. D Wawrzycka, I Sobczak, G Bartosz, T Bocer, S Ułaszewski1 & Andr´e Goffeau. FEMS Yeast Res. 

2010;10(7):828-38. 


249


The NBD domains of the MRP6/ABCC6 

oStuni anGela, MiGlionico rocchina, Monnè MaGnuS, 

Salvia antonella, caStiGlione Morelli Maria antonietta and 

BiSaccia fauStino 

Dept. of Science, University of Basilicata, 85100 Potenza, Italy 

Multidrug resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging 

to the ABC energy-dependent efflux pumps family which members share many 

characteristic structural features, including two membrane-spanning domains and 

two nucleotide-binding domains (NBD1 and NBD2) that function cooperatively 

but not equally bind and hydrolyze ATP [1]. In this family MRP6 plays an important 

physiological role as demonstrated by the fact that mutations in this gene cause 

Pseudoxanthoma elasticum (PXE) in humans, a recessive genetic disorder affecting 

connective tissues characterized by progressive mineralization of elastic fibers [2]. In 

the present study, NBD1 and NBD2 of MRP6 has been expressed in Escherichia coli, 

purified and structurally and functionally characterized. The CD spectra demonstrate 

the presence of α-helices, β-strands and turns. Both polypeptides are shown to be 

biologically active. NBD2 binds ATP with an affinity similar to NBD1 but the ATPase 

activity is significantly different in the isolated NBDs. The mixture of NBD2 and NBD1 

exhibited an activity similar to the NBD2 alone, indicating that NBD1 and NBD2 form 

a heterodimer with the latter limiting ATP hydrolysis. These findings suggest that 

NBD1 has a higher tendency to form an active homodimer, which is also supported 

by in silico analysis of energy-minimized dimers of the homology models of the two 

domains. 

1. Slot AJ, Molinski SV, Cole SP. Mammalian multidrug-resistance proteins (MRPs). Essays Biochem. 

2011;50(1):179-207. 

2. Kavukcuoglu NB, Li Q, Pleshko N, Uitto J. Connective tissue mineralization in Abcc6-/- mice, a model 

for pseudoxanthoma elasticum. Matrix Biol. 2012;31(4):246-52. 


250


HYPOxIA AND AMYLOID-β PEPTIDE MIGHT 

SYNERGISTICALLY CONTRIBUTE TO CELLS 

DYSFUNCTION IN ALZHEIMER’S DISEASE 

Padula anna, Baracca aleSSandra, SGarBi Gianluca, 

Solaini Giancarlo 

Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna, 

Via Irnerio 48 40126 Bologna 

Hypoxia can cause the alteration of energy metabolism and may have a critical role in 

neurodegeneration, including Alzheimer’s Disease (AD). Neural hypoxia can be a direct 

consequence of hypoperfusion, a common vascular component among the AD risk 

factors. It has been reported that hypoxia is intimately involved in the accumulation of 

amyloid-β (Aβ) peptides by activating the amyloidogenic pathway [1]. Aβ is a powerful 

mitochondrial toxin, especially affecting cytochrome c oxidase (COX) and causing a 

severe oxidative stress [2]. We previously demonstrated that COX activity in neurons 

of Alzheimer patients was strongly decreased without affecting the ATP synthesis rate 

[3], concluding that a decrease of COX activity might not be sufficient to reduce ATP 

synthesis under normoxic conditions. We found that in the presence of hypoxia (1.5% 

pO 2 ) COX activity of PC12 cells is about 45% reduced becoming the rate limiting step 

of OXPHOS. Given this, we hypothesized that hypoxia and Aβ might synergistically 

affect COX activity and induce mitochondrial dysfunction. As cellular model of AD we 

used neuron-like PC12 cells exposed to 10µM Aβ and low oxygen tensions for 24h. 

The combined exposure to hypoxia and Aβ peptide enhanced the dysfunction of COX 

with respect to sole hypoxia, inducing depletion of intracellular ATP with a moderate 

but statistically significant additive effect. Additionally, it was observed a major 

increase in ROS generation. These results indicate a synergistic effect of hypoxia 

and Aβ that might be critical to promoting cell death and neurodegeneration. Studies 

exposing N2a cells stably expressing human APP695 to hypoxia are in due course to 

validate the above results. 

[1] Moussavi Nik SH et al. J Alzheimer Dis 2012; 28:515-30 

[2] Aleardi AM et al. Bioenerg Biomembr 2005; 37(4):207-25 

[3] Bosetti F et al. Neurobiol aging 2002; 23:371-6 


251


VDAC1 topology in the outer mitochondrial membrane 

Guarino franceSca 1§ , toMaSello Marianna flora 2§ , 

MeSSina anGela 1 , MaGrì andrea 1 and de Pinto vito 1 

1 Department of Biological, Geological and Environmental Science, Sect. 

Biochemistry and Molecular Biology, University of Catania and National Institute 

of Biostructures and Biosystems, Sect. of Catania, 

2 Department of Pharmacological Science, viale A. Doria 6, 95125 Catania, Italy 

Voltage Dependent Anion Channel isoform 1 (VDAC1) is the main protein located in 

outer mitochondrial membrane, acting as mediator of cytosolic and mitochondrial 

functions (1). Its structure, recently solved, confirmed a β-barrel shape organized 

in 19 β-strands and a N-terminal region arranged in α-helix probably important to 

regulate channel functionality. However the sidedness of VDAC1 inside the membrane 

still remains unresolved (2). This issue is essential in order to define the structural 

determinants of the VDAC pore involved in interactions with cytosolic and mitochondrial 

proteins. We approached this matter expressing, in HeLa cells, a recombinant protein 

hVDAC1 carrying a C-terminal tag including the hemagglutinin-tag (HA), the specific 

caspase 3/7 cleavage site (DEVD) and 7 histidine residues (7His). The peculiarity of 

this composite tag resides in the DEVD amino acidic sequence which can be cleaved 

upon apoptosis induction by staurosporine. If the C-terminus of VDAC1 is exposed 

to the cytosolic side, we expect to lose the His tag as a consequence of the DEVD 

cleavage. As a result only the HA can be detected at mitochondrial level. Conversely if 

the C-terminus is oriented towards the IMS, the DEVD is not cleavable and both tags 

will be detected in mitochondria. Preliminary data by confocal microscopy showed 

that this strategy is able to define the in vivo sidedness of the VDAC1 pore. 

References 

1) Shoshan-Barmatz, V., De Pinto, V. et al. (2010) Mol Aspects Med. 31, 227-85. 

2) McDonald B. M. et al. (2009) FEBS Lett. 583, 739-42 

§ These two authors equally contributed to this work. 


252


Specific β-strands contacting the N-terminal domain of 

VDAC1 are important for the gating properties of the pore 

MaGrì andrea 1 , reina SiMona 1 , Guarino franceSca 1 , 

PaPPalardo anna Maria 1 , MilleSi Salvatrice 1 , Benz roland 2 , 

ceccarelli Matteo 3 , de Pinto vito 1 and MeSSina anGela 1 

1 Department of Biological, Geological and Environmental Sciences, Section of 

Molecular Biology, University of Catania, and National Institute for Biomembranes 

and Biosystems, viale A. Doria 6, 95125 Catania, Italy; 

2 Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, 

University of Würzburg, D-97078 Würzburg, Germany 

VDAC (Voltage Dependent Anion selective Channel) is considered an important knot 

in the cellular cross talk between the mitochondrion and the rest of the cell. The 

structure of VDAC1 isoform has been recently solved in mammals. Researches are 

now aimed to define at a molecular level its peculiar gating property, the voltagedependence, 

highly relevant in the bioenergetic metabolism. VDAC1 N-terminal 

sequence has always been the subject of considerable interest because itsdeletion 

did not affect the correct targeting of the protein to mitochondria but had serious 

effects on the pore-forming activity and the voltage-dependence of the protein (1-2). 

It has been reported as an incomplete amphipathic alpha-helix apposed to the inner 

side of the pore wall. In this work we focus our attention onto the beta-strands that 

take contact with the N-terminal domain. The exchange of the whole VDAC1 betabarrel 

with the homologous VDAC3 beta-barrel shows that the chimeric protein in 

reconstituted systems loses completely voltage-dependence, despite the presence 

also in VDAC3 of V143 and L150 residues (2). VDAC1 mutants completely lacking 

either the beta-strand 9 or both beta-strands 9 and 10 were expressed, refolded 

and reconstituted in artificial bilayers. In these experiments the mutant lacking the 

beta-strand 9 (where V143 is located) shows smaller pores but a normal voltagedependence. 

The mutant lacking both beta-strands 9 and 10 shows instead a peculiar 

voltage-dependence resulting in a fully asymmetric behavior. We used restrained 

Molecular Dynamics simulations to model the protein missing beta-strands 9 and 10. 

The results obtained support the experimental data. Our data point out the notion that 

the voltage dependent gating of VDAC1 is a complex phenomenon involving both the 

N-terminus moiety and some specific b-strands in the pore wall. 

References 

1. De Pinto V, et al (2007) Chembiochem 8:744-756 

2. Reina S, et al (2010), FEBS Letters 584: 2837-2844 

Acknowledgements: National funding sources (PRIN, PRA, FIRB-MERIT) are gratefully acknowledged 


253


Are VDAC levels in Drosophila melanogaster mitochondria 

regulated by alternative splicing of 5’-UTRs? 

iMPellizzeri aGata antonina rita 1 , accardi roSita 2 , reina SiMona 1 , 

MaGrì andrea 1 , PaPPalardo anna Maria 1 , toMMaSino MaSSiMo 2 , 

de Pinto vito 1 and MeSSina anGela 1 

1 Department of Biological, Geological and Environmental Science, Sect. 

Biochemistry and Molecular Biology, University of Catania (ITALY) and 2 International 

Agency for Research on Cancer, World Health Organization, 69372 Lyon (France) 

The porin in Drosophila melanogaster produces two splice variant transcripts that 

are termed 1A-VDAC and 1B-VDAC. These transcripts comprise the same coding 

sequence but have a different 5’-untranslated region. The biological significance of 

the two forms of VDAC mRNA is nowadays unknown. In this work, we have studied 

the influence of the two 5’-untranslated regions, 1A-5’UTR and 1B-5’UTR, on the 

expression and translation of the VDAC and reporter genes in yeast and fly cells. 

Transformation of Delta-porin cells with a construct carrying the 1A-VDAC results in 

the expression of the corresponding protein and complementation of defective cell 

phenotype. In contrast, no protein was detected in Delta-porin cells transformed by 

the plasmid expressing the 1B-VDAC. Identical results were obtained using hybrid 

constructs containing the two 5’-UTR regions in front of the reporter gene GFP. In D. 

melanogasterembryonal cells S2, 1B-5’UTR has an inhibitory effect on the translation 

of the reporter luciferase, while 1A-5’UTR luciferase is efficiently synthesised. We 

hypothesize that a little uORF present in the 1B-5’UTR can be responsible of its 

mis-translation. In addition, the overexpression of 1A-VDAC in these cells does not 

lead to an increase of VDAC protein in comparison to control, while they showed 

accumulation of the 1B-VDAC mRNA. 

We conclude that the production of the alternative 1B-VDAC mRNA can be 

stimulated by the overexpression of 1A-VDAC and contribute to the quantitative 

control of the intracellular levels of mitochondrial VDAC protein. 

References 

1) Messina, A. et al. (1996) Lett , 9-13. 

2) Oliva, M. et al. (2002) Mol.Gen.Genomics , 746-756. 


254


Short-term effect of 3,5-diiodo-L-thyronine on fatty acid 

oxidation rate and bioenergetic parameters in liver cells of 

hypothyroid rats 

cavallo aleSSandro 1 , Priore Paola 1 , Gnoni GaBriele vincenzo 1 , 

PaPa SerGio 2 , zanotti franco 3 , Gnoni antonio 3 

1 Laboratory of Biochemistry, Department of Biological and Environmental Sciences and 

Technologies, University of Salento,Via Prov.le Lecce-Monteroni,73100 Lecce, Italy. 

2 Institute of Biomembranes and Bioenergetics, CNR, 70124 Bari, Italy. 

3 Department of Medical Biochemistry, Physics and Biology, University of Bari, 70124 Bari, Italy. 

3,5-diiodo-L-thyronine (T 2 ), previously considered only a catabolic product of thyroid 

hormone metabolism, has been recently shown to play an important role on energy 

metabolism (1-2). In this study, we investigated the effect of acute administration of T 2 

to hypothyroid rats on hepatic fatty acid oxidation rate and bioenergetic parameters. 

Coupled (state 3), uncoupled (state 4) and FCCP-stimulated respiration, which, as 

compared to control, were reduced in hypothyroid mitochondria, were noticeably 

increased within 1h following i.p. injection of T 2 . The hormone effect was more 

pronounced in succinate- with respect to glutamate/malate-energized mitochondria. 

T 2 enhancement of fatty acid oxidation rate was assessed by using palmitoyl-CoA 

(+carnitine) or palmitoylcarnitine as respiratory substrate. No significant change 

in respiratory control index and ADP/O ratio was observed. The activities of the 

mitochondrial respiratory chain complexes, especially Complex II, were increased in 

T 2 -treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and 

hydrolysis direction, were enhanced. The total rate of fatty acid oxidation, followed by 

conversion of [ 14 C] palmitate to 14 CO 2 and ketone bodies, was higher in hepatocytes 

isolated from T 2 -treated rats. This increase occurs in parallel with the enhanced 

activity of carnitine palmitoyltransferase-I (CPT-I), the rate limiting enzyme of fatty acid 

β-oxidation, thus suggesting CPT-I activity as a possible target of T 2 action. Taken 

together, these results indicate that T 2 rapidly promotes the ability of mitochondria 

to import and oxidize fatty acids, and contribute to explain recent findings showing 

that T 2 may powerfully reduce adiposity and body weight gain (2), and reverse hepatic 

steatosis without inducing thyrotoxicity. 

1. Cavallo A, Gnoni A, Conte E, Siculella L, Zanotti F, Papa S, Gnoni GV. (2011) 3,5-Diiodo-L-Thyronine 

increases FoF 1 -ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats. J 

Bioenerg Biomembr 43: 349-357. 

2. Lanni A, Moreno M, Lombardi A, de Lange P, Silvestri E (2005) 3,5-diiodo-thyronine powerfully reduces 

adiposity by increasing the burning of fats. FASEB J 19: 1552-1554. 


255


Glioblastoma multiforme: antitumoral in vitro effect by 

induction of collapse of mitochondrial membrane potential 

claudia Martini 1 , BarBara coSta 2 , eleonora da Pozzo 1 , 

Sara Bendinelli 1 , Pietro caMPiGlia 3 , aleSSia BertaMino 4 , 

iSaBel GoMez-Monterrey 4 , SaBrina taliani 5 , franceSca SiMorini 5 , 

ettore novellino 4 , federico da SettiMo 5 . 

1 Dep of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa; 

2 Dep of Human Morphology and Applied Biology, University of Pisa; 

3 Dep of Pharmaceutical Science, Division of BioMedicine, University of Salerno; 

4 Dep of Pharmaceutical and Toxicological Chemistry, University of Naples Federico II; 

5 Dep of Pharmaceutical Science, University of Pisa. 

Glioblastoma Multiforme (GBM) resistance to genotoxic agents is generally due to a 

very efficient genoma repair system [1]. A possible window of intervention that triggers 

programmed cell death and bypasses the DNA damaging-activated apoptotic pathways 

could be represented by the induction of the collapse of mitochondrial membrane 

potential (ΔΨm), the well-known event that causes cytoplasmatic release of potent 

proapoptotic factors. 

The purpose of this study was to evaluate in a cell model of human GBM the antitumoral 

effects mediated by drugs that cause ΔΨm dissipation. The strategy was to increase the 

mitochondrial outer membrane permeabilization by pharmacological activation of two 

distinct targets: the 18 KDa Translocator Protein (TSPO), which is a crucial component 

of the mitochondrial multiprotein complex pore (MPTP) [2], and p53 tumor suppressor 

protein, which can induce formation of mitochondrial membrane pores following 

oligomerization of pro-apoptotic proteins belonging to Bcl2 family [3]. 

We showed that the TSPO activation by the TSPO selective ligand PIGA induced 

prolonged opening of the MPTP with consequent ΔΨm dissipation and reduction 

of tumor cell survival. In tumor cells, the activation of p53 functions, as the gene 

transactivation, by the ISA27 ligand increased transcription of the gene encoding 

the pro-apoptotic protein PUMA, a p53 target gene. In parrallel, we observed ΔΨm 

dissipation and reduction of cell survival. The combined pharmacological activation of 

the two intracellular pathways converging on mitochondria caused a synergic effect on 

induction of ΔΨm collapse which resulted in a more effective anti-tumor in vitro activity. 

References 

[1] Pilkington GJ et al. Approaches to mitochondrially mediated cancer therapy Semin Cancer Biol. 

2008;18:226-35. 

[2] Galiegue S et al. The peripheral benzodiazepine receptor: a promising therapeutic drug target.Curr Med 

Chem. 2003;10:1563-72. 

[3] Galluzzi L, et al., Targeting p53 to mitochondria for cancer therapy. Cell Cycle. 2008;7 1949-55. 


256


Comparative analysis of mitochondrial dysfunctions in 

foetus-derived fibroblasts from Down syndrome-affected 

subjects with/without congenital heart defects 

ScriMa roSella 1 , Piccoli claudia 1 , izzo antonella 2 , BonfiGlio ferdinando 2 , 

Manco roSanna 2 , quarato Giovanni 1 , cela olGa 1 , riPoli Maria 1 , 

PriSco M. 3 , Gentile flaviana 4 , calì Gaetano 4 , conti anna 2 , nitSch lucio 2,# , 

caPitanio nazzareno 1,# 

1 Dpt. of Clinical and Experimental Medicine, University of Foggia; 

2 Dpt. of Cellular and Molecular Biology and Patology, University of Naples Federico II; 

3 Dpt. of Biological Sciences, University of Naples Federico II; 

4 Institute of Experimental Endocrinology and Oncology, National Research Council, Naples 

Down syndrome is the commonest aneuploidy in humans leading in 50 % of newborns 

to defective heart development. The upregulation of Hsa21 genes in hearts of trisomic 

human fetuses causes the downregulation of genes coding for proteins involved in the 

OXPHOS and in mitochondrial biogenesis. In this study we combined functional, structural 

and gene expression analyses to verify the occurrence of mitochondrial dysfunctions in 

primary foetal fibroblasts from trisomic subject without and with congenital heart defect. 

As compared with euploid-derived samples trisomic fibroblasts unveiled impairment of 

the mitochondrial respiratory activity, specific inhibition of complex I, enhanced ROS 

production and increased level of intramitochondrial calcium. These mitochondria-related 

alterations were more markedly observed in fibroblasts derived from cardiopathic subjects 

and were rescued following treatment with the mitochondrial calcium uniporter inhibitor 

ruthenium red. Measurement of the mtDNA copy number/cell resulted in a significant 

reduction in trisomic fibroblasts wherein altered cristae morphology was also observed. 

Gene expression analysis confirmed in trisomic fibroblasts upregulation of the Hsa21 

genes RIP140, DYRK1A and RCAN1 and dysregulation of the mitochondria-linked genes 

PGC-1α, NRF1 and IMMT. Our findings support the proposal of a pathogenetic model 

whereby overexpression of the repressor protein RIP140 causes dowregulation of PGC- 

1α, a key gene in mitochondrial biogenesis and function. The upregulation of DYRK1A and 

RCAN1 might further reduce PGC-1α expression via the CaN/NFATc pathway specifically 

in cardiopathic trisomic subjects. The reduction in PGC-1α might affect mitochondrial 

biogenesis, by decreasing NRF1, dysregulate intracellular Ca 2+ homeostasis, via PPARγ, 

and cristae pattern and integrity, by IMMT. Most notably these effects appear to be 

exacerbated in the context of a specific genetic background in cardiopathic trisomic 

subjects thereby resulting in a more pronounced chronic pro-oxidative state which in 

turn could further contribute to impair a normal heart development. The availability of a 

number of drugs, enabling to correct the afore-mentioned altered pathways in trisomic 

subjects, underlines the clinical relevance of our findings. 


257


Retinoids and PKA signal transduction interaction regulate 

activity and level of complex I in human keratinocytes 

1 Scacco Salvatore, 1 PaPa franceSco, 1 nicaStro annarita, 

1 Sardaro nicola, 2 liPPoliS roSa, SiGnorile anna, 1,2 PaPa SerGio. 

1. Department of Basic Medical Sciences, University of Bari “A. Moro”, Italy. 

2. Institute of Bioenergetics and Biomembranes, CNR, Bari, Italy. 

All-trans-retinoic acid (ATRA) is one of the biologically active metabolites of vitamin A, 

which plays an important role in cell differentiation and proliferation (MB. Reeves et al. 

2007). The molecular basis of its action has not been fully elucidated. It was previously 

shown (F. Papa et al. 2007) that ATRA treatment of normal human keratinocytes 

resulted in growth suppression, increase of complex I content and reduction of the 

NADH-UQ oxidoreductase enzymatic activity. These effects were associated with 

enhanced level of GRIM-19. It has been observed that in ATRA treated cells significant 

increase of the ubiquitous Serine/Threonine phosphatase (PP2A) activity, showing an 

impact on the cAMP-PKA signal transduction pathway. In the current study, we have 

investigated the induction of cAMP-PKA signaling, by dibutyryl cyclic AMP or okadaic 

acid, on complex I activity. 

The results show that the co-incubation of normal human keratinocytes with ATRA and 

okadaic acid for 72 hours, results in no change of both activity and level of complex 

I. Exposure of 72 hours ATRA treated cells to okadaic acid for a short-term (1 hour) 

increase the activity of complex I without any variation of protein level. These results 

show that promotion of cAMP-PKA signaling, by dibutyryl cyclic AMP or okadaic acid, 

can reverse the effects ATRA treatment on complex I activity. These observations 

indicate an interplay between ATRA and PKA signal transduction in the regulation of 

cellular bioenergetics. 

The cellular level of mature complex I depends on two opposite processes: 

protein biogenesis and protein degradation. It is possible that PP2A mediates 

dephosphorylation the GRIM-19 protein, prevents its degradation by proteasome, 

favoring the subunit accumulation. 


258


Proteomic expression profile in mitochondria keratinocytes 

ATRA treated 

1 liPPoliS roSa, 2 PaPa franceSco, 2 nicaStro annarita, 2 Sardaro 

nicola, 2 Gnoni antonio and 2 Scacco Salvatore 

1. Istituto di Bioenergetica e Biomembrane, CNR, Bari, Italy. 

2. Dipartimento di Scienze Mediche di Base, University of Bari “A. Moro”, Italy. 

All-trans-retinoic acids (ATRA) is a group of metabolites related to vitamin A that 

influences the gene expression and protein production. (S. Gibbs et al., 1996). The 

molecular basis of their action has not been fully elucidated. Mitochondrial proteome 

has to be considered as a no static entity that shows characteristic changes according 

to the functional state of the cell. A wide-search proteomic approach was used to 

monitor the effect of ATRA on the exponentially growing mitochondria keratinocytes 

protein profile. By this approach a comprehensive overview of keratinocyte 

mitochondria proteomic profile of was obtained. Respect to control, in ATRA treated 

cells, a great number of mitochondria proteins, is up and down regulated. It has been 

shown that ATRA cell-treatment affect proteins witch are synthesized in very low 

amount, as reported for regulatory proteins. 

Two proteins spots, down regulated in mitochondria of keratinocytes ATRA treated, 

are identified as ATP synthase subunit Beta, component of complex III, and protein 

disulfide-isomerase A6. The significant decrease of ATP synthase point the possible 

impairment of oxidative phosphorylation by retinoid acid. Western blot analysis 

revealed, also, in the same cells, significant increase of GRIM-19 expression, an 

indispensable component of mitochondrial complex I that plays an essential role in 

the mitochondrial function. These results indicate an effect of ATRA on the expression 

of mitochondrial OXPHOS complexes. 


259


ExTRAMITOCHONDRIAL OxIDATIVE PHOSPHORYLATION: 

A NEW MODEL FOR COUPLING BETWEEN RESPIRATORY 

COMPLExES AND F o F 1 -ATP SYNTHASE 

ravera Silvia, Panfoli iSaBella, Bartolucci Martina, calzia daniela, 

aleSSandro Morelli 

Biochemistry Lab. Dep.t of Pharmacology, University of Genova, Genova Italy. 

F 1 F o -ATP synthase, produces more than 85% of cellular ATP through oxidative 

phosphorylation (OXPHOS), by a rotary catalytic mechanism. 

Recent papers report a more pervasive expression of ATP synthase than expected 

[Reviewed in 1]. Isolated myelin vesicles [2] and retinal rod outer segment disks [3] 

consume oxygen and synthesize ATP when energized with NADH, Succinate, fumarate, 

3-ketoglutarate and citrate, contain catalytically respiratory complexes sensitive to 

the common inhibitors [1,2] and all of the glycolytic and the Krebs Cycle enzymes [4]. 

Data were also confirmed by TEM and confocal microscopy, using antibody against 

the OXPHOS proteins and fluorescent probes, as MitoTracker and JC-1 [2,3]. 

Oxygen consumption by Plasmablasts (PB), cells enriched in endoplasmic reticulum, 

was significantly higher when stimulated with NADH than in quiescent blasts (B) 

(Ravera et al. Unpublished data). Moreover PB displayed a significantly different WB 

signal ratio of ATP synthase vs. ANT with respect to B. 

Consistently with the cited evidence of an extramitochodnrial OXPHOS, an upgrading 

of the current model for proton (H + ) transfer from respiratory complexes to F o is 

proposed. In line with evidence suggesting that H + move within the bilayer, rather than 

exchanging with the aqueous phase [5] , we postulate a phase separation among 

H + transfer from F o to the respiratory complexes. H + would reside in the proteolipid 

membrane, while ATP synthesis takes place in the aqueous phase. This model is 

consistent with recent structural data [6-7]. 

[1] I. Panfoli et al., Expert Rev Prot 8 (2011) 231-239. 

[2] S. Ravera, et al., Int. J. Biochem. Cell Biol. 41 (2009) 1581-1591. 

[3] I. Panfoli, et al., Int. J. Biochem. Cell Biol. 41 (2009) 2555-2565. 

[4] I. Panfoli, Biochimie 93 (2011) 1565-1575. 

[5] T.H. Haines. PNAS 80 (1983) 160-164. 

[6] R.G. Efremov, L.A. Sazanov. Nature 476 (2011) 414-420. 

[7] W.C. Lau, J.L. Rubinstein. Nature 481 (2012) 214-218. 


