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Mahidol University Annual Research Abstracts, Vol. 31

AMINOPEPTIDASE REACTIVITY IN THE DIGESTIVE TRACT OF AN ADULT HALIOTIS ASININA LINNAEUS (NO. 654) Pornrut Rabintossaporn, Pornchun Saitongdee, Prapee Sretarugsa, Prasert Sobhon Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. E-mail : [email protected] Key words : H. asinina, digestive tract, histology, leucine aminopeptidase The leucine aminopeptidase reactivity in the digestive tract of an adult Haliotis asinina was studied by enzyme-histochemical method in order to investigate the main site of protease activity. The digestive system of H. asinina consists of buccal region with a pair of salivary glands, a pair of buccal pouches and a buccal mass; esophageal pouch; crop; stomach with digestive glands; appendix; intestine; and anus. Histologically, the epithelium of the digestive tract is pseudostratified columnar type, which is composed of three kinds of cells, including granulated cells, non-granulated cells, and mucus cells. The subsets of these three types of cells vary from region to region. Aminopeptidase reactivity was located principally within the cytoplasm of granulated cells in esophagus, esophageal pouch, the proximal part of the stomach and the third intestinal region, which implies that these sites are responsible for the synthesis and secretion of peptidase for the digestion of proteinaceous nutrients. (This research was supported financially by the Thailand Research Fund.)

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follicular cells closely attached the early stage oocytes. The results indicate that the cerebral ganglion may be one of the main sources of ELH-producing cells. (This research was supported financially by the Thailand Research Fund)

A TRANSPOSASE-LIKE GENE ISOLATED FROM BURKHOLDERIA PSEUDOMALLEI BY DDRT-PCR TECHNIQUE. (NO. 656) Choeycharoen A, Vinichnaiyapark N, Vichchathorn P, Tunpiboonsak S, Tungpradabkul S. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Burkholderia mallei, Burkholderia pseudomallei and Burkholderia thailandensis are in the same genus and share some interesting properties. The former species is the zoonostic agent of glander, while the second is an etiologic agent of melioidosis. The latter is a non-virulent species. We were therefore interested the virulence factors which are unclear until now. Technique for identification of differential display gene expression using RT-PCR or DDRTPCR between a virulent and a non-virulent species was applied. In this report, the entire coding sequence of a transposase-like gene was first identified in Burkholderia pseudomallei NF10/38. The gene was 100% homologous to the Tn10 transposase gene from Salmonella typhi. In order to compare the gene localization among the three species (B. mallei, B. pseudomallei and B. thailandensis), Southern blot hybridization was performed. The hybridization pattern revealed differences in gene distribution and in copy number with the virulent species showing a higher copy number than in the non-virulent species. This finding indicate an indirect relationship between a transposase and the virulent nature of the bacteria. (Published in J Gen Appl Microbiol 2003; 49: 59-61. Supported by the Thailand Research Fund, National Science and Technology Development Agency, Thailand and Faculty of Graduate Studies, Mahidol University.)

EGG-LAYING HORMONE IMMUNOREACTIVITY IN THE NERVOUS GANGLIA AND REPRODUCTIVE ORGAN OF THAI ABALONE, HALIOTIS ASININA LINNAEUS (NO. 655) Porncharn Saitongdee, Somjai Apisawetakan, Peter J Hanna, Prasert Sobhon Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria 3174, Australia. E-mail : [email protected] or [email protected] Key words : ganglia, ovary, immunocytochemistry Distribution of egg-laying hormone (ELH) immunoreactivity was investigated in the cerebral and pleuropedal ganglia, and gonad of Thai female abalone, Haliotis asinina Linneus. By using mouse antiserum raise against recombinant fusion protein of aELH with glutahione-S-transferase from pGEX-2T, the immunocytochemistry on sections of the ganglia demonstrated that ELH immunoreaction was expressed in neurosecretory cells (NS), neurites which lies along the neuropil, and blood sinus embedded in the connective tissue sheath of the ganglion. The number of positive NS and the intensity of staining in cerebral ganglia was more than in pleuropedal ganglia. In the ovary, the ELH-immunoreactive could be detected scarcely in the cells of capsule and trabeculae and in the

ANTIMALARIAL 9-ANILINOACRIDINE COMPOUNDS DIRECTED AT HEMATIN

1 1 2

(NO. 657)

3

Auparakkitanon S , Noonpakdee W , Ralph RK , Denny WA , 1 Wilairat P Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Cancer Research and Developmental Biology, School of Biological Sciences, 3Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand. Antimalarial 9-anilinoacridines are potent inhibitors of parasite DNA topoisomerase II both in vitro and in situ. 3,6-Diamino substitution on the acridine ring greatly improves parasiticidal activity against Plasmodium falciparum by targeting DNA topoisomerase II. A series of 9-anilinoacridines were investigated for their abilities to inhibit -hematin formation, to form drug-hematin complexes, and to enhance hematin-induced lysis of red blood cells. Inhibition of -hematin formation was minimal with 3,61

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diamino analogs of 9-anilinoacridine and greatest with analogs with a 3,6-diCl substitution together with an electron-donating group in the 1'-anilino position. On the other hand, the presence of a 1'-N(CH3)2 group in the anilino ring produced compounds that strongly inhibited b-hematin formation but which did not appear to be sensitive to the nature of the substitutions in the acridine nucleus. The derivatives bound hematin, and Job's plots of UV-visible absorbance changes in drug-hematin complexes at various molar ratios indicated a stoichiometric ratio of 1:2. The drugs enhanced hematin-induced red blood cell lysis at low concentrations (<4 µM). These studies open up the novel possibility of development of 9-anilinoacridine antimalarials that target not only DNA topoisomerase II but also hematin formation, which should help delay the rapid onset of resistance to drugs acting at only a single site. (Published in Antimicrob Agents Chemother 2003; 47: 3708-12. Supported by the Thailand Research Fund.)

Faculty of Science

istry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Enzyme-based metabolic mechanisms of insecticide resistance were investigated, comparing a deltamethrin-susceptible parent stock and resistant colonies of Anopheles minimus species A using biochemical assays. The control parent colony was determined susceptible to the diagnostic lethal concentration of deltamethrin (0.05%), whereas the 6 resistant test populations at selected 4, 8, 12, 14, 16, and 18 filial generations (F4, F8, F12, F16, and F18) demonstrated varying levels of tolerance/resistance to deltamethrin. Expression of levels of non-specific esterases, monooxygenases, and glutathione S-transferases (GSTs) were measured. Results indicated that monooxygenase activity was consistently elevated in resistantselected test populations compared to the parent colony and increased as resistance intensified from F8 to F18. There was a 5-fold increase in monooxygenase in the F18 generation compared to the parental stock. Fluctuations in alpha and beta-esterase activity, measured by hydrolysis of alpha and beta-naphthylpropionate, provided no conclusive evidence of an association with pyrethroid resistance in this mosquito species. GSTs were not elevated in the 6 resistant test populations. Based on our results, it appears likely that the development of physiological resistance to deltamethrin in laboratory, resistant-selected generations of An. minimus is primarily associated with increased detoxification by over-expression of monooxygenases. The oxidases are the major contributors to pyrethroid resistance and the importance of kdr has yet to be convincingly determined. This finding represents the first report from Thailand of this metabolic mechanism of resistance in anophelines. (Published in J Vector Ecol 2003; 28: 108-16. Supported by Thailand Research Fund.)

ANTI-METASTATIC EFFECTS OF AQUEOUS EXTRACT OF HELIXANTHERA PARASITICA.

(NO. 658)

1 2 2 2

Lirdprapamongkol K , Mahidol C , Thongnest S , Prawat H , 2,3 1 1 4 Ruchirawat S , Srisomsap C , Surarit R , Punyarit P , Svasti 1,5 J. Laboratory of Biochemistry, 2Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok 10210, Thailand; 3Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 4 Department of Pathology, Pramongkutklao College of Medicine, Bangkok 10400, Thailand; 5 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Metastasis, the spread of cancer in body, is a major cause of death. We have screened anti-metastatic activity of aqueous and dichloromethane extracts of several not previously studied Thai herbs, using an in vitro invasion test. This involves the in vitro invasion of HCC-S102, a hepatocellular carcinoma cell line derived from a Thai patient, through a reconstituted-basement membrane (Matrigel). The aqueous extract of a plant (Helixanthera parasitica) revealed a significant inhibitory effect on the cancer cell invasion, and showed antioxidant activity. The aqueous extract was partially purified by silica gel column chromatography, and the highest anti-metastatic activity fraction showed 83% inhibition of invasion with low cytotoxic effect. However, anti-metastatic activity was not associated with the antioxidant activity of the aqueous extract. (Published in J Ethnopharmacol 2003; 86: 253-6. Supported by Chulabhorn Research Institute and the Thailand Research Fund.)

1

CHARACTERIZATION OF A RICE -GLUCOSIDASE HIGHLY EXPRESSED IN FLOWER AND GERMINATING SHOOT. (NO. 660)

1 3

Opassiri R, 1Cairns JRK, 2Akiyama T, 1Wara-Aswapati O, Svasti J, 4Esen A.

Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; 2Department of Low Temperature Science, Hokkaido National Agricultural Experiment Station, Sapporo 062-8555, Japan; 3Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 4 Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA. The cDNAs for two -glucosidase (E.C. 3.2.1.21) isozymes from rice (Oryza sati L.), designated bglu1 and bglu2, were cloned and sequenced. The cDNA sequences for bglu1 and bglu2 included open reading frames encoding 504 and 500 amino acid precursor proteins, respectively. Both of these enzymes appeared to enter the secretory pathway, as judged by their N-terminal signal sequences. Southern blots using gene-specific probes indicated that bglu1 and bglu2 were single copy genes. The bglu1 and bglu2 mRNAs were highly expressed in the shoot during germination, with a similar time course. However, differences were seen in expression

1

BIOCHEMICAL DETECTION OF PYRETHROID RESISTANCE MECHANISMS IN ANOPHELES MINIMUS IN THAILAND. (NO. 659) Chareonviriyaphap T , Rongnoparut P , Chantarumporn P , 1 Bangs MJ.

1 1 2 1

Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand; 2Department of Biochem-

Mahidol University Annual Research Abstracts, Vol. 31

in mature plants, where bglu1 was highly expressed in flowers, but bglu2 was not. A recombinant thioredoxin fusion protein produced from the bglu1 cDNA in redox-deficient Escherichia coli (BGlu1) hydrolyzed p-nitrophenol -D-glucoside, -D-fucoside, and other p-nitrophenol -D-glycosides, and was strongly inhibited by glucono1,5-lactone. It also hydrolyzed some natural glucosides, including the rice-derived pyridoxine-5-O-b-D-glucoside, and hydrolyzed and transglycosylated short -(13) and -(14) linked gluco-oligosaccharides. Based on the results, possible functions of BGlu1 include hydrolysis and recycling of oligosaccharides generated from rapid cell wall expansion during seed germination and flower expansion, and release of the coenzyme pyridoxine from its glucose-conjugated storage form. (Published in Plant Sci 2003; 165: 627-38. Supported by Thailand Research Fund and TRF Royal Golden Jubilee PhD Program.)

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CLONING OF CYTOCHROME P450, CYP6P5, AND CYP6AA2 FROM ANOPHELES MINIMUS RESISTANT TO DELTA-METHRIN. (NO. 662) Rongnoparut P, Boonsuepsakul S, Chareonviriyaphap T, Thanomsing N. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Two new genes in the cytochrome P450 (CYP6) family 6 with complete coding sequences were cloned and sequenced from deltamethrin-resistant Anopheles minimus, a major malaria vector in Thailand. CYP6P5 encodes a protein of 508 amino acids, while CYP6AA2 contains 505 residues. Each encoded protein contains a hydrophobic N-terminal region and a highly conserved heme-binding region typical of P450s. Alignments of deduced amino acid sequences with other insect P450 genes indicate a high degree of identity to insect CYP6 genes. Comparative mRNA expression studies using semi-quantitative RT-PCR analysis indicated that the relative amount of CYP6AA2 transcript was greater in the deltamethrinresistant An. minimus compared to the susceptible strain. The expression of CYP6AA2 in deltamethrin-resistant mosquitoes is associated with development of deltamethrin resistance in An. minimus mosquito. The CYP6P5 transcript is equally expressed in both resistant and susceptible mosquitoes. (Published in J Vector Ecol 2003; 28: 150-8. Supported by Thailand Research Fund.)

CLONING, EXPRESSION AND CHARACTERIZATION OF A FUNCTIONAL CDNA CLONE ENCODING GERANYL-GERANYL DIPHOSPHATE SYNTHASE OF HEVEA BRAZSILIENSIS. (NO. 661)

1 Takaya A, 1 Zhang YW, 2 Asawatreratanakul K, 3 Wititsuwannakul D, 4Wititsuwannakul R, 1Takahashi S, 1Koyama T.

1

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan; 2Department of Chemistry, Thaksin University, Songkla, Thailand; 3Department of Biochemistry, Mahidol University, Bangkok, Thailand; 4Department of Biochemistry, Prince of Songkla University, Hat-Yai, Thailand. Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent Km values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni 2+-affinity column were 24.1, 6.8, 2.3, and 11.5 µM, respectively. The enzyme shows optimum activity at approximately 40°C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower. (Published in Biochim Biophys Acta 2003; 1625: 214-20. Supported by Ministry of Education, Science and Culture of Japan, Japan Society for the Promotion of Science, and the Asahi-, Heiwa-Nakajima, Sumitomo- and Goho-Life Science Foundations.)

CO-MUTATION OF HPV16 E6 AND E7 GENES IN THAI SQUAMOUS CERVICAL CARCINOMAS.

(NO. 663)

1

Vaeteewoottacharn K, 2Jearanaikoon P, 1Ponglikitmongkol M.

1

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Department of Clinical Chemistry, Faculty of Medical Technology, Khon Kaen University, Khon Kaen 40002, Thailand. Infection with high-risk HPV16 is well associated with invasive cervical carcinomas. Variations in the E6 gene of HPV16 are shown to correlate with both the geographical areas and the oncogenicity. Here, we have characterized HPV16 DNA variants in the E6 and E7 coding regions of 31 Thai cervical squamous cell carcinomas. Five groups of E6 variants were identified. A mutation from T to G at nucleotide 178, leading to a change from aspartate to glutamate (D25E), was the most common variation accounting for 70%. Interestingly, 90% of these E6 (D25E) variants coincided with a specific type of E7 mutation, N29S. This is the first finding of coordinated change between E6 (D25E) and E7 (N29S) in HPV, which might indicate a specific variant for the Thai population. These comutations may not only represent an area-specific strain but may also be valuable for studying the virus and developing a suitable vaccine in Thailand. (Published in Anticancer Res 2003; 23: 1927-31. Supported by Mahidol University and the National Science and Technology Development Agency, Thailand.)

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DEPENDENCE OF METASTATIC CANCER CELL INVASION ON MLCK-CATALYZED PHOSPHORYLATION OF MYOSIN REGULATORY LIGHT CHAIN. (NO. 664) Tohtong R, Phattarasakul K, Jiraviriyakul A, Suthiphongchai T. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. The role of myosin phosphorylation by myosin light chain kinase (MLCK) in regulating the invasiveness of metastatic cancer cells was investigated using the Dunning rat prostatic adenocarcinoma cell line, Mat Ly Lu, and in vitro invasion assay. Treatment with MLCK inhibitors resulted in marked reduction of invasiveness, which was principally due to impaired cellular motility, whereas the ability to survive and proliferate, to adhere to matrix, and to secrete gelatinases were minimally affected. (Published in Prostate Cancer Prostatic Dis 2003; 6: 212-6. Supported by Mahidol University)

Faculty of Science

EFFECT OF N-TERMINAL TRUNCATION OF PLASMODIUM FALCIPARUM DIHYDROFOLATE REDUCTASE ON DIHYDRO-FOLATE REDUCTASE AND THYMIDYLATE SYNTHASE ACTIVITY.

(NO. 666)

1,2 1 2

Wattanarangsan J , Chusacultanachai S , Yuvaniyama J , 1 1 Kamchonwongpaisan S , Yuthavong Y.

1

National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand; 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. In Plasmodium falciparum and other protozoa, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by a single gene and expressed as a bifunctional protein on a single polypeptide chain. We have exploited truncation mutants to explore the roles of N-terminal amino acids of pfDHFR on DHFR and TS activities and have shown that Gln-4, Val-5, and Cys-6 are crucial for pfDHFR activity. We also demonstrated that correct conformation of pfDHFR, but not its activity, is required for TS activity. (Published in Mol Biochem Parasitol 2003; 126: 97-102. Supported by Ministry of University Affairs, Thailand; Faculty of Graduate Studies, Mahidol University; and Thailand Tropical Diseases, EU, Wellcome Trust and MMV programs.) FUNCTIONAL STUDY OF INTRACELLULAR P-GP- AND MRP1-MEDIATED PUMPING OF FREE CYTOSOLIC PIRARUBICIN INTO ACIDIC OR GANELLES IN INTRINSIC RESISTANT SIHA CELLS. (NO. 667) Laochariyakul P , Ponglikitmongkol M , Mankhetkorn S.

1 1 1 2

DOCKING AND DATABASE SCREENING REVEAL NEW CLASSES OF PLASMODIUM FALCIPARUM DIHYDROFOLATE REDUCTASE INHIBITORS

(NO. 665)

1 1

Rastelli G, 1Pacchioni S, 2Sirawaraporn W, 2Sirawaraporn R, Parenti MD, 1Ferrari AM.

1

Dipartimento di Scienze Farmaceutiche, Universita di Modena e Reggio Emilia, Modena 41100, Italy; 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Plasmodium falciparum dihydrofolate reductase (PfDHFR) is an important target for anti-malarial chemotherapy. Unfortunately, the emergence of resistant parasites has significantly reduced the efficiency of classical antifolate drugs such as cycloguanil and pyrimethamine. In this study, an approach toward molecular docking of the structures contained in the Available Chemicals Directory (ACD) database to search for novel inhibitors of PfDHFR is described. Instead of docking the whole ACD database, specific 3D pharmacophores were used to reduce the number of molecules in the database by excluding a priori molecules lacking essential requisites for the interaction with the enzyme and potentially unable to bind to resistant mutant PfDHFRs. The molecules in the resulting "focused" database were then evaluated with regard to their fit into the PfDHFR active site. Twelve new compounds whose structures are completely unrelated to known antifolates were identified and found to inhibit, at the micromolar level, the wild-type and resistant mutant PfDHFRs harboring A16V, S108T, A16V + S108T, C59R + S108N + I164L, and N51I + C59R + S108N + I164L mutations. Depending on the functional groups interacting with key active site residues of the enzyme, these inhibitors were classified as N-hydroxyamidine, hydrazine, urea, and thiourea derivatives. The structures of the complexes of the most active inhibitors, as refined by molecular mechanics and molecular dynamics, provided insight into how these inhibitors bind to the enzyme and suggested prospects for these novel derivatives as potential leads for antimalarial development. (Published in J Med Chem 2003; 46: 2834-45. Supported by University of Modena and WHO-TDR Program.)

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131, Thailand. We sought to determine the efficiency of the intracellular functional P-gp- and MRP1-mediated pumping of THP into acidic organelles in SiHa cells and etoposide-resistant SiHa/VP16 cells. The expression of both MDR1 and MRP1 genes of SiHa and SiHa/ VP16 cells was clearly shown by using RT-PCR. The functional studies of both intracellular functional P-gp- and MRP1-mediated pumping were performed by using THP in a conventional spectrofluorometer, and they demonstrated that SiHa and SiHa/VP16 cells are good models to illustrate the functional role of intracellular P-gp and MRP1 in the transport of free cytosolic drug into acidic organelles. The functional P-gp and MRP1 proteins were identified both on plasma membranes and on intracellular vesicle membranes. Within the limit of experimental error, similar efficiencies in THP transport were observed in the two proteins at both locations in SiHa and SiHa/VP16 cells. The P-gp- and MRP1-mediated pump coefficient (kav), Michealis-Menten's constant (Kmv), and maximal pumping rate (Vmaxv) values of those located on vesicular membranes were 1.87 +/- 0.30 pL.cell-1.s-1, 1.63 +/- 0.21 mM, and 4.95 +/- 0.45 nM.s 1 , respectively. Drug retention inside acidic organelles (C vmon) of

Mahidol University Annual Research Abstracts, Vol. 31

SiHa cells was significantly higher than that of SiHa/VP16 cells, perhaps a consequence of slower movement of recycling endosomes and (or) lysosomes to the cell membrane of SiHa cells, leading to distended organelles and cell death. Our results suggest that intracellular P-gp and MRP1 proteins play an important role in the transport of free drug from cytosol to cytoplasmic acidic organelles. (Published in Can J Physiol Pharmacol 2003; 8: 790-9. Supported by the Medical Scholars Program, Mahidol University, the Royal Thai Government, and the National Science and Technology Development Agency (NSTDA), Thailand.)

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HB KODAIRA II [146(HC3)HISGLN] DETECTED IN THAILAND. (NO. 668) Ngiwsara L , Srisomsap C , Winichagoon P , Fucharoen S , 1,2 Svasti J.

1 1,2 1 3 3

deduced ORF3 protein sequence identified six potential transmembrane helices and three ectodomains containing multiple sites for potential N-linked and O-linked glycosylation. N-terminal sequence analysis of mature gp116 and gp64 proteins indicated that each was derived from ORF3 by proteolytic cleavage of the polyprotein between residues Ala228 and Thr229, and Ala1127 and Leu1128, located at the C-terminal side of transmembrane helices 3 and 5, respectively. Comparison with the deduced ORF3 protein sequence of Australian gill-associated virus (GAV) indicated 83% amino acid identity in gp64 and 71% identity in gp116, which featured two significant sequence deletions near the N-terminus. Database searches revealed no significant homology with other proteins. Recombinant gp64 expressed in E. coli with and without the C-terminal transmembrane region was shown to react with antibody raised against native gp64 purified from virions. (Published in J Gen Virol 2003; 84: 863-73. Supported by Mahidol University, the National Center for Genetic Engineering and Biotechnology, Thailand, the Thailand Research Fund, TRF PhD Golden Jubilee program and the Australian Centre for International Agricultural Research.)

Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand; 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3 Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom, Thailand. Hb Kodaira [146(HC3)HisGln (CACCAA)], a variant with increased oxygen affinity, was first described in a Japanese male in 1992. Hb Kodaira II [146(HC3)HisGln (CACCAG)], with the same phenotype, has recently been reported. We now describe another case of Hb Kodaira II in a healthy female Thai. All three cases of Hb Kodaira were found in Asians, but the evolutionary origin of these cases has not been investigated. With improved techniques of detection by HPLC and IEF, Hb Kodaira may be found in non Asiatic populations. (Published in Hemoglobin 2003; 27: 37-9. Supported by Chulabhorn Research Institute and Thailand Research Fund.)

IN VITRO SUSCEPTIBILITY OF TRICHOMONAS VAGINALIS TO AT-SPECIFIC MINOR GROOVE BINDING DRUGS. (NO. 670) Chavalitshewinkoon-Petmitr P , Ramdja M , Kajorndechakiat 1 2 3 4 S , Ralph RK , Denny WA , Wilairat P.

1 1 1

Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400; 2Cancer Research and Developmental Biology, School of Biological Sciences, 3Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; 4 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400. Trichomoniasis is one of the most common sexually transmitted diseases, with around 120 million world-wide suffering from Trichomonas vaginalis-induced vaginitis every year. Although trichomoniasis can be treated with metronidazole, the prevalence of metronidazole-resistant T. vaginalis seems to be increasing. Since the percentage of AT base pairs in T. vaginalis DNA (71%) is very much higher than in human cells, in this study a series of bisquaternary quinolinium salt compounds with high AT-binding specificity were tested for their antitrichomonal activities. Minimum inhibitory concentrations (MICs) were determined for these compounds against a local strain of T. vaginalis in culture. Among 14 bisquaternary quinolinium compounds tested, an N-ethyl derivative was the most effective drug against T. vaginalis, being nearly as potent (MIC = 0.16 µM) as metronidazole (MIC = 0.096 µM), and with low toxicity towards human cells. The nature of the substitution at the quinolinium quaternary centre appears to be important in terms of effectiveness of bisquaternary compounds against the parasite. In contrast, no clear relationships could be seen for substituents on the quinolinium ring; Me and Cl substituted analogues showed higher activity against trichomonads, whereas OMe, NHMe and NH 2 substituents decreased activity. (Published in J Antimicrob Chemother 2003; 52: 287-9. Supported by Mahidol University and Thailand Research Fund.)

IDENTIFICATION AND ANALYSIS OF GP116 AND GP64 STRUCTURAL GLYCOPROTEINS OF YELLOW HEAD NIDOVIRUS OF PENAEUS MONODON SHRIMP. (NO. 669) Jitrapakdee S , Unajak S , Sittidilokratna N , Hodgson RA , 3 3 2 1,2 Cowley JA , Walker PJ , Panyim S , Boonsaeng V.

1 1,2 1,2 1,2 3

CENTEX Shrimp, 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3CSIRO Livestock Industries, Long Pocket Laboratories, Indooroopilly, Queensland, Australia. Yellow head virus (YHV) is a major agent of disease in farmed penaeid shrimp. YHV virions purified from infected shrimp contain three major structural proteins of molecular mass 116 kDa (gp116), 64 kDa (gp64) and 20 kDa (p20). Two different staining methods indicated that the gp116 and gp64 proteins are glycosylated. Here we report the complete nucleotide sequence of ORF3, which encodes a polypeptide of 1666 amino acids with a calculated molecular mass of 185 713 Da (pI = 6.68). Hydropathy analysis of the

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INHIBITION OF PLASMODIUM FALCIPARUM PROLIFERATION IN VITRO BY ANTISENSE OLIGODEOXYNUCLEOTIDES AGAINST MALARIAL TOPOISOMERASE II. (NO. 671) Noonpakdee W , Pothikasikorn J , Nimitsantiwong W , Wilairat 1 P.

1 1 2 1

Faculty of Science

possible approaches for the design of new drugs to overcome antifolate resistance. (Published in Nature Struct Biol 2003; 10: 357-65. Supported by Thailand Tropical Diseases, WHO-TDR, EU, Wellcome Trust and MMV Programs.)

Department of Biochemistry, 2Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

INVOLVEMENT OF ERK1/2 IN INVASIVENESS AND METASTATIC DEVELOPMENT OF RAT PROSTATIC ADENOCARCINOMA. (NO. 673) Suthiphongchai T, Promyart P, Virochrut S, Tohtong R, Wilairat P. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Extracellular signal-regulated kinase (ERK) activation has been implicated in cell motility and invasion. In this study, we demonstrated that the steady-state levels of activated ERK1/2 correlated with the degree of invasiveness and metastatic potential of three Dunning cancer cell lines, originating from the same parental tumor. Inhibition of mitogen-activated protein kinase kinase 1 (MEK1), an upstream regulator of ERK1/2, with PD98059 resulted in a dosedependent reduction of invasiveness with different IC50 values in the three Dunning cell lines. These results suggest that ERK is, at least in part, responsible for regulating invasiveness and may underlie the differences in the metastatic ability of the cell lines. (Published in Oncology Res 2003; 13: 253-9. Supported by Mahidol University and Thailand Research Fund.)

The development of new effective antimalarial agents is urgently needed due to the ineffectiveness of current drug regimes on the most virulent human malaria parasite Plasmodium falciparum. Antisense (AS) oligodeoxynucleotides (ODNs) have shown promise as chemotherapeutic agents. Phosphorothioate AS ODNs against different regions of P. falciparum topoisomerase II gene were investigated. Chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was exposed to phosphorothioate AS ODNs for 48 h and growth was determined by flow cytometric assay or by microscopic assay. Exogenous delivery of phosphorothioate AS ODNs between 0.01 and 0.5 µM significantly inhibited parasite growth compared with sense sequence controls suggesting sequence specific inhibition. This inhibition was shown to occur during maturation stages, with optimal inhibition being detected after 36 h. These results should prove useful in future designs of novel antimalarial agents. (Published in Biochem Biophys Res Commun 2003; 302: 659-64. Supported by the Thailand Research Fund.)

INSIGHTS INTO ANTIFOLATE RESISTANCE FROM MALARIAL DHFR-TS STRUCTURES.

(NO. 672)

1 2 2

Yuvaniyama J , Chitnumsub P , Kamchonwongpaisan S , 2 1 3 Vanichtanankul J , Sirawaraporn W , Taylor P , Walkinshaw 3 2 MD , Yuthavong Y.

1

ISOLATION OF NISIN-PRODUCING LACTOCOCCUS LACTIS WNC 20 STRAIN FROM NHAM, A TRADITIONAL THAI FERMENTED SAUSAGE.

(NO. 674)

1 1 1

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2BIOTEC, National Science and Technology Development Agency, Pathumthani 12120, Thailand; 3Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, UK. Plasmodium falciparum dihydrofolate reductasethymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide

Noonpakdee W , Santivarangkna C , Jumriangrit P , Sonomoto 2 1 K , Panyim S.

1

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Laboratory of Microbial Science and Technology, Division of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan. A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus . Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121°C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the sub-

Mahidol University Annual Research Abstracts, Vol. 31

stitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented (Published in Int J Food Microbiol 2003; 81: 137-45. Supported by Thailand Research Fund, National Research Council of Thailand and Japan Society for the Promotion of Science.) MOLECULAR CLONING, EXPRESSION AND CHARACTERIZATION OF CDNA ENCODING CIS-PRENYLTRANSFERASES FROM HEVEA BRASILIENSIS. (NO. 675) Asawatreratanakul K , Zhang YW , Wititsuwannakul D , 4 1 4 Wititsuwannakul R , Takahashi S , Rattanapittayaporn A , 1 Koyama T.

1 1,2 1 3

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Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan; 2Department of Chemistry, Thaksin University, Songkla, Thailand; 3Department of Biochemistry, Mahidol University, Bangkok, Thailand; 4Department of Biochemistry, Prince of Songkla University, Hat-Yai, Thailand. Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1,4-polymerization of isoprene units has been identified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cisprenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 x 103 - 1 x 104 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber. (Published in Eur J Biochem 2003; 270: 4671-80. Supported by Ministry of Education, Science and Culture of Japan, and the Asahi-, Heiwa-Nakajima-, Sumitomo- and Goho-Life Science Foundations.) MOLECULAR DYNAMICS SIMULATION OF THE HUMAN APODIHYDROFOLATE REDUCTASE: AN INVESTIGATION OF AN UNSTABLE ENZYME

(NO. 676)

1 1 1 2

Structure and dynamics data of the Homo sapiens apodihydrofolate reductase (hDHFR) have been investigated by molecular dynamics (MD) simulation. The MD simulation for a total 300 ps was performed for the system containing the apo-hDHFR with explicit water molecules. The last 250 ps MD trajectory of the equilibrated system was generated and 25 sampling structures of the apo-hDHFR were analyzed. The statistical quantities of the simulation reveal significant differences between the apo-enzyme and the complex form. The differences include the mainchain mobility, the solvent accessibility, the secondary structures, the phenylalanine movement and the hydrogen bond may relate to the stability of the enzyme. The tertiary folding from the secondary structure motif reveals a less compact for the apo-enzyme structure with respect to the X-ray structure of the hDHFR-folate complex. In addition, intramolecular hydrogen bond involving the backbone protons dramatically decreases. Comparisons between the unbound and ligandbound enzyme obtained from the X-ray, NMR and MD data are discussed. (Published in Mol Simulation 2003; 29: 111-21. Supported by Thailand Research Fund Rachadapisek Sompoj Endownment and National Science and Technology Development Agency, Thailand.) NOVEL MUTATIONS IN A THAI PATIENT WITH METHYLMALONIC ACIDEMIA. (NO. 677) Champattanachai V , Ketudat Cairns JR , Shotelersuk V , 1 1 1 Keeratichamroen S , Sawangareetrakul P , Srisomsap C , 4 1,5 Kaewpaluek , Svasti J. Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand; 2Schools of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, Nakorn Ratchasima 30000, Thailand; 3Department of Pediatrics, 4 Department of Forensic Medicine Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; 5Department of Biochemistry and Center for Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. A Thai patient with methylmalonic acidemia (MMA) and no methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) activity in leukocytes in the presence of deoxyadenosyl cobalamin (mut0) was found to be heterozygous for two novel mutations: 1048delT and 1706_1707delGGinsTA (G544X), inherited from her mother and father, respectively. The proband was also heterozygous for the polymorphism, A499T, which did not affect the activity of recombinant MCM. (Published in Mol Genet Metab 2003; 79: 300-2. Supported by Chulabhorn Research Institute and the Thailand Research Fund.)

1 1 1,2 3

PHOSPHATIDYLSERINE EXPOSURE IN FAS TYPE I CELLS IS MITOCHONDRIA-DEPENDENT

(NO. 678)

Uthaisang W , Nutt LK , Orrenius S , Fadeel B.

1

1

2

2

2

Sompornpisut P , Wijitkosoom A , Parasuk V , Sirawaraporn S.

1

Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm 171 77, Sweden.

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Previous studies have demonstrated that Fas-triggered activation of effector caspases and subsequent nuclear apoptosis either is mitochondria-independent (type I cells) or relies on mitochondrial amplification of the initial stimulus (type II cells). We show herein that Bcl-2 overexpression in a prototypic type I cell line (SKW6.4) promotes mitochondrial generation of ATP and blocks Fas-triggered plasma membrane externalization of phosphatidylserine (PS). Moreover, overexpression of Bcl-2 attenuates macrophage engulfment of Fas-triggered cells. Fas-mediated DNA fragmentation, on the other hand, remains unaffected in SKW6.4-bcl-2 cells. These studies thus demonstrate that PS externalization and clearance of cell corpses are mitochondria-dependent events, and show that these events can be dissociated from other features of the apoptotic program, in Fas type I cells. (Published in FEBS Lett 2003; 545: 110-4. Supported by Ministry Staff Development Project, the Ministry of University Affairs, Thailand, Swedish Research Council, Swedish Society for Medical Research, and the Jeansson Foundation.)

Faculty of Science

were highly active against all of these, with the meta-bromo again being the strongest, having an IC 50 of 37 nM against V1/S, compared to > 5000 nM for pyrimethamine itself and 1.1 nM for WR99210. (Published in Org Biomol Chem 2003; 1: 960-4. Supported by WHOTDR Program and the Wellcome Trust.)

ROLE OF THE STATIONARY GROWTH PHASE SIGMA FACTOR RPOS OF BURKHOLDERIA PSEUDOMALLEI IN RESPONSE TO PHYSIOLOGICAL STRESS CONDITIONS. (NO. 680) Subsin B , Thomas MS , Katzenmeier G , Shaw JG , 1 4 Tungpradabkul S , Kunakorn M. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Division of Genomic Medicine, University of Sheffield Medical School, Sheffield, UK; 3Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Thailand; 4Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand. The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed. The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress. By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase. (Published in J Bacteriol 2003; 185: 7008-7014. Supported by the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, and Thailand Research Fund Royal Golden Jubilee Ph.D. Program.)

1 1 2 3 2

PYRIMETHAMINE ANALOGS AS STRONG INHIBITORS OF DOUBLE AND QUADRUPLE MUTANTS OF DIHYDROFOLATE REDUCTASE IN HUMAN MALARIA PARASITES. (NO. 679) Sardarian A , 1Douglas KT , Read M , Sims PF , Hyde JE , 3 4 4 Chitnumsub P , Sirawaraporn R , Sirawaraporn W.

1 1 1 2 2 2

School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK; 2Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, UK; 3National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10400, Thailand; 4 Department of Biochemistry, Faculty of sxcience, Mahidol University, Bangkok 10400, Thailand. Pyrimethamine acts against malarial parasites by selectively inhibiting their dihydrofolate reductase-thymidylate synthase. Resistance to pyrimethamine in Plasmodium falciparum is due to point mutations in the DHFR domain, initially at residue 108 (S108N), with additional mutations imparting much greater resistance. Our previous work, the development of a simple rational drug design strategy to overcome such resistance, used suitable meta-substituents in the pyrimethamine framework to avoid the unfavorable steric clash with mutant side chains at position 108. Interestingly, the metachloro analog of pyrimethamine not only overcame the resistance due to S108N, but also that contributed by the more remote mutation, C59R. The present work improves on this by means of other meta-substituents. Against wild type DHFR, double mutant types A16V + S108T and C59R + S108T, and the highly pyrimethamine/ cycloguanil-resistant quadruple-mutant form N51I + C59R + S108N + I164L, pyrimethamine itself gave Ki values of 1.5, 2.4, 72.3 and 859 nM, respectively. The meta-substituted analogs, especially the meta-bromo analog, were much more powerful inhibitors of these DHFRs, including the quadruple-mutant form (meta-bromo analog, Ki 5.1 nM). For comparison, the dihydropyrazine antifolate, WR99210, gave Ki values of 0.9, 3.2, 0.8 and 0.9 nM, respectively. Ki values were also measured against recombinant human DHFR, as were their activities against the growth of Plasmodium falciparum cultures bearing the double mutations (FCB and K1 strains) and quadruple mutation (V1/S) and the wild type (3D7). The meta-analogs

RUBBER BIOSYNTHESIS BY A HEVEA LATEX BOTTOM-FRACTION MEMBRANE. (NO. 681) Wititsuwannakul D , Rattanapittayaporn A , Wititsuwannakul 2 W. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai 90112, Thailand. Washed bottom-fractions particles (WBPs), that is, intact membrane-bound vesicles, prepared from the bottom fraction of centrifuged fresh latex, were shown to be active in in vitro rubber biosynthesis (RB) assays. The RB activity catalyzed by WBP enzymes was confirmed by enzyme parameter criteria. A washed bottomfraction particle membrane (WBM), prepared from WBPs, was even more active in in vitro RB activity. Mg2+ was a cofactor required for RB enzymes, and the cation chelators EDTA and EGTA inhibited the RB process. The temperature had a strong effect on RB stimulation during the heat pretreatment of the WBM before RB assays. A detergent also proved to a strong RB stimulator. The anion detergent SDS, above the critical micelle concentration, was a strong stimulator of WBM activity. This was not observed with the nonionic detergents Triton X-100 and Tween 20. Various temperature ranges used for WBM preincubation showed a sharp rise in RB activity above 70°C. The temperature required for a sharp rise in RB was

1 1 1

Mahidol University Annual Research Abstracts, Vol. 31

lowered substantially in the presence of SDS. It was down to 40°C with the combined preincubation but remained constant with an even higher RB activity. Greater rubber formation at a lower temperature was observed in the presence of SDS. A synergistic effect for RB stimulation appeared during the heat pretreatment of WBM in the presence of SDS. (Published in J Applied Polymer Sci 2003; 87: 90-6. Supported by National Science and Technology Development Agency, Thailand, and Thailand Research Fund.)

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lipophilic chalcones, in particular those with 4'-hydroxyl-substituted B rings and hetero/polyaromatic A rings. In contrast, chalcones with good antimalarial activity have alkoxylated B rings and electrondeficient A rings. Visualization of the steric and electrostatic fields generated from comparative molecular field analysis (CoMFA) indicate that the ring A of chalcones make a more significant contribution to antileishmanial activity while both rings A and B are important for antimalarial activity. Despite different requirements, two alkoxylated chalcones (8, 19) were identified which combined good antimalarial and antileishmanial activities. (Published in Bioorg Med Chem 2003; 11: 2729-38. Supported by the Thailand Research Fund, National University of Singapore and WHO-TDR Program.)

STATUS OF RED CELL MEMBRANE PROTEIN PHOSPHORYLATION IN THALASSEMIA (NO. 682) Rutaiwan Tohtong, Pornpimol Metheenukul and Prapon Wilairat Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. The steady-state levels of -spectrin phosphorylation in HbH (-thalassemia 1/-thalassemia 2), HbH/HbConstant Spring (thalassemia 1/HbCS, hereafter called HbH/HbCS) and nonsplenectomized -thalassemia/HbE (hereafter called -thal/HbE) red cells were quantitated using Western hybridization. Phosphorylation of -spectrin serine and threonine residues from thalassemic samples was not significantly different from normal control. However, tyrosine phosphorylation was higher than normal control in HbH (p<0.01), HbH/HbCS (p<0.05) and -thal/HbE (p<0.05) samples. Tyrosine phosphorylation of -spectrin was observed only in the presence of vanadate, a phenomenon not hitherto reported. As tyrosine kinase activity has been linked to oxidative stress, loss of membrane lipid asymmetry and procoagulant activity of the red cell membrane, the observed increase in -spectrin tyrosine phosphorylation of the thalassemic red cells is likely, at least in part, to account for these parameters. (Published in ScienceAsia 2002; 28: 313-7. Supported by Mahidol University and Thailand Research Fund.)

