Abstract conference mbsmb 2009

Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 25

ABSTRACTS OF THE33RD ANNUAL CONFERENCE

OF THE MALAYSIAN SOCIETYFOR BIOCHEMISTRY AND

MOLECULAR BIOLOGY

27TH & 28TH AUGUST 2008,ISTANA HOTEL, KUALA LUMPUR


Poster 1

AFFINITY CHROMATOGRAPHY TO STUDY INSECT GLUTATHIONE S-TRANSFERASES

ZAZALI ALIAS1, ALAN CLARK, DING XUE, MILANA MALIUK AND RAMAVATI PAL1Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia

School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

In insects, there are six families of GST (ΩΘ∑εΔΖ). In Drosophila these are encoded by 42 genes. It is becoming clear that these enzymes areimportant, not only for their detoxication functions but also for their roles in intracellular signalling (Δ) and intermediary metabolism (ΩZ).This work is concerned with the design and use of affinity chromatography media to achieve two contrasting aims. The first is to purifyquantitatively as possible, all of the insect GSTs at one time so that the complex responses of the whole GST proteome to environmentalchanges may be monitored. The second aim is to design affinity media that may be used to isolate preferentially the members of singlefamilies, so that the catalytic properties of the enzymes may be studied. Two types of affinity matrix have been employed. The immobilizedGSH matrix absorbs Sigma (S1) and Delta families (D1,D2,D3). When S-substituted GSH (attached via the glutamate N) were used asligands, short chain aliphatic substituents did not generate successful media, nor did polar substituents such as carboxy methylene andcarbamido methylene. More successful were aromatic and long chain aliphatic residues. S-hexyl-GSH matrix captures Omega and Epsilon aswell as Delta and Sigma -class GSTs.The S-(2,4-dinitrophenyl)-GSH matrix clearly strongly enriched in Epsilon-class GSTs.

Poster 2

STEADINESS OF GENE EXPRESSION IN SERIAL PASSAGEOF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

HAFIZAH A.H.1, ZAITON Z.1, CHUA K.H.1, NUR FARIHA M.M.3, HAYATI A.R.3, ZULKHAIRI A.4 AND ZALEHA A.M.2

1Department of Physiology, Medical Centre, Universiti Kebangsaan Malaysia2Department of Obstetric & Gynecology, Medical Centre, Universiti Kebangsaan Malaysia,

3Department of Pathology, Medical Centre, Universiti Kebangsaan Malaysia4Department of Human Anatomy, Faculty of Medicine and Allied Sciences, Universiti Putra Malaysia

Human umbilical vein endothelial cells (HUVEC) are frequently cultured for various passages in order to obtain large number of cells forresearch in the various area of vascular diseases including atherosclerosis and drug effects involving endothelial cells. The steadiness characteristicand phenotype of the cultured HUVEC is essential to be maintained throughout the experiment in order to obtain reliable data. However, untiltoday the gene expression profile of the cultured HUVEC has not been clarified yet. The goal of this study was to determine the gene expressionprofile of cultured HUVEC after serial passage. HUVEC were obtained by collagenase perfusion of the large vein in the umbilical cord andcultured in medium M200 supplemented with Low Serum Growth Supplement. The confluence HUVEC was then passage several times. TotalRNA of the cultured HUVEC was extracted using TRI-REAGENT®. All primers were designed to be human specific by using Primer 3 software.Real-Time PCR reaction mixes were composed of SYBR green, 5μm of each primer, 1 μl cDNA and Taq DNA polymerase. The specificity of thereaction was confirmed by melting curve analysis and verified by agarose gel electrophoresis. Expression level of each gene was then normalizedwith GAPDH. The endothelial cell-associated genes; platelet endothelial cell adhesion molecules (PECAM), VE-cadherin, endothelial cell nitricoxide synthase (eNOS), CD34, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), vonWillebrand factor (vWF) were all expressed by cultured HUVEC and did not change significantly with serial passage. These findings havedemonstrated our current culture system can maintained the phenotype of the cultured HUVEC in serial passage and suggested that our culturedHUVEC are suitable for the cellular model to study various vascular diseases including atherosclerosis.

Poster 3

SELECTION OF HIGH-PRODUCING MAMMALIAN CELL LINES BASED ON FLUORESCENTINTENSITY USING A RAPID AUTOMATED CLONAL SELECTION METHOD

JO LYNN-KHOO, JONG HANG SIONG, HEILLY CHONG AND ZULKEFLIE ZAMROD

Department of Protein Science, Inno Biologics Sdn Bhd,Lot 1, Persiaran Negeri BBN, Putra Nilai, 71800 Nilai, Negeri Sembilan

Mammalian cells such as NS0 and CHO transfected with transgene are often used for the production of recombinant proteins for therapeutics andindustrial purposes. Here, we describe a novel method of recovering high-producing cells using ClonePix FL, a rapid automated selection ofmammalian cell colonies. We have used ClonePix FL to isolate desirable clones based on fluorescent intensity. NS0 cells were transfected withGFP genes and allowed to grow in DMEM supplemented with 10% FBS, 2 mM of glutamine and 800 μg/ml of G418 selection antibiotic. Uponconfluence, cells were transferred into semi-solid CloneMediaTM at 500 cells/ml and plated into Genetix 6-well plates. Cells were allowed togrow into colonies of 50–100 cells per colony at 37 °C. Cell growth was monitored using Genetix Imaging software until colonies reachedoptimal sizes on day 14th. The ‘Interior Median Intensity’ was detected using FITC filter at 200 ms exposure. As a result, a total of over 3000colonies were identified and 30 of the top 5% of high-expressing colonies with the intensity of 9000 FU being the highest were marked forpicking by the software. The selected fluorescent colonies were then automatically picked from the semisolid medium and dispensed into sterile96-well plates by mean of robotic mechanism. The isolated colonies with the intensity of over 9000 FU, showed senescence in 96-well culture,while those with intensity between 300 and 5000 FU continued to grow in media. The picked cells were examined under fluorescent microscopeto confirm the expression of GFP protein. Additional validation was carried out using quantitative RT-PCR (QRT-PCR). Previously, 3 to 6months were required to obtain and identify high-producing cells using limiting dilution method, with ClonePix system, we were able to obtainclones from single cells in approximately 3 weeks by combining the fluorescent detection of protein expression with the ‘cloning’ of cells to givemonoclonal populations into one single step. This has greatly improved our workflow i.e., timeline, labor and overall efficiency of cell selectionbased on fluorescent. The risk of contamination is also mitigated since imaging and picking were performed in the machine’s controlled sterileenvironment. In conclusion, we believe this novel automated method of growing and picking colonies of cells will greatly improve the quality andefficiency of cell line development. The ability of ClonePix FL to screen and select colonies based on several other parameters such as size,roundness and proximity to neighboring cells or colonies is useful to recover cells with specific criteria.