260

Nucleotidi, 

Acidi Nucleici, 

Genomi 

NAG

Nucleotidi, Acidi Nucleici, Genomi NAG 1 

Recurrent copy number abnormalities and copy neutralloss 

of heterozygosity in colorectal cancer revealed by 

genome-wide SNP array 

BarreSivincenza 1,2 , MuSSo nicolò 1,2 , caPizzi carMela 2 , 

Privitera Giovanna 3 , luca tonia 3 , caStorina SerGio 3,4 , condorelli daniele filiPPo 1,2 

1 Department of Chemical Sciences, Section of Biochemistry, University of Catania, Italy, 

2 Laboratory on Complex Systems, Scuola Superiore di Catania, University of Catania, Italy; 

3 Fondazione Mediterranea “G.B. Morgagni”, Catania, Italy; 

4 Department of Bio-Medical Sciences, University of Catania, Italy 

Two forms of genetic instability have been observed in colorectal cancer (CRC): 

chromosomal instability, that is present in 85% cases and is characterized by 

structural and numerical chromosomal abnormalities (aneuploidy); and microsatellite 

instability (MSI), characterized by a deficiency of the mismatch repair system and 

associated to a normal or quasi-normal karyotype. In the present study we analysed 

a series of 51 CRC samples and their related normal mucosae by high resolution 

genomic arrays (Affymetrix SNP 6.0 arrays) and set up a series of bioinformatics tools 

that allow the generation of a rapid report on broad (>25% of a chromosomal arm) 

and focal somatic copy number abnormalities (BCNAs and FCNAs respectively), 

somatic homozygous deletions, focal high level amplifications, and regions of loss of 

heterozygosity (LOH). MSI tumors represented 14% of our CRC series and showed 

a median values of somatic BCNAs of 3 (range 1-6), while microsatellite stable (MSS) 

tumors showed a median value of 12 (range 0-25). On the basis of results obtained in 

mucosa samples an arbitrary threshold could be adopted to distinguish absent/very 

low chromosomal instability (CIN-, number of BCNAs < 3 BCNAs) from moderate/ 

high chromosomal instability (CIN+, number of BCNAs > 3 BCNAs). According to this 

definition four CRC different groups can be distinguished: MSS/CIN+ (76% of total 

samples), MSS/CIN- (10%), MSI/CIN- (8%), MSI/CIN+ (6%). Therefore simple analysis 

of BCNAs number confirms that, in the majority of cases, chromosomal instability 

and microsatellite instability are mutually exclusive, but suggests the existence of 

a small tumor subgroup with double genomic instability (MSI/CIN+). No correlation 

was observed between the number of tumor associated CNAs and the number of 

somatic copy neutral-LOH regions, suggesting that different mechanisms underlie 

such chromosomal abnormalities. Analysis of recurrent CNAs identified somatic 

homozygous deletions that cannot be ascribed to regions of inherent fragility. 

Category: Nucleotidi, acidi nucleici e genomi 

Keyword: Colorectal cancer, SNP array, copy number abnormalities 

Poster Presentation 


262


A siRNA approach specifically targets the mutant allele and 

reduces mutant collagen in Brtl mice, a murine model for 

osteogenesis imperfecta 

Gioia roBerta 1 , rouSSeau Julie 2,3 , lieuBeau Blandine 4 , 

heyMann doMinque 2,3 , BeSio roBerta 1 , roSSi antonio 1 , 

Marini Joan c 5 , trichet valerie 2,3 , and forlino antonella 1 

1 Department of Molecular Medicine, Section of Biochemistry, University of Pavia, Italy 

2 INSERM, UMR 957, Nantes, France 

3 Université de Nantes, Nantes Atlantique Universités, Laboratoire de physiopathologie de la Résorption Osseuse et 

Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, Nantes, France 

4 ONIRIS, Nantes-Atlantic College of Veterinary Medicine, Food Science and Engineering, F-44307, 

Immuno-endocrinology unit, INRA U707, F-44307 Nantes, France 

5 Section of Connective Tissue Disorders, Bone and Extracellular Matrix Branch (BEMB), NICHD, NIH, Bethesda, MD. 

Classical osteogenesis imperfecta (OI) is a dominant “brittle bone disease” caused 

mainly by mutations in type I collagen genes. Severe phenotype in patients could be 

converted to a milder outcome by a specific suppression of the mutated allele. Aim of 

our work was to evaluate the feasibility of a silencing gene therapeutic approach for 

OI treatment. The OI Brtl mouse carrying a typical glycine substitution (Gly349Cys) in 

a col1a1 allele, was used. 

Six silencing RNAs (siRNA) and two short hairpin (shRNA) were designed to target 

the mutated col1a1 exon 23 of Brtl by differential positioning of the point mutation 

mismatches. HEK cells and primary Brtl fibroblasts were used for transfection 

experiments. Expression levels of luciferase in HEK cells and of total col1a1 and 

mutant and WT alleles in Brtl fibroblasts were evaluated by means of qPCR. Collagen 

from Brtl cells was analyzed by SDS-PAGE. 

In transfected HEK cells, only two siRNAs did not change luciferase activity when the 

luciferase gene contained the wild-type col1a1 exon 23 sequence within its 3’-UTR, 

whereas they enabled a 2-fold decrease of the luciferase activity when luciferase gene 

contained the mutated col1a1 exon 23 sequence. These two siRNAs, transfected in 

Brtl fibroblasts enabled a 60% reduction of the mutated col1a1 transcript versus a 

25% reduction of the wild-type one as measured by allele specific qPCR. Additionally, 

shRNAs corresponding to these two effective and selective siRNA were cloned in 

a lentiviral vector and one of them enabled a stable 45% specific suppression of 

the mutated col1a1 without effecting the WT allele in Brtl fibroblasts and caused 

50% reduction of the mutant collagen. Our data support the use of shRNAs as a 

strategy to specifically suppress the mutant allele making appealing the modification 

of mesenchymal stem cells of OI patients for autografts transplantation. 

Grant support: AFMTHELETON 16017, France to AF and VT. 


263


Uncovering the physiological role of human cytidine 

deaminase through the characterization of its functional 

variants 

carPi franceSco Martino a , Micozzi daniela a , Pucciarelli Stefania a , 

Polzonetti valeria a , vincenzetti Silvia B 

a University of Camerino, School of Biosciences and Biotechnology, 

Via Gentile III da Varano, Camerino 

b University of Camerino, School of Veterinary Medical Sciences, 

Via Corconvallazione, Matelica 

Pyrimidine pro-drugs must be converted into active compounds through a series of 

reactions involving the enzymes of the pyrimidine salvage pathways. Inter-individual 

variation in the activity of these metabolizing enzymes can affect the extent of pro-drug 

activation and, as a result, act on the efficacy of chemotherapy treatment. Two Single- 

Nucleotide Polymorphisms (SNPs) in the cytidine deaminase gene were detected: one, 

the 79A>C SNP resulting in a K27Q change (1, 2), and the 208G>A SNP producing an 

alanine to threonine substitution (A70T) within the conserved catalytic domain (3). In 

this work we focused on the 79A>C SNP and its products named Q27 and K27 CDA 

variants in order to highlight their functional role. As a result, the lysine residue in 27 

position establishes an ionic interaction with a glutammic acid residue at 24 position 

that is responsible for a greater structural stability of the variant carrying the K27 with 

respect to the Q27 variant. Furthermore, the presence of a glutamine residue in 27 

position, instead of lysine, is responsible for the major catalytic efficiency towards 

the natural substrates and towards the antileukemic agent cytarabine. In addition we 

assessed the kinetic properties of the recombinant heterozygous human CDA (Q/K 

variant) by a dual cloning on the plasmid pETDuet-1 in order to get a recombinant 

plasmid expressing simultaneously both CDA genes. The Q/K CDA obtained was able 

to form a tetramer giving rise to five isoforms of cytidine deaminase formed by the 

combination of the two monomers (Q27 and K27). However, Q/K CDA resulted to be 

a weaker catalyst with respect to the Q27 and K27 CDA variants. 

References 

Laliberte J, Momparler RL. Cancer Res. (1994) 54, 5401–5407. 

Kühn K, et al. (1993) Biochem Biophys Res Commun. 190, 1–7. 

Yue L, et al.. Pharmacogenetics. (2003)13, 29-38. 


264


Regulation of The P2Y-Like Receptor GPR17 by 

Agonist-Induced Desensitization in transfected 

1321N1 cells 

daniele S 1) , trincavelli Ml 1) , fuMaGalli M 2) , GiacoMelli c 1) , 

aBBracchio MP 2) , Martini c 1) 

1) Dept. of Psychiatry, Neurobiology, Pharmacology and Biotechnology, 

University of Pisa, Pisa, Italy 

2) Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, 

Dept. of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy 

GPR17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDPglucose) 

and cysteinyl-leukotrienes (cysLTs, as LTD 4 ) 1 . By bioinformatic analysis, two 

distinct binding sites have been hypothesized to be present on GPR17 and, recently, 

we have demonstrated that these sites communicate with each other with a clear 

hierarchy 2 . Both cysLT and purinergic ligands induced a time- and concentrationdependent 

GPR17 loss of response; this phenomenon was accompanied by receptor 

internalization. Upon agonist removal, receptor resensitization occurred with the 

typical kinetics of GPCR. Moreover, activation of GPR17 by UDP-glucose (but not 

viceversa) induced a partial heterologous desensitization of LTD 4 -mediated responses, 

suggesting that nucleotides have a hierarchy in producing desensitizing signals 2 . 

In this work, we investigated the role of GRK and beta-arrestins in the regulation 

of GPR17 responses, in 1321N1 cells transfected with HA-tagged GPR17. Both 

purinergic and cysLT agonists induce a time-dependent receptor phoshorylation on 

Threonine and Serine residues. Both GPR17 agonists induce GRK2 translocation 

from cytoplasm to plasma membrane, and the physical association between GRK2 

and GPR17, but these effects were more marked with the cysLT ligand with respect 

to the purinergic agonist. GRK2 appears to be essential in LTD 4- induced GPR17 

homologous desensitization, while it is not primarily involved in GPR17 homologous 

and heterologous desensitization induced by UDP-glucose. Both ligands activate 

intracellular ERK phosphorylation with a different kinetics and through a G proteindependent 

and G protein-independent mechanism for LTD 4 and UDP-glucose, 

respectively. 

At the light of these results we speculate that GPR17 activation by cysLTs and 

purinergic ligands directs the receptor towards different intracellular route. 

1 Ciana P et al. EMBO J. 2006 25:4615-277 

2 Daniele S. et al. J Pharmacol Exp Ther. 2011 338:559-67. 


265


NAD BIOSYNTHESIS ENZYMES FROM THE BACTERIAL 

WORLD AS VALUABLE TOOLS FOR VITAMIN B3 

MONONUCLEOTIDES QUANTIFICATION IN MAMMALS 

carradori rita 1 , Mori valerio 1 , aMici adolfo 1 , 

orSoMando GiuSePPe 1 , Sorci leonardo 1 , Mazzola franceSca 1 , 

Bocci Paola 2 , ruGGieri Silverio 2 , raffaelli nadia 2 

1 Department of Clinical Sciences (DISCO), Section of Biochemistry, and 

2 Department of Nutritional, Environmental and Agricultural Sciences (D3A), 

Polytechnic University of Marche, Via Ranieri, 67, 60131 Ancona, Italy 

The pyridine mononucleotides NMN and NAMN have been for a long time considered 

as vitamin B3-derived NAD precursors, with no other biological function in mammals. 

However, recent mounting evidence suggests a direct role of NMN as a key 

intermediate in various molecular processes, like those affecting axon viability or 

glucose-related metabolic disorders, underscoring the need for suitable quantitative 

detection methods. The analytical determination of these mononucleotides in 

biological samples has been so far hampered by their low amounts and chemical 

properties. To date, only a few NMN- and NaMN-detection methods, mainly relying 

on radioactivity, fluorimetry, or sophisticated LC/MS analyses, have been reported, 

sometimes with contrasting results. Therefore, the pathophysiological relevance of 

these molecules is still unclear. Here we report on two novel enzyme-coupled assays 

of wide applicability for the determination of NMN and NaMN in mammalian systems. 

These methods are based on the fluorimetric detection of the mononucleotides, either 

after direct chemical derivatization, or after their enzymatic conversion to NAD and its 

subsequent quantitation by a cycling assay. We exploit NAD biosynthetic enzymes 

from bacterial sources with peculiar catalytic properties that our laboratory first 

contributed to identify and characterize, i.e. i) NaMN adenylyltransferase of NadD 

family specifically converting NaMN into deamido-NAD, ii) NMN deamidase and 

NMN synthetase responsible for the specific interconversion of NMN into NaMN and 

viceversa, respectively. Both methods resulted to be as sensitive as the LC/MS based 

assays, with the advantage of being much faster and less expensive. They allowed 

us to perform a preliminary metabolic profiling of pyridine mononucleotides in mouse 

tissues and sera. Remarkably, they can be further employed for measuring all known 

NMN/NAMN synthesizing enzyme activities, whose differential contribution to NAD 

synthesis in physio-pathological conditions is a matter of increasing interest. 


266


Involvement of cN-II in prodrug metabolism 

daniela nicole filoni 1 , nicoletta SchiBeci natoli Scialli 1 , 

roSSana PeSi 2 , SiMone alleGrini 1 , Maria Grazia tozzi 2 

1 Dipartimento di Chimica e Farmacia, Università di Sassari; 

2 Dipartimento di Biologia, Università di Pisa 

Cytosolic 5’-nucleotidase/phosphotransferase II (cN-II), specific for 6-oxypurine 

monophosphates, is a widespread enzyme with the highest activity in tissues 

characterized by a high turnover of nucleic acids, as in human colon carcinoma and 

malignant lymphocytes 1 . As a member of the oxypurine cycle, cN-II is involved in the 

regulation of intracellular concentration of IMP, AMP, GMP and also PRPP, therefore in 

the regulation of purine and pyrimidine de novo and salvage synthesis 2 . 

Clinical interest in cytosolic 5’-nucleotidase II has been increasing enormously in 

these last years, not only because its mRNA level seems to be a prognostic factor 

in adult acute myeloid leukemia, but also for its possible involvement in anticancer 

and antiviral prodrug metabolism, as underlined by in vitro studies 3,4 . CN-II, in 

fact, not only could be responsible for resistance to nucleoside analogues with 

its catabolic function, but it might also take part in their activation pathway by its 

phosphotransferase activity. Notwithstanding many researches have been carried 

out in order to unravel this point, several contrasting aspects remain to be solved. 

For this reason we performed ex vivo experiments, using human embryonic kidney 

cells overexpressing, or not, cN-II treated with three different nucleoside analogs: 

cytarabine, fludarabine and gemcitabine. In fact, even if the role of cN-II in nucleoside 

analogue resistance has been extensively studied, further investigation is required 

to throw light on several aspects which still remain unclear; in addition, there is little 

evidence from ex vivo studies that 5’-nucleotidases actually phosphorylate (deoxy) 

nucleoside analogues. 

Very preliminary results seem to suggest not only that cN-II is directly involved in 

prodrug metabolism, but also new cytotoxic strategies for those prodrugs considered 

chain terminators. 

1. Tozzi et al., 1991, Arch. Biochem. Biophys. 

2. Ipata and Tozzi, 2006, Purin. Signall. 

3. Galmarini et al., 2001, Leukemia. 

4. Jordheim and Dumontet, 2007, BBA. 


267


Differential Roles of SNAI2/SLUG Transcription Factor in 

the Epithelial and Stromal Compartments of the Human 

Prostate Cancer 

Sorrentino carlo 1,3 , tuPone Maria Grazia 1,3 , di Meo Serena 1,3 , 

MuSiani Piero 1,3 , di ilio carMine 2,3 , di carlo eMMa 1,3 

1 Section of Anatomic Pathology and Molecular Medicine, Department of Medicine 

and Sciences of Aging, and 2 Department of Biomedical Sciences, “G. d’Annunzio” 

University of Chieti-Pescara, Chieti, Italy; 

3 Ce.S.I. Aging Research Center, “G. d’Annunzio” University Foundation, Chieti, Italy. 

Introduction. SNAI2/SLUG is a zinc-finger transcription factor that acts as a master 

regulator of cell migration, by triggering epithelial-mesenchymal transition (EMT) 

during embryonic development and tumor metastasization, and also regulates cellcycle 

progression and survival. In this study, we investigated its role in prostate cancer 

(PCa) under normal and androgen deprivation therapy (ADT) conditions. 

Methods. Laser capture microdissection and real-time RT-PCR were used to determine 

SLUG gene expression levels in normal and cancerous prostatic epithelium and 

stroma, differentiating neoplastic foci with low versus high Gleason grade. Epithelial 

and stromal cells were isolated from cancerous and normal prostate specimens from 

55 untreated and 35 ADT-treated patients who underwent radical prostatectomy 

for PCa. Immunostainings and confocal microscopy were used to visualize SLUG 

expression at the protein level. 

Results. SLUG gene expression levels were significantly (P


The Embryonic Stem Cell Marker SOx2 is Down-Regulated 

in Prostate Cancer and Up-Regulated by Androgen 

Withdrawal 

di Meo Serena 1,3 , Sorrentino carlo 1,3 , tuPone Maria Grazia 1,3 , 

MuSiani Piero 1,3 , di ilio carMine 2,3 , di carlo eMMa 1,3 

1 Section of Anatomic Pathology and Molecular Medicine, Department of Medicine 

and Sciences of Aging, and 2 Department of Biomedical Sciences, “G. d’Annunzio” 

University of Chieti-Pescara, Chieti, Italy; 

3 Ce.S.I. Aging Research Center, “G. d’Annunzio” University Foundation, Chieti, Italy. 

Introduction. SOX2 transcription factor is essential to maintain self-renewal of 

undifferentiated embryonic stem cells (ESC) and plays a role in differentiation, 

proliferation and apoptosis. Consistent with the hypothesis that stemness and 

embryonic pathways may reactivate during oncogenesis, SOX2 is over-expressed in a 

variety of tumors and also associated with cancer aggressiveness. In prostate cancer 

(PCa), however, its role is still unclear, since contrasting data are reported about its 

expression in cell lines and human samples. 

Methods. We used laser capture microdissection followed by real-time RT-PCR 

to determine SOX2 gene expression levels in the normal and cancerous prostatic 

epithelium and stroma, and discriminate neoplastic foci with low versus high Gleason 

grade. Epithelial and stromal cells were isolated from cancerous and normal prostate 

specimens from 55 untreated and 35 androgen deprivation therapy (ADT)-treated 

PCa patients who underwent radical prostatectomy for PCa, and normal prostate 

specimens from 12 patients who had undergone cystoprostatectomy for bladder 

cancer (control patients). 

Results. SOX2 gene expression level was significantly (P


Correlation between Slug transcription factor and miR-221 

in breast cancer cells 

laMBertini eliSaBetta, lolli andrea, vezzali federica, 

Penolazzi letizia, GaMBari roBerto and roBerta Piva 

Department of Biochemistry and Molecular Biology, 

University of Ferrara, 44121 Ferrara, ITALY 

Breast cancers and their metastatic progression are mainly directed by epithelial to 

mesenchymal transition (EMT), a phenomenon supported by specific transcription 

factors and miRNAs only in part known. 

In order to investigate a possible correlation between Slug transcription factor and 

miR-221, we performed gene silencing against Slug and miR-221 in aggressive breast 

cancer cell lines and evaluated by qRT-PCR and Western blot the expression of genes 

involved in supporting the breast cancer phenotype. 

We showed that Slug silencing significantly decreased the level of miR-221, positively 

affected the expression of epithelial markers including Estrogen Receptor α, 

E-cadherin and TRPS1, and caused a decrease of vimentin and a strong inhibition of 

cell migration evaluated by wound healing assays. We demonstrated that miR-221 is 

a Slug target gene, and identified by chromatin immunoprecipitation a specific region 

of miR-221 promoter that is transcriptionally active and is involved in the binding 

of Slug “in vivo”. Complete knockdown of miR-221 expression by transfection with 

antagomiR-221, causes a significative decrease of Slug expression suggesting that 

Slug is not a direct target of miR-221, but rather that a negative regulator of Slug 

could be a miR-221 target. However, miR-221 knockdown significantly attenuated 

the gap closing in the cells, but not as much as that observed in Slug-repressed 

cells. In addition, rescue experiment with pre-miR-221 did not balance siSlugmediated 

effects, suggesting that Slug silencing could significantly protect cells from 

progression towards an aggressive phenotype or metastatic stimuli that, in this case, 

are represented by miR-221 overexpression. 

For the first time the evidence of a functional link between Slug and miR-221 in breast 

cancer cells is reported. Therefore, in the context here analyzed we may conclude 

that miR-221 expression is, in part, dependent on Slug and that Slug plays a more 

important role than miR-221 in cell migration and invasion. 


270


Preclinical testing of PNP inhibitors analogues of 

Immucillin-G for the therapy of Lesch-Nyhan disease: 

preliminary in vitro studies 

Baldini eva 1 , JacoMelli GaBriella 1 , corelli federico 2 , 

Micheli vanna 1 

1 Dipartimento di Biotecnologie, 

2 Dipartimento Farmaco Chimico Tecnologico - Università di Siena 

Lesch-Nyhan Disease (LND) is a rare X-linked genetic disease characterized by 

hyperuricaemia, gout and severe neurological syndrome. Hypoxanthine-guanine 

phosphoribosyltransferase (HPRT) deficiency occurs causing uric acid, hypoxanthine 

and xanthine accumulation. Uric acid excess is commonly managed by xanthine 

oxidase inhibitors, yielding in xanthine and hypoxanthine production. The biochemical 

basis of the neurological pathology is still unclear, and no effective therapy is available. 

Deazaguanine derivatives inhibiting human purine nucleoside phosphorylase (PNP) 

were used in clinical trials to lower uric acid in gouty patients. This study is aimed at 

testing the reliability of PNP inhibitors as a therapy for LND urate and hypoxanthine 

excess. 

A 9-deazaguanosine analogue of Imucillin-G (Semeraro et al. J. Med. Chem 2006; 

compound 1a) was used as PNP inhibitor. PNP inhibition and apparent kinetic 

constants were measured in crude lysates from normal erythrocytes and fibroblasts 

by HPLC-linked methods (Micheli et al. BBA 2002). Nanomolar 1a concentrations 

markedly decreased Vmax not affecting Km ino suggesting non-competitive inhibition. 

Primary cultures of control subjects skin fibroblasts were grown with DMEM plus 

10% h.i. FCS, 1% penicillin/ streptomycin, at 37°C and 5 % CO 2 , in the presence or 

absence of 1a (10 nM and 1 µM) and of 20 µM inosine for 24, 48 and 72 h. Perchloric 

extracts obtained from medium and cells were then analysed by HPLC. No significant 

reduction of viable cells was demonstrated by MTT test and no remarkable alteration 

in cell nucleotide pattern was found after any incubation condition, while 1 µM 1a 

caused hypoxanthine and xanthine decrease together with inosine and guanosine 

increase in the culture medium. 

Present data demonstrate effective PNP inhibition by low 1a concentration, with no 

appreciable toxicity. Studies are in progress in HPRT-deficient cells, in view of a new 

therapeutic strategy against oxipurine accumulation. 

Acknowledgements: this study is funded by LND Famiglie Italiane ONLUS 


271


Tobramycin inhibits interleukin-8 gene expression and 

acts as suppressor of premature termination codons in the 

absence of NMD 

altaMura nicola 1 , GaMBari roBerto 2 , BorGatti Monica 2 

1 Institute of Biomembrane and Bioenergetics, CNR, 

Via Amendola 165/A, 70126 Bari, Italy; 

2 Department of Biochemistry and Molecular Biology, 

University of Ferrara Via Fossato di Mortara, 74, 44121 Ferrara, Italy; 

Aminoglycosides are widely used for the treatment of cystic fibrosis (CF), a fatal, 

autosomal, recessive genetic disease characterized by persistent pulmonary 

infection and extensive lung inflammation. In addition to their role as antibiotics, 

aminoglycosides have been shown to act as suppressors of nonsense mutations that 

generate premature translation termination (PTCs). PTCs constitute the molecular 

basis of many genetic diseases, including CF (5-10% of cystic fibrosis alleles contain 

PTCs) as lead to the synthesis of truncated non-functional or partially functional 

protein. Suppression of translation terminations at PTCs (read-through) has been 

developed as a therapeutic strategy to restore full-lenght protein in several genetic 

diseases. Phenotypic consequences of PTCs can be exacerbated by the nonsensemediated 

mRNA decay (NMD) pathway that detect and degrade PTC containing 

mRNA. Modulation of NMD, therefore, is also of interest as a potential target for the 

suppression therapy. Tobramycin is an aminoglycoside antibiotic, normally used to 

treat Pseudomonas aeruginosa pulmonary infection in CF patients. In the present 

study we have investigated, on one hand, the properties of Tobramycin as a modulator 

of the expression of IL-8 associated with inflammation of the CF airway pathology in 

CF IB3-1 cells induced with pro-inflammatory cytokine TNF-α. On the other hand, by 

using the yeast as a genetic system, we have examined the ability of Tobramycin to 

suppress PTCs as a function of the presence or absence of NMD. Results demonstrate 

that Tobramycin is a good inhibitor of IL-8 expression and exhibits read-through ability 

on PTCs in conditions in which NMD is not operating in the cell. 

Acknowledgements. This work was supported by contribution from FFC (Italian Cystic Fibrosis Research 

Foundation) to M.B. (FFC#2/2010), MIUR (Italian Ministry of University and Research), Fondazione Cariparo 

(Cassa di Risparmio di Padova e Rovigo), CIB and Telethon GGP10124. 


272


Peptide nucleic acids targeting miR-221 modulate p27 Kip1 

expression in breast cancer MDA-MB-231 cells 

BroGnara eleonora 1 , faBBri enrica 1 , aiMi faBio 2 , alex 2 , 

Bianchi nicoletta 1 , finotti aleSSia 3 , BreveGlieri Giulia 3 , 

BorGatti Monica 1 , corradini roBerto 2 , Marchelli roSanGela 2 , 

GaMBari roBerto 1,2 

1 BioPharmaNet, Department of Biochemistry and Molecular Biology, 

Ferrara University, Ferrara, Italy; 

2 Department of Organic and Industrial Chemistry, University of Parma, Italy; 

3 Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy 

of Thalassaemia, Biotechnology Center, Ferrara University, Ferrara, Italy; 

Peptide Nucleic Acids (PNAs) are DNA mimics where pseudo-peptide backbone 

is composed of N-(2-aminoethyl)glycine units. PNAs were found to be excellent 

candidates for antisense and antigene therapies. MicroRNAs (miRNAs, miRs) are a 

family of small noncoding RNAs that regulate gene expression by sequence-selective 

targeting of mRNAs, leading to a translational repression or mRNA degradation. 

MicroRNAs playing a crucial role in the initiation and progression of human cancer are 

defined as oncogenic miRNAs (oncomiRs). In many types of tumors, high levels of 

miR-221 is found, associated with inhibition of expression of its target p27 Kip1 mRNA, 

which encodes a very important onco-suppressor protein. In this study we describe 

the activity of a PNA targeting micro-RNA 221, associated to breast cancer. We first 

analyzed the expression of miR-221 and p27 Kip1 mRNA in different breast cancer cell 

lines and found that high expression of miR-221 and low production of p27 Kip1 are 

present in MDA-MB-231 cells. PNA against miR-221 were designed in order to bind 

very efficiently to the target RNA strand and to undergo efficient uptake in target cells. 

