Faculty of Science - Mahidol University

Mahidol University Annual Research Abstracts, Vol. 33 267 

EGG-LAYING-HORMONE IMMUNOREACTIVITY 

IN THE NEURAL GANGLIA AND OVARY OF 

HALIOTIS ASININA LINNAEUS (NO. 682) 

Saitongdee P., Apisawetakan S., Anunruang N. Poomthong T., 

Hanna P., Sobhon P 

Department of Anatomy, Faculty of Science, Mahidol University, 

Bangkok, Thailand. 

Immunoreactivity against the abalone egg-laying hormone 

(aELH) was detected in the fine granules of type 1 and 2 

neurosecretory (NS) cells, neurites in the neuropil, and blood sinuses 

in the connective tissue sheath of the cerebral, pleuropedal, and 

visceral ganglia of the tropical abalone, Haliotis asinina Linnaeus. 

The number of positive NS cells, and the intensity of staining in the 

ganglia, varied and might be related to the stage of ovarian cycle. 

At any stage, positive cells were most numerous in the pleuropedal, 

and least numerous in the visceral ganglion. In addition, several 

cells of the statocyst and associated nerves also exhibited the 

immunoreactivity. In the ovary, the most intense reactivity was 

detected in the follicular and granular cells adjacent to mature 

oocytes, in the trabeculae and the ovarian capsule. The cytoplasm 

of mature oocytes was also moderately stained. The results indicate 

that the cerebral, pleuropedal, and visceral ganglia are the main sites 

of aELH-producing cells. The ovary may also produce aELH locally. 

(Invert Neurosci. 2005 Apr 1; [Epub ahead of print]) 

CHROMATIN ORGANIZATION AND BASIC 

NUCLEAR PROTEINS IN THE MALE GERM 

CELLS OF RANA TIGERINA (NO. 683) 

Manochantr S, Sretarugsa P, Chavadej J, Sobhon P. 



The process of chromatin condensation during 

spermiogenesis in Rana tigerina is similar to the 

heterochromatization in somatic cells, where 30 nm fibers are 

coalesced together into a dense mass in spermatozoa without 

changing their initial size and nucleosomal organization. This 

conclusion was supported by the finding that the full set of core 

histones (H2A, H2B, H3, H4) are still present in sperm chromatin, 

but histone H1 is replaced by its variant, H1V. Rabbit anti-sera were 

raised against histone H3, H1, H1V, and H5 (H1 variant in chick 

erythrocyte). Anti-histone H1 antiserum cross-reacted with histone 

H1V, which implied the presence of a common epitope. Anti-histone 

H1V and H5 also showed cross-reaction with each other but not 

with histone H1, which implied the presence of a common epitope 

not shared by histone H1. Immunocytochemical studies, using the 

above antibodies as probes, showed that histones H3 is present in 

all steps of spermatogenic and spermiogenic cells, and somatic cells 

including red blood cells, Sertoli cells, and Leydig cells, while histone 

H1 is present in all of the cells mentioned except in spermatozoa 

where it is replaced by histone H1V. Histone H1V appears in the 

early spermatids starting from spermatid 1 (St1), and it persists 

throughout the course of spermatid differentiation into spermatozoa. 

Histone H1V is also found in chromosomes of metaphase 

spermatocyte and red blood cells. Thus histone H1V may cause the 

final and complete condensation of chromatin in Rana spermatozoa, 

a process which is similar to the heterochromatization occurring in 

somatic cells such as metaphase chromosome and chick erythrocyte 

nucleus. 

(Mol Reprod Dev. 2005 Feb;70(2):184-97.) 

BASIC NUCLEAR PROTEIN PATTERN AND 

CHROMATIN CONDENSATION IN THE MALE 

GERM CELLS OF A TROPICAL ABALONE, 

HALIOTIS ASININA (NO. 684) 

Suphamungmee W, Apisawetakan S, Weerachatyanukul W, 

Wanichanon C, Sretarugsa P, Poomtong T, Sobhon P 



The basic nuclear proteins (BNPs) in spermatozoa of a 

tropical abalone, Haliotis asinina, were composed of a majority of 

protamine-like (PL) protein and a small amount of histones H1 and 

H4. Abalone H1 and PL proteins exhibited strong immunological 

cross reactivities among themselves as well as with chick H5 and 

calf thymus H1. Thus, all these proteins may belong to the same 

family. Immunolocalization by indirect immunofluorescence and 

immunoelectron microscopy indicated that H1 and H4 were present 

in all steps of the male germ cells, however, with decreasing amount 

in late stage cells, particularly spermatids and spermatozoa. On the 

other hand, PL was present in middle step cells (secondary 

spermatocytes) with increasing amount in spermatids and 

spermatozoa when the chromatin became tightly packed. Thus, PL 

may be involved in the condensation of chromatin in the spermatozoa 

of this species. 

(Mol Reprod Dev. 2005 Feb;70(2):211-21.) 

PERCOLL GRADIENT-CENTRIFUGED CAPACI- 

TATED MOUSE SPERM HAVE INCREASED 

FERTILIZING ABILITY AND HIGHER CONTENTS 

OF SULFOGALACTOSYLGLYCEROLIPID AND 

DOCOSAHEXAENOIC ACID-CONTAINING PHOS- 

PHATIDYLCHOLINE COMPARED TO WASHED 

CAPACITATED MOUSE SPERM (NO. 685) 

Furimsky A, Vuong N, Xu H, Kumarathasan P, Xu M, 

Weerachatyanukul W, Bou Khalil M, Kates M, Tanphaichitr N 



Although Percoll gradient centrifugation has been used 

routinely to prepare motile human sperm, its use in preparing motile 

mouse sperm has been limited. Here, we showed that Percoll 

gradient-centrifuged (PGC) capacitated mouse sperm had markedly 

higher fertilizing ability (sperm-zona pellucida [ZP] binding and in 

vitro fertilization) than washed capacitated mouse sperm. We also 

showed that the lipid profiles of PGC capacitated sperm and washed 

capacitated sperm differed significantly. The PGC sperm had much 

lower contents of cholesterol and phospholipids. This resulted in 

relative enrichment of male germ cell-specific 

sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC 

capacitated sperm, and this would explain, in part, their increased 

ZP-binding ability compared with that of washed capacitated sperm. 

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268 

Analyses of phospholipid fatty acyl chains revealed that PGC 

capacitated sperm were enriched in phosphatidylcholine (PC) 

molecular species containing highly unsaturated fatty acids (HUFAs), 

with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant 

HUFA (42% of total hydrocarbon chains of PC). In contrast, the 

level of PC-HUFAs comprising arachidonic acid (20:4n-6), 

docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated 

sperm was only 27%. Having the highest unsaturation degree among 

all HUFAs in PC, DHA would enhance membrane fluidity to the 

uppermost. Therefore, membranes of PGC capacitated sperm would 

undergo fertilization-related fusion events at higher rates than washed 

capacitated sperm. These results suggested that PGC mouse sperm 

should be used in fertilization experiments and that SGG and DHA 

should be considered to be important biomarkers for sperm fertilizing 

ability. 

(Biol Reprod. 2005 Mar;72(3):574-83.) 

BABYLONIA AREOLATA (SPOTTED BABYLON), 

IN ACUTE AND SUBCHRONIC CADMIUM 

POISONING (NO. 686) 

Tanhan P, Sretarugsa P, Pokethitiyook P, Kruatrachue M, 

Upatham ES 



Histopathological alterations in 6- to 8-month-old juvenile 

spotted babylon, Babylonia areolata, from acute and subchronic 

cadmium exposure were studied by light microscopy. The 96-h 

LC(50) value of cadmium for B. areolata was found to be 3.35 mg/ 

L, and the maximum acceptable toxicant concentration (MATC) was 

1.6 mg/L. Snails were exposed to 3.35 and 0.08 mg/L (5% of MATC) 

of cadmium for 96 h and 90 days, respectively. After exposure the 

gill, the organs of the digestive system (proboscis, esophagus, 

stomach, digestive gland, and rectum), and the foot were analyzed 

for cadmium accumulation. The results showed that most digestive 

organs had a high affinity for cadmium. The main target organ was 

the stomach, which could accumulate on average 1192.18 microg/g 

dry weight of cadmium. Cadmium was shown to accumulate to a 

lesser extent in the digestive gland, gill, rectum, esophagus, 

proboscis, and foot. Histopathological alterations were observed in 

the gill and digestive organs (proboscis, esophagus, stomach, and 

rectum). The study showed that the stomach and gill were the primary 

target organs of both acute and subchronic exposure. Gill alterations 

included increased size of mucous vacuoles, reduced length of cilia, 

dilation and pyknosis of nuclei, thickening of basal lamina, and 

accumulation of hemocytes. The epithelial lining of the digestive 

tract showed similar alterations such as increased size of mucous 

vacuoles, reduced length of cilia, and dilation of nuclei. In addition, 

fragmentation of the muscle sheath was observed. 

(Environ Toxicol. 2005 Apr;20(2):142-9.) 

UNUSUAL GERM CELL ORGANIZATION IN THE 

SEMINIFEROUD EPITHELIUM OF A MURID 

RODENT FROM SOUTHERN ASIA, THE GREATER 

BANDICOOT RAT, BANDICOTA INDICA (NO. 687) 

Worawittayawong P., Leigh CM., Cozens G., Peirce EJ., Setchell 

BP., Sretarugsa P., Dharmarjan A., breed WG. 



In the greater bandicoot rat, Bandicota indica, of southeast 

Asia, nine cell associations were documented in the testicular 

seminiferous epithelium. In about 10% of the tubule cross sections 

two or more cell associations occurred and, furthermore, some of 

the generations of germ cells within the cell associations were 

sometimes either out of phase, or missing, in the tubule cross 

sections. These features, together with the fact that this species has 

a highly pleiomorphic sperm head shape, are somewhat reminiscent 

of those of the seminiferous epithelium in humans and some other 

primates but not of common laboratory rodents. This species could 

thus be a good model for investigating irregular patterns of 

spermatogenesis in naturally occurring wild species of rodent. 

(Int J Androl. 2005 Jun;28(3):180-8.) 

METAL-INDUCED OXIDATIVE DAMAGE IN 

CULTURED HEPATOCYTES AND HEPATIC 

LYSOSOMAL FRACTION : BENEFICIAL 

EFFECT OF A CURCUMIN/ABSINTHIUM 

COMPOUND (NO. 688) 

Barreto R., Kawakita S., Tsuchiya J., Minelli E., Pavasuthipaisit 

K. 



Objective : Metals undergo redox cycling and there is 

increasing evidence of free radical generation and oxidative injury 

in the pathogenesis of liver injury and fibrosis in metal storage 

diseases. The aim of the present study was to test a natural 

hepatoprotective compound in metal-induced liver injury. 

Methods : Hepatocytes were isolated from Wistar rats by 

collagenase perfusion method and cultured as such and also with 

alpha-linolenic acid (LNA)-bovine serum albumin (BSA). 

Hepatocytes were then cultured with a graded dilution of PN-M001 

(100 microg/mL and 200 microg/mL), which is a curcuma/ 

absinthium-containing compound, or sylibin (100 microg/mL) 

dissolved in dimethyl sulfoxide for 10 min before the addition of 

metallic salts (iron, copper and vanadium). Lysosomal fractions were 

prepared for lysosome fragility tests in which beta-galactosidase 

activity and lactate dehydrogenase (LDH) leakage were measured, 

as well as oxidative damage tests in the presence of hydrophilic and 

lipophilic free radical generators. Quenching activity by 1,1diphenyl-2-picrylhydrazyl 

(DPPH) was also assessed. 

Results : Malonildialdehyde accumulation in the medium 

showed a direct time-course increase with incubation time. Both 

PN-M001 and sylibin showed a significant protective effect against 

all challenge metal ions, as expressed by the half inhibition 

concentration (IC(50)) against lipid peroxidation. However, on a 

molar ratio, sylibin seemed to be more effective than PN-M001 in 

Fe-induced peroxidative damage (P < 0.05). Both test compounds, 

irrespective of the concentration, significantly reduced the LDH and 

beta-galactosidase concentration in the lysosomal fractions. As 

compared with untreated lysosomal fractions challenged with the 

two peroxide radicals generators, either PN-M001 or sylibin exerted 

significant protection However, PN-M001 was significantly better 

than sylibin in suppressing acid phosphatase enzyme activity. Both 

compounds showed comparable and significant DPPH radicalscavenging 

activity. 

Conclusion : These data support the potential clinical 

application of curcumin-containing compounds. 

(Chin J Dig Dis. 2005;6(1):31-6.) 

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Faculty of Science


COMPLETE DEFICIENCIES OF COMPLEMENT 

C4A AND C4B INCLUDING 2-BP INSERTION IN 

CODON 1213 ARE GENETIC RISK FACTORS OF 

SYSTEMIC LUPUS ERYTHEMATOSUS IN THAI 

POPULATIONS (NO. 689) 

Ittiprasert W., Kantachuvesiri S., Pavasuthipaisit K., 

Versertniyom O., Chaomthum L. Totemchokchyakarn K., 

Kitiyanant Y. 



The complement component C4 is encoded by two genes: 

C4A and C4B on human chromosome 6p in the major 

histocompatibility complex (MHC). Most studies have linked the 

deficiencies in C4 with systemic lupus erythematosus (SLE) in 

Angio-Irish, North American, Black American, Mexican American, 

Australian and Japanese populations. Null alleles at either locus 

(C4AQ0 or C4BQ0) are relatively common in Americans occurring 

at the C4A and C4B loci in approximately 10% and 16% of normal 

individuals, respectively. In the present study, we extensively 

examined the possible association between homozygous C4Q0 and 

SLE in a large cohort of Thai populations diagnosed as SLE and 

further attempted to identify the genetic basis of C4Q0. One hundred 

and eighteen cases of SLE patients and 145 matched controls were 

genotyped by touchdown PCR. The results confirmed the previous 

studies that 5.93% (7/118) of C4 null genes: 2.54% (3/118) of 

C4AQ0 and 3.39% (4/118) of C4BQ0 were found in SLE patients. 

In contrast to other studies, we found no cases of C4 null genes in 

normal control (0 from 145 samples). To further investigate the 

genetic basis of C4 deficiency, all genomic DNAs were also analyzed 

for 2-bp (TC) insertion at codon 1213 in exon 29 which is a common 

mutation in many C4A null genes and a novel 1-bp deletion (C) at 

codon 522 in exon 13 that is common in most C4B null genes. Both 

mutation results in a flame-shift mutation and premature stop codon 

using sequence specific primers PCR (SSP-PCR) and direct 

sequencing. The results showed that there was 2-bp insertion in exon 

29 of mutant C4B gene in one SLE patient carrying C4AQ0. There 

was no 2-bp insertion in exon 29 of both C4A and C4B genes in 

normal individual and the rest of SLE patients. All patients with 

C4AQ0 exhibited more than 5 ACR criteria including malar rash, 

oral ulcers, renal disorder, immunological disorder, anti-nuclear 

antibody, without hematological disorder. In contrast, all of C4BQ0 

SLE patients showed 5 or 6 ACR criteria including hematological 

disorder, malar rash, oral ulcers, renal disorder, immunological 

disorder and anti-nuclear antibody. A patient who possesses C4AQ0 

and 2-bp insertion in exon 29 of mutant C4B showed 9 ACR criteria 

but no discoid rash and hematological disorder. In conclusion, both 

C4AQ0 and C4BQ0 are the strong predisposing factors for SLE in 

Thais. It was supported by the absence of either C4A or C4B deletion 

in healthy control. We suggested that the different racial and genetic 

backgrounds could alter the thresholds for requirement of C4A or 

C4B protein levels in immune tolerance and regulation. 

(J Autoimmun. 2005 Aug 25(1):77-84.) 

PLATELET- ACTIVATING FACTOR ENHANCE- 

MENT OF CALCIUM INFLUX AND INTER- 

LEUKIN-6 EXPRESSION, BUT NOT PRODUC- 

TION, IN HUMAN MICROGLIA (NO. 690) 

Sattayaprasert P., Choi HB, Chongthammakun S., McLarnon 

JG 



Calcium-sensitive fluorescence microscopy and molecular 

biology analysis have been used to study the effects of plateletactivating 

factor (PAF) on intracellular calcium [Ca2+]i and IL-6 

expression in human microglia. PAF (applied acutely at 100 nM) 

elicited a biphasic response in [Ca2+]i consisting of an initial rapid 

increase of [Ca2+]i due to release from internal stores, followed by 

a sustained influx. The latter phase of the [Ca2+]i increase was 

blocked by SKF96365, a non-selective store-operated channel (SOC) 

inhibitor. RT-PCR analysis showed PAF treatment of microglia 

induced expression of the pro-inflammatory cytokine IL-6 in a timedependent 

manner which was blocked in the presence of SKF96365. 

However, ELISA assay showed no production of IL-6 was elicited 

at any time point (1-24 h) for microglial exposures to PAF. These 

findings suggest that PAF stimulation of human microglia induces 

expression, but not production, of IL-6 and that SOC-mediated 

[Ca2+]i influx contributes to the enhanced expression of the 

cytokine. 

(J. Neuroinflammation 2005. Apr 15;2(1):11.) 

SYNTHETHIC PEPTIDE USED TO DEVELOP 

ANTIBODIES FOR DETECTION OF POLY- 

HEDRIN FROM MONODON BACULOVIRUS 

(MBV) (NO. 691) 

Satidkanitkul A., Sithigorngul P., Sang-oum W. 


Bangkok, Thailand 

The use of previously published primers to amplify the 

monodon baculovirus (MBV) polyhedrin gene sequence by 

polymerase chain reaction (PCR) from post larvae (PL) of Thai 

Penaeus monodon resulted in failure. As a result, the putative 

polyhedrin protein of MBV was isolated from infected PL by 

homogenization, differential centrifugation and density gradient 

centrifugation with verification by transmission electron microscopy 

(TEM). By SDS-PAGE, a single major protein band at 58 kDa was 

obtained from the putative polyhedrin fraction and this corresponded 

to a previous report of the molecular weight of polyhedrin from 

MBV. When used for N-terminal sequence analysis, the putative 

polyhedrin protein yielded a sequence of 25 amino acids (M F D D 

S M M M E N M D D L S G D Q K M V L T L A) that did not 

correspond to the deduced amino acid sequence derived from a 

previous report of a putative MBV polyhedrin gene amplicon. 

Despite this, a synthetic peptide of our 25 amino acid sequence 

(25Pmbv) was conjugated with bovine serum albumin and used as 

an antigen for antiserum production in mice. Using 

immunohistochemistry with tissue sections of PL infected with MBV 

or other viruses, the mouse anti-25Pmbv antiserum showed strong 

immunoreactivity to occlusion bodies of MBV only. It also showed 


270 

strong reactivity to the 58 kDa putative polyhedrin protein in Western 

blots. Altogether, the results suggest that the 58 kDa protein is Thai 

MBV polyhedrin and that a previously reported MBV polyhedrin 

gene sequence may represent another protein or polyhedrin from a 

different variety of MBV. 

(Dis Aquat Org. 2005 65:79-84.) 

SURVIVAL AND INTEGRATION OF SOX-1-GFP 

MOUSE EMBRYONIC STEM CELLS IN AUDITORY 

BRAIN STEM SLICE CULTURES (NO. 692) 

A. Glavaski-Joksimovic, C. Thonabulsombat, A. Jonsoon, M. 

Eriksoon, B. Palmgren , M Hagglund an dOlivius. 



For a clinical therapeutic approach the use of stem cells in 

a cell replacement therapy is promissing for the regeneration of 

sensory function. Previously we have used embryonic neuronal tissue 

and stem cells to initiate a neuronal repair paradigm into the inner 

ear. Here we have studied the differentiation and incorporation of 

Sox1-GFP mouse embryonic stem-(ES) cell into the rat postnatal 

brainstem slice culture. Sox-1 is the earliest known specific marker 

of neuroectoderm in the mouse embryo. GFP is not detectable in 

undifferentiated ES cells but becomes evident after neural 

differentiation that allows direct analysis of the conversion of 

pluripotent ES cells into neuroectoderm in vitro. Previously it was 

shown that in adherent monoculture Sox1-GFP mouse ES cells can 

differentiate into neurons and glial cells. We have implanted Sox1- 

GFP mouse ES cells into auditory brainstem slice from SD postnatal 

rats (P 11-15). Using tissue chopper 300-ตm-thick slices 

encompassing vestibulocochlear nerve and cochlear nucleus were 

prepared and propagated using membrane interface methods 

described by Stoppini. After placing slices on membranes the 

cochlear nucleus was labeled by DiI. In vitro transplantation was 

conducted at day 5±2 in culture. A total volume of 0.5ตl of Sox1 

cell suspension in concentration of 1x10 4 /ตl was deposited next to 

the vestibulocochlear nerve and cochlear nucleus. Two weeks after 

Sox-1 cell transplantation co-cultures were fixed and immunostained 

with antibodies raised against neuronal and glial markers. We have 

demonstrated that following deposition next to the slices Sox1 cells 

actively migrate toward the cochlear nucleus pre-labeled with DiI. 

The morphological and immunohistochemical data indicated that 

grafted Sox1 cells were able to differentiate into neurons and glial 

cells by two weeks after implantation. These results demonstrate 

that organotypic slice co-cultures of the auditory brainstem with 

Sox1 cells present a useful model to study the regeneration of the 

respective neurons and that co-cultured Sox1 cells generate neurons 

and glial cells capable of integrating into the brain circuitry. 

(Poster Presentation at Inner Ear Biology Meeting, Germany. 

September 2005) 

EVIDENCE FOR THE PRESENCE OF NONODON- 

SLOW GROWTH AGENT IN THE PACIFIC WHITE 

SHRIMP (NO. 693) 

G. Anatasomboon, A. Akrajamorn, W. Panphut, W. Saeng-Oum, 

K. Sritunyaluksana and B. Withachumnarnkul 



In Thailand, the relatively sudden, nation-wide occurrence of 

unusually slow-growth in the black tiger shrimp Penaeus monodon 

(called monodon slow growth syndrome or MSGS) was a major factor 

in causing the majority of Thai shrimp farmers to convert to farming 

the Paci.c white shrimp Penaeus vannamei. One possible cause of 

MSGS was an infectious agent that could be referred to as monodon 

slow growth agent (MSGA). Thus, morphological and ultrastructural 

studies were carried out to search for pathogens with MSGS shrimp . 

Although a number of known viral, bacterial and protozoan 

pathogens were found, a substantial fraction of the MSGS shrimp 

were free of known pathogens or unusual histopathology. In addition 

to known viruses, some unknown spherical viral-like particles (~25 

nm) were frequently detected by transmission electron microscopy 

(TEM) in the lymphoid organ and gills of MSGS shrimp. Lymphoid 

organ extracts (LOE) passed through 0.45 μm membrane .lters induced 

MSGS in healthy P. monodon suggesting that the unknown viral 

particles might be associated with MSGS. In another test, Paci.c white 

shrimp P. vannamei were co-cultured with MSGS P. monodon and 

they grew normally. However, membrane .ltered LOE extracts from 

these co-cultured P. vannamei caused MSGS when injected into 

healthy P. monodon. These .ndings suggest that a pathogen called 

MSGA exists, that it is possibly a virus(es) located in the lymphoid 

organ of both P. vannamei and P. monodon, and that P. vannamei 

may act as an unaffected carrier. 

(Poster presentation at World Aqualculture, May 2005. Bali, 

Indonesia) 

RESEARCH PROGRESS ON MONODON-SLOW 

GROWTH SYNDROME (MSGS) IN THAILAND 

T.W. Flegel and B. Withyachumnarnkul 

Faculty of Science 

(NO. 694) 


Bangkok, Thailandã 

During 2002, slow growth of farmed P. monodon (monodon 

slow growth syndrome or MSGS) was reported throughout shrimp 

growing areas of Thailand and .gures indicated that annual 

production volume was down by approximately 36%. Although the 

etiology is still uncertain, a working case de.nition of MSGS has 

been developed for surveillance and epidemiological purposes . By 

this de.nition, a suspected population must have a coef .cient of 

variation (CV = Standard deviation/Mean) of more than 35% by weight 

and absence of hepatopancreatic parvovirus (HPV) or of other severe 

hepatopancreatic infections by known agents while also complying 

with any 3 out of the 5 following gross signs: 1) unusually dark color, 

2) average daily weight gain of less than 0.1 g/day at 4 months, 3) 

unusually bright yellow markings, 4) “bamboo” abdominal segments, 

5) brittle antennae. Preliminry trials using bacteria -free .ltrates from 

slow growing P. monodon caused slow growth in laboratory injection 

experiments with P. monodon but not with P. vannamei. This 

suggested that an infectious agent might be involved . An initial 

survey suggested that known pathogens were unlikely to be the cause. 

A new type of yellow head virus (YHV) was found in these shrimp, 

but preliminary tests revealed that was not correlated with the 

problem. Another candidate virus is lymphoid organ vacuolization 

virus (LOVV) .rst described in several captured P. monodon 

broodstock from Thailand by DV Lightner (personal communication) 

in late 2002. Ultracentrifuge bands of tissue homogenates from MSGS 

shrimp have shown 2 previously unreported viral-like particles, and 

extracts of these bands give products by random RT-PCR but not by 

PCR, suggesting possible involvement by an RNA virus (es). As of 

now, there is no tested management approach to tackle this problem. 



However, it is well known that exotic viruses can move easily 

amongst crustacean species. Implementing the following 

recommendations might help to reduce the impact of slow growth 

syndrome. Imported crustaceans and especially exotic species should 

be reared separately from native species particularly at the hatchery 

phase and importations should follow the full ICES protocol with 

the addition of co-habitation tests employing important, endemic 

crustacean species. This will reduce the risk of importing exotic viral 

pathogens that may damage local aquaculture or .sheries. 


Indonesia) 

PROSTAGLANDINS IN THE POLYCHAETE 

Perinereis nuntia AND THEIR RECEPTORS IN 

THE OVARY OF THE BLACK TIGER SHRIMP 

Penaeus monodon. (NO. 695) 

P. Poltana, T. Lerkitkul, G. Anantasomboon, W. Wannapapho, 

K. Wongprasert, P.J.W. Olive and B. Withyachumnarnkul 



Nereid polychaetes e.g. Perinereis nuntia brevicirris are an 

important fresh feed for Penaeus monodon broodstock; the worms 

are believed to contain substance(s) that stimulate ovarian maturation 

of the shrimp. In addition to essential polyunsaturated fatty acids, 

the polychaetes may contain certain hormones, such as 

prostaglandins (PGs), that could stimulate ovarian maturation in the 

shrimp. This study was aimed at isolating PGs from adult (atokous) 

P. nuntia and finding their receptors in the ovarian tissue of P. 

monodon. Cultured P. nuntia were extracted for PGs, using ethanol 

as solvent; the extract was analyzed by fast-performance liquid 

chromatography (FPLC), and by comparing with standard PGs, the 

substance was found to be PGF2α, which was present at a 

concentration of 0.66 ng/g wet weight of the polychaete. Using mass 

spectrophotometry (MS), the molecular weight was found to be 368.5, 

which could be either PGF2α methylester or 15(S)-15-methyl PGF2α. 

To study PG receptors, ovaries were isolated from sexually mature 

P. monodon and divided into two parts; one for paraf.n sections 

followed by immuno-peroxidase identi.cation of the receptors and 

the other for sodium dodecyl sulfate-polyacrylamide gel 

electrophoresis (SDS-PAGE) for Western immunoblotting with speci.c 

PG antibodies. The immuno-peroxidase technique revealed PGF2α 

receptor, or FP receptor, activity in the ovarian tissues. For the previtellogenic 

stage, the reaction was predominantly localized 

throughout the cytoplasm and in the cell membrane and nuclear 

membrane of the primary oocytes. For the vitellogenic stage, the 

activity was similarly localized, but with less intensity, and it was 

not observed within the cortical rods. In the spent stage, the activity 

could be similarly observed in the remaining oocytes, as well as in 

the irregular-shaped primary oocytes. The SDS-PAGE and Western 

immunoblotting revealed an FP protein band at 47 kDa in stages I 

and II and at 51 kDa in stage III and IV ovaries. By using an enhanced 

chemiluminescence (ECL) glycoprotein detection kit, no glycoprotein 

was observed in the 51 kDa band. The .ndings suggest that the 

polychaete P. nuntia contains PGF2α and this hormone might be an 

exogenous source of PGs that stimulate ovarian maturation in P. 

monodon broodstock. 


Indonesia.) 

VIBRIO BACTERIN AND CARBOXYMETHYL 

b-1,3-GLUCANS PROTECT Penaeus monodon FROM 

Vibrio harveyi INFECTION (NO. 696) 

K. Wongprasert, S. Somapa Klannukarn, K. Khanobdee, P. 

Meeratana, P. Pongtippatee-Taweepreda and B. 

Withyachumnarnkul 



The aim of this study was to determine effects of a vibrio 

bacterin, with or without carboxymethyl β-1,3-glucans (CMBG), on 

the protection of Penaeus monodon against Vibrio harveyi infection. 

It also aimed to determine the mechanism underlying any observed 

protection. The study was carried out in 2 phases. In the .rst phase, 

healthy juvenile shrimp were exposed to short-term treatment in 

concrete tanks and in the second phase, they were exposed to long - 

term treatment in commercial ponds. In both phases, they were 

subsequently challenged with V. harveyi. In the .rst phase, the shrimp 

were fed for 10 days with commercial pellets top-dressed with a 

formalin-killed V. harveyi bacterin alone, with CMBG alone or with 

bacterin plus CMBG. They were then challenged with virulent V. 

harveyi and the relative percent survival (RPS) was determined. The 

shrimp haemolymph was also examined to determine haemocyte 

counts, phagocytic index and bactericidal and phenoloxidase 

activities. In the second commercial pond phase, shrimp groups were 

given the same feeds for two months before challeng with virulent 

V. harveyi and calculation of RPS. Controls in both test phases 

comprised shrimp fed with commercial shrimp pellets plus coated 

with un-supplemented coating material.. In the .rst-phase tank trials, 

shrimp groups receiving bacterin alone, CMBG alone or the 

combination survived better than shrimp in the control group . After 

10 days of treatment, total haemocyte counts and counts of haemocyte 

types (i.e., hyalinocytes, semi-granulocytes and granulocytes) were 

signi.cantly increased in the treated shrimp groups when compared 

to the control group. Levels of phagocytosis, bactericidal activity of 

mixed cell and haemocyte fractions and prophenoloxidase activity 

of haemolymph lysate supernatant .uids were also signi.cantly higher 

in the treated shrimp groups. Treated shrimp groups from the 

commercial ponds were also protected against V. harveyi when 

compared to the control group . For all the parameters tested, there 

were no signi.cant differences among the shrimp groups treated with 

bacterin alone, CMBG alone or combined bacterin plus CMBG. Thus, 

either bacterin or CMBG could protect shrimp against V. harveyi 

challenge and this correlated with stimulation of the shrimp cellular 

and humoral defense factors. 


Indonesia) 

SEROTONIN IMMUNOREACTIVITY IN THE 

CENTRAL NERVOUS SYSTEM AND OVARIES 

OF THE BLACK TIGER SHRIMP, Penaeus monodon 

(NO. 697) 

K. Wongprasert, S. Asuvapongpatana and B.Withyachamnarnkul 




272 

Serotonin (5-hydroxytryptamine, 5HT) is involved in modulation of 

behavior and physiological functions of both vertebrates and 

invertebrates. It has been reported that 5HT induced ovarian 

maturation and spawning in certain shrimp species, but the study 

has not yet extended to the black tiger shrimp Penaeus monodon. 

To explore the role of 5HT in P. monodon, we used 

immunohistochemical method to localize 5HT-immunoreactive cells 

in the central nervous system and in the ovaries of P. monodon 

broodstock. It revealed that 5HT-positive reactions appeared in cell 

bodies of the brain and thoracic ganglia, as well as in ventral nerve 

cord and nerve fibers surrounding the ganglia. The positive reaction 

was also found in the ovaries of pre-vitellogenic stage (stage I, II), 

within the follicular cells but not in oogonia, and on the cell 

membrane of mature oocytes (stage IV). It is therefore possible that 

5HT, released from the thoracic ganglia might act directly on the 

ovaries of P. monodon. 


Indonesia) 

CHARACTERIZATION OF TRYPSIN-LIKE 

PROTEOLYTIC ACTIVITY FROM THE 

THELYCUM OF THE Penaeus monodon FEMALE 

(M340). (NO. 698) 

Kruevaisayawan H, Vanichviriyakit R, Murphy M, Hennebold 

J, Basak A, Weerachartyanukul W, Sobhon P, Tanphaichitr N. 



In penaeoid shrimp, sperm packaged in spermatophores in 

the male are inserted into and stored in the thelycum of the female 

prior to their release at the spawning time. Our published work 

indicates that the thelycum plays an active role in endowing 

fertilizing competence to sperm during their residence in the 

thelycum. After a prolonged period in the thelycum, the 

spermatophore surface is disrupted and sperm are released into the 

thelycum lumen for further molecular and physiological 

modifications. We hypothesize that the thelycum may secrete 

proteases that digest the spermatophore capsule, thus allowing the 

release of sperm into the lumen. The objective of this study was to 

investigate the presence of serine proteases in the thelycum lumen. 

Serine protease activities of the thelycal fluid (TF) were determined 

using four fluorogenic peptidyl–4–methylcoumaryl–7–amides 

(MCA) substrates; Boc–QAR–MCA, pERTKR–MCA, Boc–RVRR– 

MCA, and Suc–AAPF–MCA. Our results revealed the highest 

specific activity of trypsin–like protease(s) based on their specificity 

to Boc–QAR–MCA, a trypsin substrate (< 170 U/1 μ g protein). This 

protease activity was also reduced by trypsin inhibitors; soy bean 

trypsin inhibitor (SBTI, < 80%), 4–amidino phenyl methane 

sulfonyl fluoride (APMSF, < 75%), and N–α–tosyl–L–lysine 

chloromethyl ketone (TLCK, < 45%). These TF trypsin–like 

proteases were partially purified by SBTI–affinity column 

chromatography. Peak trypsin–like activity was eluted by 0.1 M 

sodium acetate, pH 3.0, and corresponded to two major bands with 

molecular weights of < 30 and 31.5 kDa following SDS–PAGE and 

silver staining. The doublet bands were cut and subjected to tryptic 

digestion and liquid chromatography/mass spectrometry (LC/MS). 

The MS results showed 41 spectra with no homology to any proteins 


in publicly accessible databases. In order to verify whether these 

proteins are novel, their peptide sequences are being obtained via 

MS/MS. This work is funded by National Science and Technology 

Development Agency, Higher Education Commission, Thailand 

(HK, WW, NT, PS), Thailand Research Fund (RV, PS) and the 

National Institutes of Health, U.S.A. (JH: NCRR RR00163 and 

NICHD HD42000). 

(Poster presentation at The 38 th Annual Meeting for The Society 

for The Study of Reproduction. July 2005. Quebec, Canada.) 

EXPRESSION AND POSSIBLE ROLE OF 

ARYLSULFATASE A (AS-A) IN MOUSE OVARY 

(T605). (NO. 699) 

Anupriwan A, Costa Santos D, Schenk M, Kongmanas, K, 

Carmona E, Sretarugsa P, Tanphaichitr N. 



AS-A is known as a lysosomal enzyme in somatic cells and 

an acrosomal enzyme in the sperm. In mature sperm, AS-A is also 

present on the cell surface and it functions together with its binding 

ligand, seminolipid, in egg binding. The aim of this study is to 

determine whether AS-A expresses in the female reproductive tissues 

such as the ovary. Using an antibody directed monospecifically to 

AS-A, immunocytochemistry studies revealed selective AS-A 

staining in the corpora lutea of superovulated mouse ovary sections. 

Isolated corpora lutea also showed AS-A desulfation activity on an 

AS-A 2 s artificial substrate, p-nitrocatecholsulfate (NCS)- 5.8 U/ 

mg protein, a value twice that of sperm surface AS-A. In order to 

synchronize development of all corpora lutea in the ovary, 

pseudopregnant mice were used in subsequent studies. Serum 

progesterone (P4) and luteal cell morphology were used as parameters 

for monitoring corpus luteum development. Both immunoblotting 

and immunocytochemistry revealed increasing expression of AS-A 

from Day 4 to Day 8 of pseudopregnancy, when the P4 level was 

rising and reached the maximum, respectively. The extent of AS-A 

expression continued to increase after the sharp drop of serum P4 

and morphological observation of luteolysis. Coincident with AS-A 

expression in luteal cells was the appearance of 20-hydroxysteroid 

dehydrogenase, which is responsible for the conversion of 

progesterone to 20-hydroxyprogesterone, a known mechanism of 

luteolysis. An increase in AS-A expression was also observed 

following luteolysis induction in Day 4 pseudopregnant mice with 

PGF2- injection. Notably, AS-A expression preceded that of active 

caspace-3, an apoptosis marker. All of these results suggested that 

AS-A was involved in the luteolysis process. Since cerobroside 

sulfate and seminolipid, known AS-A 2 s natural substrates, were 

not present in isolated corpora lutea, we suggest that AS-A may 

function like AS-B or AS-C (due to their similar 3-D structures) in 

desulfating glycan sulfates or estrone sulfate, respectively. Our 

structural modeling work indicated that estrone sulfate docked into 

AS-A 2 s active site pocket with minimum energy. Estrone sulfate 

desulfation would yield estrone, which may then be converted to 

estradiol, with a possible role in enhancing luteolysis. 

(Poster presentation at The 38 th Annual Meeting for The Society 

for The Study of Reproduction. July 2005. Quebec, Canada.) 



SULFOGALACTOSYLGLYCEROLIPID (SGG) 

PREFERENTIALLY LOCALIZES IN THE SPERM 

LIPID RAFTS : ATOMIC FORCE MICROSCOPIC 

APPROACHES (NO. 700) 

Wattana Weerachatyanukul, Nongnuj Tanphaichitr, Linda 

Johnston 



In this report, we examined the morphology and 

distributiontion of SGG and itsanalog, sulfogalactosylceramide 

(SGC, with the two hydrocarbon chains being C18:1 and C24:0) in 

lipid mixtures, using atomic force microscopy (AFM). SGC/SGG 

(0.2-1 mole ratio) was localized in small islands that were uniformly 

distributed throughout dimyristoyl phosphatidylcholine (DMPC)/ 

Cholesterol (Chol) monolayers 3:4:2 mole ratio mimicking sperm 

raft compositions). The islands are 0.5-2 nm higher than the 

surrounding monolayer. Increase in height corresponded with an 

increasing amount of sulfogalactolipids added. Interestingly, both 

sulfogalactolipids aggregated to form larger subdomains when the 

amount of Chol was lower (from 1 to 0.5 and 0.2) in the lipid 

mixtures. In the absence of Chol, SGC subdomains appeared as a 

flower-like pattern and SGG subdomains as a round globular-like 

pattern. This suggests that Chol may interact with sulfogalactolipids, 

and uniformly disperses them within liquidordered domains. 

Supported lipid bilayers of dipalmitoyl phosphatidylcholine (DPPC) 

/ palmitoyldocosahexaenoyl phosphatidylcholine (PDPC) / Chol 

(2:2:1), prepared by the vesicle fusion method, revealed liquidordered 

microdomains (made up of DPPC/Chol) that were about 1 

to 3 μm in size and ~1.5 nm higher than the surrounding fluid phase 

(containing mainly PDPC). Addition of SGC/SGG to this lipid 

mixture led to an alteration of domain morphology to become smaller 

domains (~0.3-0.5 μm), without affecting the height of these domains. 

When affinity purified anti-SGG was added to localize 

sulfogalactolipids in supported bilayers, SGC/SGG was found to be 

distributed in the LC phase (as gauged from the increased height of 

LC domains from 1.5 to 3 μm), although certain portion of SGC could 

also be detected in the fluid phase. These results confirmed our 

previous biochemical evidence indicating the presence of 

sulfogalactolipids in sperm raft lipids. 

(Oral presentation at the 22 nd MST Annual Conference. Journal of 

Microscopy Society of Thailand 2005, 19(1): 28.) 

ULTRASTRUCTURAL CHANGES OF THE 

CRUSHED SCIATIC NERVE FIBERS AFTER 

Pueraria mirifica TREATMENT IN RATS. (NO. 701) 

Chompoopong S, Phasukdee N, Chongthummakun , 

Wongtawatchai T, Siriphorn A, Chaiworakul P. 



The aim of this study was to determine the ultrastructural 

changes of the crushed sciatic nerve fibers after Pueraria mirifica 

treatment in male Sprague Dawley rats.Pueraria mirifica (white 

Kwao Krua ) found on the Northern Thailand, is an indigenous Thai 

herb with a long history of domestic consumption as a rejuvenating 

herb to promote youthfulness in both woman and men. Pueraria 

mirifica like soy bean and sprouts, is in group of phytoestrogen 

containing significant high level of isoflavones. Our previous study, 

motor function was assessed behaviorally using the toe spread reflex 

and the sciatic functional index (SFI), a non-invasive method of 

assessing the functional status of the sciatic nerve based on 

measurement of the animal’s walking tracks, expressed in units of 

functional deficit. SFI was significantly higher (less functional 

deficit) in the herbal extract treated than in saline treated rats after 

Day 6 up to 30 days. This study confirmed the SFI by using the 

transmission electron microscopic study in the ultrastructural 

changes of the crush / Pueraria mirifica treated sciatic nerve at Day 

7. The results showed that the dispersed myelin and degenerating 

axons were also found in myelinated nerve fibers of the distal 

segments eventhough they were seen as the increased intact 

myelinated axon at the light microscopic study. In the rat sciatic 

nerve crush model, Wallerian degeneration occurred up to 30 days. 

Our study suggests that the increase in the number of myelinated 

axons in the distal segments were enhanced by Pueraria mirifica 

but some degenerating appearance of the myelin sheath and axons 

still remained at the ultrastructural study. Pueraria mirifica may 

act as a potential neuroprotective agent for peripheral nerve injury 

but it might be investigated deeply further before manipulating them 

to serve the need for alternative medicine. 

(Oral presentation at the 22 nd MST Annual Conference. Journal of 

Microscopy Society of Thailand 2005, 19(1): 35-40.) 

PROCESS OF CHROMATIN CONDENSATION IN 

ASIAN BANDICOOT RAT (Babdicota indica) (NO. 702) 

P. Worawittayawong, P. Sretarugsa, C.M. Leigh, Sirikul 

Manochan, W. Weerachatyanukul, Prasert Sobhon, William G. 

Breed 



Bandicoot rat, Bandicota indica is one of the rodent species 

that commonly occurs in the rice fields of Thailand. The aims of the 

present study are 1) to study the organization and condensation of 

chromatin of germ cells during spermiogenesis by electron 

microscopy; 2) to localize the basic nuclear proteins during 

spermiogenesis by immunocytochemistry and immunoelectron 

microscopy; 3) to characterize the sperm basic nuclear proteins 

profile by acid urea triton-X gel electrophoresis. 

Immunocytochemistry and immunogold analysis clearly 

demonstrated that histones remained throughout late spermiogenesis 

whereas protamine was observed only in spermatozoa Fig.1-4). This 

histone remnant was not detected within the nuclear vacuoles. 

Histones were also present in epididymal sperm as demonstrating 

by acid urea gel. It was also apparent that there were transition protein 

(TP)(Fig. 5&6) and protamines in epididymal sperm, however, the 

amount of TP proteins seem to be much higher than protamines as 

compared by the intensity of Coomassie Blue staining. These results 

were in marked contrast to other mammals studied so far, where 

protamine was shown to be a predominant basic protein in mature 

sperm. 

(Poster presentation at the 22 nd MST Annual Conference. Journal 

of Microscopy Society of Thailand 2005, 19(1): 146-147.) 


274 

IMMUOLOCALIZATION OF THE HIGH 

POTENTIAL VACCINE CANDIDATE ANTIGENS 

IN THE TEGUMENT OF Fasciola gigantica (NO. 703) 

W. Khawsuk, S. Sriburee, N. Soonkhlang, C. Wanichanon, V. 

Viyanant, P. Sobhon 



Fasciola gigantica is the causative parasite of the fasciolosis 

in the cattle and human. A new way that is chosen to control the 

infection is vaccine treatment. Most of proteins that used as vaccine 

candidate are the excretory-secretory (ES) proteins. The high 

potential vaccine candidates that found in the ES antigens are 

glutathione Stransferase (GST), fatty acid binding protein (FABP) 

and cathepsin L (Cat L). ES is secreted from gut epithelium, excretory 

system and tegument of the parasite. The tegumental antigen is very 

interesting because it is directly exposed to the host immune 

responses. Therefore, tegumental antigen might be easily to attack 

by the vaccine treatment. This study has identified the location of 

the high potential antigen, GST, FABP and Cat L, in the tegument 

of newly excysted juvenile, 4-week juvenile and adult F. gigantica. 

The parasites were fixed with 4% paraformaldehyde and 0.5% 

glutaraldehyde. Cryosections were stained by immunofluorescence 

technique. LR White ultrathin sections were stained by immunogold 

labeling technique and used natural infected serum (CIS), laboratory 

infected serum (MIS) and polyclonal antibody against GST, FABP 

and Cat L as primary antibodies. The results showed that positive 

staining of CIS and MIS were found abundantly in the tegument. 

CIS was localized mainly in G2 granule of all stage parasites and 

some in G0 and G1 granule. MIS was found in G0 and G1 granules, 

and at the glycocalyx coating of the tegument. GST and FABP were 

fairly found in the matrix of the tegument but not in the granule or 

membrane. Cat L was found only on the glycocalyx coating of the 

tegument. These results suggested that the high potential antigens 

were found in the tegument, which may be released to be ES antigens. 

This study may be used as the basic knowledge for vaccine 

development against tropical fasciolosis or for the immunodiagnosis. 



HISTOLOGY AND ULTRASTRUCTURE OF THE 

MEHLIS’ GLAND-OOTYPE COMPLEX IN 

Fasciola gigantica (NO. 704) 

A. Meepool, C. Wanichanon, V. Viyanant, P. Sobhon 



The Mehlis’ gland-ootype complex is located at the midline 

of the parasite body between the testis and the uterus. It is an oval 

shave organ, which is composed of a group of cells surrounding the 

ootype and its accessory ducts. The Mehlis’ gland cells are unicellular 

exocrine gland located around the ootype which are classified into 

two types: Mehlis’ gland type 1 and 2 cells. Their cell bodies are 

located at the periphery of the gland, and they send their cytoplasmic 

processes toward the central part of the gland and enter the ootype 

wall to open at the ootype lumen where the eggshell is being formed. 

The cytoplasmic processes of the Mehlis’ gland cells are running 

between the processes of the parenchymal cells, which help to 

support them in positions. Furthermore, the parenchymal cells send 

their cytoplasmic processed to contact with the basement membrane 

of the ootype wall and its accessory ducts to supply nutrients to 

their epithelial cells. In addition to the Mehlis’ gland cells, there are 

two types of the nerve cells, type I and II, and muscle cells in the 

central part of the gland. The accessory ducts are including the 

vitelline reservoir, the median vitelline duct, the oviduct, the Laurer’s 

canal and the proximal part of the uterus. The median vitelline duct 

extends from the vitelline reservoir to the central part of the Mehlis’ 

gland and merges with the oviduct to become the ootype, and the 

ootypecontinue into the proximal part of the uterus. 



EFFECTS OF PSEUDOEPHEDRIEN ADMINIS- 

TRATION ON GLUTAMATE/N-METHYL-D- 

ASPARTATE RECEPTOR IMMNOREACTIVITY 

IN RAT HIPPOCAMPUS (NO. 705) 

S. Nudmamud-Thanoi, Thanoi S. and Sobhon P. 




Objectives: This study is aimed to investigate the effects 

of acute and chronic pseudoephedrine administration on glutamate/ 

N-methyl-D-aspartate (NMDA) receptors in hippocampus which is 

the area involved in learning and memory. 

Materials and Methods: Sprague Dawley male rats (250- 

280 g) were divided into 3 groups as acute, chronic, and control 

groups with 10 animals each. To examine as acute effect of 

pseudoephedrine, animals were administered intragastically at the 

dose of 120 mg/kg. The chronic effect was examined by treated 

pseudoephedrine intragastically at the dose of 80 mg/kg, once daily 

for 15 days. The animals in control group were administered 

intragastically with vehicle. We employed immunohistochemistry 

to determine the alteration of glutamate/M-methyl-D-aspartate 

receptors subunit 1 (NMDAR1) immunoreactivity in rat 

hippocampus following actue and chronic pseudoephedrine 

administration. 

Results : Immunohistochemistry demonstrated MNDAR1 

immunoreactive cells in all principal neuronal populations of the 

hippocampus. Immunoreactivity was accordingly limited to neurons, 

was strong in pyramidal cells of cornu ammonis fielsd 1-3 (CA1-3), 

and was especially strong in granule cells of dentate gyrus. MNDAR1 

immunodensity in three experimental groups were compared by 

analysis of variance (ANOVA) with Dunnett post hoc tests. 

NMDAR1 immunodensity was significantly increased above control 

in dentate gyrus in acute (p


HIGH DOSE PSEUDOEPHEDRINE ADMINIS- 

TRATION CAN INDUCE APOPTOSIS IN 

SEMINIFEROUS TUBULES ON MALE RATS 

S. Nudmamud-Thanoi, Thanoi, S. and Sobhon, P. 

(NO. 706) 



Objective : The present study is aimed to investigate the 

effects of pseudoephedrine, a sympathomimetic drug, on the 

reproductive system for understanding the mechanisms and 

increasing the knowledge of using the drug. 

Materials and Methods : Sprague Dawley male rats (250- 

280g) were divided into 3 groups as acute, chronic, and control 

groups with 10 animals each. To examine an acute, effect of 

pseudoephedrine, amimals were administered intragastically at the 

dose of 120mg/kg. The chronic effect was examined by treated 

pseudoephedrine intragastically at the dose of 80 mg/kg, once daily 

for 15 days. The animals in control group were administered 

intragastically with vehicle. After treatment, animals were sacrificed 

and apoptotic activites within the seminiferous tubules were studied 

using the TUNEL (TdT-mediated dUTP Nick End Labeling) assay. 

Results : The present study showed that acute 

administration of high dose pseudoephedrine can induce apoptotic 

activites within seminiferous tubules. In contrast, animals treated 

with lower dose of pseudophedrine chronically showed a small 

amount of apoptotic cells inside the seminiferous tubules. 

Qualitatively, the apoptotic actiivites were involved in every stage 

of sperm development inside the seminiferous tubules especially 

the spermatogonia. These results indicate that acute administration 

of high dose pseudoephedrine could induce apoptosis within the 

seminiferous tubules of male rats. Apoptosis that caused by 

pseudoephedrine may be due to a sudden increased adrenergic 

vasoconstriction after a single high dose administration. 

(Poster presentation at the 28 th Annual Meeting of the Society of 

Anatomy. April, 2005. Ubonratchatani, Thailand.) 

CHANGES IN EPIDERMAL STRUCTURE AND 

PROTEIN EXPRESSION DURING MOLTING 

CYCLE OF THE BLACK TIGER SHRIMP 

(Penaeus monodon) (NO. 707) 

Promwikorn W., Khunthongpan S., Kirirat P. Intasaro, P. 

Thaweethasewee P. and Withyachmunarnkul B. 



Background : In shrimp, the cycle of post-embryonic 

growth and development is characterized by a unique molting 

process. Epidermis plays an important role in the molting process. 

Although it has long been studied over seventy years, the regulatory 

mechanism of the molting cycle of the black tiger shrimp (Penaeus 

monodon), which is well-known as an important agricultural export 

product of Thailand, is far from attention. We therefore have been 

investigating regulatory mechanism of the molting cycle in the black 

tiger shrimp. 

Objective: To investgate changes in epidermal structure 

and protein expression during molting cycle of the black tiger shrimp 

by histochemistry and two dimension gel electrophoresis. 

Materials and methods: Healthy black tiger shrimps 

(Penaeus monodon), 10-20 g body-weight, were transferred from 

natural farms to culture in aquariums at Dept. Anatomy, PSU., where 

they were fed three times a day with commercial food. Eachshrimp 

were examined for molting stages at least once a day. Epidermal 

and sub-epidermis morphology of each molting stage were 

histologically investigates by staining with Masson’s trichromes, 

and periodic acid Schiff’s reagents, respectively. Epidermal protein 

expression was analysed by two dimension gel electrophoresis and 

silver stained. 

Results and discussions: It was found that the height of 

epidermis increases during the period of mid-premolt throughout 

early-postmolt stages. Glycoprotein deposition in sub-epidermis 

region is also increase corresponding to the above period. 



STRUCTURE AND PROTEIN PROFILES OF THE 

ANTENNAL GLAND OF THE BLACK TIGER 

SHRIMP (Penaeus monodon). (NO. 708) 

Asuvapongpatana S, Wongprasert K, Buranajitpirom D, 

Namwong W, Weerchatyanukul W., Apisawetakan S., 

Sritunyalucksana K, Withayachumnarnkul B. 