SYNTHESIS OF SOLUTION-PHASE COMBINATORIAL LIBRARY OF 4,6-DIAMINO-1,2-DIHYDRO-1,3,5-TRIAZINE AND IDENTIFICATION OF NEW LEADS AGAINST A16V+S108T MUTANT DIHYDROFOLATE REDUCTASE OF PLASMODIUM FALCIPARUM. (NO. 684) Vilaivan T , Saesaengseerung N , Jarprung D , Kamchonwong2 3 2 paisan S , Sirawaraporn W , Yuthavong Y.

1 1 1 1

Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10400, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. An efficient method to synthesize solution-phase combinatorial library of 1-aryl-4,6-diamino-1,2-dihydro-1,3,5-triazine was developed. The strategy involved an acid-catalyzed cyclocondensation between arylbiguanide hydrochlorides and carbonyl compounds in the presence of triethyl orthoacetate as water scavenger. A 96-membered combinatorial library was constructed from 6 aryl biguanides and 16 carbonyl compounds. Screening of the library by iterative deconvolution method revealed two candidate leads which are equally active against wild-type Plasmodium falciparum dihydrofolate reductase, but are about 100-fold more effective against the A16V+S108T mutant enzyme as compared to cycloguanil. (Published in Bioorg Med Chem 2003; 11: 217-24. Supported by Graduate School and Department of Chemistry, Chulalongkom University, European Union, Thailand Research Fund and WHO-TDR Program.)

STRUCTURE-ACTIVITY RELATIONSHIPS OF ANTILEISHMANIAL AND ANTIMALARIAL CHALCONES. (NO. 683) Liu M , Wilairat P , Croft SL , Tan AL , Go ML.

1 1 2 3 4 1

Department of Pharmacy, National University of Singapore, Singapore 117543; 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK; 4Institute of Cell and Molecular Biology, National University of Singapore, Singapore 117609. A series of oxygenated chalcones which have been evaluated earlier for antimalarial activity (Plasmodium falciparum K1) were tested for antileishmanial activity against Leishmania donovani amastigotes. A comparison of structure-activity relationships reveal that different physicochemical and structural requirements exist for these two activities. Antileishmanial activity is associated with less

THE BIOTIN ENZYME FAMILY: CONSERVED STRUCTURAL MOTIFS AND DOMAIN REARRANGEMENTS. (NO. 685) Jitrapakdee S , Wallace JC.

1 1 2

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Department of Molecular Biosciences, University of Adelaide, Adelaide, South Australia 5005 Australia.

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The biotin carboxylase family is comprised of a group of enzymes that utilize a covalently bound prosthetic group, biotin, as a cofactor. These enzymes, which include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonylCoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase, are found in diverse biosynthetic pathways in both pro-karyotes and eukaryotes. The reactions catalyzed by most members of this group of enzymes share two common features: (1) carboxylation of biotin, apparently via the formation of a carboxyphosphate intermediate, followed by (2) transcarboxylation of CO2 from biotin to specific acceptor molecules to yield different products. Structural determinations by NMR and X-ray crystallography, complemented by mutagenesis studies, have identified some motifs that are structurally or catalytically important. Analysis of the amino acid sequences of a number of biotin carboxylases not only shows remarkable similarities within certain domains but also that there appears to have been domain rearrangements between groups of carboxylases. Acyl-coenzyme A derivatives, which bind either as substrates or as allosteric regulators of the biotin carboxylases, do not appear to share any of the CoA binding motifs that have been identified in other CoA-SH/acyl-CoA binding proteins. Further comparisons of biotin-dependent carboxylases with other groups of enzymes in the protein data bank reveal that this family of biotin enzymes has strong similarities in specific domains to a number of ATP-utilizing enzymes and to the lipoyl-containing enzymes. These structural homologies are so extensive as to be highly suggestive of evolutionary relationships between biotin carboxylases and these other enzymes. (Published in Curr Protein Peptide Sci 2003; 4: 217-29.)

Faculty of Science

MOLECULAR EVOLUTION OF TEPHRITID FRUIT FLIES IN THE GENUS BACTROCERA BASED ON THE CYTOCHROME OXIDASE I GENE1 (NO. 687) W., Jamnongluk1, V., Baimai 1,2 and P., Kittayapong 1,2 *

1

Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University *Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] Key words : Bactrocera spp., cytochrome oxidase I gene, molecular evolution Fruit flies of the genus Bactrocera (Diptera: Tephritidae) are one of the major economically important insects in Asia and Australia. Little attention has been given to analyses of molecular phylogenetic relationships among Bactrocera subgenera. By using mitochondrial cytochrome oxidase I gene (COI) sequences, the phylogenetic relationships among four subgenera, Asiadacus, Bactrocera, Hemigymnodacus, and Zeugodacus, were investigated. Nucleotide diversity within subgenera ranged from 11.7 to 12.4%, and the net divergence among subgenera ranged from 11.2 to 15.7%. Phylogenetic trees calculated from both maximum parsimony and neighborjoining phylogenetic analysis methods were highly congruent in terms of tree topologies. Phylogenetic analysis of mitochondrial COI sequences suggests that tephritid fruit fly species, which attack cucurbit plants, that is, Asiadacus, Hemigymnodacus and Zeugodacus, were more closely related to each other than to fruit fly species of the subgenus Bactrocera, which attack plants of numerous families. Our data supports previous classification of Bactrocera based on morphological characters. However, the phylogetic tree showed the polyphyletic of fruit flies in subgenus Zeugodacus. Possible causes of speciation among fruit flies species in this genus were also discussed. (Genetica, (2003), 119: 19-25. Supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training (BRT 139026) and the Thailand Research Fund (RGJ/PHD0095/2541, RTA/01/ 2541, and BGJ/16/2543)

TRANSGLUCOSYLATION OF TERTIARY ALCOHOLS USING CASSAVA BETAGLUCOSIDASE. Svasti J, Phongsak T, Sarnthima R.

(NO. 686)

Center for Excellence in Protein Structure and Function, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from pnitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis. (Published in Biochem Biophys Res Commun 2003; 305: 470-5. Supported by Thailand Research Fund and Ministry of University Affairs, Thailand.)

PRODUCTION AND CHARACTERIZATION OF A MONOCLONAL ANTIBODY AGAINST 28.5 kDa TEGUMENT ANTIGEN OF FASCIOLA GIGANTICA

(NO. 688)

Kulathida Chaithirayanon1, Chaitip Wanichanon1, Suksiri Vichasri-Grams 2, Pissanee Ardseungneon 2, Rudi Grams c, Vithoon Viyanant 2,3, Edward Suchart Upatham 2, Prasert Sobhon1

1

Department of Anatomy; Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; 2Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; 3Faculty of Allied Health Science, Thammasat University, Pathumthani 12121, Thailand. Key words : Fasciola gigantica; Tegument; 28.5 kDa antigen; Monoclonal antibody A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hy-

Mahidol University Annual Research Abstracts, Vol. 31

bridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F.gigantica. This MoAb was found to be of the isotype IgG 1, k-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, wholebody extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development. © 2002 Elsevier Science B.V. All rights reserved. (Acta Tropica. (2002), 84 1-8)

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the citrate synthase gene (gltA), and the 190-kDa outer membrane protein gene (ompA). Results indicated that the three Thai rickettsiae detected in this study represent new rickettsial genotypes and form a separate cluster among the spotted fever group rickettsiae. (Journal of Medical Entomology, (2003), 40: 230-237. Supported by Institut de Recherche pour le Developpement (IRD), France and the TRF/BIOTEC Special Program for Biodiversity Research and Training (BRT 139026)

THE INDOCHINESE-SUNDAIC FAUNAL TRANSITION AT THE ISTHMUS OF KRA : AN ANALYSIS OF RESIDENT FOREST BIRD SPECIES DISTRIBUTIONS (NO. 690) Jennifer B. Hughes1+, Philip D. Round2 and David S. Woodruff1*

1

Ecology, Behavior, Evolution Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 920930116, USA, 2Department of Biology, Mahidol University, Bangkok, Thailand. Key words : Grographical range limits, biogeographical boundaries, phytogeography, Malaysia, Thailand Aim : To establish the geographical position of the biogeographical transition between Indochinese and Sundaic faunas using distributional data for the best-documented taxon, the birds. Methods : Distributional data of 544 resident forest and forest edge bird species of Thailand and the Thai-Malay peninsula were examined at 45 sites spanning 15° of latitude from northernmost Thailand to the southern peninsular Malaysia. Sites were grouped into 23 degree or half-degree latitudinal zones and avifaunal similarity coefficients were calculated between each zone. Results : A Mantel test revealed a significant transition between northern Indochinese and southern Sundaic (Indomalay) avifauna assemblages just north of the Isthmus of Kra (10°30¢ N). Northern and southern range limits of 152 species (> 269 species and subspecies combined) lie between 11° and 13° N. Main conclusions : This transition between zoogeographical subregions is not coincident with the widely recognized transition between floristic provinces which is traditionally placed 400-500 km further south at the Kangar-Pattani line, but is associated with a change from wet seasonal evergreen dipterocarp rain forest to mixed moist deciduous forest north of the Isthmus of Kra in the northern Thai-Malay peninsula. Climatological and ecological factors associated with the distribution of forest types today are reviewed and it is hypothesized that the avian transition tracks the northern phytogeographical boundary. Palaeogeographical factors, including hypothetical Neogene seaways, which may account for the historical development of both phytogeographical and avifaunal transitions are also described. (Journal of Biogeography.2003 (30): 569-580)

MOLECULAR EVIDENCE FOR NOVEL TICKASSOCIATED SPOTTED FEVER GROUP RICKETTSIAE FROM THAILAND (NO. 689) S., Hirunkanokpun1, P., Kittayapong1,2 *, J. P., Cornet3 and J. P., Gonzalez3

1

Center for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University; 3RCEVD-CVD, Institute of Sciences and Technology for Development, Mahidol University. *Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] mahidol.ac.th Key words : rickettsiae, spotted fever group, tick Ticks are of considerable medical and veterinary importance because they directly harm the host through their feeding action and indirectly through vectoring many bacterial pathogens. Despite many ticks being known from Thailand, very little is known about the bacteria they may harbor. We report here the results of a survey of tick-associated bacteria in Thailand. A total of 334 individuals representing 14 species of ticks in five genera were collected from 10 locations in Thailand and were examined for the human pathogens, Borrelia, Francisella, Rickettsia, and the common arthropod endosymbionts, Wolbachia, by polymerase chain reaction (PCR) assay using specific primers. Rickettsial DNA was detected in 30% (9/30) of Amblyomma testudinarium (Koch, 1844) collected from Khao Yai National Park, Nakhon Nayok Province and 16.84% (16/95) of Haemaphysalis ornithophila (Hoogstraal and Kohls, 1959) collected from Khao Yai National Park, Nakhon Nayok Province and Khao Ang Rue Nai Wildlife Sanctuary, Chachoengsao Province. Rickettsial DNA was not detected in any of other tick species and no DNA of Borrelia, Francisella, or Wolbachia was detected in any of 14 tick species. Phylogenetic relationships among the rickettsiae detected in this study and those of other rickettsiae were inferred from comparison of sequences of the 17-kDa antigen gene,

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MOLECULAR PHYLOGENY OF WOLBACHIA ENDOSYMBIONTS IN SOUTHEAST ASIAN MOSQUITOES (DIPTERA: CULICIDAE) BASED ON WSP GENE SEQUENCES (NO. 691) T., Ruang-areerate 1, P., Kittayapong 1,2 *, V., Baimai 1,2 and S.L., O'Neill3

1

Faculty of Science

BIOCHEMICAL AND HISTOPATHOLOGICAL EFFECTS OF GLYPHOSATE HERBICIDE ON NILE TILAPIA (OREOCHROMIS NILOTICUS)

(NO. 693)

W. Jiraungkoorskul 1, E.S. Upatham2, M. Kruatrachue 1, S. Sahaphong3, S. Vichasri-Grams1, P. Pokethitiyook1 Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Faculty of Science, Burapha University, Chonburi 20131, Thailand; 3Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Key words : Oreochromis niloticus; Roundup; histopathology; aminotransferase; alkaline phosphatase. In Oreochromis niloticus that had been exposed for 3 months to sublethal concentrations (5 and 15 ppm) of the commercial glyphosate herbicide (C3H8NO5P) Roundup, the organs exhibited varying degrees of histopathological change. In the gills filament cell proliferation, lamellar cell hyperplasia, lamellar fusion, epithelial lifting, and aneurysm were observed. In the liver there were vacuolation of hepatocytes and nuclear pyknosis. Kidney lesions consisted of dilation of Bowman's space and accumulation of hyaline droplets in the tubular epithelial cells. The structural damages could be correlated to the significant increase (p < 0.05) in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities in the second and third months of exposure. The results indicated that long-term exposure to glyphosate at sublethal concentrations had adverse effects on the histopathological and biochemical alterations of the fish. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18 : 260-267, 2003. (Wiley Periodicals, Inc.Environ Toxicol (2003) 18: 260-267.)

1

Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University; 3Department of Zoology and Entomology, School of Life Sciences, The University of Queensland, Brisbane 4072, Australia. Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] Key words : mosquitoes, molecular phylogeny, Wolbachia Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insectassociated Wolbachia form a monophyletic clade in the aProteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups. (Journal of Medical Entomology, (2003), 40: 1-5. Supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training (BRT 139026) and the Thailand Research Fund (RTA/01/2541)

WOLBACHIA INFECTION COMPLEXITY AMONG INSECTS IN THE TROPICAL RICE-FIELD COMMUNITY (NO. 694) MOLECULAR PHYLOGENY OF TEPHRITID FRUIT FLIES IN THE BACTROCERA TAU COMPLEX USING THE MITOCHONDRIAL COI SEQUENCES (NO. 692) W., Jamnongluk1, V., Baimai 1,2 and P., Kittayapong 1,2 *

1

P. Kittayapong 1,2 *, W., Jamnongluk1,2, A. Thipaksorn 1,2, J. R. Milne2 and C. Sindhusake3

1

Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University *Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] We compared sequences of the mitochondrial cytochrome oxidase I gene of eight species of the Bactrocera tau complex using Bactrocera dorsalis, Bactrocera pyrifoliae (Genome, (2003), 46: 112-118. Supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training (BRT 139026) and the Thailand Research Fund (RGJ/PHD/0095/2541, RTA/01/ 2541, and BGJ/16/2543)

Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University; 3Entomology and Zoology Division, Ministry of Agriculture and Cooperatives, Bangkok, Thailand. Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] Key words : horizontal transfer, rice pests, Wolbachia Wolbachia are a group of intracellular bacteria that cause reproductive alterations in their arthropod hosts. Widely discordant host and Wolbachia phylogenies indicate that horizontal transmission of these bacteria among species sometimes occurs. A likely means of horizontal transfer is through the feeding relations of organisms within communities. Feeding interactions among insects within the rice-field insect community have been well documented in the past. Here, we present the results of a polymerase chain reaction-based survey and phylogenetic analysis of Wolbachia strains in

Mahidol University Annual Research Abstracts, Vol. 31

the rice-field insect community of Thailand. Our field survey indicated that 49 of 209 (23.4%) rice-field insect species were infected with Wolbachia. Of the 49 infected species, 27 were members of two feeding complexes: (i) a group of 13 hoppers preyed on by 2 mirid species and parasitized by a fly species, and (ii) 2 lepidopteran pests parasitized by 9 wasp species. Wolbachia strains found in three hoppers, Recilia dorsalis, Nephotettix malayanus and Nisia nervosa, the two mirid predators, Cyrtorhinus lividipennis and Tytthus chinensis, and the fly parasitoid, Tomosvaryella subvirescens, were all in the same Wolbachia clade. In the second complex, the two lepidopteran pests, Cnaphalocrocis medinalis and Scirpophaga incertulas, were both infected with Wolbachia from the same clade, as was the parasitoid Tropobracon schoenobii . However, none of the other infected parasitoid species in this feeding complex was infected by Wolbachia from this clade. Mean (±SD) genetic distance of Wolbachia wsp sequences among interacting species pairs of the hopper feeding complex (0.118±0.091 nucleotide sequence differences), but not for the other two complexes, was significantly smaller than that between noninteracting species pairs (0.162±0.079 nucleotide sequence differences). Our results suggest that some feeding complexes, such as the hopper complex described here, could be an important means by which Wolbachia spreads among species within arthropod communities. (Molecular Ecology, (2003), 12: 1049-1060. Supported by the TRF/ BIOTEC Special Program for Biodiversity Research and Training (BRT 139026) and the Thailand Research Fund (BGJ/16/2543).

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HISTOPATHOLOGICAL CHANGES IN THE GASTROINTESTINAL TRACT OF FISH, PUNTIUS GONIONOTUS, FED ON DIETARY CADMIUM

(NO. 696)

M. Kruatrachue1, N. Rangsayatorn 2, P. Pokethitiyook2, E.S. Upatham3, S. Singhakaew 2 Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, and Mahidol University International College, Nakhonpathom 73170, Thailand; 2Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3 Faculty of Science, Burapha University, Chonburi 20130, Thailand. Cadmium is a well-known heavy metal pollutant. The important food sources of fish, phytoplanktons or invertebrates accumulate cadmium through the food chain. Fish also accumulate heavy metals in their tissues by absorption along the gill surface and gut tract wall to higher concentration levels than in their environment (Handy 1993). The adverse effects of cadmium have been studied in many fish species and in numerous target organs such as the liver, kidney and gills, (Shukla and Pandey 1988). But there is little on what is possibly the main target organ of toxic dietary cadmium, namely the gastrointestinal tract (GI). Maage and Julshamn (1993) hypothesized that the intestine is the main organ of regulation in the uptake of dietary metal in Atlantic salmon. The strong regulatory capacity of the intestine in relation to dietary metals makes this organ valuable in assessing toxic responses to dietary cadmium. Puntius gonionotus are commonly found throughout Thailand, in rivers, streams, floodplains, and reservoirs. They are also common market fish and they feed on both plant and animal matter. They have been developed as a laboratory test mode in Thailand for many years. Therefore, the aim of the present work was to study the histopathological changes in the intestine of the common silver barb, P. gonionotus, fed cadmium-contaminated cyanobacteria, Spirulina platensis. (Blackwell Publishing Ltd, molecular Ecology, (2003) 12: 10491060.)

1

ENHANCEMENT OF THE EFFICACY OF A COMBINATION OF MESOCYCLOPS ASPERICORNIS AND BACILLUS THURINGIENSIS VAR. ISRAELENSIS BY COMMUNITY-BASED PRODUCTS IN CONTROLLING AEDES AEGYPTI LARVAE IN THAILAND (NO. 695) P., Kosiyachinda 1,2, A., Bhumiratana3 and P., Kittayapong 1,2 * Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University;3Department of Biotechnology, Faculty of Science, Mahidol University. *Author for correspondence: Dr. P. Kittayapong. E-mail : [email protected] Key words: Bacillus thuringiensis var. israelensis (Bti), Aedes aegypti, Mcsocyclops asperidornis Prolonged efficacy of a combination of bacteria (Bacillus thuringiensis var. israelensis [Bti] and copepods (Mesocyclops aspericornis) in controlling immature forms of Aedes aegypti in peridomestic water containers was achieved by adding various products from local villages as supplementary food for copepods. In all experiments, 100 first-instar larvae were added into the breeding containers every day for eight weeks. Combinations of biological control agents and each local supplementary food were applied once at the beginning of the experiment. At the end of the experiment, the average number of mosquito larvae in containers with a combination of copepods and Bti with one gram of rice grain had decreased to only 0.5% of that with no control agent. In comparison, the average numbers of mosquito larvae in containers with Bti only, or copepods only, were approximately 10% and 33% of those in containers with no control agents, respectively. In addition, the number of copepods in containers with mosquito larvae and supplementary food was at least three times higher than those with mosquito larvae alone.

1

RECORDS OF RICE FIELD ODONATA FROM THAILAND (NO. 697) A., Thipaksorn 1,4 , P., Kittayapong 1,2 * , J. R., Milne 2 , V., Thirakhupt2, C., Sindhusake3 and S., Poonchaisri 3 Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University; 3Corp Protection Research and Development office, Department of Agriculture, Ministry of Agriculture and Cooperatives, Bangkok; 4Biology section, Mahidol Wittayanusorn School, Nakhonpathom. *Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] Key words : odonata, rice field, Thailand Records (1998-2000) of 29 spp., from 47 localities in 36 provinces are listed, and the abundance of some spp. is discussed. (Notulae Odonatologicae. (2003), 6: 13-24. Supported by the TRF/ BIOTEC Special Program for Biodiversity Research and Training (BRT139026)

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THE COINCIDENCE OF THRIPS AND DISPERSED POLLEN IN PNRSV-INFECTED STONEFRUIT ORCHARDS ­ A PRECONDITION FOR THRIPSMEDIATED TRANSMISSION VIA INFECTED POLLEN (NO. 698) J R Milne1* and G H Walter2

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Faculty of Science

Key words : Thailand, Birds, Mammals, Fragmentation, Hunting; Protected areas Montane evergreen forests in northern Thailand are high in biodiversity and becoming increasingly fragmented. We studied fragmentation and wildlife response in two contiguous wildlife sanctuaries. Om Koi still maintained relatively large patches (>400 ha) with some connectivity while Mae Tuen was comprised mainly of small and isolated patches (<100 ha). Mae Tuen lost 2640 ha of montane evergreen forest between 1954 and 1996 compared to a loss of 888 ha in Om Koi. We compared the wildlife between four forest patches in Mae Tuen, with four in Om Koi finding nine mammals and 89 birds in Mae Tuen and mammals and 119 birds in Om Koi. Om Koi still supports populations of large mammals and frugivorous birds extipated in Mae Tuen. The results document the high rate of fragmentation in protected areas that often interact synergistically with other pressures to reduce biodiversity. Crown Copyright © 2002 Published by Elsevier Science Ltd. All rights reserved. (Biological Conservation., (2003), 107: 155-164.)

Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; 2Department of Zoology and Entomology, The University of Queensland, Queensland 4072, Australia. Key words : PNRSV, stonefruit, nectarine, peach, plum, Thysanoptera, flowers, Thrips imaginis, Thrips australis, Thrips tabaci Recent laboratory studies have demonstrated that Prunus necrotic ringspot virus (PNRSV) (family Bromoviridae) can be readily transmitted when thrips and virus-bearing pollen are placed together on to test plants. For this transmission mechanism to result in stonefruit tree infection in the field, PNRSV-bearing pollen must be deposited onto surfaces of stonefruit trees on which thrips also occur. In a previous paper, we demonstrated that almost all pollen in a PNRSV-infected Japanese plum orchard in southeastern Queensland was deposited onto flowers, whereas few grains occurred on leaves and none on stems. Here, we results of our investigation of thrips species composition, distribution and abundance on stonefruit trees in the same study area as our previous pollen deposition study. We collected a total of 2010 adult thrips from 13 orchard during the 1989, 1991 and 1992 flowering seasons of which all but 14 were in the suborder Terebrantia. Most (97.4%) terebrantian thrips were of three species, Thrips imaginis, Thrips australis and Thrips tabaci. Thrips tabaci as well as species mixtures that included T. imaginis, T. australis and T. tabaci have been shown to transmit PNRSV via infected pollen in laboratory tests. Adult thrips were frequently collected from flowers but rarely from leaves and never from stems. Large and significant differences in numbers of T. imaginis, T. australis and T. tabaci adults in flowers occurred among orchards and between seasons. No factor was conclusively related to thrips numbers but flowers of late-flowering stonefruit varieties tended to hold more thrips than those of early-flowering varieties. Our results indicate that the common thrips species present on stonfruit trees in the Granite Belt are also ones previously shown to transmit PNRSV via infected pollen in the laboratory and that these thrips are concentrated in stonefruit flowers where most stonefruit pollen is deposited. These result contribute to mounting circumstantial evidence that stonefruit flowers may be inoculated with PNRSV via an interaction of thrips with virus-bearing pollen and that this transmission mechanism may be an important cause of new tree infections in the field.

RIBOSOMAL RNA CHARACTERIZATION OF NON-TRANSCRIBED SPACER AND TWO INTERNAL TRANSCRIBED SPACERS WITH 5.8S RIBOSOMAL RNA OF PERKINSUS SP. FOUND IN UNDULATED SURE CLAMS (PAPHIA UNDULATA) FROM THAILAND (NO. 700) Supannee Leethochavalit1*, E. Suchart Upatham2, Kwang-Sik Choi3, Pichan Sawangwong 4, Kashane Chalermwat 4, Maleeya Kruatrachue5 Institute of Marine Science, Burapha University, Bangsaen, Chonburi 20131, Thailand; 2Faculty of Science, Department of Biology, Burapha University; Bangsaen, Chonburi, 20131, Thailand; 3Faculty of Applied Marine Science, College of Ocean Science, Cheju National University, 1 Ara 1-Dong Jeju City JejuDo 690-76-56, S. Korea; 4Faculty of Science, Department of Aquatic Science, Burapha University, Bangsaen, Chonburi 20131, Thailand; 5Faculty of Science, Department of Biology, Mahidol University, Rama 6 road, Payathai, Bangkok 10400, Thailand. Key words : Perkinsus sp., Paphia undulata, nucleotide sequence, non-transcribed spacer, internal transcribed spacer 1, internal transcribed spacer 2 The genetic divergence of Perkinsus sp.found in the undulated surf clam (Paphia undulata) from the Gulf of Thailand and other known Perkinsus species was examined using the non-transcribed spacer and two internal transcribed spacers with 5.8S rRNA gene. The sequences of non-transcribed spacer (NTS) and internal transcribed spacer region (ITS) that includes the 5.8S rRNA gene flanked by ITS1 and ITS 2 (ITS1-5.8S-ITS2) were cloned and sequenced. The sequences were compared with those of Perkinsus olseni form Australia, P. atlanticus from Korea. P. marinus and P. andrewsi from the United States and P. qugwadi from Canada. The lengths of the obtained nucleotide sequences of NTS, ITS-1 5.8S rRNA and ITS-2 were 1,167, 183, 159 and 371 bp, respectively. The nucleotide sequences of NTS and ITS-5.8S rRNA of Thai Perkinsus and P. olseni showed 98.69% and 99.85% identity, re1

FRAGMENTATION AND WILDLIFE IN MONTANE EVERGREEN FORESTS, NORTHERN THAILAND

(NO. 699)

Anak Pattanavibool, Philip Dearden Department of Geography, University of Victoria, Victoria, BC. Canada V8W 3P5

Mahidol University Annual Research Abstracts, Vol. 31

spectively. When compared with P. atlanticus identities were 96.27 and 99.71%, in P. marinus 75.38 and 94.88% and in P. andrewsi 46.55 and 86.23%. The nucleotide sequences of ITS-5.8S rRNA between Thai Perkinsus and P. chesapeaki showed an identity of 87.05%. This is the first report of the occurrence of Perkinsus sp in the Gulf of Thailand. (Journal of shellfish Research., (2003),Vol. 22:431-434.)

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Wolbachia are endosymbiotic bacteria that cause reproductive alterations in numerous arthropod species. Using a PCRbased method, we found that, out of 33 odonate species, four species were infected with Wolbachia. This finding represents the first record of Wolbachia infection in tropical odonates. Identical wsp gene sequences were found in Wolbachia­infected common odonate species, Agriocnemis f. femina, collected from different locations in Thailand. The infection frequencies in several natural populations suggest that replacement of uninfected populations by Wolbachia­ infected ones has recently occurred in this damselfly species.

DENGUE AND ITS VECTORS IN THAILAND: CALCULATED TRANSMISSION RISK FROM TOTAL PUPAL COUNTS OF AEDES AEGYPTI AND ASSOCIATION OF WING-LENGTH MEASUREMENTS WITH ASPECTS OF THE LARVAL HABITAT (NO. 701) D.Strickman1 and P. Kittayapong2,3 *

1

HISTOPATHOLOGICAL ALTERATIONS OF WHITE SEABASS, LATES CALCARIFER, IN ACUTE AND SUBCHRONIC CADMIUM EXPOSURE (NO. 703) S. Thophon1, M. Kruatrachue1, E.S. Upathama, P. Pokethitiyook1, S. Sahaphong2, S. Jaritkhuan3

1

Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, MD, U.S.A.; 2Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 3Department of Biology, Faculty of Science, Mahidol University. *Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] Key words : Aedes aegypti, dengue vector, transmission risk Working in a village dengue focus in Chachoengsao Province, Thailand, aedine mosquito larvae and pupae were counted in all containers of 10 houses per month. The wings of female Aedes aegypti (L.) emerging from pupae were measured. Number of pupae and size of emerging females increased in containers with qualities that favored availability of larval food sources (e.g., uncovered containers). The small size of most mosquitoes compared with those raised in the laboratory indicated that the larval population as a whole was under nutritional stress. Applying the number of pupae per house and measurement of air and water temperature with an existing model, the risk of dengue transmission was greatest in May and June. The estimated number of female Ae. aegypti per house was well above the threshold for increasing transmission in all months but December through February. A phased approach to sampling immature aedine mosquitoes in Thailand is proposed, which would consist of routine surveillance of larval index and occasional total counts with measurement of wing size. Such a system would combine the benefits of the simple application of larval surveillance with the valuable data gathered from pupal counts and wing measurements.

Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3 Departments of Medical Science and Aquatic Science, Faculty of Science, Burapha University, Chonburi 20131, Thailand. Key words : Acute, Subchronic, Cadmium, Lates calcarifer; Histopathology, Gills, Liver, Kidney Histopathological alterations to white seabass, Lates calcarifer aged 3 months in acute and subchronic cadmium exposure were studied by light and scanning electron microscopy. The 96-h LC50 values of cadmium to L. calcarifer was found to be 20.12 + 0.61 mg/l and the maximum acceptable toxicant concentration (MATC) was 7.79 mg/l. Fish were exposed to 10 and 0.8 mg/l of Cd (as CdCl2H2O) of 96 h and 90 days, respectively. The study showed that gill lamellae and kidney tubules were the primary target organs for the acute toxic effect of cadmium while in the subchronic exposure, the toxic effect to gills was less than that of kidney and liver. Gill alterations included edema of the epithelial cells with the breakdown of pillar cell system, aneurisms with some ruptures, hypertrophy and hyperplasia of epithelial and chloride cells. The liver showed blood congestion in sinusoids and hydropic swelling of hepatocytes, vacuolation and dark granule accumulation. Lipid droplets and glycogen content were observed in hepatocytes at the second and third month of subchronic exposure. The kidney showed hydropic swelling of tubular cell vacuolation and numerous dark granule accumulation in many tubules. Tubular degeneration and necrosis were seen in some areas. © 2002 Elsevier Science Ltd. All rights reserved. (Environmental Pollution., (2003),121: 307-230.)

MOLECULAR EVIDENCE OF WOLBACHIA INFECTION IN NATURAL POPULATION OF TROPICAL ODONATES (NO. 702) A., Thipaksorn1, W., Jamnongluk1 and P., Kittayapong 1,2 *

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KINETICS OF BASIC DYE (METHYLENE BLUE) BIOSORPTION BY GIANT DUCKWEED (SPIRODELA POLYRRHIZA) (NO. 704) P.Waranusantigul 1 , P.Pokethitiyook 1 , M.Kruatrachue 1,2, E.S.Upatham1,3

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Center for Vectors and Vector- Borne Diseases, Faculty of Science, Mahidol University; 2Department of Biology, Faculty of Science, Mahidol University. *Author for correspondence: Dr. P. Kittayapong. E-mail: [email protected] Key words : Tropical odonate, population replacement, Wolbachia infection

Department of Biology. Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Mahidol University International College, Mahidol University, Nakhonpathom 73170, Thailand. 3 Faculty of Science, Burapha University, Chonburi 20131, Thailand.

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Key words : Biosorption; Basic dye; Methylene blue; Spirodela polyrrhiza Wastewater containing pigments and or dyes can cause serious water pollution problems in the form of reduced light penetration and photosynthesis, and toxicity from heavy metals associated with pigments and/or dyes. Laboratory investigations, of the potential use of dried Spirodela polyrrhiza biomass as an adsorbent for the basic dye methylene blue from aqueous solution were conducted. A series of experiments were undertaken in an agitated batch adsorber to assess the effect of the system variables, i.e. sorbent dosage, pH, and contact time. The results showed that as the amount of the dried S. polyrrhiza increased, the percentage of dye sorption increased accordingly. At pH 2.0 the sorption of dye was not favorable, while the sorption at other pHs (3.0-11.0) was remarkable. There was no significant difference in the dye concentration remaining when the pH was increased from 3.0 to 11.0. The dye removal time was influenced by the initial dye concentration, and the process followed the first­order rate kinetics. The rate constants for intraparticle diffusion were 1.00 and 3.27 mg/g/min 1/2 for 300 and 500 mg/l of dye, respectively. © 2003 Elsevier Science Ltd. All rights reserved. (Environmental Pollution, (2003), 125: 385-392.)

Faculty of Science

partment of Mathematics, Mahidol University, Bangkok, 10400 3 Thailand; Laboratory of Computational Physics, Institute of Applied Physics and Computational Mathematics, Beijing, China. Key words : Control volume, Heat transfer, Fluid flow, Solidification, Numerical method A single domain enthalpy control volume method is developed for solving the coupled fluid moving finite element method is developed for solving a fluid flow and heat transfer with solidification problem arising from the continuous casting process. The governing equations consist of the continuity equation, the NavierStokes equations and the convection-diffusion equation. The formulation of the method is cast into the framework of the PetrovGalerkin finite element method with a step test function across the control volume and locally constant approximation to the fluxes of heat and fluid. The use of the step test function and the constant flux approximation lead to the derivation of the exponential interpolating functions for the velocity and temperature fields within each control volume. The exponential fitting makes it possible to capture the sharp boundary layers around the solidification front. The method is then applied to investigate the effect of various casting parameters on the solidification profile and flow pattern of fluids in the casting process. ( International Journal of Computational Fluid Dynamics, 2003 Vol. (not known), pp. 1-8 . The National Metal and Materials Technology Center, National Science and Technology)

A MOVING FINITE ELEMENT METHOD FOR SIMULATING THE FLOW OF MOLTEN STEEL AND FLUX IN THE CONTINUOUS CASTING PROCESS (NO. 705) Jutatip Archapitak1, Benchawan Wiwatanapataphee1, Yong Hong Wu2, Yong Hong Wu2, I Ming Tang2

1

Faculty of Science, Mahidol University, Rama VI, Bangkok 2 10400; Department of Mathematics, Curtin University of Technology, Perth, Australia. Key words : moving finite element, weight residual method, continuous casting A moving finite element method is developed for solving a fluid flow problem arising from the continuous steel casting process. The governing equations consist of the continuity equation and the Navier-Stokes equations. The formulation of the method is cast within the framework of the weighted residual method. A numerical technique based on the moving finite element method is developed to simulate the flow of molten steel and flux in the casting process. (Computational Mathematics and Modeling.pp. 21-28. The Thailand Research Fund (TRF) and National Metal and Materials Technology Center National Science and Technology Development)

A NUMERICAL STUDY OF THE TURBULENT FLOW OF MOLTEN STEEL IN A DOMAIN WITH A PHASE-CHANGE BOUNDARY (NO. 707) B Wiwatanapataphee 1, Y.H. Wu2, J. Archapitak1, P.F.Siew2 , B. 1 Unyong

1

Department of Mathematics, Mahidol University, Bangkok 10400 Thailand; 2Department of Mathematics and Statistics, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia. Key words : Finite element method, Turbulent flow, Heat transfer, Solidification, Continuous casting A numerical algorithm, based on the Galerkin finite element method and the enthalpy formulation, is developed for solving the coupled turbulent fluid flow and heat transfer problem in a domain with a moving phase-change boundary. The governing equations consist of the continuity equation, the Navier-Stokes equations, the energy equation and the modified K-e equation. The formulation of the method is cast into the framework of the Bubnov-Galcrkin finite element method. A numerical study shows that the developed numerical algorithm is stable and capable of capturing the rapid change of temperature and velocity near the phase-change boundary. (Journal of Computational and Applied Mathematics. IN PRESS The National Metal and Materials Technology Center, National Science and Technology)

AN ENTHALPY CONTROL VOLUME METHOD FOR TRANSIENT MASS AND HEAT TRANSPORT WITH SOLIDIFICATION (NO. 706) Yong Hong Wu1, Benchawan Wiwatanapataphee 2 and Xijun Yu3

1

Department of Mathematics and Statistics, Curtin University of 2 Technology, G.P.O. Box U 1987 Perth, WA 6845, Australia; De-

Mahidol University Annual Research Abstracts, Vol. 31

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ELECTRONIC STRUCTURE OF ORDERED AND SORDERED FE3 PT (NO. 708) Zs Major1, S B Dugdale1 , T Jarlborg2, E Bruno3, B Ginatempo3, J B Staunton4 and J Poulter5

1

Department of Physics, University of Warwick, Coventry CV4 2 7AL, United Kingdom. Department of Mathematics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. 3 Dipartimento di Fisica and Unita, INFM, Universita di Messina, Italy. We describe a first-principles theoretical formalism for the magnetic response of paramagnetic metals in which all relativistic effects such as spin-orbit coupling are included. In particular, the easy axis and dependence upon wave vector q of the paramagnetic spin susceptibility (q) can be calculated. To illustrate we apply the method to two transition metals, yttrium and scandium. In each case we find (q) to peak at a wave vector q = (0,0,0.57) /c, coincident with a Fermi surface nesting vector, and to have an easy axis perpendicular to q. Since (q) plays a key role in determining the interaction between magnetic impurities in these metals, these results are consistent with the helical antiferromagnetic order found in many dilute rare-earth Y alloys. Conversely, the easy axis for the response to a uniform magnetic field, q = 0, lies along the c axis. (Physical Review B 68, 134412 (2003). The EPSRC (UK).

H H Wills Physics Laboratory, University of Bristol, Tyndall 2 Avenue, Bristol BS8 1TL, UK; Departement de Physique de la Mati?re Condense, University de Geneve, 24 quai Emest 3 Ansermet, CH-1211 Geneve 4, Switzerland Dipartimento di Fisica and Unity INFM, University di Messina, Salita Sperone 4 31, 98166 Messina, Italy. Department of Physics, University of 5 Warwick, Coventry CV4 7AL, UK. .Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. The electronic structure of invar alloys (i.e. materials in which the near absence of thermal expansion is observed) has been the focus of much study, owing both to the technological applications of these materials and interest in the fundamental mechanism that is responsible for the effect. Here, calculations of magnetic Compton profiles are presented for ordered and disordered Fe3Pt alloys. Using linear muffin-tin orbital and KKR methods, the latter incorporating the coherent potential approximation to describe the substitutional disorder, the electronic band structure and measurable quantities such as the Fermi surface topology are presented. (Journal of Physics:Condensed Mater 15 (2003)3619-3629. THE EPSRC (UK) and the Instituto Nazionale di Fisica della Materia (PAIS ELMAMES) (Italy) , The Royal Society)

AB INITIO CALCULATIONS OF INCOMMENSURATE ANTIFERROMAGNETIC SPIN FLUCTUATIONS IN HCP IRON UNDER PRESSURE (NO. 711) V. Thakor,1 J.B. Staunton,1 J.Poulter,2 S. Ostanin,1 B. Ginatempo,3 and Ezio Bruno. 3

1

THE CRITICAL TEMPERATURE OF THE TWO-DIMENSIONAL ±J ISING SPIN GLASS

(NO. 709)

Department of Physics, University of Warwick, Coventry CV4 2 7AL, United Kingdom; Department of Mathematics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 3 Dipartimento di Fisica and Unita, INFM, Universita di Messina, Italy. We present ab initio calculations of the static paramagnetic spin susceptibility for hcp iron at finite temperatures and for a range of pressures. The dominant magnetic fluctuations in hcp Fe are found to be incommensurate antiferromagnetic, characterized by wave vector qinc = (0.56,0.22,0). We show that q inc is linked to a Fermi-surface nesting feature. For the lowest pressure ~ 16 GPa at which hcp Fe forms, we find that these modes become unstable below a Neel temperature (TN) of 69 K. TN rapidly diminishes with increasing pressure. We therefore predict that hcp Fe will be found to have an incommensurate spin-density-wave-ordered state over a small pressure range starting with the onset of hcp phase. We note the coincidence with the superconductivity recently found in material. (Physical Review B 67, 180405(R) (2003) The EPSRC (UK).

R Sungthong and J Poulter Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. We have studied the two-dimensional ± J Ising model on the square lattice with a view to determine whether a spin glass can exist at small finite temperatures. By mapping the Ising model onto an ensemble of non-interacting lattice fermions, we have shown that the critical temperature for the spin glass phase transition appears to vanish. This result applies for all concentrations of negative bonds where the ground state is a spin glass. ( Journal of Physics A: Mathematical and General 36 (2003) 63476353. The Development and Promotion of Science and Technology Talents programme (Thailand).