A polyarginine-PNA conjugate targeted against miR-221 (Rpep-PNA-a221) showed 

both very high affinity for complementary nucleic acids and efficient uptake within 

target cells without the need of transfection reagents. Unmodified PNA with the same 

sequence displayed very poor cellular uptake. Consistently, only Rpep-PNA-a221 

strongly inhibited miR-221. Targeting miR-221 by PNA resulted in (a) lowering of the 

hybridization levels of miR-221 measured by RT-qPCR, (b) up-regulation of p27 Kip1 , 

mRNA and protein, measured by RT-qPCR and Western Blot analysis. The major 

conclusion of this study is that efficient delivery of anti-miR PNA through a suitable 

peptide carrier (Rpep-PNA-a221) leads to inhibition of miR-221 activity, altering the 

expression of miR-221 regulated functions in breast cancer cells. 


273


Transcription factors (TFs) negatively regulating γ-globin 

gene transcription: BCL11A is down-regulated during 

mithramycin induction of erythroid cells from β-thalassemia 

patients 

finotti aleSSia 1 , Bianchi nicoletta 1 , zuccato criStina 1 , BreveGlieri 

Giulia 1 , laMPronti ilaria 1 , BroGnara eleonora 1 , GaMBerini Maria 

rita 2 , BorGatti Monica 1 , GaMBari roBerto 1 

1 Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy 

of Thalassemia, Biotechnology Center, Ferrara University, Ferrara, Italy; 

2 Department of Reproduction and Growth, Paediatric and Adolescent Unit, 

S. Anna Hospital, Ferrara, Italy. 

Induction of fetal hemoglobin (HbF) is considered a new promising strategy to treat 

β-thalassemia, where the production of adult hemoglobin (HbA) is impaired by 

mutations affecting the β-globin gene. Three major loci (Xmn1-HBG2 single nucleotide 

polymorphism, HBS1L-MYB intergenic region on chromosome 6q, and BCL11A) 

contribute to high HbF production. These data are in line with a general concept that was 

followed using DNA binding drugs (DBD) or oligonucleotide decoys for transcription 

factors (decoy-ODN) able to inhibit TF/DNA interactions (the transcription factor decoy 

strategy, TFD); if the human β-globin genes are under negative transcriptional control 

caused by transcription factors exerting repressor activities, then targeting these TFs 

might be therapeutically relevant. Putative repressors of β-globin gene transcription 

are Oct-1, MYB and BCL-11A. 

We have studied the expression of BCL-11A during HbF induction of erythroid 

precursor cells (ErPC) from β-thalassemia patients treated with mithramycin. The 

levels of β-globin and BCL-11A mRNA were assayed by quantitative real-time RT- 

PCR; the production of hemoglobin by HPLC. 

The results obtained suggest that increase of β-globin gene expression and HbF 

production is preceded by a sharp decrease of BCL-11A levels in ErPC from 

β-thalassemia patients treated with 20 nM MTH. 

These results suggest that BCL-11A levels are associated with β-globin gene 

expression. Therefore, disrupting the bindings of the BCL-11A transcriptional complex 

to the β-globin gene promoter provides a novel approach for inducing expression of 

the β-globin genes. 

Acknowledgements. This work was supported by a grant by MIUR (Italian Ministry of University and 

Research), Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), CIB, by Telethon GGP10124. 

This research is also supported by Associazione Veneta per la Lotta alla Talassemia (AVLT) Rovigo. 


274

Neurochimica 

Neu

Neurochimica NEu 1 

Trimethyltin (TMT) treatment of PC-12 cells induces the 

release of group IIA secretory phospholipase A 2 (GIIA PLA 2 ). 

BiaGioni anGeli e. 1 , corvino v. 2 , MarcheSe e. 2 , Michetti f. 2 , 

nardicchi v. 1 , Bianconi M. 1 and Goracci G. 1 

Department of Internal Medicine, Section of Biochemistry, University of Perugia 1 and 

Institute of Anatomy and Cell Biology, Catholic University, Rome 2 

TMT is a neurotoxic organtin compound that induces neurodegeneration particularly in 

hippocampus (Geloso et al. 2011). Treatment of undifferentiated PC12 cells with TMT 

causes dysregulation of intracellular Ca 2+ and apoptotic cell death (Misiti et al. 2008; 

Piacentini et al. 2008). The same experimental model has been used to investigate 

on the effect of TMT on PLA 2 activity. PC12 cells express various PLA 2 types and 

among them Ca 2+ -dependent group IIA isoform (GIIA) is mainly localized in PC12 

mitochondria and its release from rat brain mitochondria, when membrane potential 

is reduced, was demonstrated (Macchioni et al. 2004) suggesting its involvement in 

neuronal cell damage (Goracci et al. 2010). In these series of experiments, cells were 

treated with TMT (0.5 – 5 M) for 5, 24 or 48 hours. PLA 2 activity was assayed in 

PC12 lysates and in culture medium using a fluorogenic substrate (PED6) by a new 

developed procedure. The treatment with TMT caused significant decrease of cellular 

PLA 2 activity which was dose and time dependent and it was accompanied by a 

parallel increase of enzyme activity in the culture media without significant changes of 

LDH activity. The released PLA 2 activity was almost completely blocked by a specific 

inhibitor of GIIA (Chiricozzi et al. 2010). It can be concluded that GIIA participates to 

the molecular events involved in neuronal cell death induced by TMT intoxication. 

Particularly, the released enzyme may cause the propagation of the toxic effect of TMT 

to the other cells of brain tissue contributing to the aggravation of neurodegeneration. 

Chiricozzi E. et al.(2010) J Neurochem 112, 1574-1583. 

Geloso M. C. et al. (2011) Neurochem Int. 

Goracci G. Et al. (2010) Mol Neurobiol 41, 274-289. 

Macchioni L. et al. (2004) J Biol Chem 279, 37860-37869. 

Misiti F. et al. (2008) Neurochem Int 52, 1092-1099. 

Piacentini R.et al.(2008) J Neurochem 105, 2109-2121. 


276


Pyruvate administration prevents cognitive deficits in a Tg 

animal model of Alzheimer’s disease 

iSoPi eliSa 1 , SenSi l. Stefano 1,2,3 

1 Molecular Neurology Unit - Ce.S.I., Chieti, Italy; 

2 Department of Neuroscience & Imaging, “G. D’Annunzio” University, Chieti, Italy; 3 

Departments of Neurology & Pharmacology, 

University of California Irvine, Irvine, California, USA 

Mitochondrial and metabolic dysfunctions play an important role in Alzheimer’s 

disease (AD) development and progression. Pyruvate, an energy substrate, has been 

shown to counteract Aβ-driven deregulation of glucose metabolism and is also able to 

protect cultured neurons against Aβ toxicity. In this study, we evaluated the potential 

neuroprotective effects of long-term administration of pyruvate in a AD transgenic 

mouse model, the 3xTg-AD mouse, that develops Aβ- and tau- dependent pathology. 

3xTg mice were treated with pyruvate [500 mg/kg, i.p; starting at the 3 months of age 

(m.o.a.; 3 times per week)] and compared with control mice receiving an IP saline 

solution. Analysis of memory performance with the Morris Water Maze (MWM) test 

revealed that pyruvate is effective in preventing short and long-term memory deficits in 

3xTg-AD mice at 12 m.o.a. No effects were seen as far as intraneuronal accumulation 

of Aβ or the appearance of hyperphoshorylated tau, thereby suggesting that pyruvate 

is likely to work on the AD-like deregulation of glucose metabolism in the brain of 

3xTg-AD mice. Overall these findings indicate that pyruvate can be a valuable, safe, 

and cheap tool to modify the progression of AD-related cognitive deficits. 


277


Microarray analysis of human neuroblastoma cells exposed 

to iron, β(1-42)-amyloid or the β(1-42)-iron complex 

Granzotto alBerto 1 , zatta Paolo 2 , SenSi l. Stefano 1,3,4 

1 Molecular Neurology Unit - Ce.S.I., Chieti, Italy; 

2 National Research Council, Biomedical Technology Institute (CNR-ITB), 

Metalloproteins Unit, Department of Biology, University of Padua, Padua, Italy; 

3 Department of Neuroscience & Imaging, “G. D’Annunzio” University, Chieti, Italy; 4 

Departments of Neurology & Pharmacology, 

University of California Irvine, Irvine, California, USA 

β-amyloid (Aβ) misfolding and deposition play a critical role in Alzheimer’s disease 

development and progression. Aβ aggregation is largely influenced by the presence 

of both endogenous and exogenous metal ions. In this study we performed 

microarray analysis of 35,129 transcripts in a neuronal-like cell line (SH-SY5Y) that 

was exposed to Aβ, Fe, or Aβ-Fe conjugates for 24 hours. Compared to Aβ or Fe, 

Aβ-Fe exposure resulted in the selective down- and up-regulation of 693 and 537 

transcripts, respectively. Analysis of gene expression profiles with Ingenuity Pathway 

Analysis (IPA) revealed that most of the differentially expressed genes are involved 

in cellular morphology maintenance. We observed under-expression of molecules 

associated with pathways that regulate neuronal structural integrity (TUBB, PP2A, 

RAB1A, INA). We also found an up-regulation of transcripts controlling enzymes 

and molecules (i.e.: PAK1, CD81, CRMP1, KLK8, MT3, and PLXNA2) that promote 

morphological abnormalities in hippocampal pyramidal neurons and in the mossy 

fibers. Aβ-Fe was also found to modify, to a lesser extent, transcripts involved in 

apoptotic pathways (BAK1, Bcl-2), in the cell response to oxidative stress (MT3, NFκB), 

calcium signaling (ACCN2), and glutamatergic neuro-transmission (GRIN2B). In 

summary, present findings suggest that Aβ-Fe can have a selective role in deregulating 

neuronal morphology and functioning. 


278


PLGA-NPs for Enzyme Replacement Therapy of 

Lysosomal Storage Diseases 

Polchi a. 1 , tancini B. 1 , toSi G. 2 , Severini G.M. 3 , MaGini a. 1 , 

urBanelli l. 1 , Bortot B. 3 , dolcetta d. 4 , vandelli M.a. 2 , forni f. 

2 and eMiliani c. 1 

1 Dept. of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy. 

2 Dept. of Pharmaceutical Sciences, & Dept. of Biomedical Sciences, University of 

Modena and Reggio Emilia, Italy. 

3 Dept. of Molecular Medicines, Institute of Maternal and Child Health IRCCS Burlo 

Garofolo, Trieste, Italy. 

4 “Mauro Baschirotto” Institute for Rare Diseases, B.I.R.D. Foundation, Vicenza, Italy. 

Lysosomal storage diseases (LSD) are a group of inherited disorders caused by 

the deficiency of lysosomal enzymes. There are no cures for LSD and treatment 

is mostly symptomatic. Enzyme Replacement Therapy (ERT) is safe and effective, 

but inefficient in the treatment of neurological manifestation, as exogenous enzymes 

cannot cross BBB. Polymeric nanoparticles (NPs) represent one of the most innovative 

non-invasive approaches for drugs-delivery. In particular, the use of nanocarriers for 

macromolecule delivery may allow to overcome some limits of the macromolecule 

therapeutic application. NPs can be modified to target specific cells and/or cross 

membrane barriers, i.e. BBB. Due to their biodegradability and biocompatibility, 

polylactide-co-glycolide (PLGA) NPs are one of the most widely applied systems 

for drug delivery. Here, we explored the use of acid alpha-glucosidase (GAA) loaded 

PLGA-NPs in Pompe Disease (PD) ERT. PD is a LSD characterized by the accumulation 

of high levels of glycogen particularly in cardiac, respiratory and skeletal muscles. So 

far PD treatment consisted of the administration of Myozyme®, a recombinant human 

GAA. Despite its efficacy, this therapy is expensive and requires long term infusion 

every 2 weeks. GAA-NPs may provide a suitable alternative to improve the stability/ 

integrity of the enzyme and avoid the risk of immunoreaction. The pharmacological 

efficacy of GAA-NPs was assessed on cultured PD fibroblasts. Our results showed 

that GAA-NPs were able to correct 50% of the enzymatic deficiency after a single 

administration (instead of the 12% obtained with the free enzyme). Moreover, the 

internalized GAA was correctly processed to the mature form, indicating that the 

enzyme reached lysosomes. These results indicate that PLGA- NPs may represent a 

useful drug-delivery tool applicable to a wide range of LSDs. Suitable modifications of 

the NPs may extend their application also to disorders with neurological involvement. 

Acknowledgments: Work supported by ELA Foundation (Agreement n. 2011-037C1B) 


279


Mass spectrometry based metabonomics of the 

cerebrospinal fluid in Multiple Sclerosis patients 

PieraGoStino daMiana 1,2 , di ioia Maria 3 , roSSi claudia 1,2 , zucchelli Mirco 1 , 

taSSoni valentina 1,2 , di ilio carMine 1,2 , luGareSi aleSSandra 1,3 , 

Sacchetta Paolo 1,2 , urBani andrea 1,3 , del Boccio Piero 1,2 

1 Centro Studi sull’Invecchiamento (Ce.S.I.), Università “G. d’Annunzio”, Chieti, Italy 

2 Dip. di Scienze Sperimentali e Cliniche, Università “G. d’Annunzio”, Chieti, Italy 

3 Dip. di Neuroscienze ed Imaging, Università “G. d’Annunzio”, Chieti, Italy 

4 Dip. di Medicina Interna, Università di Roma Tor Vergata, Roma, Italy 

Multiple Sclerosis (MS) is a chronic inflammatory disease of the Central Nervous 

System (CNS), with an autoimmune pathogenesis, characterized by loss of myelin 

sheath of axons. While the etiology of MS is still unclear, a favored hypothesis 

suggests that one factor contributing to the development of autoreactive T-cells is 

a cross-reactive immune response between viral components and CNS antigens 

(“antigenic mimicry”) 1 where possible mediators are proteins and metabolites. 

Moreover, several studies propose that the imbalance between the myelin damage 

and remyelination can be influenced by hormonal depending metabolic alteration. 

Nowadays, the advancement in mass spectrometry and bioinformatics tools seems 

to be a winning combination to get information that can lead to the discovery and 

identification of new molecular markers and metabolic imbalance in pathological 

condition. In a recent work 2 we reported an LC-MS based lipidomics application in 

MS revealing altered serum phosphocolines and lyso-phosphocolines levels in MS 

subjects, demonstrating a metabolic imbalance, although the sample is taken far 

from the site of inflammation. Here we aimed to identify a metabolic alteration in the 

CNS of MS subjects, employing a combined targeted/untargeted mass spectrometry 

based metabonomics platform. In particular we focused our attention on investigating 

CSF phosphocolines patterns by MALDI-TOF-MS technology. Lipidomics data were 

combined to amino acids and carnitines levels in CSF in a preliminary cross-sectional 

investigation of MS affected patients vs patients with other neurological diseases. 

Finally a tentative of correlation between experimental results and clinical data was 

made showing preliminary evidence for the discovery of characteristic CSF metabolic 

“fingerprint pattern” in MS disease. 

This work is supported by FISM Research Fellowship Cod.2010/B/14. 

References 

1. R. Hohlfeld and H. Wekerle. Proc Natl Acad Sci U S A, 101 Suppl 2 (2004), pp. 14599–14606. 

2. Del Boccio P. et al. J Proteomics. 2011 Nov 18;74(12):2826-36. 


280


Increase of hippocampal aluminum is linked to the 

progression of amyloid pathology in a triple transgenic 

mouse modelof Alzheimer’s disease 

ciavardelli doMenico 1, 2* , conSalvo ada 1, 3* , Gatta valentina 1, 4 , 

roSSi claudia 1, 3 , loredana renna 2 , Sacchetta Paolo 3 , 

di ilio carMine 3 5, 6 

, SenSi Stefano 


2 Faculty of Engineering, Architecture, and Motor Science, Kore University, Enna, Italy; 

3 Experimental and Clinical Science Department, “G. d’Annunzio” University, 

Chieti-Pescara; 4 Functional Genomic Unit, Ctr. of Excellence on Aging, Chieti, Italy; 

5 Mol. Neurol., Dept. of Neurosci. and Imaging, Chieti-Pescara, Italy; 

6 Dept. of Neurol., Univ. of California Irvine, Irvine, CA. 

Brain deregulation of endogenous (Fe, Cu, Zn) and exogenous metals (Al) contributes to 

severalneurodegenerative diseases. Al is a known neurotoxin that has been implicated 

in Alzheimer’s disease (AD). Previously we found that, Al is significantly increased (at 

14 months of age, m.o.a.) in the cerebral cortex of female triple transgenic AD mice 

(3xTg-AD), an AD model overexpressing human mutant APP, PS1 and phosphorylated 

tau. In this study, we analyzed Al levels in the hippocampus of 3xTg-AD mice at 14 

m.o.a. and found the metal increased when compared with age-matched wild-type 

(WT) mice. Suggesting the age dependency of the phenomenon, Al was not detected 

in the hippocampi of mice of both strains at 7 m.o.a.. Interestingly, hippocampal Al 

concentrations showed a significant positive correlation with the increased levels of 

human Aβ 1-40 and Aβ1-42 present in the 3xTg-AD mice, suggesting a functional 

link between changes in Al uptake and the development of the amyloid pathology. In 

order to assess the molecular basis of this age-dependent increase of hippocampal 

Al uptake, we evaluated, using microarray analysis, changes in expression levels of 

genes known to be involved in brain Al influx such as the transferrin receptor (TFR), 

the monocarboxylate transporters (MCTs), and the cystineglutamatetransporter 

(Xc-). When analyzing hippocampi of WT and 3xTg-AD mice at 3 and 12 m.o.a., we 

found a significant up-regulation of MCT1 and Xc- in 3 months old 3xTg-AD mice 

while this up-regulation was lost in AD mice at 12 m.o.a.. In contrast, TFR and MCT2 

were up-regulated in young and old AD mice. We found that TRF and MCT2 mRNA 

levels increased with aging, matching the age-related increase in hippocampal Al. 

Our findings suggest that Al homeostasis is linked to the progression of the amyloid 

pathology, likely through an altered expression of TFR and MCT2. 

* These authors equally contributed to the study. 


281


Diabetes-induced myelin abnormalities are associated with 

an altered lipid pattern: protective effects of LxR activation 

Mitro nico 1 , cerMenati Gaia 1 , aBBiati federico 1 , 

BrioSchi eliSaBetta 1 , de faBiani eMMa 1 , creStani Maurizio 1 , 

Garcia-SeGura luiS-MiGuel 2 , MelcanGi roBerto coSiMo 1 , 

caruSo donatella 1 

1 Department of Pharmacological and Biomolecular Sciences, Università degli studi di Milano; 

2 Instituto Cajal, C.S.I.C 

Diabetic peripheral neuropathy (DPN) represents a considerable medical problem 

because its molecular mechanism is still obscure. This pathology induces 

neurological complications on both nerve function and structure, such as myelin. 

Myelin is a biological membrane characterized by high lipid content and contributes 

to the correct function of the nervous system. The particular characteristics of the 

lipids present in the sheath provide the electrical insulating property required for the 

saltatory propagation of the nervous influx. Recently it has been highlighted the role of 

Sterol Regulatory Element Binding Factor-1c (SREBF-1c), a gene involved in fatty acid 

synthesis, in the regulation of lipid metabolism during peripheral nerve myelination. 

Liver X Receptors (LXRs), a ligand activated transcription factors belonging to the 

nuclear receptors superfamily, directly regulate the expression of SREBF-1c. In 

addition, LXRs activation induces expression of several genes regulating cholesterol 

homeostasis and modulating lipid metabolism. 

In a streptozotocin treated rat model of diabetic neuropathy, analysis of sciatic 

nerve myelin lipids revealed that diabetes alters myelin’s phospholipid, fatty acid 

and cholesterol content in a pattern that can modify membrane fluidity. Reduced 

expression of relevant genes in the fatty acid biosynthetic pathway and decreased 

levels of the transcriptionally active form of the lipogenic factor SREBF-1c were found 

in diabetic sciatic nerve as well as the expression of myelin’s major protein, myelin 

protein zero (P0). In addition, we confirmed that diabetes induces sciatic nerve myelin 

abnormalities, primarily infoldings that have previously been associated with altered 

membrane fluidity. In a diabetic setting, synthetic activator of the nuclear receptor LXR 

increased SREBF-1c function and restored myelin lipid species and P0 expression 

levels to normal. These LXR-modulated improvements were associated with restored 

myelin structure in sciatic nerve and enhanced performance in functional tests such 

as thermal nociceptive threshold and nerve conduction velocity. 

These findings demonstrate an important role for the LXR-SREBF-1c axis in protection 

from diabetes-induced myelin abnormalities. 


282


Distinct modulation of endocannabinoids upon kainic acidinduced 

seizures 

filoMena fezza 1,2# , Maria criStina Marrone 1# , nicola Mercuri 1,2 , 

Silvia Marinelli 3* and Mauro Maccarrone 1,4* 

1 European Center for Brain Research (CERC)/Fondazione Santa Lucia, Rome, 

Italy; 2 University of Rome “Tor Vergata”, Rome, Italy; 3 Fondazione EBRI-Rita Levi 

Montalcini, Rome, Italy; 4 University of Teramo, Teramo, Italy. 

# These authors contributed equally to this work. *Equally senior authors 

There are several endogenous neuroprotective mechanisms, among which 

endocannabinoids (ECs) play a role in various models of epilepsy. Indeed, it has 

been shown that increased levels of ECs protect against kainic acid (KA)-induced 

seizures. However, the mechanisms underlying this effect and its age-dependence 

are as yet unknown. To shed light on the molecular events responsible for KA-induced 

neurotoxicity, we investigated which step of the metabolic pathways that regulate 

cellular levels of ECs may be responsible for the neuroprotection exerted by these 

substances in the hippocampus of young and adult KA-treated rats. To this aim, 

we combined biochemical assays and electrophysiological recordings. We found 

that both anandamide (AEA) and e (), together with the AEA-biosynthetic enzyme 

NAPE-PLD, significantly increased in the hippocampus of young KA-treated rats, 

while decreasing in adults. In contrast, the levels of the other major endocannabinoid, 

2-arachidonoylglycerol, were higher in the hippocampus of adult compared to young 

rats, suggesting distinct age-dependent profiles of ECs upon KA insult. 

We also found that the type-1 cannabinoid receptor (CB 1 R) agonist WIN 55,212-26 

significantly reduced epileptiform burst duration induced by KA in hippocampal slices. 

Remarkably, the CB 1 R antagonist SR141716 reversed this effect, but did not affect 

per se the duration of the population spike while worsening KA-induced epileptiform 

bursting. Take togheter, these data suggest that there are age-specific alterations of 

ECs induced by KA, and support that the increase of ECs levels in the hippocampus 

of KA-treated rats might be an inhibitory mechanism against epileptiform discharges. 


283


Agonist-induced desensitization of GPR17 in 

oligodendrocyte precursor cells: a role in the maturation to 

myelinating cells? 

trincavelli Ml 1) , daniele S 1) , fuMaGalli M 2) , Bonfanti e 2) , 

GiacoMelli c 1) , aBBracchio MP 2) , Martini c 1) 

1) Dept. of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, 

Italy 2) Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Dept. of 

Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy 

GPR17 is a P2Y-like receptor that responds to both uracil nucleotides (as UDP- 

1 glucose) and cysteinyl-leukotrienes (cysLTs, as LTD ) and has been described as a key 

4 

player in oligodendrocyte differentiation. In oligodendrocyte precursor cells (OPCs), 

GPR17 expression is restricted to early stages of differentiation and segregated from 

that of mature myelin proteins to the point that mature oligodendrocytes no longer 

express the receptor2,3 . The changes in GPR17 expression during olygodendrocyte 

differentiation suggest that this receptor may undergo regulatory mechanisms such as 

desensitization, internalization and down regulation that regulate its responsiveness 

over time. 

On this basis, we analyzed GPR17 responses to UDP-glucose and LTD and its 

4 

regulation at different stages of cell differentiation in primary rat OPCs. 

In primary pre-oligodendrocytes, when GPR17 expression is maximal, the receptor 

underwent homologous desensitization. Activation of GPR17 by UDP-glucose (but 

not viceversa) induced a partial heterologous desensitisation of LTD -mediated 

4 

responses, suggesting that nucleotides have a hierarchy in producing desensitising 

signals. 

By Real-time PCR we demonstrated that expression of GPR17 at different stages of 

OPC differentiation was related with the expression of GRK2, suggesting this kinase 

could play a role in the regulation of receptor functioning during differentiation. 

Since GRK expression and functioning have been demonstrated to be altered in several 

neurodegenerative diseases, including multiple sclerosis4 , studies are in progress to 

evaluate the role of GRK2 in the regulation of GPR17 functional responsiveness during 

OPC maturation, both under physiological and pathological conditions. Determination 

of the regulatory mechanisms controlling GPR17 responses over time is pivotal to find 

new ways to foster OPC differentiation and myelin repair in demyelinating diseases. 

1 Ciana P et al. EMBO J. 2006 25:4615-277 

2 Lecca D. et al. PLoS ONE 2008 3:3579-593. 

3 Fumagalli M. et al. J. Biol. Chem. 2011 286:10593-604. 

4Vron A. et al. J. Immunol. 2005 174:4400-406. 


284


Effects of Plasma from Patients Affected by Mild Cognitive 

Impairment and Alzheimer’s Disease on Cultured 

Endothelial Cells 

2 Mazzanti l, 2 nanetti l, 2 raffaelli f, 3 luzzi S, 3 Provinciali l, 

1 di PriMio r, 2 viGnini a, 1 Salvolini e 

1 Dipartimento di Scienze Cliniche e Molecolari - Istologia, 

Università Politecnica delle Marche, Ancona, Italy 

2 Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche - 

Biochimica, Università Politecnica delle Marche, Ancona, Italy 

3 Dipartimento di Medicina Sperimentale e Clinica, Clinica Neurologica, 


There is accumulating evidence that Alzheimer’s disease (AD) can have a vascular 

contribution. In particular, an endothelial dysfunction may impair nitric oxide (NO) 

production and cause cerebral hypoperfusion. Blood flow impairment can be provoked 

also by an increased production of reactive oxygen species (ROS). Objective: The aim 

of the present study was to investigate the effect of plasma from subjects affected by 

AD and mild cognitive impairment (MCI), on human aortic endothelial cells (HAECs) 

in vitro, since endothelial dysfunction has been suggested to be an early event in 

patients affected by AD. Plasma samples were obtained from 27 AD patients, 15 MCI 

subjects, and 19 age- and sex-matched healthy subjects. After a short incubation 

period the following parameters were evaluated: NO release, superoxide dismutase 

(SOD) and Na + /K + -ATPase activities, membrane fluidity, and thiobarbituric acidreactive 

substances (TBARS) production. 

The exposure to MCI plasma provoked a decrease in NO release, more pronounced in 

the presence of AD plasma. Our data on SOD and Na + /K + -ATPase activities show a 

similar trend, since the lowest values were recorded after incubation with AD plasma. 