Objective : To study the structure and protein profiles of 

the antennal gland of the black tiger shrimp (Penaeus monodon) 

Materials and Methods : Juvenile shrimp, 20-25 g, were 

anesthetized on ice. Their anternnal glands were removed and divided 

into 2 groups. The first group was for studying under light 

microscopy, and sections were for studying the protein profile. The 

antennal glands were homogenized with lysis buffer (20 mM Tris, 

pH 7.4 and 100 mM NaCl). The homogenate was centrifuged at 

800xg, at 4 °c for 20 min, to pellet the muclei. The supernate was 

centrifuged at 100,000xg for 1 h, and the antennal gland lysate was 

collected. The pellet was incubated on ice with detergent (NaCl/Pi, 

1% triton X-100, 2mm EDTA, 1mM PMSF) for 30 min to solubileze 

the muclear membrane. The membrane solution was centrifuged at 

100,000xg, at 4 °c for 1 h, and the supernate (solubilized membrane 

lysate) was collected. The antennal gland lysate and solubilized 

membrane lysate were run on 12.5% SDSPAGE followed by staining 

with Coomassie blue or silver. 

Results : The antennal gland of P. monodon comprised 3 

parts: coelomosac, labyrinth and bladder. The coelomosac is 

surrounded by the lavyrinth and are supplied by the antennal artery. 

The coelomosac cells were similar to podocytes of mammalian 

kidney, having a large nucleus with abundant vacuoles in the 

cytoplasm. The labyrinth cell is cuboidal with concentric nycleus 

and brush border at the apical end of the cell. The labyrinth cells 

might be divided into two stages: the secretory and non secretory 

secretory stages. In the secretory stage, the cell is cuboid with the 

nucleus closed to the apical surface and the brush border is slough 

off. In the non-secretory stage, the cuboidal cell is covered with 

brush border and the nucleus was at the center of the cell. The 

proteins of the antennal gland were distributed between 10-150 kDa, 

with main components were proteins at molecular weights of 53, 

40, 30, 22 kDa. 




276 

THE STUDENT’S OPINION ON THE BASIC 

ANATOMY COURSE ACTIVITITES AT THE 

FACULTY OF SCIENCE, MAHIDOL UNIVERSITY 

DURING ACADEMIC YEAR 2004-2005. (NO. 709) 

S. Rojananuangnit, Thanabulsombat, C., Leardkamolkarn, V. 

and Pulsawat, J. 



Background : Basic anatomy course at the Faculty of 

Science, Mahidol University is a compulsory course assigned for 

health science students including second year medical-technology, 

radio-technology, pubic health students and first year nursing, sportscience 

and abnormal-audio students. The course is 3 credit hour 

teaching, composes of lecture and laboratory demonstration from 

dissected cadavers by lab instructors or student self study. After the 

lab practice, the students are evaluated by the paper test questions 

and lab specimen identification. Beside the direct learning activities, 

students are encouraged to participate in the supportive course 

activities which are related to the religion concerning of using human 

death bodies as the subject for studying. Such activities, during 

academic year 2004-2005, were co-organized by courses instructors 

and students, under the supports of the Dean of the Faculty of Science 

and some Buddhist practitioners. The supportive activities included 

the playing tributes and respects to the cadavers whom their bodies 

are used in this course. 

Objectives: This study aims to evaluate the student’s 

opinion to the course teaching activities and the supportive course 

activity which most of them were organized according to the 

Buddhist believes. 

Materials and Methods: The questions concerning the 

opinion and satisfaction on the course activities were distributed to 

all the students taking Basic Anatomy course as a compulsory course 

during academic year 2004-2005. The papers were collected and 

statistic evaluated. 

Results: The overall responded students (total 323) included 

33 male (10.22%), 290 female students (89.78%) were the first year 

students (total 225; 69.66%) and the second year students (total 98; 

30.39%). Most students were satisfied with the learning activities, 

as given in the lecture and the laboratory, at the moderate level 

(59.4%) and suggested that the laboratory lesson should be modified 

(8.6%). However, the students were most satisfied with the overall 

activities in the supportive course activities that were religious 

performed including the format and all the aspects played in Buddhist 

cultural activities. They believed that these activities related to the 

Basic Anatomy learning. However, the students preferred to perform 

these activities at less than 5 hours. 



INFECTION OF DENDRITIC CELLS BY 

DENGUE-2 VIRUS INDUCES PROTEINS 

INVOLVED IN DENGUE PATHOGENESIS (NO. 710) 

N. Luplerdlop, M. Dorothee, V. Leardkamolkarn, GJ Paul, D. 

Mathieu, Y. Hans, V. Francisco 




Objective : Dengue virus (DV) is the etiologic agent of 

one of the most important human emerging disease of these last 

decades. From 1970 up to now, the four subtypes of DV have 

constantly and largely been spread in inter-tropical regions of the 

world and have been involved in a growing number of deaths 

annually. The critical clinical outcome of the disease is the 

hemorrhagic fever (DHF) phase that corresponds to the endothelium 

disruption and the subsequent plasma extravasation. Despite of 

numerous studies most of the mechanisms associated to dengue 

pathogenesis remain unclear. In this work we tried to study cellular 

and soluble factors potentially involved in dengue pathogenesis, 

especially during DHF. 

Methods : Primary dendritic and endothelial cells, the 

principal targets for DV, were infected with Dengue 2 virus.To asses 

the cellular and soluble factors induced by the Dengue infection, 

cells and supernatants and were collected in both a dose response 

and kinetic manner. A large set of complementary technological 

approaches were used, FACS, Western blotting, ELISA, 

Immunohistochemistry, confocal microscopy, etc. 

Results : The in vitro obtrained results providing 

biomarkers involved in the Dengue pathogenesis mechanisms, 

probably induced during DHF are currently prepared for publication. 

They will be showed and discussed the day of presentation. 

Conclusion : In our experiments, we found proteins from 

the dendritic and endothelial cells that affect both the increase 

premeaaility and the intracellular structure disorganization of the 

endothelial cells. These biomarkers are currently examined ex vivo 

and should be extended in large scale study to assess their DHF 

prognostic value and their interest in the design of new therapeutic 

approaches. 

(Poster presentation at RGJ-Ph.D. Congress VI, TRF Annual 

Meeting. Chonburi, Thailand) 

REGULATION OF MT1 – MMP FUNCTIONALITY 

BY OVEREXPRESSION OF Vb3 INTEGRIN IN 

HUMAN BREAST CANCER CELLS. (NO. 711) 

K. Borrirukwanit, M.A. Lafleur, T. Blick, J. Prince, M. Waltham, 

V. Leardkamonkarn 



Objectives : Both Vβ3 integrin and MT1-MMP play 

important roles in tumor development and angiogenesis. The overall 

aim of this study was to elucidate the influence of Vβ3 integrin on 

collagen-stimulated MMP-2 activation and tumourigenicity in breast 

cancer cells. 

Methods : Stable β3 integrin expression was established 

in 3 different breast cancer cell lines by transfection. The highly 

invasive BT549 and MDA-MB-231cell lines both express 

endogenous MT1-MMP, and either lack endogenous Vβ3 integrin 

or show very low Vβ3 integrin expression, respectively, The MCF- 

7 cell line is poorly invasive, and lacks both MT1-MMP and Vβ3 

integrin, and was transfected of MT1-MMP either alone or in 

combination with β3 integrin. Functional expression of Vβ3 

integrin in all cell lines was determined by flow cytometry, 

immunofluorescence and adhesion assay. Gelatin zymography was 

used to determined MMP-2 activation, and MT1-MMP-2 activation, 



and MT1-MMP and Vβ3 integrin were monitored by Western blot. 

ECM remodeling was examined by collagen gel invasion and 

contraction. For tumor growth analysis, MCF-7 cells from each group 

were injected into the mammary fat pads of nude mice, and tumour 

sizes were records for up to 50 days after they became palpable. 

Results : Overexpression of β3 integrin increased cellular 

adhesion to ECM substrates including collagen type I, IV, VN and 

FN in both invasive cell lines, and caused increased expression of 

alpha V integrin and MT1-MMP at cell surface as determined by 

FACS analysis. Interestingly, overexpression of β3 integrin 

suppressed the collagen-induced MMP-2 activation in cells 

expressing either endogenous (transcriptionally regulated by 

collagen) or ectopic (regulated at the cell surface by collagen) MTI- 

MMP. This correlated with reduced TIMP-2 levels at cell surface, 

but could not be rescued by addition of recombinant TIMP-2. MTI- 

MMP deetion mutants lacking the cytoplasmic tail were also 

repressed by co-transfection with β3 integrin and MTI-MMP. Despite 

the inhibition of MMP-2-activation, stable β3 integrin expression 

enhanced the invasion of the BT-549 cells through 3D collagen gels, 

enhanced collagen gel contraction by MDA-MB-231 cells, and 

cooperated with MTI-MMP to cause increased growth of the MCF- 

7 cells in immunocompromised mice. 

Conclusions : These data indicate important effects of aVβ3 

integrin on MTI-MMP functionality which appear to be independent 

of MMP-2 activation, causing enhanced cell invasion, ECM 

remodeling and tumor growth. This is in contrast to an apparently 

indirect suppression of MMP-2 activation in these cells. The 

targeting of β3 integrin, in the comtext of MTI-MMP, may offer 

therapeutic potential in cancer. 


Meeting. Chonburi, Thailand.) 

FOCAL ADHESION KINASE AND SRC 

PHOSPHORYLATIONS IN HGF-INDUCED 

PROLIFERATION AND INVASION OF HUMAN 

CHOLANGIOCARCINOME CELL LINE, HuCCa-1 

U. Pongchairerk, JL Guan and V. Leardkamolkarn 

(NO. 712) 



Background : Human cholangiocarcinoma (CCA) is a high 

incidence liver cancer that causes high mortality rate inThailand. 

The cancer has poor prognosis due to the delay in detection and the 

resistance to chemotherapy, To overcome the difficulties in handling 

of this carcinoma, it is necessary to study for the details information 

of the cancer and its association in all aspects. 

Objective : To investigate the association between focal 

adhesion kinase (FAK) and Src induced by hepatocyte growth factor 

(HGF) in signaling of cancer progression, and study the role of FAK 

in HuCCA-1 cell proliferation and invasion. 

Materials & Methods : HuCCA-1 cells were confirmed 

for their characteristic by immunofluorescent staining of cytokeratin 

19 and RT-PCR for HGF and c-met mRNA expression. 

Immunoprecipitation and Western blotting were performed to study 

FAK and Src phosphorylation induced by HGF. 

Coimmunoprecipitation was assayed to investigate the association 

between FAK and Src. The cells were treated by a novel Src inhibitor 

(AZM555130) to study the effect of FAK inhibition in HuCCA-1 

cell proliferation and invasion by using MTT and invasion assays, 

respectively. 

Results : HGF enhanced HuCCA-1 cell proliferation and 

invasion. It also induced FAK and Src phosphorylation and their 

interaction in time dependent manner. Src was proved to be an 

upstream signaling molecule to FAK. An inhibitor decreased the 

level of FAK phosphorylation in association with the reduction of 

HuCCA-1 cell proliferation and invasion. 

Conclusion : FAK is a substrate of activated Src and FAK 

play a significant role in signaling pathway of cancer progression, 

especially in cholangiocarcinoma which usually highly responsive 

to HGF induction. 


Meeting. Chonburi, Thailand) 

IN VITRO AND IN VIVO ANALYSIS OF THE 5’ 

NONTRANSLATED REGIONS (5’NTRs) OF 

DENGUE TYPE 2 VIRUS (NO. 713) 

W. Sirigulpanit, RM’ Kinney, V. Leardkamolkarn 



Objective : To study structural and functional relation of 

the 5’NTR of Dengue Type 2 Virus. 

Methods : An infectious cDNA clone of DEN 2 virus was 

used to mufagenize its 5’NTR. Full-length RNA transcripts of the 

variant infectious cDNA clones were transfected into LLC-MK 2 cells. 

Cells were harvested and tested for the presence of the presence of 

dengue virus antigens by IFA. The characterization of the wild-type 

virus and its derived mutants were performed in both LLC-MK 2 

and C6/36 cells. Mouse neurovirulence assay was also investigated 

by inoculating outbred ICR mice with the progeny viruses. The 

secondary structure of 5’NTR RNAs were analyzed by using version 

3.1 of MFOLD program developed by Zuder M et al (1991). The 

viral protein synthesis was analyzed by labeing with 35 S-Methionine 

and in vitro translation was performed by rabbit reticulocyte lysate 

system in the presence of 35 S-Methionine and the translation products 

were separated in 12.5% SDS-PAGE gel. 

Results : The single point-substitution mutants produced 

similar plaques size and gave the same viral yield as the wild-type 

virus. Most of the mutants were mouse-virulent viruses, except for 

some mutants were intermediate virulence viruses. The multiplesubstitution 

and multiple-deletion mutants failed to initiate infection 

in LLC-MK 2 , BHK-21 and C6/36 cells. Interestingly, some single 

and double-point deletion mutants presented dengue virus antigens 

by IFA but failed to produce plaques in permissive cells. Comparison 

between the predictive RNA secondary structure of the 5’NTR of 

the wild-type and the mutants indicated that a single pointsubstitution 

mutants maintain the same overall secondary structure 

of the wild-type. Their viral infectivity and viral replication were 

not affected. In contrast, a single and double-point deletion mutants 

that maintain the secondry structure were a little different from that 

of the wild-type but their viral infectivity and viral replication were 


278 

significantly reduced. Furthermore, the secondary structure of the 

multiple-substitution and multiple-deletionmutants were quite 

different from that of the wild-type. These mutants were not viable. 

Transcriptional and translational efficiency of the wild-type and its 

derived mutants were consequently investigated. The preliminary 

result indicated that the single point-substitution mutant had 

efficiency translation in a rabbit reticulocyte lysate system. 

Conclusion : Mutation that disrupts the 5’NTR of Den-2 

virus cause defective in viral replication and/or viral translation 

induced by secondary structure alteration. 


Meeting. Chonburi, Thailand.) 

INVOLVEMENT OF SPECIFIC PROTEINS (SP1/SP3) 

AND NUCLEAR FACTOR Y IN BASAL TRANSCRIP- 

TION OF THE DISTAL PROMOTER OF THE RAT 

PYRUVATE CARBOXYLASE GENE IN b-CELLS 

(NO. 714) 

Sunyakumthorn P 1 , Boonsean T 1 , Boonsaeng V 1 , Wallace JC 2 , 

Jitrapakdee S 1 

1 Department of Biochemistry, Faculty of Science, Mahidol 

University, Bangkok; 2 School of Molecular and Biological 

Sciences, University of Adelaide, Adelaide, Australia . 

Key words : gene transcription, nuclear factors, pyruvate 

carboxylase. 

Pyruvate carboxylase plays diverse roles in different 

biosynthetic pathways, including glucose-induced insulin secretion 

in pancreatic β-cells. We have localized the control region of the 

P2 promoter by generating a series of 50-nested deletion constructs, 

and both 25- and 9-bp internal deletion constructs, as well as by 

performing site-directed mutagenesis. Transient transfections of 

these constructs into INS-1 cells identified a CCAAT box and a GC 

box that are located at -65/-61 and -48/-41, respectively, as the 

important determinants. Disruption of the GC box resulted in a 4fold 

reduction of the reporter activity, while disruption of the 

proximal CCAAT box (-65/-61) but not of the distal CCAAT box (- 

95/-91) increased the reporter activity by 3-fold. Simultaneous 

disruptions of both the GC box and the CCAAT box reduced the 

reporter activity to a level that was close to that of the single GC 

box mutation. Electrophoretic mobility shift assays (EMSAs) using 

nuclear extracts from INSA-1 cells demonstrated that Sp1 and Sp3 

bind a GC box while the nuclear factor Y was shown to bind the 

proximal but not the distal CCAAT box. 

(Published in Biochem Biophys Res Commun 2005; 329: 188-96. 

Supported by Thailand Research Fund, Third World Academy of 

Sciences, and Australian Research Council.) 

THE PEROXISOME PROLIFERATOR-ACTIVATED 

RECEPTOR GAMMA REGULATES MURINE 

PYRUVATE CARBOXYLASE GENE EXPRESSION 

IN VIVO AND IN VITRO. (NO. 715) 

Jitrapakdee S 1,2 , Slawik M 1 , Medina-Gomez G 1 , Campbell M 1 , 

Wallace JC 3 , Sethi JK 1 , O’Rahilly S 1 , Vidal-Puig AJ 1 . 


1 Cambridge Institute of Diabetes, Endocrinology and 

Metabolism, and Department of Clinical Biochemistry, 

University of Cambridge, Cambridge, UK; 2 Department of 

Biochemistry, Faculty of Science, Mahidol University, Bangkok; 

3 School of Molecular and Biomedical Science, University of 

Adelaide, Adelaide, Australia. 

Key words: control of gene expression, murine pyruvate carboxylase 

gene, peroxisome proliferator-activated receptor gamma. 

Pyruvate carboxylase (PC) plays a crucial role in various 

metabolic pathways including gluconeogenesis, lipogenesis and 

glucose-induced insulin secretion. Here we show for the first time 

that the PC gene is transcriptionally regulated by peroxisome 

proliferator activated receptor gamma (PPARγ) in vitro and in vivo 

in white and brown adipose tissue. PC mRNA and protein are 

markedly increased during differentiation of 3T3-L1 cells and HIB- 

1B, in parallel with the expression of the adipogenenic transcription 

factors, CCAAT-enhancer binding protein α (C/EBPα), PPARγ1 and 

PPARγ2. Tumor necrosis factor alpha (TNFα), a cytokine that blocks 

differentiation of 3T3-L1 cells, suppresses PC expression. Cotransfection 

studies in 3T3-L1 preadipocytes or HEK293T cells with 

a 2.3 kb promoter fragment of mouse PC gene linked to a luciferase 

reporter construct and with plasmids overexpressing RXRα/PPARγ1 

or RXRα/PPARγ2, showed a 6-8 fold increase above the basal 

promoter activity. Furthermore, treatment of these transfected cells 

with PPARγ agonist doubled the promoter activity. Mutation of the 

putative PPRE (-386/-374) of this 2.3 kb PC promoter fragment 

abolished PPARγ response. Gel shift and chromatin 

immunoprecipitation assays demonstrate that endogenous PPARγ 

binds to this functional PPRE of the PC promoter. Mice with targeted 

disruption of the PPARγ2 gene display approximately 50-60% 

reduction of PC mRNA and protein in white adipose tissue. Similarly 

in brown adipose tissue of PPARγ2 deficient mice subjected to cold 

exposure, PC mRNA was 40% lower than that of wild type mice. 

Impaired in vitro differentiation of white adipocytes of PPARγ2 

knock-out mice was also associated with a marked reduction of PC 

mRNA. Our findings identify PC as a PPAR γ regulated gene and 

suggest a role for PPARγ regulating intermediary metabolism. 

(Published in J Biol Chem 2005; 290: 27466-76. Supported by 

Wellcome Trust, MRC UK, Royal Society UK, and Fritz Thyssen 

Foundation Germany. ) 

CRYSTALLIZATION AND PRELIMINARY X-RAY 

CRYSTALLOGRAPHIC ANALYSIS OF 2-METHYL- 

3-HYDROXYPYRIDINE-5-CARBOXYLIC ACID 

(MHPC) OXYGENASE FROM PSEUDOMONAS SP. 

MA-1. (NO. 716) 

Oonanant W 1 , Sucharitkul J 1,2 , Yuvaniyama J 1 , Chaiyen P 1 . 

1 Department of Biochemistry and Center for Excellence in 

Protein Structure and Function, Faculty of Science, Mahidol 

University, Bangkok; 2 Department of Biochemistry, Faculty of 

Dentistry, Chulalongkorn University, Bangkok. 

Key words: 2-methyl-3-hydroxypyridine-5-carboxylic acid 

oxygenase, X-ray crystallographic data. 

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) 

oxygenase (MHPCO) catalyzes the conversion of an aromatic 

substrate, MHPC, to an aliphatic compound, α-(N- 



acetylaminomethylene)-succinic acid, and is involved in the 

degradation of vitamin B6 by the soil bacterium Pseudomonas sp. 

MA-1. Using only FAD as a cofactor, MHPCO is unique in 

catalyzing hydroxylation and subsequent aromatic ring cleavage 

without requiring a metal-ion cofactor. Here, the crystallization of 

MHPCO is reporte3d together with preliminary X-ray 

crystallographic data. An MHPCO crystal obtained by hanging0drop 

vapour diffusion diffracted X-rays to 2.25 Å resolution and 

belonged to the triclinic space group P1, with four molecules per 

asymmetric unit. 

(Published in Acta Cryst 2005; F61: 312-4. Supported by Mahidol 

University, Thailand Research Fund and TARUN/Thailand Tropical 

Diseases Research Program.) 

STUDIES ON THE TRANSGLUCOSYLATION 

REACTIONS OF CASSAVA AND THAI ROSEWOOD 

BETA-GLUCOSIDASES USING 2-DEOXY-2-FLUORO- 

GLYCOSYL-ENZYME INTERMEDIATES. (NO. 717) 

Hommalai G, Chaiyen P, Svasti J. 

Center of Excellence for Protein Structure and Function, 

Department of Biochemistry, Faculty of Science, Mahidol 

University, Bangkok. 

Key words: beta-glucosidase, cassava, Thai rosewood. 

beta-Glucosidases from cassava and Thai rosewood can 

synthesize a variety of alkyl glucosides using various alcohols as 

glucosyl acceptors for transglucosylation. Both enzymes were 

inactivated by 2-deoxy-2-fluoro-sugar analogues to form the covalent 

glycosyl-enzyme intermediates, indicating that the reaction 

mechanism was of the double-replacement type. The trapped enzyme 

intermediates were used for investigating transglucosylation 

specificity, by measuring the rate of reactivation by various alcohols. 

The glucosyl-enzyme intermediate from the cassava enzyme showed 

a 20- to 120-fold higher rate of glucose transfer to alcohols than the 

glucosyl-enzyme intermediate from the Thai rosewood enzyme. 

Kinetic analysis indicated that the aglycone binding site of the 

cassava enzyme was hydrophobic, since the enzyme bound better to 

more hydrophobic alcohols and showed poor transfer of glucose to 

hydrophilic sugars. With butanol, transglucosylation was faster with 

the primary alcohols than with the secondary or tertiary alcohol. 

Studies with ethanol and chloro-substituted ethanols indicated that 

the rate of transglucosylation was significantly faster with alcohols 

with lower pKa values, where the reactive alkoxide was more readily 

generated, indicating that the formation of the alkoxide species was 

a major step governing the formation of the transition state in the 

cassava enzyme. 

(Published in Arch Biochem Biophys 2005; 442: 11-20. Supported 

by Thailand Research Fund.) 

THE REDUCTASE OF P-HYDROXYPHENY- 

LACETATE 3-HYDROXYLASE FROM 

ACINETOBACTER BAUMANNII REQUIRES P- 

HYDROXYPHENYLACETATE FOR EFFECTIVE 

CATALYSIS. (NO. 718) 

Sucharitakul J 1 , Chaiyen P 1 , Entsch B 2 , Ballou DP 2 . 

1 Department of Biochemistry and Center for Excellence in 

Protein Structure and Function, Faculty of Science, Mahidol 

University, Bangkok; 2 Department of Biological Chemistry, 

University of Michigan, Michigan, USA. 

Key words: Acinetobacter baumannii, p-hydroxyphenylacetate 3hydroxylase 

reductase, two-protein system. 

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) from 

Acinetobacter baumannii catalyzes hydroxylation of HPA to form 

3,4-dihydroxyphenylacetate. It is a two-protein system consisting 

of a smaller reductase component (C 1 ) and a larger oxygenase 

component (C 2 ). C 1 is a flavoprotein containing FMN, and its 

function is to provide reduced flavin for C 2 to hydroxylate HPA. 

We have shown here that HPA plays important roles in the reaction 

of C 1 . The apoenzyme of C 1 binds to oxidized FMN tightly with a 

K d of 0.006 μM at 4°C, but with a K d of 0.038 μM in the presence of 

HPA. Reduction of C 1 by NADH occurs in two phases with rate 

constants of 11.6 and 3.1 s -1 and K d values for NADH binding of 2.1 

and 1.5 mM, respectively. This result indicates that C 1 exists as a 

mixture of isoforms. However, in the presence of HPA, the reduction 

of C 1 by NADH occurred in a single phase at 300 s -1 with a K d of 25 

μM for NADH binding at 4°C. Formation of the C 1 -HPA complex 

prior to binding of NADH was required for this stimulation. The 

redox potentials indicate that the rate enhancement is not due to 

thermodynamics (E° m of the C 1 -HPA complex is -245 mV compared 

to an E° m of C 1 of -236 mV). When the C 1 -HPA complex was reduced 

by 4(S)-NADH, the reduction rate was changed from 300 to 30 s -1 , 

giving a primary isotope effect of 10 and indicating that C 1 is 

specifically reduced by the pro-(S)-hydride. In the reaction of 

reduced C 1 with oxygen, the reoxidation reaction is also biphasic, 

consistent with reduced C 1 being a mixture of fast and slow reacting 

species. Rate constants for both phases were the same in the absence 

and presence of HPA, but in the presence of HPA, the equilibrium 

shifted toward the faster reacting species. 

(Published in Biochemistry 2005; 44: 10434-42. Supported by 

Mahidol University, Thailand Research Fund, Commission of 

Higher Education Staff Development Project, Ministry of Education, 

Thailand, BIOTEC, NSTDA, Thailand, and NIH, USA.) 

ARGINOSUCCINATE SYNTHETASE DEFICIENCY: 

MUTATION ANALYSIS IN 3 THAI PATIENTS. 

(NO. 719) 

Wasant P 1 , Viprakasit V 2 , Srisomsap C 3 , Liammongkolkul S 1 , 

Ratanarak P 1 , Sathienkijakanchai A 1 , Svasti J 3,4 . 

1 Division of Medical Genetics, Department of Pediatrics, Faculty 

of Medicine Siriraj Hospital, Mahidol University, Bangkok; 

2 MRC Molecular Haematology Unit, Weatherall Institute of 

Molecular Medicine, University of Oxford, Oxford, UK; 

3 Laboratory of Biochemistry, Chulabhorn Research Institute, 

Bangkok; 4 Department of Biochemistry, Faculty of Science, 

Mahidol University, Bangkok. 

Key words : arginosuccinate synthetase deficiency, mutation 

analysis, Thai patients. 

Remarkable improvements in public health, nutrition, 

hygiene, and availability of medical services in the last 20 years 

have significantly reduced infant and childhood mortality in 

Thailand. Therefore, many rare and previously unidentified genetic 

disorders, which, in the past, usually led to the death of affected 

infants before a definitive diagnosis, have now been increasingly 

recognized. Recently, we identified three unrelated patients from 

Thailand who suffered from citrullinemia, one of the inherited types 


280 

of urea cycle disorders. All were diagnosed within their first few 

weeks of life. Biochemical analyses, including plasma amino acid 

and urine organic acid profiles, are consistent with arginosuccinate 

synthetase (ASS) deficiency. Extensive mutation study by direct 

genomic sequencing of ASS demonstrated a homozygous G117S 

mutation in one patient and homozygous R363W mutations in the 

other two families. 

(Published in Southeast Asian J Trop Med Public Health 2005; 36: 

757-61. Supported by Thailand Research Fund.) 

EFFICIENT PHOTOCLEAVAGE OF LYSOZYME 

BY A NEW CHIRAL PROBE. (NO. 720) 

Buranaprapuk A 1,2 , Chaivisuthangkura P 3 , Svasti J 4 , Kumar CV 1 

1 Department of Chemistry, University of Connecticut, 

Connecticut, USA; 2 Department of Chemistry, Faculty of 

Science, Srinakharinwirot University, Bangkok; 3 Department of 

Biology, Faculty of Science, Srinakharinwirot University, 

Bangkok; 4 Department of Biochemistry, Faculty of Science, 


Key words: lysozyme, photocleavage, pyrene. 

Photocleavage of lysozyme and bovine serum albumin 

(BSA) by L-phenylalanine-1(1-pyrene)methylamide (PMA-L-Phe) 

is reported here. The chiral probe, PMA-L-Phe, has a positively 

charged side chain, while the previous probes carried a free carboxyl 

group. The yield of lysozyme cleavage by PMA-L-Phe is increased 

to 57% when compared to the previous probes, while the yield of 

BSA cleavage is reduced to 6)-beta-d-glucopyranoside 

and compound 2, 

6,2',4',5'-tetramethoxy-7-hydroxy-7-O-beta-d-apiofuranosyl-(1— 

>6)-beta-d-glucopyranoside (dalnigrein7-O-beta-d-apiofuranosyl- 

(1—>6)-beta-d-glucopyranoside). The beta glycosidase removes the 

sugar from these glycosides as a disaccharide, despite its initial 

identification as a beta-glucosidase and beta-fucosidase. 

(Published in Phytochem 2005 Aug 9; [Epub ahead of print]. 

Supported by Thailand Research Fund and Suranaree University 

of Technology.) 

VANILLIN SUPPRESSES IN VITRO INVASION 

AND IN VIVO METASTASIS OF MOUSE BREAST 

CANCER CELLS. (NO. 723) 

Lirdprapamongkol K 1,3 , Sakurai H 1,2 , Kawasaki N 1 , Choo MK 1 , 

Saitoh Y 1 , Aozuka Y 1 , Singhirunnusorn P 1 , Ruchirawat S 4 , Svasti 

J 3,5 , Saiki I 1,2 . 

1 Division of Pathogenic Biochemistry, Institute of Natural 

Medicine, Toyama Medical and Pharmaceutical University, 

Toyama, Japan; 2 The 21 st Century COE Program, Toyama 

Medical and Pharmaceutical University, Toyama, Japan; 

3 Laboratory of Biochemistry, Chulabhorn Research Institute, 

Bangkok; 4 Laboratory of Natural Products, Chulabhorn 

Research Institute, Bangkok; 5 Department of Biochemistry, 

Faculty of Science, Mahidol University, Bangkok. 

Key words : anti-metastatis, invasion, vanillin. 




Vanillin, a food flavoring agent, has been reported to show 

anti-mutagenic activity and to inhibit chemical carcinogenesis. In 

this study, we examined the effect of vanillin on the growth and 

metastasis of 4T1 mammary adenocarcinoma cells in BALB/c mice. 

Mice orally administered with vanillin showed significantly reduced 

numbers of lung metastasized colonies compared to controls. In 

vitro studies revealed that vanillin, at concentrations that were not 

cytotoxic, inhibited invasion and migration of cancer cells and 

inhibited enzymatic activity of MMP-9 secreted by the cancer cells. 

Vanillin also showed growth inhibitory effect towards cancer cells 

in vitro. However, vanillic acid, a major metabolic product of vanillin 

in human and rat, was not active in these in vitro activity assays. 

Our findings suggest that vanillin has anti-metastatic potential by 

decreasing invasiveness of cancer cells. Since vanillin is generally 

regarded as safe, it may be of value in the development of antimetastatic 

drugs for cancer treatment. 

(Published in Eur J Pharm Sci 2005; 25: 57-65. Supported by JSPS- 

NRCT Collaborative Scientific Research Fellowship Program.) 

RETROSPECTIVE STUDY OF PATIENTS WITH 

SUSPECTED INBORN ERRORS OF METABOLISM 

AT SIRIRAJ HOSPITAL, BANGKOK, THAILAND 

(1997-2001) (NO. 724) 

Wasant P 1 , Vatanavicharn N 1 , Srisomsap C 2 , Sawangareetrakul 

P 2 , Liammongkolkul S 1 , Svasi J 3 . 

1 Department of Pediatrics, Faculty of Medicine, Siriraj Hospital, 

Mahidol University, Bangkok; 2 Laboratory of Biochemistry, 

Chulabhorn Research Institute, Bangkok; 3 Department of 

Biochemistry, Faculty of Science, Mahidol University, Bangkok. 

Key words : inborn errors of metabolism, quantitative plasma amino 

acid analysis, Siriraj Hospital. 

This retrospective clinical study was carried out with 

suspected inborn errors of metabolism (IEM) at Siriraj Hospital 

during 1997-2001. The authors investigated 114 patients by 

quantitative plasma amino acid analysis. All patients were 

categorized into 2 major groups: 1) positive diagnoses for IEM, 2) 

negative diagnoses for IEM. The two groups were investigated, 

studied including statistical analysis. The authors found that most 

IEM ascertained through plasma amino acid analysis were smallmolecule 

diseases (74.3%) and amino acid disorders consisted of 

the most frequent disorders. The presented data demonstrated that 

the ratio of positive diagnoses to all patients studied was 1:8. 

Epidemiological data showed there were more male than female 

patients. Onset of diseases occurred predominantly during the first 

month of age, and was rarely found after 3 years of age. There were 

histories of consanguinity in half of the IEM patients. The most 

common presenting symptom was acute metabolic encephalopathy 

and specific signs for small-molecule disorders included 

hepatomegaly, unusual urine odor, acidosis, hyperammonemia, 

alteration of consciousness, and ketosis/ketonurea. These signs or 

symptoms indicated further metabolic investigations. Comparison 

of the data from Thailand with other countries showed both 

similarities and differences to the Caucasian population. Thus, 

further studies in IEM are much needed for the Thai population. 

(Published in J Med Assoc Thai 2005; 88: 746-53. Supported by 

Chulabhorn Research Institute and Thailand Research Fund.) 

MALARIAL (PLASMODIUM FALCIPARUM) 

DIHYDROFOLATE REDUCTASE-THYMIDY- 

LATE SYNTHASE: STRUCTURAL BASIS FOR 

ANTIFOLATE RESISTANCE AND DEVELOP- 

MENT OF EFFECTIVE INHIBITORS. (NO. 725) 

Yuthavong Y 1 , Yuvaniyama J 2 , Chitnumsub P 1 , Vanichtanankul 

J 1 , Chusacultanachai S 1 , Tarnchompoo B 1 , Vilaivan T 3 , 

Kamchonwongpaisan S 1 . 

1 National Center for Genetic Engineering and Biotechnology, 

National Science and Technology Development Agency, 

Pathumthani; 2 Center of Excellence for Protein Structure and 

Function and Department of Biochemistry, Faculty of Science, 

Mahidol University, Bangkok; 3 Department of Chemistry, 

Faculty of Science, Chulalongkorn University, Bangkok . 

Key words : dihydrofolate reductase, Plasmodium falciparum, 

thymidylate synthase. 

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) 

from Plasmodium falciparum, a validated target for antifolate 

antimalarials, is a dimeric enzyme with interdomain interactions 

significantly mediated by the junction region as well as the 

Plasmodium-specific additional sequences (inserts) in the DHFR 

domain. The X-ray structures of both the wild-type and mutant 

enzymes associated with drug resistance, in complex with either a 

drug which lost, or which still retains, effectiveness for the mutants, 

reveal features which explain the basis of drug resistance resulting 

from mutations around the active site . Binding of rigid inhibitors 

like pyrimethamine and cycloguanil to the enzyme active site is 

affected by steric conflict with the side -chains of mutated residues 

108 and 16, as well as by changes in the main chain configuration. 

The role of important residues on binding of inhibitors and substrates 

was further elucidated by site-directed and random mutagenesis 

studies. Guided by the active site structure and modes of inhibitor 

binding, new inhibitors with high affinity against both wild-type 

and mutant enzymes have been designed and synthesized, some of 

which have very potent antimalarial activities against drug -resistant 

P. falciparum bearing the mutant enzymes. 

(Published in Parasitology 2005; 130: 249–59. Supported by Medicines 

for Malaria Venture, EU INCO-DEV, Wellcome Trust, Thailand 

Tropical Diseases Program and BIOTEC, NSTDA, Thailand .) 

STOICHIOMETRIC SELECTION OF TIGHT- 

BINDING INHIBITORS BY WILD-TYPE AND 

MUTANT FORMS OF MALARIAL (PLASMODIUM 

FALCIPARUM) DIHYDROFOLATE REDUCTASE. 

(NO. 726) 

Kamchonwongpaisan S 1 , Vanichtanankul J 1 , Tarnchompoo B 1 , 

Yuvaniyama J 2 , Taweechai S 1 , Yuthavong Y 1 . 



Pathumthani; 2 Department of Biochemistry, Faculty of Science, 


Key words : dihydrofolate reductase, Plasmodium falciparum, tightbinding 

inhibitors. 


282 

A simple method for screening combinatorial and other 

libraries of inhibitors of malarial (Plasmodium falciparum) 

dihydrofolate reductase (PfDHFR) has been developed, based on 

the affinities of the inhibitors with the enzyme. In the presence of 

limiting amounts of the enzyme, a number of inhibitors in the library 

were bound to extents reflecting the relative binding affinities. 

Following ultrafiltration and guanidine hydrochloride treatment to 

release bound inhibitors, the amounts of free and bound inhibitors 

could be determined by high-performance liquid chromatography 

and liquid chromatography-mass spectrometry. The differences in 

the patterns reflected the binding of high-affinity components 

compared with the other members in the library. A good correlation 

was found between the inhibition constants (K i values) and the extent 

of binding of inhibitors to wild-type, double (C59R+S108N) and 

quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well 

as human DHFR. In addition to identifying lead components of the 

libraries with high affinities (low K i values) and stabilities (low k off 

rates), this simple method also provides an alternative way for quickly 

and accurately calculating enzyme binding affinities of inhibitors 

in combinatorial chemical libraries. 

(Published in Anal Chem 2005; 77: 1222-7. Supported by EU INCO- 

DEV, Wellcome Trust, MMV Program, WHO/TDR, Thailand Tropical 

Diseases Research Program, and BIOTEC, NSTDA, Thailand.) 

EFFECT OF METALLOPROTEASE INHIBITORS 

ON INVASION OF RED BLOOD CELL BY 

PLASMODIUM FALCIPARUM. (NO. 727) 

Kitjaroentham A, Suthiphongchai T, Wilairat P. 



Key words : invasion, metalloprotease inhibitor, Plasmodium 

falciparum. 

For successful invasion, the malaria merozoite needs to 

attach to the red blood cell membrane, undergo reorientation, form 

a junction of the apical end with the host membrane, and internalize. 

Malaria proteases have been implicated in the invasion process, but 

their specific cellular functions remain unclear. To demonstrate the 

involvement of metalloprotease in the process of Plasmodium 

falciparum merozoite entry into host red blood cell, schizont-infected 

red blood cells and parasitophorous vacuolar membrane-enclosed 

merozoite structures were treated with 1,10-phenanthroline, a metal 

chelator, resulting in a reduction of invasion with IC(50) value of 

25 and 29 μM, respectively. Absence of an accumulation of schizont 

stages after treatment with 1,10-phenanthroline indicated that the 

inhibitory effect was not due to suppression of merozoite release 

from red blood cells, but on the invasion step. Although treatment 

with GM6001, a well-known inhibitor of the mammalian matrix 

and disintegrin metalloprotease family, was less effective, 

nevertheless this study points to the importance of metal-requiring 

protease in the process of invasion of host red blood cell by the 

malaria parasite. 

(Published in Acta Trop 2005 Sep 14; [Epub ahead of print]. 

Supported by Thailand Research Fund.) 

MAPPING ANTIMALARIAL PHARMACOPHORES 

AS A USEFUL TOOL FOR THE RAPID DISCOVERY 

OF DRUGS EFFECTIVE IN VIVO : DESIGN, 

CONSTRUCTION, CHARACTERIZATION, AND 

PHARMACOLOGY OF METAQUINE (NO. 728) 

Dascombe MJ 1 , Drew MG 2 , Morris H 3 , Wilairat P 4 , 

Auparakkitanon S 4 , Moule WA 3,5 , Alizadeh-Shekalgourabi S 3,5 , 

Evans PG 2,3 , Lloyd M 3 , Dyas AM 3 , Carr P 5 , Ismail FM 3 . 

1 Faculty of Life Sciences, The University of Manchester, 

Manchester, UK; 2 School of Chemistry, University of Reading, 

Reading, UK; 3 The Medicinal Chemistry Research Group, The 

School of Pharmacy and Chemistry, Liverpool John Moores 

University, Liverpool, UK; 4 Department of Biochemistry, Faculty 

of Science, Mahidol University, Bangkok; 5 Department of 

Chemistry, University of Hertfordshire, Hatfield, UK. 

Key words : antimalarial pharmacophore, metaquine, Plasmodium 

falciparum. 

Resistant strains of Plasmodium falciparum and the 

unavailability of useful antimalarial vaccines reinforce the need to 

develop new efficacious antimalarials. This study details a 

pharmacophore model that has been used to identify a potent, soluble, 

orally bioavailable antimalarial bisquinoline,metaquine (N,N’-bis(7chloroquinolin-4-yl)benzene-1,3-diamine) 

(dihydrochloride), which 

is active against Plasmodium berghei in vivo (oral ID 50 of 25 μmol/ 

kg) and multidrug-resistant Plasmodium falciparum K1 in vitro (0.17 

μM). Metaquine shows strong affinity for the putative antimalarial 

receptor, heme at pH 7.4 in aqueous DMSO. Both crystallographic 

analyses and quantum mechanical calculations (HF/6-31+G) reveal 

important regions of protonation and bonding thought to persist at 

parasitic vacuolar pH concordant with our receptor model. 

Formation of drug-heme adduct in solution was confirmed using 

high-resolution positive ion electrospray mass spectrometry. 

Metaquine showed strong binding with the receptor in a 1:1 ratio 

(log K = 5.7 +/- 0.1) that was predicted by molecular mechanics 

calculations. This study illustrates a rational multidisciplinary 

approach for the development of new 4-aminoquinoline 

antimalarials, with efficacy superior to chloroquine, based on the 

use of a pharmacophore model. 

(Published in J Med Chem. 2005; 48: 5423-36.) 

INDUCTION OF HUMAN CHOLESTGEROL 

7ALPHA- HYDROXYLASE IN HEPG2 CELLS 

BY 2,4,6-TRIHYDROXYACETOPHENONE (NO. 729) 

Charoenteeraboon J 1 , Nithipatikom K 2 , Campbell WB 2 , 

Piyachaturawat P 3 , Wilairat P 1 , Rongnoparut P 1 . 


University, Bangkok; 2 Department of Pharmacology and 

Toxicology, Medical College of Wisconsin, Wisconsin, USA; 

3 Department of Physiology, Faculty of Science, Mahidol 



Key words : cholesterol 7alpha-hydroxylase, HepG2 cell, 2,4,6trihydroxyacetophenone. 



In animal the plasma cholesterol-lowering activity of 2,4,6trihydroxyacetophenone 

(THA) is due to enhanced cholesterol 

7alpha-hydroxylase (CYP7A1) activity. We have examined the effect 

of THA on CYP7A1 activity and mRNA level in HepG2 cells. THA 

stimulated CYP7A1 activity in a concentration- and time-dependent 

manner. After exposure for 24 h, 1 μM THA induced CYP7A1 

activity 160+/-8% and mRNA level 166+/-21% (mean+/-S.E.M.) 

of control. Moreover THA antagonized the inhibitory regulation of 

chenodeoxycholic acid on CYP7A1 mRNA expression. These results 

indicated that THA increases CYP7A1 activity in human HepG2 

cells by stimulating mRNA transcription. 

(Published in Eur J Pharmacol 2005; 515: 43-6. Supported by 

Thailand Research Fund and NIH, USA.) 

CHARACTERIZATION OF DELTAMETHRIN 

RESISTANCE IN FIELD POPULATIONS OF 

AEDES AEGYPTI IN THAILAND. (NO. 730) 

Yaicharoen R 1 , Kiatfuengfoo R 1 , Chareonviriyaphap T 2 , 

Rongnoparut P 3 . 

1 Department of Parasitology, Faculty of Medical Technology, 

Mahidol University, Bangkok; 2 Department of Entomology, 

Faculty of Agriculture, Kasetsart University, Bangkok; 



Key words : Aedes aegypti, deltamethrin, resistance mechanism. 

Five field collections of adult Aedes aegypti mosquitoes 

from different areas in Bangkok and Pathum Thani provinces were 

subjected to susceptibility tests against deltamethrin. Low levels of 

resistance were detected among all populations tested (RR 50 = 8- 

17.2) compared to the susceptible strain, Bora (French Polynesia). 

Among the five populations tested, the BKH (Bang Khen, Bangkok) 

and PSC (Phasicharoen, Bangkok) populations showed a higher level 

of deltamethrin resistance than the other three populations (RR 50 of 

BKH = 17.2, and of PSC = 13.6) and cross-resistance to DDT was 

observed in these strains. Biochemical analysis showed a significant 

elevation of mixed function oxidases enzyme activity in all 

populations. There was an elevation of non-specific esterases in all 

populations except BKL, and there was no consistent association of 

glutathione S-transferases with deltamethrin and DDT resistance, 

although not all populations were bioassayed for DDT. The partial 

cDNA sequence of the para-type voltage-dependent sodium channel 

(IIS4-IIS6) was determined for BKH and PSC populations. Common 

amino acid substitution, leucine to phenylalanine in the IIS6 region, 

found for insects including Anopheles gambiae was not found in 

either the BKH or the PSC populations. However, two other amino 

acid substitutions (proline substituted with serine at position 64 in 

the PSC population and leucine with phenylalanine at position 69 

in the BKH population) were found in the IIS5-IIS6 inter-segment 

region sequenced. The role these substitutions play in target site 

resistance is uncertain at this time. 

(Published in J Vector Ecol 2005; 30: 144-50. Supported by 

Thailand Tropical Diseases Research Program and Commission on 

Higher Education Staff Development Project, Thailand.) 

CYTOCHROME P450 GENES: MOLECULAR 

CLONING AND OVEREXPRESSION IN A 

PYRETHROID-RESISTANT STRAIN OF 

ANOPHELES MINIMUS MOSQUITO. (NO. 731) 

Rodpradit P 1 , Boonsuepsakul S 1 , Chareonviriyaphap T 2 , Bangs 

MJ 3 , Rongnoparut P 1 . 


University, Bangkok; 2 Department of Entomology, Kasetsart 

University, Bangkok; 3 US Naval Medical Research Unit, Jakarta, 

Indonesia. 

Key words : Anopheles minimus, cytochrome P450 monooxygenase, 

deltamethrin. 

We previously determined that physiological resistance in 

a laboratory-selected pyrethroid-resistant Anopheles minimus species 

A Theobald mosquito is associated with increased detoxification 

via a P450-mediated mechanism. A CYP6 gene, CYP6AA3, was 

subsequently cloned and found overexpressed in 2 resistant mosquito 

generations (F13 and F19). We report herein the cloning of CYP6P7 

and CYP6P8 genes with full coding sequences from the same An. 

minimus mosquito colony strain. CYP6P7 and CYP6P8 encode 

proteins, each with 509 amino acids. CYP6P7 had the closest (81%) 

amino acid identity with Anopheles gambiae CYP6P2. CYP6P8 

genes had 79% identity with An. gambiae CYP6P1. Using 

semiquantitative reverse transcription-polymerase chain reaction 

analysis, the mRNA expression level of CYP6P7 presented 

approximately 2- and 4-fold increases in F19 and F25 deltamethrinresistant 

populations, respectively, compared with the parent 

susceptible strain. CYP6P8 mRNA expression levels were not 

significantly different between the 3 filial generations. The 

overexpression of CYP6AA3 mRNA was greater than that of 

CYP6P7 in F19 and F25 resistant populations. The relative increase 

of both CYP6AA3 and CYP6P7 mRNA was correlated with 

increased resistance to deltamethrin in An. minimus. 

(Published in J Am Mosquito Control Assoc 2005; 21: 71-9. 

Supported by Mahidol University, Thailand Research Fund and 

Ministry of University Affairs, Thailand.) 

GENETIC STRUCTURE AND GENE FLOW 

AMONG AEDES AEGYPTI (DIPTERA : 

CULICIDAE) POPULATIONS FROM CENTRAL 

THAILAND. (NO. 732) 

Sukonthabhirom S 1 , Rongnoparut P 2 , Saengtharatip S 3 , 

Jirakanjanakit N 4 , Chareonviriyaphap T 1 . 

1 Department of Entomology, Kasetsart University, Bangkok; 


University, Bangkok; 3 Office of Dengue Control, Department 

of Communicable Disease Control, Ministry of Public Health, 

Nontaburi; 4 Center for Vaccine Development, Institute of Science 

and Technology for Research and Development, Mahidol 

University, Nakhon Pathom. 

Key words : Aedes aegypti, isozymes, gene flow. 

Isozymes from five wild-caught populations of Aedes 

aegypti (L.) were compared using starch gel electrophoresis to 

estimate rates of gene flow between and among geographically close 


284 

mosquito populations. Ae aegypti were collected from five different 

locations in Bangkok, Thailand. One collection was obtained from 

central Bangkok (Huai-Khwang); the other four samples were 

obtained from surrounding areas (Districts Latkrabang, Ratburana, 

Laksi, and Bangkok Noi). Based on 24 loci (17 enzyme systems), 

only minor genetic differentiation was observed between all five 

populations. The highest percentage of polymorphic loci (34.3) was 

observed from the central Bangkok populations; the least percentage 

of polymorphism (20.0) was seen from Laksi (north Bangkok). The 

study indicates that a large effective migration rate exists among all 

five populations. No fixed genetic differences were detected. 

(Published in J Med Entomol 2005; 42: 604-9. Supported by 

Thailand Tropical Diseases Research Program.) 

MORPHOLOGICAL VARIATIONS OF ANOPHELES 

MINIMUS A IN TAK PROVINCE, THAILAND . 

(NO. 733) 

Jaichapor B 1 , Kengluecha A 1 , Rongnoparut P 2 , Rueda LM 3 , Jones 

JW 1 , Sithiprasasna 1 . 

1 Department of Entomology, AFRIMS, Bangkok; 2 Department 

of Biochemistry, Faculty of Science, Mahidol University, 

Bangkok; 3 Walter Reed Biosystematics Unit, Department of 

Entomology, Walter Reed Army Institute of Research, Suitland, 

Maryland, USA. 

Key words : Anopheles minimus, Tak Province, wing morphological 

variation. 

Anopheles minimus Theobald is one of the major vectors 

of malaria throughout the Oriental Region, and it’s complex consists 

of at least 2 sibling species (A and C) in Thailand. This study aimed 

to determined the morphological variations of wings of An. minimus 

A and to clarify the specific status of An. minimus in Ban Khun 

Huay, Ban Pa Dae, and Ban Tham Seau, Mae Sot district, Tak 

Province, Thailand. Anopheline larvae were collected from the fields 

between October 2002 and September 2003, allowed to emerge to adults 

in the laboratory and identified by morphological and molecular 

characterization. About 1,715 of female An. minimus A were separated 

into 8 groups based on their wing scale patterns. Polymerase Chaim 

Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) 

assay (ribosomal DNA ITS2) confirmed the identification of An. 

minimus A in all 8 groups. 


609-15. Supported by TRF/BIOTEC Special Program for Biodiversity 

Research and Training and CNRS France.) 

DIFFERENTIAL LOCALIZATION OF HPV16 E6 

SPLICE PRODUCTS WITH E6-ASSOCIATED 

PROTEIN. (NO. 734) 

Vaeteewoottacharn K 1,2 , Chamutpong S 1 , Ponglikitmongkol M 1 , 

Angeletti PC 2 . 


University, Bangkok; 2 Nebraska Center for Virology, School of 

Biological Sciences, University of Nebraska-Lincoln, Nebraska, 

USA. 

Key words : cellular localization, HPV16 E6, human 

papillomavirus. 

High-risk Human Papillomavirus (HPV) is the etiological 

agent associated with the majority of anogenital cancers. The 

primary HPV oncogenes, E6 and E7, undergo a complex splicing 

program resulting in protein products whose purpose is not fully 

understood. Previous mouse studies have confirmed the existence 

of a translated product corresponding to the E6*I splice product. In 

terms of function, the translated E6*I protein has been shown to 

bind to E6 protein and to E6 associated protein (E6AP). E6*I has 

an inhibitory effect on E6-mediated p53 degradation in E6 expressing 

cells. In order to analyze the relationship between E6*I and fulllength 

E6 in relation to localization, we created a series of green 

fluorescent protein (GFP) fusion products. The localization of these 

proteins with reference to E6AP in vivo remains unclear. Therefore, 

we investigated the cellular distribution of different forms of E6 

with reference to E6AP. E6 and E6*I proteins, expressed from a 

wild type E6 gene cassette, were dispersed in the nucleus and the 

cytoplasm. Whereas, the E6 splice donor mutant (E6MT) was 

primarily localized to the nucleus. E6*I protein and E6AP were 

found to co-localize mainly to the cytoplasm, whereas the colocalization 

of full-length E6 protein and E6AP, if at all, was found 

mainly at the perinuclear region. These results suggest a functional 

relationship between the E6*I and full-length E6 protein which 

correlates with their localization and likely is important in regulation 

of the E6-E6AP complex. 

(Published in Virol J 2005; 2: 50. Supported by Mahidol University, 

Thailand National Science and Technology Agency, and Nebraska 

Center for Virology, USA.) 

