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288 EFFECTS OF WATER-SOLUBLE ANTIOXIDANTS AND MAPKK/MEK INHIBITOR ON CURCUMIN- INDUCED APOPTOSIS IN HL-60 HUMAN LEUKEMIC CELLS. (NO. 746) Banjerdpongchai R 1 , Wilairat P 2 . 1 Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai; 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok. Key words : apoptosis, curcumin, water-soluble antioxidant Curcumin is the main biologically active phytochemical compound in turmeric. It has been shown to have anticarcinogenic activity. The aims of the study were to identify the mechanism of apoptosis of HL-60 human promyelocytic leukemic cells induced by curcumin and to determine the effects of water-soluble antioxidants, ascorbic acid, Trolox (a water-soluble form of vitamin E), glutathione (GSH) and N-acetylcysteine (NAC) on this process. HL-60 cells were incubated with curcumin for 24 h and apoptotic cells were quantitated by flow cytometry following staining with annexin V-FITC and propidium iodide. Curcumin-treated HL-60 cells produced reactive oxygen species as detected by the dichlorofluorescein fluorescent assay. Apoptosis occurred via the mitochondria pathway as curcumin reduced mitochondrial membrane potential in a dose-dependent manner. In the presence of 10 μM curcumin, vitamin C (56 nM-5.6 μM) inhibited apoptosis of HL-60 cells; GSH at low concentration (1 μM) reduced apoptosis but had no effect at higher concentrations (10, 100 μM); and Trolox and NAC at 10 and 100 μM, respectively, enhanced apoptosis, but this effect was abolished at higher concentration (1 mM) of NAC. MAPKK/MEK inhibitor PD98059, enhanced curcumin-induced HL- 60 apoptotic cell death. (Published in Asian Pac J Cancer Prev 2005; 6: 282-5. Supported by the Thailand Research Fund.) PHOTOINHIBITION AND RECOVERY IN OXYGENIC PHOTOSYNTHESIS : MECHANISM OF A PHOTOSYSTEM-II DAMAGE AND REPAIR CYCLE. (NO. 747) Yokthongwattana K 1 , Melis A 2 . 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 2 Department of Plant and Microbial Biology, University of California, Berkeley, California, USA. This Chapter provides highlights on the mechanism of a photosystem II (PS II) damage and repair cycle in chloroplasts. Photo-oxidative damage to the PS II reaction center is a phenomenon that occurs in every organism of oxygenic photosynthesis. Through the process of evolution, an elaborate repair mechanism was devised, one that rectifies this presumably unavoidable and irreversible photoinhibition and restores the PS II charge separation activity. The repair process entails several enzymatic reactions for the selective removal and replacement of the inactivated D1/132 kD reaction center protein (the chloroplast-encoded psbA gene product) from the massive (>1,000 kD) H 2 O-oxidizing and O 2 -evolving PS II holocomplex. This repair process is unique in the annals of biology, nothing analogous in complexity and specificity has been reported Faculty of Science in other biological systems. Elucidation of the repair mechanism may reveal the occurrence of hitherto unknown regulatory and catalytic reactions for the selective in situ replacement of specific proteins from within multi-protein complexes. This may not only have significant applications in photosynthesis and agriculture but also in medicine and other fields. . (Published in Demmig-Adams B, Adams III WW, Mattoo AK (eds) Photoprotection, Photoinhibition, Gene Regulation and Environment. Advances in Photosynthesis Series, Springer, The Netherlands 2005; pp. 175-91. Supported by USDA National Research Initiative.) ENZYMATIC PROPERTIES OF WILD-TYPE AND ACTIVE SITE MUTANTS OF CHITINASE A FROM VIBRIO CARCHARIAE, AS REVEALED BY HPLC-MS (NO. 748) Suginta W 1 , Vongsuwan A 1 , Songsiriritthigul C 1,2 , Svasti J 3 , Prinz H 4 . 1 School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakorn Ratchasima; 2 National Synchrotron Research Center, Nakorn Ratchasima; 3 Department of Biochemsitry and Center for Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok; 4 Max-Planck Institute for Molecular Physiology, Dortmund, Germany. Key words: active site, HPLC-MS, Vibrio carchariae The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of a and β anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated β-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher M r chitooligomers, in the order of (Glc-NAc) 6 >(GlcNAc) 4 > (GlcNAc) 3 , and showed no activity towards (Glc-NAc) 2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues – Glu315 and Asp392 – by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed »20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme. (Published in FEBS J 2005; 272: 3376-86. Supported by Thailand Research Fund, Suranaree University of Technology and German Academic Exchange Service.) PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