As concerns endothelial membrane fluidity, it was deeply affected by the exposure 

to MCI plasma, and even more following incubation with AD plasma. Finally, we 

observed an enhanced TBARS production after incubation with MCI and AD plasma. 

Our results confirm the hypothesis that endothelial dysfunction is an early event in the 

progression towards AD, thus highlighting the importance of precocious diagnosis. 


285


MMP-9 isoforms in serum of Multiple Sclerosis patients 

trentini aleSSandro 1 , Manfrinato Maria criStina 1 , 

dallocchio franco 1 , Baldi eleonora 4 , tola Maria roSaria 4 , 

caStellazzi MaSSiMiliano 3 , taMBorino carMine 3 , Granieri enrico 3 , 

fainardi enrico 2 , Bellini tiziana 1 

1 Section of Biochemistry and Clinical Biochemistry, Department of Biochemistry and 

Molecular Biology, University of Ferrara, via Luigi Borsari 46, Ferrara I-44100, Italy 

2 Neuroradiology Unit, Department of Neurosciences and Rehabilitation, Azienda 

Ospedaliera-Universitaria, Arcispedale S. Anna, Corso della Giovecca 203, Ferrara 

I-44100, Italy 3 Section of Neurology, Department of Medical and Surgical Sciences 

of the Communication and Behaviour, University of Ferrara, Arcispedale S. Anna, 

Corso della Giovecca 203, Ferrara I-44100, Italy 4 Neurology Unit, Department of 

Neurosciences and Rehabilitation, Azienda Ospedaliera-Universitaria, Arcispedale 

S. Anna, Corso della Giovecca 203, Ferrara I-44100, Italy 

Objectives: Gelatinases (MMP-9 and MMP-2) have emerged as potential biomarkers 

for monitoring Multiple Sclerosis (MS) disease activity (1, 2). Recently, Bellini et al. (3) 

identified in serum the presence of a TIMP-resistant MMP-9 isoform, that escaping 

the physiological regulation by its inhibitor could be detrimental in inflammatory 

conditions. The objective was to investigate serum concentrations of TIMP-resistant 

MMP-9 in MS patients and inflammatory (OIND) and non-inflammatory (NIND) controls 

to elucidate its potential relevance as biomarker in the disease. 

Methods: The presence of TIMP-resistant MMP-9 was evaluated in serum samples 

from 48 relapsing-remitting (RR) MS patients (active (N=22) and stable (N= 26)), in 30 

OIND patients and in 28 NIND patients by an activity assay system adapted by us (3). 

Results: There were no differences in serum levels of TIMP-resistant active MMP-9 

among RRMS OIND and NIND nor between patients categorized on the basis of MRI 

disease activity. However, the relative amount of TIMP-resistant MMP-9 activity was 

lower in active RRMS than in OIND (p< 0.05) and MRI stable RRMS patients (p< 0.01). 

Discussion: Our preliminary results suggest that TIMP-resistant MMP-9 isoform 

that escapes from the physiological control is more represented in MS than in other 

inflammatory conditions and might be related to the remission of the disease. These 

findings indicate that this active MMP-9 isoform could be implicated in processes 

regulating proteolytic activity in MS. 

Conclusion: The determination of TIMP-resistant MMP-9 levels might be useful to 

discriminate between RRMS patients in active phase of disease and inflammatory 

controls as well as to monitor disease activity. 

1. Fainardi E. et al Mult Scler 2006; 12: 294-301 

2. Fainardi E. et al. Mult Scler 2009; 15: 547-554 

3. Bellini T. et al J Biochem 2012; 151: 493-499 


286


HOMOCYSTEINE AND OxIDATIVE STRESS IN ACUTE 

STROKE 

nanetti l.*, viGnini a.*, raffaelli f., Giulietti a., § Bartolini M., 

§ Perozzi c., § SilveStrini M., § Provinciali l., Mazzanti l. 

§ Department of Clinical and Sperimental Science, Università Politecnica delle Marche, Italy, 

*Department of Clinical Science, Biochemistry Section Università Politecnica delle Marche, Italy 

Homocysteine is a sulfhydryl-containing amino acid derived from the essential amino 

acid methionine. 

Ischemic stroke is a heterogeneous syndrome caused by multiple disease 

mechanisms, resulting in a disruption of cerebral blood flow with subsequent tissue 

damage. An alteration of the physiologic balance between production and degradation 

of enzymatic activity involved in the regulation of tissue oxidative status may play a 

role in influencing the results of the ischemic condition. A recent study by our group 

proposed that cerebral ischemia causes the release of nitric oxide (NO) from the 

vascular endothelium. An elevated circulating concentration of the sulfur-containing 

amino acid homocysteine, hyperhomocysteinemia, produces complex changes. In 

the peripheral circulation, these changes include oxidative stress. 

Our aim in this study was to investigate the possible correlations among homocysteine 

plasma levels, oxidative stress parameters and clinical evolution of stroke. 

Fifty patients with large-vessel ischemic stroke were studied. Biochemical 

determinations, performed at entry (T0) and then repeated one month after stroke (T1). 

Homocysteine levels were significantly increased at T0 with respect to T1 and 

showed a significant positive correlation with the expression of oxidative stress 

markers and a negative correlation with indicators of protective anti stress activity. A 

significant increase of antioxidant activity occurred from T0 to T1 and changes were 

associated with the severity of clinical conditions. In particular, the extent of reduction 

of homocysteine plasmatic levels and of oxidative stress markers and contemporary 

increase in anti stress biochemical activities were associated with the reduction of 

NIHSS scores. 

These findings, besides confirming an involvement of oxidative stress in influencing 

the evolution of stroke, suggest a role for homocysteine as a potentially modifiable 

biochemical alteration able to modulate some mechanisms involved in the production 

of ischemic damage. 

References 

Hankey GJ et al. 1999 

Nanetti L. et al, 2008 

Nanetti L. et al, 2005 


287


2D PAGE detection of Neurofilament Light Chain (NF- 

L) aggregates in Cerebrospinal fluid (CSF) and serum of 

patients with Amyotrophic Lateral Sclerosis (ALS) 

Sardaro nicola**, ruGGieri Maddalena*, Savino laura*, 

Scacco Salvatore**, nicaStro annarita**, tortelli roSanna*, leante 

roSaria*, lonGo ivana*, livrea Paolo* and SiMone iSaBella laura* 

* Department of Neuroscience and Sense Organs, University of Bari, Italy 

**Department of Basic Medical Sciences (SMB), University of Bari, Italy 

Introduction: Amyotrophic lateral sclerosis (ALS) is characterised by selective 

death of upper motor neurons in the cerebral cortex and lower motor neurons in the 

brainstem and spinal cord. Neurofilament (Nf) aggregates are a common feature of 

many neurodegenerative disorders and Cerebrospinal fluid (CSF) and serum levels of 

different NF subunits as light (NF-L) and heavy (NF-H) chains have been investigated 

as a potential disease biomarker. Nf aggregates in CSF and serum may mask Nf 

epitopes, preventing accurate quantification. 

Aim: to individualize NF-L aggregates in the CSF and serum samples of Amyotrophic 

Lateral Sclerosis (ALS) patients and neurological controls and to characterize 

aggregate protein components. 

Methods: We used many pre-analytical methods to solubilize Nf aggregates, 

immunoblotting for qualitative analysis and 2D Native/SDS PAGE protein separation 

as semi-quantitative method. NF-L was evaluated in pooled serum and corresponding 

CSF samples of ALS patients. Pool of serum and CSF of subjects with other 

neurodegenerative diseases, such as Alzheimer’s disease and spinocerebellar ataxia, 

have been used as control. 2D immunoblot analysis was performed with anti NF-L 

, anti-NFH (SMI34 and SMI35), anti total-tau (t-TAU) and anti phospho-tau (pTAU) 

antibodies. Immunodetection was revealed by chemiluminescence (ECL) method. 

Results: 2D PAGE method revealed, at the same molecular weight, aggregates 

constituted by NF-L and p-TAU.The presence of these aggregates was higher in CSF 

and serum of ALS patients if compared to control patients. 

Conclusion: NF-L aggregates are present in CSF and serum samples of patients with 

ALS and other neurodegenerative disorders. NF-L and p-TAU seem to be the only of 

tested proteins that concur to the aggregate formation. 2D-PAGE is applicable for a 

semi-quantification of NFL in CSF and serum samples but further studies, in different 

stages of disease, are needed to ameliorate the technique strength and to better 

investigate the role of this protein aggregates. 


288


microRNA mediated lysosomal enzymes down-regulation 

in cell of peripheral system from Alzheimer’s disease 

patients 

tiriBuzi roBerto 1 , criSPoltoni lucia 1 , antonio orlacchio 2,3 , 

zaMPolini Mauro 4 , Serena Porcellati 1 , aleSSandro datti 1 , Martino SaBata 1 , 

orlacchio aldo 1, 5 . 

1 Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia 

Molecolare, Università di Perugia. 2 Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Roma. 

3 Dipartimento di Medicina dei Sistemi, Università “Tor Vergata”, Roma, 4 Neuropsicologia Clinica, 

Ospedale S. Giovanni Battista, Foligno. 5 GeBiSa Research Foundation, Perugia. 

Background: Alzheimer’s disease (AD), the most common form of dementia in the 

elderly, is characterized by neurofibrillary tangles, extracellular amyloid β plaques and 

neuroinflammation. New evidences have shown that lysosomal system could represent a 

crossroad in which etiological factors in AD pathogenesis converge. To better understand 

the relevance of lysosomal enzymes in cells of peripheral system, we investigated the 

expression of Cathepsins B, D and S and β-Hexosaminidase, β-Galactosidase and 

α-Mannosidase in monocytes and lymphocytes from AD patients. 

Methods: Sample collection: All subjects enrolled fulfil the NINCDS-ADRDA criteria for 

probable AD. Peripheral blood was collected and monocytes were isolated from PBMCs 

using the Monocyte Isolation Kit II (Miltenyi Biotec). 

Lysosomal enzymes evaluation: β-hexosaminidase, α-mannosidase and β-galactosidase 

enzymatic activities were measured using commercially available substrates. Cathepsin 

B, D and S were evaluated by immunoblotting. 

Gene expression: Real-time RT-PCRs were performed using specific Assay-On- 

Demand assay for human CTSB, CTSS, CTSD, GLB1, MAN2B1, HEXB, HEXA, TFEB, 

SPI1 and 18S rRNA genes (Applied Biosystems). 

miRNAs level: For analysis of hsa-miR-128, hsa-miR-124a and hsa-mir-155 expression, 

real-time RT-PCR were carried out using specific PCR primer set (Exiqon). 

microRNA transfection: 15 nM of miR-128-1 duplex or 15 nM of anti-miR124a and antimiR155 

were used for in vitro transfections. 

Results: Data obtained revealed that Cathepsins B, D, S β-Hexosaminidase, 

β-Galactosidase and α-Mannosidase together with the transcription factors TFEB and 

PU.1 decreased as protein amount and gene expression level in both monocytes and 

lymphocytes from AD patients respect to healthy subjects. Moreover, mircroRNA analysis 

and in vitro experiments of gain and loss of function indicate that this down-regulation 

was a direct consequence of the miR-128-1 up-regulation founded in AD related cells. 

Conclusion:Globally, our data contribute to clarify the mechanism under the impairment 

of the endosomal-lysosomal system involved in the pathogenesis of Alzheimer’s disease 

and point to the miR-128 with the TFEB and PU.1 TFs as key players. 


289


HPLC ASSAY OF MOUSE HYDROxYINDOLE-O- 

METHYLTRANSFERASE (HIOMT) IN RETINAL 

HOMOGENATES 

Betti l., PaleGo l., criStofaro M., lucacchini a., deMontiS G, 

Giannaccini G. 

Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology- University of Pisa 

Hydroxyindole-O-methyltransferase (EC 2.1.1.4; HIOMT) is the final step enzyme of 

the melatonin synthesis pathway. HIOMT follows a Bi-Bi kinetic using S-adenosyl- 

L-methionine (SAM) and N-acetyl serotonin (NAS) as the substrates. This protein 

is predominantly expressed in the vertebrate pineal gland, where it produces 

melatonin from NAS by the activation of the rate-limiting enzyme aryl-alkyl-amine-Nacetyltransferase 

(AA-NAT) which undergoes the circadian regulation. Beside the main 

pineal origin of melatonin, there is also evidence concerning its synthesis in the rodent 

and human retina. On the other hand, the measurement of retinal HIOMT activity is 

critical since the enzyme is a low-abundant protein in this tissue. As well, HIOMT 

displays a high “per se” variability, species- and strain-dependent in rodents: its gene 

has been mapped inside a high-frequency meiotic recombination chromosome site, 

the pseudoautosomal region (PAR), a short region of homology between the X and Y 

chromosomes (1). 

We thus present herein a more robust HPLC method, modified from the original one (2), 

to assess HIOMT in the mouse retina. Retinal homogenates, obtained from AJ mice, 

were incubated with 50 µM NAS, 2-25 µM 3 H-SAM (20.6 – 1.65 Ci/mmol). Melatonin 

was then extracted twice in chloroform, final extracts dried under high-vacuum, resuspended 

in mobile phase and injected into the chromatograph for the isocratic 

chromatography. One ml elution fractions were collected for 30 min per sample, ovendried 

and radioactivity counted by a β-counter, after adding the scintillation liquid 

cocktail. 

Under present conditions, melatonin eluted at fractions 18-20. Retinal HIOMT V max 

and K m were 29-37 fmol/h/mg protein and about 10 µM in the mouse strain evaluated. 

The present method increases the specificity of HIOMT assay, being particularly 

appropriate for studying the enzyme regulation in retina. 

1) Kasahara T. et al., 2010, PNAS, 107(14) : 6412-17. 

2) Bernard M. et al., 1995, Brain Res., 696 : 37-48. 


290


RP-LC/UV-PDA MEASUREMENT OF THE 

ANTIDEPRESSANT TRAZODONE AND ITS MAJOR 

METABOLITE m-CPP IN HUMAN SERUM 

PaleGo l., Betti l., criStofaro M., MaSala i., zanotti S., Pacciardi 

B., Belli S., luchini f., Mauri M., lucacchini a., Giannaccini G. 


Trazodone, (2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazole[4,3-a] 

pyridine-3(2H)-one, TRZ, is a multifunctional antidepressant with hypnotic properties, 

effective in the treatment of patients with mood disorders and insomnia. The main 

mechanism of action of TRZ consists in its antagonism on 5-HT 2A/2C receptors, the drug 

being, at the same time, a low-affinity blocker of the 5-HT reuptake site (SERT). TRZ 

has been found to interact also with histamine and α-adrenergic receptor subtypes, 

albeit the drug pharmacology has not been fully elucidated, especially its effects on 

sleep function. Even if TRZ is a well-tolerated psychotropic agent, the monitoring 

of this compound and meta-chlorophenyl-piperazine (m-CPP, metabolite) in treated 

patients can be relevant for dose adjustment, therapy personalisation, especially 

when abnormal metabolism and low compliance are suspected (non-responders) or 

in case of polypharmacy (1). We present herein an easy, reliable, accurate RP-LC/ 

UV-photodiode array (PDA) method for determining trazodone and m-CPP in human 

serum suitable for therapeutic monitoring. The drug extraction from serum is a liquidliquid 

procedure using n-exane and buspirone as the internal standard (IS); specificity 

and accuracy are monitored by UV-PDA detection; for peak integration and analysis, 

the PDA is set at the TRZ and m-CPP λ max = 213 nm and buspirone λ max = 240 nm. 

Both compounds and the IS are separated within 12 min, with satisfactory extraction 

recoveries (85-100%), linearity (r coefficients > 0.99) and intraday/interday relative 

standard deviation (RSD) (within 10%). TRZ and m-CPP LOQ values account for by 

12 µg L -1 and 20 µg L -1 , respectively. In addition, the proposed method shows no 

significant elution interferences with a series of psychoactive/antidepressant drugs. 

The use of PDA spectral library additionally monitors potential interferences, making 

the procedure particularly useful for TRZ and m-CPP measurement in serum of 

patients co-treated with other CNS compounds. 

1) Mercolini L. , et al., 2008, J Pharmaceutical Biomed Analysis, 47 : 882-87. 


291


Fast one-step liquid chromatography determination of 

purine compounds in blood with photodiode array detector 

Gueli Maria concetta, SaleMi GiuSePPe 

Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche (BioNEC), 

Università degli Studi di Palermo. 

Uric acid (UA), a polyedric functional compound, is a strong natural antioxidant in 

humans where it is the end product of purine metabolism. The rational process for this 

study was the clinical interest of the metabolites as markers for energy disturbance 

in ischemia/hypoxia, antioxidant capacity, and disease activity in vivo. Unfortunately, 

reports about changes in UA levels in the neurodegenerative disorders have been 

conflicting. 

We propose a pratical one-step high performance liquid chromatography (HPLC) 

method with photodiode array detector (PDA) for the simultaneous determination of 

hypoxanthine (HX), UA, and xanthine (X) in human plasma. 

Blood samples were obtained from the healthy adult volunteers (University workers/30). 

For stability study, 200 µL of plasma was deproteinized through a Millipore-Amicon 

Ultracel. 

Waters-HPLC system consisted of a 600E Pump; 2998 PDA; Empower TM2 Data 

Software; Atlantis T3 analitycal column, 3µm; a 10µl injection volume. The m.f. was 

a 40 mmol/L potassium phosphate buffer, pH 2.2 at a flow rate of 1.0 mL/min. The 

spectral range of the PDA was 200-400 nm. The optimal wavelength for detecting a 

mixture of standards was 254 nm. 

We have obtained an execellent base-line separation of HX/UA/X from the interfering 

compounds of plasma samples, in a short elution time of less than 10min. The RT 

for HX/UA/X were 5.5; 6.6; 8.7 min, respectively. The chosen wavelength of 254 nm 

provides a higher sensitivity with a clean chromatogram. The reported reference 

ranges from 30 healthy normal subject were 1.2-17.9 µM, 151-442 µM, and 0.2-5.8 

µM, for HX/UA/X respectively. Peaks were identified by matching the RT against those 

of authentic standards, and by spiking standard solutions to ultrafiltrated plasma. This 

HPLC-PDA system is very useful tool for the assay of UA and purine derivatives both 

in research assays and in clinical laboratories. 


292


SERUM SEROTONIN AND MELATONIN IN PATIENTS WITH 

MOOD/ANxIETY AND SLEEP DISORDERS TREATED WITH 

THE ANTIDEPRESSANT TRAZODONE 

criStofaro M., Betti l., PaleGo l., MaSala i., zanotti S., Pacciardi 

B., Belli S., luchini f., Mauri M., lucacchini a. , Giannaccini G. 


Sleep disorders have very high incidence in psychiatric diseases, being however 

often overlooked in the clinical practice. Alterations in the duration, pace and quality 

of sleep acquire diagnostic and prognostic significance in relation to the psychiatric 

disorder associated. Corollary to that is the noteworthy relevance of sleep disorders’ 

management either in reducing the suffering of patients or in improving their quality of 

life, by affecting the course of the related psychiatric disease as well as by preventing 

its relapse. Nevertheless, an impediment to the good treatment of sleep disorders 

connected to psychiatric diseases is represented by the multiple variables involved. 

Aim of this research was to evaluate the clinical/biochemical effects of trazodone, 

a multifunctional antidepressant drug which also displays good hypnotic properties 

(1): the serotonergic and melatonergic metabolism was determined in patients with 

mood/anxiety disorders under trazodone therapy for sleep disturbances through the 

measurement of serotonin (5-HT) and melatonin serum levels before (T 0 ) and after (T 1 ) a 

treatment with low drug doses (8-20 mg/day) for the hypnotic action. Serum, obtained 

from peripheral venous blood withdrawn between 8.30-9.00 a.m., was extracted with 

ethanol (5-HT) and dichloromethane (melatonin), dried under high-vacuum and stored 

at -20°C until assay. An isocratic reversed-phase liquid chromatographic (RP-LC) 

method was applied, using C18 pre- and analytical columns equilibrated in a mobile 

phase composed by a mixture methanol-phosphate buffer pH 5 (25:75 v/v) at the 

constant flow rate of 1 ml min -1 . Analyte peaks were revealed by means of a PED 

electrochemical detector and amperometric measurement. 

Preliminary results, obtained in seven subjects, showed, as expected, inter-individual 

variance concerning T 0 and T 1 serum melatonin and 5-HT. A trend towards the 

reduction of both analytes mean concentrations at T 1 was observed, needing, 

however, confirmation through the extension of examined cases. 

1) Stahl S., 2009, CNS Spectr. 14 (10) : 536-46. 


293

Proteine 

PRO

Proteine PRo 1 

Effect of Mucuna Pruriens leaves extract on human 

keratinocytes Proteomic profiling 

cortelazzo aleSSio 1 , laMPariello lucia 2 , Sticozzi claudia 3 , 

Guerranti roBerto 1 3, 4 

, valacchi GiuSePPe 

1 Department of Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry 

University of Siena, Siena, Italy; 

2 Department of Chemistry, University of Siena, Siena, Italy; 

3 Department of Evolutionary Biology, University of Ferrara, Ferrara, Italy; 

4 Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea. 

The plant-derived compounds are an important source of drugs for various diseases. 

The medicinal plant Mucuna pruriens (Mp), is the most popular drug in Ayurvedic 

system of medicine. Its different preparations (from seeds), have been used as 

therapeutic medicine for several diseases such as rheumatoid arthritis, diabetes, 

atherosclerosis, nervous disorders and Parkinson disease [1]. Plants from Nigeria 

seeds were grown in Botanical Garden, University of Siena, the leaves were collected, 

dried, pulverized and extracted with methanol. The extracts were examined in vitro 

for their antioxidant activity. Being the skin one of the main target of exogenous 

oxidative stress (UV, O 3 , smoke, etc) [2] and being several skin pathologies related to 

increased oxidative stress [3]. In this study, keratinocytes (HaCaT) viability by means 

of differently diluted samples has been measured and changes in proteins expression 

have been evaluated after Mp leaves methanolic extract (MpLE) treatment. Proteins 

from cells lysates where analyzed by SDS-PAGE and 2-DE. Protein bands and spots 

were evidenced by silver staining and divided in 3 groups based on the molecular 

weight as follows: 1 (100-250 KD), 2 (37-100 KD) and 3 (10-37 KD). The analysis 

revealed that after MpLE treatment there were 29, 26 and 7 new spots in groups 1, 

2 and 3, respectively. In addition, the treatment showed the loss of 39, 134 and 77 

original in groups 1, 2 and 3, respectively. Of note, the MpLE treatment significantly 

decreased the levels of 4HNE-protein adducts especially for group 2. These results, 

suggest that the use of MpLE might be helpful in the treatment of oxidative stressrelated 

skin diseases. 

References: 

1. Misra L. et al. Indian Journal of Biochemistry and Biophysics. 2007, 44, 56-60. 

2. Valacchi G. et al. FEBS Lett . 2000, 466,165-168. 

3. Briganti F. and Picardo M. J. Eur. Acad. Dermatol. Venereol. 2003, 17,663-669. 


296


Proteomic analysis of human plasma proteome changes 

derived from in vitro proteolytic activity of Echis carinatus 

venom 

cortelazzo aleSSio 1 ,Guerranti roBerto 1 , 

hoPe-onyekwere nnadozie 1 , 

furlani eMiliano 1 , Bini luca 2 , leoncini roBerto 1 

1 Department of Internal medicine, Endocrine-Metabolic Sciences and Biochemistry, 

University of Siena, Siena, Italy 

2 Department of Molecular Biology, University of Siena, Siena, Italy 

In this study we investigated the in vitro effects of snake venom peptidases from 

the Viperidae family on human plasma proteins. To carry out our experiments we 

used Echis carinatus venom (EV). It is a complex mixture of proteases besides 

peptides, toxins and platelet aggregation inhibitors [1]. EV contains two well-known 

metalloproteinases which are prothrombin activator: ecarin and carinactivase. The 

effects of EV on the coagulation process are also represented by the breakdown of 

the fibrinogen [2]. In this work, we prepared the plasma from an healthy volunteer and 

then the plasma was depleted using ProteoPrep ® Blue Albumin & IgG Depletion Kit. 

Depleted plasma was incubated with EV or with saline solution at 60 min at 37 °C. 

2-DE was performed to analyze the complex mixture of entire proteins and fragments 

produced by the proteolytic activity. Human plasma shows significant changes in 

proteins involved in blood coagulation but also in different physiological mechanisms, 

when it was treated with the snake venom. Each protein spot was analyzed using 

ETTAN MALDI-TOF and mass fingerprinting searching was carried out using MASCOT. 

After protein identification we studied their similarity using BLAST and Prosite. 

Moreover, we investigated the proteases activities using CutDB and MEROPS. The 

identified plasma proteins can be linked to a specific pathological processes. Their 

dysfunction, caused by protease activity, leads to disease associated protein. These 

results give us new information about human plasma proteome, proteases and snake 

venom poison. This information can help us to understand the role of these three 

moieties in medical application. 

References: 

1. Calvete J. J., Sanz L., Gutiérrez J. M. Venoms, venomics, antivenomics. FEBS Lett. 2009, 583, 1736- 

1743. 

2. Guerranti, R., Ogueli, I. G., Bertocci, E. Proteomic analysis of the pathophysiological process involved in 

the antisnake venom effect of Mucuna pruriens extract. Proteomics 2008, 8, 402-412. 


297


BETA2-GLYCOPROTEIN I SELECTIVELY INHIBITS THE 

PROCOAGULANT FUNCTIONS OF THROMBIN: A NOVEL 

PHYSIOLOGICAL ANTICOAGULANT MECHANISM IN 

HAEMOSTASIS 

de filiPPiS vincenzo 1 , Pozzi nicola 1 , acquaSaliente laura 1 , 

fraSSon roBerta 1 , Banzato aleSSandra 2 , arcovito aleSSandro 3 , 

ScaGlione Giovanni luca 4 , de criStofaro raiMondo 4 , PenGo vittorio 2 

1 Laboratory of Protein Chemistry, Department of Pharmaceutical and 

Pharmacological Sciences and 

2 Department of Cardiac, Thoracic and Vascular Sciences, 

University of Padua, Padua, Italy. 

3 Institute of Biochemistry and Clinical Biochemistry and 

4 Institute of Internal Medicine, Haemostasis Research Centre, Catholic University, 

School of Medicine, Rome, Italy. 