ANTI-METASTATIC EFFECTS OF CURCUSONE B, 

A DITERPENE FROM JATROPHA CURCAS. (NO. 735) 

Muangman S, Thippornwong M, Tohtong R. 



Key words : curcusone B, Jatropha curcas, invasion. 

A new approach to cancer therapy in recent years has been 

to target the metastatic process. The anti-metastatic potential of 

curcusone B, a diterpene isolated from Jatropha curcas Linn. 

(Euphorbiaceae), a herbal plant that has been used in traditional 

folk medicine in many tropical countries, was investigated against 

4 human cancer cell lines. Treatment with non-cytotoxic doses of 

curcusone B resulted in a strong reduction of in vitro invasion, 

motility and secretion of matrix-metalloproteinases (MMP) of the 

cancer cells, whereas the ability to adhere to a Matrigel-coated 

surface was variably sensitive to curcusone B treatment. Curcusone 

B, thus, effectively suppresses the metastatic processes at doses that 

are non-toxic to cells, which may be of therapeutic benefit for the 

treatment of metastatic cancers. 

(Published in In Vivo 2005; 19: 265-8.) 




SILENCING OF YELLOW HEAD VIRUS 

REPLICATION IN PENAEID SHRIMP 

CELLS BY DSRNA. (NO. 736) 

Tirasophon W 1 , Roshorm Y 1 , Panyim S 1,2 . 

1 Institute of Molecular Biology and Genetics, Mahidol 

University, Salaya, Nakorn Pathom; 2 Department of 

Biochemistry, Faculty of Science, Mahidol University, Bangkok. 

Key words : Penaeid shrimp, RNAi, yellow head virus. 

RNA interference (RNAi) has been shown to inhibit viral 

replication in some animals and plants. Whether the RNAi is 

functional in shrimp remains to be demonstrated. In vitro transcribed 

dsRNAs of YHV helicase, polymerase, protease, gp116, and gp64 

were transfected into shrimp primary cell culture and found to inhibit 

YHV replication. dsRNA targeted to nonstructural genes (protease, 

polymerase, and helicase) effectively inhibited YHV replication. 

Those targeted structural genes (gp116 and gp64) were the least 

effective. These findings are the first evidence that RNAi-mediated 

gene silencing is operative in shrimp cells. This could be a powerful 

tool for studying gene function and to develop effective control of 

viral infection in shrimp. 

(Published in Biochem Biophys Res Commun 2005; 334: 102-7. 

Supported by Thailand Research Fund.) 

BURKHODERIA PSEUDOMALLEI. (NO. 737) 

Tungpradabkul S. 



Key words : Burkhoderia pseudomallei, genome structure, 

identification. 

Meliodosis undoubtedly qualifies as an emerging infection 

because there is no doubt that it has been recognized more frequently 

during the past two decades, both within established endemic areas 

and elsewhere. This is probably largely because of greater familiarity 

with the organism and the disease it causes and of improvements in 

laboratory and facilities. Our current understanding of the molecular 

structure in the genomic level of B. pseudomallei and other related 

species such as B. cepacia and B. mallei has also increased a possible 

way for rapid diagnosis. However, the culture method remains the 

method of choice, not only because of its reliability, but also because 

of its simplicity and cost. Its only drawback is that it takes up a 

great deal of time before a definitive diagnosis can be made. To 

help speed up the time needed for bacterial identification, a 

combination of the culture method with anyone of the immunological 

and molecular diagnostic methods discussed in this article could be 

used and results evaluated. 

(Published in Encyclopedia of Diagnostic Genomics and 

Proteomics, Marcel Dekker Inc, 2005, pp 165-9. Supported by 

Mahidol University, Thailand Research Fund, National Science and 

Technology Development Agency of Thailand.) 

CITRATE SYNTHASE MUTANTS OF AGROBAC- 

TERIUM ARE ATTENUATED IN VIRULENCE 

AND DISPLAY REDUCED VIR GENE INDUCTION. 

(NO. 738) 

Suksomtip M 1 , Liu P 2 , Anderson T 2 , Tungpradabkul S 1 , Wood 

DW 2 , Nester EW 2 . 


University, Bangkok; 2 Department of Microbiology, University 

of Washington, Seattle, Washington, USA. 

Key words : Agrobacterium tumefaciens, citrate synthase, vir gene. 

A citrate synthase (CS) deletion mutant of Agrobacterium 

tumefaciens C58 is highly attenuated in virulence. The identity of 

the mutant was initially determined from its amino acid sequence, 

which is 68% identical to Escherichia coli and 77% identical to 

Brucella melitensis. The mutant lost all CS enzymatic activity, and 

a cloned CS gene complemented a CS mutation in Sinorhizobium. 

The CS mutation resulted in a 10-fold reduction in vir gene 

expression, which likely accounts for the attenuated virulence. When 

a plasmid containing a constitutive virG [virG(Con)] locus was 

introduced into this mutant, the level of vir gene induction was 

restored to nearly wild-type level. Further, the virG(Con)complemented 

CS mutant strain induced tumors that were similar 

in size and number to those induced by the parental strain. The CS 

mutation resulted in only a minor reduction in growth rate in a 

glucose-salts medium. Both the CS mutant and the virG(Con)complemented 

CS strain displayed similar growth deficiencies in a 

glucose-salts medium, indicating that the reduced growth rate of 

the CS mutant could not be responsible for the attenuated virulence. 

A search of the genome of A. tumefaciens C58 revealed four proteins, 

encoded on different replicons, with conserved CS motifs. However, 

only the locus that when mutated resulted in an attenuated phenotype 

has CS activity. Mutations in the other three loci did not result in 

attenuated virulence and any loss of CS activity, and none were able 

to complement the CS mutation in Sinorhizobium. The function of 

these loci remains unknown. 

(Published in J Bacteriol 2005; 187: 4844-52. Supported by Higher 

Education Staff Development Project, Ministry of Education, 

Thailand and NIH, USA.) 

COMBINATION OF DSB COEXPRESSION AND 

AN ADDITION OF SORBITOL MARKEDLY 

ENHANCED SOLUBLE EXPRESSION OF SINGLE- 

CHAIN FV IN ESCHERICHIA COLI. (NO. 739) 

Sandee D 1 , Tungpradabkul S 2 , Kurokawa Y 3 , Fukui K 1 , Takagi 

M 4 . 

1 Department of Biotechnology, Graduate school of Engineering, 

Osaka University, Osaka, Japan; 2 Department of Biochemistry, 

Faculty of Science, Mahidol University, Bangkok; 3 Department 

of Bioscience, Fukui Prefectural University, Fukui, Japan; 

4 School of Materials Science, Japan Advanced Institute of 

Science and Technology, Ishikawa, Japan . 

Key words : Dsb protein, single-chain Fv, soluble expression. 


286 

Many eukaryotic proteins have been produced successfully in 

Escherichia coli. However, not every gene can be expressed 

efficiently in this organism. Most proteins, especially those with 

multiple disulfide bonds, have been shown to form insoluble protein 

or inclusion body in E. coli. An inactive form of protein would 

require an in vitro refolding step to regain biological functions. In 

this study, we described the system for soluble expression of a singlechain 

variable fragment (scFv) against hepatocellular carcinoma 

(Hep27scFv) by coexpressing Dsb protein and enhancing with 

medium additives. The results revealed that overexpression of 

DsbABCD protein showed marked effect on the soluble production 

of Hep27scFv, presumably facilitating correct folding. The optimal 

condition for soluble scFv expression could be obtained by adding 

0.5M sorbitol to the culture medium. The competitive enzyme-linked 

immunosorbent assay (ELISA) indicated that soluble scFv expressed 

by our method retains binding activity toward the same epitope on 

a hepatocellular carcinoma cell line (HCC-S102) recognized by intact 

antibody (Ab) (Hep27 Mab). Here, we report an effective method 

for soluble expression of scFv in E. coli by the Dsb coexpression 

system with the addition of sorbitol medium additive. This method 

might be applicable for high-yield soluble expression of proteins 

with multiple disulfide bonds. 

(Published in Biotechnol Bioeng 2005; 91: 418-24. Supported by 

Science and Technology Incubation Program in Advanced Region, 

Japan Science and Technology Corporation.) 

CONSTRUCTION OF SCFV DERIVED FROM A 

TUMOR-ASSOCIATED MONOCLONAL ANTIBODY 

HAVING TUMORICIDAL ACTIVITY ON HUMAN 

HEPATOCELLULAR CARCINOMA. (NO. 740) 

Tungpradabkul S 1 , Sandee D 1 , Puthong S 2 , Laohathai K 2 . 


University, Bangkok; 2 Institute of Biotechnology and Genetic 

Engineering, Chulalongkorn University, Bangkok. 

Key words : single-chain variable fragment, tumor-associated 

monoclonal antibody, tumoricidal effect. 

A mouse monoclonal antibody (Mab-HepTAA43), 

classified as an anti-tumor-associated antigen, was raised by 

immunizing BALB/c mice with the Thai human hepatocellular 

carcinoma S102 (HCC-S102) cell line cells using hybridoma 

techniques. The Mab-HepTAA43 reacted with and markedly 

inhibited the growth of human hepatocellular carcinoma cell lines 

as well as a tumor mass in an animal model. Human hepatoma 

transplanted into nude mice did not show metastasis after 20 

injections amounting to a total of about 4mg of the Mab over 1month 

period. A single-chain variable fragment (scFv molecule 

derived from the Mab was constructed by phage display method. 

DNA sequence analysis of the active variable regions of both heavyand 

light-chains of the cDNA clone was subsequently performed. 

The scFv43 molecule contains a V(L) kappa type and a unique V(H) 

sequence having 88% amino acid homology to that of Mab-MAK B 

raised against tumor-associated antigen. Immunohistochemical 

staining on frozen sections of paired hepatoma (NCI-I) and normal 

liver tissue from the same individual showed that both scFv43 and 

Mab-HepTAA43 antibodies reacted with hepatoma but not with 

normal liver tissue. The results suggest that scFv43 may be useful 

in the immunotherapy of hepatocellular carcinoma. 

(Published in Mol Immunol 2005; 42: 713-9. Supported by Mahidol 

University, Chulalongkorn University; Thailand National Science 

and Technology Development Agency.) 

PURIFICATION AND CHARACTERISATION OF 

KERATINASE FROM A THERMOTOLERANT 

FEATHER-DEGRADING BACTERIUM. (NO. 741) 

Suntornsuk W 1 , Tongjun J 1 , Onnim P 1 , Oyama H 2 , 

Ratanakanokchai K 3 , Kusamran T 4 , Oda K 2 . 

1 Department of Microbiology, Faculty of Science, King 

Mongkut’s Institute of Technology Thonburi, Bangkok; 

2 Department of Applied Biology, Faculty of Textile Science, 

Kyoto Institute of Technology, Kyoto, Japan; 3 Department of 

Biochemical Technology, School of Bioresources and Technology, 

King Mongkut’s Institute of Technology Thonburi, Bangkok; 




Key words : Bacillus licheniformis, keratinase, thermotolerance 

Isolation and identification of a thermotolerant featherdegrading 

bacterial strain from Thai soil as well as purification and 

properties of its keratinase were investigated. The thermotolerant 

bacterium was identified as Bacillus licheniformis. The keratinase 

was purified to homogeneity by three-step chromatography. The 

purified enzyme exhibited a high specific activity (218 U mg -1 ) with 

86-fold purification and 25% yield. The enzyme was monomeric 

and had a molecular mass of 35 kDa. The optimum pH and 

temperature for the enzyme were 8.5 and 60°C, respectively. The 

enzyme activity was significantly inhibited by PMSF and partly 

inhibited by EDTA and iodoacetamide, but was stimulated by metal 

ions. It hydrolysed soluble proteins with a relative activity of 4– 

100% and insoluble proteins, including keratins, with a relative 

activity of 3–35%. Therefore, the enzyme could improve the 

nutritional value of meat- and poultry-processing wastes containing 

keratins, collagen and gelatin. 

(Published in World J Microbiol Biotech 2005; 21: 1111– 

7.Supported by Japan Society for Promotion of Science and National 

Research Council of Thailand.) 

QUINONE REDUCTASE INDUCERS IN 

AZADIRACHTA INDICA A. JUSS FLOWERS, 

AND THEIR MECHANISMS OF ACTION. (NO. 742) 

Sritanaudomchai H 1 , Kusamran T 1 , Kuakulkiat W 2 , 

Bunyapraphatsara N 2 , Hiransalee A 3 , Tepsuwan A 4 , Kusamran 

WR 4 . 


University, Bangkok; 2 Department of Pharmacognosy, Faculty 

of Pharmacy, Mahidol University, Bangkok; 3 Department of 

Medical Science, Ministry of Public Health, Bangkok; 4 Chemical 

Carcinogenesis Section, Research Division, National Cancer 

Institute, Bangkok. 

We have previously shown that the flowers of neem tree 

(Azadirachta indica A. Juss, family Meliaceae), Thai variety, 

strongly induced the activity of glutathione S-transferase (GST) while 

resulting in a significant reduction in the activities of some 

cytochrome P(450)-dependent monooxygenases in rat liver, and 

possess cancer chemopreventive potential against chemicallyinduced 

mammary gland and liver carcinogenesis in rats. In the 

present study, 2 chemicals possessing strong QR inducing activity 

were fractionated from neem flowers using a bioassay based on the 

induction of QR activity in mouse hepatoma Hepa 1c1c7 cultured 

cells. Spectroscopic characteristics revealed that these compounds 



were nimbolide and chlorophylls, having CD (concentration required 

to double QR specific activity) values of 0.16 and 3.8 mug/ml, 

respectively. Nimbolide is a known constituent of neem leaves, but 

was found for the first time here in the flowers. Both nimbolide and 

chlorophylls strongly enhanced the level of QR mRNA in Hepa 1c1c7 

cells, as monitored by northern blot hybridization, indicating that 

the mechanism by which these constituents of neem flowers induced 

QR activity is the induction of QR gene expression. These findings 

may have implication on cancer chemopreventive potential of neem 

flowers in experimental rats previously reported. 

(Published in Asian Pac J Cancer Prev 2005; 6: 263-9. Supported 

by Terry Fox Run Research Fund, Oncological Society of Thailand 

and Department of Medical Services, Ministry of Public Health.) 

USE OF A REMOTE SENSING-BASED GEO- 

GRAPHIC INFORMATION SYSTEM IN THE 

CHARACTERIZING SPATIAL PATTERNS FOR 

ANOPHELES MINIMUS A AND C BREEDING 

HABITATS IN WESTERN THAILAND (NO. 743) 

Rongnoparut P 1 , Ugsang DM 2 , Baimai V 3 , Honda K 2 , 

Sithiprasasna R 4 . 


University, Bangkok; 2 RS & GIS FoS, Asian Institute of 

Technology, Pathum Thani; 3 Center for Vectors and Vectorborne 

Diseases, Faculty of Science, Mahidol University, 

Bangkok; 4 Department of Entomology, US Army Medical 

Component, AFRIMS, Bangkok. 

Key words : Anopheles minimus, breeding habitat, remote sensing 

A remote sensing (RS)-based Geographic Information 

System (GIS) was used to characterize the breeding habitats of 

Anopheles minimus species A and C in five different districts of 

Kanchanaburi Province in western Thailand. The GIS and RS were 

used to monitor the area for the presence and absence of An. minimus 

A and C in five major land areas, forest, agriculture, urban, water 

and bare land. The results show that An. minimus A survives both 

in dense canopy forest and in open fields where agriculture is 

dominant. A scatter plot of land-use/land-cover for An. minimus, 

considering proximities to the forest and proximities to agriculture, 

suggests that An. minimus A has a wider habitat preference, ranging 

from dense canopy forest to open agricultural fields. A scatter plot 

for An. minimus C, on the other hand, showed a narrow habitat 

preference. A scatter plot for proximities performed on separate 

populations of An. minimus species A, one in the north and the other 

in the south, showed that there was an association in the northern 

population with the forest and in the southern population with 

agricultural areas. There were no statistically significant differences 

in the scatter plot of proximities to urban areas and water bodies 

with the An. minimus A north, south and An. minimus C. LANDSAT 

TM satellite data classification was used to identify larval habitats 

that produce An. minimus A and C and analyze proximities between 

land-use/land-cover habitats and locations of larval habitats. An. 

minimus A has a wide habitat preference, from dense canopy forest 

to open agricultural fields, while An. minimus C has a narrow habitat 

preference. 


1145-52. Supported by TRF/BIOTEC Special Program for Diversity 

Research and Training and CNRS, France.) 

HIGHER PLANT-LIKE FLUORESCENCE 

INDUCTION AND THERMOLUMINESCENCE 

CHARACTERISTICS IN CYANOBACTERIUM, 

SPIRULINA MUTANT DEFECTIVE IN PQH2 

OXIDATION BY CYTB6/F COMPLEX. (NO. 744) 

Ruengjitchatchawalya M 1 , Kovacs L 2 , Mapaisansup T 1 , Sallai 

A 2 , Gombos Z 2 , Ponglikitmongkol M 3 , Tanticharoen M 1,4 

1 School of Bioresources and Technology, King Mongkut’s 

University of Technology Thonburi, Bangkok; 2 Institute of Plant 

Biology, Biological Research Center, Hungarian Academy of 

Sciences, Hungary; 3 Department of Biochemistry, Faculty of 

Science, Mahidol University, Bangkok; 4 National Center for 

Genetic Engineering and Biotechnology, National Science and 

Technology Development Agency, Pathumthani. 

Characterization of the photosynthetic electron transport 

in a mutant of Spirulina platensis, generated by chemical 

mutagenesis, demonstrated that the electron transfer from the 

plastoquinone (PQ) to cytochrome b6/f was slowed. 

Thermoluminescence (TL) measurements suggested the presence of 

reversed energy flow via PQ, which resulted in an emergence of the 

plant-like after-glow TL band at 45 degrees C that could be enhanced 

by the transthylakoidal pH gradient and could be eliminated by an 

uncoupler, FCCP. The localization of the changes in the electron 

transport of the mutant cells measured by various methods revealed 

that the re-oxidation of the PQ pool is hampered in the mutant 

compared to the wild-type cells. The reduction in energy migration 

was localized between PQ and PS I reaction centers. 

(Published in J Plant Physiol 2005; 162: 1123-32. Supported by 

BIOTEC, NSTDA and Hungarian Science Foundation.) 

AN EASY METHOD FOR GENERATING DELETION 

MUTANTS IN AGROBACTERIUM TUMEFACIENS 

USING A SIMPLE REPLACEMENT VECTOR. 

Suksomtip M, Tungpradabkul S. 

(NO. 745) 



Key word : Agrobacterium tumefaciens, citrate synthase gene, 

deletion mutants 

We have developed a method to make precise mutations in 

the A. tumefaciens genome at frequencies high enough to allow direct 

identification of mutants by PCR or other screening methods rather 

than by selection. This method utilized a novel pUC18-based gene 

replacement vector that was used as a donor plasmid carrying the 

desired mutation in the target cell. Two sites of single-crossover 

occurred resulting in efficient replacement of the wild type allele on 

the chromosome with the modified sequence. The usefulness of 

this method was demonstrated by making deletion mutants in citrate 

synthase genes of A. tumefaciens. 

(Published in ScienceAsia 2005; 31: 349-57. Supported by 

Commission on Higher Education, Ministry of Education.) 


288 

EFFECTS OF WATER-SOLUBLE ANTIOXIDANTS 

AND MAPKK/MEK INHIBITOR ON CURCUMIN- 

INDUCED APOPTOSIS IN HL-60 HUMAN 

LEUKEMIC CELLS. (NO. 746) 

Banjerdpongchai R 1 , Wilairat P 2 . 

1 Department of Biochemistry, Faculty of Medicine, Chiang Mai 

University, Chiang Mai; 1 Department of Biochemistry, Faculty 

of Science, Mahidol University, Bangkok. 

Key words : apoptosis, curcumin, water-soluble antioxidant 

Curcumin is the main biologically active phytochemical 

compound in turmeric. It has been shown to have anticarcinogenic 

activity. The aims of the study were to identify the mechanism of 

apoptosis of HL-60 human promyelocytic leukemic cells induced 

by curcumin and to determine the effects of water-soluble 

antioxidants, ascorbic acid, Trolox (a water-soluble form of vitamin 

E), glutathione (GSH) and N-acetylcysteine (NAC) on this process. 

HL-60 cells were incubated with curcumin for 24 h and apoptotic 

cells were quantitated by flow cytometry following staining with 

annexin V-FITC and propidium iodide. Curcumin-treated HL-60 

cells produced reactive oxygen species as detected by the 

dichlorofluorescein fluorescent assay. Apoptosis occurred via the 

mitochondria pathway as curcumin reduced mitochondrial 

membrane potential in a dose-dependent manner. In the presence 

of 10 μM curcumin, vitamin C (56 nM-5.6 μM) inhibited apoptosis 

of HL-60 cells; GSH at low concentration (1 μM) reduced apoptosis 

but had no effect at higher concentrations (10, 100 μM); and Trolox 

and NAC at 10 and 100 μM, respectively, enhanced apoptosis, but 

this effect was abolished at higher concentration (1 mM) of NAC. 

MAPKK/MEK inhibitor PD98059, enhanced curcumin-induced HL- 

60 apoptotic cell death. 

(Published in Asian Pac J Cancer Prev 2005; 6: 282-5. Supported 

by the Thailand Research Fund.) 

PHOTOINHIBITION AND RECOVERY IN 

OXYGENIC PHOTOSYNTHESIS : MECHANISM 

OF A PHOTOSYSTEM-II DAMAGE AND REPAIR 

CYCLE. (NO. 747) 

Yokthongwattana K 1 , Melis A 2 . 


University, Bangkok; 2 Department of Plant and Microbial 

Biology, University of California, Berkeley, California, USA. 

This Chapter provides highlights on the mechanism of a 

photosystem II (PS II) damage and repair cycle in chloroplasts. 

Photo-oxidative damage to the PS II reaction center is a phenomenon 

that occurs in every organism of oxygenic photosynthesis. Through 

the process of evolution, an elaborate repair mechanism was devised, 

one that rectifies this presumably unavoidable and irreversible 

photoinhibition and restores the PS II charge separation activity. 

The repair process entails several enzymatic reactions for the selective 

removal and replacement of the inactivated D1/132 kD reaction 

center protein (the chloroplast-encoded psbA gene product) from 

the massive (>1,000 kD) H 2 O-oxidizing and O 2 -evolving PS II 

holocomplex. This repair process is unique in the annals of biology, 

nothing analogous in complexity and specificity has been reported 


in other biological systems. Elucidation of the repair mechanism 

may reveal the occurrence of hitherto unknown regulatory and 

catalytic reactions for the selective in situ replacement of specific 

proteins from within multi-protein complexes. This may not only 

have significant applications in photosynthesis and agriculture but 

also in medicine and other fields. . 

(Published in Demmig-Adams B, Adams III WW, Mattoo AK (eds) 

Photoprotection, Photoinhibition, Gene Regulation and 

Environment. Advances in Photosynthesis Series, Springer, The 

Netherlands 2005; pp. 175-91. Supported by USDA National 

Research Initiative.) 

ENZYMATIC PROPERTIES OF WILD-TYPE 

AND ACTIVE SITE MUTANTS OF CHITINASE 

A FROM VIBRIO CARCHARIAE, AS REVEALED 

BY HPLC-MS (NO. 748) 

Suginta W 1 , Vongsuwan A 1 , Songsiriritthigul C 1,2 , Svasti J 3 , Prinz 

H 4 . 

1 School of Biochemistry, Institute of Science, Suranaree 

University of Technology, Nakorn Ratchasima; 2 National 

Synchrotron Research Center, Nakorn Ratchasima; 3 Department 

of Biochemsitry and Center for Protein Structure and Function, 

Faculty of Science, Mahidol University, Bangkok; 4 Max-Planck 

Institute for Molecular Physiology, Dortmund, Germany. 

Key words: active site, HPLC-MS, Vibrio carchariae 

The enzymatic properties of chitinase A from Vibrio 

carchariae have been studied in detail by using combined HPLC 

and electrospray MS. This approach allowed the separation of a 

and β anomers and the simultaneous monitoring of 

chitooligosaccharide products down to picomole levels. Chitinase 

A primarily generated β-anomeric products, indicating that it 

catalyzed hydrolysis through a retaining mechanism. The enzyme 

exhibited endo characteristics, requiring a minimum of two 

glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed 

that chitinase A had greater affinity towards higher M r 

chitooligomers, in the order of (Glc-NAc) 6 >(GlcNAc) 4 > (GlcNAc) 3 , 

and showed no activity towards (Glc-NAc) 2 and pNP-GlcNAc. This 

suggested that the binding site of chitinase A was probably composed 

of an array of six binding subsites. Point mutations were introduced 

into two active site residues – Glu315 and Asp392 – by site-directed 

mutagenesis. The D392N mutant retained significant chitinase 

activity in the gel activity assay and showed »20% residual activity 

towards chitooligosaccharides and colloidal chitin in HPLC-MS 

measurements. The complete loss of substrate utilization with the 

E315M and E315Q mutants suggested that Glu315 is an essential 

residue in enzyme catalysis. The recombinant wild-type enzyme 

acted on chitooligosaccharides, releasing higher quantities of small 

oligomers, while the D392N mutant favored the formation of 

transient intermediates. Under standard hydrolytic conditions, all 

chitinases also exhibited transglycosylation activity towards 

chitooligosaccharides and pNP-glycosides, yielding picomole 

quantities of synthesized chitooligomers. The D392N mutant 

displayed strikingly greater efficiency in oligosaccharide synthesis 

than the wild-type enzyme. 

(Published in FEBS J 2005; 272: 3376-86. Supported by Thailand 

Research Fund, Suranaree University of Technology and German 

Academic Exchange Service.) 



MOLECULAR HYDROGEN PRODUCTION BY A 

THERMOTOLERANT RUBRIVIVAX GELATINOSUS 

USING RAW CASSAVA STARCH AS AN ELECTRON 

DONOR (NO. 749) 

Mahakhan P 1 , Chobvijuk C 1 , Ngmjarearnwong M 1 , 

Trakulnalermsai S 1 , Bucke C 2 , Svasti J 3 , Kanlayakrit W 4 , 

Chitradon L 1 . 

1 Department of Microbiology, Faculty of Science, Kasetsart 

University, Bangkok; 2 School of Biosciences, University of 

Westminster, London, UK; 3 Department of Biochemistry, 

Faculty of Science, Mahidol University, Bangkok; 4 Department 

of Biotechnology, Faculty of Agro-industry, Kasetsart University, 

Bangkok. 

Key words : hydrogen production, Rubrivivax gelatinosus, raw 

cassava starch. 

Thirteen strains of phototrophic purple non-sulfur bacteria 

selected from 226 isolates showed the ability to digest raw cassava 

starch at elevated temperature, 40°C, under illuminated anaerobic 

conditions. A selected strain, designated as SB24, produced more 

amylolytic enzyme activities toward raw and cooked cassava starch, 

when grown in cooked starch than when grown in raw starch. SB24 

showed photoproduction of molecular hydrogen using raw cassava 

starch as an electron donor, when incubated with illumination under 

anaerobic conditions at 40°C. In small scale (23 mL) culture, SB24 

produced hydrogen from raw cassava starch after 20-24 h of 

cultivation, with a 3-fold higher rate of H 2 production and a 3-fold 

higher total accumulation of hydrogen at 72 h, as compared to when 

it used malate as an electron donor. In the larger scale reactor 

containing a 5.5 liter culture of SB24, hydrogen was produced at an 

earlier time using raw cassava starch than using malate. In addition, 

the highest rate of H 2 production (38.79 mL H2 liter culture -1 h -1 ) by 

SB24 with raw cassava starch was 7 times higher than that with 

malate, while the total volume of H 2 accumulated with raw cassava 

starch (4.61 liters of H 2 at 90 h) was almost two-fold higher than 

with malate. Raw starch from rice, sticky rice, corn and mungbean, 

could also be used as electron donors for H 2 production by SB24 at 

40°C. From morphological and biochemical characteristics, 

comparison of 16S rDNA sequence, and comparative studies of the 

bacterial characteristics, SB24 was found to be a thermotolerant 

anoxygenic phototrophic purple non-sulfur bacterium, Rubrivivax 

gelatinosus. 

(Published in ScienceAsia 2005; 31: 415-24. Supported by Thailand 

Research Fund and KURDI.) 

PROTEOMIC PROFILING OF CHOLANGIO- 

CARCINOMA CELL LINE TREATED WITH 

POMIFERIN FROM DERRIS MALACCENSIS. (NO. 750) 

Svasti J 1,2 , Srisomsap C 1 , Subhasitanont P 1 , Keeratichamroen 

S 1 , Chokchaichamnankit D 1 , Ngiwsara L 1 , Chimnoi N 1 , 

Pisutjaroenpong S 1 , Techasakul S 1,3 , Chen ST 4 . 

1 Chulabhorn Research Institute, Bangkok; 2 Department of 

Biochemistry, Faculty of Science, Mahidol University, Bangkok; 

3 Department of Chemistry, Faculty of Science, Kasetsart 

University, Bangkok; 4 Institute of Biological Chemistry, 

Academia Sinica, Taipei, Taiwan. 

Key words : cholangiocarcinoma, pomiferin, proteomic profiling 

Pomiferin, a prenylated isoflavonoid from Derris 

malaccensis with strong anti-fungal and anti-oxidant activities, 

showed cytotoxic activity towards human cholangiocarcinoma cells 

(HuCCA-1), with IC 50 of 0.9 mg/mL. Pomiferin caused apoptosis, 

detectable by DNA fragmentation. Two- dimensional PAGE showed 

increased expression of 12 proteins, namely glucose-regulated 

protein 75 (grp 75), calcyclin (S100A6), degraded cytokeratin 19, 

ATP synthase D, ribosomal protein P0, degraded cytokeratin 18 (two 

spots pI/MW 6.03/29.9 and pI/MW 4.66/21.5), cofilin, annexin A1, 

triose phosphate isomerase, peroxiredoxin-1, calgizzarin, and 

profilin. In contrast, cytokeratins (CK) 7, 18 and 19 were downregulated, 

and were shown by 1-DE immunodetection to be degraded. 

(Published in Proteomics 2005; 5: 5404-9. Supported by 

Chulabhorn Research Institute and Thailand Research Fund.) 

THE MOLECULAR BASIS OF MUCOPOLYSAC- 

CHARIDOSIS TYPE I IN TWO THAI PATIENTS. 

(NO. 751) 

Cairns JRH 1,2 , Keeratichamroen S 1 , Sukcharoen S 3 , 

Champattanachai V 1 , Ngiwsara L 1 , Lirdprapamongkol K 1 , 

Liammongkolkul S 4 , Srisomsap C 1 , Surarit R 1,5 , Wasant P 4 , Svasti 

J 1,3 . 

Laboratory of Biochemistry, Chulabhorn Research Institute, 

Bangkok; Schools of Chemistry and Biochemistry, Institute of 

Science, Suranaree University of Technology, Nakorn 

Ratchasima; Department of Biochemistry and Center for Protein 

Structure and Function, Faculty of Science, Mahidol University, 

Bangkok; Department of Physiology and Biochemistry, Faculty 

of Dentistry, Mahidol University, Bangkok. 

Key words : molecular basis, mucopolysaccharidosis type I, Thai 

Two Thai patients diagnosed with Hurler syndrome 

(mucoploysaccharidosis type I, MPS I) were found to have no 

detectable α-iduronidase (E.C. 3.2.1.76) activity in leukocytes, while 

normal Thai children all had significant activity, with a mean of 

135 ± 30 nmol/mg/28 h. One patient was heterozygous for A75T 

(311G>A) and S633L (198C>T) mutation, previously reported to 

cause MPS I, together with 9 other heterozygous polymorphisms 

also found in normal controls. The other patient had the previously 

described frameshift mutation 252insert C and a new nonsense 

mutation E299X (983G>T). 


1308-12. Supported by Chulabhorn Research Institute and Thailand 

Research Fund.) 

MY EXPERIENCES AS AN IUB TRAVEL FELLOW. 

Svasti J. 

(NO. 752) 



Key words: IUB, travel fellow 


290 

I was awarded an IUB Travel Fellowship in 1976, the second 

time they were awarded. The fellowship helped me to see how 

science works and to make contacts that lasted throughout my life. 

It was also the start of my involvement in international scientific 

organizations, which has been an important aspect of my work ever 

since. Travel grants to attend meetings provide much needed 

encouragement and are vitally important in developing young 

scientists. 

(Published in IUBMB Life 2005; 57: 255.) 

THIRTY YEARS OF SCIENCEASIA, JOURNAL 

OF THE SCIENCE SOCIETY OF THAILAND. (NO. 753) 

Svasti MRJ. 



Key words : Journal of the Science Society of Thailand; ScienceAsia 

ScienceAsia celebrates its 30 th anniversary as the research 

journal of the Science Society of Thailand. It fulfils the criteria of 

the Thailand Research Fund, the National Science and Technology 

Development Agency, and the Commission for Higher Education 

as an international journal published in Thailand. Some 90% of the 

Editorial Board members are full professors. About 15-20% of the 

papers submitted comes from overseas corresponding authors, while 

the remainder comes from more than 20 universities and government 

agencies in Thailand. 

(Published in ScienceAsia 2005; 31: 1-3.) 

EXPRESSION PURIFICATION CRYSTALLI- 

ZATION AND PRELIMINARY CRYSTALLO- 

GRAPHIC ANALYSIS OF CHINASE A FROM 

VIBRIO CARCHARIAE (NO. 754) 

Songsiriritthigul C 1,2 , Yuvaniyama J 3 , Robinson RC 4 , Vongsuwan 

A 1 , Prinz H 5 , Suginta W 1 . 

1 School of Biochemistry, Suranaree University of Technology, 

Nakorn Ratchasima; 2 National Synchrotron Research Center, 

Nakorn Ratchasima; 3 Department of Biochemistry and Center 

for Protein Structure and Function, Faculty of Science, Mahidol 

University, Bangkok; 4 Institute of Molecular and Cell Biology, 

Proteos, Singapore; 5 Max-Planck Institute for Molecular 

Physiology, Dortmund, Germany. 

Key words : chitinase A, crystallization, Vibrio carchariae 

Chitinase A of Vibrio carchariae was expressed in 

Escherichia coli M15 host cells as a 575-amino-acid fragment with 

full enzymatic activity using the pQE60 expression vector. The 

yield of the highly purified recombinant protein was approximately 

70 mg per litre of bacterial culture. The molecular mass of the 

expressed protein was determined by HPLC/ESI–MS to be 63 770, 

including the hexahistidine tag. Crystals of recombinant chitinase 

A were grown to a suitable size for X-ray structure analysis in a 

precipitant containing 10% (v/v) PEG 400, 0.1 M sodium acetate 

pH 4.6 and 0.125 M CaCl 2 . The crystals belonged to the tetragonal 

space group P422, with two molecules per asymmetric unit and unit 

cell parameters a = b = 127.64, c = 171.42 angstrom . A complete 

diffraction data set was collected to 2.14 angstrom resolution using 

a Rigaku/MSC R-AXIS IV ++ detector system mounted on an RU- 

H3R rotating-anode X-ray generator. 

(Published in Acta Cryst 2005; F61: 895-8. Supported by Suranaree 

University of Technology and National Synchrotron Research 

Center.) 

CO-EXPRESSION OF BACILLUS THURINGIENSIS 

CRY 4BA AND CYT2AA2 IN ESCHERICHIA COLI 

REVEALED HIGH SYNERGISM AGAINST AEDES 

AEGYPTI AND CULEX QUINQUEFASCIATUS 

LARVAE. (NO. 755) 

Promdonkoy B 1 , Promdonkoy P 1 , Panyim S 2 






Key words : Bacillus thuringiensis, crystal toxin, synergism 

Cry4Ba is a delta-endotoxin produced by Bacillus 

thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic deltaendotoxin 

produced by B. thuringiensis subsp. darmstadiensis. 

Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti 

larvae (LC 50 = 140 ng ml -1 ) but virtually inactive to Culex 

quinquefasciatus larvae. Cyt2Aa2 expressed in E.coli exhibited 

moderate activity against A. aegypti and C. quinquefasciatus larvae 

with LC 50 values of 350 and 250 ng ml -1 , respectively. Co-expression 

of both toxins in E. coli dramatically increased toxicity to both A. 

aegypti and C. quinquefasciatus lalrvae (LC 50 = 7 and 20 ng ml -1 , 

respectively). This is the first report to demonstrate that Cry4Ba 

and Cyt2Aa2 have high synergistic activity against C. 

quinquefasciatus larvae. 

(FEMS Microbiol Lett. 2005 Sep 14; [Epub ahead of print]). 

A SIMPLE DUAL SELECTION FOR 

FUNCTIONALLY ACTIVE MUTANTS OF 

PLASMODIUM FALCIPARUM DIHYDRO- 

FOLATE REDUCTASE WITH IMPROVED 

SOLUBILITY (NO. 756) 

Japrung D. 1 , Chusacultanachai S. 1 , Yuvaniyama J. 2,3 , Wilairat 

P. 2 , Yuthavong Y. 1 




Mahidol University, Bangkok; 3 Center for Excellence in Protein 

Structure and Function, Faculty of Science, Mahidol University, 

Bangkok. 

Key words : active mutant, dihydrofolate reductase, dual selection 

Sufficient solubility of the active protein in aqueous solution 

is a prerequisite for crystallization and other structural studies of 

proteins. In this study, we shave developed a simple and effective 



in vivo screening system to select for functionally active proteins 

with increased solubility by using Plasmodium falciparum 

dihydrofolate reductase (pfDHFR), a by using Plasmodium 

falciparum dihydrofolate reductase (pfDHFR), a well-know malarial 

drug target, as a model. Prior to the dual selection process, pfDHFR 

was fused to green fluorescent protein (GFP), which served as a 

reporter for solubility. The fusion gene was used as a template for 

construction of mutated DNA libraries of pfDHFR. Two amino acids 

with large hydrophobic side chains (Y35 and F37) located on the 

surface of pfDHFR were selected for site-specific mutagenesis. 

Additionally, the entire pfDHFR gene was randomly mutated using 

error-prone PCR. During the first step of the dual selection, mutants 

with functionally active pfDHFR were selected from two libraries 

by using bacterial complementation assay. Fluorescence signals of 

active mutants were subsequently measured and five mutants with 

increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G 

+ F37L and Y35L + F37R from the site–specific mutant library and 

K27E from the random mutant library, were recovered. The mutants 

were expressed, purified and characterized as monofunctional 

pfDHFR following excision of GFP. Our studies indicated that all 

mutant pfDHFRs exhibited kinetic properties similar to that of the 

wild-type protein. For comparison of properties similar to that of 

the wild-type protein. For comparison of protein solubility, the 

maximum concentrations of mutant enzymes prior to aggregation 

were determined. All mutants selected in this study exhibited 3- to 

6-fold increases in protein solubility compared with the wild-type 

protein, which readily aggregated at 2 mg/ml. The dual selection 

system we have developed should be useful for engineering 

functionally active protein mutants with sufficient solubility for 

functional/structural studies and other applications. 

(Published in Protein Eng Des Sel 2005; 18: 457-64.) 

ELECTRON MICROSOPIC STUDIES ON 

LOCALIZATION OF LEAD IN ORGANS 

OF TYHPA ANGUSTIFOLIA GROWN ON 

CONTAMINATED (NO. 757) 

Thanawan Panich-pat 1 , Peerasak Srinives 2 ,*, Maleeya 

Kruatrachue 3 , Prayad Pokethitiyook 3 ,Suchart Upatham 4 , and 

Guy R. Lanza 5 

1 Faculty of Liberal Arts and Science, Kasetsart University, 

Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand; 2 

Department of Agronomy, Faculty of Agriculture, Kasetsart 

University, Kamphaeng Saen Campus, Nakhon Pathom 73140, 

Thailand; 3 Department of Biology, Faculty of Science, Mahidol 

University, Bangkok 10400, Thailand. 4 Faculty of Science, 

Burapha University, Chonburi 20130, Thailand; 5 Environmental 

Sciences Program, University of Massachusetts, Amherst, MA 

01003, USA. * E-mail: agrpss@ku.ac.th 

Key words : Typha angustifolia, narrow-leaved cattail, lead 

accumulation 

A greenhouse study was conducted to observe the 

localization of lead in narrow-leaved cattail, Typha angustifolia. 

Light and transmission electron microscopic studies were performed 

on root, rhizome and leaf of the cattail grown in control (75 kg dry 

weight of soil with no added lead) and in the same weight of soil 

amended with 20,000 mg lead nitrate. At 15 and 90 days after 

planting, most lead was accumulated in root cells around vacuoles 

and slowly transported to leaves. In the lead-contaminated soil, parts 

of the root cell wall were damaged at the end of the experiment. 

Lead was deposited in the rhizome near the cell wall. Similar deposits 

were observed in the roots and rhizomes suggesting that lead was 

transported and localized in a similar area, whereas the leaf cells 

accumulated lead in the chloroplasts. 

(ScienceAsia, 2005, 31, 49-53;ADB) 

INVASIVE PHYTOPHAGOUS PESTS ARISING 

THROUGH A RECENT TROPICAL EVOLU- 

TIONARY RADIATION : THE BACTROCERA 

DORSALIS COMPLEX OF FRUIT FLIES (NO. 758) 

Anthony R. Clarke, 1 Karen F. Armstrong, 2 Amy E. Carmichael, 

1 John R. Milne, 3 s. Raghu, 4 George K. Roderick, 5 and David K. 

Yeates 6 

1 School of Natural Resource Sciences. Queensland University 

of Technology, Brisbane, Qld 4001, Australia; E-mail: 

a.clarke@qut.edu.au; ae.cannichael@qut.edu.au ; 2 Soil, Plant 

and Ecological Sciences Division, Lincoln University, 

Canterbury, New Zealand; E-mail: annstron@lincoln.ac.nz ; 3 

Department of Biology, Faculty of Science, Mahidol University, 

Bangkok 10400, Thailand; E-mail: frjnn@mucc.mahidol.ac.th 

; 4 Alan Fletcher Research Station, Queensland Department of 

Natural Resources & Mines and CRC for Australian Weed 

Management, Sherwood, Qld 4075, Australia; E-mail: 

raghu.s@nnn.qld.gov.au ; 5 Environmental Science, Policy and 

Management, Division of Insect Biology, University of 

California, Berkeley, California 94720-3112. E-mail: 

roderick@nature.berkeley.edu ; 6 Australian National Insect 

Collection, CSIRO Entomology, Canberra ACT 2601, Australia; 

E-mail: david.yeates@csiro.au. 

Key words diagnostics, larval host range, invasion biology. 

The Bactrocera dorsalis complex of tropical fruit flies 

(Diptera: Tephri- tidae: Dacinae) contains 75 described species, 

largely endemic to Southeast Asia. Within the complex are a small 

number of polyphagous pests of international significance, 

including B. dorsalis sensu stricto, B. papayae, B. carambolae, and 

B. philip- pinensis. Most species within the complex were described 

in 1994 and since then substantial research has been undertaken in 

developing morphological and molecular diagnostic techniques for 

their recognition. Such techniques can now resolve most taxa 

adequately. Genetic evidence suggests that the complex has evolved 

in only the last few million years, and development of a phylogeny 

of the group is considered a high priority to provide a framework 

for future evolutionary and ecological studies. As model systems, 

mating studies on B. dorsalis S.s. and B. cacuminata ha! ve substantially 

advanced our understanding of insect use of plant-derived 

chemicals for mating, but such studies have not been applied to 

help resolve the limits of biological species within the complex. 

Although they are commonly regarded as major pests, there is little 

published evidence documenting economic losses caused by flies 

of the B. dorsalis complex. Quantification of economic losses 

caused by B. dorsalis complex species is urgently needed to prioritize 

research for quarantine and management. Although they have been 

documented as invaders, relatively little work has been done on the 

invasion biology of the complex and this is an area warranting further 

work. 

(Annu. Rev. Entomol 2005, 50:293-319) 


292 

HISTOPHATHOLOGICAL ALTERATIONS IN THE 

EDIBLE SNAIL, BABYLONIA AREOLATA (SPOTTED 

BABYLON), IN ACUTE AND SUBACUTE CADMIUM 

(NO. 759) 

P. Tanhan, 1 P. Sretarugsa, 2 P. Pokethitiyook, 1 M. Kruatrachue, 1 

E. S. Upatham 3 

1 Department of Biology, Faculty of Science, Mahidol University, 

Bangkok 10400, Thailand; 2 Department of Anatomy, Faculty of 

Science, Mahidol University, Bangkok 10400, Thailand; 3 Faculty 

of Science, Burapha University, Chonburi 20131, Thailand. 

Key words: cadmium; BABYLONIA AREOLATA; histopathology 

Histopathological alterations in 6- to 8-month-old juvenile 

spotted babylon, Babylonia areolata, from acute and subchronic 

cadmium exposure were studied by light microscopy. The 96-h LC 50 

value of cadmium for B. areolata was found to be 3.35 mg/L, and 

the maximum acceptable toxicant concentration (MATC) was 1.6 

mg/L. Snails were exposed to 3.35 and 0.08 mg/L (5% of MATC) of 

cadmium for 96 h and 90 days, respectively. After exposure the gill, 

the organs of the digestive system (proboscis, esophagus, stomach, 

digestive gland, and rectum), and the foot were analyzed for cadmium 

accumulation. The results showed that most digestive organs had a 

high affinity for cadmium. The main target organ was the stomach, 

which could accumulate on average 1192.18 _g/g dry weight of 

cadmium. Cadmium was shown to accumulate to a lesser extent in 

the digestive gland, gill, rectum, esophagus, proboscis, and foot. 

Histopathological alterations were observed in the gill and digestive 

organs (proboscis, esophagus, stomach, and rectum). The study 

showed that the stomach and gill were the primary target organs of 

both acute and subchronic exposure. Gill alterations included 

increased size of mucous vacuoles, reduced length of cilia, dilation 

and pyknosis of nuclei, thickening of basal lamina, and accumulation 

of hemocytes. The epithelial lining of the digestive tract showed 

similar alterations such as increased size of mucous vacuoles, 

reduced length of cilia, and dilation of nuclei. In addition, 

fragmentation of the muscle sheath was observed. 

(Environ. Toxicol 2005. 20:142-149; ADB) 

DISPERSAL OF THE DENGUE VECTOR AEDES 

AEGYPTI WITHIN AND BETWEEN RURAL 

COMMUNITIES (NO. 760) 

Laura C. Harrington, Thomas W. Scott, Kriangkrai 

Lerdthusnee, Russell C. Coleman, Adriana Costero, Gary G. 

Clark, James J. Jones, Sangvorn Kitthavee, Pattamaporn 

Kittayapong, Ratana Sitthiprasasna and John D. Edman 

Department of Entomology, Cornell University, Ithaca, New 

York; Department of Entomology, University of California, 

Davis, California; Department of Entomology, Armed Forces 

Research Institute for Medical Sciences, Bangkok, Thailand; 

Medical Entomology Section, Laboratory of Parasitic Diseases, 

National Institutes of Allergy and Infectious Diseases, National 

Institutes of Health, Bethesda, Maryland; Dengue Branch, 

Centers for Disease Control and Prevention, San Juan, Puerto 

Rico; Center for Vectors and Vector-Borne Diseases and 

Department of Biology, Faculty of Science, Mahidol University, 


Knowledge of mosquito dispersal is critical for vector-borne disease 

control and prevention strategies and for understanding population 

structure and pathogen dissemination. We determined Aedes aegypti 

flight range and dispersal patterns from 21 mark-release-recapture 

experiments conducted over 11 years (1991-2002) in Puerto Rico 

and Thailand. Dispersal was compared by release location, sex, age, 

season, and village. For all experiments, the majority of mosquitoes 

were collected from their release house or adjacent house. Intervillage 

movement was detected rarely, with a few mosquitoes moving 

a maximum of 512 meters from one Thai village to the next. Average 

dispersal distances were similar for males and females and females 

released indoors versus outdoors. The movement of Ae. aegypti 

was not influenced by season or age, but differed by village. Results 

demonstrate that adult Ae. aegypti disperse rel! atively short 

distances, suggesting that people rather than mosquitoes are the 

primary mode of dengue virus dissemination within and among 

communities. 

(Am. J. Trop. Med. Hyg. 2005, 72(2): 209-220.) 

ACUTE AND SUBCHRONIC TOXICITY OF 

LEAD TO THE SPOTTED BABYLON, BABYLONIA 

AREOLATA (NEOGASTROPODA, BUCCINIDAE) 

(NO. 761) 

P. Supanopas 1 , P. Sretarugsa 2 *, M. Kruatrachue l , P. 

Pokethitiyook 1 and E. S. Upatham 3 

1 Departments of Biology and 2 Anatomy, Faculty of Science, 

Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand; 3 

Faculty of Science, Burapha University, Chonburi 20130, 

Thailand 

Key words : Babylonia areolata, lead, acute toxicity 

The acute and subchronic toxicity of lead was determined 

in the SpOtted babylon, Babylonia areolata. The 96-h static bioassay 

was conducted to estimate the median lethal concentration (LC 50 ). 

The snails were exposed to lead nitrate (Pb[NO 3 ] 2 ). The LC 50 values 

for 24, 48, 72. and 96 h were 29.31. 14.64. 12.44, and 10.50 mg Pb/ 

L, respectively. In the subchronic experiment, the snails were exposed 

to 0.5 mg Pb/L (10% MATC, the maximum acceptable toxicant 

concentration) of lead nitrate for 3 mo. Lead accumulation was 

found in different organs with the greatest accumulation in the 

stomach and lesser in the esophagus, gill, rectum, digestive gland, 

proboscis, and foot. The histopathologic alterations in the digestive 

system and gills of B. areolata were studied by light microscopy. 

The general tissue alterations were decrease in length of cilia, 

decrease in acidophilic granules, slight distension of nuclei, and the 

los! s of heterochromation. There were increases of mucous vacuoles, 

damaged ciliated cells, and enlargement of vacuoles. 

(J. of Shellfish Res. 2005, 24(1): 91-98; ADB.) 




TOXICITY AND ACCUMULATION AND OF LEAD 

AND CADMIUM IN THE FILAMENT GREEN 

ALGA CLADOPHORA FRACTA (O.F. MÜLLER 

ex VAHL) KÜTZING: A LABORATORY STUDY 

(NO. 762) 

Chantana Lamai 1 , Maleeya Kruatrachue 2 , Prayad 

Pokethitiyook 1 , E. Suchat Upatham 2 , and Varasaya 

Soonthornsarathool 1 

1 Department of Biology. Faculty of Science. Mahidol University, 

Bangkok 10400. Thailand. 2 Faculty of Science. Burapha 

University, Chonburi 20130. Thailand. E-mail: 

scmkt@mahidol.ac.th 

Key words : Cladophora fracta, lead, cadmium. 

The toxicity and accumulation of the heavy metals, lead 

(Pb) and cadmium (Cd) in a common filamentous green alga, 

Cladophora fracta, were studied. C. fracta were cultured in a 

modified Chu No. 10 medium, which was supplemented with 

5,10,20,40 or 80 mg/L of Pb orO.5,1, 2,4 or8 mg/L of Cd, and were 

separately harvested after 2, 4, 6 and 8 days. The toxicity symptoms 

of Pb and Cd to C. fracta showed damage and reduced number of 

chloroplasts, disintegrated cell wall and death. There were significant 

decreases-in the relative growth and total chlorophyll content when 

the exposure time and concentration were increased. The 

accumulation study showed that there were significant increases of 

metal levels in algal tissue when the exposure time and concentration 

were increased. The bioconcentration factor (BCF) of Pb was higher 

than that of Cd at the same duration, suggesting that the accumulation 

potential of C. fracta for Pb was higher than that for Cd. 

(ScienceAsia, 2005, 31: 121-127; ADB.) 

HISTOP A THOLOGICAL RESPONSES OF NILE 

TILAPlA, OREOCHROMIS NILOTICUS TO 

CONTAMINATED SEDIMENTS OF MAE KLONG 

RIVER TRIBUTARIES, THAILAND. (NO. 763) 

P. Peebua, Kruatrachue, P. Pokethitiyook, P. Kosiyachinda, and 

W. T rinachartvanit. 

Department of Biology; Faculty of Science, Mahidol University, 

Bangkok 10400, Tahiland. E-mail: scmkt@nlahidol.ac.th 

Living organisms in environment polluted with toxicants 

could likely be used as biomarkers. Nile tilapia (OreochrO111is 

Niloticus), a bottom feeder in Mae Klong River, Southwestern 

Tl1ailand, polluted by heavy metals and organochlorine 9 

compounds, might be a useful tool for this purpose. Therefore, one 

of the objectives of the present study was to quantify Endosulfan, 

and heavy metals in sediment samples collected from the Mae Klong 

River tributaries. Another objective was to investigate the 

contamination-related histopathology and pollutant accumulation 

in Nile tilapia exposed experimentally to these sediments. Five 

stations on tributaries of the Mae Klong River were chosen for 

sediment sample collections. Nile tilapia (10-12 g in weight) were 

exposed to these sediments for one month. Fish organs (gill and 

muscle) and sediments were analyzed for Endosulfan and heav)’ 

metals. Endosulfan was analyzed according to US EPA RCRA SW- 

846 method 3620 and 8080A. Heavy metals were analyzed according 

to US EP A RCRA SW-846 method 6020. In addition, the 

histopathological conditions also were determined to link to assess 

relationships if existed. The results showed abnormalities of gills 

(lamellar cell hyperplasia and fusion), liver (vacuolated hepatocytes 

and pyknotic nucleus), and kidney (dilation of Bowman’s space, 

and hyaline droplet accumulation). No recognizable changes were 

observed in muscle tissue. Endosulfan concentrations varied among 

sampling sites, but the residues were not detected in fish tissues at 

the minimum detectable limit (0.005 ppb). Pb and Cr were detected 

in sediments and fish tissues. Even though, histopathological changes 

were found, no strong relationship between polluted xenobiotics and 

histopathological conditions could be confirmed statistically. 