Poster 4

THE RELEASE OF RECOMBINANT N PROTEIN OF NIPAH VIRUS FROM ESCHERICHIA COLI BYBEAD-MILLING

SITI SARAH MOHD. NOOR1, WEN SIANG TAN2,3, TAU CHUAN LING3,2 AND BENG TI TEY1,3,*

1Institute of Bioscience, 2Faculty of Biotechnology and Molecular Science,3Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Nipah virus (NiV) is grouped in the genus of Henipavirus which belongs to the Paramyxoviridae family. The virus was reported to causerespiratory and neurological syndromes and deaths in swine and its transmission to human has claimed more than 100 human lives inMalaysia. The genome of NiV is approximately 18.2 kb in size and contains six genes encoding for six structural proteins. NiV possess alipid bilayer membrane overlying a shell of viral matrix protein (M). The fusion protein (F) and attachment protein (G) embedded withinthe lipid membrane. The nucleocapsid protein (N) associated with the large protein (L) and phosphoprotein (P) are tightly bound to thesingle helical strand of genomic RNA at the core of the virion. The N protein is the most abundant structural protein and the essentialcomponent of the viral helical nucleocapsid. Antigenicity study shows that the antibodies in the sera of naturally infected swine andhumans are primarily against the C-terminal portion of the N protein. Hence, the N protein has a great potential in development of adiagnostic reagent. The coding region of NiV N gene isolated from a swine has been cloned and the recombinant N protein has beensuccessfully expressed in Escherichia coli (E. coli). However, the recombinant N protein expressed in E. coli is an intracellular protein.Therefore, cell disruption is an essential prerequisite to its recovery. Mechanical disruption method such as bead mill is preferred for largescale disruption of cells. Hence, the aim of this study was to optimize a mechanical cell disruption process to release the recombinant Nprotein from E. coli. The cell disruption was carried out in a Dynomill Type MultiLab bead mill loaded with 0.3 mm Zirconia beads in abatch mode for a retention time of 17 minutes. The bead loading and biomass concentration used in this study were 80% (w/v) and 10%(w/v) respectively. The result showed that the highest yield of NP was obtained at impeller tip speed 10 m/s, in which a yield of 2.8446mg/g cell was achieved. While at impeller tip speed 14 m/s, a relatively low yield of 0.4536 mg/g cell was obtained. A drastically increasein temperature at operation of 14 m/s may has resulted in the denaturation of recombinant N protein. At 14 m/s, the rate of heat dissipationwas higher than the rate of heat transfer from disruption chamber to cooling jacket because of the limited heat transfer area provided.

Poster 5

BILIRUBIN LOWERING ACTION OF ORTHOSIPHON STAMINEUS IN TEMPORARILYHYPERBILIRUBINEMIC RATS

FAIZAH MOHD FAIZUL, NORHANIZA AMINUDIN, HABSAH ABDUL KADIR AND SAAD TAYYAB

Biomolecular Research Group, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur

The present study was undertaken to investigate the lowering potential of aqueous extract from Orthosiphon stamineus (OS) inexperimentally jaundiced rats. The rats weighing 150-200g were administered a single dose of phenylhydrazine (5 mg/kg body weight)intraperitoneally to induce jaundice. Blood was collected from the tail on the fifth day of phenylhydrazine treatment and after aqueousextract administration. The aqueous extract from OS in doses of 50, 500 and 1250 mg/ kg body weight, was orally administered to the ratsfor three days. The treatment showed a decline in the bilirubin concentration reaching to a normal level. Use of a smaller dose (50 mg/kgbody weight) of aqueous extract resulted in the reduction in bilirubin level from 2.53 ± 0.16 mg/dl to 1.12 ± 0.17 mg/dl. Higher doses of500 and 1250 mg/kg body weight were found to be more efficient in reducing the bilirubin level from 2.44 ± 0.12 to 0.52 ± 0.12 mg/dl andfrom 2.67 ± 0.29 to 0.32 ± 0.21 mg/dl, respectively. The extract used with the experimental doses was found to be non-toxic as shown bythe brine shrimp toxicity test. These results suggest the bilirubin lowering potential of OS in experimentally jaundiced rats.

Poster 6

BIOASSAY GUIDED ISOLATION OF CYTOTOXIC COMPOUNDS FROM HYDROCOTYLE VULGARIS

TEE SHIN LEONG1, ONG HEAN CHOOI2, LIM YANG MOOI1 AND ANTHONY HO SIONG HOCK1,*

1Faculty of Engineering & Science, Universiti Tunku Abdul Rahman, Setapak, 53300 Kuala Lumpur,2Institute of Biological Sciences, Universiti Malaya, 50603 Kuala Lumpur

* [email protected]

Hydrocotyle vulgaris is a local medicinal plant that is traditionally used for treating wounds and as a diuretic herb. However, the cytotoxicproperties of this plant have not been extensively studied. Preliminary investigations showed promising cytotoxic activity in the crudeethanolic fractions. Therefore, the current study attempted to isolate cytotoxic compounds from the stem and root of Hydrocotyle vulgarisusing a bioassay–guided isolation method. Gravity column chromatography, thin layer chromatography and high performance liquidchromatography were used for fractionation. Cytotoxic activity was tested against a human erythromyeloblastoid leukemia cell line, K-562, using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphemyl-tetrazolium bromide) cell viability assay. The hexane fraction of stemand roots showed the strongest cytotoxic activity with cell viabilities of 1.33% ± 0.57 and 2.38% ± 0.56 upon treatment with 50μg/ml ofextract. Further fractionation of the hexane fraction of the stem and roots gave 3 and 5 fractions respectively showing promising cytotoxicactivity. These fractions will be purified to identify the active constituents.


Poster 7

DETECTION OF HEMOCYANIN FROM HEMOLYMPH OF BANANA SHRIMP(FENNEROPENAUES MERGUIENSIS) BY ELISA

ACHAREE JIEWKOK AND PRAPAPORN UTARABHAND

Department of Biochemistry, Faculty of science, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand

Hemocyanin (HC) is a copper binding protein found in two phyla, mollusks and arthropods. As being the main protein component inhemolymph, HC typically represents up to 95% of the total amount of protein. HC has multifunctions such as oxygen carrying, ecdysonetransporting and being a precursor of anti-fungal peptides. Furthermore, HC was converted to be phenoloxidase like enzyme by SDStreatment as similar as phenoloxidase (PO) isolated from hemocytes. Physiological role of PO is important, as it is involved in immuneresponse of crustacean. This indicates that the HC may also play an important role in immune response. In this study, HC was purified fromhemolymph of banana shrimp (Fenneropenaues merguiensis) by ultracentrifugation and preparative PAGE. The purified HC showed twobands with Mr of 75 and 79 kDa in SDS-PAGE. Anti-HC polyclonal antibody raised against the purified HC in an albino rabbit showed a highspecificity in Western blotting analysis to the purified HC. The antibody was used to develop an enzyme linked immunosorbent assay(ELISA) which then applied to quantitate HC levels in the hemolymph of F. merguiensis. By means of ELISA quantification will be useful infuture measurement of F. merguiensis HC against potential shrimp pathogens, which would also be helpful in controlling shrimp diseases.