Background: β2glycoprotein I (β2GpI) is an abundant plasma protein (0.1-0.5mg/ 

ml) involved in the pathogenesis of the antiphospholipid syndrome, but whose 

physiological function is still elusive. Methods: The effect of β2GpI on the procoagulant 

(fibrin generation and platelet aggregation) and anticoagulant (protein C activation) 

functions of thrombin was investigated by enzyme inhibition assays, surface 

plasmon resonance (SPR), turbidimetric and immuno-cyfluorimetric analysis. Results: 

β2GpI does not affect the affinity of the enzyme for some active-site inhibitors, like 

p-aminobenzamidine and the N-terminal domain 1-47 of hirudin. Direct analysis of 

β2GpI-thrombin interaction by SPR yields a K d =34nM. Displacement experiments, 

carried out with specific binders at thrombin exosite 1 (hirugen and HD1 aptamer) 

or exosite 2 (fibrinogen γ’-peptide and HD22) and with thrombin mutants (Arg73Ala 

and Arg101Ala) having one of the two exosites selectively compromised, indicate 

that both exosites of the protease are involved in β2GpI binding. β2GpI significantly 

prolongs the clotting time in fibrin generation assays and hinders platelets aggregation 

(IC 50 =0.36μM) by inhibiting cleavage of PAR1 on intact platelets (IC 50 =0.32μM) and in 

solution. β2GpI does not alter the ability of thrombin to generate the anticoagulant 

protein C, with or without thrombomodulin added. Conclusions: β2GpI binds to 

thrombin at both exosites, while leaving the protease active site accessible. β2GpI 

inhibits the key procoagulant properties of thrombin, without affecting its unique 

anticoagulant function. These results are unprecedented and pave the way to the 

discovery of a novel physiological anticoagulant pathway in haemostasis. 


298


Osteopontin and Systemic Sclerosis: preliminary results of 

Osteopontin secretion by sclerodermic fibroblasts 

landi G., corallo c., vannoni d., Giordano n., leoncini r. 

Department of Internal Medicine, Endocrine, Metabolic Sciences and Biochemistry, 

University of Siena, Italy 

Osteopontin (OPN) is a glycoprotein expressed by a large variety of tissues, and 

plays an important role in extracellular matrix remodeling. Recent studies reported 

high levels of OPN in serum and dermis of patients affected by Systemic Sclerosis 

(SSc) and suggest that OPN could play an important role in the development of the 

disease (1) . The aim of this study is to evaluate the contribution of fibroblast in the 

expression and secretion of OPN in SSc. For this study we isolated sclerodermic 

fibroblasts from lesional (SSc) and non lesional (NA) skin of sclerodermic patients. 

Cells were cultured in a serum free medium and treated with Bleomycin (BLEO 50mU/ 

ml) and endothelin 1 (ET-1 100nM). After 72h culture media supernatants were 

centrifuged and collected for analysis. OPN secretion was evaluated with Human 

Osteopontin ELISA kit (ABCAM, Cambrige). This assay employs an antibody specific 

for human Osteopontin coated on a 96-well plate. Preliminary results showed similar 

OPN levels on untreated fibroblasts. Statistically relevant differences in culture media 

OPN concentration were found when the cells were treated with Bleomycin more 

than ET-1. These preliminary results suggest that high OPN levels already outlined in 

recent studies could be related with the pathogenesis of scleroderma and also the in 

vitro model of induced dermal fibrosis by bleomycin better simulate the behavior of 

sclerodermic fibroblasts in vivo. 

1. Wu M, et al. Osteopontin in systemic sclerosis and its role in dermal fibrosis. J Invest Dermatol. 2012 

Jun;132(6):1605-14. 

Keywords: Systemic Sclerosis, Osteopontin, Dermal Fibrosis, Bleomycin, Endothelin 1 


299


Antimicrobial peptides derived from tripsin digestion of 

bovine lactoferrin 

anna rita lizzi 1 , veronica carnicelli 1 , franceSchini nicola 1 , 

PieraGoStino daMiana 2 and antonio di Giulio 1 

1 Dept. of Biotechnological and Applied Clinical Sciences University of L’Aquila 

2 Dept. of Experimental and Clinical Sciences, University of Chieti-Pescara, Italy 

Bovine lactoferrin (bLf) is an iron-binding glycoprotein consisting of 689 amino acids, 

found in different biological fluids of mammals it exerts several biological functions 

among which antimicrobial, antiviral and anti-inflammatory activities. Lactoferrin is a 

dominant whey protein in milk throughout lactation and is able to bind two ferric ions 

[1]. When this protein is digested at acidic pH by proteases, it yields highly active 

antimicrobial peptides that inhibit the growth of a number of Gram-negative and 

Gram-positive bacteria, including strains that were resistant to native Lf [2]. 

The aim of this study was to investigate if the tripsin hydrolysis of whole bLf , as well 

as the iron-depleted protein apo-bLf, releases other, not yet well known, antimicrobial 

peptides (AMPs). After hydrolysis the peptide pools was divided in two fractions by 

Centricon filters (cut off 5 kDa). The fractions, together with the parent proteins, were 

tested for: i) the antimicrobial activity versus several E. coli and P. aeruginosa bacterial 

strains; ii) the perturbing effect on bacterial by TEM microscopy and iii) the ability 

in inducing fluorescent probes release of from synthetic vesicles (liposomes). The 

peptide pool (MW < 5 kDa) derived from apo-bLF hydrolysis, which resulted the more 

active sample, separated by HPLC and analysed by mass spectroscopy revealed the 

presence of several small peptides. In this moment we focus our attention on a 24 

amino acids peptide that seems to be the responsible for the high antimicrobial ability 

of the pool. 

1. Lizzi et al., Mini-Rev. Med. Chem., 9:687 -695, 2009. 

2 Tomita et al., J. Dairy Sci., 74: 4137-4142, 1991 


300


The allergen Mus m 1.0102: evidence of aggregation 

ferrari elena, caSali eManuela and alBerto SPiSni 

Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali - S.Bi.Bi.T.- 

Università degli Studi di Parma 

Protein aggregation is relevant to a variety of biotechnological processes: from the 

formulation of protein drugs like vaccines to the production and storage of high quality 

proteins. Here we present an investigation on the potential aggregation of the allergen 

Mus m1.0102 and its mutant C138S. The experimental conditions allowed to identify 

the formation of covalent multimers as well as of soluble aggregates: the former, 

induced by thermal denaturation of Mus m1.0102, while the latter are observed in non 

denaturating conditions for both proteins. 

Heat denatured proteins were analyzed by SDS-PAGE and revealed that, in the case 

of Mus m1.0102, Cys138 is involved in an interchange process with the only disulfide 

bridge present in the protein, an intermolecular disulphide bridge formation, playing a 

role in covalent aggregation (1). 

Dynamic Light Scattering (DLS) was used to study the aggregation state of Mus 

m1.0102 and its mutant in non denaturating conditions. The two proteins appeared 

in two distinct size populations: at proteins concentration >=2 mgr/ml, the smaller 

size fraction showed a hydrodynamic diameter corresponding to the estimated 

monomeric mass, while the larger size fraction corresponded to a higher order 

aggregate. Following dilutions up to 0,5mgr/ml, the intensity of the peaks of the native 

allergen resulted extremely monodisperse, while the mutant low-size intensity peak 

became progressively polydisperse, being the result of a mixture of mono-, di- and 

trimeric protein form. 

In conclusion, the process of covalent aggregation was acknowledged only after thermal 

denaturation of the native protein, while the equilibrium between the monomers and 

a minor aliquot of soluble aggregates was appreciated in native conditions for both 

proteins. Worth considering is that: the dilution of the mutant sample incremented 

the oligomers population, an interesting finding as transient dimers are expected to 

stimulate the allergenic reaction (2). 

1) S. Roefs and K. De Kruiff (1994) Eur. J. Biochem. 226, 883-889. 

2) M. Li, et al. (2008) J. Biol. Chem. 283, 22806-22814. 


301


Functional characterization by site-directed mutagenesis 

of human oxidosqualene cyclase (OSC) expressed in Pichia 

pastoris 

viola franca 1 , Balliano Gianni 1 , ruf arMin 2 , taraMino Silvia 1 , 

di Bartolo GiuSePPe 1 , oliaro-BoSSo SiMonetta 1 

1 Dipartimento di Scienza e Tecnologia del Farmaco – Università di Torino 

2 F. hoffman-La Roche AG, Pharma research Discovery Chemistry, 

4070 Basel, Switzerland 

In sterol biosynthesis, oxidosqualene cyclase (OSC) catalyzes the most outstanding 

step of the pathway: the shaping of the totally open triterpene intermediate 

2,3-oxidosqualene, generated by the mevalonate pathway, into lanosterol, the first 

policyclic precursor of cholesterol. Recently a new interest for this enzyme has been 

raised by an inverse docking study that identifies OSC as the highest-rank potential 

target of PRIMA-1, a small molecule activating mutant p53 protein, restoring its tumor 

suppressor function[1]. Moreover antiproliferative and antiangiogenic activity of Ro 

48-8071, one of the most potent inhibitors of OSC, has been recently shown [2,3]. 

Previous mutagenesis studies on the yeast OSC and on the homologue bacterial 

enzyme SHC have shown that some amino-acidic residues, not belonging to the 

active site and localized in an hydrofobic channel hypotized to allow the access to 

active site, are essential for maintaining the catalytic activity of the enzyme [4]. The 

crystal structure of human OSC shows the presence both of the hydrofobic channel 

and of a more polar channel allowing a chain of water molecules to reach the E459 

residue at active site : a possible way of re-protonation of catalytic D455. 

In this study we present the effects of the mutagenesis of 7 different amino-acids of 

human OSC belonging to the active site, or to the hydrophobic channel or to the polar 

one. Our results confirm the role of the polar channel, since the modifications of E459 

and of the network of H-bond connecting it to K462 and Q394 completely suppress 

the catalytic activity of OSC. 

1. Grinter S Z et al, J. Mol. Graph. Mod. 29 (2011) 795–799 

2. Meda C et al , FEBS J. 278 (2011), PP11.92 

3. Staedler D et al, J. Med. Chem. 55 (2012) 4990−5002 

4. Oliaro-Bosso et al, PLoS ONE 6(7): e22134. doi:10.1371 


302


The polyQ protein ataxin-3 protects against stress 

conditions 

BonanoMi, Marcella; PaStori, valentina; invernizzi, Gaetano, 

reGoneSi, Maria elena; tortora, Paolo 

Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano (Italy) 

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the 

expansion beyond a certain threshold of a polyglutamine (polyQ) tract in the protein 

ataxin-3 (AT3). AT3 consists of a globular N-terminal Josephin domain and of a 

flexible C-terminal tail containing the poly-Q tract. The pathology results from protein 

misfolding and intracellular accumulation of fibrillar amyloid-like aggregates. The loss 

of function resulting from misfolding might also be involved in the mechanisms of 

pathogenesis. AT3 is a conserved and ubiquitous protein known to bind polyubiquitin 

chains and to function as a deubiquitinating enzyme. It seems to be involved in 

different cellular pathways, i.e. aggresome formation and ubiquitin-proteasome 

pathway, but the physiological role is still poorly understood. To investigate possible 

functions of AT3 in cellular pathways that respond to altered protein homeostasis, 

we constitutively expressed several AT3 variants in Pichia pastoris, a methylotrophic 

yeast lacking proteins homologous to AT3. Growth assays showed that expression 

of wild type AT3 or the sole Josephin domain allows yeast growth even under 

stress conditions, such as heat-shock or ER stresses (dithiothreitol, tunicamycin). 

In contrast, the expression of a catalytically inactive variant could not restore yeast 

viability, which suggests that the deubiquitinating activity of AT3 is required for the 

protective effect. We also created a transgenic C. elegans strain over-expressing wild 

type AT3 in neurons under the control of a pan-neuronal promoter unc-119. Based 

on body bends counting, preliminary studies suggest that under stress condition C. 

elegans over-expressing wild-type AT3 shows an increased motility compared with 

wild type. Our data suggest that AT3, thanks to its deubiquitinating activity, displays a 

protective effect against stress conditions. 


303


Structural characterization of dioicin 1, novel type 1 

ribosome-inactivating protein from leaves of Phytolacca 

dioica L. 

ruSSo roSita, di Maro antiMo, chaMBery anGela and 

Parente auGuSto 

Dipartimento di Scienze e Tecnologie Ambientali, Biologiche ed Farmaceutiche, 

Seconda Università di Napoli, Via Vivaldi 43, I-81100 Caserta, Italy 

Ribosome inactivating-proteins (RIPs; EC 3.2.2.22) are single-chain (type 1) or 

heterodimeric cytotoxic (types 2 and 3) enzymes, endowed with rRNA N-β-glycosidase 

activity [1] . RIPs remove a site-specific, single adenine residue (A 4324 in the case of rat 

liver ribosomes) from the highly conserved sarcin/ricin loop of the 28S rRNA, thus 

arresting protein synthesis [2] . The genus Phytolacca is known to be a rich source of 

several type 1 RIP [3,4] : i) PAP I, II and III from leaves of Phytolacca americana L.; ii) PD- 

S 1-3 from seeds; PD-L 1-4 from leaves of adult plants of P. dioica L.; dioicin 1 e dioicin 

2 from leaves of young plants (up to three years) and from developing leaves of adult 

plants [4] . In the last experimental system, dioicin 2 is always present, while dioicin 1 is 

expressed up to day seventeen. The fact that P. dioica synthesizes and accumulates 

RIPs, indirectly indicates that their presence might be beneficial to plant fitness. 

Here we report the determination of structural and functional properties of dioicin 1, 

isolated according to Parente et al. [4] . The purity of the protein was verified by SDS- 

PAGE and ESI/ Q-TOF MS analysis. Then, using a strategy which combines Edman 

degradation and mass spectrometry (ESI/Q-TOF), the 80% of dioicin 1 primary 

structure was determined. 

The available primary structure of dioicin 1 is highly identical to PAP-II from Phytolacca 

icosandra L.. Furthermore, the completion of its primary structure will allow us to 

establish phylogenetic relationships with all known RIPs from Phytolaccaceae. 

References: 

[1] Girbes et al. (2004) Mini Rev. Med. Chem. 4, 461-476. 

[2] Endo, Y. and Tsurugi, K. (1988) J. Biol. Chem. 263, 8735-8739. 

[3] Peumans WJ et al. (2001) FASEB J. 15, 1493-1506. 

[4] Parente et al. (2010) Toxic plant proteins Lord, JM & Hartley, MR (Eds). Springer, Heidelberg, pp 79-106. 


304


PDZ Domains and Membrane Nucleolin in the Engineering 

and Production of Chimeric Toxins Against Cancer Cells 

franceSco GianSanti, luana di leandro, SaBrina Mei, 

annaMaria ciMini, rodolfo iPPoliti 

Department of Health, Life and Environmental Sciences, University of L’Aquila, 

I-67100 L’Aquila, Italy. 

In the research of new engineered toxins to target cancer cells, we have studied PDZ 

protein domain as possible tools cellular targeting of the ribosome inactivating protein 

saporin. PDZ domains are found in proteins involved in cellular processes, from the 

signal transduction to the transport and particularly in synapse formation in mammals. 

They recognize and bind short peptide sequences located at the C-terminus of a protein. 

Recognition of the C-termini represents a ‘non-invasive’ interaction, very well suited 

to mediate organization of transport, localization, sorting and spatial arrangement of 

proteins exposing their individual C-terminal tails that can be recognized and handled 

by various PDZ domains. We have focussed our attention to the PDZ domain from 

hCASK (Human calcium/calmodulin-dependent serine protein kinase) that has been 

demonstrated to bind extracellular CD98 in epithelial cells, recognized as a marker 

for several human tumors. We produced fusions of hCASK-PDZ with saporin and 

tested it on human glioblastoma cells. These constructs proved to be toxic, with 

increasing activity as a function of the number of PDZ domains, and induce cell death 

by apoptotic mechanisms. Furthermore we are exploring nucleolin expressed on the 

cell membrane of cancer cells as a specific target of human glioblastoma, since its 

membrane expression in gliomas is stricltly correlated with their malignancy and can 

be exploited to deliver toxic substances. We show in this work preliminary data on a 

DNA construct carrying the gene of the saporin toxin, linked to the AS1411 aptamer, 

specific for nucleolin, Toxicity on glioblastoma cells is reported. Possible applications 

of these targeting strategies will be discussed. 


305


Analisi proteomica della frammentazione delle proteine 

plasmatiche ed urinarie 

in pazienti soggetti a trapianto renale 

furlani eMiliano 1 , leoncini roBerto 1,2 , ScaPellato carlo 2 , 

cenSurato coStantina 3 , Guerranti roBerto 1,2 

1 Dipartimento Medicina Interna, Scienze Endocrino-Metaboliche e Biochimica, 

Università di Siena 

2 U.O.C. Laboratorio Patologia Clinica, Azienda Ospedaliera Universitaria Senese 

3 U.O.C. Nefrologia Dialisi e Trapianto, Azienda Ospedaliera Universitaria Senese 

Nel plasma e nelle urine sono presenti proteine intere e relativi frammenti creati dalle 

proteasi fisiologiche, potenzialmente importanti nella diagnostica di laboratorio poiché 

la proteolisi può essere alterata da varie condizioni patologiche. 

In un recente studio su pazienti con insufficienza renale cronica (IRC), sono stati 

identificati dei peptidi, tra i quali frammenti di albumina. 1 La determinazione 

dell’albumina nelle urine è fondamentale nei pazienti con patologie nefrologiche fra 

cui l’Acute Kidney Injury (AKI). Riguardo ai pazienti sottoposti a trapianto renale, 

molti di essi vanno incontro ad una forma di AKI che consiste in una ritardata ripresa 

funzionale dell’organo o Delayed Graft Function (DGF). Inoltre negli ultimi anni è fiorita 

la ricerca di nuovi biomarker di AKI e DGF più precoci ed efficienti di quelli tradizionali. 

Lo scopo di questa indagine preliminare è duplice: individuare i frammenti 

dell’albumina urinaria modificati rispetto al plasma per gettare luce sull’attività 

proteasica nel contesto del trapianto renale; confrontare lo schema dei frammenti di 

albumina (plasmatica e urinaria) tra le due categorie di pazienti che vanno incontro 

o non vanno incontro a DGF per valutarne un possibile utilizzo come marcatore di 

recupero dell’organo. 

Per questo abbiamo effettuato l’elettroforesi bidimensionale su gel delle proteine 

plasmatiche e urinarie di 6 soggetti trapiantati. Su tutti i campioni inoltre è 

stato eseguito un immunoblot con lo stesso Ab anti-albumina impiegato nelle 

determinazioni nefelometriche per evidenziare eventuali differenze nel pattern dei 

frammenti proteolitici. 

I nostri risultati evidenziano alcune differenze fra i frammenti plasmatici e quelli urinari, 

così come tra i pazienti con DGF e quelli senza DGF. Questi dati preliminari per essere 

confermati hanno bisogno di ulteriori studi con un maggior numero di campioni. 

1. Metzger, J. et al. Urinary excretion of twenty peptides forms an early and accurate diagnostic pattern of 

acute kidney injury. Kidney Int. 78, 1252–1262 (2010). 


306


Implications of Lamin and Emerin mutations in Dilated 

Cardiomyopathies 

Bollati Michela 1 , favalli valentina 2 , arBuStini eloiSa 3 , 

BoloGneSi Martino 1 

1 Department of BioSciences, University of Milano, Milano, Italy 

2 Centre for Inherited Cardiovascular Diseases, 

Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 

Lamin A/C is a nuclear-specific type V intermediate filament protein building up the 

major structural components of the nuclear lamina. Defects in the Lamin gene have 

been linked to more than ten different diseases known as laminopathies; these are 

associated to cardiac complications that often turn into dilated cardiomyopathy 

(DCM), a major cause of congestive heart failure [1]. Within the nuclear lamina, Lamin 

interacts with several nuclear membrane proteins, thus providing a new key target for 

the study of the effects of DCM-related mutations. Among interactors, Emerin has 

been proved to bind directly to lamin, and mutations in the Emerin gene cause DCM, 

similarly to Lamin mutations. So far, no structures of full length Lamin and Emerin 

have been reported, hampering part of the structure-based drug research. In this 

context, we designed novel constructs of full length Lamin and of the Emerin Laminbinding 

domain, that have been purified; crystallization and co-crystallization trials are 

currently ongoing. In parallel, we solved the crystal structures of two LMNA Coil2B 

domain mutants (E347K and R335W) that allow checking the effects of mutations in 

terms of structure variability of the affected protein, and help studying overall protein 

stability and the implications of mutants in the context of DCM [2]. 

1. Arbustini, E. et al., Autosomal dominant dilated cardiomyopathy with atrioventricular block: a lamin A/C 

defect-related disease, J. Am. Coll. Cardiol. 39 (2002) 981–990. 

2. Bollati, M. et al. Structures of the lamin A/C R335W and E347K mutants: implications for dilated 

cardiolaminopathies. Biochem Biophys Res Commun. 418 (2012) 217-21. 


307


From Genome to Antigen: a Structural Reverse Vaccinology 

approach to potential Burkholderia pseudomallei antigens 

laSSaux Patricia 1 , Gourlay louiSe 1 , Peri claudio 2 , ferrer navaro 

Mario 3 , conchillo Solé oScar 3 , daura xavier 3 , lonGhi renato 2 , 

coloMBo GiorGio 2 , and BoloGneSi Martino 1 . 

1. Department of BioSciences, University of Milan, Milan, Italy. 

2. Department of Computational Biology, Istituto di Chimica del Riconoscimento 

Molecolare, Consiglio Nazionale delle Ricerche, Milano, Italy. 

3. Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Spain. 

Vaccine development has recently been changed by the availability of genomes 

from many pathogens; additionally, new perspectives in vaccine use have emerged, 

such as targeting adults, combining antigens, using new method of administration, 

stimulating innate immune response, helping cure of chronic infections or defending 

the population from bioterrorism. 

In this context we focused on Burkholderia pseudomallei, an antibiotic resistant 

pathogen, listed as a biological threat agent of class 3, responsible of Melioidosis, 

a disease, affecting both humans and animals, first described in 1912. In the last 

ten years Melioidosis has become endemic in tropical areas of Southeast Asia and 

Northern Australia, with high mortality rates, its diagnosis being difficult since the 

symptoms mimic other different infections. 

Based on B. pseudomallei genomic information, we applied an improved Reverse 

Vaccinology 1 approach to select potential protein antigens to be tested as vaccine 

candidates. Our approach foresees the application of functional genomics, protein 

microarrays, bioinformatics/computational biology, complemented by structural 

biology studies 2 . Indeed since immunity is enhanced by recognition of an epitope, 

part of a protein in our case, its three-dimensional structure, stability and related 

dynamics are key factors guiding antigen selection. 

To date, 28 constructs have been designed in our lab for heterologous expression in 

E. coli as GST or His-tagged fusions proteins; protein putative antigens have been 

crystallized and 3D structures solved. The progress made to date is here-reported; the 

B.pseudomallei OppA target will be presented to describe the whole new methodology 

and its implications. 

1. Rappuoli R. (2000) Reverse Vaccinology. Current Opinion in Microbiology. 3:445–450 

2. Gourlay LJ, Santi I, Pezzicoli A, Grandi G, Soriani M and Bolognesi M. (2009) Group B Streptococcus 

pullulanase crystal structures in the context of a novel strategy for vaccine development. J. Bacteriol. 

191, 3544-3552. 


308


The role of the molten globule state in the formation of 

amyloid-like structures by human α 1 -acid glycoprotein 

BaldaSSarre Maurizio 1 , Scirè andrea 1 , Barth andreaS 2 , 

Galeazzi roBerta 1 and tanfani faBio 1 

1) Dipartimento di Scienze della Vita e dell’Ambiente, 


2) Department of Biochemistry and Biophysics, 

Stockholm University, Stockholm, Sweden 

α 1 -Acid glycoprotein (AGP) is a heavily glycosilated plasmatic protein belonging to the 

immunocalin family, a sub-group of the lipocalin family involved in immune response. 

The protein structure features a central β-barrel forming a hydrophobic binding site for 

a wide number of biologically-active exogenous and endogenous compounds, while 

the oligosaccharides allow for the interaction with, and recognition by, specific cell 

types of the immune system. Fourier-Transform infrared spectroscopy of temperatureinduced 

denaturation of AGP revealed the presence of a molten globule-like folding 

intermediate (MG) at neutral and acidic pH values, with no such intermediate being 

detected in alkaline or strongly acidic environments. Interestingly, further heat 

denaturation of the MG state obtained at acidic pH values initiated aggregation of the 

polypeptide chains into intermolecular, anti-parallel β-sheets. This is accompanied by 

the typical spectral changes occurring in the absorption and fluorescence emission 

spectra of the amyloid-binding dyes Congo Red and Thioflavin T, respectively, 

suggesting formation of fibrils featuring a cross-β architecture. Positive results were 

also obtained upon analyses of the aggregated samples with transmission electron 

and polarized light microscopies. 

In order to probe in greater detail the connection of MG and the aggregation of 

AGP, time-resolved infrared spectra were collected with millisecond resolution after 

instant release of protons by the photoreactive “caged” compound 1-(2-nitrophenyl) 

ethyl sulphate. A change in the time constant of antiparallel β-sheet formation could 

be measured, allowing us to assess the effect of temperature and pH on the MGdriven 

aggregation of AGP. The data suggest that partial or complete exposure of the 

aggregation-prone regions could trigger AGP assembly into amyloid structures. 


309


Analysis of p27 Kip1 nuclear metabolism in retinoic-treated 

neuroblastoma cells 

BencivenGa d*, BorGia a*, traMontano a, caldarelli i, Pirozzi a, 

oliva a, della raGione f and Borriello a 


Second University of Naples, Via De Crecchio 7, 80138, Naples; 

* These authors equally contributed to the study 

p27 Kip1 is a cyclin-dependent kinase (CDK) inhibitor (CKI) belonging to the Cip/Kip 

family, that also includes p21 Cip1 and p57 Kip2 . The protein regulates cell proliferation 

by modulating the activity of CDKs under several conditions, including cell-to-cell 

contact inhibition and treatment with a large number of antiproliferative drugs. In 

addition to the modulation of cell proliferation, p27 Kip1 controls cell differentiation, 

cell motility and interaction with the substrate, gene transcription and DNA duplication. 

p27 Kip1 activity is controlled by its concentration, its distribution among different 

cellular complexes, and its cellular localization. A number of evidence demonstrated 

that the metabolism and, in turn, the interactors of the CKI are modulated by its posttranslational 

modifications and mainly by phosphorylation. 

Several different residues in human p27 Kip1 have been demonstrated to be 

phosphorylated, namely Ser10, Tyr78, Tyr88, Tyr89, Thr157, Thr187 and Thr198. 

Thr187 phosphorylation by active CDK2 is required to create a phosphodegron 

necessary for Skp2 binding and the subsequent CKI ubiquitination/degradation. 

Conversely, Ser10 phosphorylation has been reported to increase p27 Kip1 stability by 

elongating its half-life. Our previous studies demonstrated that neuroblastoma cells 

treatment with all-trans retinoic acid induces a rapid neuronal-like cell differentiation 

associated to an increase of phospho(Ser10)p27 Kip1 in the nuclear compartment. 

The goal of the present study has been to identify the molecular mechanisms 

responsible of phospho(Ser10)p27 Kip1 increased stability. To this aim, we preliminary 

developed a methodology for the characterization of p27 Kip1 phosphoisoforms 

based on 2D protein separation associated to the use of well-characterized specific 

antibodies against the CKI phosphoisoforms. Our findings demonstrate that the 

increase of phospho(Ser10)p27 Kip1 stability is due to the scarce phosphorylation of 

the isoform on Thr187. At present, we are investigating whether this effect is due to a 

diminished interaction with CDK2 or to an altered binding of the protein to the kinase. 