Seasonal and sampling site variations may play important roles. 

(12 th Internatinal Symposium of Toxicity Assessment, Skaithos 

Island, Breec, June 12-17, 2005; ADB.) 

COMPARING IN VITRO TOXICITY OF 

AGROCHEMICALS CONT AMINATING 

RESERVOIRS WITH LAKE MANAGEMENT 

HERBICIDES (NO. 764) 

Trinachartvanit. W. 1 , Freeman, J. L 2 , Francis, 8.M 3 , Rayburn, 

A.L. 4 


Rama 6 Rd., Payathai, Bangkok, Thailand, Phone: (662) 201- 

5380 email: scwtc@mahidol.ac.th ; 2 Department of Crop 

Sciences, University of Illinois, 320~, 1201 W Gregory, Urbana, 

IL 61801, Phone: (217) 244-6727 E-mail: mcalistr@.uiuc.edu ; 

3 Department of Entomology, University of Illinois, 677 Morrill 

Hall, Urbana, IL, (217) 333-5136 E-mail: bfrancis@uiuc.edu ; 

4 Department of Crop Sciences, University of Illinois, 320 ERML, 

1201 W. Gregory, Urbana, IL 61801, Phone: (217) 333-4374, Email: 

araybum@uiuc.edu 

Modem agricultural practices have placed the agricultural 

community in a position of depending on agrochemicals such as 

herbicides. Through the use of herbicides, farmers have been able 

to reduce tillage practices, thereby reducing soil erosion. While 

reducing soil erosion is important to the environment, agrochemical 

use is not without negative environmental consequences. The use 

of agrochemicals has resulted in these chemicals polluting the 

surrounding waterways thus contaminating wetlands and ultimately 

river and lake systems. It is common to find levels of herbicides 

contaminating most aquatic environments in agricultural areas. This 

results in levels of specific herbicides in aquatic environments that 

are now known to cause damage to animal chromosomes. While 

much controversy has existed with respect to determining acceptable 

levels of agrochemicals contaminating these aquatic environments, 

no studies to date have compared agrochemicals with chemicals 

that are being used in the management of lakes and reservoirs. An 

agrochemical may indeed have the potential for toxicity. However, 

when compared to chemicals approved for use in lakes and reservoirs, 

the risk may be so small as to really be no risk at all. An agrochemical 

(atrazine) and chemicals approved for lake management (diquat and 

fluridone) were compared using cytotoxicity and whole cell 

clastogenicity to detennine which of the chemicals has the greatest 

potential to damage cells and chromosomes. Chinese Hamster Ovary 

cells were exposed to chemicals at various levels. The chemicals 

were rank ordered by their ability to induce cell and chromosome 

damage. A lake management herbicide, diquat, was found to be the 

most cytotoxic and genotoxic herbicide over an agrochemical, 

atrazine. 

(12 th International Symposium on Toxicity Assessment, Skaithos 

Island, Greec, June 12-17, 2005.) 


294 

A CYTOLOGICAL STUDY OF BLACK FLY 

SIMULIUM SIAMENSE (DIPTERA: SIMULIIDAE) 

(NO. 765) 

Unchalee Lualon, Chaliow Kuvangkadilok and Visut Baimai 

Department of Biology, Faculty of Science, Mahidl University, 

Bangkok, 10400 

The polytene chromosomes of the Simulium siamensis from 

7 sites collected in western, northern Thailand were analyzed. At 

the least two cytotypes are recognized based primarily on two fixed 

inversions (IS-2 and IS-2,3) in the IS arm. Cytotype A represent 

two populations from Kaeng Krachan National Park, Petchaburi 

province. All males of cytotype A were IS-2 homozygotes (IS2-2) 

whereas all females were IS2,3 heterozygotes (IS-st/2,3). Therefore 

there is indication of sex-linkage associated with inversion sequence 

in this cytotype. Cytotype B consisting of three populations from 

Mae Hong Son province and two populations from Chaiyaphum 

province, showed nine floating inversions (IS-1, IS-4, IS-5, IL-1, 

IIL-2, IIL-3, IIIS-1 and IIIL-1) in five chromosome arms with low 

frequencies ranging from 0.01-0.27. All inversions in all populations 

except for the Huay Yuak population conformed to Hardy-Weinberg 

equilibrium (p>0.05). 

(14 th International Symposium on Genetics, Genetics: Fromn Basics 

to Molecular Technology: March 11-13, 2005: BRT.) 

CYTOGENETICS OF BLACK FLY SIMULIUM 

TANI (DIPTERA : SIMULIIDAE) FROM 


Ubon Tangkawanit 1 , Chaliow Kuvangkadilok 1 , Peter H. Adler 2 

and Visut Baimai 1 

1 Depart ment of Biology, Faculty of Science, Mahidol University, 

Bangkok, Thailand, 10400; 2 Department of Entomology, 

Clemson University, South Carolina, USA. 

The plytene chromosomes of the Simulium tani from 5 sites 

collected in northern eastern and southern Thailand were analyzed. 

At least two cytotypes are recognized based primarily on one fixed 

inversion (IIIL-5,6) in the IIIL arm. Cytotype A represents one 

population from Morkpha waterfall, Doi Suthep-Pui National Park, 

Chiang Mai province which is monomorphic. S. tani cytotype B 

consists of one population from southern Thailand (Muang Tuad 

waterfall, Surat Thani province) and three populations from eastern 

Thailand (Chanthaburi and Rayong provinces). All individuals of 

cytotype B are homozygous for IIIL-5,6. They also showed six 

floating inversion (IS-5, IS-6, IL-1, IIP-1, IIIL-7, and IIIL-10) in 

five chromosome arms with low frequencies ranging from 0.02-0.43. 

All inversion in all populations except for the Muang Tuad 

population conformed to Hardy-weinberg equilibrium (p>0.05). 




CYTOTAXONOMY OF BLACK FLIES (DIPTERA: 

SIMULIIDAE) FROM THAILAND (NO. 767) 

Chaliow Kuvangkadilok 1 Chainarong Boonkemtong 2 Suwannee 

Phayuhasena 1 and Visut Baimai 1 

1 Department of Biology , Faculty of Science , Mahidol University, 

Bangkok, 10400; 2 National Center for Genetic Engineering and 

Biotechnology 113 Thailand Science Park, Phaholyothin Rd., 

Klong 1 , Klong Luang Pathumthani, 12120. 

Polytene chromosomes of eighteen Simulium species 

belonging to eight species-groups within four subgenera, i.e., 

subgenera Gomphostilbia, Nevermannia, Simulium and 

Montisimulium are described and the standard maps for the speciesgroups 

are presented. All Simulium species have a haploid number 

of three chromosomes (n=3) with decreasing in length from 

chromosomes to chromosomes ²²². The banding patterns and the 

positions of landmarks of the polytene chromosomes are species 

specific which can be used to identify the species especially the 

morphologically similar species. Eight species have low frequencies 

of paracentric inversions with no indication of sex linkage. This 

result showed no evidence of a sibling species complex within any 

taxon. Further cytological studies should provide more useful 

knowledge of species complexes of these Simulium species. 



SPECIES DIVERSITY, DISTRIBUTION AND 

PARASITES OF BLACK FLY LARVAE (DIPTERA: 

SIMULIIDAE) IN NORTHERN THAILAND (NO. 768) 

Sanae Jitklang 1 Chailow Kuvangkadilok 1 , Peter H.Adler 2 and 

Visut Baimai 1 


Bangkok, 10400; 2 Department of Entomology, Clemson 

University, South Carolina, USA. 

Species diversity , species distribution and parasites of black 

fly larvae collected from 10 northern province of Thailand in rainy 

season, winter and summer are presented. Based on the external 

morphological characters and polytene chromosome analyses, a total 

of 27 black fly species was identified. The abundance of twenty-six 

Simulium species was found in winter. However, twenty-four and 

twenty-three species were also presented in rainy season and summer, 

respectively. Additionally, these species had different distribution. 

Some species werw widely distributed in many provinces while a 

few species was restricted to one province. Moreover, preliminary 

study showed three types of parasites, i., nematodes, microsporidia 

and fungus in some Simulium larvae. 





PHYLOGEOGRAPHY OF BLACK FIY SIMULIUM 

TANI (DIPTERA : SIMULIIDAE) INFERRED 

FROM MITOCHONDRIAL DNA SEQUENCE 

(NO. 769) 

Pairot Pramual 1 Chaliow Kuvankadilow 1 Visut Baimai 1 and 

Catherine Walton 2 


Bangkok, 10400; 2 Faculty of Life Sciences, University of 

Manchester, Oxford Road M13 9PT England 

Phylogeography of 147 individuals from 16 populations of 

the black fly Simulium tani from Thailand was inferred using 

cytochrome oxidase ² gene of mitochondrial DNA. The mtDNA 

revealed high genetic differentiation between the major regions of 

north, east and central/south Thailand. Mismatch distributions 

indicate population expansions during the mid-Pleistocene and the 

late-Pleistocene suggesting that current population structure and 

diversity may be due to the species response to Pleistocene climatic 

fluctuations. Cytological investigation revealed that populations from 

the south and east have a fixed chromosomal inversion difference 

from the north and central. Since these populations also share 

ecological characteristics it suggests the inversion is involved in 

ecological adaptation. 



PHENOTYPIC AND GENOTYPIC COMPARISONS 

REVEAL A BROAD DISTRIBUTION AND 

HETEROGENEITY OF HEMOLYSIN BL GENES 

AMONG BACILLUS CEREUS ISOLATES (NO. 770) 

Suwicha Thaenthanee 1 , Amy C. Lee Wong 2 , Watanalai 

Panbangred 1 

1 Department of Biotechnology, Faculty of Science, Mahidol 

University, 2 Food Research Institute, Department of Food 

Microbiology and Toxicology, University of Wisconsin, 1925 

Willow Drive, Madison, USA. 

Key words : Hemolysin BL; Bacillus cereus; Heterogeneity, PCR 

The presence of hemolysin BL (HBL; components L 2 , L 1 

and B) –encoding genes (hblC, hblD, and hblA) from 339 Bacillus 

cereus strains isolated in Thailand was determined. PCR analysis 

showed that all three hbl genes were detected in 222 strains (65.5%). 

Two one or no hbl genes were detected in 3(0.9%), 6 (1.8%) and 

108 (31.8%) strains, respectively. Among the 222 strains in which 

all three hbl genes were detected 210 (61.9%) displayed 

discontinuous hemolysis (DH) characteristic of HBL producers, 

while 12 (3.5%) showed continuous hemolysis (CH) on sheep blood 

agar. Among strains in which none of the hbl genes was detected 

97 (28.6%) displayed CH while 11 (3.2%) did not show hemolytic 

activity. Three strains in which two hbl genes were detected showed 

CH hblC was present in five of six strains where only one hbl gene 

was detected and all of them (designated SS-00-15, TG-00-06, TG- 

00-14, F-00-25, and NR-01-49) showed DH. The Hpall restriction 

profiles for PCR fragments amplified from the hblC-A region in 

these five strains using hblC forward (FHC) and hblA reverse (RHA 2 ) 

primers displayed heterogeneous patterns, which indicated sequence 

variation. Western blot analysis using polyclonal antibodies (Pab) 

raised against HBL components purified from strain F837/76 showed 

that three of the five strains produced all three but not L 2 . The 

production of L 2 by these five strains was further analyzed using the 

Oxoid RPLA test. Three strains gave high titers (>64) whereas strains 

TG-00-06 and TG-00-14 showed lower titers of 16 and 32, 

respectively. The data show that HBL-encoding genes are widely 

distributed among B. cereus isolated in Thailand and there is high 

degree of heterogeneity in both the genes and proteins. This is the 

first report of a B cereus strain showing DH in which all three hbl 

genes and their proteins were not detected by both PCR primers and 

antibodies derived from prototype and type strains. The data also 

suggest that the L 2 component from strains TG-00-06 and TG-00- 

14 may be antigenically very different from that of most B. cereus 

isolates. 

(International Journal of Food Microbiology (2005)) 

CONSTRUCTION OF THAI WORDNET LEXICAL 

DATABASE FROM MACHINE READABLE 

DICTIONARIES (NO. 771) 

Patanakul Sathapornrungkij and Charnyote 

Pluempitiwiriyawej 

Department of Computer Science, Faculty of Science, Mahidol 

University. patanakul@yahoo.com, cccpt@mahidol.ac.th 

Key words : Lexical Databases, WordNet 

We describe a method of constructing Thai WordNet, a 

lexical database in which Thai words are organized by their 

meanings. Our methodology takes WordNet and LEXiTRON 

machine-readable dictionaries into account. The semantic relations 

between English words in WordNet and the translation relations 

between English and Thai words in LEXiTRON are considered. Our 

methodology is operated via WordNet Builder system. This paper 

provides an overview of the WordNet Builder architecture and reports 

on some of our experience with the prototype implementation. 

(Proceeding of the 10 th Machine Translation Summit, pp. 87-92, 

September 12-16, 2005, Phuket, Thailand.) 

ROBOT ARM CONTROLLING BY USING THAI 

VOICE COMMANDS (NO. 772) 

Pornpanomchai, C., Saengsopee T. and Wongseree T. 


University. E-mail : cccpp@mahidol.ac.th 

Key words : Human voice commands, Robotic control, Thai speech 

recognition, Feature Extraction 

This research proposed a robot arm controlling by using 

real time Thai voice commands. The paper separated into three parts: 

The first part described motivation, Thai speech recognition 

technique and literature reviews. The second part demonstrated 

system model and implementation. The final part showed the 

experimental result and conclusion. The accuracy of the study is 

around 91.67% in the quiet room, 89.17% in an office environment 

and 65.83% in a noisy room. 


296 

MULTI-VERSION AND EVOLUTION SUPPORT 

FOR MULTIDIMENSIONAL DATABASE SCHEMA 

Jarernsri L. Mitrpanont and Somchart Fugkeaw 

(NO. 773) 


University ccjlm@mahidol.ac.th, somchartf@yahoo.com 

Key words : Multidimensional schema, Schema Evolution, Schema 

Versioning Technique, Multi-Schema Version 

This paper proposes an evolution approach for 

multidimensional database (MDB) schemata based on objectedoriented 

and schema versioning technique. A schema evolution 

framework and two versioning algorithms, namely, Forward Schema 

Versioning algorithm and Backward Schema Versioning Algorithm 

are proposed to support the MDB schema changes and schema 

version control respectively. Our approach satisfies the full scale 

support in both forward schema evolution and backward schema 

versioning. Finally, the prototype system called SEMAN (Schema 

Evolution MANager) is developed to handle the MDB schema 

modification as well as to control the multi-schema versions based 

on our proposed model. 

(Proceedings of the 23 rd IASTED International Multi-Conference 

on Databases and Applications, pp. 151-156, February 14-16, 2005, 

Innsbruck, Austria.) 

CHARACTERIZATION OF SAMADHI STATE 

DURING MEDITATION BY HEART RATE 

VARIABILITY ANALYSIS (NO. 774) 

Sukanya Phongsuphap 1 , Yongyuth Phongsuphap 2 , Pakorn 

Chantanamasta 3 , Chidchanok Lursinsap 4 

1 Department of Computer Science, Faculty of Science, Mahidol 

University ccsps@mahidol.ac.th ; 2 Health Care Reform Project, 

National Health Security Office ; 3 Department of Medicine, 

Faculty of Medicine Ramathibodi Hospital, Mahidol University; 

4 Department of Mathematics, Faculty of Science, Chulalongkorn 

University, 

Key words: Biosignal Processing, Heart Rate Variability, Meditation 

The main objective of this work is to investigate the heart 

rate variability spectral parameters including the very-low frequency 

power (VLF), the low frequency power (LF), and the high frequency 

power (HF) for determining the Samadhi state during meditation. 

The experiment was performed on 5 subjects in the experimental 

group and 20 subjects in the control group. During 5-month period 

of data collection, we obtained 133 and 117 segments of RR-interval 

data in the Samadhi state and the normal state respectively. Results 

showed that a combination of VLF, LF, and HF had a high capability 

for discriminating the Samadhi state and the non-Samadhi state. 

We obtained an accuracy of 96.8% by Fisher discriminant function 

analysis. The characteristic patterns of the Samadhi state are as 

follows. The VLF was very low, while the HF was higher than normal. 

The LF was remarkably high. In particular, a norrowed band with 

high amplitude peak around the frequency 0.1 Hz appeared. In 

contrast, during ordinarily quiet sitting, the VLF was very high, while 

the HF was low, and the LF was relatively lower than the Samadhi 

state. The results indicated that Samadhi state might have the 

following health benefits: resetting baroreflex sensitivity and 

increasing parasympathetic tone. 

(Proceeding of the fifth International Workshop on biosignal 

Interpretation, pp. 139-142, September 6-8, 2005, Tokyo, JAPAN. 

The Thailand Research Fund (MRG4780135) 

PERFORMANCE STUDY AND DEVELOPMENT 

STRATEGIES ON THE SENDER-INITIATED 

MULTICAST (NO. 775) 

Visoottiviseth Vasaka 1 , Kido Hiroyuki 2 , Iida Katsuyoshi 2 , 

Kadobayashi Youki 2 , and Yamaguchi Suguru 2 

1 Computer Science Department, Mahidol University, Thailand 

E-mail : ccvvs@mahidol.ac.th ; 2 Graduate School of Information 

Science, Nara Institute of Science and Technology, Japan E-mail: 

{hiro-kid, katsu, youki-k, suguru}@is.naist.jp 

Key words : Routing, Multicast, Small Group Communication, 

Forwarding States Reduction 

Although IP Multicast offers efficient data delivery for large 

group communications, the most critical issue delaying widespread 

deployment of IP Multicast is the scalability of multicast forwarding 

state as the number of multicast groups increases. Sender-Initiated 

Multicast (SIM) was proposed as an alternative multicast forwarding 

scheme for small group communications with incremental 

deployment capability. The key feature of SIM is in its Preset mode 

with the automatic SIM tunneling function, which maintaining 

forwarding information states only on the branching routers. To 

demonstrate how SIM increases scalability with respect to the 

number of groups, in this paper we evaluate the proposed protocol 

both through simulations and real experiments. As from the network 

operator’s point of view, the bandwidth consumption, memory 

requirements on state-and-signaling per session in routers, and the 

processing overhead are considered as evaluation parameters. Finally, 

we investigated the strategies for incremental deployment. 

(The Institute of Electronics, Information and Communication 

Engineers (IEICE) Transactions on Communication, VOL.E88-B, 

No.4, Special Section on Internet Technology V, pp. 1395-1402, 

April 2005.) 

A DELAY-DIFFERENTIAL EQUATION MODEL 

OF THE FEEDBACK-CONTROLLED HYPOTHA- 

LAMUS-PITUITARY-ADRENAL AXIS IN HUMANS 

Lenbury Y., Pornsaward P. 


(NO. 776) 

Department of Mathematics, Faculty of Science, Mahidol 

University, Bangkok 10400, Thailand. E-mail 

scylb@mahidol.ac.th., ppornsup@hotmail.com 

Key words : cortisol secretion, delay-feedback controlled system, 

Hopf bifurcation, nonlinear model 

The present work develops and analyses 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 a qualitative and 

mathematical frame work for a better understanding of the delayed 

responsive mechanisms as well as the dynamic variations in different 

pathological situations. 

(Mathematical Medicine and Biology, 22(1)(2005) 15-33.) 

MATHEMATICAL MODELS FOR PRESSURE 

CONTROLLED VENTILATION OF OLEIC ACID- 

INJURED PIGS (NO. 777) 

Crooke P.S. 1 , Kongkul K. 2 , Lenbury Y. 2 , Adams A.B. 3 , Carter 

C.S. 3 , Marini J.J. 3 , Hotchkiss J.R. 3 

1 Department of Mathematics, Vanderbilt University, Nashville, 

TN 37240, USA. E-mail : pscrooke@math.vanderbilt.edu; 

2 Department of Mathematics, Faculty of Science, Mahidol 

University, Bangkok 10400, Thailand. E-mail : 

konvika@tsu.ac.th, scylb@mahidol.ac.th; 3 Pulmonary and 

Critical Care Medicine, Regions Hospital and University of 

Minnesota, St. Paul MN 55101 USA. E-mail : 

hotchkissjr@upmc.edu 

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) lunginjured 

pigs. Experimental data are used to find parameters in the 

mathematical models and were collected in two forms. Firstly, the 

Pe-V curves for healthy and lung injured pigs were constructed; 

these dta are used to compute compliance functions for each animal. 

Secondly, dynamic data from pressure controlled ventilation for a 

variety of applied pressures are 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 Pe-V curves and the resistive pressures 

during inspiration change from a linear relation Pr = RQ to nonlinear 

relation Pr = RQ where Q 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 22 (2005) 99-112) 

DELAY EFFECT IN MODELS OF POPULATION 

GROWTH (NO. 778) 

Giang D.V. 1 , Lenbury Y. 2 , Seidman T.I. 3 

1 Hanoi Insititue of Mathematics, 18 Hoang Quoc Viet, 10307 

Hanoi, Vietnam, E-mai dvgiang@math.ac.vn ; 2 Department of 

Mathematics, Faculty of Science, Mahidol University, Bangkok 

10400, Thailand. E-mail : scylb@mahidol.ac.th; 3 Department 

of Mathematics and Statistics, University of Maryland Baltimore 

County, Baltimore, MD 21250 USA. E-mail 

Seidman@math.umbc.edu 

Key words : Delay differential equations, Comparison theorem Oneparameter 

semi-group 

Frist we systematize earlier results on the global stability 

of the model x + μx = f(x(‘ – τ)) of population growth. Second, we 

investigate the effect of delay on the asymptotic behavior when the 

nonlinearity f is a unimodal function. Our results can be applied to 

several population models [Elements of Mathematical Ecology, 2001 

[7]; Appl. Anal. 43 (1992) 109-124; Math. Comput. Modelling, in 

press; Funkt. Boil. Med. 256 (1982) 156-164; Math. Comput. 

Modelling 35 (2002) 719-731; Mat. Stos. 6 (1976) 25-40] because 

the function f does not need to be monotone or differentiable. 

Specifically, our results generalize earlier result of [Delay Differential 

Equations with Applications in Population Dynamics 1993], since 

our function f may not be differentiable. 

(Journal of Mathematical Analysis and Applications 305 (2005) 

631-643.) 

THREE-DIMENSION MAGNETOTELLURIC 

INVASION : DATA SPACE METHOD (NO. 779) 

Siripunvaraporn W. 1 , Egbert G. 2 , Lenbury Y. 3 , Uyeshima M. 4 

1 Department of Physics, Faculty of Science, Mahidol University, 

Bangkok 10400, Thailand. E-mail : scwsp@mahidol.ac.th; 

2 College of Oceanic and Atmospheric Sciences, Oregon State 

University, Corvallis OR 97331, USA; 3 Department of 

Mathematics Faculty of Science, Mahidol University, Bangkok 

10400, Thailand. E-mail scylb@mahidol.ac.th; 4 Earthquake 

Research Insitute, University of Tokyo, 1-1-1 Yayoi, Bunkyoku, 

Tokyo 113-0032, Japan. 

Key words : Magnetotellurics, Data-space method, 3D inversion, 

Occam’s inversion 

A three-dimensional magnetotelluric (MT) minimum 

structure inversion algorithm has been developed based on a dataspace 

variant of the Occam approach. Computational costs associated 

with construction and inversion of model-space matrices make a 

model-space Occam approach to 3D MT inversion impractical. These 

difficulties are overcome with a data-space approach,where matrix 

dimensions depend on the size of the data set, rather than the number 

of model parameters. With the transformation to data space it 

becomes feasible to invert modest 3D MT data sets on a PC. To 

reduce computational time, a relaxed convergence criterion is used 

for the iterative forward modeling code used to compute the 

sensitivity matrix. This allows reduction in computational time by 

more than 70% without affecting the inversion results. Numerical 

experiments with synthetic data show that reasonable fits can be 

obtained within a small number of iterations, with a few additional 

iterations required to eliminate unnecessary structure and find the 

model with minimum norm. 

(Physics of the Earth and Planetary Interiors. 150 (2005) 3-14.) 


298 

A LATTICE BOLTZMANN METHOD FOR 

MODELING THE DYNAMIC POLE-TO-POLE 

OSCILLATIONS OF MIN PROTEINS FOR 

DETERMINING THE POSITION OF THE 

MIDCELL DIVISION PLANE (NO. 780) 

Ngamsaad W. 1 , TriampoW. 1 , Kanthang P. 1 , Tang I.M. 1 , Nuttawut 

N. 1 , Modjung C. 1 , Lenbury Y. 2 


Bangkok 10400, Thailand. E-mail ; wtriampo@yahoo.com, 

scimt@mahidol.ac.th; 2 Department of Mathematics, Faculty of 

Science, Mahidol University, Bangkok 10400, Thailand. E-mail 

: scylb@mahidol.ac.th 

Key words : Latice Boltzmann method, Bacteria, E.coli, Cell 

division, Min proteins, MinCDE oscillation 

Determining the middle of the bacteria cell and the proper 

placement of the septum is essential to the division of the bacterial 

cell. In E. coli, this process depends on the proteins MinC MinD, 

and MinE. Here, the lattice Boltzmann method (LBM) is used to 

study the dynamics of the oscillations of the min proteins from pole 

to pole. This determines the midcell division plane at the cellular 

level. The LBM is applied to the set of deterministic reaction 

diffusion equation proposed by Howard et al. to describe the 

dynamics of the Min proteins. The LBM results are in good 

agreement with those of Howard et al. and agree qualitatively with 

the experimental results. Our good results indicate that the LBM 

can be an alternative computational tool for simulating problems 

dealing with complex biological systems that can be described by 

using the reaction-diffusion equations. 

(Journal of the Korean Physical Society 46(4) (2005) 1025-1030.) 

MODELING OF THE DYNAMIC POLE-TO-POLE 

OSICLLATIONS OF THE MIN PROTEINS IN 

BACTERIAL CELL DIVISION : THE EFFECT OF 

AN EXTERNAL FIELD (NO. 781) 

Modchang C. 1 , Kanthang P. 1 , Triampo W. 1 , Ngansaad W. 1 , 

Nuttawut N. 1 , Tang I.M. 1 , Sanguansin S. 2 , Boondirek A. 2 , 

Lenbury Y. 2 


Bangkok 10400, Thailand. E-mail : wtriampo@yahoo.com, 

scimt@mahidol.ac.th; 2 Department of Mathematics, Faculty of 

Science, Mahidol University, Bangkok 10400, Thailand. E-mail 

: scylb@mahidol.ac.th 

Key words : External fields, Bacteria, E. coli, Cell division, Min 

proteins, MinCDE oscillation 

One of the most important steps in the developmental 

process of the bacteria cells at the cellular level is the determination 

of the middle of the cell and the proper placement of the septum, 

these being essential to the division of the cell. In E. coli, this step 

depends on the proteins MinC, MinD, and MinE. Exposure to a 

constant electric field may cause the bacteria’s cell-division 

mechanism to change, resulting in an abnormal cytokinesis. To see 

the effects of an external field e.g., an electric or magnetic field on 

this process, we have solved a set of deterministic reaction diffusion 

equations, which incorporate the influence of an electric field. We 

have found some changes in the dynamics of the oscillations of the 

min proteins from pole to pole. The numerical results show some 

interesting effects, which are qualitatively in good agreement with 

some experimental results. 

(Journal of the Korean Physical Society, 46(4) (2005) 1031-1036) 

A NOTE ON ASYMPTOTIC STABILITY 

CONDITIONS FOR DELAY DIFFERENCE 

EQUATIONS (NO. 782) 

Kaewong T. 1 , Lenbury Y. 2 , Niamsup P. 1 


1 Department of Mathematics, Chiang Mai University, Huaykaw 

Rd., Muang Chiangmai 50200 Thailand. E-mail : 

scipnmsp@chiangmai.ac.th; 2 Department of Mathematics, 

Faculty of Science, Mahidol University, Bangkok 10400, 

Thailand. E-mail : scylb@mahidol.ac.th 

We obtain necessary and sufficient conditions for the 

asymptotic stability of the linear delay differenc eequation x n+1 + 

pΣ N J=1 x n-k+(j-1)1 = 0, where n = 0, 1,2,…, p is a real number, and k, 1, 

and N are positive integers such that k > (N-1)1. 

(International Journal of Mathematics and Mathematical Sciences 

7 (2005) 1007-1013.) 

NONLINEAR STABILITY ANALYSES OF PATTERN 

FORMATION ON SOLID SURFACES DURING 

ION-SPUTTERED EROSION (NO. 783) 

Pansuwan A. 1 , Rattanakul C. 1 , Lenbury Y. 1 , Wollkind D.J. 2 , 

Harrison L. 2 , Rajapakse I. 2 



pansuwan@alpha.tu.ac.th. sccrt@mahidol.ac.th. 

scylb@mahidol.ac.th; 2 Department of Mathematics, Washington 

State University, Pullman WA 99164-3113, USA. E-mail : 

dwollkind@wsu.edu. 

Key words : Ion-sputtered erosion, Pattern formation on metallic 

or semiconductor surfaces, Kuramoto-Sivashinsky equation, 

Nonlinear stability analyses 

The development of spontaneous stationary equilibrium 

patterns on metallic or semiconductor solid surfaces during ionsputtered 

erosion at normal incidence is investigated by means of 

various weakly nonlinear stability analyses applied to the appropriate 

governing equation for this phenomenon. In particular, that process 

can be represented by a damped Kuramoto-Sivashinsky nonlinear 

partial differential time-evolution equation for the interfacial 

deviation from a planar surface which includes a deterministic ionbombardment 

arrival term and is defined on an unbounded spatial 

domain. The etching of coherent ripples, rhombic arrays of 

rectangular mounds or pits, and hexagonal lattices of nanoscale 

quantum dots or holes during this erosion process is based upon the 

interplay between roughening caused by ion sputtering and 

smoothing due to surface diffusion. Then, the theoretical predictions 

from these analyses are compared with both relevant experimental 

evidence and numerical simulations as well as placed in the context 

of some recent pattern formation studies. 

(Mathematical and Computer Modelling 41 (2005) 939-964.) 



CONTROLLABILITY AND STABILITY OF THE 

PERTURBED CHEN CHAOTIC DYNAMICAL 

SYSTEM (NO. 784) 

Plienpanich T. 1 , Niamsup P. 1 , Lenbury Y. 2 

1 Department of Mathematics, Chiang Mai University, Huaykaw 

Rd., Muang Chiangmai 50200, Thailand. E-mail : 

scipnmsp@chiangmai.ac.th; 2 Department of Mathematics, 


Thailand. E-mail : scylb@mahidol.ac.th 

Key words : Perturbed Chen chaotic dynamical system; Controlling 

chaos; Synchronization 

In this paper, we study perturbed Chen chaotic dynamical 

system. Firstly, we study the sufficient conditions of parameters 

which guarantee that the equilibrium points of perturbed Chen 

chaotic dynamical system are asymptotically stable. Secondly, we 

study methods for controlling chaos such as feedback control and 

bounded feedback control that suppress the chaotic behavior to 

unstable equilibrium points. Finally, we present chaos 

synchronization of perturbed Chen chaotic dynamical system by 

using active control and adaptive control. 

(Applied Mathematics and Computatons. Inpress) 

KINETIC MODELING OF LIPOPROTEIN 

PEROXIDATION INITIATED BY COPPER 

AND AZO COMPOUNDS (NO. 785) 

Yanarojana S. 1 , Chantharaksria U. 1 , Wilairat P. 2 ,Lenbury Y. 3 

1 Department of Pharmacology, Mahidol University, Bangkok 

10400, Thailand. E-mail scuct@mahidol.ac.th; 2 Department of 

Chemistry, Mahidol University, Bangkok 10400, Thailand. Email 

scpwr@mahidol.ac.th; 3 Department of Mathematics, 

Mahidol University, Bangkok 10400, Thailand. E-mail: 

scylb@mahidol.ac.th 

Key words : Lipid Perixidation, Tocopherol, Atherosclerosis, 

Kinetic Modeling, Simulation 

Oxidation of low density lipoprotein (LDL) has been 

postulated as the main cause of atherogenesis resulting in formation 

of foam cells and triggering of various pathways leading to the 

development of the disease. Therefore, antioxidants would naturally 

be expected to attenuate the progress of atherosclerosis α-Tocopherol 

(α-TocH) is the most abundant form of vitamin E in nature and the 

major antioxidant of biological membranes , α-TocH is also present 

at a much higher concentration than other antioxidants in plasma 

lipoproteins. The large amount of α-TocH present in LDL leads to 

the expectation that lipid peroxidation would be strongly inhibited 

by α-TocH. However, advanced atherosclerotic plaques are not 

deficient in antioxidant vitamin C and E, despite the occurrence of 

massive lipid oxidation. Thus conventional mechanism associated 

with the antioxidant properties of α-TocH can not explain why lipid 

peroxidation occurs in atherosclerotic lesions in the presence of 

compounds that are usually considered to be antioxidants. A kinetic 

model was developed and applied to explore the mechanism of lipid 

peroxidation process under various conditions and to examine the 

possible occurrence of lipid peroxidation in the presence of α- 

tocopherol. The model incorporated many factors, which we believe 

to play important roles in the different outcome of the process, that 

were not included in previous models. As a result, the numerical 

simulation was capable of illustrating that lipid peroxidation in the 

lipoprotein particle can occur in the presence of vitamin E ( α-TocH) 

and C under certain conditions, which include high initiation rate, 

high initial α-TocH level, low ratio of [vitamin C]/[α-TocH], and 

small lipoprotein particles. The kinetic scheme developed in this 

study defined the type of relationship that α-TocH in an environment 

exhibits either pro- or anti-oxidative property. Thus, antioxidant 

and pro-oxidant effects of tocopherol merely depend on the condition 

in which its properties are exhibited. 

(ScienceAsia. Inpress) 

STUDY OF PATTERN SELECTION ON ION- 

SPUTTERED SURFACES BY WEAKLY 

NONLINEAR STABILITY ANALYSIS (NO. 786) 

Lenbury Y. 1 , Pansuwan A. 1 , Rattanakul C. 1 , Wollkind D.J. 2 , 

Harrison L 2 , Rajapakse I. 2 



scylb@mahidol.ac.th. pansuwan@alpha.tu.ac.th. 

sccrt@mahido.ac.th; 2 Department of Mathematics, Washington 

State University, Pullman WA 99164-3113, USA. E-mail : 

dwolkind@wsu.edu. 

One of the methods found suitable for predicting pattern 

selection in nonlinear phenomena is a weakly nonlinear stability 

analysis that, although incorporating the nonlinearities of the relevant 

model system, basically pivots a pertubation procedure about the 

critical point of linear stability theory [reviewed by Wollkind et.al. 

(1994)]. The advantage of such an approach over strictly numerical 

procedures is that it allows one to deduce quantitative relationships 

between system parameters and stable patterns which are valuable 

for experimental design and difficult to accomplish using simulation 

alone. The development of spontaneous stationary equilibrium 

patterns on metallic or semiconductor solid surfaces during ionsputtered 

erosion at normal incidence can be investigated by means 

of various weakly nonlinear stability analyses applied to the 

appropriate governing equation for this phenomena. 

(Asian Mathematicl Conference 2005. National University of 

Singapore Singapore, 20-23 July 2005.) 

NONLINEAR DELAY DIFFERENTIAL EQUATIONS 

FOR POPULATION MODELS : ASYMPTOTIC 

STABILITY AND NONTRIVIAL PERIODICITY 

Giang D.V. 1 , Lenbury Y. 2 , Seidman T.I. 3 

(NO. 787) 

1 Hanoi Institute of Mathematics, 18 Hoang Quoc Viet 10307 

Hanoi, Vietnam. E-mail : dvginag@math .ac.vn.; 2 Department 

of Mathematics, Mahidol University, Bangkok 10400, Thailand. 

E-mail : scylb@mahidol.ac.th.; 3 Department of Mathematics and 

Statistics, University of Maryland, Baltimore Country, 

Maryland, USA. E-mail : seidman@math.umbc.edu 

We investigate the effect of delay on the asymptotic behavior 

of the model x + μx = f(x(‘ – τ)) 


300 

Of population growth when the nonlinearity is unimodal 

function. The existence of a nonconstant periodic solution when 

the delay is large enough is then discussed by using Holf bifucation 

and non-ejective fixed point theory of Browder (F. Brower, a further 

generalization of Schauder fixed point theorem, Duke Math J.32 

(1965), 575-578). The ω-limit set of other positive solutions is a 

closed periodic orbit or a set of unstable positive equilibrium points. 

We also applied knots theory to study periodic solutions. 

(Conference on Differential & Difference Equations and 

Applications. Florida Institute of Technology, USA. 1-5 August 

2005.) 

PATTERN FORMATION IN SIGNAL TRANS- 

DUCTION MECHANISM INVOLVING (NO. 788) 

Lenbury Y., Rattanakul C. 

Department of Mathematics, Mahidol University, Bangkok 

10400, Thailand. E-mail : scylb@mahidol.ac.th. 

sccrt@mahidol.ac.th 

We present the result of our investigation on pattern 

formation arising in the signal transduction mechanism which 

involves the G-protein. The process may be represented by two 

action diffusion equations involving one stimulating hormone and 

one inhibitor. The analysis is done by the weakly nonlinear stability 

analysis which incorporates the nonlinearities of the relevant model, 

while pivoting a perturbation procedure about the critical point of 

the linear stability analysis. The method allows one to deduce 

quantitative relations between system parameters and stable patterns 

which are valuable for clinical interpretation and difficult to 

accomplish usng simulation alone. 

(10 th Annual Meeting in Mathematics, Naresuan University, 

Thailand. 12-13. May 2005.) 

EXACT ALGORITHM FOR SPIN-CORRELATION 

FUNCTIONS OF THE TWO-DIMENSIONAL +J 

ISING SPIN GLASS IN THE GROUND STATE 

J. Poulter 1 and J.A. Blackman 2 

(NO. 789) 


University, Rama 6 Road, Bangkok 10400, Thailand; 

2 Department of Physics, University of Reading P.O. Box 220, 

Reading RG6 6AF, England. 

Key words : Exact algorithm, spin correlation, Ising spin glass 

We introduce an exact algorithm for the computation of 

spin correlation functions for the two dimensional +J Ising spin glass 

in the ground state. Unlike with the transfer matrix method, there is 

no particular restriction on the shape of the lattice sample, and unlike 

Monte Carlo based methods it avoids extrapolation from finite 

temperatures. The computational requirement depend only on the 

number and distribution of frustrate plaquettes. 

MATHEMATICAL OF BLOOD FLOW IN THE 

STENOSED CORONARY ARTERY WITH 

POROELASTIC WALL (NO. 790) 

Poltem D., Wiwatanapataphee B and Lenbury Y 

Department of Mathematics, Faculty of Scinece, Mahidol 

University, Bangkok, Thailand. 

The coronary artery disease (CAD) caused by the artery 

wall clog is a very common disease. This clog affects the decreasing 

in the blood volume in the artery. The coronary artery bypass grafting 

(CABG) has been widely used for patients with serve CAD. The 

successful of the CABG is necessary to understand blood flow in 

the stenosed artery whose wall is characterized as the porous 

elasticity. Our model applies a straight axisymmetric arterial 

segment, and computational domain consists of the lumen region 

and the intima (wall)region. The governing equations of blood flow 

in the lumen region are continuity and the Navier-Stokes equations 

and in the initima region those are the continuity and the Brinkman’s 

equations. Based on Bubnov-Galerkin finite element formulation 

an efficient numerical algorithm is developed for the solution of the 

model. 

(9 th Annual Naitonal Symposium on Computational Science and 

Engineering, Mahidol University, 22-25 March 2005, Bangkok, 

Thailand.) 

MATHEMATICAL MODEL AND NUMERICAL 

SOLUTIONS OF WAVE PROPAGATION IN 

SHALLOW WATER (NO. 791) 

Srimongkol S., Wiwatnapataphee B. 




Numerical studies have been undertaken to model various 

aspects of wave propagation in shallow water. The aim of this paper 

is on the construction of a mathematical model. To study the wave 

propagation in shallow water, an efficient algorithm is developed 

with in the framework of Lagrange-Galerkin finite element method. 

The result indicates that our model and numerical technique are 

capable to describe the wave pattern under some circumstantial 

conditions, such as effect of wind force and gravity force. 

(9 th Annual Naitonal Symposium on Computational Science and 

Engineering, Mahidol University, 22-25 March 2005, Bangkok, 

Thailand.) 

MININJECTIVITY AND KASCH MODULES (NO. 792) 

Nguyen Van Sanh, Somchit Chotchaisthit and Kar Ping Shum 


University. E-mail : frnvs@mahidol.ac.th 

Key words : quasi-minijective, self generator, annihilator, 

minannihilator, minsymmetric 

Let R be an associate ring with identity. A right R-module 

M is called mininjective if every homomorphism from a simple right 



idela of R to M can be extended to R. We now extend this notion to 

modules. We call a module N an M-minijective module if every 

homomorphism from a simple M- cyclic submodule of M to N can 

be extended to M. In this note, we charactrize quasi-mininjective 

modules and show that for a finitely generated quasi-mininjective 

module M which is a Kasch module, there is a bijection between the 

class of all maximal submodules of M and the class of all minimal 

left ideals of its endomorphism ring S = End(M) if an donly if l S r M (K) 

= K for any simple left ideal K of S. The results obtained by 

Nihcolson and Yousif in mininjective rings are generalized 

(East-west J. of Mathematics: Vol 5, No. 2 pp113-122.) 

ON THE STRUCTURE OF SERIAL ARTINIAN 

MODULES (NO. 793) 

Nguyen Van Sanh 


University, E-mail : frnvs@mahidol.ac.th 

Key words : artinian module, uniserial, serial indecomposable, 

quasi-injective, quasi-projective 

Let R be an associate ring with identity. A right R-module 

M is called uniserial if the lattice of submodules is linear. M is called 

serial if it is a direct sum of uniserial modules. The main result in 

this note is the following Theorem : Let M be an Artinian right Rmodule. 

Then the following conditions are equivalent : 

(1) M is uniserial and S = End(M) is left Artinian; 

(2) Every indecomposable module in σ [M] is quasi-injective; 

(3) Every indecomposable module in σ[M] is quasi-projective; 

(4) Every indecomposable quasi-projective module in σ[M] is 

quasi injective; 

(5) Every indecomposable quasi-injective module in σ[M] is 

quasi projective 

This theorem generalizes a result of Muller in 1969 which 

saying that a right Artinian ring R is generalized uniserial if and 

only if every indecomposable right R-module is quasi-injective. 

RING OVER WHICH EVERY CYCLIC MODULE 

HAS A S-CS INJECTIVE HULL (NO. 794) 

Maliwan Tunapan, Sarapee Chairat, Chitlada Somsup and Dinh 

Van Huynh 


University, E-mail : frnvs@mahidol.ac.th 

Key words : cyclic module, e-CS injective hull, q2fd-ring, einjective, 

CSI-ring, CSE-ring, semilocal, von Neumann regular 

A ring R is called a right qfd-ring if R/I has finite Goldie 

dimension for any right ideal I of R. In 2003, C. Faith studied a 

class of rings such that the injective hull of every cyclic right Rmodule 

is Σ-injective. He called such a ring a ring CSI-ring. In this 

research work, we study the class of ring over which every cyclic 

ringht R-module has a Σ -CS injective hull. We call them right CSErings. 

The key Lemma in our note is that any right CSE-ring right 

qfd, and therefore any right CSE-ring is right Goldie. By a result of 

Camillo in 1975, we can see that any commutative CSE-ring is 

Noetherian. The goal of our research is considerig in the case of 

non-commutativity. Our main result is that a right CSE-ring R is 

Noetherian under one of the following conditions: 

(1) R is semilocal; 

(2) R or R/rad (R) is right Kash; 

(3) R/rad (R) is von Neumann regular 

We also showed that a ring R is right Noetherian if the 

injective hull of any countable generated semisimple right R-modules 

in ?Σ -CS. 

ON THE RELATIONS BETWEEN THE CLASSES 

OF WEAKLY QF3-RINGS AND HEREDITARY 

RINGS (NO. 795) 

Maliwan Tunapan, Phan Dan and Nguyen Van Sanh 


University, E.-mail : frnvs@mahidol.ac.th 

Key words : weakly QF3-ring, hereditary ring, injective, projective 

Menabu Harada (1965, 1978, 1980) has studied some 

generalizations of QF-rings by introducting two following conditions: 

(*) Every non-small right R-module contains a non-zero 

injective submodule; 

(**) Every non-cosmall right R-module contains a non-zero 

projective direct summand; 

(***) Every indecomposable injective module E is hollow, 

i.e., every proper submodule is small lin E. 

The main results in our report are the following 2 Theorems 

: Theorem 1 Let R be a right perfect right weakly QF-3 ring. The 

following conditions are equivalent : 

(1) R is right hereditary; 

(2) e i ,R/e i ,J t is injective for each primitive idempotent e i 

and for any integer t; 

(3) R is Morita equivalent to direct sum of ring of uppertriangular 

matrices over a divisions rings 

Theorem 2 Let R be a right perfect ring. If J 2 (R) = 0 or R 

is right hereditary then the following conditions are equivalent: 

(1) The condition (*) holds; 

(2) The condition (**) holds and each e i ,R contains an 

unique minimal submodule; 

(3) R is right weakly QF-3. 

WATER QUALITY AND BREEDING HABITATS 

OF ANOPHELLINE MOSQUITO IN NORTH- 

WESTERN THAILAND (NO. 796) 

Ampornpan K. 1 , Pratap S. 2 , Montip Tiensuwan 3 ., James W.J. 1 , 

and Ratana S. 1 

1 Department of Entomology, Armed Forces Research Institute 

of Medical Sciences., Bangkok; 2 Department of Tropical 

Hygiene, Faculty of Tropical Medicine, Mahidol University, 

Bangkok; 3 Department of Mathematics, Faculty of Science, 



302 

Malaria transmission is dependent upon many hydrologydriven 

ecological factors that directly affect the vectorial competence. 

Including the presence of suitable habitats for the development of 

anopheline lavae. Larval habitats were identified and characterized 

at three malaria endomic villages (Ban Khum Huay, Ban Pa Dae. 

amd Ban Tham Seau) in Mae Sot district, in northwestern Thailand 

between July 2002 and June 2003. The Global Positioning System 

(GPS) was used to provide precise locational data for the spatial 

distribution of anopheline mosquito larvae and their habitats. Ten 

habitats categories were identified. Eighteen adult Anopheles species 

were identified from larvae in all the surveyed habitats. An minimus 

was the most common species throughout the year. The relationship 

between eight abiotic variables (temperature, hardness, carbon 

dioxide, dissolved oxygen, nitrate, phosphate, silica and pH) and 

the abundance of four major species of malaria vectors (An. (Cel.) 

dirus, An. (Cel.) minimus. An. (Cel.) maculatus, and An. (Cel.) 

sawadwongporni), and sic species of non-vectors (An. (Cel.) kochi, 

An. (Cel) jamesii, An. (Ano.) peditaeniatus, An. (Ano.) barbirostris, 

An. (Ano.) campestris, and An (Cel.) vagus) larvae was investigated. 

The results from the multiple regression models suggest that 

hardness, water temperature and carbon dioxide are the best predictor 

variables associated with the abundance of An. minimus larvae (p < 

0.001); temperature and silica concentration for An. jamesii larvae 

(p < 0.001); dissolved oxygen and silica concentration for An. 

campestris larvae (p < 0.001); an pH and silica concentration for 

An. vagus larvae (p < 0.001). We could not identify key 

environmental variables for An. maculates, An. sawadwongporni, 

An. peditaeniatus, and An. barbirostris. 

(Water Quality of Anopheline Habitats 36(1) (2005) 46-53.) 

SCREENING FOR DIABETIC RETINOPATHY IN 

RURAL AREA USING SINGLE-FIELD DIGITAL 

FUNDUS IMAGES (NO. 797) 

Paisan R. 1 , NattaponW. 2 , Edchai P 3 , Montiph Tiensuwan 4 

1 Department of Ophthalmology, Rajavithi Hospital, Bangkok, 

Thailand; 2 Department of Ophthalmology, Lerdsin Hospital, 

Bangkok, Thailand; 3 Director of Kabeheang Community 

Hopsital, Surin, Thailand, 4 Department of Mathematics, Faculty 

of Science, Mahidol University, Bangkok, Thailand. 

Key words : Diabetic retinopathy screening. Digital screening, 

Nonmydriatic fundus camera 

Objective : To evaluate the practicability of using singlefield, 

2.3 million-pixel, digital fundus images for screening of 

diabetic retinopathy in rural areas. 

Material and Method : All diabetic patients who regularly 

attended the diabetic clinic at Kabcheang Community Hospital, 

located at 15 kilometers form the Thailand –Cambodia border, were 

appointed to the hospital for a 3 day diabetic retinopathy screening 

programme. The fundi of all patients were captured in single-field, 

45 o ,. 2.3 million-pixel images using nonmydriatic digital fundus 

camera and them sent ot a reading center in Bangkok. The fundi 

were also examined through dilated pupils by retinal specialist at 

this hospital. The grading of diabetic retinopathy from two methods 

was compared for and exact agreement. 

Results : The average duration of single digital fundus 

image capture was 2 minutes. The average file size of each image 

was 750 kilobytes. The average duration of single image 

transmission to a reading center in Bangkok via satellite was 3 

minutes; via a conventional telephone line was 8 mimutes. Of all 

150 patients, 130 were assessed for and agreement between dilated 

fundus examination and digital fundus images in diagnosis of 

diabetic retinopathy. The exact agreement was 0.87, the weighted 

kappa statistics was 0.74. The sensitivity of digital fundus images 

in detecting diabetic retinopathy was 80%, the specificity was 96%. 

For diabetic macular edema the exact agreement was 0.97, the 

weighted kappa was 0.43, the densitivity was 43%, and the 

specificity was 100%. 

Conclusion : The image capture of the nonmydriatic digital 

fundus camera is suitable for screening of diabetic retinopathy and 

single-field digital fundus images are potentially acceptable tools 

for the screening. The real-time image transmission via telephone 

lines to remote reading center, however, may not be practical for 

routine diabetic retinopathy screening in rural areas. 

(Journal of The Mediacl Association of Thailand 88(2)(2005) 176- 

180.) 

APPLICATION OF MULTIPLE CRITERIA 

DECISION MAKING TO AIR POLLUTION 

DATA FROM BANGKOK, THAILAND (NO. 798) 

Montip Tiensuwan 1 , Satinee Lertprapai 1 , and Bimal K. Sinha 2 


University, Bangkok, Thailand; 2 Department of Mathematics 

and Statistics University, of Maryland, Baltimore Country, USA. 

In this study we concentrate on the air pollution data 

measured as carbon monoxide, nitrogen dioxide, sulfur dioxide and 

ozone from ten monitoring stations in Bangkok, Thailand and apply 

Multiple Criteria Decision Making (MCDM) method to compute 

an overall air pollution index for these stations and compare them. 

We also study robustness of these overall indices. 

(The 4 th Annual Hawaii International Conference on Statistics. 

Sheraton Waikiki Hotel, USA. 9-11 January 2005) 

PERCOLALTION (NO. 799) 

Kit Tyabandha and Jirabhat Yokrattanasak 

Department of Mathematics, Mahidol University, Bangkok, 

Thailand. E-mail : sckty@mahidol.ac.th 

Key words : percolation, statistical physics 


Percolationis the study of transitions between two phases. 

The classical percolation theory began with the works by 

Hammersley in 1957, where he considered liquid flowing through 

networks of tubes. Another branch of the theory of percolation comes 

from the work by Ising in 1925 in the field of atomic- and quantum 

physics. A good introduction and reviews of existing literature is 

written by Stauffer and Aharony in 1985. The word ‘percolation’ is 

an adaptation from the percolator, which is a machine for making 

coffee. This study is in the stage of survey to cover as large ground 

as possible. Later it is planned that specifically some unexplored 

areas be defined and studied. 



