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286 Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form of protein would require an in vitro refolding step to regain biological functions. In this study, we described the system for soluble expression of a singlechain variable fragment (scFv) against hepatocellular carcinoma (Hep27scFv) by coexpressing Dsb protein and enhancing with medium additives. The results revealed that overexpression of DsbABCD protein showed marked effect on the soluble production of Hep27scFv, presumably facilitating correct folding. The optimal condition for soluble scFv expression could be obtained by adding 0.5M sorbitol to the culture medium. The competitive enzyme-linked immunosorbent assay (ELISA) indicated that soluble scFv expressed by our method retains binding activity toward the same epitope on a hepatocellular carcinoma cell line (HCC-S102) recognized by intact antibody (Ab) (Hep27 Mab). Here, we report an effective method for soluble expression of scFv in E. coli by the Dsb coexpression system with the addition of sorbitol medium additive. This method might be applicable for high-yield soluble expression of proteins with multiple disulfide bonds. (Published in Biotechnol Bioeng 2005; 91: 418-24. Supported by Science and Technology Incubation Program in Advanced Region, Japan Science and Technology Corporation.) CONSTRUCTION OF SCFV DERIVED FROM A TUMOR-ASSOCIATED MONOCLONAL ANTIBODY HAVING TUMORICIDAL ACTIVITY ON HUMAN HEPATOCELLULAR CARCINOMA. (NO. 740) Tungpradabkul S 1 , Sandee D 1 , Puthong S 2 , Laohathai K 2 . 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 2 Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok. Key words : single-chain variable fragment, tumor-associated monoclonal antibody, tumoricidal effect. A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4mg of the Mab over 1month period. A single-chain variable fragment (scFv molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavyand light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma. (Published in Mol Immunol 2005; 42: 713-9. Supported by Mahidol University, Chulalongkorn University; Thailand National Science and Technology Development Agency.) PURIFICATION AND CHARACTERISATION OF KERATINASE FROM A THERMOTOLERANT FEATHER-DEGRADING BACTERIUM. (NO. 741) Suntornsuk W 1 , Tongjun J 1 , Onnim P 1 , Oyama H 2 , Ratanakanokchai K 3 , Kusamran T 4 , Oda K 2 . 1 Department of Microbiology, Faculty of Science, King Mongkut’s Institute of Technology Thonburi, Bangkok; 2 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, Japan; 3 Department of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s Institute of Technology Thonburi, Bangkok; 4 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok. Faculty of Science Key words : Bacillus licheniformis, keratinase, thermotolerance Isolation and identification of a thermotolerant featherdegrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg -1 ) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60°C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4– 100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin. (Published in World J Microbiol Biotech 2005; 21: 1111– 7.Supported by Japan Society for Promotion of Science and National Research Council of Thailand.) QUINONE REDUCTASE INDUCERS IN AZADIRACHTA INDICA A. JUSS FLOWERS, AND THEIR MECHANISMS OF ACTION. (NO. 742) Sritanaudomchai H 1 , Kusamran T 1 , Kuakulkiat W 2 , Bunyapraphatsara N 2 , Hiransalee A 3 , Tepsuwan A 4 , Kusamran WR 4 . 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 2 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok; 3 Department of Medical Science, Ministry of Public Health, Bangkok; 4 Chemical Carcinogenesis Section, Research Division, National Cancer Institute, Bangkok. We have previously shown that the flowers of neem tree (Azadirachta indica A. Juss, family Meliaceae), Thai variety, strongly induced the activity of glutathione S-transferase (GST) while resulting in a significant reduction in the activities of some cytochrome P(450)-dependent monooxygenases in rat liver, and possess cancer chemopreventive potential against chemicallyinduced mammary gland and liver carcinogenesis in rats. In the present study, 2 chemicals possessing strong QR inducing activity were fractionated from neem flowers using a bioassay based on the induction of QR activity in mouse hepatoma Hepa 1c1c7 cultured cells. Spectroscopic characteristics revealed that these compounds PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