310


Analysis of erythrocyte membranes by different proteomic 

methodologies in Rett Syndrome 

cortelazzo aleSSio 1,5 , Guerranti roBerto 1 , leoncini Silvia 2,5 , 

SiGnorini cinzia 2 , landi GiacoMo 1 , Pecorelli aleSSandra 2 , 

valacchi GiuSePPe 3 , de felice claudio 4 , ciccoli lucia 2 , 

hayek JouSSef 5 

1 Department of Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry 

University of Siena, Siena, Italy; 2 Department of Pathophysiology, Experimental 

Medicine and Public Health University of Siena, Siena, Italy; 3 Department of 

Evolutionary Biology, University of Ferrara, Ferrara, Italy; 4 Neonatal Intensive Care 

Unit, University Hospital AOUS, Siena, Italy; 5 Child Neuropsychiatry Unit, 

University Hospital AOUS, Siena, Italy 

Rett syndrome (RTT), with a frequency of ~ 1:10000–1:15000 females, is a rare and 

complex neurodevelopmental disorder [1] which presents as a neuroregression 

following a normal development for 6–18 months with loss of cognitive, social, and 

motor skills in a typical 4-stage regression. To date, mutations in 3 main genes (MeCP2 

» CDKL5 > FOXG1) have been related to RTT clinical features. A wide phenotypical 

heterogeneity is well-known in the disease which includes at least 4 major different 

clinical presentations, i.e., typical, preserved speech (PSV) early seizure (ESV) and 

congenital variants [1]. Recently, our group has shown erythrocyte shape changes in 

typical RTT patients and find to be related to membrane oxidative stress [2]. better 

characterize the erythrocytes in RTT, a classical proteomic analysis was used to 

establish a reference map of membrane proteins in the disease and compared with 

the proteins pattern in healthy controls. Preanalytical study indicated that the storage 

by freezing at -80°C can cause protein degradation with different protein pattern 

profile compared to the reference map. Differential extraction procedures were set 

up allowing separation of integral membrane proteins from peripheral species and 

to determine the most appropriate extraction method. thorough analysis of protein 

patterns in RTT patients will be of major help in defining of the most efficient strategy 

for identifying individual proteins and to investigate on possible relationships between 

erythrocyte protein patterns and clinical phenotype and/or disease severity. 

References: 

[1] Neul JL, Kaufmann WE, Glaze DG, et al. Ann Neurol. 2010;68:944-50. 

[2] Ciccoli L, De Felice C, Paccagnini E, et al. Biochimica et Biophysica Acta. 2012;511-20. 

The research has been funded by Tuscany Region [“Antioxidant (ω-3 Polyunsatured Fatty Acid, lipoic acid) 

supplementation in Rett syndrome: a novel approach to therapy”]. 


311


Single residue contribution to the self-aggregation and 

activity of an antifungal immunoglobulin derived peptide 

Pertinhez thelMa 1 , ciociola tecla 2 , conti Stefania 2 , 

Polonelli luciano 2 , SPiSni alBerto 2 

1 Laboratorio C.I.M., Tecnopolo Parma; 2 Dipartimento di Scienze Biomediche, 

Biotecnologiche e Traslazionali – S.Bi.Bi.T., Università di Parma, Italia 

Antimicrobial peptides are important effector molecules of the innate immune system. 

Peptides derived from the human immunoglobulin (Ig) proteolysis exert fungicidal 

activity against Candida albicans, in vitro and in vivo (1). The decapeptide N10K 

(NQVSLTCLVK) derived from IgG1 exhibited significant activity, EC 50 of 1.0 – 2.8 

µM against different fungal strains and showed no hemolytic or cytotoxic effects on 

human red blood cells and mammalian cells. 

N10K sequence is characterized by the alternation of hydrophobic/hydrophilic 

residues and displayed the ability to spontaneously self-assembly into rich β-sheet 

structure in aqueous solution or in the presence of C. albicans. Electron micrographs 

of negatively stained N10K demonstrated the presence of a network of fibril-like 

structures. In order to critically establish the relevance of each residue in the selfaggregation 

and fibril formation processes we analyzed N10K using alanine-scanning, 

by Circular Dichroism, Fluorescence Spectroscopy and Electron and Transmission 

Microscopy. 

The alanine substitution showed increased, unaltered or decreased candidacidal 

activity in comparison to parental peptide. For instance, replacement of cysteine, that 

hampers disulphide bond formation, determined the most significant reduction of 

candidacidal activity: in fact the absence of pre-alignment of the monomers reduces 

the entropic cost of assembly, and consequently the formation of intermolecular 

H-bonds. Substitution of the positively charged residue makes the peptide highly 

hydrophobic lowering its solubility and activity. 

Overall, sequence analysis of the propensity of cross β-aggregation, amino acid 

volume, H-bond potential, hydrophobicity was exploited to devise an explanation 

of the contribution of individual residue in both, self-aggregation and candidacidal 

activity. 

(1) L. Polonelli, T. Ciociola, W. Magliani, P.P. Zanello, T. D’Adda, F. De Bernardis, S. Arancia, E. Gabrielli, E. 

Pericolini, A. Vecchiarelli, T.A. Pertinhez, A. Spisni, S. Conti (2012) “Peptides of the Constant Region of 

Antibodies Display Fungicidal Activity.” Plos One 7, e34105. 


312


Nucleophosmin binds G-quadruplex regions 

at ribosomal DNA 

chiarella S. 1 , ScaGlione G.l. 2 , lo Sterzo c. 1 , di Matteo a. 1 , 

arcovito a. 2 , federici l. 3 

1 Dipartimento di Scienze Biochimiche, “Sapienza” Università di Roma, 00185 Roma. 

2 Istituto di Biochimica e Biochimica Clinica, 

Università Cattolica del Sacro Cuore di Roma, 00168 Roma. 

3 Ce.S.I. Centro Scienze dell’Invecchiamento e Dipartimento di Scienze Sperimentali 

e Cliniche, Università di Chieti “G. D’Annunzio”, 66013 Chieti. 

Nucleophosmin (NPM1) is an abundant phospho-protein that plays a key role 

in ribosome biogenesis. NPM1 binds nucleic acids and has intrinsic RNAase and 

chaperone activities. Although mainly localized at nucleoli, NPM1 continuously 

shuttles between the nucleus and the cytoplasm to fullfill its functions. 

The gene encoding NPM1 is mutated in 50–60% of normal kariotype acute myeloid 

leukemia (AML) patients. A distinctive feature of NPM1 mutants is their stable and 

aberrant localization in the cytoplasm of leukemic cells. All mutations lead to critical 

changes at the NPM1 C-terminus: i) loss of tryptophan residues 288 and 290 (or 290 

alone), which destabilizes the C-terminal domain; ii) generation of a new NES motif, 

which reinforces the nuclear export of NPM1 protein. Both alterations are critical 

for perturbing NPM1 mutant traffic and for the underlying aberrant accumulation of 

NPM1 in the cytoplasm of leukemic cells. 

Nucleoli are complex structures assembled around tandem arrays of rDNA genes and 

here we investigate the structural determinants of NPM1 nucleolar localization. We 

show that a domain encompassing the last 70 residues of the protein (NPM1-C70) 

binds with high affinity oligonucleotide sequences with G-quadruplex structure found 

at the non-coding strand of ribosomal DNA. Importantly the most common leukemic 

NPM1 variant C-terminal domain is unfolded and completely loses this activity. This 

is the consequence of G-quadruplex binding domain destabilization, since mutations 

aimed at refolding the leukemic variant also result in rescuing the G-quadruplex 

binding activity and nucleolar localization. In conclusion this work establishes the 

structural bases for understanding NPM1 nucleolar localization and its impairment by 

AML-associated mutations. 


313


Proteomic profile of benign tumours of parotid glands 

donadio elena 1 , GiuSti laura 1 , ventroni tiziana 1 , da valle ylenia 1 , 

Seccia veronica 2 , Sellari franceSchini Stefano 2 , 

lucacchini antonio 1 

1 Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, 


2 Department of Otorhinolaryngology, 1st ENT Unit, Azienda Ospedaliero- 

Universitaria Pisana,Pisa, Italy 

In the great variety of benign tumours of the parotid glands, a pre-operative diagnosis 

that distinguish the different isotypes is often difficult. In particular, Warthin’s tumour 

shows clinical features that do not provide a sufficient basis for a precise diagnosis. 

Identification of potential prognostic biomarkers would surely improve the required 

accuracy in diagnosis. Proteome analysis has been shown to be a useful tool for 

studying human diseases and for identifying new prognostic, diagnostic, and 

therapeutic markers: we therefore decided to explore proteomic pattern of the fine 

needle aspiration fluid of pleomorphic adenoma (PA) and warthin’s tumour (WT). 

Thirty-two patients were enrolled, 19 were classified as PA, 13 as WT. Immediately 

after surgical removal of the gland, a fine needle biopsy was made on the tumoural 

nodule and on the surrounding tissue. This fluid was centrifuged and the proteins 

were precipitated using the TCA/Acetone method. Two pools belonging to the PA and 

WT samples were made and each pool was loaded in triplicate. After 2D separation, 

the protein patterns of the PA and WT samples were compared by using Progenesis 

SameSpot (NonLinear Dynamics) to search differentially expressed protein spots. 

Proteins of interest were submitted to nanoLC-ESI-MS-MS analysis for protein 

identification. A total of 68 spots differentially expressed were found with fold variation 

> 2. Among the proteins identified by mass spectrometry, many immunoglobulins as 

Ig alpha-1 chain C region, Ig kappa chain C region, Ig lambda-2 chain C regions, Ig 

kappa chain C region, resulted to be up-regulated in WT pool with respect to adenoma 

probably due to the lymphomatosum origin of the WT tumour. 

Our preliminary results may suggest the potential applicability of the proteomic 

approach to define a potential panel of protein biomarkers useful in the diagnosis of 

benign tumour of parotid glands. 


314


Interaction of bacterial glutathione transferase with hemin 

allocati nerino 1 , MaSulli Michele 1 , federici luca 1,2 , 

di ilio carMine 1,2 

1 Dipartimento di Scienze Sperimentali e Cliniche and 2 Ce.S.I., Centro Scienze 

dell’Invecchiamento, Università ‘G. d’Annunzio’, Chieti, Italy. 

Glutathione transferases (GSTs, EC 2.5.1.18) are pivotal enzymes for detoxification 

processes in eukaryotes and prokaryotes (1, 2). GSTs catalyse glutathione conjugation 

to a broad spectrum of compounds. In addition, functions not related to detoxification 

have been discovered for GSTs including steroid biosynthesis, signal transduction 

and intracellular transport (1). 

Hemin has been shown to be hazardous to the cell membrane because it inserts 

into the lipid bilayer and binds to membrane proteins. These interactions may cause 

membrane changes, which may lead to cellular lysis. 

The ability of several eukaryotic GSTs from pluricellular and unicellular organisms 

to interact with hemin has been widely investigated (3, 4), and this compound was 

shown to bind the enzyme with high affinity (, 4). 

To verify if this interaction is a prerogative of eukaryotic GSTs or it has a general 

significance, we studied the binding of hemin to Proteus mirabilis (PmGST), a 

representative bacterial GST belonging to the Beta class. The interaction was studied 

by means of inhibition assays and fluorescence spectroscopy.Results obtained 

indicate that PmGST binds hemin efficiently, with a considerable quenching of the 

intrinsic fluorescence of PmGST. Furthermore, hemin was found to be a strong 

inhibitor of GST conjugation activity. 

1. Hayes JD, Flanagan JU, Jowsey IR (2005) Annu Rev Pharmacol Toxicol, 45: 51-88. 

2. Allocati N, Federici L, Masulli M, Di Ilio C (2009) FEBS J, 276: 58-75. 

3. Caccuri AM, Aceto A, Piemonte F, Di Ilio C, Rosato N, Federici G (1990) Eur J Biochem, 189: 493-497. 

4. Liebau E, Dawood KF, Fabrini R, Fischer-Riepe L, Perbandt M, Stella L, Pedersen JZ, Bocedi A, Petrarca 

P, Federici G, Ricci G (2009) J Biol Chem, 284: 22133-22139. 


315


Characterization of Staphylococcus lugdunensis biofilm 

formation in different growth conditions: role of Isd 

proteins on biofilm formation 

MiSSineo antonino, di Poto antonella, ciPolla lina, fuGazza 

anGela, Bertaina valentina, venuto annunziata, Pietrocola 

GiaMPiero, rindi SiMona and SPeziale Pietro. 

Molecular Medicine Department, University of Pavia, “A.Castellani” 

Biochemistry Section 

S. lugdunensis is a skin commensal that acts as an atypically virulent pathogen 

causing infections which resemble those caused by S. aureus, expecially acute 

and highly destructive cases of native valve endocarditis that often require surgical 

treatment in addition to antimicrobial therapy. It is also responsible for several types 

of biofilm-related infections. Despite the genetic presence of icaADBC homologues, 

poly-N-acetylglucosamine (PNAG) is not a major component of S.lugdunensis biofilm 

matrix suggesting that biofilm is mainly composed by proteinaceous factors. The 

genome sequence analysis of S. lugdunensis strain N920143 shows that it is the only 

staphylococcal species other than S. aureus that possesses a locus encoding four 

iron-regulated surface determinant proteins (IsdK, IsdJ, IsdC, IsdB) involved in iron 

acquisition from haemoglobin. In this study a collection of S.lugdunensis strains was 

analysed upon their biofilm formation ability in a rich medium (TSB, Tryptic Soy Broth), 

or in iron-starvation (RPMI). All the strains examined make a proteinaceous biofilm 

sensitive to trypsin, proteinase K and DNase I treatment while sodium metaperiodate 

and dispersin B have no effect. The addition of 100 µM FeCl 3 diminishes the amount 

of biofilm formation in RPMI suggesting a possible role of Isd proteins in the process. 

The expression of Isd proteins on bacterium surface was demonstrated by Western 

blot; moreover, IgG raised against them significantly decrease in a dose-dependent 

manner biofilm formation. The deletion of the entire Isd locus or IsdC gene alone 

cause a statistical significant decrease (P


Nasu-Hakola disease: a pilot study for the research of 

possible biomarkers 

1,2 Giuliano Serena, 2,3 MontalBetti lorenza, 1 floriani anna M 

1 Bardoni anna, 1 Salvini roBerta 

1 Dept.of Molecular Medicine, University of Pavia 

2” Fondazione Laura Fossati” ONLUS 

3 Dept. of Public Healthy, Neuroscience, Experimental and Forensic Medicine, 

University of Pavia 

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy or 

Nasu-Hakola disease (NHD), is a genetically heterogeneous, recessively inherited 

disorder [1]. The molecular genetic background of NHD was characterized by 

identifying mutations in two genes, DAP12 or TREM2. DAP12 is a transmembrane 

adaptor protein that is associated with the cell surface receptor TREM2 [2,3]. 

The histological hallmarks of NHD are cystic bone lesions, osteoporotic features, and 

loss of white matter in the brain [1]. This combination of symptoms is unique to this 

disease, which, as indicated by Montalbetti et al. [4] is underestimated in Italy. Patients 

are likely to go unrecognized partly because they are considered to be affected by 

other kinds of dementia or by fibrous dysplasia and, partially, because the highest 

prevalence of NHD is found in Finland and Japan, and is generally unknown outside 

these countries. 

To facilitate the recognition of NHD patients is necessary obviously to identify possible 

biomarkers of the disease. To this a proteomic approach (2-DE and MS) was used 

on plasma samples of six individuals from an Italian family. 2-DE analysis was thus 

performed on plasma from a subject with homozygous mutation in TREM2, 3 subjects 

with heterozygous mutation in the same gene, and 2 healthy subjects. 

The comparative analysis showed the presence of some proteins differently expressed. 

They were identified by gel matching and mass spectrometry. A few inflammatory 

proteins were classified (Haptoglobin, Alpha-1-acid glycoprotein 1) and others 

involved in different neurodegenerative diseases (Transrtyretin, Clusterin ) could be 

detected. 

This work was supported by ” Fondazione Laura Fossati” ONLUS 

[1] Verloes A et al. J Med Genet. 1997; 34:753-7. 

[2] Paloneva J et al. Nat Genet. 2000; 25:357-61. 

[3] Paloneva J et al. Am J Hum Genet. 2002; 71:656-62. 

[4] Montalbetti Let al. Funct Neurol. 2004; 19:171-9. 


317


A Proteomic Approach To Study Malignant Pleural 

Mesothelioma 

da valle ylenia 1 , GiuSti laura 1 , donadio elena 1 , cireGia federica 1 , 

ventroni tiziana 1 , Bonotti aleSSandra 2 , foddiS rudy 3 , 

criStaudo alfonSo 2 , lucacchini antonio 1 



2 Occupational Medicine of University Hospital of Pisa, Italy 

3 Department of Endocrinology and Metabolism, Orthopaedics and Traumatology, 

University of Pisa, Italy 

Malignant mesothelioma is a highly aggressive cancer, unresponsive to chemotherapy, 

radiotherapy, or surgical resection, and it can affect any kind of serosal membrane 

(pleura, peritoneum, pericardium and even the tunica vaginalis testis). Malignant 

pleural mesothelioma (MPM) is a rare thoracic malignancy, that is directly linked 

to asbestos exposure so the male gender is associated with the majority of MPM 

diagnoses. MPM represents less than 1% of known cancers but its incidence will 

continue to increase significantly with severe public health consequences. 

We applied a proteomic approach to define proteomic profiles of MPM tissues. 

Biopsies were obtained from 18 patients and classified in MPM (n=10), and hyperplasia 

(n=8). Protein extracts were separated using two-dimensional electrophoresis. After 

comparison of MPM and hyperplasia protein profiles, a total of 114 protein spots were 

found to be differentially expressed, each exhibiting ≥ 2 fold-change (either increase 

or decrease) of mean value spot intensity. Spots of interest were cut and proteins 

identified by nanoLC-ESI-MS-MS. Calretinin, Prelamin A/C, Desmin, Fructosebisphosphate 

aldolase A, myosin light chain 3, myosin light chain 2 ventricular/ 

cardiac muscle isoform, myosin light chain 6B and Creatine kinase M-type exhibited 

≥ 3 fold-change between MPM and hyperplasia samples. The different expression 

was validated by western blot analysis and was compared with Carcinoma samples 

(n=10) as a positive control. This is the first study performed on MPM biopsies and 

shows the potentiality of proteomic approach to define a potential panel of protein 

biomarkers useful in the diagnosis of MPM. 

This research was supported in part by grant from the “Ministero Salute, Ricerca 

Finalizzata 2009”. 


318


Potential thyroid cancer biomarkers from fine needle 

aspiration in vivo and in post surgery lesions 

cireGia federica 1 , GiuSti laura 1 , da valle ylenia 1 , raGo tiziana 2 , 

niccolai filiPPo 2 , MoliSano anGelo 2 , di coScio Giancarlo 3 , iacconi 

Pietro 4 , tonacchera MaSSiMo 2 , lucacchini antonio 1 . 


University of Pisa; Pisa, Italy. 2 Department of Endocrinology and Metabolism, 

Orthopedics and Traumatology, Occupational Medicine, University of Pisa, Pisa, Italy. 

3 Department of Oncology Section of Cytopathology, University of Pisa, Pisa, Italy. 

4 Department of Surgery, University of Pisa, Pisa, Italy. 

The annual incidence of thyroid cancer has been reported to range between 1.2 and 

2.6 cases per 100,000 in men and between 2.0 and 3.8 cases per 100,000 in women. 

Fine-needle aspiration (FNA) biopsy is the technique usually applied to distinguish 

benign from malignant thyroid nodules. However, cytological analysis can’t always 

allow a proper diagnosis of thyroid nodules. Therefore an improvement on the 

diagnostic capability of FNA could avoid unnecessary thyroidectomy. 

In a previous study (1) we examined FNA collected immediately after total thyroidectomy. 

We investigated the global changes of FNA protein patterns of two variants of 

malignant papillary thyroid cancer (PTC): classical variant PTC (cPTC) and tall cell 

variant PTC (TCV). Differences in protein expression between cancer and controls 

were identified using two-dimensional electrophoresis and mass spectrometry. With 

the present work we tested the possibility to transfer these findings on a larger number 

of pre-surgical thyroid FNA samples. We collected 184 FNA in vivo. For each sample 

a cytological analysis was performed and a histological analysis was also carried out 

for the patients that underwent surgical intervention. FNAB were classified as benign, 

cPTC and TCV. 

We evaluated by ELISA assay and western blot analysis levels of five proteins, 

previously found up-regulated in cancer respect to controls; these proteins were: 

L-lactate dehydrogenase B chain, Ferritin heavy chain, Ferritin light chain, Annexin 

A1, and Moesin. 

We confirmed the significant differences in expression of these proteins supporting 

the diagnostic relevance of these putative biomarkers. Moreover, taken together, our 

studies indicate that thyroid FNA analysis is a useful tool in the diagnostic classification 

of the disease. 

This research was supported by grant from the “Progetto Salute 2009, Regione 

Toscana”. 

1.Giusti L, et al. J Proteome Res. 2008;7(9):4079-88. 


319


Homocysteine induces insulin fibrillation and loss of 

function: A possible link with both insulin resistance and 

cutaneous amyloidosis* 

1 Paoli Paolo, 1 caSelli anna, 1 cirri Paolo, 1 Santi alice, 1 luti 

SiMone, 2 GiorGi claudia, 2 ciofi SiMone, 3 SBrana franceSca, 

4 tiriBilli Bruno, 1 caMici Guido 

1 Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Firenze 

2 Department of Chemistry, University of Florence, Via della lastruccia 3, 50019 Sesto F.no 

3 CNR, Istituto di Biofisica, via de Marini 6, 16149 Genova 4 Istituto dei Sistemi Complessi— 

Istituto Nazionale delle Ricerche, via Madonna del piano 10, 50019, Sesto F.no 

Amyloidoses are pathologies characterized by extra- or intracellular fibrillar protein 

accumulation. Protein fibrillation initiates when folded proteins are destabilized 

adopting non-native conformations more prone to aggregate. The amorphous 

aggregates evolve to lead high ordered fibrils. 

A particular form of sub-cutaneous amyloidosis was observed in diabetics who inject 

insulin repeatedly in the same skin site. The analyses performed on the nodules 

formed at the injection site showed the presence of amyloid fibrils containing insulin, 

but subjects did not develop signs of systemic amyloidosis. Up to now, the causes 

that induce the transformation of the native structure of insulin in the fibrillar form are 

unknown. 

Some authors demonstrated that insulin down-regulates the expression of 

cystathionine β-synthase favouring the accumulation of homocysteine (Hcy), an 

amino-acid generated in the pathway of cysteine synthesis. Hence, the chronic 

excess of circulating insulin, frequently observed in diabetics, could contribute to 

develop hyper-homocysteinemie, which is associated with insulin resistance. This 

suggests that high plasma levels of HCy interfere with the insulin function. 

Our experiments demonstrated that HCy binds to insulin, and that this complex 

displays a lower activity than that of native insulin on the insulin receptor. In addition, 

we observed that Hcy causes a partial insulin unfolding, favouring its aggregation. 

The obtained aggregates bind Thioflavine T and Congo Red, and are characterized by 

an high β-sheet content. Finally, AFM analysis of HCy-treated insulin demonstrated 

that these aggregates contain worm-like protofibrils. These findings suggest that Hcy 

contribute to the development of insulin resistance in vivo. We think that high insulin 

levels due to repeated insulin injection in the same site determine a local increase of 

Hcy level that, in turn, leads to insulin aggregation. 

*Work supported by the Fondazione Cassa di Pistoia and Pescia for funding. 


320


CERATO-PLATANIN AND CERATO-POPULIN INDUCE 

MAPK ACTIVATION IN PLANE AND ARABIDOPSIS LEAVES 

luti S. 1 , Bruno G. 2 , Baccelli i. 4 , Bernardi r. 3 , Martellini f. 1 , 

Scala a. 4 and PazzaGli l. 1 

1 Dipartimento di Scienze Biochimiche, Università degli Studi, Viale Morgagni 

50, 50134 Firenze, Italy. 2 Entomon s.a.s via di Ripoli 254, 50126 Firenze, 

Italy. 3 Dipartimento di Biologia delle Piante Agrarie, Università degli Studi, Via della 

Piagge 23, 56124 Pisa, Italy. 4 Dipartimento di Biotecnologie Agrarie, Università degli 

Studi, Via della Lastruccia 10, 50019 Sesto Fiorentino, Italy. 

Cerato-platanin (CP) and cerato-populin (Pop1) are small proteins produced by the 

phytopathogenic fungi Ceratocystis platani and C. populicola, respectively. CP and 

Pop1 behave as PAMPs (Pathogen associated Molecular pattern), since they elicit 

typical defense responses in various host and non-host plants such as the induction 

of synthesis of phytoalexins and the induction of the hypersensitive response that 

culminates with a programmed cell death of the infected tissues. CP structure has 

been solved by NMR and it is a double ψ-β-barrel protein. Pop1 has a similar fold but 

circular dichroism shows differences in the secondary structure. Recently, it has been 

shown that CP and Pop1 are able to induce H 2 O 2 and NO synthesis and enhance 

the transcription of several proteins related to defense such as: PR1 (unknown); 

PR2 (β-1,3 glucanase), PR5 (thaumatin), APX (Ascorbate peroxidase), ThiF protein 

(adenylyltransferase) and the WRKY transcription factor. 

The aim of this work is to investigate the activation of another typical defence response: 

the activation of MAPK cascade in host (plane) and non-host (Arabidopsis) leaves. 

The leaves were treated with 1.5nmol/10 µL of CP and Pop1. After 15min, 30min 

and 1, 3, 6, 12, 24 and 48 hours of incubation, plane leaves were subjected to protein 

extraction by a three steps method using acetone and phenol. Sample were analyzed 

by SDS-PAGE and western blot using anti-Phospho-ERK1/2 human antibodies which 

specifically recognized the MAPK3 and MAPK 6 of plants. Protein were extracted from 

Arabidopsis treated leaves by TRIS buffer containing tween and protease inhibitors. 

Samples were assayed as above reported. Results show the activation of MAPK 3/6 

after 1-3 hours of incubation in CP-treated leaves and after 6-9 hours in Pop1-treated 

leaves. Results clearly show that CP and Pop1 determine activation of MAPKs which 

peculiar to defense responses either in plant and in animal tissues. 


321


Protein machinery involved in cyt c maturation 

in gram-negative bacteria 

eva di Silvio 1 , adele di Matteo 2 , carlo travaGlini-allocatelli 1 

Dip. Scienze Biochimiche “A. Rossi-Fanelli”, Sapienza - Università di Roma 1 and 

Istituto di Biologia e Patologia Molecolari del CNR,Roma 2 

The c-type cytochromes are widespread electron carrier proteins found in all domains 

of life and are characterized by a covalently bound heme (Fe-protoporphyrin IX) 

linked to the CXXCH motif of the apoprotein. In the human pathogen Pseudomonas 

aeruginosa, the biogenesis of Cytc is mediated by Ccm proteins (CcmABCDEFGHI) 

called System I. We have already characterized PaCcmG and PaCcmH, both involved 

in thio-reduction pathway of apocytc, from a functional and structural point-ofview. 