COMPARISON OF CAVITY MODIFICATION AND 

COMMUNITY INVOLVEMENT AS STRATEGIES 

FOR HORNBILL CONSERVATION IN THAILAND 

(NO. 800) 

Pilai Poonswad 1 , Chumpol Sukkasem 2 , Somnoi Phataramata 3 , 

Sumsuding Hayeemuida 4 , Kamol Plongmai 1 , Phitaya Chuailua 1 , 

Preeda Thiensongrusame 1 , Narong Jirawatkavi 1 

1 Department of Microbiology, Faculty of Science, Mahidol 

University, Rama VI Road, Bangkok 10400, Thailand; 2 National 

Park, Wildlife and Plant Conservation Department, National 

Park and Wildlife Research Division, Natural Resource 

Conservation Office, Khao Yai National Park, Bangkok, 

Thailand; 3 National Park, Wildlife and Plant Conservation 

Department, National Park and Wildlife Research Division, 

Natural Resource Conservation Office, Sukarin National Park, 

Bangkok, Thailand; 4 National Park, Wildlife and Plant 

Conservation Department, National Park and Wildlife Research 

Division, Natural Resource Conservation Office, Budo-Sungai 

Padi National Park, Bangkok, Thailand. 

Key words : Cavity modification; Community involvement; 

Hornbill conservation; 

We monitored breeding success of hornbills in Thailand 

for four sympatric species at Khao Yai National Park during 1981– 

2002 and six species at Budo-Sungai Padi National Park during 

1994–2002. Within a 60 km2 study area at Khao Yai, the well 

protected area, use of available cavities ranged from 94% in 1984 

to 50% in 1993. Competition for nesting cavities was 40% of cavities 

available indicating the shortage of suitable cavity. We monitored 

the cavity condition and modified 48 cavities and, between 1996 

and 2000, these contributed annually 23–45% of nestings that were 

successfully fledged (n = 352). Within the 90 km 2 at Budo Mountain, 

with heavy human disturbance, use of available cavities for nesting 

decreased from 81% in 1997 to 30% by 2001 and of successful 

fledging from 96% in 1997 to 55% in 1999, but both increased 

once our involvement with local communities had eradicated 

poaching and reduced disturbance. Competition for nesting cavities 

here was 26% of cavities available indicating the effect of poaching. 

Both strategies were successful in the short term but long-term 

management of cavities in large forest trees is required at Khao Yai 

and of forest disturbance at Budo. 

(Journal of Biological Conservation. 122 (2005), pp. 385-393.) 

A BOTANICAL INVENTORY OF A TROPICAL 

SEASONAL FOREST IN KHAO YAI NATIONAL 

PARK, THAILAND: IMPLICATIONS FOR FRUIT– 

FRUGIVORE INTERACTIONS (NO. 801) 

Shumpei Kitamura 1 , Shunsuke Suzuki 2 , Takakazu Yumoto 3 , 

Phitaya Chuailua 4 , Kamol Plongmai 4 , Pilai Poonswad 4 , Naohiko 

Noma 2 , Tamaki Maruhashi 5 and Chumphon Suckasam 6 

1 Center for Ecological Research, Kyoto University, 

Kamitanakami-Hirano, 520 2113, Otsu, Japan, Email: 

shumpei@ecology.kyoto-u.ac.jp ; 2 School of Environmental 

Science, University of Shiga Prefecture, 522-8533, Hikone, 

Japan; 3 Research Institute of Humanity and Nature, 602-0878, 

Takashima, Marutamachi-Kawaramachi, Kyoto, Japan; 4 

Hornbill Project, Department of Microbiology, Faculty of 

Science, Mahidol University, 10400, Bangkok, Thailand; 5 

Department of Human and Culture, Musashi University, 176- 

8534, Nerima, Tokyo, Japan; 6 Wildlife and Plant Conservation 

Department, National Park, 10900, Bangkok, Thailand, 

Key words: Frugivory; Seed dispersal, Tropical seasonal evergreen 

forest 

The diversity of plants in tropical forests makes dietary 

studies of frugivores difficult. This paper provides a botanical 

inventory of a tropical seasonal forest community in Khao Yai 

National Park, Thailand. The forest is valuable from a conservation 

perspective because it is one of the last remaining intact forests in 

northeastern Thailand, and is an important refuge for many animal 

and plant species. A 4-ha inventory plot measuring 200 × 200 m 

was established and all plants greater than or equal to 10 cm in 

diameter at breast height (dbh) were measured and permanently 

labeled. We found 1610 stems belonging to 105 species, 76 genera 

and 35 families, with a combined basal area of 142.5 m 2 . The 

community was dominated by species of Lauraceae, Cornaceae, 

Euphorbiaceae, Meliaceae, and Elaeocarpaceae. About one-third of 

the plant species (40 spp.) identified in this study were vulnerable 

to extinction because they were mostly dispersed by large frugivores, 

which were intolerant of human impact. If they disappear, these 

forests may become dominated by plant species that are dispersed 

by abiotic means and species with small-seeded fruits. 

(Journal of Biodiversity and Conservation, Vol. 14. No. 5, May 2005, 

pp. 1241-1261(21). Supported by Research Fund of the Japan 

Society for The promotion of Science (no. 10041169) and JSPS 

Research Fellowships for Young Scientists.) 

CHARACTERISTICS OF HORNBILL-DISPERSED 

FRUITS IN A TROPICAL SEASONAL FOREST IN 

THAILAND (NO. 802) 

Shumpei Kitamura 1 , Takakazu Yumoto 1 , Pilai Poonswad 2 , 

Phitaya Chuailua 2 and Kamol Plongmai 2 

1 Center for Ecological Research, Kyoto University, 

Kamitanakami-Hirano, Otsu 520-2113, Japan; 2 Hornbill 

Project, Department of Microbiology, Mahidol University, 

Bangkok 10400, Thailand. 

Key words : Hornbill, Dispersed fruits, Tropical seasonal Forest 

Asian hornbills are primarily frugivorous. We studied the 

characteristics of fruits consumed by four sympatric hornbill species 

in Thailand: Great Hornbill (Buceros bicornis), Wreathed Hornbill 

(Aceros undulatus), Austin’s Brown Hornbill (Anorrhinus austeni) 

and Oriental Pied Hornbill (Anthracoceros albirostris). We compared 

the frequency of distribution of 11 variables for all fruit species 

collected in the study area (n = 259) and fruit species consumed by 

hornbills (n = 73). Our analysis revealed that fruits consumed by 

hornbills are: (1) large, (2) easily accessible within the canopy, (3) 

red, purple or black and (4) dehiscent or indehiscent with a thin 

husk. The range of fruit sizes eaten by hornbills in our study is 

comparable to that reported from other sites in Southeast Asia and 

Africa. The large gape width of hornbills enables them to consume 

large fruits that small frugivores would find difficult to consume. 

(Journal of Bird Conservation International, Vol. 14, Supplement 

S1, (2004) Published Online (2005) Supported by Research Fund 

of the Japan Society for the Promotion of Science (no.10041169), 

JSPS Research Fellowships for Young Scientists. and Thailand 

Hornbill Project ) 


304 

INTEGRATION OF GENETIC, SPECIES, 

POPULATION, AND LANDSCAPE INFORMATION 

FOR CONSERVATION OF HORNBILLS IN 

THAILAND : A RESEARCH PROJECT INTRO- 

DUCTION (NO. 803) 

Pilai Poonswad 1 , Anak Pattanavibool 2 , Youngyut Trisurat 3 , 

Philip Round 4 , Mathurose Ponglikitmongkol 5 , Nareerat 

Viseshakul 6 , Siriporn Thong-Aree 7 , Budsabong Kanchanasaka 7 , 

Vijak Chimchome 3 , Parntep Ratanakorn 8 

1 Department of Microbiology, Faculty of Science Mahidol 

University, Bangkok 10400 ; 2 Wildlife Conservation Society 

Thailand Program, P.O. Box 170, Laksi, Bangkok 10210; 

3 Department of Forest Biology, Faculty of Forestry, Kasetsart 

University Bangkok 10900; 4 Departmnet of Biology, Faculty of 

Science, Mahidol University, Bangkok 10400; 5 Department of 

Biochemistry, Faculty of Science, Mahidol University, Bangkok 

10400; 6 Department of Pathology, Faculty of Veterinary Science, 

Chulalongkorn University, Bangkok 10330; 7 Wildlife Research 

Division, Dept. of National Park, Wildlife, and Plant 

Conservation, Bangkok 10900; 8 Faculty of Veterinary Science, 

Mahidol University, Salaya Campus, Nakornpathom 73170. 

Key words : Genetic, Population, Hornbill 

Research and conservation of hornbills in Thailand has been 

globally recognized under the Hornbill Research Project for more 

than 20 years. Most studies have been emphasized on breeding 

biology and ecology and limited to Khao Yai National Park and 

Huai Kha Khaeng Wildlife Sanctuary. In 2003, the Project has 

launched a country-wide study on distribution and population status 

of hornbills in remaining pristine forests in Thailand. The overall 

objective is to understand the characteristics of genetics in relation 

to distribution, status of population and habitat, and threats. This is 

an effort to assist responsible/relevant authorities implement an effective 

conservation scheme for hornbills and their habitats. Various aspects 

are covered under the overall objective as the followings. For genetic 

study, the team uses either blood, feather, or tarsal scale samples to 

build a phylogenetic tree for Thai hornbills. For population genetics, 

the method is set to determine genetic diversity among populations 

of great hornbills, a widest distribution species and virtually localized 

subpopulations, from 3 priority forest landscapes; Western Forest 

Complex, Khao Yai, and Budo-Sungai Padi National Parks where 

sizes and disturbances differ. For comparative population status, 

the density estimates for hornbills will be obtained from a point 

transect technique in relation to the focal habitat type structure 

among evergreen forest of those 3 priority sites. For the distribution 

records, recce technique is used to record species occurrences in 

the rest of the protected forests. Radio telemetry is also used to 

study ranging patterns of rare and least understood species such as 

rufous-necked hornbills and plain-pouched hornbills. For habitat 

status, forest structure and composition survey has been recorded 

within 40×50m plots. After the duration of 5 years, besides 

establishing a complete database, we expect to have much 

understanding about hornbill ecology, their genetics and population 

status in order to predict the future survival of Thai hornbills and 

recommend proper-action needs to be implemented for sustainable 

conservation and management of hornbills and their habitat. 

(Oral Presentation at The 1 st Field Ecology Symposium on “Forest 

Ecology and Restoration” King Mongkut’s University of Technology 

Thonburi, Bangkok, 28-30 Jan 2005 Supported by National Center 

for Genetic Engineering and Biotechnology (BIOTEC) and Hornbill 

Research Foundation) 

CONDUCTING A SUCCESSFUL LONG-TERM 

WILDLIFE RESEARCH IN THAILAND : 

STRATEGY AND FUNDING (NO. 804) 

Pilai Poonswad 


Department of Microbiology, Faculty of Science, Mahidol 

University, Rama 6 Rd. Bangkok. 

It is very difficult to provide a typical model strategy in 

order to keep a long-term wildlife research running, particularly in 

a developing country such as Thailand where wildlife is much 

considered for its economical value, but little is care for ecological 

value. The research itself basically requires a long duration, a lot of 

man power and great amount of funding. It also involves a number 

of influential unexpected factors. To be successful and achieving 

goals such project has to be recognized by agencies of authority, 

public and private sectors. To achieve the goals, from my perspective, 

I propose following strategies: Firstly, it directly involves the 

researcher; the personal discipline, including inspiration, curiosity 

and passion, setting goals, honesty and determination. Secondly, 

the researcher must have scientific thinking process; comprising 

interrelated thinking from data emission, consistency and ethics, 

interpretation and evaluation of results, dissemination of results and 

implementation. Thirdly, social and psychological skill; it involves 

attracting public attention, arousing and convincing awareness, 

appreciation, creating trust and inviting participation. These would 

render both moral and financial supports which perpetuate the 

research and create pride among the public. The discussion is based 

on first hand experience. 

(Plenary Lecture at The 1 st Field Ecology Symposium on “Forest 


Thonburi, Bangkok 28-30 Jan 2005) 

FACTORS INFLUENCING FOOD SELECTION 

AMONG FOUR SYMPATRIC SPECIES OF 

HORNBILL AT KHAO YAI NATIONAL PARK, 


Chu-tion Kanwatanakid Savini 1 and Pilai Poonswad 2 


Rama 6 Rd., Bangkok 10400 ; 2 Department of Microbiology, 

Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 

10400. 

Asian hornbills are a frugivore species for most of the year, 

skipping to a more omnivore diet during the breeding season. Due 

to the high frugivore degree of their diet they assume an important 

seed disperser role in the forest ecology of their habitat. Result 

shows the preliminary study of an ongoing study of the feeding 

ecology of four sympatric species: Great hornbill (Bucero bicornis), 

Wreathed hornbill (Aceros undulatus, Oriental Pied hornbill 

(Anthracoceros albirostris) and Brown hornbill (Ptilolaemus 

tickelli), at Khao Yai National Park. The feeding habit and diet 

composition of each species have been monitored since January 2004 

and will continue until December 2005. This will in the end include 

two breeding seasons, January-June, and two non breeding season, 

or flocking season, from July to December. The objectives of this 

study are: (1) to define which are the factors influencing food 

selection on the base of spatial-temporal availability, chemical and 

morphological variation of the chosen food items. (2) To define 

feeding overlaps between the four sympatric species and how this 



influences their relative breeding biology and (3) define the success 

between the different species in relation to food availability and 

quality. During breeding season data on diet and feeding behaviour 

were collected after direct observation at 6 nests for each species 

(with the exception of brown hornbill where only 4 nests could be 

observed). Fruits consumed were identified as fig and non-fig fruit, 

this last one classified into species and differentiate by morphology. 

The diet during the flocking season was identified by collecting 

seed in traps set under roosting trees. Food selection of hornbill was 

measured by comparing food consumption with food availability. 

Food availability was defined monthly with phenological 

observation, base on percentage of canopy coverage, along line 

transects within the study area. Feeding bout of fruiting trees and 

nutritional analysis of selected fruits were studied in both breeding 

and non breeding season. In the end we investigated other ecological 

niches that may influence to food selection in the four observed 

species, such as height of nest, number of offspring etc. 

(Oral Presentation at The 1 st Field Ecology Symposium on “Forest 


Thonburi, Bangkok 28-30 Jan 2005. Supported by RGJ. Ph.D. and 

Thailand Hornbill Project) 

A COMPARATIVE STUDY OF FRUIT DIETS OF 

GREAT HORNBILL (BUCEROS BICORNIS) 

AND RHINOCEROS HORNBILL (BUCEROS 

RHINOCEROS) DURING THE BREEDING SEASON 

IN BUDO-SUNGAI PADI NATIONAL PARK, 

SOUTHERN THAILAND (NO. 806) 

Sukanya Chaisuriyanane 1 , Pilai Poonswad 2 , George A. Gale 1 , 

Anak Pattanavibool 3 and Wichan Eiadthong 4 

1 Division of Natural Resources Management, School of 

Bioresources and Technology, King Mongkut’s University of 

Technology Thonburi, 83 Moo 8 Thakham, Bangkhuntein, 

Bangkok 10150; 2 Department of Microbiology, Faculty of Science, 

Mahidol University, Rama 6 Rd., Bangkok 10400; 3 Wildlife 

Conservation Society (Thailand), P.O. BOX 170, Laksi, Bangkok 

10210; 4 Department of Forest Biology, Faculty of Forestry, 

Kasetsart University, Ngamwongwan Rd, Bangkok 10900 

Key words : Great Hornbill, Rhinoceros Hornbill, Fruit diets 

Fruit diets of Great hornbill (Buceros bicornis) and 

Rhinoceros hornbill (Buceros rhinoceros) during the breeding season 

were studied in Budo-SuNgai Padi National Park, Southern Thailand 

from January 2003 to August 2004. The proportion by weight of 

Great and Rhinoceros hornbill diets were 55.6% and 73.6% fig, 

41.2% and 23.8% non-fig, and 3.2% and 2.6% animal respectively. 

Great hornbill selected 20 non-fig fruit species from 12 families, 

which Polyalthia viridis, Aglaia spectabilis, Dysoxylum 

macrocarpum, Syzygium sp.1 and Canthium hirtellum were the five 

most important using on a combined ranking based on weight, 

number fruits and frequency of feeding. Rhinoceros hornbill 

selected non-fig fruit of 13 species from 8 families, which 

Oncosperma horridum, Aglaia spectabilis, Knema sp.1, Polyalthia 

viridis and Dysoxylum macrocarpum were the five most important 

using the same ranking system. This study categorized the breeding 

season into three stages; incubating phase (INC), nestling before 

female emergence phase (BEFORE) and nestling after female 

emergence phase (AFTER). Results indicated that the feeding rate 

of fig during INC (7.82 & 15.00 gram/hour of observation) and 

AFTER (7.66 & 13.07 g/h Obs.) were significantly different between 

Great and Rhinoceros hornbills. But during the BEFORE period 

feeding rate of fig (13.23 & 15.57 g/h Obs.) was not significantly 

different between hornbills. While feeding rates of non-figs during 

the three nest stages (INC 6.30 & 4.29 g/h Obs., BEFORE 12.05 & 

10.17 g/h Obs. and AFTER 6.49 & 10.83 g/h Obs.) was not 

significantly different between hornbills. This study was summarized 

only the most importance fruit diet, which is not enough to encourage 

food resources management. Thus, more study on plant community 

is needed to supplement food resources data for hornbill conservation 

and management. 

(Poster Presentation at The 1 st Field Ecology Symposium on “Forest 


Thonburi, Bangkok 28-30 Jan 2005) 

DISPERSAL OF CANARIUM EUPHYLLUM 

(BURSERACEAE), A LARGE-SEEDED TREE 

SPEICES IN A MOIST EVERGREEN FOREST IN 


Shumpei Kitamura 1 , Shunsuke Suzuki 2 , Takakazu Yumoto 3 , Pilai 

Poonswad 1 , Phitaya Chuailua 1 , Kamol Plongmai 1 , Tamaki 

Maruhashi 4 , Naohiko Noma 2 and Chumphon Suckasam 5 

1 Thailand Hornbill Project, c/o Department of Microbiology, 


10400, Thailand; 2 School of Environmental Science, The 

University of Shiga Prefecture, Hikone 522-8533, Japan 

Research Institute of Humanity and Nature, Kyoto 602-0878, 

Japan; 3 Research Institute of Humanity and Nature, 602-0878, 

Takashima, Marutamachi-Kawaramachi, Kyoto, Japan; 4 

Department of Human and Culture, Musashi University, Nerima, 

Tokyo 176-8534, Japan; 5 National Parks Division, Department 

of National Parks, Wildlife and Plant Conservation, 

Phaholyothin Rd., Chatuchak, Bangkok 10900, Thailand . 

Key words: Canarium euphyllum, Moist Evergreen Forest, Dispersal 

We investigated the dispersal of a large-seeded tree species, 

Canarium euphyllum (Burseraceae), in the moist evergreen forests 

of the Khao Yai National Park in Thailand. By combining direct 

observations of fruit consumption in tree canopies (543 h) and 

ameratrapping observations of fallen fruit consumption on the ground 

(175 d), we identified the frugivore assemblage that foraged on the 

fruits of C. euphyllum and assessed their role in seed dispersal and 

seed predation. In the canopy, our results showed that seeds were 

dispersed by a limited set of frugivores, one pigeon and four hornbill 

species, and predated by two species of squirrel. On the ground, 

seven mammal species consumed fallen fruits. A combination of 

high fruit removal rates and short visiting times of mountain imperial 

pigeons (Ducula badia) and hornbills lead us to conclude that these 

large frugivorous birds provide effective seed dispersal for this tree 

species, in terms of quantity. These frugivorous species often have 

low tolerance of negative human impacts, yet loss of these dispersers 

would have severe deleterious consequences for the successful 

regeneration of C. euphyllum. 

(Poster Presentation at The Fourth International Symposium/ 

Workshop on Frugivores and Seed Dispersal 9 – 16 July 2005 

Griffith University, Brisbane, Australia. Supported by Research 

Fund of the Japan Society for the Promotion of Science 

(no.10041169); JSPS Research Fellowships for Young Scientists. 

and Thailand Hornbill Project ) 


306 

THE BREEDING SEASON OF THREE SYMPATRIC 

RODENTS IN KHAO YAI NATIONAL PARK, THAI- 

LAND (NO. 808) 

Shunsuke Suzuki 1 , Shumpei Kitamura 2 , Masahiro Kon 1 , Pilai 

Poonswad 2 , Takakazu Yumoto 3 , Naohiko Noma 1 , and Prawat 

Wohandee 4 

1 Graduated School of Environmental Science, The University 

of Shiga Prefecture, Hikone, 522-8533, Japan; 2 Thailand 

Hornbill Project, Faculty of Science, Mahidol University, Bangkok 

10400, Thailand; 3 Research Institute of Humanity and Nature, 

Kyoto, 602-0878, Japan; 4 National Parks Division, Department 

of National Parks, Wildlife and Plant Conservation, 

Phaholyothin Rd., Bangkok 10900, Thailand. 

Key words : Rodents, Breeding season 

It has been reported that the breeding season of small 

mammals coincide with the onset of the rainy season or fruiting 

season in Southeast Asia. In the Khao Yai National Park having 

distinct rainy and dry seasons, some studies have revealed that the 

fruit availability fluctuates seasonally. The present study was made 

in order to know seasonal reproductive patterns of sympatric rodent 

species inhabiting in Khao Yai National Park, and to study the 

relationship between the rodents breeding season and precipitation 

or fruit availability. The study was carried out in the Khao Yai 

National Park in Thailand from July 2000 to April 2004. The three 

dominant rodent species, Maxomys surifer, Niviventer fulvescens 

and Rattus remotus, were studied. The seasonal captured pattern of 

juveniles suggested that all the species studied had an annual 

breeding season. The two large species, M. surifer and R. remotus, 

bred in the rainy season whereas the smallest specie, N. fulvescens, 

in the dry season. For the two large species, the number of juveniles 

was positively correlated with precipitation with zero or one month 

lag. Although the fruit availability tended to be high in the rainy 

season whereas low in the dry season, no correlation was observed 

between the fruit availability and the number of juveniles for any 

species. In contrast, the number of juveniles of N. fulvescens was 

significantly correlated with the availability of figs with one or two 

month lag. The fruits of figs were considered as important food for 

frugivorous animals in the dry season because they were available 

even in the dry season as well as in the rainy season. The results of 

camera trapping suggested that N. fulvescens preferred figs. Thus it 

is suggested that figs in the dry season may be one of important 

food item for breeding of N. fulvescens. 

(Poster Presentation at The 9 th International Mammalogical 

Congress , Sapporo, Japan 31 July – 5 August 2005. Supported by 

Research Fund of the Japan Society for the Promotion of Science 

(JSPS Research Fund for Basic Science) 

ACTIVITY PATTERNS OF TERRESTRIAL 

MAMMALS OBSERVED BY USING CAMERA 

TRAPPING (NO. 809) 

Shunsuke Suzuki 1 , Shumpei Kitamura 2 , Masahiro Kon 1 , Pilai 

Poonswad 2 , Phitaya Chuailua 2 Kamol Plongmai 2 Takakazu 

Yumoto 3 , Naohiko Noma 1 , Tamaki Maruhashi 4 and Prawat 

Wohandee 5 

1 Graduated School of Environmental Science, The University 

of Shiga Prefecture, Hikone, 522-8533, Japan. ; 2 Thailand 

Hornbill Project, Faculty of Science, Mahidol University, Bangkok 

10400, Thailand; 3 Research Institute of Humanity and Nature, 

Kyoto, 602-0878, Japan.; 4 Department of Human and Culture, 

Musashi University, Tokyo 176-8534, Japan.; 5 National Parks 

Division, Department of National Parks, Wildlife and Plant 

Conservation, Phaholyothin Rd., Bangkok 10900, Thailand. 

Key words: Terrestrial mammals, camera trapping 


Based on the data taken by Camera trapping baited with 

fruits, we described the daily activity patterns of terrestrial mammals 

in the tropical seasonal forest in Khao Yai National Park, Thailand. 

Camera trapping has been widely utilized in ecological researches 

on animals. For the data by camera trapping, there was a problem 

for the definition of one visit of an animal; pictures that have been 

taken consecutively may not necessarily represent independent 

visits.0To overcome this problem, some studies were considered 

consecutive pictures of the same species taken within a certain period 

of time as a single event. In the present study, we utilized logsurvivorship 

curve for defining an independent visit. Twelve species 

for which 30 or more visits had been recorded were classified into 

four categories based on their activity patterns. Two mammals 

(Callosciurus finlaysonii and Macaca nemestrina) were active 

unimodally with a peak around noon, and these species shares an 

arboreal habitat. Two mammals (Menetes berdmorei and Tupaia 

belangeri) were active bimodally with peaks around dawn and dusk, 

and these species are known mainly active on the ground. Five rodent 

species (Hystrix brachyura, Leopoldamys sabanus, Maxomys surifer, 

Niviventer fulvescens, and Rattus remotus) were not active during 

the daytime at all. Three Artiodactyla (Cervus unicolor, Muntiacus 

muntjak and Tragulus javanicus) were active during both the dayand 

nighttimes. In addition, we analyzed the overlap between small 

mammal species in fruit species that the animals visited. 

Consequently, it was revealed that species resembling each other in 

fruit species visiting pattern tended to be segregated temporally 

whereas species having similar activity patterns tended to visit 

different fruit species. 

(Oral Presentation at The 9 th International Mammalogical Congress 

, Sapporo, Japan 31 July – 5 August 2005. Supported by Research 

Fund of the Japan Society for the Promotion of Science (JSPS 

Research Fund for Basic Science) 

COMPARISON OF SPECIES DIVERSITY AND 

POPULATION DENSITY FOR HORNBILLS IN 

THREE DIFFERENT CONSERVATION AREAS 

IN THAILAND WITH COMMUNITY INVOLVE- 

MENT (NO. 810) 

Pilai Poonswad 1 , Siriporn Thong-Aree 2 , Anak Pattanavibool 3 , 

Vijak Chimchome 4 , Saksit Simchareon 2 , Budsabong 

Kanchanasaka 2 , Philip Round 1 Youngyut Trisurat 4 , Mathurose 

Ponglikitmongkol 1 , Nareerat Viseshakul 5 , Parntep Ratanakorn 6 , 

Prawat Wohandee 2 , Sitthichai Mudsri 2 and Nipon Sa-nguanyad 2 


University, Bangkok 10400; 2 National Park, Wildlife and Plant 

Conservation Department , National Park and Wildlife Research 

Division, Natural Resource Conservation office Bangkok 10900, 

Thailand; 3 Wildlife Conservation Society Thailand Program, P.O 

Box 170, Laksi, Bangkok 10210; 4 Department of Forest Biology, 

Faculty of Forestry, Kasetsart University Bangkok 10900; 

5 Department of Pathology, Faculty of Veterinary Science, 

Chulalongkorn University, Bangkok 10330; 6 Faculty of 

Veterinary Science, Mahidol University, Salaya Campus, 

Nakornpathom 73170. 



The forests of Thailand support 13 species of hornbills. 

Hornbills are attractive and interesting by their size, appearance and 

nesting habits. The female hornbill imprisons herself in a natural 

cavity in a large tree, leaving a small opening for her mate to feed 

her and later the chick(s). Through awareness of various ongoing 

threats to hornbill populations, including habitat destruction, 

poaching and felling of potential nest trees, we received funding 

from the National Center for Genetic Engineering and Biotechnology 

(BIOTEC) for a study of the species diversity, population sizes and 

genetic variation across the fragmented forest landscape in various 

parts of Thailand. We used point counts to determine species diversity 

and population density, and we present here the results from our 

2004 studies in three conservation areas. We recorded four species 

in the monsoon tropical forest of Khao Yai National Park (1,965 

km 2 ), including Great (Buceros bicornis), Wreathed (Rhyticeros 

undulatus), Brown (Anorrhinus austeni) and Oriental Pied Hornbills 

(Anthracoceros albirostris). Population density of all species was 

estimated at 3 individuals/10 km 2 . In the montane, dry evergreen, 

mixed deciduous and dry dipterocarp forests of Huai Kha Khaeng 

Wildlife Sanctuary (2,697 km 2 ), we recorded six species, Great, 

Wreathed, Oriental Pied, Tickell’s Brown (Anorrhinus tickelli), 

Rufous-necked (Aceros nipalensis) and Plain-pouched Hornbills (R. 

subruficollis). Population density of all species was estimated at 1 

individual/10 km 2 . Whereas, in the tropical rainforest of Budo 

Sungai-Padi National Park (189 km 2 ), where we recorded six hornbill 

species, Great, Wreathed, Rhinoceros (B. rhinoceros), Helmeted 

(Rhinoplax vigil), Bushy-crested (Anorrhinus galeritus) and Whitecrowned 

Hornbills (Berenicornis comatus), population density of 

all species was estimated at 13 individuals/10 km 2 . The differences 

in densities of hornbills between these three conservation areas may 

be due to differences in the area, type, and perhaps the quality of 

these forests. However, the high density at Budo Sungai-Padi 

National Park may have been influenced by the intensive 

conservation program during the past 10 years. Before 1994, Budo 

Sungai-Padi National Park surrounded by Muslim communities 

experienced heavy poaching of hornbill chicks for the pet trade. 

Within the last 10 years, we have succeeded in converting local 

poachers to become field research assistants and eco-tour guides. 

The former poachers assist with counts of hornbills and help to 

protect hornbills by watching nests throughout the long breeding 

cycle of up to six months during which they gather scientific 

information on nesting behaviour, food and feeding. To subsidize 

the lost income of these local assistants, we developed a “hornbill 

family adoption” program in 1998. By 2004, the 103 sponsors were 

53% Thais and 47% foreigners and they had supported to watch 

over 150 nests of six hornbill species. This program has shown clear 

and progressive development of relationships between urban, rural 

and wilderness values, using hornbills as a tool and at the same time 

ensuring their sustainable conservation. Over the 10 years, we 

estimate that more than 300 hornbill chicks have fledged successfully 

at Budo Sungai-Padi National Park. 

(Oral Presentation at The 8 th World Wilderness Congress, Alaska, 

USA, 30 September - 6 October 2005. Supported by National Center 

for Genetic Engineering and Biotechnology (BIOTEC) and Hornbill 

Research Foundation) 

HOW PROPER MANAGEMENT CAN ASSIST 

HORNBILL CONSERVATION: THE THAILAND 

EXPERIENCE (NO. 811) 



University, Rama 6 Rd., Bangkok 10400. 

Thailand possesses diverse forest habitat types ranging from 

peat swamp forest and mangrove to montane forest. Therefore, she 

supports 13 species of forest hornbills, and they are facing various 

threats. There are two considerably serious threats which affect the 

well-being of hornbill populations. These two are genuine human 

activities, including hunting hornbills for the pet trade and for food, 

and forest destruction. Although hornbill hunting is a temporary 

activity, it may nonetheless extirpate hornbill sub-populations from 

one location within a short period. While forest destruction for 

cultivation, development or land possession causes long-term and 

lasting effects on hornbill populations. Hunting is not impossible to 

manage as compared with forest destruction. To change hunting 

attitudes a number of approaches will be presented. Besides humancreated 

threats, hornbills are inevitably facing natural threats. Due 

to their inability to excavate their own nest cavities, nesting hornbills 

follow a nest-reuse regimen. Thus, another important factor, which 

directly affects nest cavities, arises from natural processes, including 

tree bark regeneration and wood decomposition. Bark regeneration 

causes closure of nest entrances, while wood decomposition causes 

nest floor sinking. Clues for the need for nest cavity monitoring 

include long-unused nests, occurrence of nesting attempts without 

sealing attempts and the abandonment of regularly used nests. These 

unsuitable conditions will result in a shortage of nest cavities, and 

consequently competition occurs. Simple modification of cavities 

to increase the chances of hornbills to breed has been successfully 

proven. 

(Keynote speaker at The 4 th International Hornbill Conference, 

Mabula Game Lodge , South Africa, 6-10 November 2005; 

Supported by the National Center for Genetic Engineering and 

Biotechnology (BIOTEC) and Hornbill Research Foundation, 

Thailand.) 

RELATIONSHIPS BETWEEN FRUITS AND 

HORNBILL ASSEMBLAGES IN LOWLAND 

DIPTEROCARP FORESTS IN SOUTHERN 


Shumpei Kitamura 1 , Pilai Poonswad 1 , Siriporn Thong-Aree 2 , 

Sitichai Madsri 2 , Takakazu Yumoto 3 

1 Thailand Hornbill Project, Department of Microbiology, 


10400, Thailand; 2 National Parks Division, Department of 

National Parks, Wildlife and Plant Conservation, Phaholyothin 

Rd., Bangkok 10900, Thailand; 3 Research Institute of Humanity 

and Nature, Kyoto 602-0878, Japan. 

Interactions among fleshy fruits and hornbill assemblages 

are presented from a 1-y study in lowland dipterocarp forests of 

Budo Sungai Padi National Park and Hala Bala Wildlife Sanctuary 

in Southern Thailand. This community-level approach allowed us 

to determine food selection by hornbills and to evaluate their role in 


308 

seed dispersal. For this, interactions between 272 fruit species and 

six hornbill species (Buceros bicornis, B. rhinoceros, B. vigil, Aceros 

comatus, A. undulatus, Anorrhinus galeritus) were studied in both 

sites. Fruit characteristics were determined for 272 species in 54 

families. Of these 272 fruit species, 123 fruit species (45%) were 

casually observed to be eaten by some frugivores. Of the123 species, 

hornbills consumed at least 50 fruit species. Fruit and seed size were 

the most important physical factors determining food selection of 

hornbills. However, for numerous fruit traits hornbills had no clear 

feeding preferences and they seemed to be quite flexible, eating 

whatever was available. This might be related to unpredictable fruit 

availability and low fruit productivity in lowland dipterocarp forests, 

which may also partially explain the dominance of the 

Dipterocarpaceae. Nevertheless each hornbill species have different 

impacts on seed dispersal. In terms of conservation, heterogeneous 

seed transport is particularly important for this severely fragmented 

lowland dipterocarp forest. (206 words) 

(Oral Presentation at The 4 th International Hornbill Conference, 

Mabula Game Lodge , South Africa, 6-10 November 2005. 

Supported by Mahidol University Government Research Fund, the 

National Center for Genetic Engineering and Biotechnology 

(BIOTEC) and Hornbill Research Foundation, Thailand.) 

HORNBILL POPULATION MONITORING IN 

TROPICAL EVERGREEN FOREST FRAGMENTS 

IN THAILAND: A PRELIMINARY RESULT (NO. 813) 

Anak Pattanavibool 1 , Pilai Poonswad 2 , Narong Jirawatkavi 2 , 

Kamol Plongmai 2 , Vijak Chimchome 3 , Pornkamol Jornburom 2 , 

Sorayut Chaikheiw 2 

1 Wildlife Conservation Society Thailand Program, PO Box 170, 

Laksi, Bangkok 10210, Thailand.; 2 Hornbill Project, c/o 


University, Bangkok 10400, Thailand; 3 Department of Forest 

Biology, Faculty of Forestry, Kasetsart University, Bangkok 10903, 

Thailand. 

Hornbill research and conservation in Thailand led by the 

Hornbill Project has been active for more than 20 years. The main 

focuses were nesting and breeding biology and ecology, and habitat 

utilization. Since 2003, the attempt to estimate population of hornbills 

has been launched in the 3 significant protected areas including Khao 

Yai National Park (KYNP), Western Forest Complex (WEFCOM), and 

Budo-Sungai Padi National Park (BUDO). The target areas differ in 

size, shape, evergreen forest connectivity, and human pressures. KYNP 

is mainly a large continuous evergreen forest tract, WEFCOM is 

composed of small to large evergreen forest patches mixed with 

deciduous forest, grassland, and human-disturbed openings, and BUDO 

is a smallest evergreen forest patches with high degree of human 

disturbance. The main objectives are to estimate density of hornbills 

in evergreen forest fragments and to set up a long-term monitoring of 

hornbill populations and its habitats to keep tracks of change in these 

key hornbill conservation areas. We use point-transect sampling 

technique to survey population. Except Budo, a transect line is 9 km in 

length and survey points is located systematically in interval of 200m 

along the transect trail. There are 45 points/transect. In total, the 

numbers of point in each sample areas are 12 transects of 540 points 

for KYNP, and 10 transects of 450 points for WEFCOM. The Budo’s 

point-transect setup is constrained by its small size, therefore, 13 

transects of 2 km each with 65 points in total were setup. Until today 2 

out of 6 surveys have been conducted and hornbill densities preliminary 


calculated. The current result shows WEFCOM’s hornbill density of 

75 birds/km 2 (95%CI of 52-108 birds/km 2 ), KYNP’s of 28 birds/km 2 

(21-38 birds/km 2 ), Budo’s of 77 birds/km 2 (43-137 birds/km 2 ). The 

rufous-necked hornbill (Aceros nipalensis) has the highest density of 

35 birds/km 2 compared to other species in WEFCOM. The eastern 

brown hornbill (Ptilolaemus tickelli austeni) and oriental-pied hornbill 

(Anthracoceros albirostris) comprise the main hornbill density in KYNP 

of 16 and 15 birds/km 2 respectively. Sample size is not enough in BUDO 

to allow estimation of density by species. The current results imply a 

pattern of high density of hornbills in evergreen forest fragments. 





Thailand.) 

COMPARISON OF THE HELMETED HORNBILL 

AND OTHER BUCEROS SPP. BY MITOCHONDRIAL 

CYTOCHROME B (NO. 814) 

Nareerat Viseshakul 1 , Siriphatr Chamutpong 2 , Suwimol 

Utarnpongsa 2 ,Pilai Poonswad 3 , Siriporn Thong-Aree 4 and 

Mathurose Ponglikitmongkol 2 

1 Department of Pathology, Faculty of Veterinary Science, 

Chulalongkorn University, Bangkok 10330, 2 Department of 

Biochemistry, Faculty of Science, Mahidol University, 


University, Bangkok 10400, 4 National Park, Wildlife and Plant 

Conservation Department, National Park and Wildlife Research 

Division, National Resource Conservation Office, Bangkok 

10900, Thailand. 

Forests of Thailand support 13 Asian hornbill species, which 

consist of 6 major generic groups i.e. Anorrhinus, Anthracoceros, 

Berenicornis, Buceros, Rhinoplax and Rhyticeros. Their 

classification is based on morphology and their breeding behavior. 

While based on the mentioned criteria, Rhinoplax or Helmeted 

Hornbill possessing a relatively large casque, was once classified 

into Buceros spp. In this report, we applied the molecular method 

of DNA sequencing in search of Buceros DNA for comparison. The 

DNA data of Buceros bicornis, B. rhinoceros and Rhinoplax vigil 

were aligned using the cytochrome b gene sequences. These three 

hornbill species were grouped into closed relatives. Although, there 

was a small variation of DNA sequences among them, however, 

Rhinoplax is confirmed for its own generic characteristics. The 

characterization of the D loop region of the mitochondrial DNA 

convinced the differentiation of the concealment among these related 

groups. Therefore, we support and propose the Helmeted Hornbill 

remains as genus Rhinoplax. 





Thailand.) 



DIFFERENTIATION OF THE BUCEROS SPP. 

BY MITOCHONDRIAL D LOOP (NO. 815) 

Mathurose Ponglikitmongkol 1 , Pattarawut Sopha 1 , Wutthipong 

Charoennitikul 1 , Pilai Poonswad 2 , Yupaporn Surapunpitak 3 

and Nareerat Viseshakul 4 


University, 2 Department of Microbiology, Faculty of Science, 

Mahidol University, Bangkok 10400, 3 National Park, Wildlife 

and Plant Conservation Department, National Park and Wildlife 

Research Division, National Resource Conservation Office, 

Bangkok 10900, 4 Department of Pathology, Faculty of 

Veterinary Science, Chulalongkorn University, Bangkok 10330, 

Thailand. 

Asian hornbills are one of the well recognized “Old world 

birds” known for their unique big beak and casque and nesting habit. 

They frequent tropical forests ranging from India, Southern China, 

Myanmar, Thailand, the Philippines, Malaysia and Indonesia. Their 

food and feeding habits make hornbills an important seed dispersal 

agent of wild plants and controlling insect population. Therefore, 

they are good indicators of the healthy forest. Of the 31 species of 

hornbills in Asia, Thailand is home to 13 species which can be 

divided into 6 major genera, including; Rhyticeros, Buceros, 

Anorrhinus, Anthracoceros, Berenicornis and Rhinoplax based on 

their morphology and breeding behavior. We report here the 

comparison of Buceros hornbill DNA using data from the maternal 

inheritance mitochondrial DNA. D3 portions of the D loop in the 

control region of Great Buceros bicornis and Rhinoceros Hornbills 

B. rhinoceros, which were characterized and compared with that of 

the Helmeted Hornbill, Rhinoplax vigil. Each of these three species 

carried its own unique repetitive sequences. The DNA sequences 

revealed the distinction among these three hornbill species, and hence 

confirming different taxonomic grouping. 





Thailand.) 

NESTING AND FLOCKING BEHAVIOR IN 

RELATION TO PHENOLOGY OF NON-FIG 

HORNBILL FOOD PLANTS AT KHAO YAI 

NATIONAL PARK, THAILAND (NO. 816) 

Kamol Plongmai 1 , Chution Savini 2 , Pilai Poonswad 3 , Pithaya 

Chuailua 1 , Prawat Wohandee 4 

1 Thailand Hornbill Project, c/o Department of Microbiology, 


10400, Thailand. ; 2 Faculty of Graduate Studies, Mahidol 

University, Rama 6 Rd., Bangkok 10400, Thailand.; 3 Department 

of Microbiology, Faculty of Science, Mahidol University, Rama 

6 Rd., Bangkok 10400, Thailand; 4 National Park, Wildlife and 

Plant Conservation Department, National Park and Wildlife 

Research Division, Natural Resource Conservation Office, Khao 

Yai National Park, Bangkok 10900, Thailand. 

Food of hornbills comprises two important components, i.e. 

fruit as the main component and animals. Fruits have been proven 

to be the main source of energy for hornbills at Khao Yai. Among 

fruits figs (Ficus spp.), particularly strangling fig, are important to 

hornbills. Most hornbills, especially at Khao Yai, consume figs in 

great proportion. These figs provide ripe fruit crops all year round, 

but wean during certain time of the year. Nevertheless the importance 

of the figs, non-fig fruits have no less importance. Many species of 

them are lipid-rich. This long-term study (1996-2003) we analyze 

phenology of 15 major species of non-fig fruit in relation to annual 

rain fall and activities of hornbills during nesting and flocking. 

Among these 15 species eleven species were important during 

hornbill nesting, while five species were important during flocking. 

Levistona speciosa produced ripe fruit crops in late flocking period 

and extended into the middle of nesting period. Cinnamomum 

subavenum showed regular cycle of seed year at every other three 

years. Whereas the rests of the species produced ripe fruit crops 

irregularly. Reproductive phenology of all 15 species (N = 111 trees) 

showed: flowering to fruiting phases ranging from 7-19 mons 

(11.7±3.2 mons); flowering phase 1-15 mons (4.7±3.0 mons); 

fruiting phase 3-6 mons (7.7±2.8 mons) and ripe fruit 1-6 mons 

(2.7±0.5 mons). Cinnamomum subavenum fruit crops showed strong 

influence on nesting, whereas Mastacia pentandra influenced 

flocking of Wreathed Hornbill (Aceros undulatus). 

(Poster Presentation at The 4 th International Hornbill Conference, 


Supported by Thailand Hornbill Project and Hornbill Research 

Foundation, Thailand.) 

CONSERVATION OF HORNBILLS IN THAILAND 


(NO. 817) 


University, Rama 6 Rd. Bangkok 10400, Thailand. 

The study of breeding biology and ecology of hornbills have 

started since 1978. Study was conducted initially in the monsoon 

evergreen forest of Khao Yai National Park (2,168 km 2 ) in 

northeastern Thailand. Four species of hornbills occurred 

sympatrically in 60 km 2 study area in Khao Yai National Park, 

namely Great (Buceros bicornis), Wreathed (Aceros undulatus), 

White-throated Brown (Anorrhinus austeni) and Oriental Pied 

Hornbills (Anthracoceros albirostris). In 1990, the research has been 

extended to Huai Kha Khaeng Wildlife Sanctuary (2,809 km 2 ) in 

the west. Four species of hornbills occurred sympatrically in a hill 

evergreen forest namely Rufous-necked (Aceros nipalensis), Tickell’s 

Brown (Anorrhinus tickelli) and Great Hornbills. Research on Plainpouched 

Hornbill (Aceros subruficollis) and sympatric species 

Oriental Pied and Great has also been conducted in a mixed 

deciduous forest of Huai Kha Khaeng Wildlife Sanctuary. To cover 

all 13 hornbill species in Thailand, in 1994, we have extended the 

study into Budo-Sungai Padi National Park (341 km 2 ) in the south. 

This park supports six species of hornbills, Great, Wreathed, 

Helmeted (Rhinoplax vigil), Rhinoceros (Buceros rhinoceros), 

Bushy-crested (Anorrhinus galeritus) and White-crowned Hornbills 

(Berenicornis comatus). We collected data on nests and nest trees, 

breeding cycle, nest site characteristics, food, flocking and home 

range. We identified the causes of nests damage and monitored intra 

and inter-specific competition. At Khao Yai, competition for nest 

cavities was 40% reflected shortage of suitable nest cavities whereas 

at Budo Mt., the competition was 26% indicating the effect of 

poaching. We also investigated the threats to the breeding of 


310 

hornbills. Subsequently, we increase breeding success and 

conservation awareness through nest cavities improvements, ceased 

poaching of hornbill chicks and getting community involvement in 

hornbill conservation. Currently supported by BIOTEC, we are 

investigating the characteristics of Phylogenetic of Thai hornbills 

and genetic variations of hornbills in fragmented forest landscapes 

and determining their population and habitat status throughout 

Thailand. 

(Oral Presentation (Special Topic) at Department of Biotechnology, 

Faculty of Science, Mahidol University, Bangkok, 4 August 2005. 

Supported by Thailand Hornbill Project, Hornbill Research 

Foundation and The National Center for Genetic Engineering and 

Biotechnology (BIOTEC) 

STUDY ON THE POLYMORPHISM OF MULTIPLE 

LOCI OF VARIABLE NUMBER OF TANDEM 

REPEATS IN MYCOBACTERIUM TUBERCULOSIS 

(NO. 818) 

1 Nat Smittipat; 1 Pamaree Billamas; 2 Manee Palittapongarnpim; 

2 Arunee Thong-On; 2 Mansuet M. Temu; 3 Prathep 

Thanakijcharoen; 3 Opart Karnkawinpong; 2 Prasit 

Palittapongarnpim 


Phatumthani, 2 Department of Microbiology, Faculty of Science, 

Mahidol University, Bangkok, and 3 Amnat Charoen Hospital, 

Amnat Charoen, Thailand. E-mail: Prasit@biotech.or.th 

Key words : VNTR, polymorphisms, Mycobacterium Tuberculosis 

Genotyping based on variable number of tandem repeats 

(VNTR) is currently a very promising tool for studying the molecular 

epidemiology and phylogeny of M. tuberculosis. Here, we 

investigated the polymorphisms of 48 loci of direct or tandem repeats 

in M. tuberculosis previously identified by our group. Thirty-nine 

loci including nine novel ones are polymorphic. Ten VNTR loci 

had high allelic diversity (Nei’s diversity values ≥0.6) and 

subsequently were used as the representative VNTR-typing set for 

comparison to IS6110-based restriction fragment length 

polymorphism (RFLP) typing. The 10-loci VNTR set, potentially 

providing >2×10 9 allele combinations, obviously showed the 

discriminating capacity over IS6110-RFLP method for M. 

tuberculosis isolates with less than 6 IS6110-hybridized bands 

whereas it had equivalent resolution to the IS6110-RFLP for the 

isolates having >5 IS6110 bands. Allelic diversity of many VNTR 

loci varied in each IS6110 type. Genetic relationships inferred from 

the 10-VNTR set supported the notion that M. tuberculosis may be 

evolved from two different lineages (high and low-IS6110 copy). In 

addition, we found that many VNTR loci had significant 

relationships to each other. These relationships could cause a 

restriction of the VNTR-typing discriminating capability to some 

extent. Our results suggested that VNTR-PCR typing is practically 

useful for application to molecular epidemiological and phylogenetic 

studies of M. tuberculosis. The discriminating power of VNTRtyping 

system could still be enhanced by the supplementation of an 

extra number of (new) VNTR loci. 

(Journal of Clinical Microbiology 43 (10), (2005): (Accepted). 


RESTRICTION FRAGMENT LENGTH POLY- 

MORPHISM STUDY OF NATION-WIDE SAMPLES 

OF MYCOBACTERIUM TUBERCULOSIS IN 

THAILAND, 1997-1998 (NO. 819) 

1 Dhanida Rienthong; 2 Pravech Ajawatanawong; 1 Somsak 

Rienthong; 3 Angkana Chaiprasert ; 1 Saijai Smithtikarn; A 

Pasakorn; 4 Prasit Palittapongarnpim. 

1 Tuberculosis Division, Department of Disease Control, Bangkok, 


Phatumthani, 3 Department of Microbiology, Faculty of 

Medicine, Siriraj Hospital and 4 Department of Microbiology, 

Faculty of Science, Mahidol University, Bangkok, Thailand. Email: 

Prasit@biotech.or.th 

Key words: RFLP, recent transmission, Mycobacterium 

tuberculosis 

Setting : During 1997-1998, a national anti-tuberculosis 

drug resistance surveillance was conducted in Thailand as a part of 

a global project. 

Objective : To evaluate the IS6110-hybridization patterns 

and the level of clustering, which was expected to be low due to the 

short duration of the sample collection. 

Design : 828 bacterial isolates were available for 

fingerprinting by standard IS6110 hybridization. 

Results : The RFLP patterns varied with geographic 

locations, ages of the patients, and resistance to rifampin and 

streptomycin. The Beijing strains were more common among 

younger patients and their prevalence appeared to decrease with the 

distance from Bangkok, while the opposite was true for the singlebanded 

isolates. Excluding isolates containing 5 or less copies of 

IS6110, 26.4 % were clustered. The clustering was more common 

among female. The clustered isolates were sometimes from different 

provinces and, if resistant to drugs, usually possessed different 

resistance profiles. 

Conclusions : The results raised a question of the validity 

of inferring the recent transmission from the clustering of IS 6110hybridization 

patterns in some settings in Thailand. Evaluating the 

level of recent transmission in a nation-wide study in a country with 

high incidence of tuberculosis should be done cautiously. 

(International Journal of Tuberculosis Lung Disease 9(5), (2005): 

576-581.) 

BURKHOLDERIA PSEUDOMALLEI INVASION 

AND ACTIVATION OF EPITHELIAL CELLS 

REQUIRES ACTIVATION OF P38 MITOGEN- 

ACTIVATED PROTEIN KINASE (NO. 820) 

P. Utaisincharoen 1 , S. Arjcharoen 1 , I. Lengwehasatit 2 , K. 

Limposuwan 1 , S. Sirisinha 1 


University; 2 Department of Biotechnology, Faculty of Science, 

Mahidol University. E-mail: scput@mahidol.ac.th 



Key words: Burkholderia pseudomallei, melioidosis, p38 MAP 

kinase, human lung epithelial cell line, IκBα 

Burkholderia pseudomallei is a causative agent of 

melioidosis. This gram-negative bacterium is able to survive inside 

the macrophages and also able to invade non-phagocytic cells 

including epithelial cells. Interaction of pathogenic bacteria to the 

host cells is frequently associated with activation of mitogenactivated 

protein (MAP) kinases signaling activity. In this study, we 

demonstrated that B. pseudomallei stimulated p38 MAP kinase of 

human alveolar lung epithelial cell line (A549). Phosphorylation of 

p38 was observed after 15 min, attained a maximal level at 60 min 

after the infection. A specific inhibitor of p38 phosphorylation, SB 

203580, was able to inhibit invasion of this bacterium into the cells 

suggesting that invasion of B. pseudomallei required activation of 

p38. In contrast, wortmannin which is a specific inhibitor of 

phosphoinositide 3-kinase (PI3-kinase) failed to inhibit the invasion. 

Moreover, SB 203580 can also interfere with IkBa degradation and 

IL-8 mRNA expression, indicating that the phosphorylation of p38 

occurred upstream of NF-kB activation. Cytochalasin D, an inhibitor 

of actin polymerization needed for internalisation of bacteria, did 

not have any effect on the phosphorylation of p38. These results 

indicate that B. pseudomallei stimulate phosphorylation of p38 

making by initial contact with the cell surface components and do 

not require internalisation and interaction with intracellular 

cytoplasmic components of the cells. 

(Microb Pathogenesis 38, (2005); 107-112.) 

