Faculty of Science - Mahidol University

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278 significantly reduced. Furthermore, the secondary structure of the multiple-substitution and multiple-deletionmutants were quite different from that of the wild-type. These mutants were not viable. Transcriptional and translational efficiency of the wild-type and its derived mutants were consequently investigated. The preliminary result indicated that the single point-substitution mutant had efficiency translation in a rabbit reticulocyte lysate system. Conclusion : Mutation that disrupts the 5’NTR of Den-2 virus cause defective in viral replication and/or viral translation induced by secondary structure alteration. (Poster presentation at RGJ-Ph.D. Congress VI, TRF Annual Meeting. Chonburi, Thailand.) INVOLVEMENT OF SPECIFIC PROTEINS (SP1/SP3) AND NUCLEAR FACTOR Y IN BASAL TRANSCRIP- TION OF THE DISTAL PROMOTER OF THE RAT PYRUVATE CARBOXYLASE GENE IN b-CELLS (NO. 714) Sunyakumthorn P 1 , Boonsean T 1 , Boonsaeng V 1 , Wallace JC 2 , Jitrapakdee S 1 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 2 School of Molecular and Biological Sciences, University of Adelaide, Adelaide, Australia . Key words : gene transcription, nuclear factors, pyruvate carboxylase. Pyruvate carboxylase plays diverse roles in different biosynthetic pathways, including glucose-induced insulin secretion in pancreatic β-cells. We have localized the control region of the P2 promoter by generating a series of 50-nested deletion constructs, and both 25- and 9-bp internal deletion constructs, as well as by performing site-directed mutagenesis. Transient transfections of these constructs into INS-1 cells identified a CCAAT box and a GC box that are located at -65/-61 and -48/-41, respectively, as the important determinants. Disruption of the GC box resulted in a 4fold reduction of the reporter activity, while disruption of the proximal CCAAT box (-65/-61) but not of the distal CCAAT box (- 95/-91) increased the reporter activity by 3-fold. Simultaneous disruptions of both the GC box and the CCAAT box reduced the reporter activity to a level that was close to that of the single GC box mutation. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from INSA-1 cells demonstrated that Sp1 and Sp3 bind a GC box while the nuclear factor Y was shown to bind the proximal but not the distal CCAAT box. (Published in Biochem Biophys Res Commun 2005; 329: 188-96. Supported by Thailand Research Fund, Third World Academy of Sciences, and Australian Research Council.) THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA REGULATES MURINE PYRUVATE CARBOXYLASE GENE EXPRESSION IN VIVO AND IN VITRO. (NO. 715) Jitrapakdee S 1,2 , Slawik M 1 , Medina-Gomez G 1 , Campbell M 1 , Wallace JC 3 , Sethi JK 1 , O’Rahilly S 1 , Vidal-Puig AJ 1 . Faculty of Science 1 Cambridge Institute of Diabetes, Endocrinology and Metabolism, and Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK; 2 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 3 School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia. Key words: control of gene expression, murine pyruvate carboxylase gene, peroxisome proliferator-activated receptor gamma. Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways including gluconeogenesis, lipogenesis and glucose-induced insulin secretion. Here we show for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator activated receptor gamma (PPARγ) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB- 1B, in parallel with the expression of the adipogenenic transcription factors, CCAAT-enhancer binding protein α (C/EBPα), PPARγ1 and PPARγ2. Tumor necrosis factor alpha (TNFα), a cytokine that blocks differentiation of 3T3-L1 cells, suppresses PC expression. Cotransfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3 kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing RXRα/PPARγ1 or RXRα/PPARγ2, showed a 6-8 fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with PPARγ agonist doubled the promoter activity. Mutation of the putative PPRE (-386/-374) of this 2.3 kb PC promoter fragment abolished PPARγ response. Gel shift and chromatin immunoprecipitation assays demonstrate that endogenous PPARγ binds to this functional PPRE of the PC promoter. Mice with targeted disruption of the PPARγ2 gene display approximately 50-60% reduction of PC mRNA and protein in white adipose tissue. Similarly in brown adipose tissue of PPARγ2 deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARγ2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identify PC as a PPAR γ regulated gene and suggest a role for PPARγ regulating intermediary metabolism. (Published in J Biol Chem 2005; 290: 27466-76. Supported by Wellcome Trust, MRC UK, Royal Society UK, and Fritz Thyssen Foundation Germany. ) CRYSTALLIZATION AND PRELIMINARY X-RAY CRYSTALLOGRAPHIC ANALYSIS OF 2-METHYL- 3-HYDROXYPYRIDINE-5-CARBOXYLIC ACID (MHPC) OXYGENASE FROM PSEUDOMONAS SP. MA-1. (NO. 716) Oonanant W 1 , Sucharitkul J 1,2 , Yuvaniyama J 1 , Chaiyen P 1 . 1 Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok; 2 Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok. Key words: 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase, X-ray crystallographic data. 2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) catalyzes the conversion of an aromatic substrate, MHPC, to an aliphatic compound, α-(N- PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