Poster 8

GENETIC VARIATION BETWEEN SELECTED IRANIAN AND MALAYSIAN RICE CULTIVARS BYUSING MICROSATELLITE MARKERS

ALI ETEMAD, M. MAHMOOD AND S.K. DAUD

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,Department of Biology, Faculty of Science, University Putra Malaysia 43400 (UPM) Serdang, Selangor, Malaysia

Rice is grown in diverse environmental conditions. Varieties from different geographical origins are expected to keep multiplicity inadaptive trades depending on the environment resulted in specific genetic structures. The application of Molecular markers can be used todetect and tract genes in breeding programs. Thus, marker assisted selection (MAS) plays important role in breeding programs. In thisstudy genetic variation among twenty six Iranian and Malaysian rice cultivars were determined using Microsatellite markers. Samples ofseeds were planted with three replication at the Department of Biochemistry, (UPM) 2007. Microsatellites are PCR based DNA markersthat are abundant, co-dominant and widely used in various organisms. A total twenty one microsatellite primers were tested in thisexperiment. The amplified PCR products were determined in 4 % Metaphor Agarose gels and 136 bands were amplified in selected(Oryza Sativa L.) cultivars. Dendogram which was constructed based on genetic distances, separating the genotypes in five clusters. In thisstudy, primers RM1 and RM 271 were produced the highest polymorphism with bands between 95 to 135 bp. In one cluster the Iranianrice cultivar namely Khazar, which have good grain quality (GQ), amylase content (AC), gelatin temperature (GT) and gel consistency(GC) is located with MR211 (Malaysian rice cultivar) which have high yield with poor grain filling efficiency, in one group. The observedgenetic diversity will be useful in choice of accessions in breeding programs and developing collection to increase the yield and grainquality as well in this strategic cereal.

Poster 9

IN SILICO COMPARATIVE PROTEOMICS ANALYSIS OF THE BURKHOLDERIA GENUS

ANNISA AKMAL ZAINAL1, KHAIRINA TAJUL ARIFIN2 AND RAHMAH MOHAMED3

1School of Biosciences & Biotechnology, Faculty of Science & Technology;2Malaysia Genome Institute, UKM-MTDC Smart Technology Centre,

National University of Malaysia, 43600 UKM Bangi, Selangor

The Burkholderia genus is classified as Gram-negative β-proteobacteria and occupy a wide range of ecological niches, which indicates theversatility of this genus. Several of the Burkholderia species are known to be disease-causing, such as B. pseudomallei and B. mallei, twoclosely related species which are the causative agents of melioidosis and glanders, respectively. B. xenovorans LB400 and B. cepaciacomplex are exploited for promoting bioremediation and plant growth, respectively. This study involved 11 proteomes of Burkholderiaspecies, i.e. B. cenocepacia AU 1054, B. cepacia AMMD, Burkholderia sp. 383, B. mallei ATCC 23344, B. xenovorans LB400,B. vietnamiensis G4, four strains of B. pseudomallei, 668, 1106a, K96243 and 1701b, and B. thailandensis E264, which were retrievedfrom the RefSeq project at the National Center for Biotechnology Information (NCBI). In order to identify unique proteins in eachBurkholderia strains used in this study, a comparative proteomics analysis was performed using local NCBI BLASTP. Each proteome wasconverted into a database using the formatdb function, and the 11 different proteomes were searched against the databases one by one. Aunique protein is defined by a protein that did not show any significant similarity with proteins from other species/strains and byidentifying unique proteins in each strain, it leads to a more focused search of novel proteins that may be involved in the adaptation toenvironment and pathogenicity of the pathogenic species of Burkholderia. This information is also hoped to contribute to our understandingof the host-pathogen relationship in those diseases and also improve our general understanding of the Burkholderia genus.


Poster 11

QUANTITATIVE EXPRESSION OF IRON SUPEROXIDE DISMUTASE IN LEGIOENLLAPNEUMOPHILA USING REAL-TIME REVERSED TRANSCRIPTASE RT-PCR

AUDREY KOW SIEW FOONG1, STACEY YONG FOONG YEE1 AND NG KIM YONG2

1School of Science, Monash University Sunway Campus Selangor; 2Malaysian University of Science and Technology, Selangor

Legionella pneumophila is a facultative intracellular parasite of protozoa in the aquatic environment. It is an opportunistic human pathogen; whena person inhales the contaminated aerosols of legionellae commonly generated from cooling towers or hot shower. In human, legionellae reside inthe alveolar macrophages and other phagocytes; multiply, disseminate to adjunction cells and causing atypical pneumonia- Legionnaires’ diseaseor a milder form of Pontiac Fever. L. pneumophila requires oxygen to grow and generates destructive reactive oxygen intermediates such ashydrogen peroxides, superoxide and hydroxyl radicals. This organism, however, possesses the (RO-) scavenging enzymes such as hydrogenperoxidase, superoxide dismutase (SOD) and catalase. In our study, we are interested in the role of the cytoplasmic iron superoxide dismutases(FeSOD) in L. pneumophila to combat with oxidative stress. The expression of FeSOD in reference strain L. pneumophila serogroup 1 (02/41),two local L. pneumophila strains (CT3C and CS1), a wild type strain of E. coli K12 and a mutated strain of E. coli QC774 (Fe- and Mn-SOD)were quantitatively measured by real-time reversed transcriptase PCR after each strains was subjected to saturations of 100% oxygen, 50:50%oxygen and carbon dioxide and 100% carbon dioxide respectively. The data showed that 20ng RNA of FeSOD was expressed in all Legionellapneumophila strains and E. coli K12 at 100% saturation of oxygen. As the oxygen saturation halved, the expression level of FeSOD decreased9.33% in L. pneumophila serogroup 1 (02/41) and an average 3.61% in 2 local L. pneumophila strains as compared with 6.29% decreased in E.coli K12. The expression of FeSOD decreased further 10% in L. pneumophila at 100% carbon dioxide saturation. The expression level of FeSODin the mutated strains of E. coli QC774 was 20% lower than other strains in all different gaseous conditions. We also subjected all the 5 bacterialstrains to 0.1mM and 1.0mM hydrogen peroxide. The expression of FeSOD increased when all bacterial strains subjected to higher concentrationof hydrogen peroxide except the reference strain L. pneumophila serogroup 1 (02/41). Similarly, the expression of FeSOD in E. coli QC477 was33% less than other strains. In summary, the FeSOD is essential to remove RO- in L. pneumophila under oxidative stress condition.