DENGUE VIRUS-ENHANCING ANTIBODY 

POSSIBLY SUPPRESSES NITRIC OXIDE 

PRODUCTION VIA IL-10 MEDIATED 

SUPPRESSIVE PATHWAY (NO. 821) 

Takol Chareonsirisuthigul 1 , Siriphen Kalayanarooj 2 , Sukathida 

Ubol 1* 

1 Department of Microbiology, Faculty of Science Mahidol 

University, Bangkok, Thailand; 2 WHO Collaborating Centre 

Case Management of Dengue/DHF/DSS, Queen Sirikit National 

Institute of Child Health, Bangkok Thailand. 

Key words : Dengue virus, Enhancing antibody, Nitric oxide 

Background: Dengue virus (DV) causes a spectrum of 

clinical manifestation ranging from asymptomatic infection, classical 

dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue 

shock syndrome (DSS). Recently, we demonstrated that patients who 

developed DHF during secondary infection generate low level of 

nitric oxide (NO) but high level of viral load and vice versa for DF 

patients. Thus, NO suppression might be one of the factors which 

determine high viremia stage in secondary DHF patients. Since this 

phenomenon occurred in secondary DHF patients but not in primary 

DHF or DF patients, therefore, we hypothesized that enhancing 

antibody may play a role on NO production suppression. In the 

presence study, we performed in vitro experiments to test the 

suppressive effect of enhancing antibody on NO production in human 

monocyte cell line infected with DV. The suppressive mechanism 

was partially investigated. 

Methods: Human monocyte cells, THP-1, were infected 

with DV in the presence or absence of enhancing antibodies. Viral 

production in supernatant was quantitated by real-time RT-PCR. 

Amount of NO in supernatant was measured by Griess reaction. Level 

of IL-10, IL-12 and IFN-ã productions were determined by ELISA. 

Results: In the presence of antibodies from DHF patients, 

enhancing viral replication was found, 3.77±1.81x10 5 versus 

3.64±2.56x10 4 copies/ml for the presence and absence of DHF 

antibody, respectively. The synthesis of NO was suppressed in the 

presence of antibody (4.43±2.62 versus 8.36±2.83 µM). IL-10 

production was 98.87±4.51 versus 6.28±0.32 pg/ml for the presence 

and absence of enhancing antibody, respectively. Level of IL-12 and 

IFN-γ productions were undetectable in our studies by ELISA. 

Conclusions: Our study demonstrated that sera from DHF 

patients enhanced replication of DV by down regulation of NO 

production. The possible mechanism of antibody mediated NO 

suppression is via IL-10 activity. Our hypothesis is supported by 

studies on Ross River virus (RRV) which IL-10 diminished NO 

production via suppression of transcription factor (STAT-1 and NFκB) 

complexes. 

IMMUNOHISTOLOGICAL CHARACTERIZATION 

OF MACROPHAGE MIGRATION INHIBITORY 

FACTOR EXPRESSION IN PLASMODIUM 

FALCIPARUM-INFECTED PLACENTAS. (NO. 822) 

Sujittra Chaisavaneeyakorn 1,5,6 , Naomi Lucchi 2 , Carlos 

Abramowsky 3 , Caroline Othoro 4 , Sansanee C. Chaiyaroj 6 , Ya 

Ping Shi 1,4 , Bernard L. Nahlen 1,7 , David S. Peterson 2 , Julie M. 

Moore 2 , and Venkatachalam Udhayakumar 1 

Division of Parasitic Diseases, National Center for Infectious 

Diseases, Centers for Disease Control and Prevention, Public 

Health Service, U.S. Department of Health and Human Services, 

Atlanta, Geolgia 30333 1 ; Center for Tropical and Emerging 

Global Diseases and Department of Infectious Diseases, College 

of Veterinary Medicine, University of Georgia, Athens, Georgia 

30602 3 , Department of Pathology, Egleston Children’s Hospital, 

Emory University School of Medicine, Atlanta, Georgia 30322 3 , 

Vector Biology and Control Research Center, Kenya Medical 

Research Institute, Kisumu, Kenya 4 ; Atlanta Research and 

Education of Foundation, Atlanta, Georgia 30033 5 , Department 

of Microbiology, Faculty of Science, Mahidol University, 

Bangkok 10400, Thailand 6 ; and Roll Back Malaria, World 

Health Organization, Geneva, Switzerland 7 

Key words: macrophage migration inhibitory factor (MIF), 

intervillous blood (IVB), Plasmodium falciparum 

Previously, we have shown that macrophage migration 

inhibitory factor (MIF) was highly elevated in the placental 

intervillous blood (IVB) of Plasmodium falciparum-infected women. 

Here, we compared the expression of MIF in placental tissues 

obtained from P. falciparum-infected and -uninfected women. 

Immunoperoxidase staining showed a consistent pattern of MIF 

expression in syncytiotrophoblasts, extravillous trophoblasts, IVB 

mononuclear cells, and amniotic epithelial cells, irrespective of their 

malaria infection status. Cytotrophoblast, villous stroma, and 

Hofbauer cells showed focal staining. Only amniotic epithelial and 

IVB mononuclear cells from P. falciparum-infected placentas 

exhibited significantly higher level of MIF expression than 

uninfected placentas. Stimulation of syncytilized human trophoblast 

BeWo cells with P. falciparum-infected erythrocytes that were 

selected to bind these cells resulted in significant increases in MIF 


312 

secretion, whereas control erythrocytes, lipopolysaccharides, and 

synthetic beta-hematin had minimal effect. These findings suggest 

that placental malaria modulates MIF expression in different 

placental compartments. 

(Infection Immunity 73, (2005); 3287-3293.) 

IN VITRO INTERACTION OF HUMAN MONOCYTE- 

DERIVED MACROPHAGES WITH PENICILLIUM 

MARNEFFEI CONIDIA (NO. 823) 

Yuttana Srinoulprasert and Sansanee Chaiyaroj 


University, Bangkok, THAILAND. 

Key words: Penicillium marneffei, pattern recognition receptors, 

human Monocyte-derived macrophage 

Objectives: Penicillium marneffei is a thermal dimorphic 

fungus, which causes an opportunistic infection, penicilliosis, in 

immunocompromised patients in south Asia and southeast Asia. 

Little is known on innate immune response to P. marneffei infection. 

We therefore attempt to evaluate initial response of macrophage to 

P. marneffei conidia. 

Methods: Human macrophages were derived from 

peripheral blood monocytes isolated from healthy volunteers. 

Adhesion between the macrophages and the fungal conidia was 

examined and specifically inhibited by monoclonal antibodies 

against pattern recognition receptors, such as mannose receptor 

(MR), Toll-like receptor (TLR)1, TLR2, TLR4, TLR6, CD14, αL, 

αM and β2 integrins. To study the consequence of interaction 

between macrophages and conidia, levels of cytokine production 

from macrophages co-cultured with the conidia were examined by 

the use of ELISA. 

Results: Binding of P. marneffei conidia to macrophages 

was significantly inhibited by monoclonal antibodies against MR, 

TLR1, TLR2, TLR4, CD14, αM and β2 integrins, in a dosedependent 

manner. Levels of TNF-1α and IL-1β?production from 

macrophages co-cultured with the conidia were significantly higher 

than that of macrophages alone. In contrast, decreased level of IL- 

10 from macrophages challenged with conidia was observed. 

Reduction in TNF-α released by macrophages stimulated with P. 

marneffei conidia was observed in the assays containing anti-TLR4 

and anti-CD14 antibodies. We also found that the production of 

cytokines (TNF-α, IL-1β, and IL-10) from macrophages was partially 

impaired when heat-inactivated autologous serum was omitted in 

the assay. 

Conclusions: These results show that P. marneffei conidia 

are recognized by variety of receptors on human macrophages for 

its initial interaction. P. marneffei conidia are able to induce TNF-α 

and IL-1β production from macrophages. However, the conidia 

seemed to suppress IL-10 secretion from macrophages. Involvement 

of the interaction between TLR4, CD14 on macrophages and conidia 

plays a role on TNF-α production. In the present study, certain serum 

components are required for the initial interaction to PRR resulting 

in cytokine production from macrophages. 


IMMUNOINFORMATICS FOR IMMUNO- 

LOGICAL ASSESMENT OF FUNGAL 

VIRULENCE FACTORS : VALIDATION OF 

CANDIDA ALBICANS VIRULENCE ANTIGENS 

(NO. 824) 

Songsak Tongchusak 1 , Sansanee Chaiyaroj 1 and V. Brusic 2 


University, Bangkok, Thailand 10400; 2 Institute for Infocomm 

research, 21 Heng Mui Keng Terrace, Singapore 119613. 

Key words: immunoinformatics, Candida albicans, Data 

warehousing 

Objectives: Candida albicans is a fungus responsible for 

opportunistic infection in humans. The infection can occur in either 

immunocompromised or immunocompetent individuals. Here we 

constructed a subject-specific database of C. albicans functional 

virulence factors and perform an immunological validation of fungal 

antigenic peptide antigens. 

Methods: C. albicans virulence protein entries and 3Dstructures 

were gathered from primary sequence databases by key 

word search and BLAST. Construction of C. albicans virulence 

factor database (CandiVF) was done by using a computational data 

warehousing program called BioWare. Prediction of HLA-DR 

binding peptide epitopes was demonstrated by Hotspot Hunter 

algorithm. For B-cell prediction, calculation of surface accessibility 

was computed by Naccess. Highly expose amino acid residues were 

visualized by Raswin. Additionally, B-cell epitopes were predicted 

from high accessibility contact residues. 

Results: The database, CandiVF contains 153 virulence 

proteins of C. albicans. This dataset comprises of 457 primary 

protein sequences. Eight structures of C. albicans were retrieved 

from PDB and 35 models were generated using homology modeling. 

These data were used to build Candida albicans virulence factors 

database or “CandiVF”. The database was integrated with 

bioinformatic tools including keyword search, BLAST, and JMOL 

structure-viewer. The CandiVF database is accessible at http:// 

sdmc.i2r.a-tar.edu.sg/Templar/DB /PFAD/. A total 211 entries of 

secretory proteins and cell wall-associated proteins were selected 

for analyses of HLA-DR binding and B-cell epitope prediction. The 

predicted peptide sequences will be presented. 

Conclusions: The use of bioinformatics tools with data 

warehousing system will provide further insight for understanding 

pathogenesis of C. albicans infections and also help the discovery 

of new targets for the development of vaccines and therapeutics 

against candidiasis. 

HOST-PATHOGEN INTERACTION AND INNATE 

IMMUNE RESPONSE OF PENICILLIUM 

MARNEFFEI INFECTION (NO. 825) 

Sansanee Chaiyaroj, Yuttana Srinoulprasert, Piyapong 

Pongtanalert 





Key words: P. marneffei, Pattern recognition receptors (PRRs), 

proinflammatory cytokines. 

Penicillium marneffei is a thermal dimorphic fungus, which 

causes an opportunistic infection, penicilliosis, in 

immunocompromised individuals. Very little is known about initial 

interaction and innate immune response between host cells and P. 

marneffei conidia. We hypothesized that P. marneffei conidia may 

adhere to extracellular matrix (ECM) and lung epithelial cells after 

inhalation. Therefore an investigation on the role of various ECM 

molecules as targets for the conidial adhesion was carried out using 

competitive binding inhibition assay to lung epithelial cells (A549). 

Chondroitin sulfate (CS) B, heparin, heparan sulfate, and chitosan 

polysulfate showed significant inhibitory effect on conidial adhesion 

to A549 cells. Similarly, the forementioned substances also inhibited 

conidial adhesion to fibronectin and laminin. Additionally, the levels 

of conidial adhesion to A549 cells decreased when the cells were 

treated with chondroitinase ABC, but not chondroitinase ACII. The 

results indicated that pattern of IdoA-(2S)GlcNAc of CSB or other 

molecules containing resemble disaccharide pattern have influence 

on the adhesion of the conidia to lung epithelial cells and ECM 

proteins. We further investigated the involvement of alveolar 

macrophage and dendritic cell once the fungal infection in lung 

alveoli. Significant role of TLR1, 2, 4, CD14 and mannose receptor 

(MR) on the initial interaction of macrophages and dendritic cells 

with the fungal conidia and yeasts was observed. TLR2, MR, and 

DC-SIGN were also involved in yeast internalization by human 

dendritic cells. Such interactions of P. marneffei conidia to these 

receptors contributed to subsequent production of TNF-α from 

macrophages. Altogether, the results demonstrated the activation 

of PRRs upon conidial adhesion leads to a release of proinflammatory 

cytokines by macrophage as well as mediating yeast endocytosis. 

INNATE IMMUNE RESPONSE OF PENICILLIUM 

MARNEFFEI INFECTION (NO. 826) 

Sansanee C. Chaiyaroj, Yuttana Srinoulprasert, and Piyapong 

Pongtanalert 


University, Bangkok, THAILAND 

Key words : Penicillium marneffei, TLRs, MR 

Penicillium marneffei is a thermal dimorphic fungus, which 

causes an opportunistic infection, penicilliosis, in 

immunocompromised individuals. Very little is known about innate 

immune response between host cells and P. marneffei conidia. We 

therefore attempt to evaluate initial responses of macrophage and 

dendritic cell once the fungal infection in lung alveoli. 

Human macrophages and dendritic cell were derived from 

peripheral blood monocytes isolated from healthy volunteers. 

Adhesion between the macrophage or the dendritic cell and the fungal 

conidia was examined and specifically inhibited by monoclonal 

antibodies against pattern recognition receptors, such as mannose 

receptor (MR), Toll-like receptor (TLR)1, TLR2, TLR4, TLR6, 

CD14, αL, αM and β2 integrins. Flow cytometric analysis was 

employed to evaluate yeast internalization by dendritic cell. To study 

the consequence of interaction between macrophages and conidia, 

levels of cytokine production from macrophages co-cultured with 

the conidia were examined by the use of ELISA. 

Significant role of TLR1, 2, 4, CD14 and mannose receptor (MR) 

on the initial interaction of macrophages and dendritic cells with 

the fungal conidia and yeasts was observed, in a dose-dependent 

manner. Levels of TNF-1α and IL-1β production from macrophages 

co-cultured with the conidia were significantly higher than that of 

the macrophages alone. In contrast, decreased level of IL-10 from 

macrophages challenged with conidia was observed. Reduction in 

TNF-α released by macrophages stimulated with P. marneffei conidia 

was observed in the assays containing anti-TLR4 and anti-CD14 

antibodies. Such interactions of P. marneffei conidia to these 

receptors contributed to subsequent production of TNF−α from 

macrophages. Additionally, TLR2, MR, and DC-SIGN also involved 

in yeast internalization by human dendritic cells. Altogether, the 

results demonstrated the activation of PRRs upon conidial adhesion 

that leads to the release of proinflammatory cytokines by macrophage 

as well as mediating yeast endocytosis. In addition, we also found 

that the production of cytokines (TNF?α? IL-1β? and IL-10) from 

macrophages was partially impaired when heat-inactivated 

autologous serum was omitted in the assay. 

These results show that P. marneffei conidia and yeasts are 

recognized by variety of receptors on human macrophage and 

dendritic cell for its initial interaction. P. marneffei conidia are able 

to induce pro-inflammatory cytokine productions from macrophages. 

However, the conidia seemed to suppress anti-inflammatory cytokine 

secretion from macrophages. Lastly, certain serum components are 

required for the initial interaction to PRR resulting in cytokine 

production from macrophages. 

(Abstract 3 rd APSMM ) 

ANTI-HIV-1 CONSTITUENTS FROM CLAUSENA 

EXCAVATA : PART II. CARBAZOLES AND A 

PYRANOCOUMARIN (NO. 827) 

Boonsong Kongkathip 1 , Ngampong Kongkathip 1 , Arunrat 

Sunthitikawinsakul 1 , Chanita Napaswat 2 and Chalobon 

Yoosook 1 

1 Natural Product and Organic Synthesis Research Unit, 

Department of Chemistry, Faculty of Science, Kasetsart 

University, Bangkok 10900,Thailand; 2 Department of 

Microbiology, Faculty of Science, Mahidol University, Bangkok 


Key words: Clausena excavata; anti-HIV-1 activity 

Three carbazole derivatives, O-methylmukonal (1), 3formy1-2,7-dimethoxycarbazole 

(2) and clauszoline J (3), and a 

pyranocoumarin, clausenidin (4), were isolated form the rhizomes 

and roots of Clausena excavats. Compound 1, isolated form this 

plant form the first time, has not been reported previously as having 

anti-HIV-1 activity. Compounds 1-4 displayed anti-HIV-1 activity 

in a syncytial assay with EC 50 values of 12, 29.1, 34.2 and 5.3 μM, 

respectively, and thus exhibited potential therapeutic index (PTI) 

values of 56.7, 8.0, 1.6 and 7.0 respectively, All isolated compounds 

demonstrated a lack of cytotoxicity against the KB and BC-1 cancer 

cell lines. 

(Phytotherapy Research 19, (2005); 728-731. Supported by the 

Thailand Research Fund under the Royal Golden Jubilee Program 

and the Biodiversity Research and Training Program.) 


314 

DETECTION OF LYMPHATIC WUCHERERIA 

BANCROFTI IN CARRIERS AND LONG-TERM 

STORAGE BLOOD SAMPLES USING SEMI- 

NESTED PCR (NO. 828) 

P. Kanjanavas 1 , P. Tan-ariya 2 , P. Khawsak 1 , A. Pakpitcharoen 1 , 

S. Phantana 3 , K. Chansiri 1 

1 Department of Biochemistry, Faculty of Medicine, 

Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, 

Thailand; 2 Department of Microbiology, Faculty of Science, 

Mahidol University, Bangkok 10400, Thailand; 3 Filariasis 

Division, Ministry of Public Health, Nontaburi, Thailand. 

Key words: Wuchereria bancrofti; Semi-nested PCR 

The semi-nested PCR was conducted for detection of 

Wuchereria bancrofti in patients’ blood. The results demonstrated 

that the semi-nested PCR could detect as little as 0.001 fg of parasite 

DNA. In addition, the primers showed no PCR amplification from 

human and other hemoparasites such as Brugia malayi, Plasmodium 

falciparum and P. vivax DNAs. This technique was used for detection 

of 18 W. bancrofti infected blood samples with a long-term storage, 

the data revealed that all samples were positive, the results obtained 

from this study clearly indicated that the semi-nested PCR is specific. 

(Molecular and Cellular Probes 19, (2005); 169-172) 

GENOTYPIC STUDY OF PHEUMOCYSTIS 

JIROVECII IN HUMAN IMMUNODEFICIENCY 

VIRUS-POSITIVE PATIENTS IN THAILAND (NO. 829) 

Suradej Siripattanapipong 1 , Jeerapun Worapong 2 , Mathirut 

Mungthin 3 , Saovanee Leelayoova 3 and Peerapan Tan-ariya 1 


University, Bangkok 10400, Thailand; 2 Department of 

Biotechnology, Institute of Science and Technology of Research 

and Development, Mahidol University, Salaya, Nakornpratom 

73170, Thailand; 3 Department of Parasitology, 

Phramongkutklao College of Medicine, Ratchawithi Rd., 


Key words: Pneumocystis jirovecii, genotypic, (HIV)-infected 

patients 

Pneumocystis jirovecii is one of the common opportunistic 

infections in human immunodeficiency virus (HIV)-infected patients 

in Thailand. Information regarding genotypic and epidemiological 

of this organism in Thai patients is not available. We analyzed the 

genotypes of 28 Pneumocystis jirovecii-positive specimens from 

bronchoalveolar lavage and sputum samples from HIV-infected Thai 

patients based on nucleotide variations of the internal transcribed 

spacer regions 1 and 2 of the rRNA gene. Thirteen genotypes were 

the same as previously reported outside Thailand. Ten genotypes, 

which included Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb and Qq, were new. 

Ir and Rp were unique and dominant types observed in HIV-infected 

Thai patients. Thirteen specimens (46.4%) were infected with a 

single type of Pneumocystis jirovecii, and fifteen (53.6%) mixed 

infections. These differences may be used as genotypic markers for 

studying the epidemiology and transmission of Pneumocystis 

jirovecii in the Thai population. 

(Clinical Microbiology, Vol.43, No.5 (2005); 2104-2110 Supported 

by the Thailand-Tropical Diseases Research Program (T-2) 


3-NITROPROPIONIC ACID (3-NPA), A POTENT 

ANTIMYCOBACTEDRIAL AGENT FRO ENDO- 

PHYTIC FUNGI : IS 3-NPA IN SOME PLANTS 

PRODUCED BY ENDOPHYTES? (NO. 830) 

Porntep Chomcheon 1 , Suthep Wiyakrutta 2 , Nongluksna 

Sriubolmas 3 , Nattaya Ngamrojanavanich 1 , Duangnate 

Isarangkul 4 , and Prasat Kittakoop 5 

1 Program of Biotechnology and Department of Chemistry, 

Faculty of Science, Chulalongkorn University, Thailand; 


University, Thailand; 3 Department of Microbiology, Faculty of 

Pharmaceutical Sciences, Chulalongkorn University, Thailand; 

4 Center for Biotechnology, Institute of Science and Technology 

for Research and Development, Mahidol University, Thailand; 

and 5 National Center for Genetic Engineering and Biotechnology 

(BIOTEC), National Science and Technology Development 

Agency (NSTDA), Thailand Science Park, Pathumthani, 

Thailand. 

Key words: 3-nitropropionic acid, antimycobacterial agent, 

endophytic fungi 

3-Nitropropionic acid (3-NPA) was found in extracts of 

several strains of endophytic fungi. 3-NPA exhibited potent 

antimycobacterial activity with the minimum inhibition 

concentration of 3.3 uM, but was inactive against NCI-H187, BC, 

KB, and Vero cell lines. Endophytes were found to produce high 

levels of 3-NPA, and therefore 3-NPA accumulated in certain plants 

may be produced by the associated endophytes. 3-NPA may be used 

as a chemotaxonomic marker for endophytic fungi. The structure of 

3-hydroxypropionic acid, a nematicidal agent, should be revised to 

3-NPA. 

(Published in Journal of Natural Products 68, (2005); 1103-1105) 

MIXED LEPTOSPIRE INFECTION OF SMALL 

MAMMALS IN THAILAND (NO. 831) 

Doungchawee G. 1* , Phulsuksombat D. 2 , Naigowit P. 3 , 

Khoaprasert Y. 4 , Sangjun N. 2 , Chalermsanyakorn P. 5 , Smythe L. 6 

1 Department of Pathobiology, Science, Mahidol University, 

Bangkok, Thailand. 2 Thai Component, Armed Force Research 

Institute of Medical Sciences, Bangkok, Thailand. 3 Research 

Center for Leptospira Laboratory, National Institute of Health, 

Nonthaburi, Thailand. 4 Agricultural Zoology Research 

Group,Department of Agriculture,Bangkok, Thailand. 

5 Department of Pathology, Ramathibodi Hospital, Mahidol 

University, Bangkok, Thailand. 6 WHO/FAO Collaborating 

Centre for Reference and Research on Leptospirosis, WHO 

Western Pacific Region and Queensland Scientific Services, 

Australia. 

Key words : Direct Immunofluorescence, pathogenic Leptospira, 

Rodent leptospirosis 

Background: In an attempt to identify the source of 

leptospires pathogenic to humans, small wild mammals were trapped 

during leptospirosis outbreaks in a rural area of the Country in 1999- 

2000 and subsequent study of an urban area in 2002. 

Methods: All rodents from the rural source were examined 

using kidney isolation and those found positive for leptospires were 

approximately 15% (190 of 1310). A developed direct 



immunofluorescent assay (DFA) was considered as an alternative 

method to leptospire culturing. The DFA evaluated in this study 

was effective for the detection of acute leptospirosis and exhibited 

high sensitivity (100%) and specificity (94%). 

Results: Among the ten different species of wild animals 

captured, R. norvegicus had the highest leptospire infection rate as 

revealed by both detection methods. Moreover, more than a half the 

proportion of cases positive by DFA had evidence of mixed infection 

with three or more different serovars of pathogenic Leptospira spp. 

in the kidneys.From 190 isolates of rural rodents,137 were serotyped 

by cross agglutinin absorption and microscopic agglutination 

tests,showing some similarities and variations to the serovar level 

when compared with the DFA. The detection of leptospires in the 

kidneys of urban rats and shrews was 33 % (14 of 42), twice that of 

rural rodents. Overall, the most commonly infecting serovars were 

autumnalis and canicola in both the rural and urban areas. 

Conclusion: This finding suggests that wild rodents and 

shrews play an important source of multiple pathogenic leptospires 

in both areas and need active surveillance measure to evaluate the 

epidemiology of the disease. 

(Oral Presentation : At the the fourth scientific meeting of the 

International Leptospirosis Society (ILS).The Central Duangtawan 

Hotel, Chiang Mai, Thailand. November 14-16, 2005.) 

INVESTIGATION ON EXTRACTED OUTER 

MEMBRANE SHEATH OF 5 LEPTOSPIRAL 

SEROVARS BY ELECTRON MICROSCOPY AND 

IMMUNOBLOT ASSAY (NO. 832) 

Prukswan Chetanachan, 1 Galayanee Doungchawee, 2 Umaporn 

Seena, 3 Mayurachat Biaklang, 3 Suchada Geawduanglek 2 and 

Pimjai Naigowit 4 

1 National Institute of Health, Department of Medical Sciences, 

Ministry of Public Health, Nonthaburi, 11000, Thailand. 

2 Department of Pathobiology, Science, Mahidol University, 

Bangkok, 10400, Thailand.; 3 Medical Biotechnological Center, 

Department of Medical Sciences, Ministry, Ministry of Public 

Health, Nonthaburi, 11000, Thailand. 4 Technical Office, 

Department of Medical Sciences, Ministry of Public Health, 

Nonthaburi, 11000, Thailand. 

The purpose of this study was to identify the structure and 

specificity of Outer Membrane Sheath (OMS) extracts of 5 different 

serovars of Leptospira spp.: autumnalis, bratislava, sejroe, 

icterohaemorrhagiae and patoc. The leptospiral outer membrane 

fraction (OMS) was prepared by 10% NaCl and 0.01% SDS 

extraction. Then, this OMS was observed under transmission 

electron microscope (TEM). The SDS separated antigens were 

transferred and immunostained with reference rabbit antisera raised 

against each serovars of interest. 

Under TEM, the swollen leptospiral cells were observed 

after 10% NaCl absorption and thin fragments were found detached 

from an axial filament after 0.01% SDS extraction. Whereas, 

immunoblot results showed these fragments were reactive with each 

serovar specific antisera and representative of outer membrane 

components. 

(Oral Presentation : At the 2005 Annual meeting of the Medical 

Biotechnology Center, by Department of Medical Science, Ministry 

of Public Health,at Rose Garden Hotel ,Nakorn Pathom Province. 

August 16-17, 2005.) 

COMPARISON IMMUNOBLOTTING ANALYSIS 

OF LEPTOSPIRA FOR SEROTYPING COMPA- 

TIBLE TO MICROSCOPIC AGGLUTINATION 

METHOD (NO. 833) 

Doungchawee Galayanee, Naigowit Pimjai and Kongtim 

Suraphol 

Department of Pathobiology, Faculty of Science, Mahidol 

University, Bangkok 10400, Thailand. 

Immunoblotting analysis of reference Leptospira using 

rabbit antiserum generated against leptospiral serovars, 

representatives of locally important serogroups,the results showed 

that all leptospires demonstrable with distinct multiband patterns 

varied with serovars. A major reactivity on immunoblot equivalent 

to MAT typing of leptospires is a diffuse broad banding of molecular 

masses between 20-30 kDa. In addition,a number of genus associated 

or Leptospira common bands exhibited on the immunoblot when 

reacted with any serovar specific polyclonal antiserum included 

multiple discrete bands (range between 14-20 kDa) and singlet or 

duplet band of 36-37 kDa. Whereas bands of 23 ,32 and 41 kDa 

were only detected in those pathogenic Leptospira investigated. 

Overall,both pathogenic Leptospira interrogans and non-pathogenic 

L. biflexa have characteristic immunoblotting patterns useful for 

differentiation and typing of Leptospira serovar and/or serogroup 

as compared with the MAT results. The advantages of 

immunoblotting using polyclonal antibodies are more flexible 

than MAT method at low risk to prepare non-viable leptospires as 

antigens and the resulting blots can be kept and tested at any 

convenience,which is considerable simplicity and accuracy of use . 

(Oral Presentation : At the 2005 Annual meeting of the Medical 

Biotechnology Center, by Department of Medical Science, Ministry 

of Public Health,at Rose Garden Hotel ,Nakorn Pathom Province. 

August 16-17, 2005.) 

IMMUNODISTINCTION OF REFERENCE 

LEPTOSPIRES ON IMMUNOBLOTS (NO. 834) 

Doungchawee G. 1 *, Naigowit P. 2 , Ekpo P. 3 , Sirawaraporn,W 4 ., 

Kongtim S. 1 

1 Department of Pathobiology, Faculty of Science, Mahidol 

University, Bangkok 10400. 2 Research Center for Leptospira 

Laboratory, National Institute of Health, Nonthaburi Province, 

Thailand. 3 Department of Immunology, Faculty of Medicine 

Siriraj Hospital, Mahidol University. 4 Department of 

Biochemistry, Faculty of Science, Mahidol University, Bangkok 


Key words: Immunoblotting, Smearlike reactivity, Serovar 

differentiation 

Background : The determination of serovar and serogroup 

status of various reference leptospires is conventionally based on 

the use of microscopic agglutination test (MAT). Accordingly,this 

test is required a number of reference antisera to react with the 

live leptospires, which known to be time-consuming, difficult for 

maintaining of organisms until optimal for the test. Considering the 

tediousness and complexity of the MAT,this would be simplified by 

the use of immunoblotting as the alterative. 


316 

Methods: SDS-PAGE and Immunoblotting analysis was 

evaluated by reacting whole cell lysates of reference leptospires 

with rabbit antisera to individual serovars. 

Results: By homologous reaction,individual immunoblots 

of whole cell lysates of various leptospiral serovars showed 

predominance of smearlike bands diffuse between 20-30 kDa 

including some other common leptospiral bands. Immunoblotting 

using specific antiserum demonstrated that this smearlike banding 

is exclusive to the serovar and relevant to the serovar determined 

antigen as confirmed by serovar specific monoclonal antibodies. 

All serovar members of the group were detectable with smearlike 

bandings by reacting with any antiserum specific to the member. 

In contrast, these smearlike reactivities were depleted when antisera 

tested were absorbed by agglutination with the homologous or related 

leptospires as example found in the reference Australis serogroup. 

The resulting reactivities by immunoblotting and MAT using 

monoclonal and polyclonal antibodies gave the same identifying 

serovars of reference leptospires studied. 

Conclusion: This smearlike banding shown on 

immunoblot is characteristic for identifying and differentiating 

serovar and/or serogroup as if determined by the standard MAT. 

The overall pattern on immunoblot is used to distinguish various 

different leptospira as well. 

(Poster Presentation : At the fourth scientific meeting of the 

International Leptospirosis Society (ILS). The Central Duangtawan 

Hotel, Chiang Mai, Thailand. November 14-16, 2005) 

INCREASED SERODIAGNOSIS OF LEPTO- 

SPIROSIS BY IGM IMMUNOBLOTTING (NO. 835) 

Doungchawee G. 1* , Petkanjanapong W. 2 , Hinjoy S. 3 , 

Wattanawong D. 4 , Midtrapanon S. 5 


University. Bangkok, Thailand; 2 National Institute of Health, 

Department of Medical Science, Ministry of Public Health; 

3 Bureau of Epidemiology, Department of Disease Control, 

Ministry of Public Health; 4 General Communicable Diseases 

Bureau, Department of Disease Control, Ministry of Public 

Health.; 5 Roiet Provincial Health Office,Roiet, Thailand. 

Key words: IgM immunoblot , Acute fatal illness, Anti-leptospiral 

antibodies 

Background: People at risk were investigated for 

leptospirosis during an incident of sudden death cases of unknown 

origin in Roiet province,a rural area of the Country. 

Methods: Serum samples from 19 workers,who had the 

same activities as those of acute fatal and un-identifiable 

illnesses,were determined for anti-leptospiral antibodies by the use 

of standard microscopic agglutination test (MAT) and IgM 

immunoblot method. 

Results: Out of 19 cases investigated, 4 (21%) were 

diagnosed for leptospirosis and the remainders were negative by the 

MAT. Individuals of the MAT positive cases were detected with 

variable levels of agglutinating antibodies against leptospira used. 

IgM immunoblot was introduced for the detection of anti-leptospiral 

antibodies and gave higher positive rate (63 % of 19) than those of 

the MAT on comparison. The overall individuals of leptospirosis 

cases showed specific reactivities against pathogenic L. interrogans 

serovars panama,new,bratislava and sejroe as performed by 

immunoblotting. 


Conclusion: All of the MAT proven leptospirosis cases 

were consistent with the specificity of the results obtained by IgM 

immunoblotting. Interestingly,the IgM immunoblot showed evidence 

of higher leptospirosis cases in comparison with those determined 

by standard MAT. This finding would suggest the potential of IgM 

immunoblot for rapid case diagnosis and alternative screening test 

of human leptospirosis with comparison to the MAT. 




VARIABLE SEROTYPES OF LEPTOSPIRE 

ISOLATED FROM SEVERE PATIENTS (NO. 836) 

Watchararattanapol A. 1* , Galayanee D. 1 , Udomsangpetch R. 1 , 

Kositanont U. 2 


University, Bangkok.; 2 Department of Microbiology, Faculty of 

Medicine Siriraj Hospital, Mahidol University, Bangkok. 

Key words: Leptospire isolate, Serotyping ,MAT, Immunoblotting 

Background : In endemic area of the country, most 

leptospirosis patients were identified with high level of agglutinating 

antibodies which sometimes gave reactivity to variable leptospires 

by using standard microscopic agglutination test (MAT). The 

findings of such antibodies developed were inconclusive to the 

previous or recent infection and made confusing diagnosis for 

clinicians and epidemiologists. The MAT of use still could not 

overcome the cross reactivity among serovars of Leptospira 

interrogans. We compared data between leptospiral isolates of 

human origin and experimental animal using MAT and 

immunoblotting in order to explore the possibility of identifying 

serovar of the isolates and interpretation of antibody results. 

Methods: Representative leptospires used were isolated 

from two severe patients and individually inoculated into rats and 

hamsters. Leptopsiral isolates and serum antibodies were both 

collected and further studied by MAT, dark field microscopy and 

immunoblotting . 

Results: The morphological characteristics of leptospires, 

derived from experimental rats and hamsters were not only shorter, 

thinner,less motile than those of human origin but also having bothended 

hooks. Serotyping by MAT of the animal derived isolates 

were reactive with the reference anti-ballum antiserum and their 

homologous sera. Whereas, those of human origin gave predominant 

reactivity with the anti-autumnalis. Determination of serum 

antibodies of both experimental animals and patients gave variable 

reactivities to a number of leptospires used on the immunoblots 

and MAT. 

Discussion: The results indicate that human leptospirosis 

cases should be infected with combined types of leptospires. 

Comparison between the culture isolates of human and animal 

sources, the data can be determined for different serotypes of the 

infecting leptospires. Therefore,the variable antibody response 

would be promising and consequent to the natural leptospirosis 

infection as being proved by this animal model. 






IDENTIFICATION OF LEPTOSPIRES IN 

PARAFFINIZED TISSUE SECTIONS BY 

IMMUNOFLUORESCENT STAINING (NO. 837) 

Kongtim S 1 , Doungchawee G 1 , Puchadapirom P 1 , Thamavit W 1 , 

Chalermsanyakorn P 2 


University. 2 Department of Pathology, Faculty of Medicine; 

Ramathibodi Hospital, Mahidol University. 

Key words : DFA, pepsin treatment, paraffinized and formalinized 

tissue 

Background : Direct immunofluorescent assay (DFA) is 

the method of successful detection of leptospiral organisms in the 

frozen tissue. However, most pathological laboratories concern and 

handle specimens as paraffinized and formalin-fixed tissues which 

is less sensitive and un-appropriate for the investigation of 

leptospirosis case. 

Methods : We performed alternatively by pretreatment 

of such formalinized kidney tissue with 1 % pepsin in 0.02 M 

hydrochloric acid at 50 o C for 2 hrs and Tween 20 ( 0.05% in 0.1 M 

PBS) and developed reaction by probing with FITC- conjugated 

rabbit anti-leptospiral antibodies. The sections were therefore 

observed under a fluorescent microscope. 

Results : Leptospires pertaining in the kidney of rat 

reservoir were determined using formalin-fixed tissues in comparison 

between samples with and without pepsin treatment. Specific 

reactivity by immunofluorescent staining of the organisms of 

interest was clearly visualized in the renal tissue sections pretreated 

with pepsin. The leptospira could be accessible in the formalinfixed 

tissues of more than a couple year storage by this modified 

method as well. 

Discussions : Pepsin treatment of tissue was useful for 

retrospective studies of animal leptospirosis and help improving 

the immunostaining of the leptospires in renal tissues available in 

routine histological and/or pathological laboratories. 




MODIFIED SILVER STAIN ENHANCING 

DETECTION OF LEPTOSPIRES IN 

FORMALINIZED KIDNEY SECTIONS (NO. 838) 

Kongtim S, Doungchawee G, Thamavit W, Puchadapirom P. 

Department of Pathobiology, Faculty of Science, Mahidol 

University. 

Key words : Leptospira, Modified silver stain, Formalin fixed tissue. 

Background : Silver staining is an alternative method 

used to detect spirochetes in fixed tissues. But, the problem of 

silver precipitation and tissue darkening interfered the detection of 

organism of interest. 

Method: We modified Warthin-Starry technique by treating 

tissue sections with 0.5 % iodine in 70 % alcohol prior to brief 

exposure to 5 % sodium thiosulfate. The tissue section was then 

impregnated in 1 % silver nitrate solution for half an hour. Finally, 

silver development of the microorganism was done by incubating 

the section with a mixture of 2 % silver nitrate, 5 % gelatin and 1% 

hydroquinone. 

Results: The results of modified silver staining of 

formalinized kidney sections from rats proven leptospirosis showed 

that the spiral- shaped bacteria were detected better than those of 

conventional silver staining method used. 

Discussions: Leptospira was considered as one of the 

most infecting agents that localized at the renal tubules of infected 

animals. The modified method of study was found to enhance the 

detection of spirocheatal organisms of rodent leptospirosis. This 

would allow a convenience and possibility of using routine 

histological samples for investigating animal leptospirosis. 




INVESTIGATION OF LEPTOSPIRA CARRIER 

POTENTIAL AMONG RODENTS IN BANGKOK 

(NO. 839) 

Uraiwan Kositanont 1 , Galayanee Doungchawee 2 , Saraphol 

Kongtim 2 , Wiaranan Pulsawat 1 , Chantra Singchai 3 , Pilaipan 

Puthavathana 1 

1 Department of Microbiology, Faculty of Medicine Siriraj 

Hospital and 2 Department of Pathobiology, Faculty of Science, 

Mahidol University, Bangkok 10700, 3 Bangkok Metropolitan 

Adminstration. 

Background : Leptospirosis is zoonosis that can be 

transmitted from animals to humans and occurs worldwide. Rat and 

other rodents, for example Rattus norvegicus, Rattus rattus and house 

mice are most important sources of human infections. Detection of 

leptospires in reservoirs would offer data in epidemiological studies 

This study was aimed to investigate leptospiral infections in rodents 

and to compare the results tested by three techniques, namely PCR, 

MAT and DFA. In addition, the presence of leptospires in tissue 

specimens from the kidney, liver, lung and brain by semi-nested 

PCR was determined. 

Materials & Methods : A total of 117 rodents were trapped 

in markets of various districts in Bangkok in years 1999 and 2002. 

Serum samples were tested for antibodies to leptospires by 

microscopic agglutination test (MAT). Frozen rat kidneys were tested 

for leptospiral antigens by direct immunofluorescence assay (DFA). 

Kidneys livers lungs and brains were tested by semi-nested PCR. 

Results : The results of three (MAT, DFA and PCR) assays 

for detection of leptospiral detction were compared. There was 100% 

concordance of results between DFA and PCR. Of 86 rodent kidney 

samples with negative antibody results, the positive results of both 

DFA and PCR were found in 23/50 samples (46%) and 8/36 samples 

(22.2%) in 1999 and 2002, respectively. Leptospiral DNA was 

detected only in kidneys, not in liver, lung and brain. 


318 

Conclusions : The results demonstrated that antigen 

detection test showed higher sensitivity than antibody detection test. 

Regardless of difficulty of sample collection, antigen detection and 

DNA detection provide the exact numbers of leptospiral infected 

rats in epidemiological data. The actual prevalence of leptospiral 

infections can be a useful indicator for leptospirosis. 

(Supported by grants from World Organization (SE THA OCD 001 

RB99) and Leptospirosis Control Office, Department of 

Communicable Disease) (Presentation At the 2005 Annual meeting 

of the Medical Biotechnology Center, by Department of Medical 

Science, Ministry of Public Health,at Rose Garden Hotel ,Nakorn 

Pathom Province. August 16-17, 2005. At the fourth scientific meeting 

of the International Leptospirosis Society (ILS). The Central 

Duangtawan Hotel, Chiang Mai, Thailand. November 14-16, 2005.) 

PATHOLOGY OF LEPTOSPIROSIS : AUTOPSY 

STUDIES (NO. 840) 

V. Boonpucknavig 1 , P. Chalermsanyakorn 2 , G. Doungchawee 3 , 

K. Niwatyakul 4 , S. Kongtim 3 

1 Department of Pathology, Bangkok Hospital, Bangkok, 

Thailand; 2 Department of Pathology, Ramathibodi Hospital, 

Mahidol University, Bangkok, Thailand.; 3 Department of 

Pathobiology, Faculty of Science, Mahidol University, Bangkok, 

Thailand.; 4 Department of Medicine, Loei Hospital, Loei 

Province, Thailand. 

Full autopsy were performed on four cases of Leptospirosis 

complicated with multiorgans involvement. Antileptospira 

antibodies were indentified by MAT and immunoblotting techniques. 

The immunoblotting data revealed antibodies against more than one 

serovar of leptospira. Histopathology, immunopathology, and 

electronmicroscopy diagnosis of 3 cases included. 

Cardiovascular System 

- Pancarditis of the heart 

- Arteritis of coronary arteries, vasa vasorum of the aorta, 

and pulmonary artery 

Respiratory System 

- Exudate and diffuse alveolar damage phase of adult 

respiratory distress syndrome of both lungs 

Urinary System 

- Acute interstitial nephritis with focal tubular necrosis of 

the kidney 

- Acute endocapillary proliferative (post infectious) 

glomerulonephritis 

- Endarteritis of the renal artery 

- Cystitis of the urinary bladder 

Hepatobiliary System 

- Focal necrosis of hepatocytes and triaditis portal tracts of 

the live 

- Acute cholecystitis of the gall bladder 

Hematopoietic System 

- Acute hemorrhagic splenitis with severe lymphoid 

depletion 

Central Nervous System 

- Acute meningitis and choroid plexitis of the brain 

In addition, one case showed acute hemorrhagic 

pancreatitis. Pathology in case 4 were overwhelmed by secondary 

fungal infection (mucormycosis). Immunofluorescence microscopy 


revealed intact and granular antigen of leptospires in perivascular 

tissue of many organs. In the kidney, leptospires were identified in 

peritubular capillaries. 

(Symposium : At the fourth scientific meeting of the International 

Leptospirosis Society (ILS).The Central Duangtawan Hotel, Chiang 

Mai, Thailand. November 14-16, 2005.) 

RESPONSE OF PATHOGENIC Leptospira 

interrogans SEROVAR CANICOLATO UVA 

IRRADIATION (NO. 841) 

Wong-ekkabut J. 1,* , Triampo W. 1,4 , Chadsuthi S. 1 , 

Doungchawee G. 2 , Krittanai C. 3 , Tang I. M. 1,4,5 


Bangkok 10400; 2 Department of Pathobiology, Faculty of 

Science, Mahidol University. ; 3 Institute of Molecular Biology 

and Genetics, Mahidol University, Nakhonpathom 73170, 

Thailand; 4 Capability Building Unit in Nanoscience and 

Nanotechnology, Faculty of Science, Mahidol University, 

Bangkok, 10400, Thailand; 5 Institute of Science and Technology 

for Research and Development, Mahidol University, 

Nakhonpathom 73170, Thailand. 

Key words : UVA, L. interrogans serovar canicola, immunoblotting, 

SDS-PAGE 

Background : According to the lesser knowledge of how 

environmental conditions, outside the host, influence the survival 

of Leptospira, the causing agent of a widely distributed zoonosis 

.Hence, an Ultraviolet-A (UVA) irradiation of the Leptospira was 

performed in order to investigate the basis of their survival and 

future application in the control of spreading of the organism. 

Methods: Leptospira interrogans serovar canicola was 

selected as a model for studying the effect of ultraviolet-A (UVA) 

radiation at variable times. Conventional microscopic agglutination 

test (MAT), dark field microscopic technique and spectrophotometry 

were used to determine the growth and viability of the organisms. 

Antigen and protein components of experimental leptospira were 

analyzed by sodium dodecylsulfate polyacrylamide gel 

electrophoresis (SDS-PAGE) and immunoblotting method 

Results: The decrease of growth and viability of the 

leptospira tested was found when exposing to the longer exposure 

duration of UVA. Considering the antigen expression of the L. 

interrogans serovar canicola, the 21 kDa antigenic component 

basically present in the unexposed control was disappeared after 24 

hr UVA-exposure as determined by immunoblotting. Whereas the 

76 kDa protein was detectable in the leptospires with 2 hr UVAexposure 

and gradually fade in those of longer exposure samples by 

SDS-PAGE analysis. Alternatively, the protein components of 

molecular masses between 56-70 kDa were observed. 

Conclusion: The change of protein and antigenic 

components of the leptospira that induced by UVA might have a 

role in the survival and growth of the organisms.Further information 

should be investigated to give more understanding and usefulness 

of ultraviolet radiation for controlling the Leptospira. 






A NEW TECHNIQUE FOR IMMUNOFLU- 

ORESCENT STAINING OF LEPTOSPIRES IN 

PARAFFIN SECTIONS (NO. 842) 

Suraphol Kongtim 1 , Galayanee Doungchawee 1 , Pranom 

Puchadapirom 1 , Wittaya Thamavit 1 , Panas Chalermsanyakorn 2 . 


University. 2 Department of Pathology, Ramathibodi Hospital, 

Mahidol University. 

Key words : FA-staining, leptospires, pepsin, paraffin sections. 

A standard immunofluorescent method was modified for 

staining of leptospires in formalin fixed, paraffin embedded tissues. 

Routine paraffin sections were deparaffinized and treated with 1% 

pepsin in 0.02 M hydrochloric acid at 50 ð C for 3 hrs. washed with 

Tween20, 0.05 % in 0.85 % normal saline, then exposed to anti 

Leptospira for 30 min, washed 2x5 min in 0.01 PBS, mounted in 

buffer glycerol, and examined under a fluorescence microscope. 

Pepsin treatment greatly enhanced subsequent staining of leptospires 

in naturally infected rat kidneys as well as culture smears. 

Leptospires in naturally infected rat kidneys were usually 

undetectable in untreated sections but clearly visible in stained 

pepsin-treated sections. Naturally infected rat kidney usually 

contained many leptospire antigens which could be stained to make 

individual leptospires prominent. The staining method could be 

employed with a single conjugated anti-leptospiral antibody for 

staining of leptospires from 23 serogroups. Also leptospires could 

be stained in tissues stored in formalin for more than 24 months and 

in 26 year old paraffin embedded tissues. 

(36 Years Ramathibodi, Toward Better Health for All. Impact 

Muangtong Thani.) 

AN INVESTIGATION OF RODENT LEPTOSPIROSIS 

IN BANGKOK, THAILAND (NO. 843) 

Niwetpathomwat A 1 , Doungchawee G 2 

1 Department of Veterinary Medicine, Faculty of Veterinary 

Science, Chulalongkorn University, Bangkok and 2 Department 

of Pathobiology, Faculty of Science, Mahidol University, 


Key words : Rodents, Leptospirosis, Direct immunofluorecense 

assay, Bangkok, Thailand 

Leptospirosis is a major public health disease throughout 

the world. The transmitted routes of infection are either direct or 

indirect contact with leptospira reservoirs, especially with rodent 

species. Disease surveillance of such reservoirs should be undertaken 

to further preventive strategies. Rodent leptospirosis was investigated 

in Bangkok during 2002 by direct immuonofluorescense assay with 

6 different FITC conjugated, rabbit anti-leptospira antibodies 

(serovar bataviae, bratislava, canicola, hebdomadis, javanica and 

pyrogenes) which was carried out to detect leptospires in the rodent’s 

kidneys. The results showed that positive findings were significantly 

different (P

320 

and 2 Service de Parasitologie, Mycologie Médicale et Maladies 

Tropicales, Hôpital E. Herriot, Hospices Civils de Lyon, and 

3 Service de Parasitologie et Maladies Tropicales, Hôpital de la 

Croix-Rousse, Hospices Civils de Lyon, France; 4 Department 

of Pathobiology, Faculty of Science, Mahidol University, 

Bangkok, Thailand; 5 Department of Parasitology, Faculty of 

Medicine, University of Indonesia, Jakarta, Indonesia; 

6 Department of Infectious, Parasitic, and Immunomediated 

Diseases, Istituto Superiore di Sanità, Rome, Italy. E-mail 

Address : scrud@mahidol.ac.th. 

Because of the lack of methods for continuous in vitro 

culture of Plasmodium vivax, little is known about drug-resistance 

mechanisms in this malaria-causing parasite. Therefore, 

identification of all the genes potentially involved in drug resistance 

and of molecular markers related to drug resistance would provide a 

framework for studying the incidence and spread of drug-resistant 

P.vivax strains. We have identified the P.vivax orthologue of the 

pfmdrl gene (pvmde1), which was shown to have a role in the drug 

resistance of Plasmodium falciparum. Comparison of the alignments 

of both nucleotide and amino acid sequences of pvmdr1 with those 

of other Plasmodium multidrug-resistance genes revealed an openreading 

frame of 4392 base pairs encoding a deduced protein of 

1464 amino acids. Nucleotide polymorphisms at 2 codons of the 

pvmdr1 gene-Y976F and F1076L __ were found in 14 of 23 P. vivax 

isolates from different areas of endemicity, including Thailand, 

Indonesia, Turkey, Azerbaijan, and French Guyana. 

(The Journal of Infectious Diseases. 2005; 191:272-7) 

NASAL IMMUNIZATION WITH A MALARIA 

TRANSMISSION-BLOCKING VACCINE CANDI- 

DATE Pfs25 INDUCES COMPLETEPROTECTIVE 

IMMUNITY IN MICE AGAINST FIELDISOLATED 

Plasmodium falciparum. (NO. 846) 

Takeshi Arakawa, 1 Ai Komesu, 1 Hitoshi Otsuki, 2 Jetsumon 

Sattabongkot, 3 Rachanee Udomsangpetch, 4 Yasunobu 

Matsumo, 5 Naotoshi Tsuji, 6 Yimin Wu, 7 Motomi Torii, 2 and 

Takafumi Tsuboi 8 

1 Division of Molecular Microbiology, Center of Molecular 

Biosciences, University of the Ryukyus, Nishihara, Okinawa 

903-0213, Japan; 2 Department of Molecular Parasitology, 

Ehime University School of Medicine, Toon, Ehime 791-0295, 

Japan; 3 Department of Entomology, Armed Forces Research 

Institute of Medical Sciences, Bangkok 10400, Thailand; 


University, Bangkok 10400, Thailand; 5 Laboratory of Global 

Animal Resource Science, Graduate School of Agricultural and 

Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 

113-8657, Japan; 6 Laboratory of Parasitic Diseases, National 

Institute of Animal Health, National Agricultural Research 

Organization, Tsukuba, Ibaraki 305-0856, Japan; 7 Malaria 

Vaccine Development Branch, National Institute of Allergy and 

Infectious Diseases, National Institutes of Health, Rockville, 

Maryland 20852, United States of America; 8 Cell-free Science 

and Technology Research Center, Ehime University, 

Matsuyama, Ehime 790-8577, Japan. E-mail Address : 

scrud@mahidol.ac.th. 

Key words : P.falciparum; transmission-blocking vaccine 

Malaria transmission-blocking vaccines based on antigens expressed 

in sexual stages of the parasites are considered one promising strategy 

for malaria control. To investigate the feasibility of developing 

noninvasive mucosal transmission-blocking vaccines against 

Plasmodium falciparum, intranasal immunization experiments with 

yeast-expressed recombinant Pfs25 proteins were conducted. Mice 

intranasally immunized with the Pfs25 proteins in the presence of a 

potent mucosal adjuvant cholera toxin induced robust systemic as 

well as mucosal antibodies. All mouse IgG subclasses except IgG3 

were found in serum at comparable levels, suggesting that the 

immunization induced mixed Th1/Th2 responses. Consistent with 

the expression patterns of the Pfs25 proteins in the parasites, the 

induced immune sera specifically recognized ookinetes, but not 

gametocytes. In addition, the immune sera recognized only the Pfs25 

proteins with native conformation, but not the denatured forms, 

indicating that mucosal immunizations induced biologically active 

antibodies capable of recognizing conformational epitopes of native 

Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture 

of the mouse immune sera and gametocytemic blood derived from 

patients infected with P.falciparum resulted in complete interference 

of oocyst development in mosquito midguts. The observed 

transmission-blocking activities were strongly correlated with 

specific serum antibody titers. Our results demonstrated for the 

first time that a P.falciparum transmission-blocking vaccine 

candidate is effective against field-isolated parasites and justify the 

investigation of noninvasive mucosal vaccination regimes for control 

of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic 

disease. 