We are now studying PaCcmI and PaCcmF, two proteins involved in the final 

steps of heme-apocytc ligation pathway and probably forming a multisubunit heme 

ligation complex together with PaCcmH. How the CcmFHI complex recognizes its 

substrates apocyt and heme remains unknown. Bioinformatic analyses suggest that 

the periplasmic domain of PaCcmI is composed of three TPR motifs (tetratricopeptide 

repeat), probably involved in the recognition of apocytc, and an α/β domain at the 

C-terminus. It has been proposed that PaCcmI may interact with apocyt through the 

TPR motifs and present it to CcmF/H complex responsible for the formation of the 

covalent bonds with heme. PaCcmI has been cloned and purified in a soluble state: 

functional characterization using equilibrium and kinetic fluorescence spectroscopy is 

in progress to demonstrate the binding with apocytc and synthetic peptides mimicking 

the C-terminal portion of apocytc. Crystals of the purified PaCcmI protein have been 

obtained; we are currently involved in production of selenomethionine substituted 

CcmI crystals in order to solve its 3D structure. 

PaCcmF is a large integral membrane protein with small periplamatic domains. 

Bioinformatics analysis method suggests that it contains 15 trans-membrane helices 

in P. aeruginosa. It has been proposed that CcmF may act as heme reductase and 

Cytc synthase. We are currently focusing on the functional role played by the largest 

soluble periplasmic domain of this membrane protein. Preliminary results suggest that 

this 100 residues domain recognizes and binds to the heme group. Altogether, these 

results may shed light to our understanding the cytochrome c biogenesis process in 

bacteria. 


322


Characterization of novel antimicrobial peptides isolated 

from the saliva of Polistes dominulus 

Martellini f. 1 , Bruno G. 4 , Pantera B. 4 , luti S. 1 , PaGni e. 2 , 

carreSi l. 1 , turillazzi S. 3 , caPPuGi G. 1 and PazzaGli l. 1 . 

1 Dipartimento Scienze Biochimiche, Università degli Studi di Firenze, Italy 

2 Scuola superiore S. Anna, Pisa, Italy 

3 Dipartimento Biologia Evoluzionistica, Università degli Studi di Firenze, Italy 

4 Entomon s.a.s via di Ripoli 254, 50126 Firenze, Italy 

The production of antimicrobial peptides (AMPs) is one of the most important innate 

defense mechanisms adopted by prokaryotes and eukaryotes against infections 

caused by microrganisms. On the basis of their structural features, AMPs are 

classified into four classes: (i) linear peptides, devoid of cysteines and forming 

α-helices, such as cecropins; (ii) peptides with an over-representation in proline and/ 

or glycine residues; (iii) peptides stabilized by intramolecular disulfide bonds, such 

as defensins; (iv) peptides composed by rare and modified aminoacids. Many AMPs 

have been found in insects; they are usually small cationic molecules, characterized 

by a low molecular weight, often forming amphipathic structures, present both in the 

hemolymph and in the saliva. 

Here we report the isolation of PolA and PolB, two AMPs from the larval saliva of the 

social wasp Polistes dominulus, a very common specie in Europe. Microbiological 

tests proved that larval saliva of P. dominulus has an antibiotic activity against Bacillus 

subtilis, Gram +, and Escherichia coli, Gram -. These antimicrobial peptides were 

purified by RP-HPLC and characterized by peptide sequencing and mass spectrometry. 

PolA has a molecular weight of 2279.50 Da and is composed of 21 aminoacids. It 

is rich in proline and its sequence has been completely determined. PolB, with a 

mass of 3700,54 Da, is formed from 35 aminoacids and has been almost completely 

sequenced. It belongs to the group of defensins and shows some similarities with a 

putative defensin of the dipter Culicoides sonorensis. In our studies we evaluated 

minimum inhibitory concentration (MIC 90) of PolA concerning B. subtilis grown on 

LB. Under the experimental conditions used we found that MIC 90 is 0,0029 mg/mL, 

thus indicates that polA presents a remarkable activity against Gram +. 

A thorough characterization of the properties of these peptides could bring significant 

goals in therapeutic. 

The complete characterization of the peptides will bring to an increase of antimicrobial 

molecules to be tested as new therapeutic agents. 


323


Membrane cholesterol is a key-modulator of the 

endocannabinoid-hydrolase FAAH 

daineSe enrico 1,2 , de faBritiiS Gianni 3 , SaBatucci annalaura 1 , 

oddi SerGio 1,2 , anGelucci clotilde B. 1 , di Pancrazio chiara 1 , 

GiorGino toni 3 , Stanley nathaniel 3 , cravatt BenJaMin 4 , Mauro Maccarrone 1,2 

1 Department of Biomedical Sciences, University of Teramo, Teramo, Italy, 2 European Center 

for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy, 3 Computational biochemistry 

and biophysics laboratory, (GRIB-IMIM), University of Pompeu Fabra, Barcelona Biomedical 

Research Park (PRBB), Barcelona, Spain, 4 Departments of Cell Biology and Chemistry, The 

Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, U.S.A. 

Fatty acid amide hydrolase (FAAH) is a representative member of membrane enzymes 

that, having as substrates lipid molecules, need to get a lipophilic compound from 

within the membrane, and to catalyze its hydrolysis by water. Moreover, FAAH is the 

enzyme playing a critical role in the modulation the endocannabinoid tone in mammals, 

degrading important neurotransmitters such as anandamide and oleamide. The latter 

compounds are involved in a number of human diseases, representing therefore a 

valuable target for biochemical and pharmacological research. 

Attempts to investigate the role of the surrounding lipids in membrane-embedded 

enzymes has not been quite successful so far, yet lipophilic compounds are expected 

to interact with the membrane in several ways. The aim of this study was to investigate 

in details the effect of membranes of different lipid compositions that on FAAH and to 

ascertain their effect on its structure, kinetic and membrane binding properties. 

Here we describe the first structure in solution of FAAH by small angle X-ray scattering 

(SAXS) analysis, and disclose new structural and functional effects of membrane 

lipids on this enzyme. Furthermore, this study represents a conceptual step forward 

to understand mechanistic details of the functional regulation of a paradigmatic 

membrane enzyme like FAAH by the surrounding lipids. To this aim, a combination 

of experimental and computational methods was integrated in a multidisciplinary 

approach. 

Overall, our findings stress the importance of membrane lipids, and in particular of 

cholesterol, for the modulation of the endocannabinoid tone within a cell, but also 

show an interesting example of the extensive modulation that the membrane lipid 

composition exerts on a characteristic and important membrane enzyme at the level 

of structure, localization and activity. 

This work was supported by Ministero dell’Istruzione, dell’Università e della Ricerca (Grant PRIN 2008). 


324


DNA binding properties of the human ZFP57 protein 

de ceSare lucia 1 , BaGlivo ilaria 1 , eSPoSito SaBrina 1 , 

rivellino aleSSia 1 , GriMaldi Giovanna 2 , riccio andrea 1 and 

Pedone Paolo v 1 . 

1 Department of Enviromental Science, University of Naples 2, 8100 Caserta, Italy. 

2 Institute of Genetics and Biophysics A.Buzzati-Traverso, CNR, 80131 Naples, Italy. 

DNA methylation is one of the epigenetic modifications involved in gene silencing, 

including that of the imprinted genes. Imprinted genes are expressed from only 

one parental allele along methylation patterns that are determined during gamete 

formation. Imprinted genes play a key developmental role and are generally organized 

in clusters controlled by CpG-rich sequences known as Imprinting Control Regions 

(ICRs). ICRs are targeted by parental allele specific DNA Methylation imprints that are 

established during late gametogenesis, manteined in zygote and somatic cells, and 

erased in primordial germ cells. 

Methyl DNA Binding Proteins (MBPs) interacts with methylated CpG and recruit on 

DNA different corepressor complexes forming transcriptionally silenced chromatin 

structures. Sequence-specific recognition of methylated DNA has been demonstrated 

for human zinc finger proteins Kaiso, ZBTB4, ZBTB38 and ZFP57. These proteins 

interact in a sequence specific manner with DNA containing methylated CpG. 

We have demonstrated that the mouse ZFP57 protein (mZFP57) containing 5 zinc 

finger domains (ZFs), recognizes methylated DNA target sequences using the 

second and the third ZFs. In this study we present our results on the human ZFP57 

(hZFP57) protein. hZFP57 contains 7 ZFs, the third and the forth being homologue 

to the second and third of the mZFP57. We demonstrated that the human protein 

sequence extending from glycine 226 to valine 288, including the third and the fourth 

ZFs, is sufficient for high affinity binding to the methylated DNA sites, and we mapped 

the critical ZF amino acid residues required for the interaction with DNA. 

Recently, mutations in hZFP57 mapping in the zinc finger region have been associated 

with transient neonatal diabetes (TND); we demonstrated that TND mutations in 

hZFP57 ZF 3 and 4 abolish DNA binding. 

References: 

1. Quenneville S. et al. Molecular Cell 44, 361-372, Nov 2011. 

2. Mackay D.J.G. et al., Nature Genetics 40, 949-951, Aug 2008. 


325


Similarities and differences of rat and human fatty acid 

amide hydrolases 

di venere alMerinda 1,2 , daineSe enrico 3,4 , fezza filoMena 2 , 

anGelucci Beatrice clotilde 3 , roSato nicola 1,2 , 

cravatt BenJaMin f 5 , finazzi-aGrò aleSSandro 6 , Mei GiaMPiero 1,2 

Maccarrone Mauro 3,4 

From the 1 Neuromed – Pozzilli Italy and 2 Department of Experimental Medicine and 

Biochemical Sciences, University of Rome “Tor Vergata”, 00133 Rome, Italy, the 

3 Department of Biomedical Sciences, University of Teramo, 64100 Teramo, Italy, 

the 4 European Center for Brain Research/Istituto di Ricovero e Cura a Carattere 

Scientifico Santa Lucia Foundation, 00143 Rome, Italy, the 5 Departments of Cell 

Biology and Chemistry, The Skaggs Institute for Chemical Biology, The Scripps 

Research Institute, La Jolla, California 92037 and the 6 Center for Integrated Research 

(CIR)/University Campus Biomedico di Roma, 00128 Rome, Italy 

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that interacts with 

neuromodulatory fatty acid amides and esters and is expressed in mammalian brain 

and peripheral tissues. It degrades important neurotransmitters such as oleamide and 

anandamide, and it has been involved in a number of human pathological conditions 

representing therefore a valuable target for biochemical and pharmacological 

research. In this study, we have investigated in vitro the structure-function relationship 

of rat and human FAAHs through several spectroscopic techniques.In particular, we 

investigated the structural features of the two proteins, both in the presence and 

absence of the irreversible inhibitor methoxyarachidonyl-fluorophosphonate. The 

results demonstrate that the structural properties of the two FAAHs are different, 

involving both secondary and tertiary structure elements of the proteins, despite their 

high sequence homology and overall similarity in temperature-dependence. 

Additionally, membrane binding and kinetic assays of both FAAHs indicate that also 

the functional properties of the two enzymes are different in their interaction with lipid 

bilayers and with exogenous inhibitors. 


326


Role of Arg403 for thermostability and catalytic activity of 

12/15-lipoxygenases 

dalla Bernardina SiMona 1 , di venere alMerinda 1 , Mei GiaMPiero 1 , 

kühn hartMut 2 , ivanov iGor 3 and roSato nicola 1 

1 Department of Experimental Medicine and Biochemical Sciences, University of Tor 

Vergata, Via Montpellier 1, 00133 Rome, Italy; 2 Institute of Biochemistry, Charité- 

Universitätsmedizin Berlin, Oudenarder Str. 16, D-13346 Berlin, Germany; 3 Institute 

of Toxycology and Pharmacology, Universitätsmedizin Rostock, Schilling Allee 70, 

D-18059 Rostock, Germany 

Lipoxygenase are metal-containing lipid peroxidizing enzymes that catalyze 

dioxygenation of polyunsaturated fatty acids to their corresponding hydroperoxy 

derivatives. 

Mammalian lipoxygenases (LOXs) have been implicated in the pathogenesis of 

inflammatory and hyperproliferative diseases but the detailed mechanism of action 

is still a matter of discussion. Mutagenesis and structural modeling suggested that 

for 12/15-LOX fatty acid substrate penetrates into the active site with its methyl 

terminus ahead, whereby the fatty acid carboxylic group might interact with Arg403 

that is localized at the entrance into the putative substrate binding pocket. X-ray 

crystallography and molecular dynamics simulations of ligand binding at the active 

site of rabbit 12/15-LOX suggested that active site inhibitors and/or substrate fatty 

acids induce conformational changes in the immediate surrounding of the catalytic 

center. In particular, external α2-helix translocates away from its original position 

to open the entrance to the binding cavity close to Arg403 partly, whereby its base 

(residues 171-176) become disordered. In ligand-free 12/15-LOX Arg403 forms an 

ionic network with the side chain of either Asp174 as found in crystal and/or Glu176 

as observed during MD-simulations. As suggested by MD-simulations this interaction 

might be disrupted upon ligand binding. 

To obtain more direct evidence for the possible role of Arg403 for the catalytic and 

structural properties of mammalian 12/15-LOX we created the Arg403Leu mutant of 

the rabbit 12/15-LOX. The data presented here suggest that Arg403Leu exchange 

affects catalytic activity and increased the susceptibility of the enzyme for substrate 

inhibition. However, it hardly impaired the affinity of the mutant towards both linoleic 

acid and arachidonic acid. Moreover, we found that Arg403Leu exchange does 

not induce major alterations in sensitivity of the enzyme for temperature-induced 

unfolding. In contrast, the tertiary structure of the enzyme protein was considerably 

destabilized as indicated by fluorescence studies. 


327


The point mutation in the apoptosis inducing factor(AIF) 

gene causing early derangement in human brain 

development specifically alters the NADH reactivity of the 

flavoprotein 

aliverti aleSSandro, Baroni Sara, Pandini vittorio, Piccolo elena, 

Sorrentino luca, vanoni Maria antonietta 

Dipartimento di Bioscienze, Università degli Studi di Milano, 

via Celoria 26, 20133 Milano, Italy. 

The apoptosis inducing factor (AIF) is a mitochondrial NAD(H)-binding flavoprotein 

that, when released from the organelle in response to various stimuli, causes cell 

death (1). In addition to its role in caspase-independent apoptosis, AIF also plays 

an essential, although poorly understood, function in maintaining the functionality 

of oxidative phosphorylation. While AIF gene inactivation is embryonic lethal in 

mammals, downregulation of its expression causes myopathies and neurological 

abnormalities (1). Very recently, two distinct point mutations in human AIF gene were 

identified as responsible of human mitochondriopathies resulting in severe early 

encephalomyopathy (2) and prenatal ventriculomegaly (3), respectively. We have 

reproduced the latter mutation in mouse AIF. The pathogenic AIF-G307E variant 

was isolated in a recombinant form and was shown to be as stable as the wild-type 

protein. This amino acyl replacement was found to significantly slow down the reaction 

between the protein and NADH that leads to the formation of a NAD + -FADH - chargetransfer 

complex, which has been previously shown to be linked to a monomer-dimer 

transition of the protein quaternary structure (1). These results suggest that the slower 

reaction with NADH of AIF-G307E in comparison to the wild-type protein might be at 

the basis of the pathogenic mechanism of the mutation. Furthermore, in the light of 

the proposed role of AIF as a switch in sensing the mitochondrial redox state and/or 

NAD(H) concentration (1), our data underscore the importance of a ready reactivity of 

AIF toward NADH for the full functional integrity of the mitochondrion. 

This work has been supported by a PRIN2008 grant awarded by the Italian Ministry of 

Education and University (MIUR) to M.A. Vanoni. 

1. Sevrioukova IF. Antioxid Redox Signal. 2011; 14: 2545-79. 

2. Ghezzi D, et al. Am J Hum Genet. 2010; 86: 639-49. 

3. Berger I, et al. Mol Genet Metab. 2011; 104: 517-20. 


328


Tissue and subcellular localization of mammalian renalase, 

a FAD-containing protein involved in the pathogenesis of 

cardiovascular diseases 

Baroni Sara 1 , ShuGrue chriStine 2 , velázquez heino 2 , 

aliverti aleSSandro 1 , Gorelick fred S. 2 , deSir Gary v. 2 

1 Dipartimento di BioScienze, Università degli Studi di Milano, 

via Celoria 26, 20133 Milano, Italy. 

2 Department of Internal Medicine, Yale University School of Medicine, 

VACHS, 333 Cedar Street, New Haven, CT 06520-8029, USA 

Renalase is a secretory protein and flavoenzyme that is ubiquitous in vertebrates 

and conserved in some other phyla. In mammals it has been shown to modulate 

cardiovascular responses, being particularly active in decreasing catecholaminergic 

tone, lowering blood pressure, and in protecting the heart against ischemic damage (1). 

Lowered renalase levels in tissue and plasma might be the basis of the cardiovascular 

complications observed in chronic kidney disease patients (1). Renalase secretion 

into the circulation is enhanced in response to stressors such as hypertension, but the 

molecular mechanism regulating its basal or stimulated secretion are unknown (2). We 

find that renalase has a signal-sequence, but this sequence is not cleaved prior to its 

secretion, suggesting that it may traffic in an atypical secretory pathway. In pig kidney, 

our immunofluorescence studies showed that renalase is exclusively expressed in 

the proximal tubule. Similar studies in human immortalized HK-2 cells, as well as on 

�pig and mouse primary cell lines, indicated that renalase is preferentially localized 

in the cytoplasm, where it shows a punctate distribution, suggestive of an organelle 

association. The identification of these subcellular compartment(s), mechanism of 

association, and renalase’s mechanism of secretion are underway. This knowledge 

could lead to novel therapies for cardiovascular and kidney diseases (3). 

This work has been supported by travel fellowships granted by the Italian Society of 

Biochemistry and Molecular Biology (SIB) and by the Consorzio Interuniversitario di 

Biotecnologie to S. Baroni. 

1. Desir GV. Curr Opin Nephrol Hypertens. 2011; 20: 31-6. 

2. Milani M, et al. J Mol Biol. 2011; 411: 463-73. 

3. Unger T, et al. Eur Heart J. 2011; 32: 2739-47. 


329


Structural analysis of Nucleophosmin C-terminal domain 

in complex with a G-quadruplex sequence from the c-MYC 

promoter 

lo Sterzo carlo 1 , Gallo anGelo 2 , Mori Mirko 2 , di Matteo adele 1 , 

Bertini ivano 2 , Banci 2 , Brunori Maurizio 1 and federici luca 3 

1 Institute of Biology and Molecular Pathology of the CNR, Department of 

Biochemical Sciences “A. Rossi Fanelli”, “Sapienza” University of Rome, 

00185 Rome, Italy. 2 Magnetic Resonance Center, Department of Chemistry, 

University of Florence, 50019 Sesto Fiorentino, Italy. 

3 Center of Excellence on Aging and Department of Biomedical Sciences, 

University of Chieti “G. D’Annunzio”, 66013 Chieti, Italy. 

Nucleophosmin (NPM1) is a nucleo-cytoplasmic shuttling protein, mainly localized at 

nucleoli, which participates to several cellular functions, including ribosome maturation 

and export, centrosome duplication and response to stress stimuli. The NPM1 gene is 

the most frequently mutated in Acute Myeloid Leukemia (AML) patients. All mutations 

map to the last exon of the gene and the encoded mutated proteins are aberrantly and 

stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal 

domain and the appearance of a new nuclear export signal. Previously, we have 

shown that the 70-residue NPM1 C-terminal domain (NPM1-C70) binds a specific 

region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. 

Here we present the solution structure of the NPM1-C70 domain and analyse its 

interaction with a c-MYC derived G-quadruplex by NMR. These data were used to 

calculate an experimentally restrained molecular docking model for the complex. The 

NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface 

between helices H1 and H2, through electrostatic interactions with the G-quadruplex 

phosphate backbone. Several residues contacting the G-quadruplex DNA are 

known to be subjected to post-translational modifications, including acetylation and 

phosphorilation, suggesting their possible regulatory role in modulating NPM1-DNA 

interactions. Finally and interestingly, we report that a 17-residue lysine-rich sequence 

at the N-terminus of the three-helix bundle, that proved necessary to increse affinity 

for the G-quadruplex partner, is disordered and does not participate directly to the 

contact surface in the complex. 


330


THERMAL AND CHEMICAL UNFOLDING OF 

METHYLTHIOADENOSINE PHOSPHORYLASE II FROM THE 

ARCHAEON SULFOLOBUS SOLFATARICUS 

cacciaPuoti Giovanna, fuccio franceSca, de leo eSter, 

BaGarolo Maria liBera and Porcelli Marina 

Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via 

Costantinopoli 16, 80138 Napoli 

5’-Deoxy-5’-methylthioadenosine phosphorylase II from Sulfolobus solfataricus 

(SsMTAPII) is a hyperthermophilic and hyperthermostable enzyme belonging to purine 

nucleoside phosporylases, ubiquitous enzymes that function in the purine salvage 

pathway. SsMTAPII is a tightly packed hexameric protein organized as a dimer-oftrimers 

with one active site per monomer. Each subunit is stabilized by two pairs of 

intrasubunit disulfide bridges and contains in its C-terminal region a CXC motif as a 

typical feature. To get information on the role played by disulfide bonds in stability 

and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S 

mutants was studied by thermal and guanidinium chloride-induced unfolding and 

analyzed by fluorescence spectroscopy, circular dichroism and SDS-PAGE. No thermal 

unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while 

in the presence of reducing agents a Tm of 100°C can be measured demonstrating 

the involvement of disulfide bridges in enzyme thermostability. Differently from the 

wild-type, C262S and C259S/C261S show complete thermal denaturation curves 

with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing 

conditions these values decrease by 4°C and 8°C respectively, highlighting the 

important role exerted by CXC disulfide on enzyme thermostability. Chemical and 

thermal unfolding of SsMTAPII are irreversible processes. Nevertheless, under 

reducing conditions we were able to highlight the reversible dissociation of the 

hexamer to monomers and to detect the reversibly unfolded species by SDS-PAGE. 

These results provide convincing evidence that disulfide bonds play a key role in 

the conformational stability of SsMTAPII. In fact, the removal of these covalent links 

weakening the highly compact structure of the enzyme allows the reversible transition 

from the native to the denatured state before covalent modifications, induced by 

high temperatures and chemical denaturants, may cause the irreversible protein 

degradation. This is the first report on the reversible unfolding of a hyperthermophilic 

oligomeric protein with disulfide bonds. 


331


Biochemical characterization of Pseudomonas aeruginosa 

HD-GYP phosphodiesterases involved 

in c-di-GMP-dependent biofilm formation 

Stelitano valentina, rinaldo Serena, Giardina GiorGio, 

caStiGlione nicoletta, caruSo Manuela, cutruzzolà franceSca. 

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, 

Sapienza University of Rome, Italy. 

Bacteria exist in nature as planktonic single-cells or in a sessile multicellular state, 

named biofilm. In the latter, the bacterial community optimizes the cell-environment 

and cell to cell communication sensing strategies. Biofilms are widely diffuse in many 

industrial, environmental and clinical settings and are less sensitive to treatments 

with antimicrobial agents compared to planktonic cells. Biofilm-forming pathogens 

are responsible of >60% of human infections: the reduced sensitivity to conventional 

antibiotics calls for new anti-biofilm therapies. A key player in biofilm development is 

the second messenger cyclic-di-GMP; the enzymes controlling its intracellular levels, 

i.e. diguanylate cyclases (DGC) and phosphodiesterases (PDE), are found only in 

bacteria, thus, they represent ideal targets to develop effective anti-biofilm strategies. 

The aim of this project is to study the biochemical/structural bases of c-di-GMP 

metabolism in the human pathogen Pseudomonas aeruginosa, main cause of death 

in cystic fibrosis patients. Despite the relevance of these enzymes, no molecular data 

on the reaction mechanism are available. We have chosen to study representative 

members of each subclass of putative DGCs and PDEs. Here we report the results 

obtained on two putative PDEs of the HD-GYP subtype (PA4781 and PA4108) whose 

ability to lower c-di-GMP levels in P.aeruginosa was previously suggested. 

Our recent biochemical studies on purified PA4108 demonstrated that this HD-GYP 

protein has c-di-GMP and pGpG (c-di-GMP degradation product) hydrolytic activity. 

On the other hand, purified PA4781 and its isolated catalytic domain are inactive as 

c-di-GMP hydrolase whereas the latter protein can cleave the intermediate pGpG. 

These results suggest that PA4781 may lower the intracellular levels c-di-GMP 

indirectly, whereas PA4108 acts as a bona fide PDE. Since PA4781 catalytic domain 

is able to reduce c-di-GMP intracellular levels also as recombinant protein in E.coli, 

the need of a physiological partner to trigger PA4781 enzymatic activity cannot be 

ruled out. 


332


Amyloidoses associated to Apolipoprotein A-I: 

analysis of the molecular bases of the disease 

rita del Giudice 1 , anGela arciello 1 , daria Maria Monti 1 , 

anGela aMoreSano 2 and renata Piccoli 1 

1 Department of Structural and Functional Biology and 2 Department of Chemical 

Sciences, University of Naples Federico II, Naples, Italy 

Variants of Apolipoprotein A-I (ApoA-I) are associated to hereditary systemic 

amyloidoses, characterised by amyloid deposition of extracellular fibrils in peripheral 

organs of patients, mainly constituted by N-terminal fragments of the protein. The 

polypeptide corresponding to ApoA-I region 1-93 is the main constituent of heart 

amyloid deposits. 

We produced a recombinant form of the fibrillogenic polypeptide, named [1-93] 

ApoA-I, that shares fibrillogenic pathway with its natural counterpart. A lipid 

environment affects the conformational state and aggregation propensity of [1-93] 

ApoA-I. Cholesterol, a natural ApoA-I ligand, and liposomes of different compositions 

induce and stabilize the polypeptide α-helical state, inhibiting aggregation (1). [1-93] 

ApoA-I recognizes specific binding sites on rat cardiomyoblasts cell membrane and 

is internalized mostly through chlatrin-coated vesicles and lipid rafts, while ApoA-I 

is internalized mostly through chlatrin-coated vesicles and macropinocytosis. Unlike 

ApoA-I, the polypeptide is degraded by lysosomes and proteasome. Fibrils of [1-93] 

ApoA-I are instead unable to enter the cells (2). The interaction of the polypeptide with 

target cells and its intracellular route is certainly relevant to the comprehension of the 

pathology molecular bases. 