Poster 12

EFFECT OF HPV E6 AND E7 PROTEINS ON THE ACTIVITY OF MHC CLASS I PROMOTER

AZAHEMY ABDULLAH1, SHAFINA HANIM HABIB1, LIM BOON KIONG2 AND ROHANA YUSOF1

1Department of Molecular Medicine and 2Department of Obstetrics and Gynaecology,Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Infection with human papillomavirus (HPV) has long been recognized as the principle causative factor for cervical cancer. Ironically, infection byHPV itself is not considered to be a reportable disease as it was found to be fairly ubiquitous among the sexually-active demography. In mostcases HPV infection is swiftly cleared, predominantly through cellular mediated immunity mechanism (CMI). HPV-infected cells will onlyundergo transformation when the virus perseveres over the immunological barrier. Presentation of antigen by MHC Class I molecule forms thefoundation for CMI and as such, it becomes fairly reasonable to imply that inhibition of MHC Class I should therefore be the core survivalstrategy for HPV. In this study, efforts were made to observe the potential ability of two viral oncoproteins, E6 and E7 in mitigating the activity ofthe promoter for HLA-A allele using dual luciferase assay system. It was found that in the presence of viral E6 and E7 proteins, the activity ofHLA-A promoter was markedly decreased at the tune of two-fold. However, given the complexity of antigen presentation mechanism, it isprobably immature to claim that virally-induced inhibition of the MHC Class I promoter at the HLA-A allele is the principle intervention juncturethat brings down CMI response in HPV-infected cells. On-going efforts at present attempts to further understand the underlying mechanismbehind the E6 and E7 inhibition, including the possible role of E6 and E7 in mitigating cellular cytokines production.

Poster 13

XANTHORRHIZOL INDUCED CASPASE-DEPENDENTAPOPTOSIS IN HUMAN BREAST CANCER CELLS MCF-7

CHEAH YEW HOONG1, TEE THIAM TSUI1, NOOR RAIN ABDULLAH2 AND AZIMAHTOL HAWARIAH LOPE PIHIE1

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi,Selangor 2Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, 50588 Jalan Pahang, Kuala Lumpur

The present study investigates the antiproliferative effect, mode of cell death and the mechanism of action of xanthorrhizol, a naturalsesquiterpenoid compound derived from the rhizome of Curcuma xanthorrhizza Roxb on human breast cancer cells, MCF-7. Xanthorrhizolshowed potent cytotoxic effect on MCF-7 cells with an IC50 value of 1.71 ± 0.16 μg/ml. The antiproliferative activity of xanthorrhizol wasdue to the induction of apoptosis in MCF-7 cells and not necrosis as demonstrated by flow cytometry analysis. Treatment of xanthorrhizolat increasing concentrations (2 μg/ml, 5 μg/ml, 10 μg/ml, 15 μg/ml and 20 μg/ml) on MCF-7 increase the population of late apoptotic cellsat 24 hours incubation. The population of necrotic cells remained unchanged from 2-10 μg/ml of xanthorrhizol treatment. The apoptosistriggered by xanthorrhizol in MCF-7 cells was associated with the down-regulation of the antiapoptotic BCL-2 protein expression whichmay be due to the high expression of p53 protein. Moreover, the proteolisis of the 35 kDa executioner procaspase-7 was detected in treatedMCF-7 cells and was proven to be induced by the active caspase-9 upon treatment with xanthorrhizol. Active caspase-7 then cleaved andinactivated the poly(ADP-ribose) polymerase (PARP-1). These results, suggested that xanthorrhizol exerted antiproliferative effects onMCF-7 cells by inducing the apoptotic pathway via the down-regulation of BCL-2 protein levels, up-regulating the p53 protein, activationof caspase-9 and caspase-7 and inactivation of PARP-1.


Poster 14

EFFECTS OF THE TREATMENT PERIOD WITH GLYCYRRHIZIC ACID ON 11β-HYDROXYSTEROIDDEHYDROGEANSE TYPE 1 ACTIVITY IN RATS

CHIA YOKE YIN1, TON SO HA1 AND KHALID BIN ABDUL KADIR2

1School of Science, 2School of Medicine and Health Sciences, Monash University Sunway Campus

Glycyrrhizic acid (GA), a known inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD), shows potential in improving metabolicdisturbances associated with insulin resistance (IR). 11βHSD1 plays a pivotal role in intracellular metabolism and glucose homeostasis. Thisstudy was conducted to determine the effects of GA on 11βHSD1 activities in different treatment periods using rat as animal models. 48 maleSprague Dawley rats used in this study were subjected to different treatment periods [e.g. 12, 24 and 48 hours] and fed ad libitum. Treated andcontrol groups were intraperitoneally administered with GA (50 mg/kg) and saline respectively. Six tissues (subcutaneous [ATS] and visceraladipose tissue [ATV], abdominal muscle [MA], quadriceps femoris [MT], liver [L] and kidney [K]) were sampled in this study. 11βHSD1activities were lower in all tissues of GA-treated rats when compared to the control rats of identical treatment periods (12, 24 or 48 hours).11βHSD1 activities decreased in all tissues for the 48 hour (p < 0.01); in all tissues (p < 0.01) except MA and MT for the 12 hour and ATVfor the 24 hour. When compared between different treatment periods, increase (p < 0.05) was seen between rats from the 12- and 24-hourgroups in all tissues and 12- and 48-hour groups in MA, L and K while decrease (p < 0.01) was seen in ATS, ATV and MT between rats fromthe 24- and 48-hour groups. Results from the 48-hour-treated rats displayed more prominent reduction, thus indicating that longer treatmentperiod may yield better results. Thus, GA could be useful for further studies on Type II diabetes as it lowers 11βHSD1 activities.