(Infection and Immunity 2005, inpress) 


MEMORY T CELL PROTECT AGAINST 

Plasmodium vivax INFECTION (NO. 847) 

Kulachart Jangpatarapongsa 1,2 , Jeeraphat Sirichaisinthop 3 , 

Jetsumon Sattabongkot 4 , Sornchai Looareesuwan 5 , Marita 

Troye-Blomberg 6 , Rachanee Udomsangpetch, 1,2, * 

1 Department of Pathobiology, Faculty of Science, 2 Department 

of Parasitology, Faculty of Medical Technology, Mahidol 

University, 3 Center of Malaria Research and Training, Ministry 

of Public Health, 4 Department of Entomology, USAMC, 

AFRIMS,5 e Department of Clinical Tropical Medicine, Faculty 

of Tropical Medicine, Mahidol University, Bangkok, Thailand, 

6 Department of Immunology, The Wenner-Gren Institute, 

Stockholm University, Stockholm, Sweden. E-mail Address : 

scrud@mahidol.ac.th. 

Key words : malaria; Plasmodium vivax, memory T cells 

Immunity induced by P.vivax infection leads to memory T 

cell recruitment which will be activated during the “relapsing period” 

or “subsequent infection”. This study aims to determine memory T 

cells in patients with acute and convalescent P.vivax infection. 

Lymphocytes from P.vivax infected patients, immune and naïve 

control were studies for immature memory T cell expressing 

CD45RO + CD27 + and the mature cells expressiong CD27: During 

acute infection a significant increase in the median percentage of 

memory T cell subsets having CD4 + were observed when compared 

to those seen in either naïve or immune controls. The high level of 

memory T cells was maintained until 60 days post treatment. 



Immune controls living in the endemic area had somewhat high level 

of memory T cells subsets having CD8 + . A ~3 fold increase of these 

cell types was shown in the acute infection and the level was 

persistent until 60 days post treatment. 

Phenotypic characterization of the peripheral lymphocytes 

during acute infection revealed that a large fraction of the 

lymphocytes carried the phenotypes suggesting a role of these cells 

in the early defense against P.vivax 

Very low levels of P.vivax specific antibody were found. 

Cell-mediated immunity may play a stronger role in the development 

of naturally acquired protection against P.vivax infection than 

humoral immunity. 

Taken together, our results provide further insighy into the 

mechanism of cell-mediated immunity to P.vivax infection which 

could be important for the future successful vaccine development 

and antimalarial drug designation. 

(In Press in Microbes & Infection 2005.) 

EFFECTS OF PRAZIQUANTEL AND ARTESUNATE 

ON THE TEGUMENT OF ADULT Schistosoma 

Mekongi HARBOURED IN MICE (NO. 848) 

Wannee Jiraungkoorskul 1 , Somphong Sahaphong 1,2 , Prasert 

Sobhon 3 , Suda Riengrojpitak 1 , Niwat Kangwanrangsan 1 

1 Department of Pathobiology and 3 Department of Anatomy, 

Faculty of Science, Mahidol University, Rama VI road, Bangkok 

10400, Thailand; 2 Mahidol University International College, 

Mahidol University Salaya Campus, Nakhonpathom 73170, 

Thailand. 

Key words: schistosomiasis; Schistosoma mekongi; praziquantel; 

artesunate; scanning electron microscopy; tegumental changes 

The effects of praziquantel and artesunate on the tegument 

of adult Schistosoma mekongi harboured in mice were compared 

using scanning electron microscopy (SEM). Forty-two mice infected 

with S. mekongi for 49 days were treated intragastrically with either 

300 mg/kg praziquantel or 300 mg/kg artesunate. Mice were 

sacrificed 1 or 3 days post-treatment. Worms were collected by 

perfusion and examined by SEM. One to 3 days after administration 

of artesunate, the tegument of S. mekongi showed severe swelling, 

vacuolization, fusion of the tegumental ridges and loss or shortening 

of the spines on the trabeculae, collapse and peeling. Praziquantel 

induced similar tegumental alterations as those observed after 

administration of artesunate, but they were less severe. Three days 

post-treatment, there was evidence of recovery only in the case of 

praziquantel. The results of our study suggest that artesunate is more 

effective than praziquantel in causing tegumental damage in adult 

S. mekongi, and provides a basis for subsequent clinical trials. 

(Parasitology International 54, (2005); 177-183) 

Eurytrema Pancreaticum: THE IN VITRO EFFECT 

OF PRAZIQUANTEL AND TRICLABENDAZOLE 

ON THE ADULT FLUKE. (NO. 849) 

Wannee Jiraungkoorskul 1, , Somphong Sahaphong 1,2 , Tawewan 

Tansatit 1 , Niwat Kangwanrangsan 1 , Siriporn Pipatshukiat 2 


University, Bangkok 10400, Thailand.; 2 Mahidol University 

International College, Mahidol University, Salaya Campus, 

Nakhonpathom 73170, Thailand. 

Key words: Eurytrema pancreaticum; praziquantel; 

triclabendazole 

The efficacy and tolerance of the 80 μg/ml praziquantel 

(PZQ) and 40 μg/ml triclabendazole (TCZ) against adult stage 

Eurytrema pancreaticum in vitro were investigated at 3, 12, and 15 

hours incubation. Motility of the flukes and histopathological 

changes were studied. Sudden paralysis and death were observed 

after exposed to PZQ as early as 3 hours incubation. In contrast, the 

TCZ treated flukes showed active mobility at all intervals. By light 

microscopic examination, severe damages in various organs such 

as tegument, muscle, and testes were observed early at 12 hours 

incubation of these drugs. PZQ caused more severe damage to flukes 

than TCZ. There were vigorous contraction of musculature, 

progressive shrinkage of circular and longitudinal muscles, 

vacuolization and disintegration of the tegument disrupting the 

worms’ outer surface including detachment of spines in the PZQ 

treatment. The cells in testes were slightly increased in size and 

followed by degeneration leaving several hollow spaces. The uterus 

and vitelline glands remained unaffected. The direct observation of 

the fluke motility and light microscopic study highly suggested that 

praziquantel was more effective than triclabendazole treatment for 

the eurytremiasis infection. 

(Experimental Parasitology 111, (2005); 142-147.) 

HISTOPATHOLOGICAL CHANGES OF LIVER, 

KIDNEY AND SPLEEN IN FISH FROM THE 

MEKONG RIVER (NO. 850) 

Kantimanee Phanwichien 1 , Apichat Pradermwong 2 and 

Kammachapol Purepong 1 

1 Department of Zoology, Faculty of Science, Kasetsart 

University; 2 Department of Pathobiology, Faculty of Science, 

Mahidol University. 

Key words : Histopathology, Fish, Liver, Kidney, Spleen, Mekong 

River 

Both cryinids and catfishes were collected from 5 stations : 

Amphur Chaing San, Chaing Rai Province, Amphur Chaing Khan, 

Leoi Province, Amphur Bungkan, Nong Khai Province, Amphur 

Muang, Nakorn Phanom Province, and Amphur Kong Chain, 

Ubonratchathani Province of the Mekong River since December, 

2001 until May, 2004. This seasonal collection was conducted to 

evaluate fish health by study the histopathological changes of liver, 

kidney and spleen. The noticible changes in all tissues were observed 

in fish feeding benthose and large older fish more severe than the 

pelagic and small young fish. The changes found in fish tissues 


322 

from all stations showed the same in the following characteristics: 

fatty change in liver cells which caused pale-yellowed liver, vacuolar 

degeneration, couldy swelling, accumulation of yellow-brown 

granules in cytoplasm, necrosis of liver cells together with central 

veins and hepatic bile ducts, including hepatic bile duct hyperplasia. 

In kidney tissue, degeneration of glomeruli, hyaline degeneration 

and necrosis of proximal epithelium, and hyperplasia of renal 

segments were noticed. The histopathological changes found in 

both tissues revealed the damages from toxicants accumulated in 

fish. In spleens, they were found hemorrharage and necrotic 

erythrocytes which the hemorrhages showed the increase 

reproducible of erythrocytes. The more severe of all changes in fish 

were observed in summer from Nakorn Phanom and Nongkhai 

Provinces, Chaing Rai, Ubonratchathani and Leoi Provinces, 

respectively. In winter, fish from Chaing Rai Province were showed 

more severe while the others showed the same level of changes. 

These changes may be caused by toxicants accumulated in benthose 

or toxins from fish pellets. 

(The Proceeding of 4 th National Environmental Conference, 19-21 

Janm 2005, the Ambassador City, Cholburi, Thailand.) 

SOCIAL ISOLATION ALTERS THE EFFECTS 

OF ETANOL IN THE RAT SOCIAL INTERACTION 

TEST (NO. 851) 

Noppamars Wongwitdecha, Benja Ngamnawakul, Haruthai 

Thaidee, Sompop Soo-ampon 

Department of Pharmacology, Institute of Science and 

Technology for Research and Development, Mahidol University, 

Thailand. 

Social isolation rearing from weaning has been reported to 

change the behaviors of the adult animals and modify the 

responsibility to many addictive drugs. However, there is no report 

concerning the effect of social isolation on the behavioral effect of 

ethanol in the social interaction test. 

Objective: The aim of the present experiments was to 

investigate the effect social isolation on the behavioral effect of 

ethanol in the rat social interaction test. 

Methods: Male Wistar rats were reared from weaning either 

singly (isolation rearing) or in groups of five-six rats/cage (social 

rearing). Four weeks later, these rats were tested for their sensitivity 

to ethanol using the social interaction test. 

Results: In the rat social interaction test, the active social 

interaction behavior of the saline-treated isolation reared rats was 

not significantly difference from the socially reared controls. 

However, the latencies of active social interaction of the salinetreated 

isolation reared rats were significantly longer than the social 

reared rats (P


PSYCOLOGICAL STRESS ALTERS THE 

EFFECTS OF ETHANOL IN THE RAT SOCIAL 

INTERACTION TEST (NO. 853) 

Wongwitdecha N 1,4 , Ngamnawakul B 1,2 , Thaidee H 1,3 , Soo-ampon 

S 1 

1 Department of Pharmacology, 2 Department of Biology, 

3 Toxicology Program, Faculty of Science, 4 Institute of Science 

and Technology for Research and Development, Mahidol 

University, Thailand. 

Key words : Psychological stress, social interaction test, ethanol 

Psychological stress from the early stage of life such as 

social isolation rearing from weaning has been shown to change the 

behaviors of the adult animals and modify the responsibility to many 

addictive agents. However, there is no report concerning the effect 

of psychological stress on the behavioral effect of ethanol in the 

social interaction test. 

Objectives : The purpose of the present experiments was 

to investigated the effects of psychological stress on the rat behavioral 

effect of ethanol in the rat social interaction test. 

Methods : Male Wistar rats were obtained from weaning 

either singly (isolation rearing) or in groups of five-six rats/cage 

(social rearing). Four weeks later, these rats were tested for their 

sensitivity to ethanol using the social interaction test. 

Result : In the rat social interaction test the active social 

interaction behavior of the saline-treated isolation reared rats was 

not significantly difference from the socially reared controls. 

However, the latencies of active social interaction of the salinetreated 

isolation reared rats were significantly longer than the social 

reared rats (P

324 

The water decoction of heartwood and stem bark of Ventilago 

harmandiana Pierre (Family Rhammnaceae) are used in Thai 

traditional medicine for the treatment of diabetes as well as wound 

and chronic inflammation. Methanolic extracts from the heart wood, 

stem bark, stem wood and pure compounds of the herb exerted strong 

inhibitory effects on the acute phase of inflammation as seen in 

ethyl phenylpropiolate (EPP)-induced ear edema as well as in 

carragenin-induced paw edema in rats. Activated macrophages 

participate actively in the inflammatory response by producing large 

amounts of pro-inflammatory cytokines such as TNF-α, 

chemoattractant cytokines such as IL-8 and pro-inflammatory 

products of arachidonic acid metabolism as well as nitric oxide. 

This study examined whether a quinone and two anthraquinone 

compounds from Ventilago harmandiana modulate the production 

of PGE 2 , tumour necrosis factor-á (TNF-á) and nitric oxide (NO) by 

LPS-activated human macrophages. The expression of the mRNA 

of COX-2 and TNF-á was also investgated. All three compounds 

from Ventilago harmandiana were shown to significantly inhibit 

the production of TNF-á and PGE 2 in a concentration-dependent 

manner without notable cytotoxic effects on these cells. The 

expression of both TNF-á and COX-2 mRNA was also significantly 

inhibited by these compounds. However, neither the production of 

NO nor iNOS mRNA expression could be detected from LPS-human 

activated macrophages. These results indicated that the inhibitory 

effects of a quinone and the two anthraquinone compounds from 

Ventilago harmandiana Pierre on the production, by activated human 

macrophages, of PGE 2 and TNF-á which is regulated at the 

transcriptional level might be attributed, in part, to their antiinflammatory 

activities. 

INHIBITORY EFFECTS OF NEUTROPHIL FUNC- 

TION AND T-LYMPHOCYTES PROLIFERTION 

BY PUERARIA MIRIFICA EXTRACTS (NO. 856) 

Payong Wanikiat 1 , Yupin Sanvarinda 1 , Taworn Jaipetch 2 , 

Vichai Reutrakul 2 

1 Department of Pharmacology, 2 Department of Chemistry, 


Thailand. 

Key words : Pueraria mirifica; Inflammation; Anti-inflammation; 

Neutrophil functions; Chemotaxis; Superoxide anion generation; 

MPO production; Elastase release 

Pueraria mirifica (P. mirifica) Airy Shaw (Legumunosae), 

a phytoestrogen – rich plant with a local name of white Kwao Krrua, 

has long been used in Thailand as a rejuvenating folk medicine. It 

has also been used by menopausal women for the hormonal 

replacement purpose. The chemical compounds found in the plant 

including Isoflavones and glycosides e.g. daidzein, genistein, 

kwakurin; Chromenes e.g. miroestrol, deoxymiroestrol, 

isomiroestrol, Comestans e.g. coumestrol and Pterocarpans e.g. 

puemiricarpene etc. It is believed that the content of phytoestrogen 

in the plants are of therapeutic benefit. Some active ingredients of 

P. mirifica such as miroestrol exhibit estrogenic like activity. 

Estrogen plays a protective role on cardiovascular system and has 

been known to exert anti-inflammatory effects both in vivo and in 

vitro by inhibiting the expression and production of various proinflammatory 

mediators. In this study, methanol and ethylacetate 

extracts of Pueraria mirifica were investigated for their antiinflammatory 

activities based on neutrophil functions, including 

chemotaxis, superoxide anion generation (SAG), myeloperoxidase 

(MPO) production, and elastase release, and on T-lymphocyte 

proliferation. Both crude extracts were primarily investigated for 

their cytotoxic effects on both neutrophils and lymphocytes using 

MTT assay. Neutrophil viability was not affected by these 

compounds (1-100 μg/ml) either at 4 hours incubation period, except 

at a concentration of 1000 μg/ml (IC 50 >1000). After incubation of 

human neutrophils with various concentrations of both methanol 

and ethylacetate extracts of Pueraria mirifica, neutrophil 

chemotaxis, SAG, MPO production, and elastase release induced 

by N-formyl-methionyl-leucyl-phenylalanine (fMLP) were strongly 

inhibited in a concentration-dependent manner. Both crude extracts 

also inhibited T-lymphocyte proliferation as quantified by [H 3 ] 

thymidine incorporation. 

These results suggested that the inhibitory effects of the 

Pueraria mirifica extracts on neutrophil functions and T-lymphocyte 

proliferation might be attributed, in part, to their anti-inflammatory 

activities. 

CONCERTED MECHANISM OF SWE1/WEE1 

REGULATION BY MULTIPLE KINASES IN 

BUDDING YEAST. (NO. 857) 

Satoshi Asano 1 , Jung-Eun Park 1 , Krisada Sakchaisri 1, 2 , Li- 

Rong Yu 3 , Sukgil Song 4 , Porntip Supavilai 2 , Timothy D Veenstra 3 

and Kyung S Lee 1 

1 Laboratory of Metabolism, Center for Cancer Research, 

National Cancer Institute, NIH, Bethesda, MD, USA. 

2 Department of Pharmacology, Faculty of Science, Mahidol 

University, Bangkok, Thailand. 3 Laboratory of Proteomics and 

Analytical Technologies, NCI-Frederick, Frederick, MD, USA. 

4 College of Pharmacy, Chungbuk National University, South 

Korea. 

Key words : Cdc5, Cdc28, G2/M transition, Swe1 

In eukaryotes, entry into mitosis is induced by cyclin Bbound 

Cdk1, which is held in check by the protein kinase, Wee1. In 

budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck 

through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is 

hyperphosphorylated prior to ubiquitin-mediated degradation. Here, 

we show that Hsl1 and Hsl7 are required for proper localization of 

Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5dependent 

Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound 

Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this 

modification served as a priming step to promote subsequent Cdc5dependent 

Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 

also facilitated Cdc5 localization to the bud neck through the 

enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 

and the polo-box domain of Cdc5. We propose that the concerted 

action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates 

is an evolutionarily conserved mechanism that is crucial for 

effectively triggering mitotic entry and other critical mitotic events. 

(EMBO J. 24, (2005) 2194-2204.) 




INTERLEUKIN-5 REDUCES THE EXPRESSION 

OF UTEROGLOBIN-RELATED PROTEIN (UGRP) 

1 GENE IN ALLERGIC AIRWAY INFLAMMATION 

(NO. 858) 

Yoshihiko Chiba 1, 2 , Achara Srisodsai 1, 3 , Porntip Supavilai 3 and 

Shioko Kimura 1 

1 Laboratory of Metabolism, National Cancer Institute, National 

Institutes of Health, Bethesda, MD 20892, USA 2 Department of 

Pharmacology, Hoshi University, Tokyo 142-8501, Japan 


University, Bangkok 10400, Thailand. 

Key words : Uteroglobin-related protein 1 (UGRP 1), Interleukin- 

5 (IL-5), Allergic asthma 

Airway inflammation is thought to play a major role in the 

pathogenesis of bronchial asthma. The precise role of individual 

inflammatory cells, mediator and asthma related genes in allergic 

lung diseases is not completely understood. The uteroglobin-related 

protein (UGRP) 1 was proposed to be an asthma candidate gene 

and play a role in regulating lung inflammation, however its precise 

function in the airways remains obscure. In this investigation, we 

used a mouse model of allergic airway inflammation to establish a 

relationship between UGRP 1 and IL-5 in airway inflammation. 

Ovalbumin (OVA) challenged mice demonstrate eosinophilia in 

airway tissues and high levels of IL-5 in bronchoalveolar lavage 

(BAL) fluid analogous to that found in bronchial asthma. 

Interestingly, these “OVA-challenged” mice show down-regulation 

of Ugrp1 expression as compared with the control group. Regression 

analysis further demonstrates a significant negative correlation 

between Ugrp1 mRNA expression in the lung and IL-5 levels in 

BAL fluid with r = 0.948 and P < 0.0001 when IL-5 levels were 

normalized by log transformation. Intranasal instillation of IL-5 to 

mice revealed an inhibitory effect of IL-5 on the expression of Ugrp1 

mRNA. Together, these results indicate an involvement of IL-5 in 

the down-regulation of Ugrp1 expression in airway inflammation 

such as allergic asthma disease. 

(Immunol Lett. 97(2005)123-129.) 

ANTIOXIDANT EFFECTS OF AQUEOUS EXTRACTS 

FROM DRIED CALYX OF HIBISCUS SABDARIFFA 

LINN. (ROSELLE) IN VITRO USING RAT LOW- 

DENSITY LIPOPROTEIN (LDL) (NO. 859) 

Vilasinee Hirunpanich 1 , Anocha Utaipat 1 , Noppawan Phumala 

Morales 2 , Nuntavan Bunyapraphatsara 3 , Hitoshi Sato 4 , Angkana 

Herunsalee 5 and Chuthamanee Suthisisang 1 

1 Department of Pharmacology, Faculty of Pharmacy, Mahidol 

University; 2 Department of Pharmacology, Faculty of Science, 

Mahidol University; 3 Department of Pharmacognosy, Faculty 

of Pharmacy, Mahidol University; 4 Department of Clinical and 

Molecular Pharmacokinetics/Pharmacodynamics, Faculty of 

Pharmaceutical Science, Showa University; 5 Medicinal Plant 

Research Institute Department of Medical Sciences, Ministry 

of Public Health, Thailand. 

Key words: Hibiscus sabdariffa; roselle; low-density lipoprotein 

(LDL) oxidation; antioxidant 

The present study quantitatively investigated the antioxidant 

effects of the aqueous extracts from dried calyx of Hibiscus sabdariffa 

LINN. (roselle) in vitro using rat low-density lipoprotein (LDL). 

Formations of the conjugated dienes and thiobarbituric acid reactive 

substances (TBARs) were monitored as markers of the early and 

later stages of the oxidation of LDL, respectively. Thus, we 

demonstrated that the dried calyx extracts of roselle exhibits strong 

antioxidant activity in Cu2+-mediated oxidation of LDL (p

326 

in thalassemia. The patients with 7/7 genotype appeared more prone 

to gallstone formation (100%) than those with 6/7 (47%) and 6/6 

(41%) genotype. These results suggested that genetic polymorphisms 

in the key UGT1A1 enzyme family influence on serum bilirubin 

level in both groups of subjects. However the high incidence of 

gallstones in patients with 6/7 and 6/6 genotypes could be caused 

by other factor such as other site of mutation, food, environmental 

factors or pathophysiology in the patients. 

(Thai Journal of Pharmacology, Jan-Apr 2005, Vol.27, No.1, p. 60. 

Proceedings of 27 th Pharmacological and Therapeutic Society of 

Thailand Meeting, 17-18 March 2005.) 

EFFECTS OF UDP-GLUCURONOSYLTRANS- 

FERASE (UGT) 1A1 AND 1A6 POLYMORPHISMS 

ON ACETAMINOPHEN PHARMACOKINETICS 

IN THAI b-THALASSEMIA/HBE PATIENTS 

(NO. 861) 

Jeeranut Tankanitlert 1 , Thad A Howard 2 , Anusorn 

Temsakulphong 1 , Pornpan Sirankapracha 3 , Noppawan Phumala 

Morales 1 , Yupin Sanvarinda 1 , Pranee Fucharoen 3 , Russell E 

Ware 3 , Suthat Fucharoen 3 and Udom Chantharaksri 1 


University, 2 Division of Hematology, St. Jude Children’s 

Research Hospital, Memphis, TN 38105, USA, 3 Thalassemia 

Research Center, Institute of Science and Technology for 

Research and Development, Mahidol University, Bangkok, 

Thailand. 

Key words: Acetaminophen metabolism, UDP-glucuronosyltransferase, 

genetic polymorphism 

Pathophysiological changes and organ damage from iron 

overload can alter drug pharmacokinetics in patients with 

thalassemia, but genetic variations may also be important. Numerous 

drugs are metabolized by UDP-glucuronosyltransferase (UGT) 

enzymes including acetaminophen (paracetamol, PCM), a commonly 

available and widely used analgesic. UGT1A1 (UGT1A*28) 

promoter polymorphisms affect bilirubin metabolism and UGT1A6 

polymorphisms (UGT1A6*2) also reduce enzymatic activity. These 

polymorphisms may contribute to the interindividual variations 

observed in PCM toxicity in the general population and in 

thalassemia. To elucidate the pharmacokinetics of PCM in 

thalassemia compared with normal subjects, peripheral blood DNA 

was extracted for UGT1A genotyping by fragment size and RFLP 

methods. Sixteen normal and nineteen thalassemic subjects were 

grouped according to UGT1A genotypes. After a single oral dose of 

1000 mg PCM, drug disposition was determined by HPLC. In normal 

subjects, the UGT1A1*28 and UGT1A6*2 polymorphisms 

significantly decreased serum levels of PCM and its metabolites, 

while in thalassemic subjects only UGT1A6*2 had significant 

effects. This is the first pharmacogenetic study in thalassemia and 

the results indicate that these subjects might be more susceptible to 

unexpected side effects while taking therapeutic doses of PCM. 


CHOLESTERYL LIONOLEATE IN LIPOPROTEINS: 

AN INDEX OF OXIDATIVE DAMAGE AND 

CLINICAL SEVERITY IN b-THALASSEMIA/HBE 

(NO. 862) 

Rataya Luechapudiporn 1, , Noppawan Phumala Morales 2 , Suthat 

Fucharoen 3 , Udom Chantharaksri 2 

1 Department of Pharmacology, Faculty of Pharmaceutical 

Sciences, Chulalongkorn University, 2 Department of 

Pharmacology, Faculty of Science, Mahidol University, Bangkok 


Key words : Cholesteryl esters, Oxidative damage, Thalassemia 

Serum markers of oxidative damage and cholesteryl esters 

were measured in lipoproteins of 30 β-thal/Hb E patients and 

compared with 10 healthy volunteers. Patients presented ironoverloading, 

a precipitous drop of α-tocopherol, and increased lipid 

peroxidation (TBARs) in both plasma and lipoproteins. Cholesteryl 

esters (CEs) in both LDL and HDL were separated and identified 

using HPLC. Cholesteryl linoleate, the most abundant CEs in 

lipoproteins was 70% lessen and some 50% reduction occurred to 

the others. The inverse relationship of the cholesteryl linoleate to 

cholesteryl oleate (CL/CO) ratios to the varying degrees of clinical 

severity suggested that the CL/CO ratio is an index of the damaged 

lipoproteins and may be used as a pathologic marker of the 

underlying iron overloading. A good correlation of NTBI and TBARs 

(r = 0.8, p < 0.01) in the LDL strongly support the contention of 

overloaded iron is responsible for initiating the lipid peroxidation 

in β-thal/Hb E. 

This study demonstrated that cholesteryl linoleate was the 

primary target of oxidative damage, induced by NTBI in the ?βthal/Hb 

E. It, thus, suggests that cholesteryl linoleate is the major 

target of oxidative damage in lipoproteins and may be used as a 

‘severity index’ of the thalassemic syndrome. 

CONTINUOUS TRACKING TASK (CTT): THE 

RELATIONSHIP OF TIME ON TASK TO THE 

EFFECTS OF SEDATIVE DRUGS (NO. 863) 

Soo-amon S 1 , Johnsen S 2 , Wongwitdecha N 1 , Plasen S 1 , 

Hindmarch I 2 


University, Bangkok, Thailand, 2 HPRU Medical Research 

Centre, University of Surrey, Guildford, UK. 

The continuous tracking task (CTT) is a psychomotor task 

assessing sensorimotor coordination and divided attention. CTT tasks 

have been shown to be sensitive to the impairing effects of 

psychoactive drugs and are often used in studies assessing behavior 

following drug administration. The aim of this retrospective study 

was to investigate the relationship between test duration (7.5 or 10 

min) and CTT performance following administration of alprazolam, 

lorazepam, promethazine and placebo. The results showed that 

alprazolam, lorazepam, and promethazine impaired performance on 

the CTT at 7.5 min and that a plateau was reached by 10 min for 

promethazine. In contrast, following placebo, performance on the 



CTT was stable at 7.5 min but had deteriorated by 10 min. These 

data suggest that performance on CTT tasks should take account of 

“time-on-task” as this appears to separate out drug induced 

impairment from task fatigue. 




MODIFICATION OF PARAOXONASE AND 

PLATELET- ACTIVATING FACTOR ACETYL- 

HYDROLASE ACTIVITES IN HEMIN-INDUCED 

LIPOPROTEIN OXIDATION (NO. 864) 

Wongroganawong D, Chalermchoung C, Unchern S, 

Chantharaksri U, Phumala Morales N. 

Department of Pharmacology, Faculty of Science, Mahidol 

University, Bangkok 10400. 

Paraoxonase (PON) and platelet-activation factor 

acetylhydrolase (PAF-AH) are antioxidant enzymes associated with 

the HDL particle. Their functions are known to protect lipoproteins 

from oxidative modification. PON activity has been showed to be 

modified in oxidized HDL and in oxidative related diseases such as 

atherosclerosis. Although PAF-AH activity has been also reported 

to be modified in several diseases, there was no evidence related to 

the oxidative stress conditions. Hemin, a degradative product of 

hemoglobin, has been reported as a potent oxidative inducer of 

lipoproteins in vitro. It has become a prime suspect to be responsible 

for lipoprotein oxidation in thalassemic patients because of its high 

concentration in the serum of patients. This study was, therefore, 

aimed to study kinetic of hemin induced oxidation of lipoproteins 

on the modification of PON (in HDL) and PAF-AH (in LDL and 

HDL) activities. The results clearly demonstrated that PON was more 

susceptible to hemin-induced oxidative stress than PAF-AH. In HDL, 

PON activity was rapidly decreased within 1 hr after incubation 

with hemin. The activity of PON was continuously decreased. Its 

activity was remained only 30 % after 10 hr of the incubation. On 

the other hand, PAF-AH activity was unchanged in HDL but only 

slightly reduced in LDL, about 20% after 10 hr of incubation. Our 

results suggested that the loss of PON activity in hemin induced 

HDL oxidation may be relevant in vivo and could have and impact 

in thalassemia. 




IN VIVO PHENOTYPING OF CY2A6 IN THAIS : 

COUMARIN VS NICOTINE (NO. 865) 

Peamkrasatam S 1,4 , Sriwatanakul K 1 , Wananukul V 2 , Sura T 2 , 

Chavalittumrong P 3 , Kamataki T 4 , Yoovathaworn K 1 


University, Bangkok Thailand, 2 Department of Medicine, Faculty 

of Medicine at Ramathibodi Hospital, Mahidol University, 

Bangkok, Thailand, 3 Department of Medicine Science, Ministry 

of Public Health, Bangkok, Thailand, 4 Laboratory of Drug 

Metabolism, Graduate School of Pharmaceutical Science, 

Hokkaido University, Sapporo, Japan. 

The association between the distribution characteristics of CYP2A6 

catalytic activityies towards coumarin and nicotine was analyzed in 

120 apparently healthy Thai volunteers. The probe drugs were given 

sequentially in the same subject with an approximate interval of 

one week, Urinary 7-hydroxycoumarin, plasma cotinine, and plasma 

nicotine were measured using HPLC assay. Genotyping for CYP2A6 

gene of the subjects was also performed. The distribution of 7-OHC 

excreted in the subsequent 8-hr urine (0.03-15 mg) after a single 

oral administration of 15 mg coumarin (Venalot®) and cotinine/ 

nicotine ratio of the plasma concentrations (0.00-13.48) 2 hr after 

chewing a piece of nicotine chewing gum containing 2 mg nicotine 

(Nicorette®) showed clearly bimodality. However, the probit plot 

of cotinine/nicotine ratio in the plasma showed the higher number 

of apparent poor metabolizers (PM), in comparison to that of 7- 

OHC excreted in the urine. Despite the discordance in the number 

of PM subjects, a correlation between the in vivo dispositions of 

coumarin and nicotine was extremely close (R=0.92). No statistically 

significant difference in the CYP2A6 activities between male and 

female subjects was found. The results confirm that phenotyping of 

CYP2A6 using coumarin and that using nicotine are not 

metabolically equivalent. Ten subjects with known CYP2A6 

genotypes were given a tablet of 15 mg coumarin again. Liver 

function test was investigated just before and24 hr after the coumarin 

administration. The results showed that coumarin at the challenging 

dose did not disturb the liver function even in the three subjects 

genotyped as CYP2A6*4/*4. Since the approximate half life of 

coumarin in human is only 2 hr, the probe drugs, therefore, could be 

theoretically given within two days consecutively. Even though both 

coumarin and nicotine can be clinically used as probe drugs in routine 

testing for CYP2A6 phenotype our results indicate that giving 

coumarin and nicotine sequentially to the same subject is a better 

protocol for a precise CYP2A6 phenotyping. 




THE ACCUMULATION OF CADMIUM IN SNAKE- 

HEAD FISH (Ophicephalus striatus) FROM 

BANGKOK METROPOLITAN MARKETS. (NO. 866) 

Sarunya Laovitthayanggoon 1 , Auratai Aramphongphan 1, 2 , 

Lakana Himakhun 3 

1 Toxicology Graduate Program, Faculty of Science, Mahidol 

University ; 2 Department of Pharmacology, Faculty of Science, 

Mahidol University ; 3 Department of Pathobiology, Faculty of 

Science, Mahidol University. 

Key words: Snakehead-fish, Cadmium and GFAAS 

The study was carried out to investigate the cadmium 

contamination in the various tissues (muscle, skin, gill and liver) of 

snakehead fish (Ophicephalus striatus), a fish with scales commonly 

found in Southeast Asia, which is economically important to 

Thailand. The samplings covered six Bangkok markets: Bangkok 

Noi, Ratchawat, Klongteoy, Yingchareon, Wongweanyai and Sainert 

markets. The cadmium level was measured by the Atomic Absorption 

Spectrophotometry (GFAAS). Results showed that the level of 

cadmium accumulation in muscle, skin, gill and liver were 0.02- 

0.10, 0.02-0.07, 0.02-0.07 and 0.1-1.7 ppm (µg/g) respectively. The 

highest level was found in the liver especially at Bangkoknoi market, 

which was around 1.7 ppm (mg/kg.), which was 0.2 ppm more than 


328 

acceptable standard in Thailand. Further investigation for detected 

cadmium level toxicity in snakehead-fish cell line is now underway. 

(Presented at: 31 st Congress on Science and Technology of Thailand 

(STT 2005), 18-20 October 2005 At Techno polis, Suranaree 

University of Technology, Nakhon Ratchasima) 

THE CONTAMINATION OF CADMIUM IN 

CATFISH (Clarius batrachus) FROM BANGKOK 

METROPOLITAN MARKETS (NO. 867) 

Udarat Boonraksa 1 , Auratai Aramphongphan 1, 2 , Lakana 

Himakhun 3 

1 Toxicology Graduate Program, Faculty of Science, Mahidol 

University; 2 Department of Pharmacology, Faculty of Science, 

Mahidol University; 3 Department of Pathobiology, Faculty of 

Science, Mahidol University. 

Key words : cadmium, catfish, GFAAS 

In this study, the cadmium contaminated in catfish (Clarias 

batrachus) tissues was analyzed, 36 samples collected from six 

markets cover Bangkok metropolitan; i.e. Bangkoknoi, 

Wongweinyai, Sainert, Yingchareon, Klongtaey and Rachawat 

markets. Various organs of fish (muscles, skin, gills and liver) were 

collected and dried by a freeze dryer, digested in nitric acid by a 

digestive microwave oven. Cadmium levels were determined by 

Graphite Furnance Atomic Absorption Spectrophotometer (GFAAS). 

The results showed that the levels of cadmium accumulation in 

muscle, skin, gills and liver were 0-0.036, 0-0.045, 0.001-0.067 and 

0.010-0.185 ppm respectively. The highest level was found in the 

liver (0.185 ppm), which is still lower than the standard level as 

determined by the Fish Inspection and Quality Control Division, 

Thailand. 


(STT 2005), 18-20 October 2005 at Technopolis, Suranaree 


GENOTOXICITY TESTING OF COMMONLY 

AVAILABLE THAI HERBS (NO. 868) 

Nanthiya Rattanakhot, Auratai Arampongphan, Patoomrat 

Toojinda, Panya Temjarean, Kumpon Sriwatanakul, 

Apanchanid Thepouyporn 

1 Toxicology graduate program, Faculty of science, Mahidol 

University; 2 Department of Pharmacology, Faculty of science, 

Mahidol University; 3 Department of Chemistry, Faculty of 

science, Mahidol University; 4 Department of Pathobiology, 

Faculty of science, Mahidol University; 5 Department of 

Nutrition of Tropical & food Science, Faculty of Tropical 

medicine, Mahidol University. 

Key words: Genotoxicity, Ames’ test, Thai herbs 

At present Thai herbs are wildly used in several Thai spas 

and hospitals for health and beauty benefits. The commonly used 

herbs include Zingiber cassumunar (Plai), Curcuma xanthorrhiza 

(Wan-chak-mod-luk), Curcuma longa (Cha-min-chan), Piper 

nigrum (Black pepper), some herbal weight loss products, and Ya- 


ayu-wattana (Thai herbal “elixir”). In this study, genotoxicity testing 

was conducted to assess the mutagenicity potential of these herbs. 

Crude extracts of Wan-chak-mod-luk, Cha-min-chan, Black pepper, 

herbal weight loss products, and Ya-ayu-wattana were extracted by 

95 % ethanol and dried by Lyophilizer. The Plai was extracted by 

steam distillation. The bacterial reverse mutation test (Ames’ test) 

using Salmonella Typhimurium TA 98 and TA 100 with and without 

an extract of rat liver homogenate (S9 fraction) were used to detect 

the mutagenic effects in this study. The results indicated no 

mutagenic effects in the herb-extract tested. However, Zingiber 

Cassumunar showed dose response in Salmonella Typhimurium TA 

98 with S9 fraction. Further studies using mammalian cell culture 

or another model of chromosome aberration are recommended for 

assessing these herbs more thoroughly. 


(STT 2005), 18-20 October 2005 at Technopolis, Suranaree 


A ROLE OF CATION EXCHANGE CAPACITY ON 

THE FORMATION OF POLYSTYRENE-CLAY 

NANOCOMPOSITES BY IN-SITU INTERCALA- 

TIVE POLYMERIZATION (NO. 869) 

S. Apiwantrakul 1 , T. Srikhirin 1,3 , D. Triampo 2,4 , R. 

Puttiworanart 5 , S. Limpanart 5 , T. Osotchan 1,3,4 , and W. 

Udomkichdecha 5 

1 Physics Department, 2 Chemistry Department, 3 Nanoscience and 

Nanotechnology Capability Building Unit, Faculty of Science, 

Mahidol University, Rama 6 Rd., Rachathevee, Bangkok 10400, 

Thailand 4 Institute of Science and Technology for Research and 

Development, Salaya Campus, Mahidol University, Nakorn 

Pathom 71730, Thailand 5 Metallurgy and Materials Science 

Research Institute, Chulalongkorn University, Phyathai Rd., 


Key words: nanocomposites, polystyrene, surfactants, 

montmorillonite, intercalation 

An effect of cation exchange capacity (CEC) on a formation 

of polystyrene clay nanocomposites is reported. Two types of 2:1 

layered silicate having different CEC’s, Wyoming (SWy, 97 meq/ 

100g of clay) and Bentonite-H (BNH, 131 meq/100g of clay) were 

investigated. The organoclay was prepared by mixing purified clay 

with octadecyldimethylammonium chloride (ODA) in aqueous 

solution. The packing of the intercalated ODA surfactant was found 

to depend on the CEC and the degree of solvent extraction. Two 

possible phases of the interlayer packing, solidlike and liquidlike, 

were detected for the extracted BNH due to the charge heterogeneity 

of the clay. The liquidlike phase shows a good affinity toward styrene 

monomer which promotes a formation of exfoliated nanocomposites. 

On the other hand, the solidlike phase shows a restricted dispersion 

in styrene monomer. The organoclay interlayer shows a limited 

expansion by the styrene monomer. This leads to the formation of 

intercalated nanocomposites. An increase in the percentage of 

organoclay loading hinders a formation of the exfoliated 

nanocomposites. 

(Journal of Applied Polymer Science, 95, 785-789 (2005) 



EFFECT OF THE SURFACTANT COVERAGE ON 

THE PREPARATION OF POLYSTYRENE-CLAY 

NANOCOMPOSITES PREPARED BY MELT 

INTERCALATION (NO. 870) 

S. Limpanart 1,2 , S. Khunthon 2 , P. Taepaiboon 3 , P. Supaphol 3 , T. 

Srikhirin 4,5,* , W. Udomkichdecha 1 

, Y. Boontongkong6 . 

1 Department of Materials Science, Faculty of Science, 

Chulalongkorn University, Phayathai, Bangkok 10330, 

Thailand; 2 Metallurgy and Materials Science Research Institute, 


Thailand; 3 Petroleum and Petrochemical College, 


Thailand; 4 Department of Physics, Faculty of Science, Mahidol 

University, Phayathai, Bangkok 10400, Thailand; 5 Capability 

Building Unit in Nanoscience and Nanotechnology, Faculty of 

Science, Mahidol University, Phayathai, Bangkok 10400, 

Thailand; 6 National Metal and Materials Technology Center, 


Klongluang, Prathumthani 12120, Thailand. 

Key words: nanocomposites, nanomaterials, polymers, clay, 

polystyrene 

The melt intercalation of polystyrene (PS) into organoclay 

with varying the degree of surfactant coverage by melt intercalation 

was investigated. The surface coverage of the organoclay was found 

to play a major role in controlling the type of the final composites 

formation. Two major types of composites were found depending 

upon the surface treatment of the organoclay. They are conventional 

composites and intercalated nanocomposites. At the high degree of 

clay’s surface coverage, the conventional composite was obtained. 

It is characterized by the micron size aggregated of the organoclay 

particle. An intercalated nanocomposite was observed in the 

organoclay with a lower surface coverage. The organoclay is 

dispersed into a smaller stack along with the intercalation of 

polystyrene into the organoclay interlayer. This is originated from 

the affinity between the organoclay surface and a molten polystyrene. 

(Material Letter, 59, 2292-2295 (2005) 

ELECTROSPINNING OF POLYSTYRENE/POLY 

(2-METHOXY-5 (2’ETHYLHEXYLOXY)-1,4-PHENY- 

LENE VINYLENE) BLENDS (NO, 871) 

Patcharaporn Wutticharoenmongkol 1 , Pitt Supaphol 1 , Toemsak 

Srikhirin 2 , Teerakiat Kerdcharoen 2 and Tanakorn Osotchan 2 

1 The Petroleum and Petrochemical College, Chulalongkorn 

University, Soi Chula1 2, Phyathai Road, Pathumwan, Bangkok 

10330, THAILAND; 2 Department of Physics, Faculty of Science, 

Mahidol University, Rama 6 Road, Ratchathewi, Bangkok 

10400, THAILAND. 

Key words: electrospinning; polystyrene; poly(phenylene vinylene) 

Ultrafine polystyrene (PS)/poly(2-methoxy-5-(2’ethylhexyloxy)-1,4-phenylene 

vinylene) (MEHPPV) fibers were 

successfully prepared by electrospinning of PS/MEH-PPV solutions 

in chloroform, 1,2-dichloroethane, and tetrahydrofuran (THF). Three 

concentrations of the solutions were prepared: 8.5, 16, and 23.5% 

(w/v), with the compositional weight ratios between PS and MEH- 

PPV being 7.5:1, 15:1, and 22.5:1, respectively. Smooth fibers only 

observed from 23.5% (w/v) PS/MEH-PPV solution in chloroform. 

Improvement in the electro-spinnability of 8.5% (w/v) PS/MEH- 

PPV solution in chloroform was achieved by addition of an organic 

salt, pyridinium formate (PF), or by addition of a minor solvent 

with high dielectric constant value. The average fiber diameters of 

the as-spun PS/MEH-PPV fibers were between 0.30 and 5.11 µm. 

(Journal: Journal of Polymer Science, Part B, Polymer Physics, 

43, 1881-1891 (2005)) 

ON THE ESTIMATION OF SOLAR ENERGETIC 

PARTICLE INJECTION TIMING FROM ONSET 

TIMES NEAR EARTH (NO. 872) 

Alejandro Saiz, 1,2 , Paul Evenson 3 , David Ruffolo 1 , and John W. 

Bieber 3 

1 Department of Physics, Faculty of Science, Mahidol Univesity, 

Bangkok 10400, Thailand; 2 Department of Physics, Faculty of 

Science, Chulalongkorn University, Bangkok 10330, Thailand; 

3 Bartol Research Institute, University, Delaware, Newark DE 

19716. 

Key words : interplanetary medium,methods numerical, solar 

terrestrial relations 

We examine the accuracy of a common technique for 

estimating the stat time of solar energetic particle injection based 

on a linear fit to the observed onset time versus 1/(particle velocity). 

This is based on a concept that the first arriving particles move 

directly along the magnetic field with no scattering. We check this 

by performing numerical simulations of the transport of solar protons 

between 2 and 2000. MeV from the Sun to the Earth, for several 

assumptions regarding interplanetary scattering and the duration of 

particle injection, and by analyzing the results using the inverse 

velocity fit. We find that, in most cases, the onset times align close 

to a straight line as a function of inverse velocity. Despite this, the 

estimate injection time can be in error by several minutes. Also, the 

estimated path length can deviate greathy from the actual path length 

along the interplanetary magnetic field. The major difference 

between the estimazted and actual path lengths implies that the first 

arriving particles cannot be viewed as moving directly along the 

interplanetary magnetic field. 

(The Astrophysical Journal, 626:1131-1137, 2005 June 20) 

RELATIVISTIC SOLAR NEUTRONS AND PROTONS 

ON 28 OCTOBER 2003. (NO. 873) 

John W. Biever 1 , John Clem 1 , Paul Evenson 1 , Roger Pyle 1 , David 

Ruffolo 2 , Alejandro Saiz 3 

1 Bartol Research Institute, University of Delaware, Newark, 

Delaware, USA.; 2 Department of Physics Mahidol University, 

Bangkok, Thailand.; 3 Department of Physics, Chulalongkorn 


The solar cosmic ray event associated with the X17.2 class 

flare of 28 October 2003. was unusal in several respects (1) Several 

high-latitude neutron monitors observed a large, highly anisotropic 


330 

spike at event onset. (2) The earliest onset was detected by stations 

viewing towards the anti-Sunward hemisphere. (3) The event 

displayed an extremely slow, protracted decay. (4) The near equatorial 

monitor in Tsumeb, Africa recorded a small increase consistent with 

a solar neutron event = 7 minutes prior to the onset at high latitudes. 

We analyze these signals and infer that relativistic solar neutrons 

were emitted over a duration of = 9 minutes, starting 7 minutes 

before the main injection of relativistic protons. 

(Gepohysical research letters, Vol 32. 2005.) 

TRANSPORT AND ACCELERATION OF SOLAR 

ENERGETIC PARTICLES FROM CORONAL 

MASS EJECTION SHOCKS (NO. 874) 

David Ruffolo 

Department of Physics, Faculty of Science, Mahidol University, 


Key words : Sun, particle emission, interplanetary medium 

After a brief overview of solar energetic particle (SEP) 

emission from coronal mass ejection (CME) shocks, we turn to a 

discussion of their transport and acceleration. The high energy SEP 

are accelerated near the Sun, and because of their well-known source 

location, their transport can be modeled quantitatively to obtain 

precise information on the injection function (number of particles 

emitted vs. time), including a determinatin of the onset time to within 

1 min. For certain events, transport modeling also indicates magnetic 

topology with mirroring or closed field loops. Important progress 

has also been made on the transport of low enery SEP from very 

strong events, which can display exhibit interesting saturation effects 

and compositional variations. The acceleration of SEP by CMEdriven 

shocks in the interplanetary medium is attributed to diffusive 

shock acceleration, but the spectrum of SEP production is typically 

modeled empirically. Recent progress has largely focused on using 

detailed composition measurements to determine fractionation 

effects of shock acceleration and even to clarify the nature of the 

seed population. In particular, there are many indications that the 

seed population is suprathermal (pre-energized) and the injection 

problem is not relevant to acceleration at interplanetary CME-driven 

shocks. We argue that the finite time available for shock acceleration 

provides the best explanation of the high-energy rollover. 

(International Astronomical Union 2005) 

FINITE TIME SHOCK ACCELERATION AND FITS 

TO ESP ION SPECTRA (NO. 875) 

Chanruangrit Channok 1,2,3 , David Ruffolo 2 , Mihir Desai 4 , and 

Glenn Mason 4 

1 Department of Physics, Chulalongkorn University, Bangkok, 

Thailand; 2 Department of Physics, Mahidol Univesity, Bangkok, 

Thailand; 3 Department of Physics, Ubonrajathanee University, 

Ubonrajathanee, Thailand; 4 Department of Physics, University 

of Maryland, Maryland, USA. 

Energetic storm particles (ESP) of various ion species have 

been shown to comprise suprathermal seed ions accelerated by 

traveling interplanetary shocks. The observed spectral rollovers at – 


0.1 to 10 MeV nucleon -1 can be attributed to the finite time available 

for shock acceleration. Using the locally measured shock strength 

parameters as inputs, the finite-time shock acceledration model can 

successfully fit the energy spectra of carbon, oxygen, and iron ions 

measured by ACE/ULEIS during 3 ESP events. The inferred 

scattering mean free path in the acceleration region ranges from 

typical interplanetary values for the weakest ESP events down to 

4.0 x 10 -3 AU for the stronget event. This is consistent with the idea 

that proton-amplified waves result from the very intense particle 

fluxes in major events. 

Work in Thailand was supported by the Commissionfor 

Higher Education, the Rachadapisek Sompoj Fund of Chulalongkorn 

University, and the Thailand Research Fund. Work at the University 

of Maryland was supported by NASA contract NAS5-30927 and 

NASA grant PC 251428. 

RELATIVISTIC SOLAR PROTONS ON 1989 

OCTOBER 22; INJECTION ALONG BOTH LEGS 

OF A LOOP (NO. 876) 

David Ruffolo 1 , Paisan Tooprakai 2 , Manit Rujiwarodom 2 , 

Thiranee Khumlumlert 3 , Maneenate Wechakama 4 , John Bieber 5 , 

Paul Evenson 5 , Roger Pyle 5 

1 Department of Physics, Mahidol University, Bangkok, Thailand; 


Thailand; 3 Department of Physics, Naresuan University, 

Phitsanulok, Thailand; 4 Department of Physics, Kasetsart 

University, Bangkok, Thailand; 5 Bartol Research Institute, 

University of Delaware, Newark, DE. 

Worldwide neutron monitor observations of relativistic solar 

protons on 1989 October 22 have proven puzzling,with an initial 

spike at some stations followed by a hump with bi-directional flows 

and a very slow decay. We analyze data from polar monitors, which 

measure the directional distribution of solar energetic particles 

(mainly protons) at rigidities of –1-3 GV. The inferred density and 

anisotropy are simultaneously fit by simulating the particle transport 

for various magnetic field configurations and determining the bestfit 

injection function near the Sun. The data are not well fit for an 

Archimedean spiral field, a magnetic bottleneck beyond Earth, or 

particle injection along one leg of a closed magnetic loop. A model 

with simultaneous injection along both legs of a closed loop provides 

the best explanation. Refined fits indicate a very low spectral index 

of turbulence, q < 1, and hence an unusually low correlation length 

of magnetic fluctuations in the loop, a parallel scatterig mean free 

path of 1.2 to 2 AU, a loop length of 4.7 +/-0.3 AU, and escape 

from the loop on a time scale of 3 hours 

TURBULENCE, DROPOUTS AND SUPPRESSION 

OF THE FILED LINE RANDOM WALK (NO. 877) 

David Ruffolo 1 , Piyanate Chuychai 1,2,3 , William H. Matthaeus 3 , 

George Rowlands 4 , 

1 Department of Physics, Mahidol University, Bangkok, Thailand; 


Thailand; 3 Bartol Research Institute, University of Delaware, 

USA. 4 Department of of Physics, University of Warwick, 

Coventry, UK. 



We employ the well-tested to two-component model oF solar wind 

turbulence to explain dropouts in impulsive SEP events over 

distances – 1 AU from the Sun, while at longer distances, much 

faster diffusive transport is found. Magnetic field lines are 

temporarily trapped in filaments defined by the small-scale topology 

of the 2D component of turbulence (fluctuations with perpendicular 

wave vectors). Within such island, the 2D component does not 

contribute to the random walk, which therefore takes place at the 

much slower rate for the slab component (fluctuations with parallel 

wave vectors). A further consideration is that both observed and 

simulated dropouts occur very sharply. We provide computational 

evidence and a theoretical explanation that strong 2D turbulence 

can inhibit diffusion due to the slab component. Therefore, while 

the dropout filament are basically difiened by the small-scale 

topology of 2D turbulence, there can be sharp trapping boundaries 

where the 2D filed is strongest. 

RECORD-SETTING GROUND LEVEL 

ENHANCEMENT ; JANUARY 20, 2005 (NO. 878) 

John W. Bieber 1 , John Clem 1 , Paul Evenson 1 , Roger Pyle 1 , Marc 

Duldig 2 , John Humble 3 , David Ruffolo 4 , Alejandro Saiz 4,5 and 

Manit Rujiwarodom 5 

1 Bartol Research Institute, University of Delaware, Newark, 

Delaware, USA. 2 Australian Antarctic Division, Kingston, 

Tasmania, Australia; 3 School of Mathematics and Physics, 

University of Tasmania, Hobart, Tasmania, Australia; 


Bangkok, Thailand; 5 Department of Physics, Faculty of Science, 

Chulalongkorn University, Bangkok, Thailand. 