ON TWO NOVEL APPLICATIONS OF RANKED SET SAMPLING (NO. 712) FIRST-PRINCIPLES RELATIVISTIC THEORY OF THE MAGNETIC RESPONSE OF PARAMAGNETIC METALS : APPLICATION TO YTTRIUM AND SCANDIUM (NO. 710) V. Thakor,1 J.B. Staunton,1 J.Poulter,2 S. Ostanin,1 B. Ginatempo,3 and Ezio Bruno. 3 Sukuman Sarikavanij, and Montip Tiensuwan Department of Mathematics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. In this paper we discuss two case studies which clearly indicate the advantages of using a ranked set sample (RSS) over

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those of a simple random sample (SRS). The applications of RSS considered here cover single family homes sales data and trees data. It is demonstrated that in each case RSS is much more efficient than SRS for estimation of population mean. (Grant: * The Institute for the Promotion of Teaching Science and Technology (IPST) **The Thailand Research Fund) (Calcutta Statistical Association Bulletin Vol. 54, March & June 2003, Nos. 233214, pp. 105-114)

Faculty of Science

ON ESTIMATION OF POPULATION VARIANCE BASED ON A RANKED SET SAMPLE (NO. 714) Montip Tiensuwan1*, and Sukuman Sarikavanij1**

1

Department of Mathematics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Ranked set sampling (RSS) is by now a well known sampling strategy which provides more efficient estimates of a population mean compared to the traditional simple random sampling (SRS). Although a lot of work on RSS has been done related to estimation of the mean, very little is known about estimation of variance. In this paper the problem of variance estimation based on RSS is studied in detail. It is shown that, based on a single cycle RSS, there does not exist an unbiased estimate of population variance. For multiple cycles in the balanced case, nonnegative quadratic unbiased estimates of population variance are derived. We compare our estimates with an unbiased estimate based on SRS for uniform, exponential and normal distributions. The results show that our proposed estimates are more efficient than the one based on SRS. (Grant: * The Thailand Research Fund * The Institute for the Promotion of Teaching Science and Technology (IPST)) (Journal of Applied Statistical Science (to appear in Vol. 12, 2003).

REPEATABILITY OF AN AUTOMATED LANDOLT C TEST, COMPARED WITH THE EARLY TREATMENT OF DIABETIC RETINOPATHY STUDY (ETDRS) CHART TESTING (NO. 713) Paisan Ruamviboonsuk1, Montip Tiensuwan2, Catleya Kunawut1, and Patcharapim Masayaanon 1

1

Department of Ophthamology, Rajavithi Hospital, Bangkok, 10400, Thailand; 2Department of Mathematics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Purpose : To evaluate the repeatability of visual acuity scores from the automated test and compare them with the Early Treatment of Diabetic retinopathy Study (ETDRS) chart. Design : Instrument validation study based on a model of repeatability study in two observations. Methods : Setting: a prospective, clinic-based, comparative study. A total of 206 participants without ocular diseases and refractive errors in their right eyes were randomly enrolled in the automated group in which 107 participants performed the automated test and the ETDRS group in which 99 participants read the ETDRS chart. All participants were tested with only their right eyes without corrections at 4 meters and came back to have the same tests 1 week later. The automated test used the Landolt rings as optotypes and was conducted by a low-ended personal computer with a 15-inch monitor and a wireless keyboard. The "letter" score calculated by counting every correct response to optotypes, and the "threshold curve" score interpreted from the optotype size at the midpoint of a visual acuity threshold curve. Results : The 95% confidence interval of test-retest of visual acuity scores from the automated test are comparable to the ETDRS chart (.143 compared with .125 for letter scores, .145 compared with .122 for threshold curve scores). The score repeatabilities, calculated from the standard deviations of test-retest, from the automated test are also comparable to the ETDRS chart (.201 compared with .177 for letter scores, .206 compared with .172 for threshold curve scores). All comparisons demonstrated no statistical difference (P > .05). Conclusions : The automated testing system in this study enables practical measuring visual acuity by the Landolt rings. The system's repeatability, which is comparable to the ETDRS chart, supports its role as an alternative tool for measuring outcome in new clinical research. Its ability to practically generate visual acuity threshold curves may also be useful in future clinical research studies. (Grant: The Research Funds of Rajavithi Hospital, grant number 18/2544) (Am J Ophthalmol 2003; 136: 662-669 ? 2003 by Elsevier Inc. All rights reserved.)

ON A COMPARISON OF TWO STANDARD ESTIMATES OF A BINOMIAL PROPORTION BASED ON MULTIPLE CRITERIA DECISION MAKING METHOD (NO. 715) Satinee Lertprapai 1, Montip Tiensuwan1, and Bimal K. Sinha 2

1

Department of Mathematics, Faculty of Science, Mahidol University Bangkok, 10400, Thailand; 2Department of Mathematics and Statistics, University of Maryland, Baltimore County, USA In this paper we consider the problem of estimation of a binomial proportion based on x~ B(n, ), n known, 0 < < 1. We compare two standard estimates T1 = x / n and T2 = [x + n /2] / [n + n ] on the basis of Multiple Criteria Decision Making (MCDM) procedure. Our recommendation is that we should use T2 rather than T1 for small values of n. (Grant: Research supported by a scholarship under the Ministry Staff Development Project of Ministry of University Affairs, Thailand. Research supported by a grant from the Thailand Research Fund(TRF) Research supported by UMBC's Presidential Research Professorship grant) (Fifth International Triennial Calcutta Symposium on Probability and Statistics, December 28-31, 2003, Department of Statistics and Calcutta Statistical Association, Department of Statistics Calcutta University, Kolkata, India)

Mahidol University Annual Research Abstracts, Vol. 31

WATER QUALITY AND LARVAL HABITATS OF MALARIA MOSQUITO IN NORTH-WESTERN THAILAND (NO. 716) Ampornphan Kengluecha1, Ratana Sithiprasasna 1, Montip Tiensuwan2, and James W. Jones 1 Department of Entomology, US Army medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok Thailand; 2Department of Mathematics, Faculty of Science, Mahidol University Bangkok, 10400, Thailand. Malaria transmission is dependent upon many hydrologydriven ecological factors that directly affect the vectorial competence including the presence of suitable habitats for development of larval anopheline stages. Larval habitats were identified and characterized in three malaria endemic villages (Ban KhunHuay, Ban PaDae and Ban Tham Seau) in northwestern Thailand between July 2002 and June 2003. Global Positioning System (GPS) was used to provide the precise locational data for spatial distribution of anopheline mosquito larvae and their habitats preference. Ten categories of habitats were identified and four major species of malaria vectors (An. Dirus, An. Minimus, An. Maculates and An. Sawadwongporni) were collected. An. Minimus was the most common species throughout the year, 18 adult Anopheles species were identified from larvae from all surveyed habitats. The relationship between eight abiotic variables (temperature, hardness, carbondioxide, dissolved oxygen, nitrate, phosphate, silica and pH) and the abudance of anopheline larvae was investigated. The results from the multiple regression model suggested hardness, water temperature and carbon dioxide to be the best predictor variables associated with the abundance of An.minimus larvae at p < 0.001. For An. Dirus, the population of immature mosquito showed significantly relationship with water pH in the breeding sites at p < 0.001. (Grant: AFRIMS) (The 52nd annual meeting of the American Society of Tropical Medicine and Hygiene; December 3-7, Philadelphia Marriott-Downtown Hotel, Philadelphia, Pennsylvania, USA)

1

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However, DPOAE levels in some Meniere's patients were normal, or near normal, in spite of hearing loss. This wasn't the case with sensorineural hearing loss of other etiologies. The purpose of this study was to compare 2f1-f2 DPOAE amplitude signal to noise ratio (SNR) between normal hearing subjects (group 1) and Meniere's patients with normal hearing (group 2), and between Meniere's patients with hearing loss (group 3) and non-Meniere's patients with hearing loss (group 4). Each group consisted of 30 ears of people between 20 and 60 year olds. The pure tone thresholds at 0.5, 1, 2, 3, 4, 6, 8 kHz between group 1 and group 2 and between group 3 and group 4 showed no significant difference. The 2f1-f2 DPOAE amplitude SNR and DP I/O functions were obtained by using Otodynamic OAEs analyzer Model ILO 9.6 DP-grams were obtained at primary frequencies (f2) = 1001-6348 Hz with primary level L1 = L2 = 75 Dbspl. DP-I/O functions were generated at f2 = 1001-6006 Hz with L1 = L2 = 45-75 Dbspl. The f2/f1 ratio was fixed at 1.21 in both measurements. The I/O function slopes were calculated by curve fitting method. There was no significant difference regarding the 2f1-f2 DPOAE amplitude SNR and 2f1-f2 DPOAE amplitude SNR I/O function between normal hearing subjects and Meniere's patients with normal hearing. The af1-f2 DPOAE amplitude SNR between Meniere'spatients with hearing loss and non-Meniere's atients with hearing loss showed significant difference at 2002 and 3174 Hz at pvalue <0.05. The 2f1-f2 DPOAE amplitude SNR I/O function between Meniere's patients with hearing loss and non-Meniere's patients with hearing loss showed significant difference only at f2 = 3003 Hz, L1 = 75 dBSPL at p-value < 0.05. There was no significant difference of I/O function slopes between normal hearing subjects and Meniere's patients with normal hearing and between Meniere's patients with hearing loss and non-Meniere's patients with hearing loss. These findings might indicate that, firstly, Meniere's patients with normal hearing should have normal outer hair cell functions similar to normal hearing subjects. Secondly, Meniere's patients with hearing loss showed better preservation of OHCs than those in non- Meniere's patients with hearing loss, despite the same pure tone thresholds. From this study, DPOAEs can be used as a part of the diagnostic tool for monitoring puter hair cell functions in Meniere's patients and predicting the prognosis of MD.

THE COMPARISON OF DISTORTION PRODUCT OTOACOUSTIC EMISSIONS BETWEEN MENIERE AND NON-MENIERE HEARING LOSS EARS (NO. 717) Chuleerat Jantrasakul 1*, Chanchai Jariengprasert 1, Krisna Lertsukprasert1, and Montip Tiensuwan2

1

(Grant: *Partial supported from Faculty of Graduate Studies, Mahidol University, Bangkok, Thailand)

Department of Otolaryngology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University; 2Department of Mathematics, Faculty of Science, Mahidol University Bangkok, 10400, Thailand. Distortion product otoacoustic emissions (DPOAEs) reflect the function of outer hair cells. This function is influenced by different variables. Among these the volume and pressure exerted by the endolymph were very important in patients with Meniere's disease (MD). Previous studies of DPOAEs in patients with MD have shown that most had a reduction of DPOAEs below population averages, as did those with sensorineural hearing loss of other etiologies.

APPLICATION OF LOG-LINEAR MODELS TO CANCER PATIENTS : A CASE STUDY OF THE NATIONAL CANCER INSTITUTE (NO. 718) Montip Tiensuwan and Pornpis Yimprayoon Department of Mathematics, Faculty of Science, Mahidol University Bangkok, 10400, Thailand. Cancer is a noninfectious disease which is on the increase throughout the world and has become a serious problem for public health in many countries, including Thailand. In Thailand, cancer has risen significantly to become a leading cause of death. The Na-

270

tional Cancer Institute where most patients are admitted. In this study, the data sets were the number of new cancer patients who were admitted between January 1999 and December 1999 at the National Cancer Institute. The main objective of this study is to test association between personal, cancer and clinical variables by using log-linear models. In each sex, a test of independence is used to find the relationships between any two variables which are chi-square tested and Cramer's V and two-dimensional log-linear models are used to obtain estimated parameters, expected frequencies and standardized residuals. For both sexes, the three-dimensional log-linear models are applied to test association between any three variables. Among these models considered, the best one was chosen by analysis of deviance. The results of chi-square tested show that most paired variables of personal, cancer and clinical variables are significantly related at p-value < 0.05. For both male and female patients, the variable site of cancer is highly related to personal variables such as race, religion, marital status, age and region, cancer and clinical variables such as diagnostic evidence, stage of diagnosis, treatment and status of last contact, which provide the highest Cramer's V value. So marital status, age and region are the most influential factors effecting the site of cancer for male and female patients. Moreover, the site of cancer also effects the method of diagnostic evidence and treatment. Since site of cancer in each sex is different, prevention for various sites of cancer should be considered for each specific sex. In addition, for male and female patients, treatment is related to age, site of cancer and stage of diagnosis. Consequently, physicians should consider these factors to select the appropriate method of treatment. (Grant: The Thailand Research Fund) (National Conference: Progress in Mathematics 2003, May 8-9, 2003, Department of Mathematics, Mahidol University and The Thailand Research Fund, Best Western Fortune Hotel, Bangkok, Thailand)

Faculty of Science

MODELING OF BONE FORMATION AND RESORPTION MEDIATED BY PARATHYROID HORMONE : RESPONSE TO ESTROGEN/PTH THERAPY (NO. 720) Chontita Rattanakul 1 , Yongwimon Lenbury 1 , Nateetip Krishnamara2, David J. Wollkind3

1

Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400 Thailand. E-mail: 2 [email protected] Department of Physiology Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, 3 Thailand. E-mail : [email protected]; Department of Mathematics Washington State University, Pullman WA 991643113, U.S.A. E-mail : [email protected] Key words : Bone remodeling, Parathyroid hormone control, Estrogen therapy Bone, a major reservoir fo body calcium, is under the hormonal control of the parathyroid hormone (PTH). Several aspects of its growth, turnover, and mechanism, occur in the absence of gonadal hormones. Sex steroids such as estrogen, nonetheless, play an important role in bone physiology, and are extremely essential to maintain bone balance in adults. In order to provide a basis for understanding the underlying mechanisms of bone remodeling as it is mediated by PTH, we propose here a mathematical model of the process. The nonlinear system model is then utilized to study the temporal effect of PTH as well as the action of estrogen replacement therapy on bone turnover. Analysis of the model is done on the assumption, supported by reported clinical evidence, that the process is characterized by highly diversified dynamics, which warrants the use of singular perturbation arguments. The model is shown to exhibit limit cycle behavior, which can develop into chaotic dynamics for certain ranges of the system's parametric values. Effects of estrogen and PTH administrations are then investigated by extending on the core model. Analysis of the model seems to indicate that the paradoxical observation that intermittent PTH administration causes net bone deposition while continuous administration causes net bone loss, and certain other reported phenomena may be attributed to the highly diversified dynamics which characterizes this nonlinear remodeling process. (BioSystems 70(1) (2003) 55-72. The Thailand Research Fund (RTA 4580005 and PHD/0016/2543.)

MODELING OF BONE FORMATION AND RESORPTION MEDIATED BY PARATHYROID HORMONE: RESPONSE TO ESTROGEN/PTH THERAPY (NO. 719) Pachara Chaisuriya 1, Sing-Cheong Ong2

1

Department of Mathematics, Faculty of Science, Mahidol University. Rama 6 Rd., Bangkok 10400, Thailand. E-mail : [email protected]; 2Department of of Mathematics, Central Michigan University, Mount Pleasant, MI 48859. E-mail : [email protected] Key words : Schur product, Hadamard product, Schur mutiplier norm. A sufficient condition for an infinite matrix having its norm as an operator on l 2 same its Schur multiplier norm is given. All known examples, which have their operator norm same as Schur multiplier norm, satisfy this condition. Some related results are proved. A formula for the distance from a give matrix to the unitaries is also given. (AMS Mathematical. Subject Classification (2000). 15A18, 47A10. Ministry of University Affairs of Thailand.)

TOTAL CURVATURE AND LENGTH ESTIMATE FOR CURVES IN CAT(K) SPACES (NO. 721) Chaiwat Maneesawarng, Yongwimon Lenbury Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400 Thailand. E-mail: [email protected], [email protected] Key words : Curvature bounded above, CAT(K), Length estimate. We introduce the notion of total curvature of curves (which agrees with the usual one in the piecewise smooth case) in spaces of Alexandrov curvature bounded above. Basic properties of total curvature, including rectifiability of curves of finite total curvature and additivity of total curvature, are then obtained. A sharp

Mahidol University Annual Research Abstracts, Vol. 31

upper estimate of a type due to Schmidt on the length of a curve in a CAT(K) space is also given in terms of its total curvature and the distance between its endpoints. (Differential Geometry and its Applications. 19 (2003) 211-222. The Thailand Research Fund (PDF/35/2543.)

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THE ANALYSIS OF HIGHER-ORDER CASCADE SYSTEMS WITH SEPARATION CONDITIONS PIVOTING ON THE SLOW COMPONENTS: APPLICATION TO A MODEL OF MIGRATION FOR SURVIVAL OF THE SPECIES (NO. 722) Thongchai Dumrongpokaphan 1, Yongwimon Lenbury 1, Philip S. Crooke 2

1

Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand. E-mail: 2 [email protected] ac.th, [email protected] Department of Mathematics, Vanderbilt University, Nashville, Tennessee 37240 U.S.A. E-mail: [email protected] Key words: Cascade systems, singular perturbation, sustained oscillation, persistence, Migration. Cascade systems, characterized by highly diversified time responses, are considered in this paper. Singular perturbation principles, which have been used to analyze relaxation oscillations in second-order dynamical systems, will be extended here to accommodate nonlinear systems in which more state variables are involved in multiscale interactions. Separation conditions will be derived for the identification of limit cycle behavior in a higher-dimensional (n>4) cascade system. It is found that when appropriate regularity and boundedness requirements are met by the slow components of the dynamical system, pivoting on the slow components can lead to separation conditions which identify limit cycle behavior as well as other dynamic behavior permitted by the model. The principle is then applied to a model of two communities coupled by migration. Through such analysis, we can examine how the mechanisms of migration, variations in reproduction, recruitment, mortality, and feeding success, exploited by interacting species, may achieve survival and coexistence of the populations concerned. (Mathematical and Computer Modelling. 38 (2003) 671-690. The Thailand Research Fund (RTA4580005 and PHD/0029/2543)

We introduce a Plane-Spin-Rotator (PSR) model as one of a myriad of non-equilibrium statistical mechanics models governed by stochastic dynamics. The system consists of a one-dimensional chain of lattice sites in which each site is attached with a spin initially in the ordered state e.g., pointing in the same direction. We incorporate the effects of a non-equilibrium phenomenon by giving the system a dynamics. Namely we put a random walker (RW) or a Brownian agent at the origin or in the middle of the lattice chain at the beginning and let it execute pure unbiased random walk to disorder i.e., to destroy the line up of the spins. The local update rule whereby the system changes periodically from one state to another is that each time step as the RW moves it has a certain probability to rotate the spin or change the angle between the x-axis and the spin. We find the nontrivial statistics of due to this and other simple stochastic (Markovian type) model such as and do not behave in sinusoidal fashion as one might expect. These functions and other can be calculated analytically exploiting the results from its "cousin" model introduced in Physical Review E, Vol. 59, no. 5 p. 5127. Excellent agreement from theoretical and Monte Carlo computer simulation results is found. (ScienceAsia. 29 (2003) 289-299. The Thailand Research Fund (TRG4580090 and RTA4580005), MTEC (MT-NS-45-POL-14-06G) MATHEMATICAL MODELS FOR PRESSURE CONTROLLED VENTILATION OF OLEIC ACID-IN JURED PIGS (NO. 724) Philip S. Crooke1, Konvika Kongkul2, Yongwimon Lenbury2, A.B. Adams3, C.S. Carter3, J.J. Marini 3, J.R. Hotchkiss3

1

Department of Mathematics, Vanderbilt University, Nashville, Tennessee 37240 U.S.A., E-mail: [email protected] 2 Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand, E-mail: 3 [email protected] Pulmonary and Critical Care Medicine, Regions Hospital and University of Minnesota, St. Paul, MN 55101 U.S.A. Key words : Oleic acid injury, mathematical model, variable compliance. One-compartment, mathematical models for pressure controlled ventilation, incorporating volume dependent compliances, linear and nonlinear resistances, are constructed and compared with data obtained from healthy and (oleic acid) lung-injured pigs. Experimental data is used to find parameters in the mathematical models and was collected in two forms. Firstly, the P -V curves for healthy e and lung injured pigs were constructed; this data is used to compute compliance functions for each animal. Secondly, dynamic data from pressure controlled ventilation for a variety of applied pressures is used to estimate resistance parameters in the models. The models were then compared against the collected dynamic data. The best mathematical models are ones with compliance functions of the form C(V) = a+bV where a and b are constants obtained from the P -V e curves and the resistive pressures during inspiration change from a where Q linear relation P = RQ to a nonlinear relation P r = RQ r is the flow into the one-compartment lung and is a positive number. The form of the resistance terms in the mathematical models indicate the possible presence of gas-liquid foams in the experimental data. (Mathematical Medicine and Biology. In Press. The Thailand Research Fund (RTA4580005).

RANDOM WALK ON A PLANE-SPIN-ROTATOR SYSTEM : CONTINUUM THEORY AND MONTE CARLO SIMULATIONS (NO. 723) Wannapong Triampo 1, Darapond Triampo 2, I. Ming Tang 1, Yongwimon Lenbury 3 Department of Physics, Faculty of Science, Mahidol University Rama 6 Rd., Bangkok 10400 Thailand. E-mail: [email protected] 2 yahoo.com, [email protected] Department of Chemistry, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 3 10400, Thailand. Department of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400 Thailand. Email: [email protected] Key words : Stochastic process, random walk, Brownian, spin, Monte Carlo, non-equilibrium statistical mechanics.

1

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NONLINEAR DELAY DIFFERENTIAL EQUATIONS INVOLVING POPULATION GROWTH (NO. 725) Yongwimon Lenbury 1, Dang Vu Giang 2 Department of Mathematics, Mahidol University, Rama 6, 2 Bangkok, 10400 Thailand. E-mail: [email protected] Hanoi Institute of Mathematics, 18 Hoang Quoc Viet, 10307 Hanoi, Vietnam. E-mail: [email protected] . Key words : Delay effect, population model, stability, unimodal function. Conditions are given on the function f such that population x(t) given by x(t) = -µx(t) + f(x(t-)) becomes extinct or remains globally stable. Our theorems are shown to be applicable to the Nicholson's model of blowflies and the population dynamics of baleen whales. In some of these cases the function f is unimodal rather than monotone. (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005).

1

Faculty of Science

Key words : Pattern formation, nonlinear stability analysis, ion sputtering The development of spontaneous stationary equilibrium patterns on ion-sputtered semiconductor surfaces is to be investigated by means of weakly nonlinear stability analyses applied to appropriate governing evolution equation of the phenomenon. The mathematical system modeling the phenomenon can be reduced to a single nonlinear partial differential time-evolution equation defined on an unbounded two-dimensional spatial domain. The etching of coherent ripples and hexagonal lattices of nanoscale quantum dots or holes on the semiconductor surface during the erosion process is based upon the interplay between roughening caused by ion sputtering and smoothing due to surface diffusion. After summarizing the results of pattern formation investigations of morphological and Turing instabilities, which bear a striking resemblance to the development described above and have been successfully quantified by the same methods, the weakly nonlinear stability analyses of a hexagonal basic platform will be performed on the model evolution equation. Then the theoretical prediction from this analysis will be compared with both relevant experimental evidence and numerical simulations as well as placed in the context of some recent pattern formation studies. The results of this work have the potential to contribute to a general understanding of pattern formation in a wide variety of materials science areas. (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005).

A DELAY-DIFFERENTIAL EQUATION MODEL OF CORTISOL SECRETION PROCESS IN HUMAN (NO. 726) Pornsarp Pornsawad, Yongwimon Lenbury Department of Mathematics, Mahidol University, Rama 6, Bangkok, 10400, Thailand. E-mail: [email protected], [email protected] Key words: Cortisol secretion, delay mechanism, nonlinear delaydifferential equations, bifurcation analysis. The present work develops and analyzes a model system of delay-differential equations which describes the core dynamics of the stress-responsive hypothalamus-pituitary-adrenal axis. This neuroendocrine ensemble exhibits prominent pulsatile secretory patterns governed by nonlinear and time-delayed feedforward and feedback signal interchanges. Formulation and subsequent bifurcation analysis of the model provide qualitative and mathematical frame work for better understanding of the delayed responsive mechanisms as well as the dynamic variations in different pathological situations. (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005).

NONLINEAR MATHEMATICAL MODELS FOR PRESSURE CONTROLLED VENTILATION WITH VARIABLE COMPLIANCE (NO. 728) Konvika Kongkul 1, Yongwimon Lenbury 1, Philip S. Crooke

1 2

Department of Mathematics, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand. E-mail: [email protected], 2 [email protected] Department of Mathematics, Vanderbilt University,Nashville, TN 37240 U.S.A. E-mail: [email protected] vanderbilt.edu Key words : Pressure controlled ventillation, variable compliance, nonlinear one-compartment models. We present mathematical models which describe the volume of a single compartment lung. The models are constructed and compared with data obtained from healthy and lung injured (oleic acid) pigs. Experimental data is used to find the parameters in the mathematical models and was collected in two forms. Firstly, the P -Vcurves for healthy and lung injured pigs were found; this data is el used to compute compliance function for each pig. Secondly, dynamic data from pressure controlled ventilation of the pigs for a variety of applied pressures is used to calculate resistance parameters in the models. The models were then compared against the collected dynamic data. It is demonstrated that the best mathematical models are ones that have compliance functions of the form C(V) = a + bV, one segment linear functions of the lung volume, where a and b are constants obtained from the P - V curves and the resistive pressures el during inspiration change from a linear relation P = RQ to a nonlinear r relation P r = R Q where Q is the flow into the one-compartment lung. Although expressions for certain outcome variables are not always obtainable in these models, asymptotic formulas for key outcome variables (tidal volume, minute ventilation, end-expiratory

WEAKLY NONLINEAR STABILITY ANALYSIS OF TURING PATTERN FORMATION ON IONSPUTTERED SEMICONDUCTOR SURFACES

(NO. 727)

Adoon Pansuwan, Yongwimon Lenbury Department of Mathematics, Mahidol University, Rama 6, Bangkok, 10400, Thailand. E-mail: [email protected], [email protected]

Mahidol University Annual Research Abstracts, Vol. 31

pressure, mean alveolar pressure and work) as a function of the physiologic and ventilator parameters are derived. (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005). THE ASYMPTOTIC STABILITY OF

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2

x - ax

n+1 n-1

+

bx

n- k

=

0

2

(NO. 731)

Piyapong Niamsup1, Yongwimon Lenbury VENTILATOR ASYNCHRONY IN PARTIAL SUPPORT, PRESSURE-CONTROLLED VENTILATION (NO. 729) Sahattaya Rattanamongkonkul, Yongwimon Lenbury Department of Mathematics, Mahidol University, Rama 6 Rd., Ba g o 1 4 0 Th ia d E- i:[email protected], n k k 0 0 , aln . mal [email protected] Key words: Partial ventilatory support, pressure-controlled ventilation, one-compartment model. In this research, we deal with the modelling of partial ventilatory support during pressure-controlled ventilation (PCV). Given the characteristics of the patient (resistance R, and compliance C), together with the airway pressures (P and PEEP), we investigate set the stability of mechanical ventilation using two different triggering options: pressure triggering and flow triggering. In each case, we are interested in the variation of tidal volume (V ) and end-expiratory T pressure (P ) as functions of the cut-off parameter ().

ex 1

Department of Mathematics, Faculty of Science, Chiangmai University, Chiangmai 50200, Thailand. E-mail: [email protected] 2 chiangmai.ac.th Departments of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400 Thailand. E-mail: [email protected] Key words : Linear delay difference equation, asymptotic stability, necessary and sufficient conditions. We give the necessary and sufficient conditions for the asymptotic stability of the linear delay difference equation x - a x + bx

n+1 n-1 2

= 0,

n-k

n = 0, 1, ...

where a and b are arbitrary real numbers and k is a positive integer greater than 1. (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Researhc Fund (RTA4580005).

(Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005).

MATHEMATICS MODELING OF ENE-TO-SIDE ANASTOMOSIS IN CORONARY ARTERY BYPASS GRAFTING (NO. 730) Benchawan Wiwatanapatapee1, Permyos Ruengsakulrach3 , Duangkamol Poltem1, Yongwimon Lenbury1, Mahosut Punpocha2

1

SIMPLE STOCHASTIC MODELLING OF MUTAGENESIS

(NO. 732)

Wannapong Triampo 1, Darapond Triampo 2, I. Ming Tang 1, Yongwimon Lenbury 3

1

Departments of Mathematics, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400 Thailand. E-mail: [email protected], [email protected], [email protected] 2 ac.th King Mongkut's Institute of Technology, Bangkok, 3 Thailand. E-mail: [email protected] The Division of Cardiac Surgery, Heart Institute, Saint Louis Hospital and Foundation. E-mail : [email protected] Key words: Coronary artery bypass grafts, end-to-side anastomosis, two-dimensional model, Bobnov-Galerkin formulation. The aims of this project are to developed a mathematical model of end-to-side anastomosis of the coronary artery bypass grafting and determine the most appropriate comparison the possibility of adverse effect of the degree of grafting. Morphometric data of the coronary artery grafts of 10, 30, 45, 60 and 90 degree are used to construct a mathematical model of the flow in the host coronary artery with any degree (50%, 60%, 70%, 90%) of severity stenosis. The model various angles of grafting is built in a simplified two-dimensional representation of real tissue geometry. Numerical solutions of velocity profiles were calculated by using Finite Element Method based on a Bobnov-Galerkin formulation. (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005).

Department of Physics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. E-mail: [email protected], 2 [email protected] Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. E-mail: 3 [email protected] Department of Mathematics, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. E-mail: [email protected] Key words : Stochastic models, mutagenesis. The kinetics of spontaneous mutagenesis induced by the stochastic action of mutagens on the DNA chain is studied. This is done by modelling the chain as a sequence of DNA-bit values (`0' for normal bases and `1' for abnormal bases) and counting the number of bit value changes resulting from the tautomeric shifts made by the mutagen as it makes a walk along the chain. It is found that the final number of DNA-bit changes increases as Öt when the walk is random and increases as t when the walk is biased. It is also found that the final number of bit changes does not grow linearly as the number of mutagens is increased (Progress in Mathematics 2003. Bangkok, Thailand. 8-9 May 2003. The Thailand Research Fund (RTA4580005).

274

MODELLING DELAY MECHANISMS IN HUMAN PHYSIOLOGY AND BIOLOGY

50

Faculty of Science

3, did not show any cytotoxic effect against KB and BC-1 cell lines (IC value > 20 µg/mL)

(NO. 733)

(Phytother. Res 17, 1101-1103 (2003) Yongwimon Lenbury 1, Dang Vu Giang 2, Pornsarp Pornsaward1, Chontita Rattanakul 1

1

Department of Mathematics, Mahidol University, Rama 6, Bangkok,10400,Thailand.E-mail:[email protected] ahidol.ac.th , 2 [email protected], [email protected] Hanoi Institute of Mathematics,18 Hoang Quoc Viet, 10307 Hanoi, Vietnam. E-mail : [email protected] Key words : Delay mechanisms, mathematical modelling, human physiology. Many types of delay mechanisms have been identified to operate in physiological and biological systems. In population dynamics, delays due to maturation time have long been recognized and modelling attempted with some success. We will discuss some new results on nonlinear differential equation population model with constant time delay. In the human physiology, delays due to late responses in hormone secretion positive (feed-forward) and negative (feed-back) control loops in a cascade system may be modelled through a Hopf bifurcation analysis leading to interesting clinical interpretation. A third type of delay action has been observed in late recovery time after cessation of drug treatments. We shall illustrate this type of delays by numerical simulations of our model of bone formation mediated by the parathyroid hormone and estrogen supplements. Such discoveries can have far reaching implications for disease diagnosis and control. (8 th National Conference in Mathematics. Bangkok, Thailand. 2123 May 2003. The Thailand Research Fund (RTA4580005) .

HORNBILLS : THEIR ECOLOGICAL ROLES AND CONSERVATION (NO. 735) Pilai Poonswad , Chumpol Sukkasem , Somnoi Phataramata , 2 1 1 Sumsuding Hayeemuida , Kamol Plongmai , Phitaya Chuailua , 1 1 Preeda Thiensongrusamee , and Narong Jirawatkavi

1 1 2 2

Thailand Hornbill Project, Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. E-mail 2 :[email protected]; National Park and Wildlife Research Division, Natural Resource Conservation Office, National Park Wildlife and Plant Department, Bangkok, Thailand. Key words : Hornbill, Seed dispersers, Conservation Hornbills, although, are omnivorous but their main food consists of fruit. At Khao Yai, hornbills consumed at least 70 species of animals and 84 species of 57 genera in 30 families of fruits during their life cycle namely early nesting, nesting late nesting and flocking period. Through their habit of storing many fruits per feeding and regurgitating as they move hornbills are undoubtedly good seed dispersers. Among 30 tree families, Meliaceae, Myristicaceae, Burseraceae and Aracaceae, by their nature, depend largely on hornbills. Not only large amount of fruit they consume but hornbills also 2 move for large ranges (4-35 km ) depending on species, thus enhance seed dispersion. Therefore, in order to keep the forest ecosystem healthy, hornbills well being should be monitor for efficient conservation. Breeding success of hornbills is influenced by the availability of suitable nest cavities. Besides natural causes, human activities particularly poaching obviously affect the breeding success of hornbills. The loss of suitable condition of hornbill nest cavities can be subjected to natural processes, such as decay of wood which causes nest floor sinking, growth of plant tissue which causes narrowness or close of the nest entrance. Long-term monitoring of the breeding status was conducted between 1993-2000 for four hornbill species in Khao Yai National Park, Great Hornbill Buceros bicornis, Wreathed Hornbill Aceros undulatus, White-throated Brown Hornbill Anorrhinus austeni and Oriental Pied Hornbill Anthracoceros albirostris. Similarly between 1994-2000 six species were studied in Budo-Sungai Padi National Park, Great, Wreathed, Rhinoceros B. rhinoceros, Helmeted Buceros (Rhinoplax) vigil, Bushy-crested Anorrhinus galeritus and White-crowned Hornbills Berenicornis comatus. In Khao Yai, Great Hornbill had 80.3% breeding success with 114 chicks produced, Wreathed Hornbill had 79.4% breeding success, 54 chicks produced, White-throated Brown Hornbill had 83.8% breeding success, 71.3% chicks produced and Oriental Pied Hornbill had 92.2% breeding success, 213 chicks produced. Among 23 genera of nest trees of hornbills of Khao Yai, the important genera were Dipterocarpus (42.4%), Syzygium (23.6%) and Cinnamomum (6.1%). The need of nests improvement in Dipterocarpus was 21.1%, Syztygium was 42.5% and Cinnamomum was 30.0%. Improvement of nest cavities between 1996-2000 assisted chick production of Great Hornbill for 57.5% (of total 80 chicks), of Wreathed Hornbill for 36.1% (of total 36 chicks), of Whitethroated Brown Hornbill for 35.3% (of total 39.1 chicks) and Oriental Pied Hornbill for 10.2% (of 162 chicks). In Budo-Sungai Padi, similar to Khao Yai, the major nest trees are trees of Dipterocarpaceae (60%), but Hopea is the most important genus which accounted to

ANTI-HIV-1 LIMONOID : FIRST ISOLATION FROM CLAUSENA EXCAVATA (NO. 734) Arunrat Sunthitikawinsakul , Ngampong Kongkathip , Boonsong 1 1 2 Kongkathip , Sida Phonnakhu , John W. Daly , Thomas F. 2 2 3 3 Spande , Yuth Nimit , Chanita Napaswat , Jittra Kasisit , and 3 Chalobon Yoosook

1 1 1

Natural Products and Organic Synthesis Research Unit (NPOS), Department of Chemistry, Faculty of Science, Kasetsart Univer2 sity, Bangkok 10900, Thailand; Laboratory of Bioorganic Chem3 istry, NIDDK, NIH, DHHS, Bethesda, Maryland USA; Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Key words : Clausena excavata; limonoid; anti-HIV-1 activity; clausenolide-1-ethyl ether; coumarin A limonoid, clausenolide-1-ethyl ether(1) and two coumarins, dentain (2) and nor-dentatin (3), were isolated from Clausena excavata. Limonoid 1 was obtained from the crude ethanol extract of the rhizomes and the roots but had not previously been isolated from C. excavata and exhibited HIV-1 inhibitory activity. Coumarins 2 and 3, with their structures related to an anti-HIV-1 substance, (+)-calanolide A (4), were obtained from the crude chloroform extract of the rhizomes. Both induced toxicity to cells used in a syncytium assay for anti-HIV-1 activity. These compounds, 1-

Mahidol University Annual Research Abstracts, Vol. 31

31%. The percentage of nesting here has progressively decreased from 80.9% in 1997 through 55.8% (1998), 47.8% (1999) to a low of 36.4% in the year 2000. Breeding success was difficult to determine due to inaccessibility during a two-year military operation in the area. Between 1997-2000, it appears that at least 86 chicks fledged. Participation in the research and conservation program by villagers and urban people was considered successful in terms of almost total eradication of poaching, yet illegal logging has significantly increased, and this is now the biggest threat to the conservation of hornbills in the area. (Present at Toward the Integratin of Biodiversity Studies under the st 21 Century COE Program of Kyoto University, Kyoto, Japan 2627, March 2003.)

275

IDENTIFICATION OF GENOMIC DIFFERENCES BETWEEN PATHOGENIC AND NON-PATHOGENIC STRAINS OF LEPTOSPIRA USING REPRESENTATIONAL DIFFERENCE ANALYSIS

(NO. 736)

Padungsri Dubbs, Sathiti Sirisingha, Sansanee Chairojana Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; E-mail : [email protected] mahidol.ac.th The aim of this research is to identify genes that are present only in virulent strains of Leptospira as compared to those of nonvirulent strains using representational difference analysis. Chromosomal DNA of Leptospira interrogans serovar autumnalis, isolated from patients with severe symptoms of leptospirosis, is subjected to subtractive hybridization with chromosomal DNA, isolated from Leptospira meyeri serovar semaranga. After hybridization, the non-hybrid DNA fragments containing only tester DNA are selectively amplified. The amplified products are cloned and sequenced.

cally significant (p<0.05) decreasing of the responsiveness to Poke Weed Mitogen (PWM) of skin wart patient compared with the normal control group (Stimulation Index; SI 22.7 + 3.9 vs. 32.9 + 3), however there was no differnece in other mitogens and superatigens. The responsiveness to their crude antigen extracts stimulation could not be observed. However, one of six patients showed high proliferative responsiveness (SI = 66.83). the detection of CD4+, CD8+, CD86+ and CD152+ cells in wart patients peripheral blood mononuclear cells elucidated a statistically significant (p<0.05) decrease from the control group i.e. CD4+ (25.4+2.7% vs 30.5 + 2.3%), CD8+ (14.8+1.5% vs 17.4+1.8%), CD86+ (13+3% vs 19.1+2%) and CD152+ (8+1.7% vs 10.1+1.8%). Nevertheless, CD28+ and +T cells were not different. The detection of CD86+ and +T cells in skin wart cryosection as well as collagenase extract (infiltrating cells) compared with eyelid specimen (normal tissue control) was not different, but + T cells was found in all normal tissue collagenase extracted cell (five subjects) while it was detected in only one of ten wart subjects. The immune competence compared between nonrecurrence and recurence skin wart patients showed a statistically significant (p < 0.05) higher responsiveness of T cells mitogen stimulation in the recurrence group; Phytohemagglutinin (SI : 60.1+15.2 vs 15.4+7.3), Concanavalin A (SI; 53.5+9 vs 21.7+6). The findings of this study suggest that the decreasing of immune competence especially to PWM stimulation and the reducing number of CD86+, CD152+ cells may play some role in skin wart patients. Furthermore, a decreasing number of skin + T cells suggested a possible role in wart ocurrence.