Poster 15

PURIFICATION OF A CUTINASE VARIANT TO HOMOGENEITY VIA ION EXCHANGECHROMATOGRAPHY

CHIN IUAN SHEAU1, 2, ABDUL MUNIR ABDUL MURAD1, 2, SHEILA NATHAN1,2, NOR MUHAMMAD MAHADI1, 2 ANDFARAH DIBA ABU BAKAR1

Faculty of Science and Technology, Universiti Kebangsaan Malaysia1; UKM-MTDC Smart Technology Centre, Malaysia Genome Institute2

Cutinase, a small lipolytic enzyme secreted by the phytopathogen Glomerella cingulata, is ideally suited for the modification of syntheticfibers as it is naturally produced for the degradation of cutin, the structural polyester of plant cuticle. The natural function of cutinase as apolyesterase can be further explored to expand its capability in the biomodification of synthetic polymers. The gene encoding for this enzymehas been cloned and over-expressed in an Escherichia coli expression system as a soluble active enzyme. Modification of this cutinase at thegenetic level has been carried out by means of directed mutagenesis. One of the single-site cutinase generated, L172K was experimentallytested to be enhanced in terms of catalytic performance. Yet, this outcome has to be reassessed as the assays were carried out towards therecombinant cutinase and its variants in the form of fusion enzymes. Thus, in order to study the enzyme kinetics, it is necessary to cleave thefusion partner, Trx-tag with recombinant enterokinase, and subject to purification via Ion Exchange Chromatography (IEC). Reassessment ofthe theoretical pI value of the L172K variant revealed that with only a single amino acid substitution, the pI value of this variant shifts from6.53 (pI value of wt recombinant cutinase) to 7.59. In the first pH scouting experiment, the binding and elution buffers were adjusted from pH8.4 to pH 9.5 due to the pI value differences between the variant and the wild-type cutinase. This pH condition is sufficient to promote thebinding of L172K to the anion exchanger, yet resulted in broad elution. The pH condition was then adjusted to 9.2, to achieve selective elutionof the targeted enzyme. However, the elution of this variant was once again found in every single fraction collected. The pH of the buffers wasthen readjusted to pH 8.8, and the elution of L172K was centered to the first few fractions. These fractions were sufficiently separated fromthe eluted fractions of Trx-tag. A step-gradient elution was then performed to minimize targeted enzyme loss. We successfully directed theelution of the L172K variant, separated from Trx-tag with a prolonged constant level of the elution buffer (6% of the elution buffer with 1 MNaCl). This percentage of elution buffer (6%) was selected as it corresponds to a constant conductivity that is high enough to elute thetargeted L172K variant, yet low enough for the elution of the unwanted fusion partner.

Poster 16

EFFECTS OF GLYCYRRHIZIC ACID ON 11β-HYDROXYSTEROID DEHYDROGENASE TYPE 1 ANDTYPE 2 ACTIVITIES

CHOH LEANG CHUNG1, CHIA YOKE YIN1, TON SO HA1 AND KHALID BIN ABDUL KADIR2

1School of Science, 2School of Medicine and Health Sciences, Monash University Sunway Campus

Elevated intracellular glucocorticoid (GC) levels correlates with development of central obesity and metabolic syndrome. Microsomalenzymes, 11β-hydroxysteroid dehydrogenases (11β-HSDs) regulate intracellular GC levels. 11β-hydroxysteroid dehydrogenases exist in twoisoforms, namely type 1 (11β-HSD1) which converts inactive GC to active GC and type 2 (11β-HSD2) which catalyses the reverse reaction.In animal and clinical studies, inhibition of 11β-HSD1 improves symptoms of MetS. Glycyrrhizic acid (GA), a principle bioactive compoundin licorice, is a non-selective, reversible and competitive inhibitor for both 11β-HSD1 and 2. Therefore, GA has the potential to function as atherapeutic agent for patients with MetS. In this study, effects of GA on the activities of 11β-HSD1 and 2 in rat tissues (i.e. subcutaneous andvisceral adipose tissue, abdominal and quadriceps femoris, liver and kidney) were investigated. It was found that 11β-HSD1 and 2 activitiesdecrease in rats administered with GA orally for one week (50 mg/kg) compared to the control rats. 11β-HSD1 activities in subcutaneousadipose tissue and liver showed significant decrease (p < 0.05) while liver and kidney tissue show a significant decrease (p < 0.05) in11β-HSD2 activities. In conclusion, GA inhibits 11β-HSD activities and may therefore be used to improve MetS symptoms.


Poster 17

EXPRESSION AND PURIFICATION OF EIMERIA TENELLA RECOMBINANT GPI-ANCHOREDVARIANT SURFACE PROTEINS

CHOW YOCK PING1, WAN KIEW LIAN1,2 AND SHEILA NATHAN1,2*

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia2Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, Bangi, Selangor

Eimeria tenella infects chicken ceaca and causes coccidiosis, an enteric disease that inflicts economic losses to the world poultry industry.Glycosylphosphatidylinositol (GPI) -anchored proteins are major molecules on the Eimeria tenella surface essential for invasion. Theseproteins are believed to play an important role in Eimeria pathogenesis and can elicit strong immune responses. We have targeted a subsetof sporozoite and merozoite stage specific GPI-anchored proteins as potential molecular signature candidates of Eimeria tenella infection.Seventeen surface proteins, i.e. SAG 1 (sporozoite stage specific), SAG 10, 13, 14 (sporozoite and merozoite stage), and SAG 2, 3, 4, 5,6, 7, 8, 12, 15, 16, 18, 19, 23 (merozoite stage specific) were selected and cloned into pET 32b(+) and expressed in Escherichia coliRosetta gami (DE3). All the recombinant surface proteins were expressed as soluble protein through the induction by 0.1 mM IPTG at20 ºC for 20 hours. The expressed soluble proteins were further purified using Nickel Sepharose and will be utilized as antigens to screenimmunized chicken sera.

Poster 18

HISTOLOGICAL STUDIES ON THE PROTECTIVE EFFECT OF MUCUNA PRURIENS SEEDEXTRACT AGAINST DAMAGES CAUSED BY NAJA NAJA SPUTATRIX VENOM

N.H. TAN1, S.Y. FUNG1 AND S.M. SIM2

1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia2Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Nigeria as anti-snake bite remedy.Pretreatment with Mucuna pruriens seed extract (MPE) has been shown to protect the effect against the cardiovascular, respiratory andneuromuscular depressant effects of Naja naja sputatrix (NNS) venom. MPE pretreatment may have a direct effect on rat heart renderingthe heart more resistant to venom-induced cardiovascular depressant effect. Previous investigations indicated that the protective action ofMPE pretreatment against NNS venom involves protection against the toxic action of the venom in heart and, to a lesser extent, theneuromuscular depressant effect. Histological studies on rat organs confirmed results from the pharmacological studies: injection of NNSvenom in untreated rats caused disruption in the striations of the heart muscle but the histological damages were prevented by MPEpretreatment.