Mahidol University Annual Research Abstracts, Vol. 33 287 were nimbolide and chlorophylls, having CD (concentration required to double QR specific activity) values of 0.16 and 3.8 mug/ml, respectively. Nimbolide is a known constituent of neem leaves, but was found for the first time here in the flowers. Both nimbolide and chlorophylls strongly enhanced the level of QR mRNA in Hepa 1c1c7 cells, as monitored by northern blot hybridization, indicating that the mechanism by which these constituents of neem flowers induced QR activity is the induction of QR gene expression. These findings may have implication on cancer chemopreventive potential of neem flowers in experimental rats previously reported. (Published in Asian Pac J Cancer Prev 2005; 6: 263-9. Supported by Terry Fox Run Research Fund, Oncological Society of Thailand and Department of Medical Services, Ministry of Public Health.) USE OF A REMOTE SENSING-BASED GEO- GRAPHIC INFORMATION SYSTEM IN THE CHARACTERIZING SPATIAL PATTERNS FOR ANOPHELES MINIMUS A AND C BREEDING HABITATS IN WESTERN THAILAND (NO. 743) Rongnoparut P 1 , Ugsang DM 2 , Baimai V 3 , Honda K 2 , Sithiprasasna R 4 . 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 2 RS & GIS FoS, Asian Institute of Technology, Pathum Thani; 3 Center for Vectors and Vectorborne Diseases, Faculty of Science, Mahidol University, Bangkok; 4 Department of Entomology, US Army Medical Component, AFRIMS, Bangkok. Key words : Anopheles minimus, breeding habitat, remote sensing A remote sensing (RS)-based Geographic Information System (GIS) was used to characterize the breeding habitats of Anopheles minimus species A and C in five different districts of Kanchanaburi Province in western Thailand. The GIS and RS were used to monitor the area for the presence and absence of An. minimus A and C in five major land areas, forest, agriculture, urban, water and bare land. The results show that An. minimus A survives both in dense canopy forest and in open fields where agriculture is dominant. A scatter plot of land-use/land-cover for An. minimus, considering proximities to the forest and proximities to agriculture, suggests that An. minimus A has a wider habitat preference, ranging from dense canopy forest to open agricultural fields. A scatter plot for An. minimus C, on the other hand, showed a narrow habitat preference. A scatter plot for proximities performed on separate populations of An. minimus species A, one in the north and the other in the south, showed that there was an association in the northern population with the forest and in the southern population with agricultural areas. There were no statistically significant differences in the scatter plot of proximities to urban areas and water bodies with the An. minimus A north, south and An. minimus C. LANDSAT TM satellite data classification was used to identify larval habitats that produce An. minimus A and C and analyze proximities between land-use/land-cover habitats and locations of larval habitats. An. minimus A has a wide habitat preference, from dense canopy forest to open agricultural fields, while An. minimus C has a narrow habitat preference. (Published in Southeast Asian J Trop Med Public Health 2005; 36: 1145-52. Supported by TRF/BIOTEC Special Program for Diversity Research and Training and CNRS, France.) HIGHER PLANT-LIKE FLUORESCENCE INDUCTION AND THERMOLUMINESCENCE CHARACTERISTICS IN CYANOBACTERIUM, SPIRULINA MUTANT DEFECTIVE IN PQH2 OXIDATION BY CYTB6/F COMPLEX. (NO. 744) Ruengjitchatchawalya M 1 , Kovacs L 2 , Mapaisansup T 1 , Sallai A 2 , Gombos Z 2 , Ponglikitmongkol M 3 , Tanticharoen M 1,4 1 School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok; 2 Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Hungary; 3 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; 4 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani. Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers. (Published in J Plant Physiol 2005; 162: 1123-32. Supported by BIOTEC, NSTDA and Hungarian Science Foundation.) AN EASY METHOD FOR GENERATING DELETION MUTANTS IN AGROBACTERIUM TUMEFACIENS USING A SIMPLE REPLACEMENT VECTOR. Suksomtip M, Tungpradabkul S. (NO. 745) Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok. Key word : Agrobacterium tumefaciens, citrate synthase gene, deletion mutants We have developed a method to make precise mutations in the A. tumefaciens genome at frequencies high enough to allow direct identification of mutants by PCR or other screening methods rather than by selection. This method utilized a novel pUC18-based gene replacement vector that was used as a donor plasmid carrying the desired mutation in the target cell. Two sites of single-crossover occurred resulting in efficient replacement of the wild type allele on the chromosome with the modified sequence. The usefulness of this method was demonstrated by making deletion mutants in citrate synthase genes of A. tumefaciens. (Published in ScienceAsia 2005; 31: 349-57. Supported by Commission on Higher Education, Ministry of Education.) PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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