SUSCEPTIBILITY OF HEP-G2 CELL DURING G1/S AND G2/M CELL CYCLE PHASE TO CYTOTOXIC IMMUNE CELLS (NO. 738) Sripapa Suphacharatwong and Molvibha Vongsakul Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, E-mail : [email protected] Key words : Hep-G2, cell cycle, cytolysis Hepatocarcinoma is a major type of human cancer, apart from tumor oncogenesis, the immune response is another interesting subject for study. In this study, human hepatocellular carcinoma cell line, Hep-G2 was used as the target cell for in vitro cytotosic assay by normal peripheral blood mononuclear cells to study HepG2 cytolysis susceptibility. Three aspects were studied. First, the effect of Hep-G2 cell cycle on cytolysis susceptibility. This was studied by the detection of cyclinD1 and A in Hep-G2 cell extracts of various cell stages (chemical treated cell cycle arrest, i.e. hydroxyurea for G1/S, expected to express cyclinD1, etoposide for G2/M, expected to express cyclinA, and serum deprivation for G/0G1 arrest, expected to express cyclinD1) by western immunoblot staining with specific monoclonal antibodies and the propidium iodide staining for flow cytometry analysis was also performed. These Hep-G2 cells were used in cytolysis assay. Second, the effect of time in HepG2 cytolysis susceptibility was assayed by comparing percetage of HepG2 death between 4 hours and 18 hours of cytolysis incubation times, the Trypan blue dye exclusion and Acridine orange/Ethidium bromide staining were performed. Third, the phenotypes of cytotoxic immune cells or killer cells of peripheral blood mononuclear cells were studied by immunocytochemistry staining of experimental cell cytospin preparation with monoclonal antibody to CD4, CD8 and CD56. This study elucidated that there was a weak effect of Hep-G2 cell stage on cytolysis susceptibility; i.e. Hep-G2 cell at a

ANALYSIS OF PERIPHERAL BLOOD NOMONUCLEAR CELLS AND INFILTRATING CELLS OF CUTANEOUS WARTS (NO. 737) Potjaman Phumeesat and Molvibha Vongsakul Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, E-mail : [email protected] Key words : Immune cell capability, Human cutaneous wart, in vitro Cutaneous warts in a human are tiny skin infections usually caused by viruses of the human papillomavirus (HPV) family. The most common effected areas are fingers, hands arms and feet. In this study, the immune cells of cutaneous skin wart patients were investigated. Three aspects were studied. First, the immune competence of cutaneous skin wart patients was studied by using proliferation assay of peripheral blood mononuclear cells stimulated with mitogens, superantigens, common antigen and crude wart extract. Second, the identification of peripheral blood mononuclear cells was done by immunocytochemistry staining with CD4, CD8, CD28, CD152, CD86 and +T cell monoclonal antibodies. Third, the investigation of CD86+ and +T populations in skin wart lesions, immunohistochemistry staining of wart cryosection and collagenase extract were performed. The results of this study showed statisti-

276

G2/M stage demonstrated the high percentage of cytolysis susceptibility over G1/S and G0/G1 stage. The results of the cytotoxic assay were 39+1.00% vs 35+3.61% vs 26+2.00%. In addition, the percent of dead Hep-G2 cell in 18 hours of cytolysis incubation time was statistically higher than 4 hours incubation (63+1.00% vs 39+1.00%). Moreover, the phenotypes of peripheral blood mononuclear killer cells in Hep-G2 conjugate were CD56+ > CD8+ >CD4+ (59+0.8%>32+1.4% > 9+1.5%). The findings of this study suggest that tumor cell cycle stage possible have the different effect in cytolysis susceptibility by cytotoxic immune cells or killer cells of the immune response, which is expected to be Cd56+ cell.

1

Faculty of Science

Department of Microbiology, Faculty of Science, Mahidol Uni2 versity and Laboratory of Immunology, Chulabhon Research Institute, Bangkok, Thailand. Two recently produced monoclonal antibodies were used to develop an antigen capture enzyme-linked immunosorbent assay (ELISA) for rapid diagnosis of Penicillium marneffei. The method was evaluated with 53 patients with culture confirmed penicilliosis and 240 controls. The diagnostic sensitivity, specificity, and accuracy of the ELISA were 92.45, 97.5 and 96.59% respectively. (Journal of Clinical Microbiology, Jan 2003, Vol. 41, No. 1, p. 432434.)

IMPROVED GREEN FLUORESCENT PROTEIN REPORTER GENE-BASED MICROPLATE SCREENING FOR ANTI-TUBERCULOSIS COMPOUNDS BY UTILIZING AN ACETAMIDASE PROMOTER (NO. 739) Chartchai Changsen , Scott G. Franzblau , and Prasit 1,2 Palittapongarnpim Department of Microbiology, Faculty of Science, Mahidol Uni2 versity Rama 6 Road, Bangkok 10400, Thailand; Institute for Tuberculosis Research College of Pharmacy, University of Illi3 nois at Chicago, Chicago, Illinois 60612 USA, and National Center for Genetic Engineering and Biotechnology, Pathumthani, 12120, Thailand. The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high throughput antimicrobial drug screening. However, screening for antituberculosis compounds using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal to noise ratio. Therefore and alternative promoter was evaluated for enhanced fluorescence during microplate-based culture and for response to 18 established antimicrobial agents using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains, H Rv, H Ra and 37 37 Erdman were transformed with pFPCA1, containing a red-shifted 2 gfp gene driven by the acetamidase promoter of M. smegmatis mc 155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct with signal: noise ratios of 20.6 -27.8 and 3.8 - 4.5, respectively. The MICs of 18 established antimicrobial agents against all strains carrying pFPCA1 in the GFPMA were within one to two two-fold dilutions of those determined by either fluorometric or visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild type and pFPCA1 transformants in MABA. This optimized GFP assay is sufficiently simple, robust and inexpensive (no reagent costs) to be utilized in routine high throughput screening for anti-tuberculosis compounds. (Antimicrobial Agents and Chemotherapy 2003 Dec. page 36823687.)

1 1,2 2

LEVELS OF MACROPHAGE INFLAMMATORY PROTEIN 1 (MIP-1) AND MIP-1 IN INTERVILLOUS BLOOD PLASMA SAMPLES FROM WOMEN WITH PLACENTAL MALARIA AND HUMAN IMMUNODEFICIENCY VIRUS INFECTION (NO. 741) Sujittra Chaisavaneeyakorn , Julie M. Moore , Lisa Mirel , 4 3 Caroline Othoro , Juliana Otieno, Sansanee C. Chaiyoroj , Ya 1,4 1,6 1 Ping Shi , Bernard L. Nahlen , Altaf A. Lal and Venkatachalam 1 Udhayakumar Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, 3 Geolgia 303331, Center for Tropical and Emelging Global Diseases and Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Ath2 ens, Georgia 30602; Department of Microbiology, Faculty of Sci4 ence, Mahidol University, Bangkok 10400, Thailand; Center for Vector Biology Control and Research, Kenya Medical Research 5 Institute; and Ministry of Health, New Nyanza Provincial Gen6 eral Hospital; Kisumu, Kenya; and Rollback Malaria, World Health Organization, Geneva, Switzerland Macrophage inflammatory protein-1 (MIP-1) and MIP1 play an important role in modulating immune responses. To understand their importance in immunity to placental malaria (PM) and in human immunodeficiency virus (HIV)-PM coinfection, we investigated levels of these chemokines in the placental intervillous blood plasma (IVB plasma) and cord blood plasma of HIV-negative PM-negative, HIV-negative PM-positive, HIV-positive PM -negative women, the MIP-1 concentration in IVB plasma was significantly elevated in HIV-negative PM-positive women. The MIP-l level was not altered in association with either infection. The IVB plasma levels of MIP-l and MIP-1 positively correlated with the cord blood plasma levels of these chemokines. As with IVB plasma, only cord plasma from PM-infected mothers had significantly elevated levels of MIP-1 and MIP-1 levels in PM-positive women were positively associated with parasite density and malaria pigment levels. Regardless of HIV serostatus, the IVB MIP-1 level was significantly lower. In women with PM-associated anemia. In summary, an elevated level of MIP-1 was associated with PM. HIV infection did not significantly alter these two chemokine levels in IVB plasma. (Clinical and Diagnostic Laboratory Immunology, July 2003, Vol. 10, No. 4, p. 631-616)

1 1,2 3 1

ANTIGEN DETECTION ASSAY FOR IDENTIFICATION OF PENICILLIUM MARNEFFEI INFECTION (NO. 740) Sansanee C. Chaiyaroj , Runglawan Chawengkirttikul , Stitaya 1,2 1 Sirisinha , Pramuan Watkins , and Yuttana Srinoulprasert

1,2 1

Mahidol University Annual Research Abstracts, Vol. 31

RELATIONSHIP BETWEEN PLASMA INTERLEUKIN-12 (IL-12) AND IL-18 LEVELS AND SEVERE MALARIAL ANEMIA IN AN AREA OF HOLOENDEMICITY IN WESTERN KENYA (NO. 742) Sjittr Chaisavaneeyakorn , Caroline Othoro , Ya Ping Shi , 4 2 1 Juliana Otieno , Sansanee C. Chaiyaroj , Altaf A, Lal , and 1 Venkatachalam Udhayakumar

1 1,2 3 1, 3

277

Division of Parasitic Disease, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, 2 Georgia 30333; Department of Microbiology, Faculty of Science, 3 Mahidol University, Bangkok 10400, Thailand; Vector Biology and Control Research Center, Kenya Medical Research Insti4 tute and Ministry of Health, New Nyanza, Provincial General Hospital, Kisumu, Kenya. In this study, we investigated whether levels of interleukin-12 (IL-12) and IL-18 in plasma are associated with severe malarial anemia outcomes in an area of holoendemicity in western Kenya. We compared plasma IL-12 and IL-18 levels in six groups of children grouped into the categories aparasitemic, asymptomatic, mild malaria, high-density uncomplicated malaria (UC), moderate malarial anemia (MMA), or severe malarial anemia (SMA). IL-12levels were significantly reduced in children with SMA (P < 0.05) but not in other groups compared to children in the aparasitemic control group. IL-18, a cytokine known to be critical for the induction of gamma interferon along with IL-12, was produced more frequently (70%) in children with UC (P = 0.06) than in children in the aparasitemic control group (32%). However, in the SMA group the IL-18 response rate declined to 30%, which was similar to that in the aparasitemic control group, which showed a 32% response rate. This finding suggests that the IL-18 response may be impaired in children with SMA. In summary, the results from this study support the hypothesis that impairment of IL-12 and/or IL-18 response may contribute to the development of severe malarial anemia in areas of holoendemicity for malaria. (Clinical and Diagnostic Laboratory Immunology, July 2003, Vol. 10, No. 3, P. 362-366.)

ing to degree of sulfation. Our study demonstrated high levels of conidial adhesion to chondroitin sulfate B (CSB), heparin, heparan sulfate (HS), and highly sulfated chitosan (CP-3). Significant inhibitory effect of adhesion to A549 lung carcinoma cells was achieved by these substances. The similar effect was not observed when other chondroitin sulfates (A, C, and D), chitosan and pentosan polysulfate were used. Our results suggested that disaccharide backbone as well as sulfated side chains have influence on fungal adhesion to human lung epithelial cells. We further verified if the inhibitory structure of glycosaminoglycans can affect the adhesion of conidia to laminin and fibronectin. CSB, heparin, HS and CP-3 could significantly inhibit the adhesion of the fungal spore the fibronectin. Nevertheless the levels of inhibition were not comparable to those observed when these compounds were employed to inhibit adhesion to A549 cells. The results indicated that fibronectin and laminin are not the only adhesion receptors on the surface of human lung epithelium. Additionally, we will also present data on the characteristics of laminin adhesion. (103 Annual Meeting American Society for Microbiology Washington D.C., Convention Center, Washington, D.C. May 8-22, 2003.)

rd

CREATION OF A ZYMOGEN

1,2 1

(NO. 744)

2

Parit Plainkum , Stephen M. Fuchs , Suthep Wiyakrutta , and 1,3 Ronald T. Raines

1

Department of Biochemistry, University of Wisconsin, Madison, 2 Wisconsin 53706-1544, USA, Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 3 10400, Thailand. Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1396, USA. Key words : Zymogen, Ribonuclease A, Plasmepsin II Cells produce proteases as inactive zymogens. Here, we demonstrate that this tactic can extend beyond proteases. By linking the N anc C termini of ribonuclease A, we obstruct the active site with the amino acid sequence recognized by plasmepsin II, a highly specific protease from Plasmodium falciparum. We generate new N and C termini by circular permutation. In the presence of plasmepsin 3 II, a ribonuclease zymogen gains - 10 -fold in catalytic activity and maintains high conformational stability. We conclude that zymogen creation provides a new versatile strategy for the control of enzymatic activity, as well as the potential development of chemotherapeutic agents. (Nature Structural Biology, volume 10, number 2, February 2003.)

3-111 CHARACTERIZATION OF ADHESIVE TARGETS OF PENCILLIUM MARNEFFEI CONIDIA (NO. 743) Y. Srinoulprasert, Sanasanee Chaiyaroj Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Penicilliosis is a disseminated an progressive infection caused by a dimorphic fungus, Penicillium marneffei. Very little is known about route of infection and initial interaction of P. marneffei conidia with host cells. We hypothesize that the initial infection may be an adhesion of conidia to extracellular matrices and lung epithelial cells. We investigated role of various extracellular matrix components (chondroitin sulfates, heparin, and heparan sulfate) as a target for P marneffei conidia adherence using a direct adhesion assay. These molecules were also used to block the binding of the fungal spores to potential host target cells. In addition, highly sulfated glycosaminoglycans were tested for the inhibitory effects ow-

CRYSTALLIZATION AND PRELIMINARY X-RAY CRYSTALLOGRAPHIC ANALYSIS OF D-PHENYLGLYCINE AMINOTRANSFERASE FROM PSEUDOMONAS STUTZERI ST201 (NO. 745) Palangpon Kongsaeree , Chariwat Samachart , Poramaet 3 3 3 Laowanapiban , Suthep Wiyakrutta , and Vithaya Meevootisom

1 1,2 1,2

Department of Chemistry, Facultyof Science, Mahidol Univer2 sity, Rama VI Road, Bangkok 10400, Thailand; Center for Protein Structure and Function, Faculty of Science, Mahidol uni3 versity, Rama VI Road, Bangko 10400, Thailand and Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.

278

Key words : D-Phenylglycine aminotransferase, X-ray crystallography, Pseudomonas stutzeri D-Phenylglycine aminotransferase (D-PhgAT) catalyzes the reversible transamination of D-phenylglycine to L-glutamate with 2-oxoglutarate as the amino-group acceptor. Crystals of substrate free Pseudomonas stutzeri D-PhgAT bound to the cofactor pyridoxal5'-phosphate (PLP) were obtained by the hanging-drop vapour diffusion method using ammonium sulfate as a precipitant. The crystals belong to space group P3 21 or P3 21, with unticell parametrs a 1o 2 = b = 75.155, c = 147.554 A . The asymmetric unit contains one molecule of D-PhgAT and has a solvent content of 50.0%. A complete native X-ray diffraction data set was collected from a single o crystal at 100 K to a resolution of 2.3 A . (Acta Crystallographica Section D Biological Crystallography 2003; 59(Pt 5): 953-4.)

Faculty of Science

ANTIMALARIAL DIHYDROISOCOUMARINS PRODUCED BY GEOTRICHUM SP., AN ENDOPHYTIC FUNGUS OF CRASSOCEPHALUM CREPIDIOIDES (NO. 747) Palangpon Kongsaeree , Samran Prabpai , Nongluksna 2 1 2 Sriubolmas , Chanika Vongvein , and Suthep Wiyakrutta

1 1 1

Department of Chemistry and Center for Protein Structure and Function, Faculty of Science, Mahidol University, Rama VI Road, 2 Bangkok 10400, Thailand; Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phyathai Road, Bangkok 10300, Thailand and Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. Key words : Antimalarial, Dihydroisocoumarins, Endophytic fungus Three novel dihydroisocoumarin derivatives with antimalarial, antituberculous, and antifungal activities have been isolated by bioassayguided fractionation from an endophytic fungus, Geotrichum sp., collected from Crassocephalum crepidioides. Structures were established as 7-butyl-6, 8-dihydroxy-3(R)-pent-11enylisochroman-1-one, and 7-but-15-eny-6-8-dihydroxy-3(R)-pent11-enylisochroman-1-one, and 7-butyl-6-, 8-dihydroxy-3 (R) pentylisochroman-1-one using spectroscopic data. (Internation Publication. J. Nat Prod 2003; 66(5): 709-11.)

A SPECTROPHOTOMETRIC ENZYMATIC CYCLING METHOD USING L-GLUTAMATE DEHYDROGENASE AND D-PHENYLGLYCINE AMINOTRANSFERASE FOR DETERMINING LOW LEVELS OF L-GLUTAMATE (NO. 746) Wanida Khampha, Vithaya Meevootisom, and Suthep Wiyakrutta Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, Thailand. Key words : D-phenylglycine aminotransferase, Glutamate, Substrate cycling This report describes a new continuous spectrophotometric method capable of determining low levels of L-glutamate. The assay is based on substrate cycling between L-glutamate dehydrogenase (GIDH) and the novel enzyme D-phenylglycine aminotransferase (D-PhgAT). In this system, GIDH converts L-glutamate to 2oxoglutarate with concomitant reduction of NAD+ to NADH. The 2-oxoglutarate is recycled back to L-glutamate in a transamination reaction catalysed by D-PhgAT using D-4-hydroxyphenylglycine as an amino donor which is converted to 4-hydroxybenzoylformate. Both NADH and 4-hydroxybenzoylformate strongly absorb UV light 3 3 -1 -1 at 340 nm (340 nm = 6.22 x 10 and 8.90 x 10 L.mol .cm , respectively). By this method, the signal amplification effect of the cycling reactions is further enhanced by the combined absorption of the two accumulating reaction products. The calibration curve was linear from 0.2 to 20 µM L-glutamate, with a detection limit of 0.14 µM and a relative standard deviation of 5.1%. Average food samples can be significantly diluted before subjected to the assay, thus reducing the untoward effects of interfering substances. Because of the unique substrate specificity of D-PhgAt, L-glutamate could be selectively determined in the presence of other common amino acids at relatively high concentrations. The procedure is simple, rapid, accurate, and should be easily automated. It was satisfactorily applied to measure L-glutamate in various kinds of food products. (1 International Symposium on Recent Advances on Food Analysis, November 5-7, 2003, Prague, Czech Republic.)

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BIOTECHNOLOGY OF PHYTOESTROGEN-RICH; P. mirifica : VI. MICRONUCLEUS TEST (NO. 748) Punya Temcharoen 1, Wandee Sutjit2 and Wichai Cherdshewasart3

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Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Program of Biotechnology, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10300; 3Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330. The mutagenic activity of White Kwao Krua plant (P.mirifica cultivar-Wichai-III) was evaluate for its chromosome damaging potential by the bone marrow micronucleus test in male rats. Animals received an oral administered with P.mirifica at the dose of 16 g/kg body weight (BW), were killed at 24, 48 and 72 hours after the treatment. Then, bone marrow cells were collected and 1,000 polychromatic erythrocytes (PCEs) per animal were analyzed for the presence of micronuclei formation. Four hundred erythrocytes were scored for the PCEs:NCEs (normochromatic erythrocytes) ratios. The results demonstrated that P.mirifica could not significantly increase the frepuency of MNPCEs in the treated animals. It could conclude that P.mirifica cultivar Wichai-III did not exhibit mutagenicity via micronucleus formation. (Poster presentation in 29 th Congress on Science and Technology of Thailand 20-22 October 2003, Golden Jubilee Convention Hall, Khon Kean University)

Mahidol University Annual Research Abstracts, Vol. 31

EFFECTS OF STEVIOSIDE ON CHROMOSOME ABERRATION IN MALE HAMSTER BONE MARROW CELLS. (NO. 749) Paweena Panyaporn 1, Punya Temcharoen2, Lukana Himakoun2, Pranom Puchadapirom2 and Chaivat Toskulkao3

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that all tested rats were found did not have significantly different on the growth rate, food intake, hematology, AST (GOT), ALT (GPT), ALP, total protein, triglyceride, creatinine and BUN in serum. Therefore, it could be concluded that the NOAEL and ADI of the liquid smoke from bagasse inwater base tested were 10 and 0.01 ml/kg b.w., respectively and the level of liquid smoke fed in Wistar rats by intragastric administration method did not showed any adverse effects. (Poster presentation in 41 st Kasetsart University Annual Conference at Kasetsart University 3-7 February 2003)

Graduate Program in Toxicology, 2Department of Patho-biology, and 3Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Key words : hamster, stevioside, chromosome aberration Stevioside , a natural non-caloric sweetener, is a major component of Stevia rebaudiana Bertoni. It has been used in a variety foods and beverages for diabetics and obese people. The cytogenetic effects of stevioside were examined on male Syrian hamster bone marrow cells in vivo . Each group of five male hamsters was exposed to stevioside 1, 5 and 10 g/kg body weight respectively, by gavages. After 24 hours, metaphase chromosomes from bone marrow cells were collected from femora and analyzed for the chromosomal aberrations. The student's T-test was used for statistical analysis. Cytogenetic damage induced by stevioside at the concentrations of 0, 1, 5 and 10 g/kg body weight were slightly increase when the dose of stevioside was increased but not statistically significant from the control group. The major change was chromosome breaks. Chromosomal rearrangement was not found even at the highest dose of stevioside (10 g/kg body weight). The concentrations of stevioside in this study were higher than the usual daily dose for humans. The research recommends that further study is necessary to predict the long term genotoxic risk of stevioside on human beings. (Presented as poster in the conference "Environmental Science, Technology and Management." At CRI on July 8-11, 2003.)

A SIMPLE IN VITRO ASSAY FOR DETERMINING THE SENSITIVITY OF Plasmodium vivax ISOLATES FROM FRESH HUMAN BLOOD TO ANTIMALARIALS IN AREAS WHERE P.vivax IS ENDEMIC

(NO. 751)

Bruce M. Russell, 1-2* Rachanee Udomsangpetch,3 Karl H. Rieckmann,1 Barbara M. Kotecka, 1 Russell E. Coleman, 2 and Jetsumon Sattabongkot2

1

Australian Army Malaria Institute, Enoggera OLD 4052, Aus3 tralia, and Department of Pathobiology, Faculty of Science, 2 Mahidol University, and Department of Entomology, U.S. Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand. The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P.falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB+ serum. We successfully carried out 34 in vitro drug assays on 39 P.vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean precentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% + 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P.vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC 50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5ng/ml, IC 50 = 0.47 ng/ml). The poor response of P.vivax to both tafenoquine (MIC = 14,000 ng/ml, IC 50 = 9,739 ng/ml) and sulfadoxine (MIC = 50,000 ng/ml, IC 50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P.vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assesing the antimalarial sensivitity of P.vivax populations and for screening new antimalarials sensitivity of P.vivax populations and for screening new antimalarials in the absence of long-term P.vivax cultures. (Antimicrobial Agents and Chemotherapy, Jan. 2003, p.170-173)

STUDY OF ACUTE AND SHORT TERM TOXICITY TEST OF LIQUID SMOKE FROM BAGASSE IN WISTAR RATS (NO. 750) Siwaporn Siwawej 1 , Namaporn Attaviroj 1 and Punya Temcharoen2

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Department of Food Science and Technology, Faculty of AgroIndustry, Kasetsart University, Bangkhen Campus, Bangkok, 10900. 2Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400. The study on acute toxicity test of liquid smoke from bagasse in water base was carried out. 1.5 and 10 ml/kg b.w. of liquid smoke were single intragastric administration to the Wistar rat and clinical sign were observe for 14 days. Results obtained indicated that all tested rats did not show either any adverse effect or death occurred. Therefore, the LD50 of liquid smoke tested was > 10 mg/kg b.w. The investiigation on short term toxicity study showed

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COMPARISON OF THE DEFORMABILITY OF RED BLOOD CELLS PARASITIZED BY Plasmodium falciparum and Plasmodium vivax.

(NO. 752)

Faculty of Science

41% of severe malaria (N=104), and 100% of cerebral malaria patient isolates (N=15); P=.001. Autoagglutination was reversed by heparin (EC50; 50 units/ml) and EGTA (EC 50; 0.01 mM). The median (range) number of IRBC in agglutinates per 1000 IRBC was significantly higher in cerebral malaria (6; 3-42) compared to severe (0; 0-52) and uncomplicated malaria (0; 0-24) (P=.01). In multivariate analyses, high parasitemia and cerebral malaria were associated independently with parasite agglutination. (In press in Journal of Infectious Diseases 2003.)

Rossarin Suwanarusk,1 Brain M. Cooke,2 Arjen M. Dondorp,3,4 Kamolrat Silamut, 3 Jetsumon Sattabongkot, 5 Nicholas J. White,3,4 and Rachanee Udomsangpetch6

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Department of Parasitology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand. 2Department of Microbiology, Monash University, Victoria 3800, Australia. 3Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 4Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, UK. 5Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. 6Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Key words : malaria, P.vivax, red blood cells, deformability Red blood cells (RBCs) must deform considerably, during their multiple passages through the microvasculature and the sinusoids of the spleen. RBCs infected with P.falciparum (Pf-IRBCs) become increasingly rigid as they mature, but avoid splenic clearance by sequestering in venules and capillaries. In contrast P.vivax does not sequester. We compared the effects of P.vivax and P.falciparum infection on RBC deformability in a laminar shear flow system. Pf-IRBCs became more rigid as the parasite matured but equivalent maturation of P.vivax resulted in a doubling of flexibility. Coincidentally, the IRBC surface area increased from 56.7 um 2+ 1.3 to 74.7 um 2+ 0.6 to 90.9 um 2+ 1.1 in ring-, trophozoite, and schizont Pv-IRBCs, respectively, whereas Pf-IRBC did not increase in size. P.vivax increases the deformability of IRBCs and thereby avoids splenic entrapment. (In press in Journal of Infectious Diseases 2003.)

In vitro EFFICACY OF ANTIMALARIAL DRUGS AGAINST P. vivax ON THE WESTERN BORDER OF THAILAND. (NO. 754) Kesinee Chotivanich,1 Rachanee Udomsangpetch, 2 Wirongrong Chierakul, 1 Paul Newton, 1,3 Ronatrai Ruangveerayuth,4 Sasithon Pukrittayakamee, 1 Sorchai Looareesuwan, 1 Nicholas J. White1,3 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 2Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand. 3Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, United Kingdom. 4Mae Sot Hospital, Tak, Thailand. Key words : P.vivax, antimalarial drugs, in vitro drug susceptibility The susceptibility of 20 isolates of P.vivax on the ThaiMyanmar border to 7 antimalarial drugs was evaluated using the schizont maturation inhibition technique. The geometric mean 50% inhibition concentration (IC 50) values were : quinine 308 ng/ml, amodiaquine 14 ng/ml, chloroquine 50 ng/ml, mefloquine 127 ng/ ml, sulfadoxine/pyrimethamine (80:1) 800/10 ng/ml, pyrimethamine 8 ng/ml and artesunate 0.5 ng/ml. Compared with P.falciparum in this area, P.vivax was more sensitive to chloroquine and artesunate, equally sensitive to quinine, and more resistant to mefloquine. (In press in American Journal of Tropical Medicine and Hygiene 2003.)

1

PLATELET-INDUCED AUTOAGGLUTINATION OF P. falciparum INFECTED RED CELLS AND DISEASE SEVERITY IN THAILAND. (NO. 753) Kesinee Chotivanich, 1 Juntima Sritabal, 1 Rachanee Udomsangpetch,2 Paul Newton,1,3 Katarzyna A. Stepniewska, 1,3 Ronatrai Ruangveerayuth,4 Sorchai Looareesuwan, 1 David J. Roberts,5 Nicholas J. White 1,3

1

EFFECTS OF ELEPHANT GARLIC VOLATILE OIL (ALLIUM AMPELOPRASUM) AND T-2 TOXIN ON MURINE SKIN (NO. 755) Sudarat Nguansangiam1, Subhkij Angsubhakorn 2, Sutatip Bhamarapravati 3 and Apichart Suksamrarn4 Department of Pathology, Bangkok Metropolitan Administration Medical College and Vajira Hospital, Bangkok; 2Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok; 3Department of Plant Science, Faculty of Science, Mahidol University, Bangkok; 4Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand. Effects of elephant garlic (Allium ampeloprasum) volatile oil (GVO) and trichothecene (T-2) were studies in Swiss albino mice. The animals were 1) topically applied GVO, 2) topically applied T-2 toxin, 3) topically applied GVO followed by T-2 toxin (GVO/T-2) and 4) T-2 toxin application followed by GVO (T-2/GVO) on the right footpad. All animals were observed by Langerhans cell

1

Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 2Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand. 3Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, United Kingdom. 4Mae Sot Hospital, Tak, Thailand. 5 National Blood Service, John Radcliffe Hospital, Oxford, United Kingdom. Key words : Erythrocyte, Platelet, P.falciparum, Malaria The relationship of platelet mediated autoagglutination of P.falciparum Infected red cells (IRBC) to disease severity was investigated in 182 Thai patients With falciparum malaria. Autoagglutination was evident in 43% of uncomplicated malaria (N=63),

Mahidol University Annual Research Abstracts, Vol. 31

enumeration and pathological changes of the footpad on days 1, 3, 5 and 7. The number of Langerhans cells in the GVO treated group (1,097±33/mm2 to 1,624±19/mm2) was not significantly different when compared with the corresponding control left footpad (1,143±33/mm2 to 1,627±21/mm2). Langerhans cells density in T-2 toxin treated group (629±29/mm2 to 1,090±31/mm2) was reduced by 20-35% of the opposite control footpad (962±40/mm 2 to 1,392±29/mm2). Furthermore, GVO/T-2 and T-2/GVO treated mice showed a decrease in Langerhans cell number than a single T-2 toxin treated group. While Langerhans cells in T-2 toxin, GVO/T-2 and T-2/GVO groups revealed a smaller cell size with shortening dendritic processes when compare to the normal control group. Histopathology finding of the footpad skin in T-2 toxin treated group revealed epidermal desquamation and necrosis with edema and inflammatory cell infiltration. While GVO/T-2 and T-2/GVO showed a similar sequence but a lesser severe degree. These findings suggested that GVO both in pre-and post-treatment could protect-2 toxin induced epidermal damage in a mouse footpad. (Southeast Asian J Trop Med Public Health 2003 (in press)

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cations and adverse events, vital signs and body weight were performed. Blood samples were drawn to determine CD4+ and CD8+ cell counts, percent CD4+ and CD8+. Determination for plasma HIV1 RNA was also done. Genotypic drug resistance testing study was also conducted to determine emergence of drug mutation in volunteers. Results, Discussion and Conclusion: From the 260 volunteers who started in this extension study, 186 volunteers continued and remained until the 232-week duration. Changes in the number of CD4+ T cells by Area-Under-Curve-Minus-Baseline (AUCMB) determine whether a volunteer is a responder or a nonresponder to the REMUNE® treatment. The responders, 51.08% of the 186 volunteers, showed increasing in the number of CD4+ cells and stabilizing the level of HIV-1 RNA copies at week 232. The mean body weight also increased by 1.04 kg at week 232 compared to day 1. Results in the genotypic-drug resistance testing indicate no evidence of mutation that could lead to resistant strain. Standard treatment suggests of using the antiretroviral when CD4+ cell count is lower than 300 cells/mm3. Therefore REMUNE® can be used as an immunomodulator before CD4+ cell counts of HIV-1-infected persons decline to the level that the initiation of antiretroviral drugs is recommended. The results also indicate that REMUNE® can delay the use of antiretroviral drugs, has safety profile, and the absence of drug resistance in REMUNE®-treated cohort enables the use of any available drug whenever necessary. (29th Congress on Science and Technology of Thailand (STT 2003) Khon Kaen University, Khon Kaen, Thailand October 20-22, 2003)

PRELIMINARY RESULTS FROM 232 WEEKS CLINICAL TRIAL USING REMUNETM, AN IMMUNO-MODULATOR, AS INITIAL TREATMENT FOR HIV INFECTION (NO. 756) W. Sukeepaisarncharoen 1, W.Chantratita 2, S. Kulpradist 3, V. Chandeying 4, S. Rugpao 5, B. Israngkura Na Ayudhtaya 6, W. Sirawaraporn 7, C. Boonshuyar 8 and V. Churdboonchart7 Khon Kaen University Khon Kaen, Thailand; 2 Ramathibodi Hospital, Bangkok, Thailand; 3 Vajira Hospital, Bangkok, Thailand; 4 Prince of Songkla University, Songkla, Thailand; 5Chiang Mai University, Chiang Mai, Thailand; 6 Pramongkutklao Hospital, Bangkok, Thailand; 7Faculty of Science, Mahidol University, Bangkok, Thailand; 8Faculty of Public Health, Mahidol University, Bangkok, Thailand. Key words: Immune-based therapy, Immunomodulator, delaying antiretroviral drugs, drug resistance The purpose of this extension of P2101B is to determine the immunotherapy that can delay the AIDS progression. The result of 186 volunteers was classified into two groups, responder and nonresponder, by Area-Under-the-Curve-Minus Baseline (AUCMB) that determines whether the value of AUCMB is positive or negative respectively. The responders, 51.08%, showed increasing number of CD4+ cells and stabilizing level of HIV-1 RNA copies at week 232. Drug-resistant virus also was not found by genotypic test; therefore, REMUNE can be used as an immunomodulator to delay the initiation of antiretroviral treatment and a drug therapy that can be used after immunotherapy failure. Methodology: This is P2101B Extension Study up to 232 Weeks. Volunteers previously enrolled in the 40-weeks, double-blind, randomized, placebo-controlled P2101B study who continued in the Extended Phase II P2101B study up to 132 weeks were still the same in this open-label REMUNE® extension study. The remaining 260 volunteers were grouped into nine cohorts from five clinical test sites (Khon Kaen, Pramongkut, Vajira, Chiang Mai and Songkla). These volunteers were administered with 1.0 ml of REMUNE® through intramuscular injection into the belly or triceps muscle every 12 weeks. Clinical assessment or physical examination, including medi1

BIOCHEMICAL AND HISTOPATHOLOGICAL EFFECTS OF GLYPHOSATE HERBICIDE ON NILE TILAPIA (OREOCHROMIS NILOTICUS)

(NO. 757)

Wannee Jiraungkoorskul, 1 Edward Suchart Upatham,2 Maleeya Kruatrachue,1 Somphong Sahaphong,3 Suksiri Vichasri-Grams,1 Prayad Pokethitiyook1 Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Faculty of Science, Burapha University, Chonburi 20131, Thailand; 3Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Key words : Oreochromis niloticus; Roundup; aminotransferase In Oreochromis niloticus that had been exposed for 3 months to sublethal concentrations (5 and 15 ppm) of the commercial glyphosate herbicide (C3H8NO5P) Roundup, the organs exhibited varying degrees of histopathological change. In the gills filament cell proliferation, lamellar cell hyperplasia, lamellar fusion, epithelial lifting, and aneurysm were observed. In the liver there were vacuolation of hepatocytes and nuclear pyknosis. Kidney lesions consisted of dilation of Bowman's space and accumulation of hyaline droplets in the tubular epithelial cells. The structural damages could be correlated to the significant increase (p<0.05) in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities in the second and third months of exposure. The results indicated that long-term exposure to glyphosate at sublethal concentrations had adverse effects on the histopathological and biochemical alterations of the fish.

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(Contract grant sponsor: Ministry of University Affairs and Faculty of Graduate Studies, Mahidol University in the academic year of 2000-2001. Environmental Toxicology 18: 260­267, 2003.)

Faculty of Science

-thal/Hb E patients, but not detectable in serum of non-thalassemia and found that hemin readily catalyzed free radical reaction [1]. It was previously demonstrated that free radical modulated platelet functions; therefore, we performed the study to evaluate whether hemin was responsible for platelet hyper-function in -thal/Hb E patients. The effects of hemin on platelet aggregation of normal volunteer's blood were performed in whole blood (impedance aggregation) and in platelet rich plasma (optical aggregation) using a Chronolog aggregometer. Platelet release was also measured in PRP. The results showed that hemin was dose dependently induced platelet aggregation and secretion. The synergism between hemin with ADP and collagen was demonstrated. The possible mechanism of hemin-induced platelet aggregation was under-investigated.

BIOCHEMICAL AND ULTRASTRUCTURAL ALTERATIONS IN NILE TILAPIA (OREOCHROMIS NILOTICUS) INDUCED BY GLYPHOSATE HERBICIDE. (NO. 758) Wannee Jiraungkoorskul, 1 Somphong Sahaphong, 1,3 Edward Suchart Upatham, 2,4 Maleeya Kruatrachue, 2 Prayad pokethitiyook2, Suksiri Vichasri-Grams, 2 Suda Riengrojpitak1

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Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. 2Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand .3Mahidol University International College, Mahidol University, Salaya Campus, Nakhonpathom 73170, Thailand. 4Burapha University, Chonburi 20131, Thailand. Key words : aminotransferase, alkaline phosphatase, glyphosate Nowadays, the quantities of use herbicides are increased. In Thailand, Roundup has recently become a popular herbicide used for the control of weed. It is a broad-spectrum, non-selective, postemergent herbicide that is used to kill unwanted plants in wide variety situations. Despite its long and extensive use, there are few reports on long term effects especially on biochemical and ultrastructural alterations in fish. Thus it is essential to study the changes that may occur in different organs. The main aims of the present study were to investigate the biochemical and ultrastructural alterations of Nile tilapia, Oreochromis niloticus, in response to prolonged exposure (three months) to sublethal concentrations of a commercial glyphosate herbicide, Roundup. (This study was supported by the Ministry of University Affairs and Faculty of Graduate Studies, Mahidol University in the academic year of 2000-2001.) (The 11th International Symposium of the World Association of Veterinary Laboratory Diagnosticians and OIE Seminar on Biotechnology, Bangkok, Thailand. June 22-26, 2003)

CHARACTERIZATION OF CHOLESTEROL AND CHOLESTERYL ESTERS IN THE LOW DENSITY LIPOPROTEIN (LDL) IN THALASSEMIA/HEMOGLOBIN E PATIENTS (NO. 760) Rataya Luechapudiporn , Paveena Yamanont , Piyanee 1 1 Rattanachamnong , Noppawan Phumala Morales , Suthat 2 1 Fucharoen and Udom Chantraraksri

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Department of Pharmacology, Faculty of Science, Mahidol Uni2 versity, Bangkok 10400, Thailand and Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. E-mail : [email protected] ac.th Key words : thalassemia, cholesteryl ester, oxidative stress Thalassemia/hemoglobin E (-thal/Hb E) is the most common thalassemia disease in Thailand. The common clinical complications of -thal/Hb E include atherogenesis-related vascular complications such as pulmonary thromboembolism and cerebral thrombosis. Since the oxidative modification of low-density lipoprotein (LDL) plays a key role in atherogenesis, lipid compositions of LDL and oxidative stress markers in the patients should be investigated. In this study, the oxidative modification of LDL was demonstrated by significant increase in level of TBARs (p<0.05) and significant decrease in level of vitamin E (p<0.0001). Level of total cholesterol (TC), free cholesterol (FC), cholesteryl linolenate (CLN), cholesteryl arachidonate (CA), cholesteryl linoleate (CL) and cholesteryl oleate (CO) were significantly lower in LDL of -thal/Hb. Moreover, ratio of CL and CLN to TC were significantly decreased (P<0.0001 and P<0.05 respectively) whereas Ratio of FC and CP to TC were significantly increased (P<0.05). This study shows the change of chemical composition in oxidative LDL of -thal/Hb patient. Moreover our results also demonstrate that CL, the major cholestryl ester in LDL, is the major target for oxidation in LDL of -thal/Hb E.

HEMIN : A CAUSATIVE FACTOR OF PLATELET HYPERRESPONSIVENESS IN -THALASSEMIA/ HEMOGLOBIN E PATIENTS (NO. 759) Jaroen Sarasumcun, Udom Chantharaksri, Noppawan Phumala Morales, Nuttawut Sibmoo and Supeenun Unchern Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Key words : thalassemia, platelet functions, Hemin The response of platelets in splenectomized--Thalassemia/Hemoglobin E (Sp--thal/ Hb E) patients were increased to mechanical and chemical stimulation. However, the precise cause of platelet hyperactivity in these patients has not been elucidated. Recently, our group demonstrated the present of hemin (iron (III) protoporphyrin IX), degradative product of hemoglobin in serum of

LIPID FLUIDITY OF HEMIN INDUCED OXIDATION OF LOW AND HIGH DENSITY LIPOPROTEIN

(NO. 761)

Chalermkhwon Chalermchoung, Paveena Yamanont, Udom Chantharaksri, and Noppawan Phulama Merales Department of Pharmacology, Faculty of Scinence, Mahidol University, Bangkok 10400, Thailand. E-mail : chalermkshwon @yahoo.com

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Key words : fluorescence anisotropy, fluidity, hemin Hemin, a degradative product of hemoglobin, has been reported as a potent oxidative inducer of lipoproteins in vitro. The high concentration of hemin in plasma of -thalassemia/Hb E patients may be responsible for oxidative stress in the patients. Lipid fluidity of lipoproteins is an important factor that determines their metabolism and interaction. In this study, lipid fluidity of hemininduced oxidized lipoproteins was determined using fluorescence anisotropy method. Two fluorescent probes, diphenylhexatriene (DPH) and trimethylamino-diphenylhexatriene (TMA-DPH) were used for determined lipid fluidity at core and surface of lipoproteins, respectively. Decreasing lipid fluidity at core and surface of lipoproteins, respectively. Decreasing lipid fluidity was found at both surface and core region of hemin-oxidized lipoproteins. Alteration of lipid fluidity was correlated with oxidative damage of the particles as showed by tocopherol consumption. Hemin could insert its pro-atherosclerogenic in thalassemia/Hb E by altering fluid state of lipoprotein particles.