Poster 19

THE ANTIOXIDANT ACTIVITIES AND TOTAL PHENOLIC COMPOUNDS OF FICUS DELTOIDEA

LEE CHOY LONG1, MAIZATUL HASYIMA OMAR2, HABSAH ABDUL KADIR1 AND GOH BEY HING1

1Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur2Institute of Medical Research, Jalan Pahang, 50588 Kuala Lumpur

The present study evaluated the antioxidant activity of Ficus deltoidea, a herbal medicinal plant in Malaysia. It is believed that F. deltoideapossesses many nutritional values in treating several diseases and for health care maintenance. The crude water extract of the leaves ofF. deltoidea was partitioned into petroleum ether, dichloromethane, ethyl acetate, and aqueous fractions. The antioxidant activity of thevarious fractions were tested by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and reducing power activity assay.The content of total phenolics in the fractions was determined spectrometrically according to the Folin-Ciocalteu procedure and calculatedas gallic acid equivalents (GAE/gDW ). The DPPH radical scavenging assay has revealed ethyl acetate fraction to possess the highestantioxidant activity. The IC50 value of the extract on DPPH scavenging activity was 60.0 μg/ml., comparable to butylated hydroxytoluene(BHT) and catechin that were used as positive controls. The reducing power of fractions was carried out with ascorbic acid as a standardreducing agent and ethyl acetate fraction exhibited the highest reducing power. The active fraction in ethyl acetate was then fractionated(fractions I – X) by silica gel column chromatography using gradient solvents (dichloromethane: ethyl acetate, 9:1–1: 9, v/v). The TLC-DPPH analysis showed only one DPPH radical scavenging band in the sub-fraction X and detected with Rf value of 0.22. A concentrationdependence was observed in both DPPH scavenging and reducing power activities. In addition, the antioxidant activities were closelyrelated to the content of phenolic compounds as evident by ethyl acetate fraction showing the highest value of 54.9 GAE/gDW . The datasuggest that F. deltoidea possesses the potential to be used to treat or prevent degenerative diseases where oxidative stress is implicated.


Poster 20

DETECTION OF PHYTOCHELATIN SYNTHASE IN FUNGI ASSOCIATED WITH MANGROVE

NOOR AFIZA BADALUDDIN, MARIAM TAIB, AZIZ AHMAD AND JAMILAH MOHD SALIM @ HALIM

Department of Biological Sciences, Faculty of Science and Technology,Universiti Malaysia Terengganu, 21030 Kuala Terengganu

Phytochelatins play important role in heavy metal detoxification, primarily Cd2+, where plants and some fungal species detoxify the metalsin a similar way, that is chelating these substances and decreasing their free concentrations. Phytochelatins are derived from glutathioneand synthesized by phytochelatin synthase. This study was conducted to detect the production of phytochelatin synthase in fungi isolatedfrom mangrove. Fungi associated with mangrove in Universiti Malaysia Terengganu and Tok Bali, Kelantan were isolated and identifiedby using Direct Plating and Slide Culture techniques. Altogether, 25 fungal species were identified with 7 aquatic species. These aquaticfungal isolates were screened for cadmium tolerance property by growing them on agar with different cadmium concentrations. The bestfungus that could tolerate cadmium, indicating highest production of phytochelatin was identified as Trichoderma sp. The activity of thephytochelatin synthase produced by the fungus was estimated using Ellman’s test for glutathione measurement. The result showed that theenzyme activity is directly correlated to cadmium concentration. The phytochelatin synthase appeared as a clear band on SDS-PAGE witha molecular weight of around 63.8 kDa. The implication of phytochelatins in heavy metal detoxification causes a huge potentialcommercial value towards many industries especially in waste treatment.

Poster 21

IDENTIFICATION OF THE MAIN COMPONENT OF JAMU RATUS ABSORBED INTO MILK ANDBRAINS OF SUCKLING NEONATAL RATS

NORHAFILDA ISMAIL, MUHAMAD HASNUL NAIM ABD. HAMID AND MD. SHAROM YUSOF

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor

Widespread self-prescription of post-partum formulae like Jamu Ratus and the risks it may pose to the suckling neonates are a valid causefor concern which needs to be addressed. In the present study, components of Jamu Ratus absorbed into the mammary glands of lactatingrats and their possible presence in tissues of the suckling neonates were chromatographically characterised. Prior to this, the lactatingdams were pretreated with various doses of the CHCl3 fraction of the drug (JEC), orally administered on a daily basis, for a period of upto nine days. Upon sacrifice, various tissues were taken from the dams (milk, plasma and liver) and suckling neonates (liver and brain);these were then subjected to solvent extraction prior to analyses by thin layer chromatography and HPLC. Chromatograms from HPLCanalyses indicate the presence of a quercetin-like compound, which eluted with an Rt value of 25.6 min, in the milk and livers of theexperimental dams. Significantly, this identical component was similarly detected in the brain and liver extracts of even the sucklingneonates nursed by dams given the lowest dosage of the drug. A cause for concern indeed!

Poster 22

BIOEQUIVALENCE STUDY OF TWO TABLET FORMULATIONS OF RIFAMPICIN IN HEALTHYADULT SUBJECTS

ZAMRI CHIK1, ROMA C. BASU1, ROKIAH PENDEK2, LEE TOONG CHOW3 AND ZAHURIN MOHAMED1

1Department of Pharmacology, 2Department of Medicine, Faculty of Medicine, University of Malaya,50603 Kuala Lumpur 3Info Kinetic Sdn. Bhd., Kompleks Eureka, USAINS Holding, 11800 USM, Penang

Rifampicin is a semisynthetic antibiotic derivative of rifamycin and is indicated for the treatment of different forms of tuberculosis. Toensure the efficacy and safety of a rifampicin generic product, a bioequivalence study was conducted. The objective of this study is tocompare the rate and extent of absorption of a generic rifampicin product (Siticox capsule 300 mg, manufactured by Idaman PharmaManufacturing Sdn Bhd.), and its metabolite, 25-desacetylrifampicin in oral dosage form, with the proprietary product (Rimactane®capsule 150 mg, manufactured by Novartis South Africa [PTY] Ltd), in healthy human subjects, under fasting conditions. The study wasa single dose versus double dose, randomized, two way crossover study with eight-week washout period between the two study arms. Itinvolved 14 healthy volunteers and the total number of 14 subjects were calculated to have 80% power to detect a 20% difference betweenthe preparations at p < 0.05 [1]. Subjects received either 300mg of reference or test formulation. Blood samples were collected at pre-doseand a serial of 15 samples were collected from each of the subject from 45 minutes until 24 hours post-dose. Plasma concentrations ofrifampicin and its metabolite, 25-desacetylrifampicin were analysed using a validated HPLC method. For rifampicin, the 90% confidenceinterval (CI) for the ratio test/reference for both log AUC0-∞ and Cmax were within the bioequivalence limit of 80 - 125 %. There was nostatistically significant difference in test and reference tmax between the two products, and hence the two products, Siticox and Rimactane®

are bioequivalent. For the metabolite 25-desacetylrifampicin, the 90% CI for the ratio test/reference for log AUC0-t failed to fall within thebioequivalence limit (90% CI: 79.6 -104.7), however, the non-log transformed AUC0-t result was within the bioequivalence limit of 80 –120 % (90% CI: 81.7 -106.3). The 90% CI for the ratio of the test/reference for the log Cmax was within the Malaysian NPCB (NationalPharmaceutical Control Bureau) bioequivalence limit (75-133%). Based on the above criteria, Siticox and Rimactane® are also bioequivalentfor the active metabolite. In conclusion therefore , Siticox and Rimactane® are bioequivalent and one can be substituted for the otherwithout any change in drug efficacy and safety.