Within a 6-minute span on January 20, 2005, the count 

rate registered by a neutron monitor at the sea level station of 

McMurdo, Antarctica increased by a factor of 30, while the rate at 

the high-altitude (2820 m) site of South Pole increased by a factor 

of 56. The size of the increase at McMurdo qualifies it as the largest 

observed at sea level since the famous 1956 event, while the increase 

at Southe Pole may have been the largest (in percentage terms) ever 

registered by a neutron monitor. This paper uses data from the 

“Spaceship Earth” network of neutron monitors to characterize and 

model the time evolution of cosmic ray density and anisotropy during 

the event of January 20, 2005. 

OBSERVATION OF NEUTRON AND GAAM RAY 

EMISSIN FROM THE OCTOVER 28, 2003 SOLAR 

FLARE (NO. 879) 

John Bieber 1 , John Clem 1 , Paul Evenson 1 , Roger Pyle 1 , David 

Ruffolo 2 

1 Bartol Research Insitute, University of Delaware, Newark, 

Delaware; 2 Department of Physics, Faculty of Science, Mahidol 


Recently we published an analysis of the response of the 

Tsumeb neutron monitor to the large solar flare of October 28, 2003. 

(GRL doi:10.1029/2004GL021492, 2005) We concluded that the 

flare produced neutrons over an extended interval of approximately 

seven minutes. Gamma-rays observed from the SAMPEX spacecraft 

now confirm the extremely long duration of energetic emIssion from 

this event. Both POLAR and SAMPEX have detected protons that 

may have resulted from the decay of neutrons emitted by this flare. 

We use these data to determine the energy spectrum of neutrons 

emitted by the flare, and to further refined our calculation of the 

time structure of the emission. We compare the emission time 

structure of the neutrons and gamma-rays with available optical data, 

and with the injection profile of the GeV interplanetary protons in 

an attempt to identify the source region of this energetic radiation. 

RELATIVISTIC PARTICLE INJECTION AND 

INTERPLANETARY TRANSPORT DURING THE 

JANUARY 20, 2005 GROUND LEVEL ENHANCE- 

MENT (NO. 880) 

Alejandro Saiz 1 , David Ruffolo 1 , Manit Rujiwarodom 2 , John W. 

Bieber 3 , John Clem 3 , Paul Evenson 3 , Roger Pyle 3 , Marc L. 

Duldig 4 , John E. Humble 5 



Chulalongkorn University, Bangkok, Thailand; 3 Bartol Research 

Institute, University of Delaware, Newark, Delaware, USA.; 

4 Australian Antractic Division, Kingston, Tasamania, Australia, 

5 School fo Mathematics and Physics, University of Tasamnia, 

Hobart, Tasamnia Australia. 

Besides producing the largest ground level enhancement 

(GLE) in half a century, the relativistic solar particles detected during 

the event of January 20, 2005 showed some interesting temporal 

and directional features, including extreme anisotropy. In this paper 

we analyze the time evoluation of cosmic ray density and anisotropy 

as characterized by data from the “Spaceship Earth” network of 

neutron monitors by using numerical solutions of the Fokker-Planck 

equation for particle transport. We find that a sudden change in the 

transport conditions during the event is needed to explain the data 

and we propose that this change was cuased by the solar particles 

themselves. 

(29 th International Cosmic Ray Conference Pune (2005), 101-104.) 

LARGEST GLE IN HALF A CENTURY ; NEUTRON 

MONITOR OBSERVATIONS OF THE JANUARY 20, 

2005 EVENT (NO. 881) 

John W. Bieber 1 , John Clem 1 , Paul Evenson 1 , Roger Pyle 1 , Marc 

Duldig 2 , John Humble 3 , David Ruffolo 4 , Manit Rujiwardom 5 , 

Alejandro Saiz 5,6 

1 Bartol Research Institute, University of Delaware, Newark DE 

19716, USA. 2 Australian Antarctic Division, Kingston, Tasmania, 

Australia; 3 School of Mathematics and Physics, University of 

Tasmania, Hobart, Tasmania, Australia; 4 Department of Physics, 

Faculty of Science, Mahidol University, Bangkok, Thailand; 

5 Department of Physics, Faculty of Science, Chulalongkorn 


Within a 6-minute span on January 20, 2005, the count 

rate registered by a neutron monitor at the sea level station of 

McMurdo, Antarctica increased by a factor of 30, while the rate at 

the high-altitude (2820 m) site of South Pole increased by a factor 

of 56. The size of the increase at McMurdo qualifies it as the largest 

observed at sea level since the famous 1956 event, while the increase 

at South Pole may have been the largest (in percentage terms) ever 


332 

registered by a neutron monitor. This paper uses data from the 

“Spaceship Earth” network of neutron monitors to characterize the 

time evolution of cosmic rays during the event. We also investigate 

spectral evolution using multiplicity data from a specially 

instrumented mobile monitor that was located in McMurdo Sound 

at the time of the event. 

INJECTION AND TRANSPORT OF RELATIVISTIC 

SOLAR PROTONS ALONG BOTH LEGS OF A 

CLOSED INTERPLANETARY MAGNETIC FIELD 

LOOP (NO. 882) 

David Ruffolo 1 , Paisan Tooprakai 2 , Manit Juiwarodom 2 , 

Thiranee Khumlumlert 3 , Maneenate Wechakama 4 , John W. 

Biever 5 , Pau Evenson 5 , Roger Pyle 5 



Chuylalongkorn University, Bangkok, Thailand; 3 Department 

of Physics, Faculty of Science, Naresuan University, Phitsanulok 

65000, Thailand; 4 Department of Physics, Faculty of Science, 

Kasetsart University, Bangkok 10900, Thailand.; 5 Bartol 

Research Institute, University of Delaware, Newark DE 19716, 

USA. 

Worldwide neutron monitor observations of relativistic solar 

protons on 1989 October 22 have proven puzzling, with an initial 

spike at some stations followed by a second peak, which is difficult 

to understand in terms of transport along a standard Archimedean 

spiral magnetic field or a second injection near the Sun. Here we 

analyze data form polar monitors, which measure the directional 

distribution of solar energetic particles (mainly protons) at rigidities 

of –1-3 GV. This event has the unusual properties that the particle 

density dips after the initial spike, followed by a hump with 

bidirectional flows and then a very slow decay. The density and 

anisotropy data are simultaneously fit by numerically solving the 

partial differential equation of particle transport for various magnetic 

field confirgurations and then performing numerical convolutions 

to determine the best-fit injection function near the Sun. The data 

are not well fit for an Archiedean spiral field, a magnetic bottleneck 

beyond Earth, or particle injection along one legs of a closed 

magnetic loop. A model with simultaneous injection along both 

legs of a closed loop proveds the best explanation: particles moving 

along the near leg make up the spike, those coming from the far leg 

make up the hump, and trapping the loop accounts for the slow 

decay of the particle density. The injection of particles near the 

Sun exhibits a short, strong peak, extended injection at a low level 

over = 100 minutes, and a sharp cutoff that is probably due to 

changing magnetic connection to the source. Refuined fits indicate 

a very low spectral index of turbulence q, a mean free path of 2.2 to 

3.3 AU, a loop lengh of 4.1 to 4.6 AU, and escape of relativistic 

protons from the loop on a time scale of 3 hours. The weak scattering 

is consistent with reports of weak fluctuations in magnetic loops 

and other regions other regions of high Alfven speed, while the low 

q value may indicate a lower correlation length as well. 

LONG-TERM PROLACTIN EXPOSURE 

DIFFERENTIALLY STIMULATED THE 

TRANSCELLULAR AND SOLVENT DRAG- 

INDUCED CALCIUM TRANSPORT IN THE 

DUODENUM OF OVARIECTOMIZED RATS (NO. 883) 

Nateetip Krishnamra, Kukiat Tudpor, and Narattaphol 

Charoenphandhu 

Department of Physiology, Faculty of Science, Mahidol 

University E-mail: scnks@mahidol.ac.th, scnch@mahidol.ac.th 

Key words : calcium, duodenum, prolactin 


Prolactin, having been shown to stimulate transcellular 

active and solvent drag-induced calcium transport in the duodenum 

of female rats, was postulated to improve duodenal calcium transport 

in estrogen-deficient rats. The present study therefore aimed to 

demonstrate effects of long-term prolactin exposure produced by 

anterior pituitary (AP) transplantation on the duodenal calcium 

transport in young (9-week-old) and adult (22-week-old) 

ovariectomized rats. We found that estrogen deficiency did not alter 

the transcellular active duodenal calcium transport in both young 

and adult rats fed with normal calcium diet (1.0% w/w Ca), but the 

same condition manifested a decrease in the solvent drag-induced 

duodenal calcium transport from 75.50±10.12 to 55.75±4.77 

nmol•hr -1 •cm -2 (P < 0.05) only in adult rats. Long-term prolactin 

exposure stimulated the transcellular active calcium transport in 

duodenum of young and adult AP-grafted ovariectomized rats fed 

with normal calcium diet by more than two-fold from 7.56±0.79 to 

16.54±2.05 (P < 0.001), and 9.78±0.72 to 15.99±1.75 (P < 0.001) 

nmol•hr -1 •cm -2 , respectively. However, only the solvent drag-induced 

duodenal calcium transport in young rats was enhanced by prolactin 

from 95.51±10.64 to 163.20±18.03 nmol•hr -1 •cm -2 (P < 0.001) while 

that in adult rats still showed a decreased flux from 75.50±10.12 to 

47.77±5.42 nmol•hr -1 •cm -2 (P < 0.05). Because oral calcium 

supplement has been widely used to improve calcium balance in 

estrogen-deficient animals, the effect of high calcium diet (2.0% w/ 

w Ca) was also investigated. The results showed that stimulatory 

action of long-term prolactin on the transcellular active duodenal 

calcium transport in both young and adult rats was diminished after 

feeding high calcium diet. The same diet also abolished prolactinenhanced 

solvent drag-induced duodenal calcium transport in young, 

and further decreased that in adult AP-grafted ovariectomized rats. 

We concluded that solvent drag-induced duodenal calcium transport 

in adult rats was decreased after ovariectomy. Long-term prolactin 

exposure stimulated the transcellular active duodenal calcium 

transport in both young and adult rats, while enhanced solvent draginduced 

duodenal calcium transport only in young rats. Effects of 

prolactin were abolished by high calcium diet. 

(Supported by the National Center for Genetic Engineering and 

Biotechnology (BIOTEC, to Prof. Nateetip) and the Thailand 

Research Fund (TRF, to Prof. Nateetip). 



SIGNIFICANT EFFECTS OF ESTROGEN ON 

CARDIAC MYOFILAMENT ACTIVATION 

UNDER DIABETIC COMPLICATION (NO. 884) 

Ariyaporn Thawornkaiwong and Jonggonnee Wattanapermpool 


University. E-mail: tejwt@mahidol.ac.th 

Key words: estrogen, insulin, diabetes 

Deficiency of either estrogen (E 2 ) or insulin (Ins) suppresses 

maximum myofibrillar ATPase activity to the same magnitude but 

only in ovariectomized (Ovx) hearts demonstrating myofilament Ca 2+ 

hypersensitivity. Surprisingly, no additive suppression in maximum 

myofibrillar ATPase activity was observed in diabetic-Ovx (Diab- 

Ovx) hearts in which the Ca 2+ hypersensitivity was still present. We 

therefore hypothesized that the two hormones play a similar 

regulatory mechanism in maximum myofibrillar ATPase activity but 

E 2 plays a dominant effect on myfilament Ca 2+ sensitivity. To 

investigate the potential regulatory pathway of the hormones on 

myofilament activation, relations of %?-myosin heavy chain (?- 

MHC) and maximum ATPase activity were then examined. Despite 

the same ratio of ATPase to % ?-MHC detected in Ovx and control 

groups, the ratio in Diab group was significantly increased. These 

results indicate an adaptive response of crossbridge interaction in 

Ins deficient heart which may be resulted from the presentation of 

E 2 . The same suppression of this ratio in Diab-Ovx hearts indicates 

beneficial effects of E 2 on cardiac adaptation in Diab rats. Concerning 

the proposed dominant role of E 2 in myofilament Ca 2+ 

hypersensitivity, the mobilization of intracellular Ca 2+ by 

sarcoplasmic reticulum (SR) was then focused. The SR Ca 2+ uptake 

activity and the expression of SR Ca 2+ ATPase (SERCA) were 

examined. Surprisingly, deficiency of both Ins and E 2 or of individual 

hormone caused reductions in both activity and expression of 

SERCA to the same degree. Thus, the induction in myofilament 

Ca 2+ hypersensitivity in E 2 deficient hearts may not be due to changes 

in intracellular Ca 2+ . Alterations in the myofilament apparatus could 

serve as a potential target for the hormonal regulation. Results from 

the present study clearly indicate an essential role of E 2 in cardiac 

myofilament activation especially under diabetic complication. 

(Supported by the Medical Scholar Program (MSP). 

CARDIO-PROTECTIVE EFFECTS OF EXERCISE 

TRAINING ON CHANGES IN SR Ca 2+ UPTAKE 

ACTIVITY IN OVARIECTOMIZED RATS (NO. 885) 

Jitanun Laosiripisan and Jonggonnee Wattanapermpool 



Key words : estrogen, exercise training, SR Ca 2+ uptake, SR Ca 2+ 

ATPase activity 

The use of hormone replacement therapy in postmenopausal 

women has been recently shown to induce many unfavorable effects, 

including cardiovascular defects, which then urges the studies of 

other preventive or therapeutic alternatives. In the present study, 

preventive effects of exercise training on changes in cardiac SR Ca 2+ 

uptake activity in 10-wk ovariectomized (OVX) rats were tested. A 

9-wk running program on a motor-driven treadmill was introduced 

to the exercise groups one week after surgery. The SR Ca 2+ uptake 

and SR Ca 2+ -ATPase (SERCA2a) activities were measured using 

left ventricular homogenate and SR vesicles, respectively. 

Significant suppressions in both Ca 2+ uptake and SERCA2a activities 

but increases in Ca 2+ uptake sensitivity were demonstrated in OVX 

hearts. Immunoblot analysis shows the downregulation of SERCA2a 

protein in OVX hearts with no change in the expression level of its 

regulatory protein called phospholamban (PLB). It is the 

phosphorylation of PLB either at Ser-16 or Thr-17 residues that will 

release its inhibitory action on SERCA2a. The amounts of these 

phosphorylated forms of PLB were then analysed using Western blot 

probing with individual phosphorylated-PLB antibody. There was 

a significant reduction in phospho-Thr-17-PLB in OVX hearts, in 

which exercise training could also prevent the change. These results 

indicate the cardio-protective effects of exercise training on changes 

in cardiac SR Ca 2+ uptake activities in OVX rats via restoration of 

both the amount and activity of SERCA2a protein. 

(Supported by The Thailand Research Fund, Faculty of Science, and 

Mahidol University) 

SIGNIFICANCE OF ESTROGEN ON CARDIAC 

CONTRACTILE ACTIVITY & THE BENEFIT OF 

PHYTOESTROGENS (NO. 886) 

Jonggonnee Wattanapermpool 



Key words: estrogen, cardiac contractile activity, exercise training 

An important question related to the well recognizable sex 

difference in the incidence of cardiovascular diseases is the 

significance of sex hormones on the cardiovascular system especially 

on the cardiac contractile function. Although the receptors are 

present in the cardiac myocardium, the regulatory role of estrogen 

in the cardiac contractile activity is still unclear. A series of 

experimental studies using ovariectomized rats as a model of estrogen 

deficiency have demonstrated a significant role of estrogen in 

regulating the cardiac contractile activity. Changes in the 

intracellular Ca 2+ in signaling the contraction/relaxation cycle were 

largely contributed by alterations in Ca 2+ handling process of the 

cardiac myocytes. Besides hormone replacement therapy, exercise 

training and phytoestrogens could be other alternative modes for 

the cardio-protection. 

(Supported by The Thailand Research Fund, Faculty of Science, and 

Mahidol University) 

SECRETION OF CARNITINE IN THE DISTAL 

CAPUT EPIDIDYMIDIS OF RATS PERFUSED 

IN VITRO (NO. 887) 

Chumpol Pholpramool and Kanokporn Poungpong 


University. E-mail: sccpp@mahidol.ac.th 

Key words : carnitine transporter, rat epididymis, carnitine 


334 

The mode of carnitine transport in the male reproductive tract is not 

clearly understood although carnitine plays an important role in male 

fertility. This study examined the mechanisms of [3H] L-carnitine 

secretion in a sperm-free isolated segment of the rat distal caput 

epididymidis perfused in vitro. The secretion varied with the 

perfusion rate from 0.25 to 1.35 ?l/min in a curvilinear fashion being 

4.00 + 0.63 pmol/min.cm at the perfusion rate of 0.78 + 0.03 ?l/ 

min, and was maintained for 3 hrs. The secretion was not altered 

when the luminal fluid contained L-carnitine at a concentration 10 

folds higher than that in the bath. Replacement of luminal Na + with 

either N-Methyl-D-glucamine or choline did not suppress the 

secretion of carnitine. Instead, it was significantly increased. 

Addition of acetyl-L-carnitine (250 ?M) in the bath failed to inhibit 

secretion. However, raising the concentration of L-carnitine in the 

bath from 30 to 500 ?M enhanced the rate of secretion up to 10 

folds. The results suggest that secretion of carnitine is carrier 

mediated. Uptake from the bath into the epididymal cells is achieved 

by a transporter with low Km and high Vmax, most likely CT1 

(OCTN2), which has been localized on the basolateral membrane 

of the epididymal tubule whilst efflux into the lumen occurs via at 

least 3 pathways: 1) CT2, probably the main route; 2) a transporter 

with high Km and Vmax; and 3) a carnitine exchanger. 

(Supported by Commission on higher Education Staff Development 

Program to K. Poungpong) 

INDUCTION OF APOPTOSIS IN THE ABSENCE 

OF CASPASE-3 BY THE FLAVONE VR-3623 (NO. 888) 

Kulawee Sujarit 1 , Orawin Prangsaengtong 1 , Samaisukh 

Sophasan 1 , Vichai Reutrakul 2 

1 Department of Physiology, 2 Department of Chemistry, Faculty 

of Science, Mahidol University, E-mail address: 

scssj@mahidol.ac.th 

Key words : VR-3623, MCF-7 cell, caspase-independent 

The mechanism of the antiproliferative activity of VR-3623, 

a flavone purified from a Thai tropical plant, Gardenia obtusifolia, 

on human MCF-7 breast cancer cells, which lack caspase-3 was 

investigated in vitro in the present study. Treatment of MCF-7 cells 

with 3 concentrations, 0.8 (GI 50 ), 2, and 8 ?M of VR-3623 for 3-72 

hrs. resulted in a dose-and time-dependent increased apoptotic cells 

as evaluated by 4’,6-diamidino-2-phenylindole (DAPI) staining and 

poly(ADP-ribose) polymerase (PARP) cleavage. The apoptotic cells 

characterized by chromatin condensation and DNA fragmentation 

increased from 8% to 66% as observed at 3 and 72 hrs., respectively, 

after incubation with 8 ?M of VR-3623. Immuno blot analysis 

showed the expression of the proteolytic PARP cleavage (85 kDa) 

at 24 and 72 hrs. Preincubation cells with 10 ?M of Z-VAD-MK, a 

pan caspase inhibitor, for 1 h. before the addition of 8 ?M of the 

flavone could alleviate cell death by 13% and completely prevent 

the cleavage of PARP. Measurement of caspases–9 and –7 activities 

in the apoptotic cells by using a fluorogenic assay showed that both 

caspases were activated, but at the low levels throughout the 

experiments. It is concluded that VR-3623 suppresses the growth of 

MCF-7 cells by induction of apoptosis and caspase-3 may not be 

essential in the apoptosis induced by this flavone. 

(Supported by the Thailand Research Fund and Deptartment of 

Physiology, Faculty of Science, Mahidol University.) 


ANTI-INFLAMMATORY AND IMMUNOMODU- 

LATORY ACTIVITIES OF STEVIOSIDE AND ITS 

METABOLITE, STEVIOL ON THP-1 CELLS (NO. 889) 

Chaiwat Boonkaewwan 1 , Chaivat Toskulkao 1 , Molvibha 

Vongsakul 2 

1 Department of Physiology and 2 Department of Microbiology, 


Thailand. E-mail address: kongchaiwat@hotmail.com; 

sccts@mahidol.ac.th; scmvs@mahidol.ac.th 

Key words : anti-inflammation, inflammatory cytokines, stevioside 

Stevioside (SVS), a natural noncaloric sweetener isolated 

from Stevia rebaudiana Bertoni, possesses anti-inflammatory and 

anti-tumor promoting properties; however, no information is 

available to explain its activity. The aim of this study was to elucidate 

the anti-inflammatory and immunomodulatory activities of 

stevioside and its metabolite, steviol (SVO). Stevioside 1 mM 

significantly suppressed lipopolysaccharide (LPS)-induced release 

of TNF-α and IL-1β and slightly suppressed nitric oxide release in 

THP-1 cells without exerting any direct toxic effect, whereas steviol 

100 µM did not. Activation of IKKβ and transcription factor NF-κB 

were suppressed by stevioside, as demonstrated by western blotting. 

Furthermore, only stevioside induced TNF-α, IL-1β and Nitric oxide 

release in unstimulated THP-1 cells. Release of TNF-α could be 

partially neutralized by anti-TLR4 antibody. This study suggested 

that stevioside attenuates synthesis of inflammatory mediators in 

LPS-stimulated THP-1 cells by interfering the IKKβ and NF-κB 

signaling pathway, and stevioside-induced TNF-α secretion is 

partially mediated through TLR4. 

(Supported by Mahidol University grants (Government Funds) and 

Ministry of University Affairs, Thailand) 

EVALUATION ON THE HEPATOPRO- 

TECTIVE EFFECTS OF THE STANDARD 

EXTRACTS FROM Curcuma longa and 

Andrographis paniculata (NO. 890) 

Surawat Jariyawat, Kanoknetr Suksen, Aporn Chuncharunee, 

Samaisukh Sophasan and Pawinee Piyachaturawat 

Department of Physiology, Graduate Program in Toxicology, 

Faculty of Science, and Department of Anatomy, Faculty of 

Medicine, Siriraj Hospital, Mahidol University. E-mail: 

scsjr@mahidol.ac.th 

Key words: Curcuma longa, Andrographis paniculata, 

hepatoprotection 

This study aims to evaluate the hepatoprotective effect of 

the standard extracts from Curcuma longa and Andrographis 

paniculata (Turmeric and AP-extracts). An oral administration of 

the extract was given to adult male mice 24 h prior to being induced 

by hepatotoxins and the leakage of hepatic enzymes in plasma was 

evaluated at 24 h later. The turmeric extract exhibited 

hepatoprotective activities against CCl 4 but not acetaminophen 

whereas the AP-extract showed hepatoprotective activities against 

both CCl 4 - and acetaminophen-induced toxicities. Changes of liver 

histopathology after treatments were well correlated with the changes 



of biochemical parameters. By using a human hepatocarcinoma 

cell line, HepG2, both of the standard extracts showed the protective 

activities against acetaminophen-induced toxicity. The results 

suggested that the AP-extract is more effective in providing 

protection against hepatotoxins. 

(Supported by a grant from the National Research Council of 

Thailand-2547) 

EVALUATION ON THE CHOLERETIC EFFECT 

OF HYDROXY ACETOPHENONES ON THE 

SECRETING SPECIES OF BILE ACIDS (NO. 891) 

Pawinee Piyachaturawat, Suparat Khamdang, Apichart 

Suksamrarn 

Department of Physiology and Graduate Program in Toxicology, 

Fac. of Science, Mahidol University; Department of Chemistry, 

Fac. of Science, Ramkhamhaeng University. E-mail: 

scppy@mahidol.ac.th 

Key words : Bile secretion, Bile acids, Hydroxyacetophenone 

Choleretic effect of hydroxy acetophenones has been 

reported to differently increase bile flow rate with varying biliary 

bile acids output as well as plasma cholesterol (EJP387: 21, 2000). 

The secretion varied from essential bile acid dependence to essential 

bile acid independence. The mechanisms by which these compounds 

exerted different actions remain unclear. This study aims to 

determine the species of bile acids in the secreted bile after receiving 

hydroxy acetophenones as bile acids provide the major force for 

bile formation. The compounds used were 2,4,6-tri, 2,6-di, 2,4-di 

and 4-mono hydroxy acetophenones (2,4,6-THA, 2,6-DHA, 2,4- 

DHA and 4-MHA). By using HPLC analysis, four major bile acids 

species were detected in normal rat bile. They were cholic, 

chenodeoxycholic, deoxycholic and ursodeoxycholic acids, 

accounting for about 70, 9, 3, and 18 % of total bile acids, 

respectively. After administration of the compounds at a dose of 

100 mg/kg, id, for 30 min, although the secreted bile acid outputs 

were markedly increased, their percent distribution of individual 

species was not different among groups and from the control. 4- 

MHA did not alter the output. It is suggested that hydroxy 

acetophenones stimulated biliary secretion of bile acids from the 

intracellular storage pool in the hepatocyte, not from newly 

synthesized bile acids. 

(Supported by Thailand Research Fund and ESTEM, MUA) 

ENDOGENOUS PROLACTIN MODULATED THE 

CALCIUM ABSORPTION IN THE JEJUNUM OF 

SUCKLING RATS (NO. 892) 

Suwimol Amnattanakul, Narattaphol Charoenphandhu, 

Liangchai Limlomwongse, and Nateetip Krishnamra 


University E-mail: scnks@mahidol.ac.th, naratt@narattsys.com 

Key words : calcium absorption, intestinal segments, prolactin 

Prolactin has been reported to stimulate intestinal calcium 

absorption in young and mature, but not aging rats. The present 

study was performed on suckling rats to elucidate the actions of 

endogenous prolactin on calcium absorption in various intestinal 

segments. Before measuring the calcium fluxes, 9-day-old rats were 

administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg 

bromocriptine, i.p., twice daily to abolish secretion of endogenous 

pro lac tin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, 

s.c. Thereafter, the 16-day-old rats were experimented upon by 

instilling the 45 Ca-containing solution into the intestinal segments. 

The results showed that, under a physiological condition, the jejunum 

had the highest rate of calcium absorption compared with other 

segments (1.4 ± 0.35 µmol·h –1 ·cm –1 , p < 0.05). The duodenum and 

ileum also manifested calcium absorption, whereas the colon showed 

calcium secretion. Lack of endogenous prolactin decreased lumento-plasma 

and net calcium fluxes in jejunum from 2.07 ± 0.31 to 

1.19 ± 0.12 and 1.40 ± 0.35 to 0.88 ± 0.18 µmol·h –1 ·cm –1 (p < 0.05), 

respectively, and exogenous prolactin restored the jejunal calcium 

absorption to the control value. Endogenous prolactin also had an 

effect on the duodenum but, in this case, exogenous prolactin did 

not reverse the effect of bromocriptine. However, neither ileal nor 

colonic calcium fluxes were influenced by prolactin. Because luminal 

sodium concentration has been demonstrated to affect calcium 

absorption in mature rats, the effect of varying luminal sodium 

concentrations on calcium fluxes in suckling rats was evaluated. 

The jejunum was used due to its highest rate of calcium absorption. 

After filling the jejunal segments with 124 (control), 80, 40 mmol/ 

L Na + -containing or Na + -free solution, increases in calcium 

absorption were found to be inversely related to luminal sodium 

concentrations in both control and bromocriptine-treated rats. The 

plasma concentration of 45 Ca under luminal sodium free condition 

was also higher than that of the control condition (2.26% ± 0.07% 

vs. 2.01% ± 0.09% administered dose, p < 0.05). However, 3 Hmannitol, 

a marker of the widening of tight junction that was 

introduced into the lumen, had a stable level in the plasma during 

an increase in plasma 45 Ca, suggesting that the widening of tight 

junction was not required for enhanced calcium absorption. In 

conclusion, calcium absorption in suckling rats was of the highest 

rate in the jejunum where endogenous prolactin modulated calcium 

absorption without increasing the paracellular transport of mannitol. 

(Supported by The Thailand Research Fund and the Graduate 

Student Grant of the Faculty of Graduate Studies, Mahidol 

University.) 

MECHANICAL VENTILATION INDUCES ALTERA- 

TIONS OF THE UBIQUITIN-PROTEASOME 

PATHWAY IN THE DIAPHRAGM (NO. 893) 

Keith C. DeRuisseau, 1 Andreas N. Kavazis, 1 Melissa A. Deering, 1 

Darin J. Falk, 1 Darin Van Gammeren, 1 Tossaporn Yimlamai, 1,2 

George A. Ordway, 3 and Scott K. Powers 1 

1 Department of Applied Physiology and Kinesiology, University 

of Florida, Gainesville, Florida; 2 Department of Physiology, 

Faculty of Science, Mahidol University, Bangkok, Thailand; and 

3 Department of Physiology, University of Texas Southwestern 

Medical Center, Dallas, Texas E-mail: sctyl@mahidol.ac.th 

Key words: respiratory muscle, proteolysis, proteasome activity 

Prolonged mechanical ventilation (MV) results in 

diaphragmatic atrophy due, in part, to an increase in proteolysis. 

These experiments tested the hypothesis that MV-induced 

diaphragmatic proteolysis is accompanied by increased expression 


336 

of key components of the ubiquitin-proteasome pathway (UPP). To 

test this postulate, we investigated the effect of prolonged MV on 

UPP components and determined the trypsin-like and 

peptidylglutamyl peptide hydrolyzing activities of the 20S 

proteasome. Adult Sprague-Dawley rats were assigned to either 

control or 12-h MV groups (n = 7/group). MV animals were 

anesthetized, tracheostomized, and ventilated with room air for 12 

h. Animals in the control group were acutely anesthetized but not 

exposed to MV. Compared with controls, MV animals demonstrated 

increased diaphragmatic mRNA levels of two ubiquitin ligases, 

muscle atrophy F-box (+8.3-fold) and muscle ring finger 1 (+19.0fold). 

However, MV did not alter mRNA levels of 14-kDa ubiquitinconjugating 

enzyme, polyubiquitin, proteasome-activating complex 

PA28, or 20S α-subunit 7. Protein levels of 14-kDa ubiquitinconjugating 

enzyme and proteasome-activating complex PA28 were 

not altered following MV, but 20S α -subunit 7 levels declined (– 

17.7%). MV increased diaphragmatic trypsin-like activity (+31%) 

but did not alter peptidylglutamyl peptide hydrolyzing activity. 

Finally, compared with controls, MV increased ubiquitin-protein 

conjugates in both the myofibrillar (+24.9%) and cytosolic (+54.7%) 

fractions of the diaphragm. These results are consistent with the 

hypothesis that prolonged MV increases diaphragmatic levels of key 

components within the UPP and that increases in 20S proteasome 

activity contribute to MV-induced diaphragmatic proteolysis and 

atrophy. 

(Supported by National, Heart, Lung, and Blood Institute Grant) 

BARAKOL EXTRACTED FROM CASSIA SIAMEA 

STIMULATES CHLORIDE SECRETION IN RAT 

COLON (NO. 894) 

Chatsri Deachapunya, Sutthasinee Poonyachoti, Watchareewan 

Thongsaard, Nateetip Krishnamra 


University E-mail: scnks@mahidol.ac.th 

Key words : Barakol, Cassia siamea, chloride secretion 

Barakol is a purified extract of Cassia siamea, a plant that 

has been used as a laxative in traditional medicine. In this study, the 

effect of barakol on anion transport across the rat colon epithelium 

was investigated. Colonic epithelium was mounted in Ussing 

chambers and bathed with Ringer’s solution. Addition of 1 mM 

barakol to the basolateral solution produced a slow increase in shortcircuit 

current (Isc) in proximal colon and distal colon by 24.5 +/- 

2.2 and 24.2 +/- 1.4 microA/cm(2), respectively. Barakol increased 

Isc in a concentration-dependent manner with an EC(50) value of 

0.4 mM. The barakol-stimulated increase in Isc was inhibited by 

subsequent treatment with 500 microM diphenylamine-2-carboxylic 

acid or 400 microM glibenclamide added to the apical solution and 

200 microM bumetanide added to the basolateral solution. 

Pretreatment of the tissues with 200 microM bumetanide, but not 

10 microM amiloride, completely abolished the barakol-increased 

Isc. Ion substitution experiments showed an inhibition of barakolstimulated 

Isc in chloride-free solution but not in bicarbonate-free 

solution. In addition, pretreatment of tissues with 10 microM 

tetrodotoxin or 10 microM indomethacin, but not 1 microM atropine 

or 10 microM hexamethonium, partially inhibited the Isc response 

by barakol. The present results demonstrated the stimulatory effect 

of barakol on the bumetanide-sensitive chloride secretion in rat colon. 

The effect of barakol was partly mediated by the stimulation of 

submucosal nerves and through the release of cyclooxygenase 

metabolites. These findings thus provide an explanation for the 

underlying mechanism of barakol as a secretagogue in mammalian 

colon. 

(Supported by The Thailand Research Fund) 


GDNF FAMILY RECEPTOR ALPHAL PHENOTYPE 

OF SPERMATOGONIAL STEM CELLS IN IMMA- 

TURE MOUSE TESTIS (NO. 895) 

Anyanee Buageaw , Meena Sukhwani , Ahmi Ben-Yehudah , 

Jens Ehmcke , Vanessa Y. Rawe , Chumpol Pholpramool , Kyle 

E. Orwig , and Stefan Schlatt 


University. E-mail: sccpp@mahidol.ac.th 

Spermatogonial stem cells (SSCs) are essential for 

spermatogenesis, and these adult tissue stem cells balance selfrenewal 

and differentiation to meet the biological demand of the 

testis. The developmental dynamics of SSCs are controlled in part 

by factors present in the stem cell niche, which is located on the 

basement membrane of seminiferous tubules situated among Sertoli 

cells. Sertoli cells produce glial cell line derived neurotrophic factor 

(GDNF) and disruption of GDNF expression results in spermatogenic 

defects and infertility. GDNF signals through a receptor complex 

that includes GDNF family receptor alpha1 (GFRA1), which is 

thought to be expressed by SSCs. However, expression of GFRA1 

on SSCs has not been confirmed by in vivo functional assay, which 

is the only method that allows definitive identification of SSCs. 

Therefore, we fractionated mouse pup testis cells based on GFRA1 

expression using magnetic activated cell sorting (MACS). GFRA1 

sorted and depleted fractions were characterized for germ cell markers 

by immunocytochemistry, and stem cell activity by germ cell 

transplantation. The GFRA1 positive cell fraction co-eluted with 

other markers of SSCs, including ITGA6 and CD9, and was 

significantly depleted of KIT positive cells. The transplantation 

results confirmed that a subpopulation of SSCs expresses GFRA1, 

but that the stem cell pool is heterogeneous with respect to the level 

of GFRA1 expression. Interestingly, POU5F1 positive cells were 

enriched nearly 15-fold in the GFRA1 selected fraction, possibly 

suggesting heterogeneity of developmental potential within the stem 

cell pool. 

(Supported by Commission on Higher Education Staff Development 

Program to A. Baugeaw) 

INTERACTIONS OF STEVIOSIDE AND STEVIOL 

WITH RENAL ORGANIC ANION TRANSPORTERS 

IN S2 CELLS AND MOUSE RENAL CORTICAL 

SLICES (NO. 896) 

Chutima Srimaroeng 1,2 , Promsuk Jutabha 3 , John B. Pritchard 2 , 

Hitoshi Endou 3 , Varanuj Chatsudthipong 1 


University; 2 Laboratory of Pharmacology and Chemistry, 

NIEHS, National Institutes of Health, Research Triangle Park, 

North Carolina, USA; 3 Department of Pharmacology and 

Toxicology, Kyorin University School of Medicine, Tokyo, Japan. 

E-mail: scvcs@mahidol.ac.th 



Key words : organic anion, organic anion transporter, renal cortical 

slices 

Our previous studies have shown that both stevioside and 

steviol inhibited transepithelial transport of para-aminohippurate 

(PAH) in isolated rabbit renal proximal tubules by interfering with 

organic anion transport system. The current study examined the 

direct interactions of stevioside and steviol with specific organic 

anion transporters. METHODS: S2 cells expressing human organic 

anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an 

intact renal epithelium were used to determine the inhibitory effect 

of stevioside and steviol on organic anion transport. RESULTS: 

Stevioside at 0.5-1 mM showed no interaction with any OAT. In 

contrast, steviol markedly inhibited substrate uptake in all S2hOAT 

cells. Steviol had low IC50 for hOAT1 (11.4 μM) and hOAT3 (36.5 

μM) similar to that of probenecid, whereas IC50 for hOAT2 (1000 

μM) and hOAT4 (285 μM) was much higher. Results obtained in 

mouse renal cortical slices were very similar; that is, stevioside was 

without inhibitory effect and steviol was a potent inhibitor of PAH 

and estrone sulfate (ES) transport. 

Conclusions : Stevioside has no interaction with human or 

mouse OATs. In contrast, steviol interacts directly with human OATs, 

in particular, hOAT1 and hOAT3, with a potency approximating 

probenecid, suggesting that the inhibition of OAT-mediated transport 

by steviol could alter renal drug clearance. 

(Supported by Royal Golden Jubilee and Thailand research fund) 

TRANSPORT OF THE NATURAL SWEETENER 

STEVIOSIDE AND ITS AGLYCONE STEVIOL BY 

HUMAN ORGANIC ANION TRANSPORTER 

(HOAT1; SLC22A6) AND HOAT3 (SLC22A8) (NO. 897) 

Chutima Srimaroeng, Varanuj Chatsudthipong, Amy G. 

Aslamkhan, John B. Pritchard 


University and Laboratory of Pharmacology and Chemistry, 

National Institute of Environmental Health Sciences, National 

Institutes of Health, Research Triangle Park, North Carolina 

(C.S., A.G.A., J.B.P.) E-mail: scvcs@mahidol.ac.th 

Key words : Organic anion transporter, steviol, stevioside 

The natural sweetening agent stevioside and its aglycone 

metabolite, steviol, have been shown to inhibit transepithelial 

transport of para-aminohippurate (PAH) in isolated rabbit renal 

proximal tubules by interfering with basolateral entry. The aim of 

the present study was to determine which of the cloned basolateral 

organic anion transporters were involved in the renal transport of 

stevioside and steviol. This question was addressed in Xenopus laevis 

oocytes expressing human organic anion transporter 1 (hOAT1), 3 

(hOAT3), and winter flounder OAT (fOat1). The parent compound, 

stevioside, had no inhibitory effect on either PAH (hOAT1) or ES 

(estrone sulfate; hOAT3) uptake. In contrast, steviol showed 

significant, dose-dependent inhibition of PAH and ES uptake in 

hOAT1- or hOAT3-expressing oocytes, respectively. The IC(50) of 

steviol for hOAT1-mediated PAH transport was 11.1 microM 

compared with 62.6 microM for hOAT3-mediated ES uptake. The 

Michaelis-Menten inhibition constants (K(i)) for steviol transport 

mediated by hOAT1 and hOAT3 were 2.0 +/- 0.3 and 5.4 +/- 2.0 

microM, respectively. Trans-stimulation of PAH efflux by steviol 

was assessed to determine whether steviol itself was transported by 

hOAT1 or hOAT3. A low concentration of 1 microM steviol increased 

the efflux of [(3)H]PAH (trans-stimulated) via both hOAT1 and 

hOAT3. In addition, it was shown by electrophysiology that steviol 

entry induced inward current in fOat1-expressing oocytes. In 

conclusion, stevioside had no interaction with either hOAT1 or 

hOAT3, whereas hOAT1, hOAT3, and fOat1 were all shown to be 

capable of steviol transport and thus, can play a role in its renal 

transport and excretion. 

(Supported by Royal Golden Jubilee and Thailand research fund) 

INVOLVEMENT OF TYROSINE KINASE AND 

PI3K IN THE REGULATION OF OAT3-MEDIATED 

ESTRONE SULFATE TRANSPORT IN ISOLATED 

RABBIT RENAL PROXIMAL TUBULES (NO. 898) 

Sunhapas Soodvilai 1 , Stephen H. Wright 2 , William H. Dantzler 2 , 

and Varanuj Chatsudthipong 1 


University; 2 Department of Physiology, University of Arizona, 

Tucson, Arizona, USA. E-mail: scvcs@mahidol.ac.th 

Key words : tyrosine kinase, PI3K, organic anion transporter 

It was shown previously that OAT3 activity was 

differentially regulated by protein kinases including MAPK, PKA, 

and PKC. The present study investigated the short-term effect of 

tyrosine kinase and phosphatidylinositol 3 kinase (PI3K) on OAT3mediated 

organic anion transport in S2 segments of renal proximal 

tubules. Genistein, a tyrosine kinase inhibitor, and wortmannin, a 

PI3K inhibitor, inhibited transport of estrone sulfate (ES), a 

prototypic substrate for OAT3, in a dose-dependent manner. 

Previously, we showed that epidermal growth factor (EGF) stimulated 

OAT3 activity via the MAPK pathway. In the present study, we 

investigated whether EGF-stimulated OAT3 activity was dependent 

on tyrosine kinase and PI3K. We showed that EGF stimulation of 

OAT3 was reduced by inhibition of tyrosine kinase or PI3K, 

suggesting that they play a role in the stimulatory process. Inhibitory 

effects also indicated that tyrosine kinase and PI3K are involved in 

the MAPK pathway for EGF stimulation of OAT3 in intact renal 

proximal tubules, with PI3K acting upstream and tyrosine kinase 

acting downstream of mitogen-activated/extracellular signalregulated 

kinase kinase (MEK) activation. 

(Supported by Supported by Royal Golden Jubilee) 

INHIBITORY EFFECTS OF CHOLERETIC 

HYDROXYACETOPHENONES ON ILEAL BILE 

ACID TRANSPORT IN RATS (NO. 899) 

Jainuch Kanchanapoo, Mrinalini C. Rao, Samaisukh Sophasan, 

Apichart Suksamrarn and Pawinee Piyachaturawat 


University . E-mail: scppy@mahidol.ac.th 

Key words : Ileum bile acid transport; Cholesterol, 

Hydroxyacetophenone 

The effects of the choleretic and cholesterol lowering 

compound, 2,4,6-trihydroxy acetophenone (THA) and its analog, 


338 

2,6-dihydroxy acetophenone (DHA), on ileal bile acid absorption 

were investigated in rats. THA inhibited taurocholate (TC) uptake 

into ileal brush border membrane vesicles (BBMV), showing a 

maximum inhibition of 50%, whereas DHA completely inhibited 

TC uptake into ileal BBMV. THA exhibited competitive inhibition 

with a K i of 9.88 mM, while DHA showed non-competitive inhibition 

with a K i of 7.65 mM. Both total and ouabain-sensitive basolateral 

membrane (BLM) Na + -K + -ATPase activities, which are essential for 

maintenance of the Na + -gradient for bile acid transport, were 

inhibited by THA and DHA in a dose-dependent manner. The 

inhibition of BLM ATPase was uncompetitive with a K i of 10.1 and 

5.0 mM for THA and DHA, respectively. Administration of THA 

or DHA (400 μmol/kg) twice a day, to hypercholesterolemic rats for 

3 weeks caused similar and marked reductions in plasma cholesterol 

to 60% of the cholesterol-fed controls. The data suggest that the 

inhibitory actions of THA and DHA on two essential components 

of ileal bile acid recycling to liver could, in part, contribute to the 

cholesterol lowering effect of the hydroxyacetophenone compounds. 

These effects on decreasing bile acid recycling, in combination with 

their potent choleretic effect, accelerating biliary excretion of bile 

acids, are responsible for the effective cholesterol lowering capacities 

of these compounds. 

(Supported by: The Royal Golden Jubilee program, (to JK, PP), a 

grant from the Thailand Research Fund (to PP, SS) and a research 

team strengthening grant from BIOTEC (to AS, PP) 

WEANLING, BUT NOT ADULT, RABBIT COLON 

ABSORBS BILE ACIDS : FLUX IS LINKED TO 

EXPRESSION OF PUTATIVE BILE ACID 

TRANSPORTERS (NO. 900) 

Dirk Weihrauch, Jainuch Kanchanapoo, Mei Ao, Roli Prasad, 

Pawinee Piyachaturawat and Mrinalini C. Rao 

Department of Physiology & Biophysics, University of Illinois 

at Chicago, Chicago, Illinois; Department of Animal Physiology, 

University of Osnabrück, Osnabrück, Germany; Department of 

Physiology, Faculty of Science, Mahidol University, Thailand. 

E-mail: scppy@mahidol.ac.th 

Key words : Apical sodium-dependent bile acid transporter (Asbt), 

Farnesoid X receptor (FXR), Ileal bile acid binding protein (iBABP). 

Intestinal handling of bile acids is age-dependent; adult, 

but not newborn, ileum absorbs bile acids, and adult, but not 

weanling/newborn, distal colon secretes chloride in response to bile 

acids. Bile acid transport involving the apical sodium-dependent 

bile acid transporter (Asbt) and lipid binding protein (LBP) is wellcharacterized 

in the ileum, but little is known about colonic bile 

acid transport. We investigated colonic bile acid transport and the 

nature of the underlying transporters/receptors. Colon from adult, 

weanling and newborn rabbits were screened by semi-quantitative 

RT-PCR for Asbt, its truncated variant t-Asbt, LBP, multidrug 

resistance protein 3 (Mrp3), organic solute transporter alpha 

(Ostalpha) and farnesoid X receptor (FXR). Asbt and LBP show 

maximal expression in weanlings and significantly less expression 

in the adults and newborns. The ileum, but not the colon, expresses 

t-Asbt. The mRNA expression of Asbt, LBP and FXR in the weanling 

colon parallels the profile in the adult ileum, a tissue designed for 

high bile acid absorption. To examine their functional role, 

transepithelial (3)H-taurocholate transport in weanling and adult 

colon and ileum was measured. Under short-circuit conditions the 


weanling colon (1.23 +/- 0.62 nmol/hr/cm (2)) and ileum (5.53 +/- 

1.20 nmol/hr/cm (2)) and the adult ileum (11.41 +/- 3.45 nmol/hr/ 

cm (2)) show net bile acids absorption. However, the adult colon 

secretes bile acids (-1.39 +/- 0.47 nmol/hr/cm (2)). We demonstrate 

for the first time that the weanling, but not adult, distal colon shows 

net bile acid absorption. Thus, the increased expression of Asbt and 

LBP in the weanling colon is associated with parallel increases in 

taurocholate absorption. This has relevance in the enterohepatic 

conservation of bile acids at a stage when ileal bile acid recycling is 

not fully developed. 

Background: Insulin resistance of skeletal muscle, a major 

tissue responsible for the peripheral disposal of glucose, is considered 

a major cause of type 2 diabetes. Recent evidence suggests that 

oxidative stress may play a role in the etiology of insulin resistance. 

The information on the direct effect of oxidative stress on insulin 

action in intact mammalian skeletal muscle is limited. Furthermore, 

as insulin action and cellular oxidant status can be modified by 

exercise training, no investigation has evaluated how exercise 

training would affect insulin-stimulated glucose transport in skeletal 

muscle subjected to oxidative stress. 

Objectives: The purposes of the present study were to 

examine the direct effect of oxidative stress on insulin action on 

skeletal muscle glucose transport and to evaluate the role of prior 

exercise training in oxidative stress- and insulin-stimulated glucose 

transport. 

Methods: Male Sprague-Dawley rats either remained 

sedentary or underwent exercise training. Isolated type I soleus (SOL) 

and type II extensor digitorum longus (EDL) were incubated in the 

medium in the absence or presence of 100 mU/ml glucose oxidase 

and/or 5 mU/ml of insulin. Thereafter, glucose transport activity 

was measured. 

Results: Hydrogen peroxide by itself activated (p


The information on the direct effect of oxidative stress on insulin 

action in intact mammalian skeletal muscle is limited. Furthermore, 

as insulin action and cellular oxidant status can be modified by 

exercise training, no investigation has evaluated how exercise 

training would affect insulin-stimulated glucose transport in skeletal 

muscle subjected to oxidatrive stress. 

Objectives : The purposes of the present study were to 

examine the direct effect of oxidative stress on insulin action on 

skeletal muscle glucose transport and to evaluate the role of rpior 

exercise taining in oxidative stress- and insulin-stimulated glucose 

transport. 

Methods : Male Sprague-Dawley rats either remained 

sedentary or underwent exercise training. Isolated type I soleus 

(SOL) and type II extensor digitorum longus (EDL) were incubated 

in the medium in the absence or presence of 100 mU/ml glucose 

oxidase and/or 5 mU/ml of insulin. Thereafter, glucose transport 

activity was measured. 

Results : Hydrogen peroxide by itself activated (p

340 

STRUCTURAL AND ANTIGENIC ANALYSIS OF 

THE YELLOW HEAD VIRUS NUCLEOCAPSID 

PROTEIN p20 (NO. 904) 

Nusra Sittidilokratna 1,2 , Natthida Phetchampai 1,2 , Vichai 

Boonsaeng 2 and Peter J Walker 3 

1 National Center for Genetic Engineering and Biotechnology 

(BIOTEC), National Science and Technology Development 

Agency (NSTDA), Phathumthani 12120, Thailand; 2 Center 

Excellence for Shrimp Molecular Biology and Biotechnology, 


Thailand; 3 CSIRO Livestock Industries, Australian Animal 

Health Laboratory, Bictora 3220, Australia. 

Key words : Yellow head virus, shrimp virus, nucleoprotein, 

bacterial expression 

Yellow head virus (YHV) is an invertebrate nidovirus that 

is highly pathogenic for marine shrimp. Nucleotide sequence 

analysis indicated that the YHV ORF2 gene encodes a basic protein 

(pl = 9.9) of 146 amiono acids with a predicted molecular weight of 

16,325.5 Da. The deduced amino acid sequence indicated a 

predominance of basic (15.1%) acidic (9.6%) and hydrophilic polar 

(34.3%) residues and high proportion proline and glycine residues 

(16.4%). The ORF2 gene was cloned and expressed in E. coli as a 

M r = 21 kDa His 6 - protein that reacted with YHV nucleoprotein 

(p20) monoclonal antibody. Segments representing the four linear 

quandrants of the nucleoprotein were also expressed in E. coli as 

GST –fusion proteins. Immunoblot analysis using YHV polyclonal 

rabbit antiserum indicated the presence of linear epitopes in all except 

the V 37 –Q 74 quadrant. Immunoblot analysis of the GST-fusion 

proteins and C-terminally truncated segments of the nucleoprotein 

allowed mapping of YHV monoclonal antibodies Y19, Y20 and YII4 

to linear epitopes in the acidic domain between amino acids I 116 and 

E 137 . The full-length nucleoprotein was expressed at high level in 

E.coli and was easily purified in quantity from the soluble cell 

fraction by Ni + - NTA affinity chromatography. 

BACTERIAL CLEARANCE RATE AND A NEW 

DIFFERENTIAL HEMOCYTE STAINING METHOD 

TO ASSESS IMMUNOSTIMULANT ACTIVITY ION 

SHRIMP (NO. 905) 

Kallaya Sritunyalucksana 1 , Warachin Gangnongiw 1 , Somwit 

Archakunakorn 2 , Daniel Fegen ,Timothy W. Flegel 1 

1 National Center for Gentic Engineering and Biotechnology 

(BIOTEC) and Centex Shrimp , and 2 Shrimp Biotecnology 

Business Unit (SBBU), Faculty of Science, Mahidal University, 

Rama VIRoad, Bangkok 10400, Thailand. 

Key words : Black tigr shrimp Penaeus⋅ mondon⋅ 

Hemocytes⋅Bacterial clearance⋅Immumostimulant 

New methods were developed to assess immunostimulant 

efficacy in the black tiger shrimp Penaeus monodon. Test Shrimp 

were fed with 2 or 4 % yeast extract (YE)-coated feed while controls 

were fed non-coated feed. After 4 wk of feeding , individual shrimp 

were assessed for total hemocvte counts(THC), the number of 

granular hemocytes (GH) and rate of bacterial clearance . For 

hemocyte counts, formalin-fixed hemolymph was stained with 1.2 

% Rose Bengal in 50 % ethanol for 20 min at room temperature . 

Some of this mixture was used for THC with a hemocytometer 

while some was smeared on a microscope slide and left to day before 

counterstaining with hematoxylin for GH counts , By this tecnicque 

,high quality smears were obtained for accurate differential 

counts.Bacterial clearance assays were used to assess the sum effect 

of humoral and cellular defense mechanisms. Vibrio harveyi was 

injected intramuscularly at 1x 10 8 cells per shrimp and hemlymph 

was collected in anticoaqulant 0,15,30 and 60 min post-injection 

for quadruplicate drop count (20 μl) on TCBS agar.Total hmocyte 

counts for shrimp fed with 4% YE were singnificantly higher 

(p

Faculty of Science - Mahidol University

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