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THE EFFECT OF G-PROTEIN ACTIVATORS, MASTOSPORAN AND COMPOUND, ON SEROTONIN SECRETION AND SIGNALLING PATHWAY IN HUMAN PLATELETS (NO. 763) Supachoke Mangmool, Supeenun Unchern, Yupin Sanvarinda and Darawan Pinthong Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Key words : G-protein activators, signaling pathway, human platelets Mastsporan and compound 48/80 have been found to accelerate guanine nucleotide exchange and GTPase activity of purified GTP-binding protein. These compounds can directly activate secretory processes of mast cells, pancreatic islets and adrenal chromaffin cells by penetrating through plasma membranes and directly stimulate membrane GTPase activity and stimulates PLC-mediated events without mediating via receptor binding. This study aims to examine whether these compounds affect secretion of both intact and permeabilized human platelets, and examine the subtype of G3 protein signaling. [ H]-serotonin labeled platelets were pre-incubated for 5 min and were activated with various concentrations of mastoparan and compound 48/80 for 3 min at room temperature or preincubated with streptolysin O (SLO) for 2 min before activation. 3 The amounts of [ H]5-HT release were determined by liquid scintillation counting. Mastoparan was found to produce a concentrationdependent increase in 5-HT release from intact platelets with an EC 50 of 20 µM. The maximal secretion was obtained at the concentration of 60 µM. Similarly, compound 48/80 caused a concentration-dependent increase in 5-HT release with maximal secretion obtained at the concentration of 400 µg/ml. Permeabilized platelets with streptolysin O significantly increase serotonin secretion. To investigate whether the observed stimulation of serotonin secretion is mediated through the G subunit of G-protein, the G-protein blocking agents i (e.g. G -sensitive pertussis toxin, benzalkonium chloride, a selective i G inhibitor, and daunomycin, a lipid bilayer stabilizer) were used. i Mastoparan- and compound 48/80-induced secretion was inhibited by preincubation with pertussis toxin only in SLO-permeabilized platelets whereas benzalkonium chloride and daunomycin did not affect mastoparan- and compound 48/80-induced secretion in both intact and SLO-permeabilized platelets. The results from this study suggested that mastoparan- and compound 48/80 promoted secretion by mechanisms involved neither the stimulation of G -subtype i of G-protein nor interfering with lipid bilayer of the membranes. The secetory event may result either from a direct fusogenic action and/or from the stimulation of putative exocytosis-linked G-protein, G . Their mechanisms on small GTPase proteins, on G and on meme e brane perturbation in human platelets remain to be elucidated. (This work is supported by Institutional Strengthening Program)

CHARACTERIZATION OF LOW DENSITY LIPOPROTEIN (LDL) IN -THALASSEMIA/ HEMOGLOBIN E PATIENTS BY THE FLUORESCENT POLARIZATION PROBE (NO. 762) Rataya Luechapudiporn , Chalermkhwon Chalermkchong , 1 2 1 Paveena Yamonont , Suthat Fucharoen , Udom Chantharaksri , 1 and Noppawan Phumala Morales

1 1 1

Department of Pharmacology, Faculty of Science, Mahidol Uni2 versity, Bangkok 10400, Thailand and Thalasssemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. E-mail : [email protected] Key words : Anisotropy, Thalassemia, Lipid fluidity, Low density lipoprotein Atherogenesis-related vascular complications such as pulmonary thromboembolism are common in -thalassemia/hemoglobin E (-thal/Hb E) patients. Since oxidative modification of low density lipoprotein (LDL) plays a key role in atherogenesis, physical properties and chemical compositions of LDL in the patients should be characterized. In this study, lipid fluidity of LDL separated from plasma of non-thalassemia and -thal/Hb E was examined by using a fluorescence polarization probe, 1, 6diphenylhexatriene (DPH). The probe is localized mainly in the lipoprotein core. The fluorescence polarization anisotropy (r) was calculated from fluorescence intensities parallel and perpendicular to the exciting polarized light plane. This r value was inversely related to lipid fluidity. The r value of LDL separated from -thal/ Hb E was significantly decreased (p<0.001), indicating less lipid fluidity of LDL in the patients. In the patients, the r value of LDL showed the a good correlation with cholesteryl linoleate ( r = 0.511, p<0.01) and a slightly correlation with cholesteryl arachidonate (r = 0.511, p < 0.01) and a slightly correlation with cholesteryl arachidonate (r = 0.376, p < 0.05). A correlation with cholesteryl linoleate : cholesteryl oleate ratio (r = 0.326, p < 0.05) was also found, indicating that alteration of lipid fluidity related to degree of oxidative stress in LDL. Lipid fluidity of LDL is an important parameter to determine its biological functions. Therefore, the vascular complication in -thal/Hb E could be explained, in part, by the changes of physicochemical properties of LDL.

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THE EFFECT OF EXEMASTANE A STEROIDAL AROMATASE INHIBITOR ON ENDOGENOUS STEROID LEVEL AND THE DETERMINATION OF EXEMASTANE IN URINE (NO. 764) Navaporn Saardpun , Tongtavach Anukarahanonta , Yupin 1 1 1 Sanvarinda , Surin Plasen , and Darawan Pinthong Department of Pharmacology, Faculty of Science, Mahidol Uni2 versity, Bangkok 10400, Thailand. National Doping Control Center, Mahidol University, Bangkok 10400, Thailand. Key words : aromatase inhibitor, exemestane detection, steroid hormone profiles Exemestane (Aromasin®) is a steroidal aromatase inhibitor which irreversibly blocks the conversion of androgens to estrogens. The use of this drug was expected to elevate androgen but decrease estrogen level. Recently, aromatase inhibitors become abusively used as doping drug and already entered in IOC doping list since 2001. However there is no strong evidence to support the benefit of this drug abuse in doping. The aims of this study are to investigate whether a single 200-mg oral administration of exemestane affects endogenous steroid hormone level, and developed a sensitive method for determination of exemestane in urine. Twenty healthy male volunteers were recruited in this study. All subjects were taken a single oral administration of 200 mg exemestane. Urine samples were collected on the day before and through 7 days after drug administration. Each sample was divided into 2 aliquots. First aliquot was taken to analyze the exemestane level by enzymatic hydrolysis, liquid phase extraction and HPLC/DAD. Second aliquot was taken to determine the endogenous steroid level using GC/MS. The procedures for the determination included solid-phase extraction, enzymatic hydrolysis, liquid phase extraction and derivatization. Exemestane level excreted in urine were shown in excretion rate (ng/hr) over time whereas the results of endogenous steroid level were shown in percentage of changing level from baseline. The method was validated in terms of reproducibility, recovery and precision in the range 10-2,000 ng/ml urine and the detection limit of 9 ng/ml. The effect of a single oral dose 200 mg exemestane on endogenous level revealed a remarkably increase in androstendione and to the lesser extent, other androgens, testosterone, epitestosterone, DHT and DHEA. The detectable level of exemestane in uring existed in short period of time. However, testosteone and estrone ratio (T/E ) increased continuously 1 through 7 days after drug administration. This ratio could be a good marker for possible use of exemestane in doping analysis. In conclusion, a single oral dose of exemestane significantly increased androgen level, which may be benefit in doping. The prolonged inhibition of aromatase inhibitors a multiple administratio may sustain the rise of androgens and can be exploited to doping.

1 1 2

MOLECULZR MECHANISM OF UTEROGLOBIN RELATED PROTEIN 1 (UGRP1) GENE INDUCTION BY INTERLEUKIN-10 AIRWAY EPITHELIAL CELLS (NO. 765) Achara Srisodsai , Yoshihiko Chiba , Faruk Sheikh , Howard A. 4 3 1 Young , Raymond P. Doneelley , Porntip Supavilai , and Shioko 2 Kimura Department of Pharmacology, Faculty of Science, Mahidol Uni2 versity, Bangkok, Thailand. Laboratory of Metabolism, National Cancer Institute, National Instiutes of Health, Bethesda, MD, 3 USA. Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, 4 Bethesda, MD. USA. Laboratory of Experimental Immunology, National Cancer Institute, Frederick, MD, USA. Key words : interleukin-10, uteroglobin related protein 1, airway epithelial cells, STA3 Objective : To understand the molecular mechanism of UGRP1 induction b IL-10 in airway epithelial cells. Methods : The effects of of IL-10 on the expression of UGRP1 mRNAs using human lung adenocarcinoma NCI-H441 cells were examined by Northen Blot analysis and the UGRP1 promoter activity was measured by transient transfection analysis. Gel shift analysis was then used to identify transcription factor(s) that may be responsible for IL-10 induction of UGRP1 gene expression. Results : It was demonstrated that IL-10 induces UGRP1 gene expression in human lung NCI-H441 cells at the tranwcriptional leel in a rapid and stable fashion. Two T/EBP binding sites located in the hUGRP1 gene promoter is responsible for IL-10 induction of hUGRP1 gene expression. IL-10R1 and IL-10R2 are expressed in H441 cells, however, STAT3 is barely activated upon IL-10 treatment. In contrast, STAT3 was strongly actiated when cells were treated with other IL-10 family cytokines such as IL-22 and IFNwhile no UGRP1 expression was induced. In vivo experiments using mouse lungs exhibited the increase of Ugrp1 mRNA by IL-10. Conclusion : These results demonstrate that IL-10 induces UGRP1 gene expression in lung epithelial cells through a T/EBP/ NKX2.1 -dependent, STAT3-independent pathway. (Supported by the Royal Golden Jubilee Scholarship and NIH Research Grant)

1 1 2 3

COUPLING MORPHOGENESIS TO MITOTIC ENTRY (NO. 766) Krisada Sakchaisri , Satoshi Asano , Li-Rong Yu , Mark J. 3 1 1 1 Shulewitz , Chong J. Park , Jung-Eun Park , Young-Wook Cho , 2 4 3 Timothy D. Veenstra , Porntip Supavlai , Jeremy Thorner , 1 Kyung S. Lee Laboratory of Metabolism Center for Cancer Research, National 2 Cancer Institute, NIH Bethesda, MD; Mass Spectrometry Cen3 ter, NCI-Frederick, Frederick, MD; Department of Molecular 4 and Cellular Biology, University of California, Berkley, CA; Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand.

1 4 1 2

Mahidol University Annual Research Abstracts, Vol. 31

Key words : Cdk1 regulation, mitotic entry, G2/M transition, Saccharomyces cerevisiae Objective : To study regulatory pathway of Swe1 in Saccharomyces cerevisiae Methods : To identify the upstream kinase(s) of Swe1, six kinases purified from Sf9 cells were subjected to kinase assay using GST-Swe1 (K473A) purified from bacterial cells as a substrate. For Swe1 modification, total cellular proteins were prepared from yeast cells both wild-type and mutants as indicated. Immunoblotting analyses were carried out using antibodies against Swe1, Myc-epitope (for Hsl1-Myc ), Cdc5- and Cla4-dependent 13 phosphorylation sites o Swe1 in vitro were mapped within tryptic peptide using mass spectrometry. Site-directed mutagenesis was used to introduce mutations (Ser to Ala or Thr to Ala) in a functional (cMyc) -tagged Swe1. The resulting constructs were integrated at the 12 URA3 locus in a strain carrying an mith1. To examine the morphology, yeast cells were fixed with formaldehyde and then examined by DIC microscopy. Results : The investigation has led to demonstrate that sequential multi-step phosphorylation by Cla4/PAK and Cdc5/Polo make Swe1 susceptible to subsequent degradation in a septin-dependent manner. Cla4-medited Swe1 phosphorylation occurs in S phase, whereas Cdc5-mediate phosphorylation happens prior to entry into mitosis. Conclusions : The septin collar serves as an organizing platform to permit Cla4 and Cdc5 to sequentially phosphorylate Swe1 and that this cumulative multi-kinase-dependent modification in critical for Swe1 degradation and therefore for proper onset of mitosis. (Supported by the Royal Golden Jubilee Scholarship and NIH Research Grant)

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ous injection of estrogen (5 µg/rat), progesterone (1 mg/rat), or estrogen plus progesterone three times a week all effectively prevented the upregulation of the 1-adrenoceptors. Western blot analyses using polyclonal antibody of 1-adrenergic receptors revealed the same pattern of changes in the protein content of the receptors in these various groups of experimental hearts as those obtained from the receptor binding assay. These results suggest a possible direct suppressive effect of ovarian sex hormones on the expression of cardiac 1-adrenergic receptors. (Published in Life Sciences 72: 1813-1824, 2003. Research Grants: The Thailand Research Fund, Faculty of Science, and Mahidol University to J. Wattanapermpool.

STUDIES OF PROLACTIN ACTION ON SOLVENT DRAG-INDUCED ACTI VE CALCIUM TRANSPORT IN RAT DUODENUM (NO. 768) Chaiyot Tanrattana and Nateetip Krishnamra Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand Key words : active calcium absorption, paracellular transport, prolactin The duodenal active calcium (Ca) absorption was composed of two pathways i.e., transcellular and solvent drag-induced paracellular pathway. We have recently shown that prolactin (PRL), with its novel role in the regulation of Ca metabolism, enhanced the transcellular component. Therefore, the present study aimed to evaluate the acute PRL effect on the paracellular active Ca transport in the duodenum of 200-250 g female Wistar rats. Ussing chamber technique was used to measure the electrical parameters and the bidirectional calcium fluxes (as calculated from the specific activity of 45Ca). The transcellular Ca transport was inhibited by adding Ca 2+ATPase inhibitor into the serosal solution. PRL in the concentrations of 200 and 600 ng/ml significantly increased the mucosa-to-serosa flux of calcium from the control value of 24.3 ± 2.36 nmol.hr-1.cm-2 to 45.42 ± 3.47 (p<0.01) and 63.82 ± 5.28 nmol.hr-1.cm-2 (p<0.001) in a dose-response manner. Higher concentrations of 800 and 1,000 ng/ml, however, returned the fluxes towards control value i.e., 53.93 ± 5.41 and 29.05 ± 2.61 nmol.hr-1.cm-2 thus showing the biphasic action of PRL. Plotting of the mucosa-to-serosa and serosa-to-mucosa flux ratio confirmed the existence of the two components of the active Ca absorption. The finding that prolactin had no effect on the jejunum while cytochasasin E (inhibitor of cytoskeleton activity) increased the flux of mannitol (a paracellular marker) in the jejunum without affecting the mucosato-serosa flux of Ca demonstrated the separate size and charge selective properties of tight junction. In contrast, duodenum mannitol flux was not affected by cytochalasin E, while calcium flux was significantly enhanced by 600 ng PRL/ml. The results suggested that (i) duodenal and jejunal tight junctions were structurally different, and (ii) PRL altered the charge selectivity of the intercellular channels without affecting the size selectivity of duodenal tight junction. Therefore, it was concluded that PRL directly and acutely enhanced the paracellular solvent drag-induced active calcium absorption in the duodenum. This paracellular transport mechanism for calcium was not directly related to the junctional transport of mannitol.

UPREGULATION OF 1-ADRENERGIC RECEPTORS IN OVARIECTOMIZED RAT HEARTS

(NO. 767)

Ariyaporn Thavornkriwong, Surin Preawnim, and Jonggonnee Wattanapermpool Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, THAILAND. Key words : 1-adrenergic receptors, ovarian sex hormones Changes in cardiac myofilament Ca2+ activation have been demonstrated in ovariectomized rats. The underlying mechanisms responsible for these changes, however, are unknown. Accordingly, we measured both density and binding affinity of cardiac 1-adrenergic receptors in sarcolemmal preparations from 10-week ovariectomized rats, pair-fed ovariectomized rats, and sham-operated control rats. Receptor protein content was also measured by immunoblotting. Deprivation of ovarian sex hormones for 10 weeks induced a significant upregulation of 1-adrenergic receptors without affecting binding affinity. The same magnitude of receptor upregulation was also detected in pair-fed ovariectomized hearts. To determine which hormone is responsible for the observed increase in 1-adrenergic receptor density, various sex hormone supplemental regimens were administered to ovariectomized rats. Subcutane-

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(Supported by The Thailand Research Fund and presented orally at the Annual Meeting of the Physiological Society of Thailand, Khonkhaen University 2003.)

Faculty of Science

INTERACTION OF HUMAN AND RAT ORGANIC ANION TRANSPORTER 2 WITH VARIOUS CEPHALOSPORIN ANTIBIOTICS (NO. 770) Suparat Khamdang a,b , Michio Takeda a, Ellappan Babu a, Rie Noshiroa, Maristela Lika Onozato c, Akihiro Tojo c, Atsushi Enomotoa, Xiu-Lin Huangd, Shinichi Karikawad, Naohiko Anzaia, Pawinee Piyachaturawat b, Hitoshi Endoua*

*

ACUTE AND CHRONIC EFFECTS OF PROLACTIN ON PASSIVE CALCIUM ABSORPTION IN SUCKLING RATS Suwimol Amnattanakul and Nateetip Krishnamra

(NO. 769)

Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand Key words : passive calcium absorption, prolactin, tight junction permeability Previous studies found that prolactin enhanced the passive calcium (Ca2+) absorption in sexually mature and young, but not in aged rats. The action depended on the presence of 80 mM or higher concentration of sodium in the lumen of the small intestine. The effects of an acute intraperitoneal and seven day subcutaneous admininistration of 2.5 mg prolactin/kg body weight on the passive Ca2+ absorption in the suckling rats were presently investigated in the in situ 5 cm intestinal segments. Electrolyte solution(0.4 ml) containing 20 mM Ca 2+ and 124 mM, 80 mM or 40 mM or sodium free solution were instilled into the intestinal segment. 3 H-mannitol and 45Ca were used to evaluate the tight junction permeability and Ca 2+ fluxes, respectively. Bromocriptine was administered (3 mg/kg body weight, twice daily) in some animals to inhibit the secretion of endogenous prolactin in an attempt to evaluate its significance. The results showed no acute effect of prolactin on passive Ca2+ absorption in the suckling rats, which was different from that of adult rats. However, the endogenous prolactin enhanced Ca2+ absorption in the duodenum and jejunum, but not ileum or colon , just like in the adult rats. In the absence of endogenous prolactin, duodenal lumen to plasma Ca2+ (CaL-P) flux was significantly reduced from 1.94 ± 0.27 to 1.23 ± 0.2 µmol/cm/hr (p<0.05). Administration of a high dose of prolactin (2.5 mg/kg body weight), in contrast to endogenous prolactin, decreased the duodenal CaL-P flux, thus, confirming the biphasic action of prolactin also seen in the adult rats. Interestingly, the reduction of luminal sodium concentration to 40 mM or absence of luminal sodium was found to enhance the jejunal calcium absorption. Furthermore, the stimulatory effect of endogenous prolactin was absent in the presence of luminal sodium of 40 mM. when paracellular transport of 3H-mannitol and 45Ca was concurrently studied in the presence of 124 mM sodium or absence of luminal sodium, it was found that they were not parallel. The sodium free electrolyte solution enhanced the paracellular absorption of 45Ca but not 3H-mannitol, suggesting that the passive absorption of Ca2+ was not regulated simply by the size-restricted tight junction. In conclusion, our studies demonstrated some characteristics of the passive calcium absorption in the sucking rats and its response to prolactin that were different from that observed in adult rats. (Supported by The Thailand Research Fund.)

Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan; bDepartment of Physiology; Faculty of Science, Mahidol University, Bangkok, Thailand; cDepartment of Nephrology and Endocrinology, University of Tokyo, Tokyo, Japan; d Kobuchizawa Laboratories, Fuji Biomedixs Co., Yamanashi, Japan Key words : Organic anion transporter; Proximal tubule; Cephalosporin antibiotic; Transport; Cell line Cephaloosporin antibiotics are thought to be excreted into the urine via organic anion transporters (OATs) and OAT can mediate nephrotoxicity by cephalosporins, particularly by cephaloridine. The purpose of this study was to elucidate the interaction of humanOAT2 and rat-OAT2 with cephalosporin antibiotics using proximal tubule cells stably expressing human-OAT2 and rat-OAT2. HumanOAT2 is localized to the basolateral side of the proximal tubule, whereas rat-OAT2 is localized to the apical side of the proximal tubule. Cephalosporins tested were cephalothin, cefoperazone, cefazolin, ceftriaxone, cephaloridine, cefotaxime, cefadroxil and cefamandole. These cephalosporins dose-dependently inhibited organic anion uptake mediated by human-OAT2 and rat-OAT2. There was no species difference observed for the effects of ATO2 with cephalosporins between human and rat transporters. Kinetic analysis revealed that the inhibitory effects for human-OAT2 were competitive. Cephaloridine significantly decreased the viability of cells stably expressing human-OAT2, human-OAT1, human-OAT3 and human-OAT4. The decreased viability of cells stably expressing human-OAT1, human-OAT3 and human-OAT4 but not humanOAT2 was reversed by probenecid. In conclusion, human-OAT2 interacts with cephalosporins, and thus, human-OAT2 may mediate the uptake of cephalosporins on the basolateral side of the proximal tubule. The interaction of human-OAT2 with cephalosporins was the weakest among the basolateral human-OATs tested. In addition, it is suggested that human-OATs mediate cephaloridine-induced nephrotoxicity. (Published in European Journal of Pharmacology 465 (2003): 1-7)

IDENTIFICATION OF A NOVEL VOLTAGEDRIVEN ORGANIC ANION TRANSPORTER PRESENT AT APICAL MEMBRANE OF RENAL PROXIMAL TUBULE (NO. 771) Promsuk Jutabha3, Yoshikatsu Kanai1, Makoto Hosoyamada 2, Arthit Chairoungdua3, Do Kyung Kim4, Yuji Iribe2, Ellappan Babu 2 , Ju Young Kim2 , Naohiko Anzai 1 , Varanuj Chatsudthipong3, and Hitoshi Endou1

1

Department of Pharmacology and Toxicology, Kyorin University, School of Medicine, Japan, 2Department of Oral Physiology, Chosun University, College of Dentistry, Korea, and 3Department of Physiology, Faculty of Science, Mahidol University.

Mahidol University Annual Research Abstracts, Vol. 31

Key words : OATV1, renal proximal tubule A novel transport protein with the properties of voltagedriven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size fractioned poly (A)+ RNA and screened for p-aminohippurate (PAH) transport in a high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transport 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 6065% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine. (The Journal of Biological Chemistry, USA., Vol. 278, No. 30, pp. 27930-38, 2003)

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determine the relative contribution of OCT1 and OCT2 for TEA transport in single S2 segments of rabbit RPT. Cimetidine and NBDTMA were high-affinity inhibitors of TEA transport in S2 segments (median IC50 values of 12.3 and 1.4 microM, respectively); in comparison, tyramine and pindolol were low-affinity inhibitors (265 and 69.3 microM, respectively). These IC50 values were sufficiently close to those for OCT2 to support the conclusion that TEA transport in the S2 segment of rabbit RPT is dominated by OCT 2. However, the profile of inhibition of tyramine (an OCT1-selective substrate) transport in single S2 segments indicated that, despite a comparatively low level of expression, OCT1 can play a dominant role in the uptake of selected OC substrates. (Am J Physiol Renal Physiol, 2003, Vol. 285 (6): F1149-59. Research supported by: The Royal Golden Jubilee & Thailand Research Fund)

MECHANISMS OF STEVIOL TRANSPORT BY HUMAN ORGANIC ANION TRANSPORTERS 1 AND 3 (hOAT1 AND 3) (NO. 773) Chutima Srimaroeng 1,2, Varanuj Chatsudthipong 2, John B. Pritchard1

1

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, NC. 2 Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Key words : hOAT1, hOAT3 The natural sweetening agent stevioside and its metabolite steviol have been shown to affect the transepithelial transport of para-aminohippuric acid (PAH) in isolated S2 segments of rabbit renal proximal tubules. Both compounds inhibit by interfering with the basolateral entry step. In humans, two organic anion transporters OAT 1 (hOAT1) and 3 (hOAT3) are strongly expressed on the basolateral membrane and regarded as the major transporters that contribute to the elimination of either endogenous or exogenous compounds. In this investigation, cloned hOAT 1 and hOAT3 expressed in Xenopus laevis ooctyes were used to establish whether one or both transporters were involved in the transport of stevioside and steviol. Steviol significantly inhibited PAH and estrone sulfate (ES) transport in a dose-dependent manner in hOAT1 or hOAT3-expressed ooctyes; whereas, stevioside had no inhibitory effect on PAH and ES transport. The IC50 of steviol for hOAT1 was 11.14 µM and its IC50 for hOAT3 was 62.64 µM. Kinetic studies showed that steviol acted as a competitive inhibitor of hOAT1 (Ki 5.80 µM) but as an non-competitive inhibitor of hOAT3 (ki 80.14 µM). A trans-stimulation study was used to investigate the transport mechanism of steviol. A low concentration of steviol, 1 µM, trans-stimulated 3HPAH transport via both hOAT1 and hOAT3. Interestingly, high concentrations of steviol demonstrated trans-inhibition of 3H-PAH transport via both hOAT1 and hOAT3. In conclusion, the parent compound, stevioside, has no interaction with either hOAT1 and hOAT3; whereas, both hOAT1 and hOAT3 mediate the transport and excretion of steviol. (Research supported by: Thailand Research Fund. Presented in: 43rd Annual Meeting "The American Society for Cell Biology", San Francisco, CA, USA. Dec. 13-17, 2003)

FUNCTIONAL MAPPING OF rbOCT1 AND rbOCT2 ACTIVITY IN THE S2 SEGMENT OF RABBIT PROXIMAL (NO. 772) Santi Kaewmokul1, Varanuj Chatsudthipong1, Kristen K. Evans2, William H. Dantzler 2, Stephen H. Wright2,

1

Department of Physiology, Faculty of Science, Mahidol University. 2Department of Physiology, University of Arizona, Tucson, AZ, USA. Key words : rbOCT1, rbOCT2, rabbit proximal tubule A strategy was developed to determine the distribution of activity mediated by the organic cation (OC) transporters OCT1 and OCT2 in rabbit renal proximal tubule (RPT). Both transporters displayed similar affinities for tetraethylammonium (TEA; in CHOK1 cells, TEA concentrations that resulted in half-maximal transport were 19.9 and 34.5 microM for OCT1 OCT2, respectively). Similarly, some OCs showed little capacity to discriminate between the two processes (IC50 values for ephedrine of 13.6 and 24.2 microM for OCT1 and OCT2, respectively). However, OCT2 had a higher affinity for cimetidine and [2-(4-nitro-2,1,3-benzoxadiazol-7-yl) aminoethyl] trimethylammonium (NBD-TMA; 1.3 and 1.4 microM, respectively) than did OCT1 (97.3 and 108 microM, respectively). Conversely, OCT1 had a higher affinity for tyramine and pindolol than did OCT2 (21.2 and 2.4 vs. 361 and 50 microM, respectively). We designated these as "discriminatory inhibitors" and used them to

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THE ROLE OF NITRIC OXIDE, ANGIOTENSIN II AND ENDOTHELIN ON RENAL FUNCTION AFTER ISCHEMIA/REPERFUSION IN RATS (NO. 774) Varanuj Chatsudthipong and Umarat Srisawat Department of Physiology, Faculty of Science, Mahidol University, Rama VI, Rajathevi, Bangkok 10400 Thailand. Key words : Nitric oxide, angiotensin II, endothelin This study evaluates involvement of nitric oxide (NO), angiotensin II and endothelin in renal function after ischemia/ reperfusion (I/R) and identifies agents that may enhance the recovery of renal function. Renal hemodynamics and handling of water and electrolytes were determined using clearance techniques before and after ischemia induced by 20 minutes of renal artery occlusion. In ischemic kidney, GFR and RBF decreased by ~69% and ~78% respectively in the first reflow period. Urine output increased ~45% and Na+ excretion increased ~13%. Without treatment, indicated parameters of kidney function slightly improved. Infusion of L-arginine and L-NAME before ischemic induction had no effect. Pretreatment with captopril (ACE-inhibitor) prevented I/R-induced changes in renal function. Similar results were observed in posttreatment with BQ-123 (ETA- receptor antagonist), SNP and losartan (AT1-receptor antagonist) during the reflow period. However, a slight decrease in mABP was obserbed in captopril, BQ-123 and SNP treatment groups but not in losartan treatment group. These results indicate changes in renal function after I/R due to lack of endogenous NO activity and increase in angiotensin and endothelin activities. This study also suggests that losartan has the most potential therapeutic use in prevention of post-ischemic decrements in renal function. (Research supported by: Faculty of Science, Mahidol University Presented in: Experimental Biology 2003, San Diego, USA. April 1115, 2003)

Faculty of Science

tively. Organic anion drugs such as pentobarbital and bromsulphalein were also tested and it was found that both rbOCT 1 and rbOCT2 can interact with these drugs. The IC50s of pentobarbital were 77.7, 19.9 µM and sulfobromopthalein were 94.6, 60.8 µM for rbOCT1- and rbOCT2 mediated uptake, respectively. These data suggest that rbOCT1 and rbOCT2 play roles in secretion of both organic cation and organic anion drugs from the body. (Research supported by: The Thailand Research Fund. Presented in: Experimental Biology 2003, San Diego, USA. April 11-15, 2003)

THE INTERACTION OF STEVIOL WITH RABBIT OCT1 AND OCT2 (NO. 776) Varanuj Chatsudthipong, Anusorn Lungkaphin, and Santi Kaewmokul Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Key words : Steviol, rbOCT1, rbOCT2 Previously we have demonstrated the interaction of steviol, a major metabolite of stevioside (a non-caloric natural sweetener), with human organic anion transporters 1 and 3 (hOAT 1 and hOAT3) expressed in renal S2 cells. These findings are consistent with our previous observations on the effect of steviol on PAH transport in isolated perfused S2 segments of rabbit renal proximal tubules (JPET, 2001). No interaction of stevioside with those transporters was observed. The present study was conducted to determine whether stevioside and/or steviol interact with rabbit organic cation transporters 1 and 2 (rbOCT1 and rbOCT2) expressed in CHO cells. Stevioside had no effect on OCT1 or OCT2-mediated TEA uptake into CHO cells. In contrast, TEA uptake via these transporters was markedly inhibited by steviol in a dose-dependent manner. A higher concentration of steviol was required to significantly inhibit TEA uptake via rbOCT 2 than via rbOCT1. The calculated Kis of steviol for rbOCT1 and rbOCT2 expressed in CHO cells were 17 and 312 µM, respectively. To investigate this further, the effect of stevioside and steviol on TEA uptake into single non-perfused S2 segments of rabbit proximal tubules was examined. Both were found to inhibit TEA uptake, but the inhibitory effect of steviol was much greater than that of stevioside (Kis of 120 vs. 603 µM, respectively). These data suggest that steviol interacts with rbOCT1 and rbOCT2 in both cell culture expression systems and intact tubules, but an interaction of stevioside with cation transporters was observed only in the intact renal tubules. (Research supported by: The Thailand Research Fund. Presented in: Experimental Biology 2003, San Diego, USA. April 11-15, 2003)

MULTISPECIFICITY OF rbOCT1 AND rbOCT2

(NO. 775)

Varanuj Chatsudthipong and Santi Kaewmokul Department of Physiology, Faculty of Science, Mahidol University, Rama VI, Rajathevi, Bangkok 10400 Thailand. Key words: rbOCT1, rbOCT2 Organic cation transporters (OCTs) play roles in the elimination of cationic drugs and endogenous metabolites in renal proximal tubules. However, there are differences in substrate specificity for these transporters. The present study was aimed at determining the specificity and affinity of various drugs for rbOCT 1 and rbOCT2 stably expressed in CHO-K1 cells. The inhibition of [3H]TEA uptake in these stable clones by organic drugs was studied. Organic cation drugs such as trimethoprim, procainamide and propanolol, categorized as an antimicrobial drug, an antiarrhythmic drug and a -adrenoceptor blocker, were tested. The IC 50s of trinethoprim, procainamide and propanolol for rbOCT 1 and rbOCT2-mediated [3H]TEA uptake were as follows: trimethoprim (5.7, 4.7 µM), procainamide (9.2, 21.2 µM) and propanolol (3.1, 76.6 µM), respec-

INTERACTION OF THE METAL CHELATOR, DMPS, WITH OAT1 AND OAT3 IN INTACT ISOLATED RABBIT RENAL PROXIMAL TUBULES (NO.777) Anusorn Lungkaphin 1, Varanuj Chatsudthipong 1, Kristen K. Evans2, Carlotta E. Groves 2, Stenphen H. Wright2, and William H. Dantzler2 Department of Physiology, Faculty of Science, Mahidol University. Department of Physiology, University of Arizona.

Mahidol University Annual Research Abstracts, Vol. 31

Key words : DMPS, OAT1, OAT3 DMPS is used clinically to increase urinary excretion of heavy metals, including mercury and arsenic. We used single S2 segments and suspensions of rabbit renal proximal tubules (RPT) to test the interaction of this anionic heavy metal chelator with basolateral transporters, OAT1 and OAT3. RT-PCR revealed expression of both transporters in single S2 segments. [(3)H]pAminohippurate ([(3)H]PAH), and estrone sulfate ([(3)H]ES) were used as specific substrates for rbOAT 1 and rbOAT3, respectively. PAH and ES were transported into non-perfused single RPT segments with Kts of 67 +/- 20 and 3.4 +/- 1.2 micro M, respectively, and into tubule suspensions with Kts of 58 +/- 17 and 7.7 +/- 2.1 micro M, respectively. Reduced DMPS (DMPSH) inhibited uptake of both substrates into single tubule segments with Kapps of 405 +/ - 49 micro M (for [(3)H]PAH and 320 +/- 66 micro M (for [(3)H]ES). Oxidized DMPS (DMPSS), the prevalent from in the blood, also inhibited uptakes of [(3)H]PAH (Kapp of 766 +/- 190 micro M) and [(3)H]ES (696 +/- 166 micro M). Inward gradients of ES, DMPSH an DMPSS trans-stimulated the 30 sec efflux of preloaded [(3)H]ES across the basolateral membrane of RPT. Similarly, DMPSH, and PAH itself, trans-stimulated the 15 sec efflux of [(3)H]PAH. In contrast, efflux of [(3)H]PAH was inhibited by presence of DMPSS in the bathing medium. These data suggest that, whereas both OAT 1 and OAT3 probably transport DMPSH, DMPSS transport may be limited to OAT3. This is the first evidence showing that both OAT1 and OAT3 can transport DMPS across the basolateral membrane of renal proximal tubules. (Am J Physiol (Renal Physiol) 2003 (in press) Research supported by: The Ministry of University Affairs.)

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sequences have 99% and 98% similarity to C. gloeosporioides in Genbank Database. The two cultures have 3 different base pairs (0.5% difference). Further study is needed on more Thai C. gloeosporioides from papaya host to see if there is any geographical relationship among samples.

ANTIBACTERIAL ACTIVITY OF Boesenbergia rotunda (L.) Mansf. AND Myristica fragans Houtt. AGAINST Helicobacteri pylori (NO. 779) Siriyaporn Juthapruth1, Warocha Mahachai 2 and Sutatip Bhamarapravati 1 Department of Plant Science, Faculty of Science, Mahidol University 2Department of Microbiology, Faculty of Medicine Chulalongkorn University. Key words : Boesenbergia rotunda, Myristica fragans, Helicobacter pylori Helicobacter pylori (HP), a gram negative bacterium, is recognized as the primary etiological agent for the development of gastritis, dyspepsia and peptic ulcer disease. In developing countries the incidence of HP infection ranges from 50­100 %. The incidence becomes higher in the older population. In Thailand, two plants, namely Boesenbergia rotunda (L.) Mansf. and Myristica fragans Houtt. have been reported for their uses with dyspepsia. Their crude extracts were reported to possess anti-H. pylori activity. This investigation developed primary cultures from samples derived from hospital patients and conduct in vitro anti-H.pylori testing on compounds from these two plants. The compounds used were pinostrobin and red oil from root of B. rotunda, and dihydroguaiaretic acid from aril of M. frograns. Claritomycin was used as a positive control. All three samples showed positive results in agar diffusion test at p< 0.05.

1

THE STUDY OF MORPHOLOGY AND MOLECULAR CHARACTERISTIC OF Colletotrichum gloeosporioides FROM PAPAYA HOST IN THAILAND (NO. 778) Pimonrut Temjitpukdee 1 , Jirapan Worapong 2 , Tavorn Vinijsanum2, and Sutatip Bhamarapravati 3 Department of Biology, 2Department of Biotechnology and 3Department of Plant Science, Faculty of Science, Mahidol University. Key words : Papaya, Colletotrichum gloeosporiodes , Anthracnose Colletotrichum gloeosporioides is the causative agent of anthracnose in Papaya (Carica papaya L.). To date there is no molecular genetic information available on C. gloeosporioides on papaya host in Thailand. In this investigation C. gloeosporioides was isolated from ripe papaya harvested from different locations in central Thailand. Single spore culture of each sample was initiated and morphological characteristics recorded. Then DNA from each single spore culture was extracted, amplified using PCR method, and sequenced. DNA sequences of all samples were then characterized and compared using Blast software. The result shows that spore size of C. gloeosporioides from Pathumthani (P) and Ayutthaya (A) are 3.8x14.1µ and 4.5x13.7µ respectively. Their growth rates are also different. (P) and (A) DNA

1

ORCHIDANTHA FOETIDA (LOWIACEAE) A NEW SPECIES FROM THAILAND (NO. 780) Thaya Jenjittikul1 & Kai Larsen2

1

Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand, E-mail address: [email protected] mahidol.ac.th. 2K. Larsen, Institute of Systematic Botany, Faculty of Science, University of Aarhus, Nordlandsvej 68, DK-8240, Denmark, E-mail address: [email protected] Key words : Orchidantha, Thailand, Taxonomy Orchidantha foetida sp. nov. from NE Thailand is described and illustrated and a key to the three species occurring in Thailand is presented. (Nordic. J. Bot. 2002. 22 (4): 405-408.)