Poster 23

COMPARISON METHOD OF DNA EXTRACTION FROM ARCHIVAL FORMALIN-FIXED,PARAFFIN-EMBEDDED TISSUES

EDWIN SHIAW CHIN HAN1, SHIRAN MOHD SIDIK2, CHEAH YOKE-KQUEEN1*, TAN GEOK-CHIN3 ANDHAYATI ABDUL RAHMAN3

1Department of Biomedical Sciences, 2Department of Pathology, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor. 3Department of Pathology, Faculty of Medicine,

Hospital Universiti Kebangsaan Malaysia, Jalan Ya’acob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur*Corresponding author : [email protected]

The archival formalin-fixed, paraffin-embedded (FFPE) tissues have been used by pathologists for decades due to its stable format forhistological analysis and long period storage capabilities. Moreover, archival FFPE tissues are proved to be an important resource formolecular genetic studies due to its large number of materials in the collection. A total of 30 FFPE blocks from the year of 2005 to 2006were assessed with each modified and adapted method. The extraction of nucleic acids from archival formalin-fixed, paraffin-embedded(FFPE) tissues enable researchers to perform various types of downstream studies that include diagnostic and retrospective moleculargenetic studies based on DNA amplification by PCR. However, the extraction of high quality nucleic acids from archival FFPE tissuescould be difficult and challenging. The ultimate aim of this study is to compare various DNA extractions from archival (FFPE) tissues. Inthis study, we have compared four protocols, including the modified enzymatic extraction method (method A), thermal cycler and Chelex-100 extraction method (method B) and heat-induced retrieval in alkaline solution extraction method (method C and D). The purity andconcentration of DNA extraction was evaluated by measuring the extracted DNA yields using biophotometry. On the other hand,amplifiable extracted DNA was evaluated through PCR amplification. The extracted DNA samples were assessed by PCR amplification ofa fragment of Cytochrome p450 2D6 gene. In this study, method A gave the highest percentage (63.3%) with the range of 1.6 to 2.0 in260/280 ratio compare to other methods. The average DNA concentration obtained in this study are 28.03 ng/ul (method A), 369.83 ng/ul(method B), 13.77 ng/ul (method C) and 33.69 ng/ul(method D). Amplification of Cytochrome p450 2D6 gene sequence was successfulperformed in 18 of 30 (60%) samples by method A, 7 of 30 (23.3%) samples in method B, 5 of 30 (16.7%) samples in method C and 13of 30 (43.3%) samples in method D. Besides that, no significant correlation was observed between the age of the FFPE with the yield,purity and amplifiable properties of the extracted DNA.

Poster 24

OVER EXPRESSION OF THE 35 KDA ITIH4 FRAGMENT: EXCLUSIVE TO SEX-STEROIDHORMONE ASSOCIATED CANCERS?

EMIDA MOHAMED1, PUTERI SHAFINAZ ABDUL-RAHMAN1, SITI ZAWIAH OMAR2 AND ONN HAJI HASHIM1

1Department of Molecular Medicine and, 2Department of Obstetrics & Gynecology, Faculty of Medicine,University of Malaya, Kuala Lumpur

Sex-steroid hormones play important roles in inducing the changes in our body known as primary and secondary sex characteristics. Theseendogenous hormones have also been strongly implicated in the etiology of some cancers. Using lectin-based electrophoretic approach, ourgroup has previously reported the elevated expression of a 35 kDa ITIH4 fragment in four different types of cancers associated with increasedlevels of sex-steroid hormones. Enhanced expression was detected in patients with breast carcinoma, endometrial adenocarcinoma, germ cellovarian carcinoma and epithelial ovarian carcinoma, relative to the control subjects. On the other hand, the differential expression was notsignificantly detected in sera of patients with nasopharyngeal carcinoma, osteosarcoma and two types of cervical carcinoma. In the presentstudy, similar approach was extended to other hormonally correlated non-cancerous cases i.e., cohorts of normal pregnant women and patientswith hydatidiform mole. Our results also demonstrated the up-regulated expression of the 35 kDa ITIH4 fragment in sera of pregnant womenand patients with hydatidiform mole. This finding indicates that the abundance of the 35 kDa ITIH4 cleavage fragment is not exclusive tocancer but is also enhanced in other conditions associated with increased levels of sex-steroid hormones.

Poster 25

HIGHLY THERMOSTABLE L2 LIPASE: PURIFICATION AND PARTIAL CHARACTERIZATION

FAIROLNIZA MOHD SHARIFF1, RAJA NOOR ZALIHA RAJA ABD. RAHMAN1, MAHIRAN BASRI2 ANDABU BAKAR SALLEH1

Enzyme and Microbial Technology Research, 1Faculty of Biotechnology and Biomolecular Sciences,2Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

The highly thermostable recombinant L2 lipase was previously screened and isolated from a hot spring in Perak, Malaysia. The lipasegene was successfully amplified, cloned and expressed into Escherichia coli system. The over-expressed L2 lipase was easily recoveredvia single step affinity chromatography with high purity and high yield purification. It has a molecular weight of 43 kDa. This enzyme wasfound to be stable at alkaline pH for 30 min where the residual activity was retained up to more than 50%. It is most active in hightemperature environment especially in temperature range of 55 to 75ºC. Effect of inhibitors and metal ions results indicated that theenzyme was a metalloenzyme.