Mahidol University Annual Research Abstracts, Vol. 33 279 acetylaminomethylene)-succinic acid, and is involved in the degradation of vitamin B6 by the soil bacterium Pseudomonas sp. MA-1. Using only FAD as a cofactor, MHPCO is unique in catalyzing hydroxylation and subsequent aromatic ring cleavage without requiring a metal-ion cofactor. Here, the crystallization of MHPCO is reporte3d together with preliminary X-ray crystallographic data. An MHPCO crystal obtained by hanging0drop vapour diffusion diffracted X-rays to 2.25 Å resolution and belonged to the triclinic space group P1, with four molecules per asymmetric unit. (Published in Acta Cryst 2005; F61: 312-4. Supported by Mahidol University, Thailand Research Fund and TARUN/Thailand Tropical Diseases Research Program.) STUDIES ON THE TRANSGLUCOSYLATION REACTIONS OF CASSAVA AND THAI ROSEWOOD BETA-GLUCOSIDASES USING 2-DEOXY-2-FLUORO- GLYCOSYL-ENZYME INTERMEDIATES. (NO. 717) Hommalai G, Chaiyen P, Svasti J. Center of Excellence for Protein Structure and Function, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok. Key words: beta-glucosidase, cassava, Thai rosewood. beta-Glucosidases from cassava and Thai rosewood can synthesize a variety of alkyl glucosides using various alcohols as glucosyl acceptors for transglucosylation. Both enzymes were inactivated by 2-deoxy-2-fluoro-sugar analogues to form the covalent glycosyl-enzyme intermediates, indicating that the reaction mechanism was of the double-replacement type. The trapped enzyme intermediates were used for investigating transglucosylation specificity, by measuring the rate of reactivation by various alcohols. The glucosyl-enzyme intermediate from the cassava enzyme showed a 20- to 120-fold higher rate of glucose transfer to alcohols than the glucosyl-enzyme intermediate from the Thai rosewood enzyme. Kinetic analysis indicated that the aglycone binding site of the cassava enzyme was hydrophobic, since the enzyme bound better to more hydrophobic alcohols and showed poor transfer of glucose to hydrophilic sugars. With butanol, transglucosylation was faster with the primary alcohols than with the secondary or tertiary alcohol. Studies with ethanol and chloro-substituted ethanols indicated that the rate of transglucosylation was significantly faster with alcohols with lower pKa values, where the reactive alkoxide was more readily generated, indicating that the formation of the alkoxide species was a major step governing the formation of the transition state in the cassava enzyme. (Published in Arch Biochem Biophys 2005; 442: 11-20. Supported by Thailand Research Fund.) THE REDUCTASE OF P-HYDROXYPHENY- LACETATE 3-HYDROXYLASE FROM ACINETOBACTER BAUMANNII REQUIRES P- HYDROXYPHENYLACETATE FOR EFFECTIVE CATALYSIS. (NO. 718) Sucharitakul J 1 , Chaiyen P 1 , Entsch B 2 , Ballou DP 2 . 1 Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok; 2 Department of Biological Chemistry, University of Michigan, Michigan, USA. Key words: Acinetobacter baumannii, p-hydroxyphenylacetate 3hydroxylase reductase, two-protein system. p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) from Acinetobacter baumannii catalyzes hydroxylation of HPA to form 3,4-dihydroxyphenylacetate. It is a two-protein system consisting of a smaller reductase component (C 1 ) and a larger oxygenase component (C 2 ). C 1 is a flavoprotein containing FMN, and its function is to provide reduced flavin for C 2 to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C 1 . The apoenzyme of C 1 binds to oxidized FMN tightly with a K d of 0.006 μM at 4°C, but with a K d of 0.038 μM in the presence of HPA. Reduction of C 1 by NADH occurs in two phases with rate constants of 11.6 and 3.1 s -1 and K d values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C 1 exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C 1 by NADH occurred in a single phase at 300 s -1 with a K d of 25 μM for NADH binding at 4°C. Formation of the C 1 -HPA complex prior to binding of NADH was required for this stimulation. The redox potentials indicate that the rate enhancement is not due to thermodynamics (E° m of the C 1 -HPA complex is -245 mV compared to an E° m of C 1 of -236 mV). When the C 1 -HPA complex was reduced by 4(S)-NADH, the reduction rate was changed from 300 to 30 s -1 , giving a primary isotope effect of 10 and indicating that C 1 is specifically reduced by the pro-(S)-hydride. In the reaction of reduced C 1 with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C 1 being a mixture of fast and slow reacting species. Rate constants for both phases were the same in the absence and presence of HPA, but in the presence of HPA, the equilibrium shifted toward the faster reacting species. (Published in Biochemistry 2005; 44: 10434-42. Supported by Mahidol University, Thailand Research Fund, Commission of Higher Education Staff Development Project, Ministry of Education, Thailand, BIOTEC, NSTDA, Thailand, and NIH, USA.) ARGINOSUCCINATE SYNTHETASE DEFICIENCY: MUTATION ANALYSIS IN 3 THAI PATIENTS. (NO. 719) Wasant P 1 , Viprakasit V 2 , Srisomsap C 3 , Liammongkolkul S 1 , Ratanarak P 1 , Sathienkijakanchai A 1 , Svasti J 3,4 . 1 Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok; 2 MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK; 3 Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok; 4 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok. Key words : arginosuccinate synthetase deficiency, mutation analysis, Thai patients. Remarkable improvements in public health, nutrition, hygiene, and availability of medical services in the last 20 years have significantly reduced infant and childhood mortality in Thailand. Therefore, many rare and previously unidentified genetic disorders, which, in the past, usually led to the death of affected infants before a definitive diagnosis, have now been increasingly recognized. Recently, we identified three unrelated patients from Thailand who suffered from citrullinemia, one of the inherited types PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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