Mahidol University Annual Research Abstracts, Vol. 31

LABILE PLASMA IRON IN IRON OVERLOAD: REDOX ACTIVITY AND SUSCEPTIBILITY TO CHELATION (NO. 1096) Breno P. Esposito 1, William Breuer 1, Pornpan Sirunkapracha 2, 2 3 Pensri Pootrakul , Chaim Hershko and Z loav Cabantchik1 Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel, 2Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Thailand, 3Department of Hematology, Shaare Zedek Medical Center, Jerusalem, Israel Iron overload conditions, such as thalassemia and hereditary hemochromatosis (HH), are characterized by the appearance of high levels of serum iron, transferrin saturation and ferritin. However, it is the presence of Non-Transferrin Bound Iron (NTBI) forms in the plasma that is believed to be responsible for catalyzing the formation of reactive oxygen species in the circulation, the accumulation of oxidation products and tissue iron overload (reviewed by Breuer et al. 200). To test this hypothesis we assessed the redox active component of NTBI, which we refer as LPI, labile plasma iron, in the plasma of normal, -thalassemia and hemochromatotic patients. LPI levels in plasma or serum were determined with the aid of the fluorogenic probe DHR (dihydrorhodamine 123) by monitoring the time dependent generation of reactive oxidant species (ROS) generated by ascorbate and blocked by iron chelators. The assay is considered specific for labile iron since: a. it uses physiological concentrations of ascorbates, b. it involves no manipulation of the serum sample that might lead to extraction of iron from transferrin and c. the signal is blocked by specific iron chelators which neither bind heme-bound iron nor inhibit peroxidases. The results indicated that LPI was essentially absent from sera of normal individuals but was present in the majority of sera of 57 -thalassemia patients at levels of 1-14 mM (mean value of 2.6+0.8 µM). The LPI levels correlated significantly (r=0.88) with those of NTBI measured either as MDCI (mobilizer-dependent chelatable iron, as described by Breuer et al. 2001b) or DCI (deferrioxamine-chelatable iron, as described by Breuer et al. 2001a) but showed 30-40% higher sensitivity. Importantly, standard oral treatment of patients with the iron chelator deferiprone (L1) raised the plasma NTBI levels (measured as DCI and MDCI) due to mobilization of iron from tissues into the circulation. However, L1 administration did not lead to an increase in LPI levels, indicating that L1-chelated iron appearing in the plasma had essentially no redox activity prompted by ascorbate. On the contrary, oral deferiprone treatment effectively reduced the LPI that was already present in the patients prior to drug treatment, to virtually undetectable levels (<0.5 µM). Thus ascorbate driven generation of oxidizing species provides a versatile means for analyzing the redox capacity of labile iron present in native plasma/serum samples and associated with NTBI. Moreover, the approach used in this work enables the assessment of LPI susceptibility to in vivo or in vitro chelation and the potential of LPI to cause tissue damage, as found in iron overload conditions. (This work was carried out in part by contract with Aferrix sponsored by the Israeli Ministry of Industry and Resources and by grants from the European Community 5 th Framework and Apotex Inc., Ont. Canada.) (Presented at the Bioiron 2003 World Congeess on Iron Metabolism, NIH, Bethesda, USA, May 4-9, 2003)

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LIVER IRON MEASUREMENT AND MAPPING USING MAGNETIC RESONANCE IMAGING

(NO. 1097)

Timothy St. Pierre 1, Paul Clark1, Wanida Chua-anusorn1, Gary Jeffrey 2, John Olynyk2, Erin Robins3, Rob Lindeman4, Pensri Pootrakul5. School of Physics and 2School of Medicine, The University of Western Australia, 3SKG Radiology, St John of God Hospital, Subiaco, WA, 4Department of Haematology, Prince of Wales Hospital, Sydney, NSW, Australia, 5Thalassemia Research Center, Institute of Sciences and Technology for Research & Development, Mahidol University, Thailand. Magnetic resonance imaging can be used for the noninvasive measurement of liver iron concentration (LIC) in tissue with iron overload via quantitative image measurement of the hydrogen proton transverse relaxation rate (R2). The potential range of LIC measurement has been further extended by the development of a biexponential R2 imaging technique. The aim of this study was to (¡) determine the accuracy and reproducibility of R2 measurements on a set of phantoms on different MR scanners, (¡¡) determine the precision of R2 measurements on the liver made on different scanners, and (¡¡¡) determine whether a universal calibration curve could be developed for the quantification of liver iron concentration. The accuracy and reproducibility of R 2 imaging on aqueous MnCI2 phantoms with a range of R2 values covering those encountered in human liver has been demonstrated on five 1.5 T whole body imaging units. The mean relaxivity value for the five scanners was 74.1 s-1 (mM)-1, with a standard deviation of 0.3 s-1 (mM)-1. The coefficient of variation between the five scanners was less than 0.5 %. For the precision measurement study, 10 volunteers (3 healthy, 2 with hemochromatosis, 5 with -thalassemia) were measured twice each on 2 different scanners a day apart. The precision of inter scanner R2 image measurement of the liver was 7.7%, with a non-significant systematic difference between scanners of 1.2%. For liver iron calibration curve development, 105 volunteers (32 with hepatitis, 23 with hemochromatosis, and 50 with thalassemia) who were undergoing liver biopsy during the course of their treatment, were recruited. The mean R2 in the right-hand side of the liver for slices of maximal cross-section was found to correlate significantly with needle liver biopsy iron concentration (Spearman rank order correlation coefficient of 0.97). A universal calibration curve was applicable to all patient groups, and covered the entire range of liver iron concentrations presented, from 0.3 to 43 mg of iron per gram dry tissue. The accuracy of the calibration curve over different ranges of LIC is estimated by calculating the standard deviation of the LIC differences between the calibration and the ranked order data yielding the following uncertainties and (ranges): +0.1 (0.3­1.8); +0.3 (1.8­0.5); +0.9 (5­20); +1.1 (20­43) mg Fe/g dry tissue. The results of this study demonstrate that hepatic R2 image measurement can be used in a machine independent manner for the non-invasive measurement of liver iron concentration, from normal liver iron levels to highly loaded liver iron levels (approx 40 mg Fe/g dry tissue). (Presented at the Bioiron 2003 World Congress on Iron Metabolism, NIH, Bethesda, Maryland, USA, May 4-9, 2003)

1

428

Institute of Science and Technology for Research and Development

Patient with -thalassemia/Hb E disease is high prevalent in Southeast Asia region. They suffer from progressive Fe overload with multiple organ dysfunction including endocrinopathy. The major source of excess body Fe is from an inappropriate increased GI dietary Fe absorption in non-transfused patient and non-regularly transfused individual. We attempted to study the thyroid function in this group of patients who received minimal or no red cell transfusion. Twenty four male and 19 female patients were included. Their ages range from 15-62 years. Twenty eight volunteers were splenectomised cases and 15 individuals with intact spleen. Their serum transterrin Fe were saturated or oversaturated. The mean+SD of serum ferritin was 2283+1003 ng/ml in male and 2759+1320 ng/ml in female. The hepatic Fe in male and female were 20.8+6.8 and 20.5+9.2 mg/g DW respectively. The low level of T3, T4, free T4 were observed in 1, 7, 8 and 6 patients respectively. Increased TSH level was detected in 6 cases. There were no sign and symptom of hypothyroid. Thirty two volunteers (21 males, 11 females) received oral Fe chelation-deferiprone at low dose -50 mg/kg/day for a period of 20-80 weeks. The measurement of serum thyroid hormones, serum ferritin and hepatic Fe concentration were performed before and at the end of chelation. Paired sample t-test and Wilcoxon signed ranks test were used. (Presented at: - 7 th International Symposium on Chelating Agents in Biomedicine Toxicology and Therapeutics, July 10-12, 2003, Pilsen, Czech Republic - 13 th International Conference on Oral Chelation in the Treatment of Thalassemia and other Diseases, July 13-15, 2003, Pilsen, Czech Republic.)

NON-INVASIVE LIVER IRON MEASUREMENT IN PATIENTS WITH -THALASSEMIA/HEMOGLOBIN E DISEASE USING MRI: MONITORING THE EFFECT OF ORAL IRON CHELATION WITH DEFERIPRONE (L1) (NO. 1098) Wanida Chua-anusorn 1 , Adam Fleming 1 , Pichest Metarrugcheep2, Pornpan Sirunkapracha3, Pensri Pootrakul3 and Timothy St. Pierre 1

1

Biophysics Programme, School of Physics, University of Western Australia, 2Neurological Institute, Bangkok and 3Thalassemia Research Centre, Institute of Sciences and Technology for Research & Development, Mahidol University, Salaya, Thailand. Non-invasive measurement of liver iron concentrations can be achieved through measurement of proton transverse relaxation rates (R2) using magnetic resonance imaging [1-3], In this study, the potential for using the R2-MRI measurement technique to monitor changes in liver iron concentration during chelation therapy was examined. R2-MRI and needle biopsy were used to measure liver iron concentrations in 10 -thalassemia/hemoglobin E patients who had not previously undergone chelation therapy. The volunteers were measured a second time after having been treated with the oral iron chelator deferiprone for a period of between 12 months and 17 months. MRI liver iron measurements were obtained by measuring the mean R2 for the largest cross sectional axial slice of the liver. The mean and standard deviation of liver iron concentrations for the 10 patients measured by R2-MRI and needle biopsy iron assay were 16.1+4.4 and 19.7+7.0 mg Fe/g dry tissue respectively before commencement of iron chelation therapy. The corresponding measurements at follow-up were 6.7+6.7 and 7.1+7.8 mg Fe/g dry tissue respectively. The mean fractional change in liver iron concentration for the 10 patients measured by R2-MRI and needle biopsy iron assay was - 61 +38 % and - 63+31 % respectively. Nine of the patients exhibited decreases in their liver iron concentrations as measured by both MRI and needle biopsy iron assay. The standard deviation of R2 within the liver image (R2) is related to the standard deviation of local iron concentrations in the tissue. The mean change in R2 for the patients was -56+23 % suggesting that the iron remaining within the liver was more uniformly distributed after the administration of chelation therapy. These results suggest that R2-MRI has the potential to be used as a clinical monitoring tool in chelation therapy trials. (Presented at the Bioiron 2003 World Congress on Iron Metabolism, NIH, Bethesda, Md, USA, May 4-9; 2003)

NONHEME IRON, PLATELET FACTOR 3 LIKE ACTIVITY AND RED BLOOD CELL VESICLES IN -THALASSEMIA/Hb E PATIENTS TREATED WITH CURCUMIN (NO. 1100) Ruchaneekorn W Kalpavidh 1, Orn-uma Sroijit 1, Noppadol Siritanaratkul 2 , Nisarat Opartkiattikul 3, Kovit Pattanapanyasat4, Chada Phusalaphong5, Suthat Fucharoen6, Department of Biochemistry, 2 Department of Medicine, 3 Department of Clinical Pathology, 4Office for Research and Development, Faculty of Medicine Siriraj Hospital, 5 The Government Pharmaceutical Organization, 6 Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University -thalassemia/HbE is a genetic disease resulting in excess a globin chains. The deposition of excess unmatched globin chains, which contain free iron, nonheme iron, and hemichromes on the membrane, can generate reactive oxygen species by several mechanisms including Fenton reaction and resulted in loss of erythrocyte membrane phospholipids asymmetry with the exposure of phosphatidylserine (PS) which provides a docking site for coagulation protein. The stressed red blood cell membrane also has a tendency to vesiculate. We, therefore, have conducted a study to evaluate the effect of natural antioxidant curcumin from rhizomes of tumeric (Curcuma longa L) on nonheme iron, platelet factor 3 like activity, and red blood cell vesicles in -thalassemia/HbE patients.

THYROID FUNCTION IN PATIENT WITH -THALASSEMIA/Hb E DISEASE AND EFFECT OF ORAL IRON CHELATION­DEFERIPRONE

(NO. 1099)

P.Pootrakul, S Issarachai, U.Athikomrattnakul, P Sirunkapracha Institute of Science and Technology for Research and Development, Mahidol University. Salaya Campus and Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Mahidol University Annual Research Abstracts, Vol. 31

Twenty-five patients received 500 mg curcumin daily for 6 months and withdrawal the treatment for 3 months, and then some patients received placebo for 2 months. The amount of nonheme iron was detected by ferrozine reaction. The PF 3 like activity was determined by measuring the activity of thrombin generated by using synthetic thrombin substrate S-2238. The red blood cell-PS positive vesicles were labeled by annexin V-fluorescein isothiocyanate (FITC) and detected by flow cytometry. -thalassemia/HbE patients who received curcumin for 6 months significantly reduced nonheme iron and PF 3 like activity approximately 40% and 33% respectively. On the contrary, there was no significantly difference in red blood cell vesicles. All parameters measured in patients after withdrawal treatment and received placebo were the same as baseline. This study suggests that curcumin can reduce nonheme iron, the target of oxidant damage. Furthermore, curcumin may have some effects in alteration of hemostatic system in these patients. (This work was supported by the Thailand Research Fund. Presented at the 9 th National Conference on Thalassemia, June 18-19, 2003, Bangkok, Thailand)

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at first transfusion, requirement of blood transfusion, spleen condition and growth development. The preliminary clinical features and hematologic data of the first 600 patients recruited in this program are as follows. 1. With strict scoring criteria including age onset and age at first transfusion, requirement of blood transfusion, spleen condition and growth development, patients were divided into 156 mild cases, 208 intermediate and 236 severe cases. 2. Mean+SD hemoblobin (Hb) and absolute Hb F levels were summarized as in the Table I.

Hemoglobin Hb (g/dl) Absolute Hb F (g/d)

-thalassemia/Hb E (Mean + S.D.) Mild Intermedia Severe 7.97+ 1.14 2.76+ 1.2 6.69+ 1.14 2.3+ 1.6 5.82+ 0.09 1.8+ 0.6

3. Concomitant inheritance of -thalassemia gene was found in 24.4% of mild cases, and absolute Hb F levels in the mild cases without -thalassemia gene interaction were higher than those who have coinheritance of -thalassemia gene (3.1+1.1 g/dl vs 1.6+0.9 g/dl). 4. Although many genotypes of -thalassemia mutations interacted with Hb E gene, there is no specific mutation in each group. The 4 bp deletion in codons 41/42 represented about 50% of total cases. There is a trend that patients with IVSI-1/Hb E had higher Hb levels than the others. (This study was supported by National Institute of Health .Presented at the 9 th International Conference on Thalassemia and Hemoglobinopathies, Palermo, Italy, October 15-19, 2003)

HEMOGLOBIN E DISORDERS: PATHOPHYSIOLOGY AND CLINICAL FINDINGS (NO. 1101) Suthat Fucharoen1, Pranee Winichagoon 1, Orapan Sripichai 1, Thongperm Munkongdee 1, Kanjana Vichittumaros 1, Pornpan Sirankapracha 1, Nopadol Siritanaratkul 2, Tanyachai Sura 3, Ampaiwan Chuansumrit 3, Issarang Nuchprayoon 4, Saranya Suwansings 5, Somchai Intarasiripong 6, Ken Abel 7, Andreas Braun7 Thalassemia Research Center, Institute of Science and Technology for Research and Development, 2Faculty of Medicine Siriraj Hospital, 3Faculty of Medicine Ramathibodi Hospital, Mahidol University, 4 Faculty of Medicine, Chulalongkorn University, 5 Chiangrai Pachanukraw Hospital, 6Maharaj Nakornrajchasima Hospital, Thailand and 7SEQUENOM, San Diego, CA, USA Beta thalassemia/Hb E is the most common b-thalassemia disease in Southeast Asia. Clinically they are very heterogeneous in severity, varying from phenotypically mild cases to severe thalassemia major, in which the patients die within the first decade of life. Previous study showed that hemoglobin levels varied from 3-13 g/dl with a mean hemoglobin 7.7+1.55 g/dl and the majority of the patients have moderate clinical severity (3). Major modifying factors demonstrated in the mild cases are coinheritance of mild +thalassemia and Hb E genes, coinheritance of µ-thalassemia gene and increased production of Hb F (4-7). However, the fact that many patients who have seemingly identical genotypes, °-thalassemia/ Hb E, and do not have a detectable -thalassemia haplotype or increased Hb F production still have a mild clinical symptom, and many patients had a very severe clinical symptom similar to homozygous °-thalassemia suggests that other modifying genetic factors may contribute to the severity of the disease. We are conducting a prospective study searching for modifying genetic factors in 1000 -thalassemia/Hb E patients. They were phenotypically divided into 3 groups, mild, intermediate and severe cases, using strict scoring criteria including age onset and age

1

SNP MAP AND HAPLOTYPE ANALYSIS OF THE BETA-GLOBIN GENE REGION IN BETA°-THAL/ HB E PATIENTS (NO. 1102) Ken Abel1, Johanna Whitacre 1, Orapan Sripichai 2, Porat Erlich3, Lindsay Farrer 3, Andreas Braun1, Suthat Fucharoen2

1 SEQUENNOM, San Diego, CA; 2Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Thailand, 3Boston University, Boston, MA

Beta-thalassemias are extremely common in South East Asia, and due to recent worldwide migrations these abnormal alleles and associated disorders becoming more common in other region. Many genetic defects responsible for beta thalassemias have been described. The mutant Hb E allele encodes a Glu to Lys amino acid substitution at codon 26, plus the potential for alternative mRNA splicing leading to a frame shift mutation. A puzzling feature of patients compound heterozygous for beta°-thalassemia/Hb E is the variable disease presentation. Patients with similar beta globin mutations display a surprising variation in disease severity ranging from nearly asymptomatic to severe, transfusion-dependent disease. The need exists to identify the modifying genetic factors, representing either novel therapeutic targets or the focus of new diagnostics to identify at-risk individuals for early intervention.

430

Institute of Science and Technology for Research and Development

comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders. (This work was supported by grants to the CAGT Research Group. D.J. was supported by the Royal Golden Jubilee Scholarship and S.F. is a Senior Research Scholar of the Thailand Research Fund.) (Published in J Biotechnology 2003; 101: 1-9.)

Using an automated, chip-based platform for genetic analysis based on mass spectrometry (MassARRAY), we are examining the extent to which genetic variation within the beta globin gene complex may contribute to thalassemia disease severity in Thai/ Chinese patients with beta°-thalassemia/Hb E disease. Toward this goal a map of single nucleotide polymorphisms (SNPs) has been constructed spanning more than 80 Kb, including all beta-like genes and extending into the locus control region (LCR). Assays were designed for>130 putative SNPs obtained from targeted resequencing efforts and from the public domain, and are being tested in individuals from different ethnic groups. Also included are assays for polymorphic sites reported to influence globin gene expression (XmnI+/ -and BP1-binding sites upstream of G-and -globin, respectively). SNPs having minor allele frequencies >0.15 are being selected at spacing of roughly 2-3 Kb. Multiplexed assays are being used in haplotype analysis for association with disease severity. These efforts will be complemented by a genome scan for other modifier alleles, comparing up to 100,000 gene-based SNP frequencies in pooled DNAs from mild and severe disease patients. (This study was supported by the National Institute of Health, USA Presented at the American Society Human Genetics, November 2003, LA, USA.)

HEMOGLOBIN H DISEASE: NOT NECESSARILY A BENIGN DISORDER (NO. 1104) David H.K. Chui, Suthat Fucharoen, Vivian Chan Department of Pathology and Molecular Medicine, McMaster University Faculty of Health Sciences, Hamilton, ON, Canada; Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Thailand; and University Department of Medicine, University of Hong Kong and Queen Mary Hospital, Hong Kong, China. Key words: Hb H; -thalassemia; genetic disease Hb H disease is a form of -thalassemia often manifested clinically as thalassemia intermedia with moderate anemia. It is commonly found in Southeast Asian, Middle Eastern, and Mediterranean populations. There is a wide spectrum of genotypes and phenotypic presentations. These range from those who appear clinically to be asymptomatic, to others who are more anemic, having significant hepatosplenomegaly and requiring occasional or even regular transfusions, to the severe Hb H hydrops fetalis syndrome that can cause death in the affected fetuses late in gestation. This hereditary disorder is usually caused by deletions removing all but one single -globin gene (deletional Hb H disease). A small proportion of patients have deletions removing 2 globin genes plus a nondeletional mutation affecting a third -globin gene (nondeletional Hb H disease). In general, nondeletional Hb H disease has a more severe clinical course than the deletional form. Review of recent literature suggests that Hb H disease is not as benign a disorder as previously thought. It can bring about growth retardation during childhood and iron overload in adults regardless of previous transfusion history, leading to hepatic, cardiac, and endocrine dysfunction. Significant anemia might occur during infections, fever, hypersplenism, or pregnancy that may necessitate the need for blood transfusions. An essential part of the maternal/ child health care should include screening the partners of all pregnant women with Hb H disease for their thalassemia carrier status, and providing these and other couples who are at risk of conceiving offspring with Hb H disease with appropriate genetic counseling. (S.F. is a Senior Research Scholar of the Thailand Research Fund. Published in Blood 2003; 101: 791-800.) HEMIN : A POSSIBLE CAUSE OF OXIDATIVE STRESS IN BLOOD CIRCULATION OF -THALAS SEMIA/HEMOGLOBIN E DISESAE (NO. 1105) Noppawan Phumala 1 , Supatra Porasuphatana 1, Supeenun Unchern 1, Pensri Pootrakul 2, Suthat Fucharoen 2, and Udom Chantharaksri1

INSERTION OF COMMON MUTATIONS INTO THE HUMAN -GLOBIN LOCUS USING GET RECOMBINATION AND AN EcoRI ENDONUCLEASE COUNTERSELECTION CASSETTE

(NO. 1103)

Duangporn Jamsai 1,2, Mikhail Nefedov1, Kumaran Narayanan 1,3, Michael Orford1,4, Suthat Fucharoen2, Robert Williamson1, Panos A. Ioannou1,4

1

CAGT Research Group, The Murdoch Childrens Research Institute, Royal Children's Hospital, Parkvill, Vic. 3052, Australia; 2Thalassemia Research Center, Institute of Science and Technology for Research and Development and Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Thailand, 3Faculty of Resource Sciences and Technology, University of Malaysia Sarawak, Kota Samarahan, 94300 Sarawak, Malaysia; 4The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus. Key words : BAC recombination; counterselection; thalassemia; A large number of mutations have been described in the human -globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human -globin locus. We report here the use of the GET Recombination system with an EcoRI/KanR counterselection cassette to facilitate the introduction of the Hb E codon 26, GAGAAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human -globin locus. The counterselection cassette was first inserted into the target sequence in the -globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacl. Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and

Mahidol University Annual Research Abstracts, Vol. 31

Department of Pharmacology, Faculty of Science, Mahidol University, Rama 6 Rd, Bangkok 10400, Thailand; 2Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. Key words : ESR; hemin; luminol; pro-oxidant; -thalassemia/Hb E A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of -thalassemia/hemoglobin E disease (-thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron(III) protoporphyrin IX). Hemin was present at levels ranging from 50 to 280µM in serum of -thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of -thal/ Hb E was demonstrated by luminal-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of -thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum hemin, r = 0.778 (P<0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p = 0.259) and 0.519 (p = 0.004), respectively, Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major prooxidant in blood circulation of -thal/Hb E. (Published in Free Radical Research 2003; 37(2): 129-135.)

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mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, CT) into the human -globin locus. We have observed the frequency of recombinant clones to be as high as l in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications. (This work was supported by grants to the CAGT Research Group. D.J. was supported by the Royal Golden Jubilee Scholarship and S.F. is a Senior Research Scholar of the Thailand Research Fund. Published in Molecular Biotechnology 2003; 23: 29-36.)

Hb KODAIRA II [146(HC3)HISGln] DETECTED IN THAILAND (NO. 1107) Lukana Ngiwsara, 1,2 Chantragan Srisomsap, 1 Pranee Winichagoon, 3 Suthat Fucharoen, 3 and Jisnuson Svasti1,2

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Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand; 2Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand; 3Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom, Thailand. Hb Kodaira [146(HC3)HisGln (CACCAA], a variant with increased oxygen affinity, was first described in a Japanese male in 1992. Recently, while our study was in progress, a second report described the same phenotypic variant Hb Kodaira II, but with a different nucleotide change (CACCAG) at codon 146 of the globin gene. We report here the first case of Hb Kodaira II in Thailand, in a healthy 31-year-old Thai female (S.P.), who came for a routine check up. She had the following hematological profile at the steady state: hemoglobin (Hb) 14.7 g/dL (normal 13.8 + 0.7); packed cell volume (PCV) 0.470 L/L (normal: 0.425+ 0.170); MCV 94.0 fL (normal: 92.5 + 3.7); MCH 29.0 pg (normal : 30.6+ 1.9); MCHC 31.0 g/dL (normal:32.6 + 0.9); RBC 5.07x10 9/L (normal: 4.5+ 0.3). Analysis of the blood sample by automated high performance liquid chromatography (HPLC) (VARIANTTM System; BioRed Laboratories, Hercules, CA, USA) revealed an abnormal Hb present at a level of 45.4% eluted earlier than Hb A, with Hb F and Hb A2 being in the normal range. The abnormal Hb could not be separated from Hb A by cellulose acetate electrophoresis at pH 8.6. However, the abnormal -globin chain migrated slightly more slowly toward the cathode than normal -globin chain on cellulose acetate. Hb Kodaira II, also showed mildly elevated red blood cell (RBC) and Hb levels, but was otherwise in normal health. The present studies confirm that the Hb Kodaira [146(HC3)HisGln] phenotype can result from two mutation, namely CACCAA in Hb Kodaira and CACCAG in Hb Kodaira II. All these cases has not been investigated. The mutation is not detectable by the traditional technique of electrophoresis at alkaline pH, where the imidazole group of His 146 is expected to be uncharged. However, it is possible, that with improved techniques of detection by HPLC and IEF, Hb Kodaira may be found in non Asiatic populations. (Published in Hemoglobin 2003; 27: 37-39.)

INSERTION OF MODIFICTIONS IN THE -GLOBIN LOCUS USING GET RECOMBINATION WITH SINGLE-STRANDED OLIGONUCLEOTIDES AND DENATURED PCR FRAGMENTS

(NO. 1106)

Duangporn Jamsai 1,2 , Michael Orford1,2, Suthat Fucharoen 2, Robert Williamson 1, and Panayiotis A. loannou 1,3

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CAGT Research Group, The Murdoch Childrens Research Institute, Royal Children's Hospital, Parkvill, Vic. 3052, Australia 2 Thalassemia Research Center, Institute of Science and Technology for Research and Development and Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Thailand, 3The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus. Key words : BAC; homologous recombination; mutagenesis; thalassemia. We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human -globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the -globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the

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sions were treated with the oral iron chelator, deferiprone 25-50 mg/ kg/d for between 17 and 86 weeks (mean 49 weeks). There were significant decreases in serum ferritin (initial mean+standard deviation 2168+1142, final 418+247 mg/l; t-test for paired samples, p.= 0.005), hepatic iron (initial 20.3+6.26, final 11.7+4.83 mg/g/dry weight; p=< 0.02), red cell membrane iron (initial 76.2+3.464, final 7.2+0.56 mmol/mg protein; p=< 0.0005) and serum non-transferrin bound iron (initial 9.0+0.56, final 5.9+0.89 mmol/l; p= < 0.0005). There was also a significant rise in serum erythropoietin (initial 240+195.l final 433.2+269.2 U/l; p=0.034). The haemoglobin level rose in three patients and transfusion requirements were reduced substantially in four patients. Serum thiobarbituric acid reactive substance (TBARS) also fell in six of eight patients. Patients generally improved clinically, with weigh gain observed. Side-effects were mild and included gastrointestinal symptoms (6) and arthralgia (1), not requiring withdrawal of the drug. One patient died at 17 weeks of therapy as a result of an intercurrent infection. His neutrophil count was normal. We conclude that deferiprone is an effective, well-tolerated iron chelator for patients with thalassaemia intermedia. Further studies are needed to determine the optimum dose and length of treatment needed to reduce iron burden to a safe level in these patients. (This study was supported by grants from the Thailand Research Fund and the Mahidol University Research Grant. Published in Br J Haematol 2003, 122: 305-310.)

TARGETED MODIFICATION OF A HUMAN -GLOBIN LOCUS BAC CLONE USING GET RECOMBINATION AND AN I-SceI COUNTERSELECTION CASSETTE (NO. 1108) Duangporn Jamsai,1,2Michael Orford,1,3 Mikhail Nefedov,1 Suthat Fucharoen,2 Robert Williamson, 1 and Panayiotis A. Ioannou 1,3

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CAGT Research Group, The Murdoch Childrens Research Institute, University of Melbourne, Royal Children¢s Hospital, Parkville, Victoria 3052, Australia; 2The Thalassemia Research Center, Institute of Science and Technology for Research and Development, and Instetute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, Thailand 3 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus. Key words : BAC; homologous recombination; thalassemia; globin locus; counterselection; restriction endonuclease; I-SceI. There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in genomic locus, using a human -globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by ISceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants. (This work was supported by grants to the CAGT Research Group. D.J. was supported by the Royal Golden Jubilee Scholarship and S.F. is a Senior Research Scholar of the Thailand Research Fund. Published in Genomics 2003; 82: 68-77.)

THE EFFECTS OF VITAMIN E ON PLATELET ACTIVITY IN -THALASSAEMIA PATIENTS

(NO. 1110)

Unchern S1, Laoharuangpanya N 2, Phumala N1, Sipankapracha P 3 , Pootrakul P 3 , Fucharoen S 3 , Wanachivanawin W4 , Chantharaksri U1 Department of Pharmacology, Faculty of Science, Mahidol University, 2Laboratory of Pathobiology, Chulaborn Research Institute, 3Thalassaemia Research Centre, Institute of Science and Technology for Research and Development, Mahidol University, and 4Department of Medicine, Faculty of Medicine, Mahidol University, Bangkok, Thailand. Key words : vitamin E, platelet hyperactivity, lipid peroxidation, oxi A double-blind, crossover, placebo-controlled study of the effect of vitamin E on platelet functions was performed on nine splenectomized and 16 non-splenectomized -thalassaemia/haemoglobin E (-thalassaemia/HbE) patients. The patients were supplemented with a daily dose of vitamin E (525 IU) for 3 months. The functions of platelets were assessed by adenosine diphosphate (ADP)induced platelet aggregation and adenosine triphosphate release. Plasma -tocopherol, plasma thiobarbituric reactive substances (TBARs) and serum ferritin levels represented patients' antioxidant status, lipid peroxidation status and iron status respectively. Before experimentation, all patients had low plasma -tocopherol levels. The splenectomized patients showed severe iron overload iron, had higher plasma TBAR levels and their platelets were more reactive to ADP than those of non-splenectomized patients. Three months of daily vitamin E supplementation resulted in a significant increase in plasma -tocopherol levels and reduction in plasma TBAR levels in all patients. Serum ferritin levels of the patients were not altered;

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CLINICAL TRIAL OF DEFERIPRONE IRON CHELATION THERAPY IN -THALASSAEMIA/ HAEMOGLOBIN E PATIENTS IN THAILAND

(NO. 1109)

Pensri Pootrakul, 1 Pornpan Sirankapracha, 1 Jongkal Sankote, 1 Udom Kachintorn, 2 Wanna Maungsub,2 Kosit Sriphen, 2 Kleonphan Thakernpol, 2 Kanit Atisuk, 2 Suthat Fucharoen, 1 Udom Chantraluksri,3 Oded Shalev4 and A.Victor Hoffbrand 5

1

Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, 2Faculty of Medicine, Siriraj Hospital, 3Faculty of Science, Mahidol University, Bangkok, Thailand, 4Hadassah University Hospital, Jerusalem, Israel, and 5 Department of Haematology, Royal Free Hospital, London, UK. Key words : deferiprone, iron chelation, haemoglobin E, thalassaemia intermedia. Nine patients with either -thalassaemia/haemoglobin E (7) or homozygous -thalassaemia (2) not requiring regular transfu-

Mahidol University Annual Research Abstracts, Vol. 31

however, vitamin E reduced the platelet reactivity of the splenectomized patients towards normal levels. The influence of vitamin E on platelet reactivity may result in delaying hypoxaemia and pulmonary occlusion that commonly occurs in splenectomized thalassaemia/HbE patients. (Published in Br J Haematol 2003; 123: 738-744.)

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Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan; 2 Division of Clinical Immunology, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan; 3Thalassemia Research Center, 4Field of Molecular Genetics and Genetic Engineering, Institute of Molecular Biology and Genetics, Mahidol University, Nakhonpathom, Thailand. Key words : erythroid progenitor cells; IFN-; apoptosis; PI3-kinase Interferon (IFN)- is a survival factor for mature erythroid progenitor cells. To elucidate related survival mechanisms, we compared the role of phosphatidylinositol 3-kinase (PI3-kinase) in the survival signals of IFN- and erythropoietin (EPO). Human erythroid colony-forming cells (ECFCs) purified from peripheral blood were used, and Ly294002 was used as a PI3-kinase inhibitor. Treating ECFCs with a high concentration of Ly294002 (50 µmol/L) in the presence of EPO and/or IFN- reduced cell viability by inducing apoptosis. However, treating cells with a lower concentration of Ly294002 (10 µmol/L) did not affect the antiapoptotic function of IFN- and abolished the antiapoptotic effect of EPO. Adding IFN- or EPO induced Bcl-x expression in ECFCs, as determined by Western blotting, and expression was suppressed in the presence of Ly294002. We also examined the phosphorylation of the protein kinase Akt, the downstream target of PI3-kinase. EPO stimulation significantly increased the phosphorylation, but IFN- did not. These results suggest that IFN- plays a role in preventing the apoptosis of erythroid progenitor cells by affecting Bcl-x expression, thereby reducing the disruption of the mitochondrial transmembrane potential via PI3-kinase pathways that are related to but distinct from the EPO pathway. (This work partially supported by grants from the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand, Kyushu University P&P grant and the Ministry of University Affairs. K.P. was supported by the MD/PhD scholar. S.F is a Senior Research Scholar of the Thailand Research Fund. Published in International J Hematol 2003; 78: 421-428.)

LABILE PLASMA IRON IN IRON OVERLOAD: REDOX ACTIVITY AND SUSCEPTIBILITY TO CHELATION (NO. 1111) Breno P. Esposito, William Breuer, Pornpan Sirankpracha, Pensri Pootrakul, Chaim Hershko, and Z. loav Cabantchik. Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Israel; Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom, Thailand; and the Department of Hematology, Shaare Zedek Medical Center, Jerusalem, Israel. Key words : iron overload; plasma non-transferrin bound iron; labile plasma iron, deferiprone Plasma non-transferrin-bound-iron(NTBI) is believed to be responsible for catalyzing the formation of reactive radicals in the circulation of iron overloaded subjects, resulting in accumulation of oxidation products. We assessed the redox active component of NTBI in the plasma of healthy and -thalassemia patients. The labile plasma iron (LPI) was determined with the fluorogenic dihydrorhodamine 123 by monitoring the generation of reactive radicals prompted by ascorbate but blocked by iron chelators. The assay was LPI specific since it was generated by physiologic concentrations of ascorbate, involved no sample manipulation, and was blocked by iron chelators that bind iron selectively. LPI, essentially absent from sera of healthy individuals, was present in those of -thalassemia patients at levels (1-16 µM) that correlated significantly with those of NTBI measured as mobilizer-dependent chelatable iron or desferrioxamine chelatable iron. Oral treatment of patients with deferiprone (L1) raised plasma NTBI due to iron mobilization but did not lead to LPI appearance, indicating that L1-chelated iron in plasma was not redox active. Moreover, oral L1 treatment eliminated LPI in patients. The approach enabled the assessment of LPI susceptibility to in vivo or in vitro chelation and the potential of LPI to cause tissue damage, as found in iron overload conditions. (This study was supported in part by the Incubator Program Aferrix and the National Research Council of Thailand. Published in Blood 2003; 102: 2670-2677.)

PREATTENTIVE PROCESSING OF LEXICAL TONE PERCEPTION BY THE HUMAN BRAIN AS INDEXED BY THE MISMATCH NEGAIVITY PARADIGM (NO. 1113) Wichian Sittiprapaporn 1 , Chittin Chindaduangratn 1, Mari Tervaniemi2, and Naiphinich Kotchabhakdi1

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THE SINGALING PATHWAYS OF ERYTHROPOIETIN AND INTERFERON- DIFFER IN PREVENTING THE APOPTOSIS OF MATURE ERYTHROID PROGENITOR CELLS (NO. 1112) Kittiphong Paiboonsukwong, 1,4 Ilseug Choi, 1 Takamitsu, 1 Yasunobu Abe,1 Junji Nishimura,2 Pranee winichagoon, 3 Suthat Fucharoen,3 Hajime Nawata, 1 Koichiro Muta1

Clinical and Research Electroneurophysiology Laboratory, Neuro-Behavioural Biology Center, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand; 2Cognitive Brain Research Unit, Department of Psychology, University of Helsinki. FIN00014, Helsinki, Finland. Key words: Event-related potential (ERP); Mismatch negativity (MMN); Memory trace Mismatch negativity (MMN) was used to investigate the processing of the discrimination between native and non-native CV syllables in tonal languages. MMN elicited by the native word was greater than that elicited by the non-native word. Hearing a native-

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Event-related potentials (ERPs) responses to infrequently presented spoken deviant syllables /pi:/ among repetitive standard / po:/ syllables were recorded in Thai subjects who ignored to these stimuli while reading books of their choices. Hemispheric predominance of this across-category change perception of vowels in consonant-vowel (CV) syllables was assessed using the low-resolution electromagnetic tomography (LORETA). The vowel across-category changes elicited a change-specific mismatch response (MMN). The MMN was predominant in the left hemisphere, emphasizing the role of the left hemisphere in speech processing already at a preattentive processing level also in CV-syllables. (Published in Chiang Mai Medical Bulletin 2003; 42(3) (Suppl.): 19 Presented at 1)The 2 nd Annual Meeting of Prince of Songkhla University, Pattani Campus 2003. 2)The 3 rd Annual International Conference of Thai Society of Behavioural Medicine and the 1 st Asian Regional Conference on Behavioural Medicine, Bangkok, 2003. 3)The Annual Meeting of "Mahidol Day", Chiang Mai University 2003)

language deviant significantly altered the elicited MMN in both amplitude and scalp voltage field distribution, reflecting the presence of a long-term memory trace for spoken words in tonal languages. (Published in Ann. N. Y. Acad. Sci. 2003; 999: 199-203)

GENDER DIFFERENCES ON PHONETIC MISMATCH NEGATIVITY ASSESSED BY LOW-RESOLUTION ELECTROMAGNETIC TOMOGRAPHY (LORETA) (NO. 1114) Wichian Sittiprapaporn, Chittin Chindaduangratn, Naiphinich Khotchabhakdi Clinical and Research Electroneurophysiology Laboratory, Neuro-Behavioural Biology Center, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand. Key words : Mismatch negativity (MMN); Speech; Low-resolution electromagnetography (LORETA) Electrophysiological approach with low-resolution electromagnetic tomography (LORETA) was used to examine gender effects on the amplitude, latency and topography of phonetic MMN in response to consonant-vowel (CV) syllables. To clarify the role of gender differences in the MMN, we compared the amplitude, latency and topography of phonetic MMNs between right-handed healthy male (12) and female (12) subjects. There were significant differences in amplitude and latency for phonetic MMNs between genders. Both genders showed very similar strongly left lateralized activation patterns. Voxel t-statistic image tests for gender differences in overall activation patterns showed the lack of significant differences in lateralization of activation pattern between genders. These data are relevance to a growing number of researches of gender effects in response to changes of phonemes in normal adults. (Published in Chiang Mai Medical Bulletin 2003; 42(3) (Suppl.): 18 Presented at 1) The 2nd Annual Meeting of Prince of Songkhla University, Pattani Campus 2003. 2)The 3 rd Annual International Conference of Thai Society of Behavioural Medicine and the 1 st Asian Regional Conference on Behavioural Medicine, Bangkok, 2003. 3) The Annual Meeting of "Mahidol Day", Chiang Mai University 2003)

INTRAOPERATIVE MONITORING OF AUDITORY FUNCTION IN TRIGEMINAL NEURALGIA (NO. 1116) Wichian Sittiprapaporn 1 , Ake Hungsasut 2 , Natthacha Jiaraninkoonchai 2, Thaveesak Chanwitthanuchit2, and Chittin Chindaduangratn1

1

Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Development, Mahidol University at Salaya Campus, Nakhon Pathom 73170; 2Department of Surgery, Ramathibordi Hospital, Mahidol University. Key words: Trigeminal Neuralgia; Intraoperative Monitoring; Brainstem Auditory Evoked Potentials (BAEP) Intraoperative monitoring of brainstem auditory evoked potentials (BAEP) has been widely employed to detect alteration of auditory function, which may respond to adjustment of surgical technique, and has successfully reduced the occurrence of postoperative hearing loss. Several potential mechanisms of injury may affect the cochlear nerve, and complete loss of BAEP is often associated with postoperative hearing loss. We report here a case of intraoperative auditory function related to the treatment for Trigeminal neuralgia. Intraoperative BAEP was monitored in patient undergoing the treatment for trigeminal neuralgia. The technique involves the placement of scalp disposable surface electrodes with lead attached and disposable subdermal needles as well as a bilateral earphone (Cz-Ai, CzAc and ground at Fz) to deliver alternating 75 dB auditory clicks at 17.5 Hz. The observational interval is 10 msec, with 2,000 target trials, and a sample rate of 10,000 Hz. BAEP waveforms suddenly disappeared completely during closure of the dura. Intraoperative monitoring of BAEP effectively identifies physiological dysfunction of the cochlear nerve, and thereby alerts the neurosurgeon to take corrective measures. The case presented here illustrates intraoperative loss of auditory function apparently during surgical manipulation. This study illustrates that Brainstem auditory potentials (BAEP) ware useful indicators of auditory during posterior fossa surgery. The intraoperative loss of auditory upon the cochlear nerve may disrupt function apparently during surgical manipulation. (Presented at The 3 rd Annual International Conference of Thai Society of Behavioural Medicine and the 1 st Asian Regional Conference on Behavioural Medicine, Bangkok, 2003)

BRAIN LATERALIZATION OF ACROSS-CATEGORY CHANGE OF VOWELS IN CONSONANTVOWEL (CV) SYLLABLES (NO. 1115) Wichian Sittiprapaporn 1 , Mari Tervaniemi 2 ,Chittin Chindaduangratn1 and Naiphinich Khotchabhakdi1

1

Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Development, Mahidol University at Salaya Campus, Nakhon Pathom 73170, THAILAND; 2Cognitive Brain Research Unit, Department of Psychology, P.O. Box 9 (Siltavuorenpenger 20 C) 00014 University of Helsinki, Helsinki, Finland. Key words: Event-related potential (ERP); vowels; lateralization

Mahidol University Annual Research Abstracts, Vol. 31

INFLUENCE OF SERVICING SATISFACTION IN SKINCARE TREATMENT TO HEALTH BEHAVIORS (NO. 1117) Wichian Sittiprapaporn 1, Saowaros Panjapaiboon 2, Piyanuch Pringprom2, and Chittin Chindaduangratn1

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Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Development, Mahidol University at Salaya Campus, Nakhon Pathom 73170, THAILAND; 2Pruksa Skincare Management Clinic, Nonthaburi 11120, THAILAND. Key words: Behaviors; Health; Skins; Skincare; Treatment The objectives of this study was to investigate the influence of servicing satisfaction factors in skincare treatment to health behaviors. This study find out the level of influence of servicing satisfaction, the correlation between influence of servicing satisfaction and variables of individual data, and the correlation between influence of servicing satisfaction and variables of experience. The data were collected from 14 skincare management clinics (approximately 325 participants each) in Thailand using questionnaires. Descriptive statistics including frequency, percentage, arithmetic mean and standard deviation, and one-way ANOVA were calculated for data analysis. The main findings were as follows: firstly, the influence of servicing satisfaction factors to the skincare treatment to health behaviors of participants in Thailand was at a high level (4.93+0.62) according to Likert scale. Secondly, the price (4.74+0.62), effectiveness (4.46+0.71), and doctor (4.31+0.70) were rank at the top three in skincare management. Thirdly, the intrinsic factor (4.31+0.42) and extrinsic factor (4.53+0.58) of influence of servicing satisfaction in skincare treatment to health behaviors were also at a high level. Finally, concerning to the variables of individual of experience, there were significant (<0.05) with respect to the following factors: genders, education, medical instrument, prices, and effectiveness. The recommendation as a result of this study are that the administrators of skincare management clinics must plan to maintain for the intrinsic factors and extrinsic factors and development for a higher level of satisfaction because the influence of servicing satisfaction in skincare treatment to health behaviors may affect the performance and the growth of skincare management clinics in Thailand. (Present at The 3 rd Annual International Conference of Thai Society of Behavioural Medicine and the 1 st Asian Regional Conference on Behavioural Medicine, Bangkok, 2003.)