Mahidol University Annual Research Abstracts, Vol. 33 289 MOLECULAR HYDROGEN PRODUCTION BY A THERMOTOLERANT RUBRIVIVAX GELATINOSUS USING RAW CASSAVA STARCH AS AN ELECTRON DONOR (NO. 749) Mahakhan P 1 , Chobvijuk C 1 , Ngmjarearnwong M 1 , Trakulnalermsai S 1 , Bucke C 2 , Svasti J 3 , Kanlayakrit W 4 , Chitradon L 1 . 1 Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok; 2 School of Biosciences, University of Westminster, London, UK; 3 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 4 Department of Biotechnology, Faculty of Agro-industry, Kasetsart University, Bangkok. Key words : hydrogen production, Rubrivivax gelatinosus, raw cassava starch. Thirteen strains of phototrophic purple non-sulfur bacteria selected from 226 isolates showed the ability to digest raw cassava starch at elevated temperature, 40°C, under illuminated anaerobic conditions. A selected strain, designated as SB24, produced more amylolytic enzyme activities toward raw and cooked cassava starch, when grown in cooked starch than when grown in raw starch. SB24 showed photoproduction of molecular hydrogen using raw cassava starch as an electron donor, when incubated with illumination under anaerobic conditions at 40°C. In small scale (23 mL) culture, SB24 produced hydrogen from raw cassava starch after 20-24 h of cultivation, with a 3-fold higher rate of H 2 production and a 3-fold higher total accumulation of hydrogen at 72 h, as compared to when it used malate as an electron donor. In the larger scale reactor containing a 5.5 liter culture of SB24, hydrogen was produced at an earlier time using raw cassava starch than using malate. In addition, the highest rate of H 2 production (38.79 mL H2 liter culture -1 h -1 ) by SB24 with raw cassava starch was 7 times higher than that with malate, while the total volume of H 2 accumulated with raw cassava starch (4.61 liters of H 2 at 90 h) was almost two-fold higher than with malate. Raw starch from rice, sticky rice, corn and mungbean, could also be used as electron donors for H 2 production by SB24 at 40°C. From morphological and biochemical characteristics, comparison of 16S rDNA sequence, and comparative studies of the bacterial characteristics, SB24 was found to be a thermotolerant anoxygenic phototrophic purple non-sulfur bacterium, Rubrivivax gelatinosus. (Published in ScienceAsia 2005; 31: 415-24. Supported by Thailand Research Fund and KURDI.) PROTEOMIC PROFILING OF CHOLANGIO- CARCINOMA CELL LINE TREATED WITH POMIFERIN FROM DERRIS MALACCENSIS. (NO. 750) Svasti J 1,2 , Srisomsap C 1 , Subhasitanont P 1 , Keeratichamroen S 1 , Chokchaichamnankit D 1 , Ngiwsara L 1 , Chimnoi N 1 , Pisutjaroenpong S 1 , Techasakul S 1,3 , Chen ST 4 . 1 Chulabhorn Research Institute, Bangkok; 2 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 3 Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok; 4 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Key words : cholangiocarcinoma, pomiferin, proteomic profiling Pomiferin, a prenylated isoflavonoid from Derris malaccensis with strong anti-fungal and anti-oxidant activities, showed cytotoxic activity towards human cholangiocarcinoma cells (HuCCA-1), with IC 50 of 0.9 mg/mL. Pomiferin caused apoptosis, detectable by DNA fragmentation. Two- dimensional PAGE showed increased expression of 12 proteins, namely glucose-regulated protein 75 (grp 75), calcyclin (S100A6), degraded cytokeratin 19, ATP synthase D, ribosomal protein P0, degraded cytokeratin 18 (two spots pI/MW 6.03/29.9 and pI/MW 4.66/21.5), cofilin, annexin A1, triose phosphate isomerase, peroxiredoxin-1, calgizzarin, and profilin. In contrast, cytokeratins (CK) 7, 18 and 19 were downregulated, and were shown by 1-DE immunodetection to be degraded. (Published in Proteomics 2005; 5: 5404-9. Supported by Chulabhorn Research Institute and Thailand Research Fund.) THE MOLECULAR BASIS OF MUCOPOLYSAC- CHARIDOSIS TYPE I IN TWO THAI PATIENTS. (NO. 751) Cairns JRH 1,2 , Keeratichamroen S 1 , Sukcharoen S 3 , Champattanachai V 1 , Ngiwsara L 1 , Lirdprapamongkol K 1 , Liammongkolkul S 4 , Srisomsap C 1 , Surarit R 1,5 , Wasant P 4 , Svasti J 1,3 . Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok; Schools of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, Nakorn Ratchasima; Department of Biochemistry and Center for Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok; Department of Physiology and Biochemistry, Faculty of Dentistry, Mahidol University, Bangkok. Key words : molecular basis, mucopolysaccharidosis type I, Thai Two Thai patients diagnosed with Hurler syndrome (mucoploysaccharidosis type I, MPS I) were found to have no detectable α-iduronidase (E.C. 3.2.1.76) activity in leukocytes, while normal Thai children all had significant activity, with a mean of 135 ± 30 nmol/mg/28 h. One patient was heterozygous for A75T (311G>A) and S633L (198C>T) mutation, previously reported to cause MPS I, together with 9 other heterozygous polymorphisms also found in normal controls. The other patient had the previously described frameshift mutation 252insert C and a new nonsense mutation E299X (983G>T). (Published in Southeast Asian J Trop Med Public Health 2005; 36: 1308-12. Supported by Chulabhorn Research Institute and Thailand Research Fund.) MY EXPERIENCES AS AN IUB TRAVEL FELLOW. Svasti J. (NO. 752) Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok. Key words: IUB, travel fellow PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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