Being patients heterozygous for the variant gene, the isolation of an amyloidogenic 

variant from patient’s plasma is impracticable. We generated a useful cell model 

by stably transfecting CHO-K1 cells to express variant L174S of ApoA-I (3). The 

variant was isolated from the culture medium following a one-step procedure and 

its lipid content was defined by mass spectrometry analyses. Two saturated and two 

monounsaturated fatty acids were identified, in higher amounts than those associated 

to the natural protein, indicating a possible role of fatty acids in trafficking and 

secretion of ApoA-I amyloidogenic variants. 

1. Monti DM et al (2010) Eur Biophys J 39, 1289-99 

2. Arciello A et al (2011) J Cell Mol Med 15, 2652-63 

3. Monti DM et al (2012) Amyloid-J. of Protein Folding Disorders, 19, 21-27 


333


Structure and metal binding properties of ZinT, an auxiliary 

component of the high affinity ZnuABC zinc trasporter 

1 alaleona flaMinia, 2 ilari andrea,, 3 BattiStoni andrea 

3 Patrizia Petrarca, 3 Serena aMMendola 1 chiancone eMilia 

1 Dipartimento di Scienze Biochimiche“A. Rossi Fanelli“,“Sapienza” 

Università di Roma, Roma; 2 Istituto di Biologia e Patologia Molecolari, CNR, Roma, 

3 Dipartimento di Biologia, Università di Roma Tor Vergata, Roma 

During infections, the correct supply of Zn to pathogenic bacteria is ensured by the 

ZnuABC high affinity zinc uptake system which is expressed upon limited availability 

of the metal. ZnuABC comprises ZnuA, a periplasmic protein that captures Zn(II) and 

delivers it to ZnuB, the membrane permease and ZnuC, the ATPase component. 

In many bacteria, another periplasmic protein, ZinT, was shown to participate in 

Zn homeostasis. ZinT contributes to Salmonella growth in zinc-poor media and 

cooperates with ZnuA in zinc recruitment during severe metal shortage. When Zn(II)bound, 

ZnuA and ZinT form a very stable 1:1 complex in accordance with the high 

homology of ZinT to the C-terminal domain of the Streptococcus pneumoniae zinc 

importer AdcA whose N-terminal domain resembles ZnuA. 

Salmonella ZinT binds Zn(II) with an affinity (Kd ~ 22 nM) resembling that of YodA, 

the E. coli protein homologous to ZinT (Kd.~ 20 nM). To gain insight into formation 

of the ZinT-ZnuA complex, the crystallization of ZinT and of the ZnuA-ZinT complex 

from S. enterica was attempted. The X-ray structure of recombinant ZinT was solved 

in the apo-form, SeZinT, and in two Zn(II)-bound forms, Zn(II)-SeZinT and Zn(II)- 

SeZinT-PEG6 containing a PEG6 molecule in the catalytic Zn(II)-binding pocket. In 

Zn(II)-SeZinT-PEG6 zinc is coordinated tetrahedrally by His153, His155, His144 and 

an oxygen atom belonging to PEG or to a water molecule, whereas in Zn(II)-SeZinT 

His144 and a water molecule are not at a coordinating distance from the metal (2.93 

Å and 2.96 Å respectively). The crystals of the ZnuA-ZinT complex obtained to date 

diffract only to low resolution (5-6 Å). Other conditions are being tested to improve the 

quality of the crystals. 

The ZinT data are discussed in the framework of the X-ray crystal structures of YodA, 

which binds two Zn(II) at the active site. 


334

Participant 

list

ACETO ANTONIO a.aceto@unich.it 

ACQUASALIENTE LAURA laura.acquasaliente.1@studenti.unipd.it 

ALBERGHINA MARIO malber@unict.it 

ALIVERTI ALESSANDRO alessandro.aliverti@unimi.it 

ALLEGRA MARIO mario.allegra@unipa.it 

ALLOCATI NERINO allocati@unich.it 

AMATI FRANCESCA francesca.amati@uniba.it 

AMOROSO MARIA ROSARIA mr.amoroso@hotmail.it 

ANASTASIA LUIGI luigi.anastasia@unimi.it 

ANGELUCCI CLOTILDE BEATRICE bcangelucci@unite.it 

ANGELUCCI STEFANIA s.angelucci@unich.it 

AQUILANO KATIA katia.aquilano@uniroma2.it 

ARDINI MATTEO matteo.a@webfusion.it 

ARESE MARZIA marzia.arese@uniroma1.it 

AVIGLIANO LUCIANA avigliano@uniroma2.it 

BAGAROLO MARIA LIBERA marialibera.venere@libero.it 

BALDASSARRE MAURIZIO m.baldassarre@univpm.it 

BALDUINI ALESSANDRA alessandra.balduini@unipv.it 

BALDUINI CESARE cesare.balduini@unipv.it 

BALLIANO GIANNI gianni.balliano@unito.it 

BARCAROLI DANIELA barcaroli@med.uniroma2.it 

BARILE MARIA m.barile@biologia.uniba.it 

BARONI SARA sara.baroni@unimi.it 

BARRA DONATELLA donatella.barra@uniroma1.it 

BARRESI VINCENZA barregi@unict.it 

BASILE ANNA abasile@unisa.it 

BATTISTONI ANDREA andrea.battistoni@uniroma2.it 

BELLELLI ANDREA andrea.bellelli@uniroma1.it 

BENATTI UMBERTO benatti@unige.it 

BERGANTE SONIA sonia.bergante@unimi.it 

BERNARDI FRANCESCO francesco.bernardi@unife.it 

BERNARDINI GIULIA bernardini@unisi.it

BERTELLI MATTEO bertellimatteo@hotmail.com 

BERTONI ALESSANDRA alessandra.bertoni@med.unipmn.it 

BETTI LAURA betti@farm.unipi.it 

BETTUZZI SAVERIO saverio.bettuzzi@unipr.it 

BIAGIONI ANGELI EMANUELA emanuela.biagioni.angeli@gmail.com 

BISACCIA FAUSTINO faustino.bisaccia@unibas.it 

BOLOGNESI MARTINO martino.bolognesi@unimi.it 

BONARDI DARIO dario.bonardi@gmail.com 

BONOMI FRANCESCO francesco.bonomi@unimi.it 

BORGATTI MONICA brgmnc@unife.it 

BORGIA ALESSIA alessiabrg@hotmail.it 

BORRIELLO ADRIANA adriana.borriello@unina2.it 

BRACCI LUISA braccil@unisi.it 

BRESCIANI ROBERTO brescian@med.unibs.it 

BRISDELLI FABRIZIA fabrizia.brisdelli@cc.univaq.it 

BROZZU ANDREA 308329@studenti.unito.it 

BRUNI PAOLA paola.bruni@unifi.it 

BRUNORI MAURIZIO maurizio.brunori@uniroma1.it 

BRUSCHETTA GIUSEPPE gbruschetta@unime.it 

BRUZZONE SANTINA santina.bruzzone@unige.it 

BUCCIARELLI TONINO t.bucciarelli@unich.it 

BUSSOLINO FEDERICO federico.bussolino@ircc.it 

BUTTARI BRIGITTA brigitta.buttari@iss.it 

CACCURI ANNA MARIA caccuri@uniroma2.it 

CALATRONI ALBERTO alberto.calatroni@unime.it 

CALDARELLI ILARIA ilaria.caldarelli@unina2.it 

CALONGHI NATALIA natalia.calonghi@unibo.it 

CALVARUSO GIUSEPPE gcalva@unipa.it 

CAMPANER STEFANO stefano.campaner@iit.it 

CAPITANIO NAZZARENO n.cap@unifg.it 

CAPPELLO ANNA RITA cappello@farmbiol.uniba.it 

CAPRARO JESSICA jessica.capraro@unimi.it

CARLETTI ERMINIA e.carletti@unich.it 

CARNICELLI VERONICA veronica.carnicelli@cc.univaq.it 

CARRADORI MARIA RITA m.r.carradori@univpm.it 

CARUSO DONATELLA donatella.caruso@unimi.it 

CASADIO RITA casadio@biocomp.unibo.it 

CASTAGNOLA MASSIMO massimo.castagnola@icrm.cnr.it 

CATTANEO FABIO fabio.cattaneo@unina.it 

CAVALLO ALESSANDRO alessandro.cavallo@unisalento.it 

CECCONI FRANCESCO francesco.cecconi@uniroma2.it 

CECI ROBERTA roberta.ceci@uniroma4.it 

CELA OLGA o.cela@unifg.it 

CENCETTI FRANCESCA francesca.cencetti@unifi.it 

CHIAPPINI PIERO p84chiappini@gmail.com 

CHIARELLA SARA sara.chiarella@uniroma1.it 

CHIARUGI PAOLA paola.chiarugi@unifi.it 

CIALABRINI LUCIA lucia.cialabrini@hotmail.it 

CIANCI ELEONORA eleonora.cianci@unich.it 

CIAVARDELLI DOMENICO d.ciavardelli@unich.it 

CIMINO FILIBERTO cimino@dbbm.unina.it 

CINI CHIARA chiara.cini@uniroma1.it 

CIREGIA FEDERICA ciregia@farm.unipi.it 

COLEMAN MICHAEL P. michael.coleman@bbsrc.ac.uk 

COMEGNA MARIKA comegna@dbbm.unina.it 

CONDORELLI DANIELE FILIPPO condorda@unict.it 

CONFORTI ERIKA eri.conforti@libero.it 

CONSALVO ADA ada.consalvo@libero.it 

CORDA DANIELA d.corda@ibp.cnr.it 

CORSETTO PAOLA ANTONIA paola.corsetto@unimi.it 

CORTELAZZO ALESSIO cortelazzo2@unisi.it 

COSTA BARBARA bcosta@farm.unipi.it 

CRESTANI MAURIZIO maurizio.crestani@unimi.it 

CRISTOFARO MARIO mario.cristofaro@for.unipi.it

CUTRUZZOLA’ FRANCESCA francesca.cutruzzola@uniroma1.it 

DA POZZO ELEONORA dapozzo@farm.unipi.it 

DA VALLE YLENIA ylenia.davalle@gmail.com 

D’ADDA DI FAGAGNA FABRIZIO fabrizio.dadda@ifom-ieo-campus.it 

D’AGUANNO SIMONA simona.d.aguanno@uniroma2.it 

DAINESE ENRICO edainese@unite.it 

DANIELE SIMONA dsimona@farm.unipi.it 

D’ASCOLA ANGELA adascola@unime.it 

D’AVENIA MORENA mdavenia@unisa.it 

DE CESARE LUCIA lucia.decesare@unina2.it 

DE COLA ANTONELLA antonelladecola@hotmail.it 

DE CRISTOFARO RAIMONDO rdecristofaro@rm.unicatt.it 

DE FILIPPIS VINCENZO vincenzo.defilippis@unipd.it 

DE FLORA ANTONIO toninodf@unige.it 

DE LAURENZI VINCENZO delaurenzi@unich.it 

DE LUCA ANASTASIA anastasia.deluca@gmail.com 

DE LUCA ANTONELLA antdeluca@unich.it 

DE MARCO MARGOT mdemarco@unisa.it 

DE PINTO VITO vdpbiofa@unict.it 

DE RASMO DOMENICO d.derasmo@ibbe.cnr.it 

DEL BOCCIO PIERO p.delboccio@unich.it 

DEL CORSO ANTONELLA adelcorso@biologia.unipi.it 

DELLA RAGIONE FULVIO fulvio.dellaragione@unina2.it 

DELOGU LUCIA GEMMA lgdelogu@uniss.it 

DI COLA DOMENICO green2@mail.chieti.com 

DI EMIDIO GIOVANNA gi.diemidio@gmail.com 

DI FRANCESCO ANDREA adifra@hotmail.it 

DI GIULIO ANTONIO antonio.digiulio@cc.univaq.it 

DI GIUSEPPE FABRIZIO fabrizio_samp@yahoo.it 

DI ILIO CARMINE diilio@dsb.unich.it 

DI LIEGRO ITALIA italia.diliegro@unipa.it 

DI MATTEO ADELE adele.dimatteo@uniroma1.it

DI MICHELE ANTONIO a.dimichele@unich.it 

DI SILVESTRE SARA saradisilvestre@gmail.com 

DI SILVIO EVA eva.disilvio@uniroma1.it 

DI TOMMASO MONIA d_monia@libero.it 

DI TOMO PAMELA pameladitomo@libero.it 

DI VITO CLARA clara.divito@unimi.it 

DOLCE VINCENZA vdolce@unical.it 

D’ONOFRIO NUNZIA nunzia.d’onofrio@unina2.it 

DUGO LAURA l.dugo@unicampus.it 

ELEUTERIO ENRICA enrica.eleuterio@istruzione.it 

EMANUELLI MONICA m.emanuelli@univpm.it 

EMILIANI CARLA emiliani@unipg.it 

EVANGELISTA DANIELA evangelistadaniela@virgilio.it 

FABBRI DANIELE daniele.fabbri10@unibo.it 

FALCIANI CHIARA falciani4@unisi.it 

FALCO ANTONIA afalco@unisa.it 

FARAONIO RAFFAELLA faraonio@dbbm.unina.it 

FAVAROLO BARTOLO b.favaloro@unich.it 

FEDERICI GIORGIO giorgio.federici@uniroma2.it 

FEDERICI LUCA lfederici@unich.it 

FERLAZZO ALIDA MARIA alferl@unime.it 

FEZZA FILOMENA filomena.fezza@uniroma2.it 

FILONI DANIELA NICOLE tchuss@hotmail.it 

FINAZZI AGRO’ ALESSANDRO finazzi@uniroma2.it 

FORLI’ FEDERICA fforli@unich.it 

FORLINO ANTONELLA aforlino@unipv.it 

FRANCESCHINI NICOLA nicola.franceschini@cc.univaq.it 

FRASSON ROBERTA roberta.frasson@inwind.it 

FURLANI EMILIANO emifu@inwind.it 

GALLETTI PATRIZIA patrizia.galletti@unina2.it 

GALLORINI MARIALUCIA marialuciagallorini@gmail.com 

GAMBARI ROBERTO gam@unife.it

GAROFALO MARIANGELA mariangela.garofalo@hotmail.it 

GELFI CECILIA cecilia.gelfi@unimi.it 

GENNARO RENATO rgennaro@units.it 

GHASEMI REZA gen_tums@yahoo.com 

GIANNACCINI GINO ggino@farm.unipi.it 

GIARDINA BRUNO bgiardina@rm.unicatt.it 

GILARDI GIANFRANCO gianfranco.gilardi@unito.it 

GIULIANO MICHELA mgiulian@unipa.it 

GIULIANO SERENA giuser07@unipv.it 

GIUNTA CARLO carlo.giunta@unito.it 

GIUSSANI PAOLA paola.giussani@unimi.it 

GIUSTI LAURA giusti@farm.unipi.it 

GNONI GABRIELE gabriele.gnoni@unisalento.it 

GRANZOTTO ALBERTO alberto.granzotto.1@studenti.unipd.it 

GRAZIANO VINCENZO wincyo@hotmail.com 

GUARNIERI CARLO carlo.guarnieri@unibo.it 

GUELI MARIA CONCETTA mariac.gueli@unipa.it 

HRELIA SILVANA silvana.hrelia@unibo.it 

IENTILE RICCARDO ientile@unime.it 

INDIVERI CESARE indiveri@unical.it 

IPPOLITI RODOLFO rodolfo.ippoliti@univaq.it 

ISOPI ELISA e.isopi@unich.it 

LAISO GIULIANA giuliana.laiso@libero.it 

LANDI GIACOMO landi.g@hotmail.it 

LATTANZIO ROSSANO rossanolattanzio@libero.it 

LEONCINI GIULIANA leoncini@unige.it 

LEONCINI EMANUELA emanuela.leoncini@unibo.it 

LO STERZO CARLO carlo.losterzo@uniroma1.it 

LOUBNA ABDELHADI loubna.abdel@unimi.it 

LUCACCHINI ANTONIO lucas@farm.unipi.it 

LUTI SIMONE simone.luti@unifi.it 

MACCARRONE MAURO mmaccarrone@unite.it

MAGGIOLINI MARCELLO marcellomaggiolini@yahoo.it 

MAGINI ALESSANDRO alessandro.magini@libero.it 

MAGNANI MAURO lucilla.magi@uniurb.it 

MAGNI GIULIO g.magni@univpm.it 

MAGNONE MIRKO mirko.magnone@unige.it 

MAGRI’ ANDREA andrea.chimbiol@yahoo.it 

MALUMBRES MARCOS mmm@cnio.es 

MANCINI FRANCESCO PAOLO mancini@unisannio.it 

MANCINI ANDREA andrea.mancini@unimi.it 

MANGONI MARIALUISA marialuisa.mangoni@uniroma1.it 

MARENGO MAURO mauro.marengo@unimi.it 

MARINELLO ENRICO marinello@unisi.it 

MARINIELLO LOREDANA loredana.mariniello@unina.it 

MARINO MARIA m.marino@uniroma3.it 

MARTELLI PIER LUIGI gigi@biocomp.unibo.it 

MARTELLINI FEDERICA federica.martellini@unifi.it 

MARTELLO EMANUELA emanuela.martello@libero.it 

MARTINI CLAUDIA cmartini@farm.unipi.it 

MARTINI FILIPPO f.martini@dsb.unich.it 

MASET FABIO fabio.maset@unipd.it 

MASULLI MICHELE masulli@unich.it 

MAZZONE MASSIMILIANO massimiliano.mazzone@vib-kuleuven.be 

MELINO GENNARO melino@uniroma2.it 

MEROLLE LUCIA lucia.merolle@unibo.it 

MESSANA IRENE imessana@unica.it 

MICHELI VANNA vanna.micheli@unisi.it 

MICOZZI DANIELA daniela.micozzi@unicam.it 

MIGGIANO RICCARDO riccardo.miggiano@pharm.unipmn.it 

MIGLIACCIO ANTIMO antimo.migliaccio@unina2.it 

MILANI SIMONA simona.milani@unimi.it 

MIRIANI MATTEO matteo.miriani@unimi.it 

MISSINEO ANTONINO antonino.missineo01@ateneopv.it

MITRO NICO nico.mitro@unimi.it 

MOIO PASQUALE marilisasulpizio@yahoo.it 

MONTECUCCO CESARE cesare.montecucco@unipd.it 

MONTI LUCA luca.monti02@ateneopv.it 

MORACCI MARCO m.moracci@ibp.cnr.it 

MORI VALERIO v.mori@univpm.it 

MOTTA CARLA carlamba@libero.it 

MURA UMBERTO umura@biologia.unipi.it 

NANETTI LAURA lnanetti@univpm.it 

NAPOLITANO MARCO marconapolitano82@libero.it 

NASTASI GIANCARLO gnastasi@htomail.it 

NEGRI ARMANDO armando.negri@unimi.it 

OLIARO BOSSO SIMONETTA simona.oliaro@unito.it 

ORLACCHIO ALDO o rly@unipg.it 

OSTUNI ANGELA angela.ostuni@unibas.it 

PACELLA STEPHANIE pacstephanie@yahoo.it 

PADULA ANNA anna.padula4@unibo.it 

PALMIERI FERDINANDO fpalm@farmbiol.uniba.it 

PALMIERI LUIGI luigi.palmieri@uniba.it 

PANEBIANCO CONCETTA concetta.panebianco@uniba.it 

PAOLI PAOLO paolo.paoli@unifi.it 

PAPA SERGIO sergio.papa@uniba.it 

PARADIES GIUSEPPE g.paradies@biologia.uniba.it 

PARENTE AUGUSTO augusto.parente@unina2.it 

PARROTTA ROSSELLA rossella.parrotta@unito.it 

PASCALE MARIA pascale@unisa.it 

PASSARO FABIANA fabiana.passaro@unina.it 

PASSI ALBERTO alberto.passi@uninsubria.it 

PEDONE PAOLO VINCENZO paolov.pedone@unina2.it 

PENNELLI ALFONSO a.pennelli@dsb.unich.it 

PERTINHEZ THELMA thelma@unipr.it 

PETRAGNANI NICOLA n.petragnani@unich.it

PETRUZZELLI RAFFAELE r.petruzzelli@dsb.unich.it 

PICCOLI RENATA piccoli@unina.it 

PICCOLI MARCO piccolimarco83@gmail.com 

PINI ALESSANDRO pinia@unisi.it 

PINOTTI MIRKO mirko.pinotti@unife.it 

PIPINO CATERINA c.pipino@unich.it 

POLCHI ALICE alicepolchi@virgilio.it 

POMPUCCI GIUSEPPE pompucci_1936@libero.it 

PORTA RAFFAELE portaraf@unina.it 

PORTANOVA PATRIZIA patrizia.port@libero.it 

POZZI VALENTINA valentinapozzi81@gmail.com 

PRIOIETTI D’EMPAIRE LUCIA hns@unife.it 

RAFFAELLI NADIA n.raffaelli@univpm.it 

RAFFAELLI FRANCESCA francesca.raffaelli@alice.it 

RAVERA SILVIA silvia.ravera@gmail.com 

REA SILVIANA silviana.rea@unina.it 

REICHHART JEAN-MARC J M.Reichhart@unistra.fr 

RIBONI LAURA laura.riboni@unimi.it 

RICCITELLI ELENA elena.riccitelli@unimi.it 

RIGANO RACHELE rachele.rigano@iss.it 

RIVELLINO ALESSIA alessia.rivellino@unina2.it 

RIVIELLO ANTONELLA riviello@hotmail.it 

RIZZI MENICO menico.rizzi@pharm.unipmn.it 

RIZZO BENEDETTA benedetta.rizzo2@unibo.it 

RIZZO ANGELA MARIA angelamaria.rizzo@unimi.it 

ROSATI ALESSANDRA arosati@unisa.it 

ROSATO NICOLA nicola.rosato@uniroma2.it 

ROSSI CLAUDIA claudia.rossi@unich.it 

ROSSI MARQUEZ GIOVANNA giovanna.rossimarquez@unina.it 

ROSSINI GIAN PAOLO gianpaolo.rossini@unimore.it 

RUGGIERI SILVERIO ruggieri@univpm.it 

SABATINI STEFANIA stefania.sabatini@uniroma4.it

SACCHETTA PAOLO ps@unich.it 

SALA GIAN LUCA gianluca.sala@unimore.it 

SALOMONI PAOLO p.salomoni@ucl.ac.uk 

SANNA FABIO sanna.fabio@yahoo.it 

SANTI ALICE alice.santi@unifi.it 

SANTUCCI ANNALISA annalisa.santucci@unisi.it 

SARDANELLI ANNA MARIA sardanelli.a@biochem.uniba.it 

SARDARO NICOLA sardaronicola@libero.it 

SARTINI DAVIDE davide_sartini@libero.it 

SARTOR GIORGIO giorgio.sartor@unibo.it 

SCACCO SALVATORE salvatore.scacco@uniba.it 

SCARFI SONIA soniascarfi@unige.it 

SCARINGI RAFFAELLA raffaella.scaringi@unimi.it 

SCHURUCHI MICHELE mscuruchi@yahoo.it 

SCOTTI LUCA l.scotti@unich.it 

SCRIMA ROSELLA r.scrima@unifg.it 

SEPPI CLAUDIO cseppi@unipv.it 

SERINI GUIDO guido.serini@ircc.it 

SIGNORELLO MARIA GRAZIA mariagrazia.signorello@unige.it 

SIGNORILE ANNA anna.signorile@biochem.uniba.it 

SILVESTRI ILARIA MARIA TERESA ilaria.silvestri@unimi.it 

SINIGAGLIA FABIOLA fabiola.sinigaglia@med.unipmn.it 

SOLAINI GIANCARLO giancarlo.solaini@unibo.it 

SPISNI ALBERTO alberto.spisni@unipr.it 

STEFANETTI CHIARA chiara.stefanetti@centrovolta.it 

STELITANO VALENTINA valentina.stelitano@uniroma1.it 

STRAZZULLI ANDREA a.strazzulli@ibp.cnr.it 

SUCCOIO MARIANGELA mariangela.succoio@gmail.com 

SULPIZIO MARILISA marilisa.sulpizio@istruzione.it 

TAGLIAZUCCHI DAVIDE davide.tagliazucchi@unimore.it 

TESORIERE LUISA luisa.tesoriere@unipa.it 

TIBONI GIAN MARIO tiboni@unich.it

TIRIBUZI ROBERTO robertotiribuzi@yahoo.it 

TORTI MAURO mtorti@unipv.it 

TORTORA PAOLO paolo.tortora@unimib.it 

TOSATTO SILVIO silvio.tosatto@unipd.it 

TOSSI ALESSANDRO atossi@units.it 

TRAINI SARA trainis@unich.it 

TRENTINI ALESSANDRO alessandro.trentini@unife.it 

TRINCAVELLI MARIA LETIZIA ltrincavelli@farm.unipi.it 

TRINGALI CRISTINA cristina.tringali@unimi.it 

TROADEC JEAN-DENIS j-d.troadec@univ-cezanne.fr 

TURCO MARIA CATERINA mcturco@unisa.it 

VANONI MARCO marco.vanoni@unimib.it 

VENERANDO BRUNO bruno.venerando@unimi.it 

VENTO RENZA r.vento@unipa.it 

VIANI PAOLA paola.viani@unimi.it 

VIECELI DALLA SEGA FRANCESCO francesco.vieceli2@unibo.it 

ZAPPAVIGNA SILVIA silvia.zappa@libero.it 

ZOCCHI LOREDANA loredana_zocchi@yahoo.it 

ZUCCHELLI MIRCO m.zucchelli@unich.it 

ZULLO ALBERTO albzullo@unisannio.it

index 

Programme ............................................................................................................. Pag. 3 

Antonini Lecture .................................................................................................. Pag. 9 

Session 1 ............................................................................................................... Pag. 11 

Session 2 ............................................................................................................... Pag. 19 

Session 3 ............................................................................................................... Pag. 25 

Session 4 ............................................................................................................... Pag. 31 

Session 5 ............................................................................................................... Pag. 37 

Session 6 ............................................................................................................... Pag. 43 

Session 7 ............................................................................................................... Pag. 49 

Session 8 ............................................................................................................... Pag. 55 

Ammine Biogene ............................................................................................. Pag. 65 

Biochimica Marina e dell’Ambiente ................................................... Pag. 71 

Biochimica Cellulare ..................................................................................... Pag. 75 

Biochimica della Nutrizione ................................................................... Pag. 143

index 

Immunologia Biochimica ......................................................................... Pag. 169 

Sviluppo Differenziamento e Apoptosi .......................................... Pag. 177 

Biotecnologie e Biochimica Industriale ........................................ Pag. 197 

Bioinformatica ................................................................................................. Pag. 205 

Enzimologia e Regolazione Metabolica ........................................ Pag. 211 

Glicobiologia ..................................................................................................... Pag. 225 

Membrane e Bioenergetica ................................................................... Pag. 241 

Nucleotidi, Acidi Nucleici, Genomi .................................................. Pag. 261 

Neurochimica ................................................................................................... Pag. 275 

Proteine ................................................................................................................ Pag. 295 

Participant list .................................................................................................. Pag. 335

Honorary President Giorgio Federici Università di ... - Sib Chieti 2012

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