Event-related potentials (ERPs) responses to infrequently presented spoken deviant syllables among repetitive standard syllables were recorded in (10) subjects who ignored to these auditory stimuli while reading books of their choices. The phonetic sounds change elicited change-specific mismatch negativity (MMN). The low-resolution electromagnetic tomography (LORETA)-MMN generator appeared in the left auditory cortex, emphasizing the role of the left hemisphere in speech processing already at a preattentive processing level also in consonant-vowel (CV)-syllables. In addition, the electrophysiological approach with LORETA was used to examine gender effects on the amplitude, latency and laterality of phonetic MMNs in response to CV syllables. To clarify the role of gender differences in the MMN, we compared the amplitude, latency and laterality of phonetic MMNs between right-handed healthy male (12) and female (12) adults. While both genders showed very similar strongly left lateralized activation patterns, there were significant differences in amplitude and latency for phonetic MMNs between genders. These data are relevance to a growing number of researched of gender effects in response to changes of phonemes in normal adults. (Presented at The 3 rd Annual Conference of the Institute of Science and Technology for Research and Development, Mahidol University 2003)

FUNCTIONAL SPECIALIZATION OF THE HUMAN AUDITORY CORTEX IN PROCESSING OF SPEECH SOUNDS (NO. 1119) Wichian Sittiprapaporn, Chittin Chindaduangratana and Naiphinich Kotchabhakdi Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Research & Development Mahidol University, Salaya, Nakhon Pathom 73170. Key words: Speech; Sound; Auditory; Cortex; perception; Asymmetry; Evoked field; Low-resolution electromagnetic tomography (LORETA); Mismatch negativity (MMN) The present study was intended to make electrophysiological investigations into the preattentive perception of native and nonnative speech sounds. In addition, it was to estimate the electric sources contributing to the scalp recorded auditory mismatch negativity (MMN) by means of low-resolution electromagnetic tomography (LORETA) of speech sounds in tonal language. For these purposes, we recorded the MMN, elicited by monosyllabic words in tonal languages and the LORETA was utilized to explore the neural electric activity. Our results suggest that the inferior frontal gyrus (BA 44/45; x, z, y = -52, 24, 22) of the left-hemisphere was predominant in the perception of native speech sounds, whereas the nonnative speech sound was dominated by the superior frontal gyrus (BA 8; x, y, z = 25, 17, 43) of the right-hemisphere, which may be explained by the specialization in processing the prosodic and emotional components of speech formed in this hemisphere. (Presented at The 3 rd Annual Conference of the Institute of Science and Technology for Research and Development, Mahidol University 2003)

AUDITORY PREATTENTIVE PROCESSING OF PHONETIC SOUNDS IN CONSONANT-VOWEL (CV) SYLLABLE (NO. 1118) Wichian Sittiprapaporn, Chittin Chindaduangratana and Naiphinich Kotchabhakdi Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Research & Development Mahidol University, Salaya, Nakhon Pathom 73170. Key words: Event-related potential (ERP); Mismatch negativity (MMN); Phoneme; Low-resolution electromagnetic tomography (LORETA); Gender

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Institute of Science and Technology for Research and Development

showed very similar strongly left lateralized activation patterns. There were significant differences in amplitude and latency for phonetic MMNs between genders. These data are relevance to a growing number of researches of gender effects in response to changes of phonemes in normal adults. (Acknowledgement: This study was supported by the University Development Communities (UDC) grant from Mahidol University, Thailand. Presented at The 29 th Congress on Science and Technology of Thailand, Khon Kaen University 2003)

BRAIN LATERALIZATION OF WITHIN- AND ACROSS-CATEGORY CHANGE OF VOWELS IN CONSONANT-VOWEL (CV) SYLLABLES (NO. 1120) Wichian Sittiprapaporn 1 , Mari Tervaniemi 2 ,Chittin Chindaduangratn1 and Naiphinich Kotchabhakdi1

1

Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Research and Development, Mahidol University at Salaya Campus, Nakhonpathom 73170, THAILAND; 2Cognitive Brain Research Unit, Department of Psychology, P.O. Box 9 (Siltavuorenpenger 20 C) 00014 University of Helsinki, Helsinki, Finland. Key words : Event-related Potential (ERP); Brain; Vowel Event-related potentials (ERPs) responses to infrequently deviant syllables /pi/ and /pi:/ among repetitive standard /po:/ syllables were recorded in Thai subjects who were asked to ignore these stimuli while reading books of their choices. Hemispheric predominance of these within- and across-category change perception of vowels in consonant-vowel (CV) syllables was assessed using the low-resolution electromagnetic tomography (LORETA). The vowel of within- and across-category changes elicited a change specific mismatch response (MMN). The MMN of within-category of vowel was predominant in both left and right temporal boles whereas the MMN of across-category of vowel was predominant in the left temporal lobe (BA 44/45), emphasizing the role of the left hemisphere in speech processing of a pre-attentive processing level also in CVsyllables. (Acknowledgement: This study was supported by the University Development Communities (UDC) grant from Mahidol University, Thailand. Presented at The 29 th Congress on Science and Technology of Thailand, Khon Kaen University 2003)

DEMONSTRATION OF AMINO ACID NEUROTRANSMITTER INNERVATION IN HUMAN PINEAL GLAND (NO. 1122) Pansiri Phansuwa-Pujito, 1 Siriwan Thammikul, 2 Paisam Sithigorngul2 and Piyarat Govitrapong 2 Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, Bangkok10110, Thailand; 2Neuro-Behavioual Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; 3Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand. The amino acid neurotransmitters, -aminobutyric acid (GABA) and glutamate (Glu). Are known to be involved in the physiological functions of the mammalian pineal gland. In order to investigate both of these innervations in the human pineal, the immunohistochemical study was performed on the human pineal glands by using monoclonal antibodies against GABA and antiserum against glutamate as probes. GABA-immunoreactive (IR) cells and nerve fibers were found throughout the gland. Some IR cells resembled neurons with long processes were found occasionally. Only a few Glu-IR nerve fibers but many Glu-1R cell bodies were demonstrated in the human pineal gland. They wee arranged with unstained cells into clusters and some of them appeared to be neuron-like cells. Therefore, the present study supports the theory of regulation by both GABA and glutamate amino acid neuortransmitters in the human pineal gland. In addition, the presence of numerous immunoreactive cells indicates paracrine or local circuit regulation in human pineal. J. Sci. Asia 2003;29:235-239.

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GENDER DIFFERENCES ON MISMATCH NEGATIVITY RESPONSES TO PHONETIC SOUNDS

(NO. 1121)

Wichian Sittiprapaporn 1, Mari Tervaniemi 2 ,Chittin Chindaduangratn1 and Naiphinich Kotchabhakdi1

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Clinical & Research Electroneurophysiology Laboratory, NeuroBehavioural Biology Center, Institute of Science & Technology for Research and Development, Mahidol University at Salaya Campus, Nakhon Pathom 73170, THAILAND; 2Cognitive Brain Research Unit, Department of Psychology, P.O. Box 9 (Siltavuorenpenger 20 C) 00014 University of Helsinki, Helsinki, Finland. Key words: Event-related potential (ERP); Mismatch negativity (MMN); Gender Electrophysiological approach with low-resolution electromagnetic tomography (LORETA) was used to examine gender effects on the amplitude, latency and topography of phonetic MMN in response to consonant-vowel (CV) syllables. To clarify the role of gender differences in the MMN, we compared the amplitude, latency and topography of phonetic MMNs between right-handed healthy male (12) and female (12) subjects. While both genders

IMPROVEMENT OF VASCULAR FUNCTION Y CHRONIC ADMINISTRATION OF A CYCLOOXYGENASE INHIBITOR IN CHOLESTEROLFED RABBITS (NO. 1123) Supath Srisawat, Laddawal Phivthong-ngam, Supeenun 2 Unchern , Udom Chantharaksri, Piyarat Govitrapong3 and Yupin 1 Sanvarinda Department of Pharmacology, Faculty of Science, Mahidol University, 2Department of Pharmacology, Faculty of Medicine, Srinakharinwirot University, Bangkok and 3Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahdiol University Salaya, Nakornpathom, Thailand.

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Mahidol University Annual Research Abstracts, Vol. 31

1. Atheroscherotic cardio- and cerebrovascular disease is a leading cause of mortality in Western countries. Aspirin-like drugs are widely used to prevent and treat these occlusive cardio- and cerebrovascular diseases. The beneficial effects of these drugs have been largely attributed to inhibition of platelet cyclo-oxygenase activity and thromoxane (TX) A2 production. We investigated the effect of an aspirin-like drug, namely indomethacin, on endothelial function, plaque and platelet aggregation and the formation of vasoactive substances during the development of atherosclerosis in cholesterol-fed rabbits. 2. Rabbits were fed 1% cholesterol (n = 8), 1% cholesterol plus 25 mg/day indomethacin (n = 8) or normal rabbit chow (control group; n = 8) for 12 weeks. Urinary excretion rates of 2,3dinor-TXB2, 6-keto-prostaglandin (PG) F1a , 8- iso-PGF 2a and nitrate were analysed at the beginning of dietary intervention and at 4 weekly intervals thereafter. At the end of the study period, platelet aggregation, aortic plaque formation and endothelium-dependent and ­independent vascular function of isolated aortic rings ex vivo wee assessed. 3. Compared with control, in the cholesterol-fed group, urinary 2,3-dinor-TXB2, 6-keto-PCF and 8-iso-PGF2a excretion and platelet aggregation were significantly increased (P < 0.05), but urinary excretion of nitrate was decreased (P < 0.05). Treatment with indomethacin significantly reduced platelet aggregation, urinary 2,3dinor-TXB2, 6-keto-PGF1a and 8-iso-PGF2a excretion (P < 0.05 vs the cholesterol-fed group) and attenuated the reduction in urinary nitrate excretion. 4. Cholesterol feeding progressively increased aortic intimal thickening and impaired endothelium-dependent vasodilator function (P < 0.05 vs control), whereas indomethacin partially prevented aortic plaque formation and restored endothelium-dependent vasodilation (P < 0.05) vs the cholesterol-fed group). 5. The present study demonstrates that indomethacin reduces the progression of atherosclerotic lesions and improves endothelium-mediated vascular responses ex vivo in cholesterol-fed rabbits. The beneficial effects of indomethacin may be due to its ability to prevent the elevation of platelet aggreation, TXA1 (measured as urinary 2,3-dinor-TXB 2 excretion) and 8-iso-PGF2a formation and to retard the decrease in endogenous nitric oxide synthesis (assessed as urinary excretion of nitrate). Despite indomethacin treatment leading to the suppression of prostacyclin biosynthesis (assessed as urinary 6-keto-PGF2a excretion), according to our data, indomethacin appears to preserve endothelial function. (Clinical and Experimental Pharmacology and Physiology (2003) 30, 405-412.)

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Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA; 3Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornpathom 73170, Thailand. The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP+) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP+mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP+ produced a concentration- and timedependent reduction in the uptake of [ 3H]dopamine. We observed a significant decrease in [ 3H]WIN 35428 binding in the intact cells with MPP+. The saturation analysis of the [ 3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (Bmax) with an increase in the dissociation equilibrium constant (Kd) after MPP + treatment. Furthermore, biotinylation assays confirmed that MPP+ reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP+ may e a contributory factor in the down-regulation of DAT may signal event leading to cellular toxicity. (Biochemical and Biophysical Research Communications 2003; 311(1):49-54.)

MELATONIN RECEPTOR EXPRESSION IN RAT CEREBRAL ARTERY (NO. 1125) Prapapun Chucharoen 1 , Banthit Chetsawant 1 , Anan Srikiatkhachorn,2 Piyarat Govitrapong 1

1

Neuro-Behavioral Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; 2Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Melatonin has been shown to act on certain vascular beds. However, the identity of melatonin receptors in the cerebral artery has not been established. In the present study, we have attempted to identify melatonin receptor mRNAs of rat cerebral artery by using a reverse transcriptase polymerase chain reaction technique. Total RNAs were extracted from the cerebral artery and different regions of male Sprague-Dawley rats. Amplification of RNAs from pineal gland, hypothalamus and cerebral artery with mt1 while amplification of RNAs from pineal gland and hypothalamus with mt2 primers resulted in products of the predicted lengths. The result indicated that mt1 but not mt 2 melatonin receptor. Further studies are necessary to understand the significance of this receptor in regulating physiological function. (Neuroscience Letters 341 (2003)259-261.)

1-METHYL-4-PHENYLPYRIDINIUM-INDUCED DOWN-REGULATION OF DOPAMINE TRANSPORTER FUNCTION CORRELATES WITH A REDUCTION IN DOPAMINE TRANSPORTER CELL SURFACE EXPRESSION (NO. 1124) Jaturaporn Chagkutip, 1,3 Roxanne A. Vaughan, 2 Piyarat Govitrapong,1,3 and Manuchair Ebadi1

1

Department of Pharmacology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA

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Institute of Science and Technology for Research and Development

rats was studied during development from birth (PO) to postnatal day 16 (P16). A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect changes in the expression of delta- (DOR) kappa- (KOR) and mu- (MOR) opioid receptor mRNAs in rat cochleae at PO, P4, P8 and P16. Expression of DOR mRNA levels steadily increased from PO to P8 with no further increases by P16. KOR mRNA was expressed at a relatively high level at PO and P4 followed y a decrease while MOR mRNA was expressed at a low level at PO and P4 followed by an increase by P8 and P16. Immunocytochemical labelling of inner ear sections revealed unique developmental and distribution patterns of opioid receptors. In the organ of Corti DOR immunoreactivity (DOR-IR) was detected in hair cells from P4. In contrast MOR-IR was present only in supporting cells at PO-P16. In the spiral ganglion all three receptor subtypes were expressed from PO on nerve cell soma and qualitatively appeared to increase with age. Also DOR-I and MORIR wee detected at P8 and P16 in nerve fibers within the spiral ganglion. In the limbus DOR-IR was detected at P8 and P16 on cells proximal to the tectorial membrane while MOR-IR was detected more distally. In general these findings demonstrate that within the inner ear each receptor subtype follows specific temporal and spatial developmental patterns, some of which may e associated to the onset of hearing. The data provide further evidence that the opioid system may play a role in the development and functioning of the inner ear. (Hearing Research 184 (2003) 1-10)

THE PRESENCE OF OPIOID RECEPTOS IN RAT INNER EAR (NO. 1126) Nopporn Jongkamonwiwat1 , Pansiri Phansuwan-Pujito 2 Puangkeaw Sarapoke 1 , Bangkok Chetsawant 1, Stefano O. Casalotti 3 , Andrew Forge 3 , Hilary Dodson 3 , Piyarat Govitrapong1

1

Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; 2Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand; 3Institute of Laryngology and Otology, University College London, Gray's Inn Road, London WCX 8EE, UK Opioid peptides have been identified in the inner ear but relatively little information is available about the expression and distribution of their receptors. The aim of the present study was therefore to identify and localize the m (MOR), d(DOR) and k (KOR) opioid receptor subtypes within the rat cochlea. The expression of these opioid receptor subtypes was determined by reverse transcriptase-polymerase chain reaction followed by nested polymerase chain reaction analysis. Amplification of RNAs from rat cerebral cortex (positive control) and rat cochlea with MOR, DOR and KOR primers resulted in products of the predicted lengths, 564, 536 and 276 bp, respectively. Restriction digestion confirmed the identity of these products. All three receptor subtypes were identified in the cochlea and further characterized by immunocytochemistry. DOR and KOR immunoreactivity was found in inner and outer hair cells, bipolar cells of the spiral ganglion and interdental cells of the limbus. In contrast, no MOR immunoreactivity was observed in the inner and outer hair cells, and interdental cells. All three types of receptor fibers were also detected in the bipolar cells and nerve fibers within the spiral ganglion. In addition, MOR ­ and KORcontaining nerve fibers were observed in the limbus. These findings are the first report of the presence of all three classical opioid receptors in the inner ear and suggest that these receptors may have both presynaptic and postsynaptic roles. (Hearing Research 181 (2003) 85-93.)

EFFECTS OF D1 AND D2-DOPAMINE RECEPTOR ACTIVATION ON MELATONIN SYNTHESIS IN BOVINE PINEALOCYTES (NO. 1128) Chorthip Santanavanich 1, Banthit Chetsawang 1, Manuchair Ebadi2, Piyarat Govitrapong 1

1

Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornpathom 73170, Thailand; 2Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, School of Medicine and Health Science, USA. Key words: Pineal gland, pinealocytes, dopamine receptors, melatonin, N-acetyltransferase Previous studies have identified and characterized D1 and D2 dopamine receptors in bovine pineal glands. The data indicate that the density of D1 dopamine receptors (974 fmol/mg protein) far exceed that of D2 dopamine receptors (37 fmol/mg protein). The objective of this study was to identify the mRNAs for both D1 and D2 dopamine receptors and to elucidate the status of dopamine and its possible involvement in the pineal function, particularly on melatonin synthesis. The expression of these dopamine receptor subtypes were determined by using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique with specific pairs of primers to amplify D1 and D2 dopamine receptor mRNAs. Amplification of RNAs from bovine striatum (positive control) and bovine pineal gland resulted in products of the predicted lengths of 231 bp for D1 and 333 bp for D2 dopamine receptors. The results indicate that both D 1 and D2 dopamine receptor mRNAs are present in the bovine pineal gland. The role of dopamine receptors was investigated by studying the effects of selective D1 and D2 dopamine agonists and antagonists on the N-acetyltransferase (NAT) activity of cultured bovine pinealocytes. The data showed that SKF-38393, a selective D1 agonist, enhanced NAT activity and increased melatonin level, and the

THE OPIOID RECEPTORS IN INNER EAR OF DIFFERENT STAGES OF POSTNATAL RATS

(NO. 1127)

Pansiri Phansuwan-Pujito 1, Ladda Saleema 2, Sujira Mukda 2, Walaiporn Tongjaroenbuangam 2, Nuanchan Jutapakdeegul 2, Stefano O. Casalotti 3, Andrew Forge 3, Hilary Dodson 3, Piyarat Govitrapong 2,*

1

Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand; 2Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus 3 Nakornpathom 73170, Thailand; Institute of Laryngology and Otology, University College London, Gray's Inn Road, London WC1X 8EE, UK There is increasing evidence that the opioid system has a role in hearing. To provide furthe evidence for such a role, the expression of opioid receptor mRNAs and proteins in the inner ear of

Mahidol University Annual Research Abstracts, Vol. 31

stimulatory effect was blocked by SCH-23390, a D 1 selective antagonist, whereas quinpirole, a selective D2 agonist, inhibited NAT basal activity and decreased the melatonin basal level. Furthermore the inhibitory effect was blocked by D 2 selective antagonists, spiperone, haloperidol and domperidone. The present results indicate that the pineal dopamine receptors have a distinct effect on pineal function. The precise mechanism whereby activation of dopamine receptors altered the NAT activity and melatonin level needs to be further delineated. (J. Pineal Res. 2003;35: 169-176.) THE OPIOID RECEPTORS IN INNER EAR OF DIFFERENT STAGES OF POSTNATAL RATS

439

(NO. 1130)

Piyarat Govitrapong 1, Sujira Mukda 1, Pansiri PhansuwanPujito 2 , Walaiporn Tongjaroenbuangam 1 , Udomsri Showpittapornchai2, Stefano O. Casalotti 3, Hilary Dodson 3, Andrew Forge3

1

IDENTIFICATION OF DOPAMINE TRANSPORTER IN BOVINE PINEAL GLAND USING [3H]GBR 12935

(NO. 1129)

Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170 Thailand, 2Department of Anatomy, Faculty of Medicine, Srinakarinwirot University, Bangkok 10110, Thailand, 3Institute of Laryngology and Otology, University College London, Gray's Inn Road, London WC1X 8EE, UK. Key words: Opioid receptors, cochlea, inner ear, hair cells, development, hearing There is increasing evidence that the opioid system has a role in hearing. To provide further evidence for such a role, the expression of opioid receptor mRNAs and proteins in the inner ear of rats was studied during development from birth (P0) to postnatal day 16 (P16). Immunocytochemical labelling of inner ear sections revealed unique developmental and distribution patterns of opioid receptors. In the organ of Corti DOR immunoreactivity (DOR-IR) was detected in hair cells from P4. In contrast MOR-IR was present only in supporting cells at P0 to P16. In the spiral ganglion all three receptor subtypes were expressed from P0 on nerve cell soma and qualitatively appeared to increase with age. Also DOR-IR and MORIR were detected at P8 and P16 in nerve fibres within the spiral ganglion. In the limbus DOR-IR was detected at P8 and P16 on cells proximal to the tectorial membrane while MOR-IR was detected more distally. In general these findings demonstrate that within the inner ear each receptor subtype follows specific temporal and spatial developmental patterns, some of which may be associated to the onset of hearing. The data provide further evidence that the opioid system may play a role in the development and functioning of the inner ear. (Acknowledgments : The authors greatly acknowledge the Wellcome Trust for supporting the collaborative research initiative grants to PG. This study was also supported in part by Thailand Research Fund to WT. International Brain Research Organization 2003)

P. Govitrapong1,2, P. Vilaipun2, and M. Ebadi1 Department of Pharmacology, Physiology and Therapeutics, School of Medicine & Health Sciences, University of North Dakota, Grand Forks, North Dakota 58203, U.S.A; 2NeuroBehavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornpathom, 73170, Thailand. Key words: Dopamine transporter, pineal gland, D1 dopamine receptors, D2 dopamine receptors, melatonin, N-acetyltransferase The mammalian pineal glands contain several neurotransmitters and receptors for amino acids, biogenic amines and peptides. Some of these, such as dopamine and D1 and D2 dopamine receptors, have been previously identified and characterized in the bovine pineal gland by our group. As a matter of fact, the density of D1 dopamine receptors in the pineal gland is higher than that of corpus striatum, suggesting that this organ must possess a high affinity dopamine transporter, which has been identified in this study by using [3H]GBR 12935 as a radiological and nomifensine to determine non specific binding. The association rate of [ 3H]GBR 12935 binding to the pineal membrane was examined as a function of time. The binding reached equilibrium within 45 min of incubation at 25?C. The specific binding was reversible and saturable. The dissociation time course of the specific [3H]GBR 12935 binding from the bovine pineal membrane was also studied. A half-life (t1/2) of 14 min was obtained. The saturation analysis of the [ 3H]GBR 12935 binding revealed a dissociation equilibrium constant (Kd) of 6.0 ± 0 0.9 nM and a receptor density (Bmax) of 6.9 ± 0.3 pmol/mg protein, which were comparable to those values obtained from bovine striatum and frontal cortex. In competitive experiments, the concentrations of drugs required to inhibit 50% of the binding (IC 50) were in descending order GBR 12909 > GBR 12935 > trans-flupenthixol > nomifensine > cis-flupenthixol > amitriptyline > imipramine > desipramine > dopamine > fluoxetine > fuvoxamine > d-amphetamine. However, nisoxetine, SCH 23390, norepinephrine and serotonin were unable to displace [3H]GBR binding. These results showed that drugs capable of blocking dopamine transporters were effective in displacing [3H]GBR binding; whereas specific norepinephrine and serotonin transporter inhibitors were less effective or ineffective. In addition, the dopamine transporter was ion dependent since sodium increased [3H]GBR binding in a concentration related manner. These results indicate that a high affinity dopamine transporter exists in the bovine pineal whose physiological functions need to be delineated and characterized in future investigations. (Journal of Pineal Research (2003)35:16-23.)

1

THE POSSIBLE ROLE OF OPIOID RECEPTORS IN THE BOVINE PINEAL GLAND (NO. 1131) Phansuwan-Pujito P1, Ebadi M2, Govitrapong P3 Department of Anatomy, Srinakharinwirot University, Faculty of Medicine, Bangkok, Thailand; 2Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, School of Medicine and Health Science, USA; 3Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Thailand. The opioidergic innervation has been suggested to involve in the stimulation of melatonin synthesis in the mammalian pineal gland. Opioidergic nerve fibers have been demonstrated in the gland together with the characterization of opioid receptors. However, the

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Institute of Science and Technology for Research and Development

ally declined towards adult stage. The present data provide further evidence of the role of opioid receptors in the development of the auditory system. (Acknowledgement: an International Collaborative Research Initiative Grant from the Wellcome Trust to Dr.Piyarat Govitrapong supported this study. IBRO Meeting 2003)

precise location of opioid receptors in the pineal gland is still unknown. In the present study, the immunohistochemical studies of opioid peptide and receptors were performed in the bovine pineal gland by using antisera raised against leu-enkephalin, and mu- and delta-opioid receptors in order to localize the opioid receptors related to the opioid peptides. Both mu- and delta-opioid receptor immunoreactivities were demonstrated predominantly in pinealocytes, and. some in neuronal-like cells and nerve fibers. Interestingly, a large number of delta-opioid receptor immunoreactive nerve fibers were observed at the periphery of the bovine pineal gland. By double labeling immunofluorescene study, a colocalization of delta-opioid receptors and leu-enkephalin was found in some pinealocytes and on nerve fibers in the parenchyma of the gland, but not in nerve fibers at periphery. However, delta-opioid receptors were observed to colocalize with the neuropeptide Y (NPY) in the several nerve fibers at the peripheral of the gland. The present results indicate that the opioidergic innervation might control melatonin synthesis in the pattern of paracrine regulation by acting on postsynaptic and presynaptic types of the opioid receptors on the pinealocytes and the nerve fibers. In addition, the presynaptic delta-opioid receptors may modulate the release of opioid peptide as well as other neurotransmitters or neuropeptides such as NPY from nerve fibers. Acknowledgement: This study was supported in part by the National Institute of Neurological Disorders and Stroke, No. 1RO1-NS3876404. (IBRO meeting)

POSTNATAL TOUCH STIMU1 LATION ACUTELY ALTERS CORTICOSTERONE LEVELS AND GLUCOCORTICOID RECEPTOR GENE EXPRESSION IN NEONATE RATS (NO. 1133) N. Jutapakdeegul1., Stefano O Casalotti 2., P. Govitrapong., N. Kotchabhakdi 1

1

Neurobehavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornpathom 73170. Thailand. 2Institute of Laryngology and Otology, UCL, London WC1X 8EE. UK. Key words: Postnatal touch stimulation, Corticosteroid hormone, Glucocorticoid receptor, Semiquantitative RT-PCR Environmental manipulation early in life can alter the development of the Hypothalamic-Pituitary-Adrenal (HPA) axis by mechanisms that are still unclear. The aim of the present work was to study the acute effects of postnatal touch stimulation, in an attempt to understand the mechanism by which touch stimulation during early period of life alters the HPA response to stress in adult animals. Rat pups were gently brushed for 15 minutes daily during the first postnatal week. Serum corticosterone levels were determined by radioimmunoassays, while glucocorticoid receptor (GR) gene expression was assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Touch stimulation induced a significant decrease (30-36%) in serum corticosteroid secretion during the first and the second postnatal day as compared to the unstimulated group. In contrast, GR gene expression in the touch stimulation group was significantly increased in several brain areas such as hippocampus (19-21%), frontal cortex (26-34%) and midbrain (15-24%). The results thus indicate that neonatal touch stimulation cause acute hormone-secretion and gene-expression changes within the period of stimulation. These changes may be the underlying cause for the permanent changes that have been observed in adult animals touch-stimulated as neonates. (Developmental Neuroscience 2003; 25: 26-33.)

OPIOID RECEPTORS GENE EXPRESSION IN THE INNER EAR OF EARLY POSTNATAL RATS (NO. 1132) Jutapakdeegul N 1, Saleema L 1, Chetsawang B1, Casalotti SO2, Govitrapong P1

1

Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom, Thailand 2Institute of Laryngology and Otology, University College London, London, United Kingdom. Several data support the hypothesis that endogenous opioid modulation of brain development is a fundamental principle of mammalian embryogenesis. Our group has previously identified subtypes of opioid receptor in the mammalian cochlea. In the present study we have determined the expression of mu, delta and kappa opioid receptor subtypes in postnatal rat cochlea by reverse transcriptase polymerase chain reaction (RT-PCR). Total RNAs was extracted from cochlear tissues of postnatal (P0, P4, P8 and P16) and adult Spraque-Dawley rats. Semi-quantitative RT-PCR analysis was performed with primers specific for the nucleotide sequences representing mu, delta and kappa opioid receptor subtypes and actin was used as an internal control. Amplification of mRNAs from rat cochlea tissues with MOR, DOR, KOR and actin primers resulted in products of the predicted lengths 564, 356, 276 and 830 bp, respectively. The results of these studies indicate that the mRNAs encoding all three opioid receptor subtypes were expressed in postnatal cochlea as early as P0 and the relative level of expressions varied during cochlea development. The lowest level of delta opioid receptor mRNAs expression was on the first day of birth (P0) and increased gradually on postnatal period towards adult stage. The level of mu opioid receptor mRNAs expression was also as same as but with delayed pattern compare to DOR. In contrast, the kappa opioid receptor mRNAs expression was highest at P0 and P4 and then gradu-

EXERCISE DURING PREGNANCY INCREASES HIPPOCAMPAL BDNF mRNA EXPRESSION AND SPATIAL LEARNING IN NEONATAL RAT PUP

(NO. 1134)

Panaree Parnpiansil1 , Nuanchan Jutapakdeegul 1 , Thyon 1 Chentanez2, Naiphinich Kotchabhakdi

1

Neuro-Behavioral Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand; 2Department of Physiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.

Mahidol University Annual Research Abstracts, Vol. 31

Physical activities for a few days can increase brain derived neurotrophic factor (BDNF) mRNA in rat hippocampus. To investigate the influence of maternal exercise during pregnancy on rat pup hippocampal BDNF mRNA, we studied its expression by semi-quantitative RT-PCR method after young pregnant rats were exercised on a motor driven treadmill. Pups of exercised mothers had significant increased hippocampal BDNF mRNA expression compared to the control rat pups at birth (on postnatal day 0) (p<0.001). In contrast, hippocampal BDNF mRNA expression in pups of exercised mothers decreased significantly on postnatal day 28 (p<0.002). Spatial learning of rat pups was examined by multiple Tmaze training for 7 consecutive days between postnatal days 40-47. Pups of exercised mothers showed significant increase in spatial learning ability as demonstrated by significant decreases in total time from starting to target and total number of errors as compared to agematched control pups during the first four days of 7 consecutive days on multiple T-maze training (p<0.05). Thus physical exercise during gestation in pregnant mother can increase hippocampal BDNF mRNA expression of postnatal pups and an improvement on spatial learning in pups from exercised dam. (Neuroscience Letter 2003; 352: 45-48) DISCRIMINATION OF AEDES AEGYPTI (DIPTERA : CULICIDAE) LABORATORY LINES BASED ON WING GEOMETRY Nuananong Jirakanjanakit 1*, Jean-Pierre Dujardin 2

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441

(NO. 1136)

Center for Vaccine Development, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand, 2IRD-CNRS, UMR 9926, Montpellier (France) NCID, DPD, Center for Disease Control and Prevention, Chamblee, Atlanta, GA Key words : geometric morphometrics, Aedes aegypti, Thailand The recent techniques of geometric morphometrics were applied to the study of four Thailand laboratory lines of the dengue vector Aedes aegypti. These lines differed by their geographic origin and the number of generations spent in the laboratory. Using phase contrast microscope, a set of sixteen landmarks of the wings could be identified. A subsequent Procrustean superposition analysis produced the corresponding size and shape variables which were statistically analyzed. Size showed a significant decrease with number of generations spent in the laboratory. Shape allowed an almost perfect reclassification of the specimens. This tool is able to accurately distinguish different laboratory lines and will be useful in field studies related to entomological surveillance and vector control. (This work was supported by IRD-DSF, France and NCID, DPD, Center for Disease Control and Prevention, Atlanta, GA.)

MULTI-COUNTRY VALIDATION STUDY OF AEDES AEGYPTI PUPAL PRODUCTIVITY SURVEY METHODOLOGY-DENGUE VECTORS IN THAILAND (NO. 1135) Barbazan Philippe ,Jirakanjanakit Nuananong , Tuntraprasart 3 4 5 Walairut , Thammapalo Suwich , Saengtharatip Seeviga , 1,2 1,2 1 1,2 Bellec Christian , Yoksan Sutee ,Gonzalez Jean-Paul.

1 1,2 1

Center for Vaccine Development - Research Center for Emerging Viral Diseases Institute of Sciences and Technology for Development Mahidol University at Salaya, 25/25 2 Phutthamonthon 4, Nakhonpathom 73170, Thailand; IRDResearch Unit 034, Institut de Recherche pour le Developpement. 3 France Department of Medical Entomology, Faculty of Tropicak 4 Medicvine, Mahidol University, Bangkok; Dengue fever control 5 division, Ministry of Public Health, Thailand; Vector Borne Disease Bureau, Disease Control Department, Ministry of Public Health, Thailand. Key words : Aedes aegypti index, Pupae, Dengue The multi-Country validation study of Aedes aegypti pupal productivity survey methodology is part of a WHO-TDR project. Principal objective is to develop a method to improve entomological routine strategy to evaluate risk of dengue virus transmission. In Thailand the study will be done from 2003 to 2004 in 5 areas representative of different of urbanisation, and climate (Khon Kaen, Chiang Mai, Surat Thani, Ratchaburi and Bangkok). In most of the containers an exhaustive collection of larvae and pupae will be done. Results of pupae survey will be compared with routine work done by MOPH teams in the frame of dengue surveillance. A development will focus on a specific and exclusive survey based only on jars used by the population to collect water and the most productive containers for A. aegypti larvae. A GIS will be used to store and analyse data and identification results. Improvement of the efficiency of control strategy will be proposed based on the study results. (Sources of Grant : WHO-TDR, CVD, IRD.)

MONITORING JAPANESE ENCHEPHALITIS VIRUS BY USING DOMESTIC SENTINEL PIGS IN KAMPHANGSAEN DISTRICT NAKHON PATHOM, THAILAND (NO. 1137) Narong Nitatpattana 1, Mimi khin1, Somnuek Palabodeewat 1, Pattanapon Thongchai 1, Philippe Babarzan1,2, Sutee Yoksan1 and Jean-Paul Gonzalez 1,2

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Center for Vaccine Development, Research Center for Emerging Viral Disease, Institute of Science and Technology for Research and Development, Mahidol University, Thailand at Salaya, 25/25 Phutthamonthon 4, Nakhon Pathom 73170; 2Research Unit 034, Institut de Recherche pour le Dé veloppement, Paris, France Key words : Japanese Enchephalitis Virus, pig, Thailand Awareness of the annual recurrence of Japanese Encephalitis Virus (JEV) infections among the people residing in the provinces of Central plain region of Thailand has promoted to investigate seroconversion rate pigs (amplifying host). Ten domestic piglets (12 weeks old) from Nakhon Pathom Province were chosen as the sentinels for the period covering January to May 2003. Activity of JE was monitored by ELISA for detection of specific JE IgM and IgG. Serodiagnosis was mainly based on the appearance of specific IgM which occurred among 5 out of 9 pigs. JEV was isolated from 2 piglets. In a piglet (RCEVD 03/01) JE virus was collected in day 0 (MID50 104.3 /ml) and day 14, and pig RCEVD 03/07 also revealed the presence of JEV on the day 0 collection (age of pig 12 weeks) JE virus viremia period of about 14 days provide ample time for the vector Culex mosquito to feed on the pigs and provoke the transmission of JEV in the human community residing in the areas. (Source of Grant: TRF, CVD, IRD.)

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be made in cord blood by the automated HPLC system, and -globin genotyping can be confirmed by gap-PCR technique. In this study we followed the levels of Hb E in newborn Hb E heterozygotes who had Hbs A, F and E with or without Hb Bart's, every 1-2 months, until the age of one year. Iron supplementation was given daily in all infants, during the age of 4-10 months old, to prevent reduction of Hb E level due to iron deficiency anemia. The levels of Hb E in 50 cord blood of Hb E heterozygote ranged between 2.1 and 10% (mean+SD 4.3+1.54%) and those of 6 cases double heterozygous for Hb E and -thalassemia 1 were also in the same range. A sharp increase in the amount of Hb E was th observed at the 8-12 weeks follow-up and was constant after the 6 month at the average amount of 29+0.28%. However, a lower level of Hb E was observed, during one year follow-up, in infants double heterozygous for Hb E and -thalassemia 1. Decreased expression E of -globin gene can be demonstrated as the Hb E/Hb A ratio in newborns, 0.54+0.11, which was constant throughout neonatal period. In comparison with normal infants a delayed decline of Hb F levels in Hb E heterozygotes was also observed during one year follow-up. The amount of Hb F at 12 weeks of age was significantly higher than those of the normal infants (34.8+6.75 vs 7.7+4.76%) th and reduced to the level closed to normal infants at the 20 week. The result showed that the delay in the switch from - to - globin may occur due to the abnormal expression of the downstream globin gene. (This study was supported by the Thailand Research Fund. Presented at the 9th International Conference on Thalassemia and Hemoglobinopathies, Palaermo, Italy, October 15-19, 2003.)

A RISK OF JAPANESE ENCEPHALITIS VIRUS DOMESTIC TRANSMISSION : VIROLOGICAL ANDENTOMOLOGICAL APPROCH (NO. 1138) Narong Nitatpattana 1, Apiwathnasorn Chamnarn2, Philippe Barbazan1,3, Leemingsawat Somjai 2, Sutee Yoksan1, and JeanPaul Gonzalez 1,3 Center for Vaccine Development, Research Center for Emerging Viral Disease, Institute of Science and Technology for Research and Development, Mahidol University, Thailand; 2 Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Thailand; 3Research Unit 034, Institute de Recherche pour le Developpement, Paris, France. Key words: Japanese encephalitis virus, Mosquito, Thailand. Japanese Encephalitis virus (JEV) is a mosquito borne flavivirus. It is a leading cause of viral encephalitis in Thailand and in the public health agenda of preventable childhood diseases. The population at risk comprise children under 15 years of age and mainly living in rural areas. In the absence of any intervention JE infection could not only result in high case fatality rate, but also surviving patients recur a nuerological defact (disabilities). The entensity of public health concern stimulated the Ministry of Public Health to introduce JE vaccine to the national immunization program in 1995. Both "Beijing" and "Nakayama" JE vaccines induce the homologous and heterologous neutralizing antibody response. Despite intense control effects JE infection continue to be of great concern especcially as viral encephalitis of unknown origin often mimicking JE infection is encountered in rural areas. The necessary for a better understanding of complex JE epidemiology and in depth knowledge of JE virus circulation appear "fundamental" in order to identify the prevalence of vaccine genotypes in Thailand, the mechanisms of the change in epidemiology from North to South and the importance to isolate local strains to serve as candidate vaccine virus for more efficient, production of immunity in the local population of Thailand. (Source of Grant: TRF, CVD, IRD. Developmental Neuroscience 2003; 25: 26-33)

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EVIDENCE FOR ROLE OF BONE MARROW ENVIRONMENT IN THE ACCELEARATED APOPTOSIS OF THALASSEMIC ERYTHROID PROGENITOR CELLS IN BONE MARROW (NO. 1140) Kittiphong Paiboonsukwong , Nattaya Sae-Ung , Pranee 2 3 2 Winichagoon , Koichiro Muta , Suthat Fucharoen

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EXPRESSION OF -AND--GLOBIN GENES IN INFANTS HETEROZYGOUS FOR HEMOGLOBIN E (NO. 1139) Praneee Winichagoon-Fucharoen , Kanjana Vichittumaros , 2 2 Pattanee Winichagoon , Chureeporn Chitchumroonchokechai , 1 Suthat Fucharoen

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Institute of Molecular Biology and Genetics, Thalassemia Research Center, Institute of Science and Technology for Research 3 and Development, Mahidol University, Thailand; Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan. Thalassemia, which is characteized by the absence or reduced synthesis of globin chains, is the most common hereditary hematological disorder. The molecular defects of this disease have been characterized in great detail but the mechanisms leading to hemolysis and ineffective erythropoiesis are not well described. Previous study showed that marrow erythroid precursor cells in -thalassemic patients were under accelerated apoptosis, which was proposed to be correlated with the extent of ineffective erythropoiesis in thalassemia. We have postulated that enhanced apoptosis in thalassemia might occur from the abnormal interaction of various growth factors. To test this hypothesis, the in vitro model of production of human RBCs from isolated peripheral blood erythroid colony forming cells (ECFCs) by negative selection has been used in combination with recombinant growth factors to promote RBC differentiation. Cell viability and apoptotic cells were determined by trypan blue exclusion test and flow cytometric analysis, respectively. Our

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Thalassemia Research Center, Institute of Science and Technol3 ogy for Research and Development and Institute of Nutrition, Mahidol University, Salaya, Nakornpathom 73170, Thailand. Hemoglobin (Hb) E, the most prevalent hemoglobinopathy in Southeast Asia, occurs from the mutation at codon 26 of the + globin gene. The abnormal gene also acts as a mild -thalassemia since an adjacent cryptic splice site in codon 25 is also activated. The interaction of Hb E and -thalassemia leads to a compound heterozygote, -thalassemia/Hb E disease, with a variable clinical presentation from a mild to a severe chronic hemolytic anemia. Neonatal screening of Hb E along with the detection of -thalassemia can

Mahidol University Annual Research Abstracts, Vol. 31

results indicated that the effect of erythropoietin (EPO) and interferon gamma (IFN-) on prevention of apoptosis of ECPCs are the same among the day 7 ECFCs from normal and the thalassemic subjects. There is no significant difference in the rate of apoptosis of erythroid progenitor cells from normal and thalassemic patients by using current cultured condition. However, our preliminary study using ECFC from bone marrow samples of -thalassemia/Hb E patient demonstrated the accelerated apoptosis in patients' erythroid cells. The possible explanation is that characteristics of ECFCs cells isolated from peripheral blood may be different from the intact erythroid cells in the bone marrow. This implies that enhanced apoptosis in the erythroid precursor cells may result from the interaction between erythroid precursor cells and environment in the bone marrow. (This study was supported partly by the National Science and Techth nology Development Agency. Presented at the 6 Conference AsiaPacific International Molecular Biology Network 2003, November 12-13, 2003, Tokyo, Japan.)

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carried out to clarify this hypothesis. This information should be useful in understanding of erythropoiesis in thalassemia, as well as in gene therapy in the future. (This work was partially supported by grants fromthe NSTDA, Thailand, Kyushu University P&P grant and the Ministry of University th Affairs, Japan. Presented at the 9 National Conference on Thalassemia, June 18-19, 2003, Bangkok, Thailand.)

HUMANISED MOUSE MODELS FOR -THALASSEMIA/Hb E DISEASE (NO. 1142) Duangporn Jamsai , Faten Zaibak , Jim Vadolas , Suthat 2 2 1 Fucharoen , Robert Williamson , and Panos Joannou

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Murdoch Childrens Research Institute, Royal Children's Hos2 pital, Australia, Thalassemia Research Centre, Institue of Science and Technology for Research and Development and Institute of Molecular Biology and Genetics, Mahidol University, nakornpathom, Thailand. HbE is the most common hemoglobin variant, caused by a substitution of Glutamic acid by Lysine at codon 26 of the -globin. This mutation also activates a cryptic splice site in exon I, resulting in weak aberrant splicing. Individuals homozygous for HbE exhibit mild thalassemia, while combination of HbE with a -thalassemia mutation results in -thalassemia/HbE disease with a highly variable severity of presentation. We have used the GET Recombination system in combination with novel counterselection markers to introduce the HbE and the codons 41/42 bp deletion mutations into a 205 kb BAC containing the entire human -globin locus. The normal and modified -globin BAC clones were used to generate transgenic mice. Transgenic mice were bred with heterozygous knockout mice carrying a deletion of the moust -globin genes. Mice hemizygous for the HbE transgene on a heterozygous knockout background have 25-35% hetrotetrameric (am2/bE2) hemoglobin and exhibit mild thalassemia phenotype. The heterotetrameric hemoglobin thus shows significant complemntation of the intermediated thalassemia phenotype that is normally gound in the heterozygous knochout mice, while miced carrying teh 4 bp deletion are similar to heterozygous knockout mic, with a -thalassemia intermedia phenotype. Further breeding of these mice has resulted in mice carrying the HbE transgene on a homozygous knockout background with 100% am2/bE2 hemoglobin. Mice expressing high level of HbE transgen survive to adulthood with mild thalassemia phenotype similar to human HbE homozygotes. These are the first mouse medels to recapitulate the main features of -thalassemia/HbE disease and should be invaluable models for the development of varius therapeutic approaches. (This study was supported partly by grants to the CAGT Research Groups and the Thailand Research Fund.) - AND -THALASSEMIA MUTATIONS IN -THALASSEMIA/Hb E PATIENT IN CHIANG RAI AND KORAJ (NO. 1143) Orapan Sripichai , Thongperm Munkongdee , Kanjana 2 2 3 Vichittumaros , Pranee Winichagoon , Saranya Suwansings , 4 4 Somchai Intarasiripong , Nittaya Visanuyothin , and Suthat 2 Fucharoen

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EFFECT OF CYCLOSPORINE A ON APOPTOSIS OF THALASSEMIA ERYTHROID PROGENITOR CELLS (NO. 1141) Nattaya Sae-ung , Pranee Winichagoon , Koichiro Muta , Suthat 2 Fucharoen

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Faculty of Medical Technology, Thalassemia Research Center, Instiute, of Science and Technology for Research and Develop3 ment, Mahidol University, Thailand, Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. The prevention of apoptosis is a key function of growth factors in regulation of erythropoiesis. Erythropoietin (EPO) has been accepted to be an essential growth factor in erythropoiesis. Many studies have also indicated that nuclear transcription-B (NF-B) activation can suppress cell death pathway and that NF-B activation is required to protect many cell types from apoptosis. NF-B activation allows free NF-B translocate from cytoplasm to the nucleus and bind to specific site of DNA to regulate expression of genes such as Bc1-x gene that expresses anti-apoptotic protein Bc1x. Previously we showed that EPO retarded apoptosis of normal erythroid progenitor cells via NF-B activation and Bcl-xinvolvement by using cyclosporine A (CsA) that blocks NF-B complex dissociation. In this study, we investigated the effect of CsA on apoptosis of thalassemic erythroid progenitor cells. Day7-erythroid colony forming cells (ECFCs) were treated with or without CsA and culo tured in the presence or in the absence of EPO for 24 hours at 37 C. Then, apoptosis was assessed from redistribution of phosphatidylserine by annexin V-FITC using flow cytometry. Cell viability was performed by using trypan blue staining. The results showed that EPO retarded apoptosis of ECFCs as compared with cells cultured without EPO. Reduction of cell viability related to dose-dependent effect of CsA (5-100 µM). We also found that CsA induced apoptosis of the cells cultured with EPO. As these results were similar to the previous study in normal erythroid progenitor cells, we suggest that EPO may retard apoptosis of both thalassemic and normal erythroid progenitor cells in the same mechanism. Further study of NF-B (p65) and Bc1-x expressions of the cells will be

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Department of Biochemistry, Faculty of Science, Thalassemia Research Center, Institute of Science and Technology for Re3 search and Development, Mahidol University, Salaya, Chiangrai 4 Pachanukraw Hospital, Muang, Chiang Rai, Maharaj Nakhonrajchasima Hospital, Muang, Nakhon Ratchasima. Beta-thalassemia/HbE is one of the common genetic diseases in Thailand. The molecular characterization of -thalassemia is absolutely necessary for molecular diagnosis as well as any genetic epidemiological study in this region. In order to determine the prevalence and molecular basis of and -thalassemias amont Thai -thalassemia/HbE patientss, a total of 166 subjects (90 from Chiang Rai and 76 from Nakhon Ratchasima (Koraj)) were enrolled in this study. The patients have been classified as mild, intermediate, and severe case according to the scoring system that includes Hb level, age onset age at first transfusion, transfusion requirement, size of spleen, and growth intermediate, and 79 (47.6%) as severe cases. Molecular basis of -thalassemia mutations was performed by reverse dot-blot hybridization (RDB) of the amplified DNA with a set of allele specific oligonucleotide (ASO)-probes to detect the common mutations. The results showed that, although there are more than 20 -thalassemia mutations in the Thai population, RDB technique using betaE (codon 26 (GA)) and 9 different ASO-probes could detect in totally 100% of the cases. Two mutations account for 94% of Chiang Rai cases (codons 41-42 (-TCTT) 55.6% and codon 17 (AT) 38.9%). In Koraj, 8 different -thalassemia mutations were identified and the common mutations included codons 41-42, 48.7%; codon 17, 22.4% and IVS-I#5 (G C), 15.8%. Two cases turnout to be + (codon -28 (AG)) -thalassemia/HbE. The results suggested that the molecular spectrum of -thalassemia mutations in -thalassemia/HbE patients from Koraj is broader than that of Chiang Rai. To study co-inheritance of -thalassemia in -thalassemia/HbE patients the specific polymerase chain reaction has also been performed to detect -thalassemia occurred from -3.7 kb, -4.2 kb, --SEA, and --THAI deletions, Hb Constant Spring (HbCS), and -3,7 triplication of the -genes ( /-). Among the 166 -thalassemia/Hb E patients, 16 subjects were heterozygous -thalassemia (13 with -3, 7 kb deletion, 1 with -4.2 kb deletion, and 2 with HbCS) -3,7 -3,7 and one subjects was omzygous -thalassemia (- /- ). The -SEA, and -THAI deletions and triplication of the -genes were not found in these subjects. The clinical features of all -thalassemia/ Hb E patients co-inherited with -thalassemia are mild. Our data showed that -thalassemia and HbCS genes interact in 38.1% of mild cases (41.7% of Chiang Rai and 33.3% of Koraj mild cases) and there are still unknown determinant (s) that may ameliorate the clinical severity of -thalassemia/Hb E patients. (This work was supported by the National Institute of Health, USA. th Presented at the 9 National Conference on Thalassemia, June 1819, 2003, Bangkok, Thailand.)

Emerging Viral Diseases (CVD-RCEVD), Institute of Science and Technology for Research and Development, Mahidol University, 3 Salaa Campus, Nakhon Pathom; Institute de Recherche pour Le Développment (IRD), France. Key words : Seroepidemiological survey, dengue outbreak, Thailand. From August 2000 to 2001, a dengue outbreak occurred in Mueang district, Ratchaburi Province, Thailand. About 800 cases of dengue infection were reported, and among them, 49.5% were clinically diagnosed as dengue hemorrhagic fever according to the WHO criteria. During the outbreak, the incidence rate of dengue infection in Hin Gong subdistrict was 2.9 per 1,000 population. A seroepidemiological survey was conducted among primary schoolchildren from July 2000 to June 2001, to monitor dengue transmission. In a baseline survey, 283 children were surveyed for dengue antibody and 71% were IgG seropositive. In June 2001, the rate of dengue infection showed an increase of 8.8% among immune children and 10.3% among naíve schoolchildren. Among 283 schoolchildren, 90 were followed up 3 times, in September and December 200, and June 2001. An increase in the rate of seroconversion was observed in the period September to December 2000, while the peak dengue outbreaks in the dry season occurred in February 2001. Serosurvey among schoolchildren appear to be early warning system, and can be advantageous in early dengue control actions, in order to break the chain of transmission before and impending epidemic. (Publication : Southeast Asian J Trop Med Public Health 2003;34(3):564-8. Sources of grant : 1. The Thailand Research Fund (TRF) for Royal Golden Jubille Ph.D research scholarship, Thailand. 2. Institut de Recherche pour le Développment (IRD-Research Unit 034) France. 3. Aventis, France.)

AEDES AEGYPTI INFECTED RATE AND CLIMATIC FACTORS CORRELATED WITH DENGUE TRANSMISSION (RATCHABURI PROVINCE, THAILAND : 2000-2001) (NO. 1145) Walairut Tuntaprasart , Philippe Barbazan , Yupha 1 1 3 Rongsriyam , Somjai Leemingsawat , Narong Nitatpattana , 3 3,4 Sutee Yoksan , and Jean Paul Gonzalez

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SEROEPIDEMIOLOGICAL SURVEY AMONG SCHOOL CHILDREN DURING THE 2000-2001 DENGUE OUTBREAK OF RATCHABURI PROVINCE, THAILAND (NO. 1144) Walairut Tuntaprasart , Philippe Barbazan , Narong 2 1 2 Nitatpattana , Yupha Rongsriyam , Sutee Yoksan , and Jean Paul 2,3 Gonzalez

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Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, E-mail : [email protected] 2 th. Department of Parasitology, Faculty of Public Health, 3 Mahidol University, Bangkok; Center for Vaccine DevelopmentResearch Center for Emerging Viral Diseases (CVD-RCEVD), Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakhon Pathom; 4 Institut de Recherche pour le Développment (IRD), France. Key words : Aedes aegypti infected rate, climatic factors, dengue transmission Dengue transmission is a consequence of the relationships between human, virus and mosquito. The prevention and control strategies usually emphasize vector control, such as source reduction and insecticide spraying. Therefore, entomological surveys using traditional larvel indices are currently used to monitor Aedes aegypti populations in terms of dengue transmission. Unfortunately, these indices seemed to have little or no correspondence with the

Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, E-mail : [email protected] 2 ac.th Center for Vaccine Development-Research Center for

Mahidol University Annual Research Abstracts, Vol. 31

actual occurrence of transmission. The main objective of this study is to identify a critical Aedes index and its correlation between other relevant factors and dengue transmission covering a one-year period in Ratchaburi Province, Thailand. During an outbreak in 2000-2001, the peaks of epidemic occurred in both the rainy and the dry seasons. The common larval indices such as House index (HI), Container index(CI), Breteau index (BI) and female mosquito density were high during the rainy season but decreased 33.4%, 55.7%, 58.1% and 25.5, respectively in the dry season. It shows that these indices could not be used for prediction of the epidemic in the dry season. Moreover, the study revealed that seasonal change of minimum ratio between indoor and outdoor temperature during 7 days was the most important predictive indicator for estimating number of infec2 tious mosquitos and showed the best fit with the actual data, R = 0.717 (p-value < 0.01). In addition, the infectious mosquito this index (IMI) was introduced as a more appropriate predictor because the trend of this index increased before the peaks of DHF occurence. Moreover, the relative risk of infected mosquitos was statistically 1.4 times higher during the dry season. This finding emphasizes the important role of infected mostquitos for a better understanding of the dengue transmissin dynamics in natural conditions. It leads to a more realistic strategy in areas at risk, helps to predict variation of dengue trasmission and improves control strategies. (Presented in 14 European Conference of the Society for Vector Ecology (SOVE), September 3-6, 2003; Bellinzona, Switzerland. Sources of grant : 1.The Thailand Research Fund (TRF) for Royal Golden Jubilee Ph.D research scholarship, Thailand. 2.Institut de Recherche pour le Développement (IRD-Research Unit 034), France.)

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MECHANICAL PROPERTIES OF GUM DEPROTEINIZED NATURAL RUBBER IN COMPARISON WITH SYNTHETIC CIS-1,4 POLYISOPRENE VULCANIZATES (NO. 1147) Nittaya Rattanasom1,2 , Uraiwan Thammasiripong 3 , Krisda Suchiva2,3 Institute of Science and Technology for Research and Development, Mahidol University (E-mail : [email protected]); 2Centre for Rubber Research and Development, Faculty of Science, Mahidol University; 3Department of Chemistry, Faculty of Science, Mahidol University.

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Key words : deproteinized natural rubber; synthetic cis-1, 4 polyisoprene; crosslink density The mechanical properties of gum deproteinized natural rubber (DPNR) and cis-1, 4 polyisoprene vulcanizates (IR) having various degree of crosslink density were compared. Whole natural rubber (WNR) vulcanizate was used as a reference for the DPNR. The mechanical properties of DPNR and IR at similar optimum crosslink density were also determined. Tensile and tear strength of DPNR and IR passed through a maximum with the crosslink density and then declined as crosslinking increased. However, DPNR showed a little higher maximum tensile strength but similar maximum tear strength to those of IR. In addition, IR exhibited an abrupt drop in tear strength at lower crosslink density and had a sharper peak than did DPNR. This might be due to the crystallizability of IR was less than that of DPNR. At the similar optimum crosslink density, DPNR yielded greater tensile strength, and crack growth resistance than those of IR. Therefore, DPNR might be an alternative raw material for producing low allergen NR products. (Contract grant sponsor: The Thailand Research Fund. Oral Presentation: "The 8th Pacific Polymer Conference" 24-27 November 2003, Queen Sirikit Convention Center, Bangkok, Thailand.)

CRACK GROWTH AND ABRASION RESISTANCE OF CARBON BLACK-FILLED NATURAL RUBBER VULCANIZATES (NO. 1146) Nittaya Rattanasom1,2, Oraphin Chaikumpollert3 Institute of Science and Technology for Research and Development, Mahidol University (E-mail: [email protected]); 2Centre for Rubber Research and Development, Faculty of Science, Mahidol University; 3Department of Chemistry, Faculty of Science, Mahidol University

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BLASTOCYSTIS ISOLATES FROM A PIG AND A HORSE ARE CLOSELY RELATED TO BLASTOCYTIS HOMINIS (NO. 1148) Umaporn Thathaisong 1 , Jeerapun Worapong2 , Mathirut Mungthin3, Peerapan Tan-Ariya1, Kwanjai Viputtigul4, Apichart Sudatis4, Adisak Noonai5, and Saovanee Leelayoova 3*

1Department of Microbiology, Faculty of Science, and 2Center for Biotechnology, Mahidol University, 3Department of Parasitology and 5Department of Microbiology, Phramongkutklao College of Medicine, 4Division of Research, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand.* Corresponding author E-mail: [email protected]

Key words: natural rubber; modulus; crosslink density The influences of parameters contributing to rubber stiffness, i.e., crosslink density, hardness and modulus on crack growth and abrasion resistance of carbon black-filled purified natural rubber (PNR) vulcanizates as well as a whole natural rubber (WNR) vulcanizate counterpart or a control were elucidated. In addition, tensile properties of PNR and WNR were also determined. PNR containing the same curative level as the control had lower stiffness and exhibited superior crack growth resistance. The results revealed that the improved crack growth resistance of PNR, compared to the control, was due to its lower crosslink density, hardness and modulus. In addition, tensile strength and abrasion resistance of PNR vulcanizates were not as sensitive as crack growth resistance to the changes of their stiffness. (Contract grant sponsor: National Metal and Materials Technology Center. Publication: J. Appl. Polym. Sci. (2003), 90, 1793) - Oral Presentation: "New Version of Science and Technology", 27-28 November 2003, Institute of Science and Technology for Research and Development)

Key words : Blastocystis, zoonotic transmission, RFLP of ssu rDNA Blastocystis has a widespread distribution in a variety of animals, which is a potential source of infection for humans. However, the contribution of zoonotic transmission remains unclear due to the absence of molecular proof of these organisms being identical to those found in humans. We report herein the similar subgroup of Blastocystis isolates from humans, pigs, and a horse using a restric-

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A NEW RECORD OF PESTALOTIOPSIS THEAE, EXISTING AS AN ENDOPHYTE ON CINNAMOMUM INERS IN THAILAND (NO. 1150) Jeerapun Worapong 1*, Suthum Inthararaungsorn 1, Gary A. Strobel2 & W. M. Hess3

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tion fragment length polymorphism (RFLP) analysis of partial smallsubunit ribosomal DNA (ssu rDNA). Additionally, sequence and phylogenic analysis of partial ssu rDNA of Blastocystis from a human, a pig, and a horse sharing a common subgroup shows that Blastocystis isolates from a pig and a horse were monophyletic and closely related to B. hominis, with 92 to 94% identity. These results suggest the possibility of zoonotic potential of Blastocystis. (Journal of Clinical Microbiology, Mar., Vol. 41(3), 2003: 967­975. This work was financially supported by Thailand-Tropical Diseases Research Program (T2) (ID 00-1-HEL-24-011).

Center for Biotechnology, Institute of Science and Technology for Research and Development, Mahidol University, Bhudthamonthon 4 Rd., Salaya, Nakornprathom 73170, Thailand; 2Department of Plant Sciences, Montana State University, Bozeman, Montana, 59717, USA. 3Department of Integrated Biology, Brigham Young University, Provo, Utah, 84602, USA. *Corresponding author E-mail: [email protected] Key words : appendaged conidia, environmental scanning electron microscopy An endophytic fungus, Pestalotiopsis theae strain CTC5, was isolated from the cambium of Cinnamomum iners at the Siri Rukhachat Medicinal Plant Garden, Salaya campus of Mahidol University, Nakornprathom, Thailand. By regular scanning electron microscopy (SEM), a spheroid-like extension of this fungus appears on the three relatively long appendages that occur on each conidium. The isolate CTC5, also frequently produces the basal appendage with the small knob at the end. The high similarities of ITS1, 5.8S rDNA and ITS2 sequences strongly confirmed that the isolate CTC5 should be classified as Pestalotiopsis theae by virtue of 99.63% homology. Pestalotiopsis theae is commonly known as a pathogen on leaves of Thea sinnensis and other plants. The combination of classical and molecular phylogenic classification may contribute a better understanding of evolutionary relationships among Pestalotiopsis isolates from diverse places and help explain relationships to their host plants. Furthermore, the new technique of environmental scanning electron microscopy is used herein to show the slimy nature of the conidiospores and that these appendages cannot be seen while the spores are in the acervulus. Mycotaxon (88), 200: 365-372. Financial assistance of Initiative Research Grant provided by Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand is greatly appreciates. Financial assistance has also been provided by the NSF, USDA, the Montana R&C Commission, and the Montana Agricultural Experiment Station. We are grateful to Mondotech (Thailand) Co., Ltd., a representative of Eppendorf, Germany, for providing MasterTaq polymerase and loaning the thermal cycler "Mastercycler® to our newly established laboratory.

A RECORD OF MUSCODOR ALBUS, AN ENDOPHYTE FROM MYRISTICA FRAGRANS IN THAILAND (NO. 1149) Kathawut Sopalun1, Gary A. Strobel2, W. M. Hess3, and Jeerapun Worapong 4 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; 2Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana 59717, USA; 3Department of Integrated Biology, Brigham Young University, Provo, Utah 84602, USA; 4Center for Biotechnology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakornprathom 73170, Thailand *Corresponding author E-mail: [email protected] mahidol.ac.th

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Key words : Mycelia Sterilia, ITS and 5.8S rDNA, azulene derivatives Isolate MFC2 is identified as Muscodor albus, an endophyte residing in the cambium of Myristica fragrans, a nutmeg tree in Thailand. This strain is characterized by the production of a whitish felt-like mycelium on PDA. It develops no fruiting structures on water agar containing sterile pieces of the host plants and other media. The MFC2 isolate is related to the family of Xylariaceae based upon 92% similarity of ITS1, 5.8S rDNA, and ITS2 sequences of Xylaria arbuscula and Hypoxylon fragiforme. High similarities of ITS1, 5.8S rDNA, and ITS2 sequences showed that the isolate MFC2 is taxonomically related to M. albus, M. roseus, and M. vitigenus by virtue of 99%, 99% and 92% homology, respectively. For GC/MS volatile fingerprinting, M. albus isolate MFC2 produces volatile compounds which are some of the major components of the volatiles of M. albus, i.e., propanoic acid and ethanol. M. albus, M. roseus and M. vitigenus each primarily produce naphthalene either exclusively or in combination with naphthalene derivatives while MFC2 mainly produces bulnesene, ledol, (-)-globulol and a number of other azulene derivatives. Because of its DNA homologies, the whitish nature of the mycelium and some similarities in the volatile compounds that are produced, the organism is recorded here as the MFC2 strain of M. albus. Mycotaxon (88), 200:239-247. Financial assistance: Initiative Research Grant of the Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand and NSF, USDA, the Montana R&C Commission, and the Montana Agricultural Experiment Station. Kathawut Sopalun was a recipient of scholarship from the Ministry of University Affairs under the Science and Technology Higher Education Development Project in Agricultural Biotechnology.

DISTRIBUTION OF FOREST GAPS ON THE MO SINGTO BIODIVERSITY RESEARCH PLOT (NO. 1151) Chanpen Wongsripueak1,2, Warren Y. Brockelman 2,3, Amnart Boonkongchart 3, Saiwaroon Boonkongchart 3 Department of Biology, Faculty of Graduate Studies, Mahidol University; 2Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170; 3National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120.

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Mahidol University Annual Research Abstracts, Vol. 31

Why are ecologists concerned with studying gaps and disturbance areas in forests? There have been many studies for many decades in tropical and temperate forests. A gap is defined as a hole or space in the forest that is caused by falling trees, snapped trees and so forth. The disturbance damage created by abiotic e.g. wind and biotic factors, e.g. fungus and insect. The shape of the forest opening is irregular and its size depends on the extent of the catastrophe. The border of gaps may be defined as the inner-most points reached by canopy foliage. Gaps and disturbed areas play an important role in forest dynamics. They affect the regeneration and species composition of the forest ecosystem. Gap environments differ from closed canopy: higher light intensity, soil nutrients and moisture. These available resources offer advantages to many kinds of plant species, especially pioneer species. The study of gap dynamics and their species composition leads to greater understanding of tropical forest dynamics. Consequently, in the year 2003, we started mapping gaps in the Mo Singto biodiversity research plot, we defined a gap as above but with most of seedlings and saplings no higher than 5 m. A total of 84 gaps on plot were mapped and their sizes were calculated using GIS. The total area of gap is 2.75% of the plot area and the size of gaps ranges between 5 m2 and 941.12 m2, with mode size about 25 to 50 m2. 20, 20, and 44 gaps were created in 2003, 2002, and before 2002, respectively. Surprisingly, gaps formed in 2003 comprise most of the total gap area (44.5%), caused by catastrophic wind-storm in the late summer season. Studies of a gap species composition, niche partitioning, and seedling recruitment are currently being undertaken.

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diameter have been censused as of 2001. There are 199 species of trees in 142 genera and 61 families, and 120 species of lianas on the plot. The mean tree density is 565.5 stems ha­1 and basal area is 29.7 m2 ha­1. At the north border of the plot lies several ha of secondary forest about 20 years old that is regenerating from slash-and burn farming carried out before the park was created in 1961. Mammals that occur regularly on the plot include sambar deer Cervus unicolor, muntjack Muntiacus muntjack, wild pig Sus scrofa, Himalayan black bear, Malayan sun bear Selanarctos malayana , dhole Cuon alpinus, Asian elephant Elephas maximus, Malayan porcupine Hystrix brachyura, pig-tailed macaque Macaca nemestrina, Slow loris Nycticebus coucang, and binturong Arctictis binturong. About 140 species of birds are resident on the plot or the surrounding area, including 4 species of hornbills, 4 species of barbets, 5 species of pittas and 8 species of woodpeckers.

USE OF THE MO SINGTO BIODIVERSITY RESEARCH PLOT FOR THE STUDY OF GIBBON (HYLOBATES LAR) RANGING BEHAVIOR (NO. 1153) Warren Y. Brockelman 1,2 , Saiwaroon Chongko 2 , Amnart Boonkongchart 2 and George Gale 3

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THE MO SINGTO LONG TERM BIODIVERSITY RESEARCH PLOT, KHAO YAI NATIONAL PARK

(NO. 1152)

W. Y. Brockelman1,2, Anuttara Natalang2, Chen Nan1, and George Gale3 Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170; 2National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120; 3 King Mongkut's University of Technology Thonburi, School of Bioresources & Technology, 83 Moo. 8 Thakham, Bangkhuntien, Bangkok 10150, Thailand. In 1996, Researchers at the Mo Singto long term gibbon study site in Khao Yai National Park, Nakhon Nayok Province, began to establish a permanent forest dynamics plot over the territory of the main gibbon study group, "Group A". The initial purpose of this plot was to enable more intensive study of gibbon diet and foraging behavior, but the plot has developed into a long-term study site for biodiversity and plant-animal relations in general. The plot is located in seasonal evergreen forest that receives 2000­3000 mm of rainfall per year, mostly during April through October; the months November through March are usually dry. The monthly mean temo perature ranges from about 15-19 C in December-January to about o 21-25 C in April-July. The plot is in hilly terrain ranging from 725 to 815 in altitude. The bedrock consists of Lower Jurassic siltstone and sandstone of the Khorat Group, with underlying Permo-Triassic volcanic rocks exposed in some of the lower streams. Soils are generally clay-loams or silt-loams except in ravines and low areas which had mostly loams. All trees over 10 cm dbh and all lianas over 3 cm

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Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170; 2National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120; 3 King Mongkut's University of Technology Thonburi, School of Bioresources & Technology, 83 Moo. 8 Thakham, Bangkhuntien, Bangkok 10150, Thailand. The 30-ha Mo Singto Plot has been placed over the home range of gibbon (Hylobates lar) group A so that the feeding and ranging behavior of the group can be studied in detail. This has permitted a complete inventory of the diet and more precise analysis of the daily range and tree use, as all trees >10 cm dbh have been mapped and identified. There are approximately 200 species of trees on the 30-ha plot, and around 120 species of woody vines. The method of following the gibbons from tree to tree during the entire active period also allows the mapping of seeds defecated by the gibbons and will permit mapping of seed shadows on the plot for all plant species dispersed by them. Gibbons are followed from their previous sleeping tree to the next night tree and all trees entered are recorded (by tree id number) along with all behaviors performed in each tree, including travel through, rest, grooming, vocal behavior, intergroup conflict behavior, feeding behavior, and defecation. Feces are collected into plastic bags and all seeds are sieved out, identified and counted. All species of seeds are tested for germination in a field laboratory. The data each month are analyzed for dietary diversity and changes, and the tree data can be input into a GIS by tree id in order to study foraging paths over the plot. Observations now in progress each month have revealed that individual gibbons utilize a total of around 200 to over 300 trees per day in the rainy season but only 100­200 trees in the cool dry season when succulent fruits are less available.

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49 direct observations of post-seed dispersal, 2,194 beetles of 53 species (9 genera) were collected. Beetles were caught more from night traps than day traps and more during the beginning of the rainy season (Apr.­Jun.) than in other seasons. However, beetles were equally abundant in valley and hill habitats. According to their dung processing and nest constructing methods, dung beetles are classified into three functional groups; tunnelers (86% of 1,818 beetles from traps), rollers (3%), and dwellers (11%). All tunnelers and rollers removed small seeds from their original positions while dwellers did not. From 49 observations, 10% (range 0­100%) of large seeds were removed by 9% (0­75%) of dung beetles that visited dung. Tunnelers of eight species (5­26 mm in length) moved those seeds into their tunnels under or short distances from dung piles with an average of 14.9 cm (range 0­105, N=32) and buried them 2.7 cm (range 0­7, N=32) under the soil. Each tunneler transported a single or two seeds during each observation. Large rollers (26 mm) removed 4­8 large seeds (mean 6.3, N=3) in their balls while dung balls of two small roller species (4 and 7 mm) contained only small seeds. These rollers rolled their balls an average of 62.8 cm away (range 5­150 cm, N=24) and buried them an average of 5.2 cm (range 0­7, N=21). Considering these removal distances and burial depths, seeds are most likely to be given the advantages of escaping from clumping and avoiding predator attraction and have a greater possibility of germinating.

DIVERSITY OF WOODY VINES ON THE MO SINGTO FOREST BIODIVERSITY PLOT, KHAO YAI NATIONAL PARK (NO. 1154) Warren Y. Brockelman 1 , J. F. Maxwell 2 , and Amnart Boonkongchart 3 Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170; 2Chiang Mai University Herbarium, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200; 3National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120 A complete census of lianas, or woody vines, over 3 cm in diameter was carried out on the Mo Singto Plot during the 2000­ 2001 dry season. A total of 120 species were identified on the plot (excluding rattans), and at least 40 more species have been found in the Mo Singto ­ Park Headquarters area. A total of 33 families were found, of which the most species-rich were Apocynaceae (14 species), Leguminosae (13 species), Rubiaceae (9 species), and Annonaceae (8 species). On the 30-ha plot, a total of 9510 genets (liana stems rooted in the ground) and 11,526 ramets (liana branches climbing trees) were enumerated. The two most abundant species were Uncaria scandens (6.2 % of genets) and Uncaria (5.0 % of genets) (both Rubiaceae). These two species are particularly aggressive in tree-fall gaps. The great majority of species are branch twiners, with some being stem twiners, tendril climbers and scramblers. The largest liana is Spatholobus harmandii (Leguminosae, Papilionoidae), which reaches 50 cm in diameter and covers a large area of the top canopy. A total of 54 species occur at a density of less than 1 stem per ha, and would thus be considered "rare". A database of liana species and their stem characters is being prepared which will allow identification of the species from vegetative characters alone.

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CHARACTERISTICS OF FRUITS CONSUMED BY THE WHITE HANDED GIBBON (HYLOBATES LAR) IN KHAO YAI NATIONAL PARK, THAILAND (NO. 1156) Chuti-on Kanwattanakid1,2, Warren Y. Brockelman 2 Department of Biology, Faculty of Graduate Studies, Mahidol University; 2Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170. Gibbons act as generalized frugivores in tropical rain forests. They play an important role in the forest ecosystem as seed dispersers. Fruit characteristics also have an important role in explaining the relationship between plants and gibbon coevolution. Feeding behavior of white-handed gibbons (Hylobates lar) was studied in Khao Yai National Park, Thailand. The main purpose of this research was to identify the diverse foods in the gibbons' diet and determine the fruit characteristics that influence the white-handed gibbon's choice. The methods of study included direct observation of gibbon behavior and morphology of fruits, leaves and other plant parts. Fecal samples were also collected. Collected fruit was analyzed for nutritional value in the laboratory at the Institute for Nutrition, Mahidol University. The method of handling of fruit by gibbons was also observed in the zoo. There were 30 families and 65 plant species collected and identified in the diet of one gibbon family. Most food diet came from trees (72%), but also from climbers (26.6%) and treelets (2.1%). The gibbons fed on 50 species of fruit with Ficus as the most consumed fruit. Young leaves, flowers, spadix and spathe were also observed to be consumed. Gibbons mostly consumed ripe fruit with bright colors (yellow, red, orange and purple), which was soft and

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POST-DISPERSAL OF SEEDS IN FECES OF WHITE-HANDED GIBBONS (HYLOBATES LAR) BY DUNG BEETLES (COLEOPTERA: SCARABAEIDAE) (NO. 1155) Janya Jadejaroen 1,2, Warren Y. Brockelman 2

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Department of Biology, Faculty of Graduate Studies, Mahidol University; 2Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170. Seeds dispersed by fruigivorous mammals like gibbons were found to be positively affected by dung beetles (Coleoptera: Scarabaeidae). They incidentally remove seeds while feeding and removing feces, to be used for brooding. The study was done between Jan.­Dec., 2000, in a primary rain forest at Khao Yai National Park, Thailand, to provide a basic knowledge of ecological interactions between these beetles and the seeds dispersed in feces of whitehanded gibbons (Hylobates lar). A total of 3,809 seeds of 42 species (27 families) were recovered from 157 samples of gibbon feces. These seeds ranged 0.5­30 mm in length. From 288 pitfall traps and

Mahidol University Annual Research Abstracts, Vol. 31

juicy. Small size (less than 10 mm) and light weight (less than 10 g) and, fruits with a single well-protected seed were found more than other fruit types to be consumed by the gibbon. The nutritional value of 6 consumed types of fruits and leaves did not differ much. These observations were supported by an experiment in the zoo which revealed that gibbons chose the suitable size and weight that fit in their hands. These results indicated that the food characteristic is one of the main factors as well as other factors such as food availability and abundance determining the gibbon's choice. However, there are many factors that influence food selection of gibbons which can explain their behavior and the territorial defense hypothesis. Study of fruit characteristics should be carried out in relation to other factors which might be important in food selection. This will explain food selection of white-handed gibbons which is important for gibbon conservation in the future.

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COMPARISON OF STRUCTURE AND SPECIES DIVERSITY OF MIXED FRUIT ORCHARDS AND NATURAL FOREST AT BAN KHIRIWONG, NAKHON SRI THAMMARAT PROVINCE, SOUTH THAILAND (NO. 1158) Warren Y. Brockelman 1,2, Panarat Charoenchai 2, and George Gale3

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Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170; 2National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120; 3 King Mongkut's University of Technology Thonburi, School of Bioresources & Technology, 83 Moo. 8 Thakham, Bangkhuntien, Bangkok 10150, Thailand The structure and canopy characteristics were measured in mixed fruit orchard (suan som rom) and natural forest in Ban Khiriwong, Nakhon Si Thammarat Province. The orchard canopy was half as high and only half as dense as that of the forest, and had less cover over the ground (85% vs. 100%). The lower understory layer was much less dense than that of the forest. The major fruit species contributing to the canopy volume were Durio zibethinus (34.2%), Parkia speciosa (16.3%), Garcinia mangostana (12.9%), Artocarpus integer (6%), Lasium domesticum (4.7%), Areca catechu (4.6%), Musa acuminata (3.6%), and Nephelium lappaceum (2.7%). A total of 20 species of plants were planted, some of them shrubs. It is recommended that more understory plants be used in the orchard to improve canopy cover and increase commercial value. 1

ECOLOGICAL STUDY OF LIANAS AND SOME VINES IN MO SINGTO BIODIVERSITY RESEARCH PLOT, KHAO YAI NATIONAL PARK (NO. 1157) Kanok Lertpanich1,2, Warren Y. Brockelman 2

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Department of Biology, Faculty of Graduate Studies, Mahidol University; 2Center for Conservation Biology, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170 The ecological study of lianas in Mo Singto Biodiversity Research Plot started with the survey of all stems of ? 3 cm dbh lianas. The 28.56 ha area contained 9510 stems (genets), which included 35 families, 116 species, plus six unknown species. The families Leguminosae and Apocynaceae had the most species. On the other hand the highest density families were Rubiaceae, followed by Apocyanaceae. The lianas of > 1cm dbh were censused in 40 sampling plots 20 x 20 m for the analysis of liana density and diversity. This dataset was also used with the constraint factors data for investigating the liana community in relation to environmental factors. The liana density of the four habitat types were not significantly different. The regression of ecological diversity (S or ln S) and ecological density (ln N) showed no differences among the four habitats, and the species richness was simply related to differences in the overall abundance among the habitats. The study found that the lianas of > 1 cm dbh had a density of 26.9 + 11.28 stems per 0.04 ha and diversity of 14.80 > 4.68 species per 0.04 ha. The SHE analysis (species richness, diversity and equitability) was performed for the > 1 cm dbh lianas in Mo Singto site dataset and found that the species conform to the logarithmic series distribution. The liana community had a log series alpha of 24.07 + 1.96 and log series x = 0.98 + 0.01 within the total 1.6 ha area. The study of the relations between lianas and environmental factor was carried out by multiple regression analysis. This analysis was used for finding the key environmental factors that determined the liana density and diversity. The results showed that there was no significant relation between environmental factors and either liana density or liana diversity.

INHERITANCE AND SELECTIVE EFFECTS OF COLOR PHASE IN WHITE-HANDED GIBBONS (HYLOBATES LAR) IN CENTRAL THAILAND.

(NO. 1159)

Warren. Y. Brockelman Center for Conservation Biology. ISTRD, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand. Key words: Hylobates lar, white-handed gibbon, color polymorphism, Thailand The white-handed gibbon Hylobates lar (Hylobatidae) is polymorphic for pelage coloration throughout its range. The frequenceis of the two color phases, brownish-black and buff, and their pattern of inheritance, are reported in a sample of 64 wild social groups and in an additional sample of newborn individuals, in Khao Yai National Park, east-central Thailand. The total sample of 318 individuals is half dark and half light phase. Color is not correlated with sex or age class. Data on parent and offspring coloration are consistent with simple Mendelian inheritance in which dark is controlled by a dominant allele. The monogamous adult pair combinations are random with respect to color, contrary to some previous reports. No evidence was found for any selection based on color; there were no differences in numbers of offspring or group size among different adult pair color combinations. Whether this polymorphism

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is maintained by balancing selection is unclear; several plausible mechanisms are discussed.2 ( 1 The Sixth Annual Meeting BIOTEC Research Units, National Center for Genetic Engineering and Biotecnology, National Science and Technology Development Agency, Ministry of Science and Technology, 11-13 December 2003, Chonburi. 2 Brockelman, W.Y. 2003. Inheritance and selective effects of color phase in white-handed gibbons (Hylobates lar) in central Thailand. Mammalian Biology)

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