Poster 26

EFFECT OF TARRENA SP. UPON MUSHROOM TYROSINASE ENZYME ACTIVITY

FARIDA HARYANI AB. AZIZ1, SYAHIDA AHMAD1,2, NORDIN HAJI LAJIS2,3, KHOZIRAH SHAARI2,3 AND FARIDAH ABAS2,4

1Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Biosciences, 3Faculty of Sciences,4Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Tyrosinase is a key enzyme involved in the biosynthesis of melanin. Recently, tyrosinase inhibitors have become increasingly important in treatingpigmentation-related disorders, via cosmetic or medical product application. In this study, 60 Malaysian traditional plants have been screened for anti-tyrosinase activity using commercial mushroom tyrosinase enzyme. The results demonstrated that Tarrena sp. showed the highest inhibition (89.5%)upon mushroom tyrosinase enzyme without cytotoxicity effect on human epidural melanocytes cells (HEMn). While, six plants showed moderateinhibition (50-79%), ten plants showed weak inhibition (<50%) and the rest showed no inhibition upon mushroom tyrosinase enzyme activity.Therefore, Tarrena sp. was selected for further analysis in this study. The Tarrena sp. methanolonic extract was then fractionated using hexane,dimethlychloromethane, ethyl acetate and methanol solvents in order to determine its active constituent. The present results showed that ethyl acetatefraction of the Tarrena sp. significantly suppressed mushroom tyrosinase enzyme activity with IC50 value of 42.14μg/ml. Thus, we believed that theethyl acetate fraction of Tarrena sp. might have the active constituents that have the potential to become the main ingredient or lead compound informulating new cosmaceutical or pharmaceutical products mainly in treating skin pigmenting disorders.

Poster 27

ANALYSIS ON CATHARANTHUS ROSEUS, GYNURA PROCUMBENS AND PERESKIA SACHAROSA LEAVESDNA EXPRESSION AND RESPOND AT DIFFERENT DOSAGE OF GAMMA IRRADIATION TREATMENT

AHMAD FARID ISMAIL1, AZLEEN MAT SHARIF1, NUR HIKMAH RAMLI1, IRA MAYA SOPHIA NORDIN1,MOHD NORHISHAM SUKUR1 AND ISHAK MAT1

1Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, USM, 11800, Penang

Herbal plants such as Catharanthus roseus, Gynura procumbens and Pereskia sacharosa have been widely used by traditional medicinepractitioners as complementary treatment for various diseases. Nowadays, although the leaves of herbals plant need to be boiled as usual forextraction of the nutrition, there are also herbal products sold as capsules or pills that have been marketed worldwide. Gamma irradiationtechnique had been used as a mean of sterilization of the herbal products prior. In this study, we investigate the differential effects of expressionprofile and quality of DNA of three different species of herbal plants after treatment with gamma irradiation at different dosages. Catharanthusroseus, Gynura procumbens and Pereskia sacharosa leaves were irradiated at the different dosages of gamma ray by using Gamma Cell Irradiator.Four dosages had been selected at 15, 25, 50 and 75 gray (gy). Leaves at 0 gray (un-irradiated) had been used as a control. The DNA was isolatedby using DNA isolation commercial kit. Electrophoresis of DNA was carried out on 1% (w/v) agarose gel. Isolated DNA was mixed with 1 μl 6xloading dye. The mixture was then loaded in each gel electrophoresis well and VC Lambda EcoR1 + Hind III had been used as a standard marker.The electrophoresis was run at 85 volts for 55 minutes. Gel Imaging (Syngene) had been used to visualize the band appeared under ultravioletconditions. DNA purity had been obtained by determining the ratio of absorbance using DNA/RNA spectrophotometer. The samples were placedin cuvette and the optical density (O.D) at 260 and 280 nanometer (nm) against blank was determined. Ratio of pure DNA analysis by usingspectrophotometer showed that there were no major differences in DNA purity for all the species using in this study. The ratio A260 / A280 rangesfrom 1.1 to 1.4, and this may indicates a pure DNA was isolated after the gamma irradiation treatment. Gel electrophoresis analysis forCatharanthus roseus and Gynura procumbens leaves DNA extraction showed high yield of DNA bands appearing for both species, whichsuggest both DNAs have higher molecular weight (MW) of DNA compared to Pereskia sacharosa. Different dosages of gamma irradiationtreatment may partially induce changes on the stability of the Catharanthus roseus, Gynura procumbens and Pereskia sacharosa DNA molecules.Future study being planned will look at the effect of gamma irradiation on gene variation and expression of the gene products.

Poster 29

STIMULATION EFFECT OF LEAD (Pb) TO MICROBIAL GROWTH OF CONSORTIUM CULTUREDURING THE INDIVIDUAL BTEX EXPOSURE

FELLIE EDWIN AMIR1, WONG KOK KEE1, ABDUL JALIL ABDUL KADER2, OTHMAN OMAR1, BRID QUILTY3 ANDSALMIJAH SURIF1

1School of Environmental & Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,43600 UKM, Bangi, Selangor, 2Faculty of Science and Technology, International Islamic University Malaysia,

50728 Kuala Lumpur, 3School of Biotechnology, Dublin City University, Dublin 9, Ireland

Groundwater contamination by toxic pollutants has been a great concern due to its importance as our major source of drinking water.Hydrocarbon benzene, toluene, ethylbenzene and xylene (collectively known as BTEX) are potential contributor in this regard, bacause of theirpresence in petrol, gasoline, diesel and petrochemical products. In this study, the toxicity of BTEX to a consortium culture (CC) and the effects oflead (Pb) to the growth were studied. The CC was capable of utilizing the individual BTEX as the sole carbon and energy source at lowconcentrations (10mg/L to 100 mg/L) but growth was inhibited at high concentration (500 mg/L). The specific growth rate of CC,in μ (hr-1), wascalculated from its growth curve during the 48 hours of exposure. A siginificant decrease in μ value was observed in each BTEX whichcorresponds to increase of the concentration (from 10 mg/L to 500 mg/L). The IC50 profile for individual BTEX was observed to be E>X>T>B.At 30 mg/L of individual BTEX (withoud Pb), growth inhibition of CC was observed in all treatment; benzene (4.09% ± 0.010), toluene (2.92%± 0.004), ethylbenzene (24.27% ± 0.008) and xylene (11.99% ± 0.005). However, at 30 mg/L of individual BTEX (with Pb at 50ppm), Pbshows a synergistic effect on the growth of CC in the treatment whereby growth stimulation was observed in benzene (24.56 % ± 0.007) andtoluene (20.76 % ± 0.011), while a growth inhibition of CC in ethylbenzene and xylene was reduced to 13.45% ± 0.003 and 9.36%, respectively.


Poster 30

CONSTRUCTION AND ANALYSIS OF SMALL-INSERT GENOMIC LIBRARIES OF EIMERIA MAXIMA

HALIMAH ALIAS1 AND KIEW-LIAN WAN1,2

1Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, 43600 UKM Bangi, Selangor DE;2School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,

43600 UKM Bangi, Selangor DE

Eimeria maxima is an ubiquitous intestinal parasite and one of the seven Eimeria species that causes avian coccidiosis. Further studies onthe E. maxima genome may facilitate the development of more effective controls of the disease. In order to initiate sequencing of theE. maxima genome, we have constructed plasmid libraries from genomic fragments of various sizes. Genomic DNA was initially shearedusing sonication before the DNA fragments were end-repaired and dephosphorylated. Subsequently, the DNA fragments were size-selected on an agarose gel, extracted and purified. DNA fragments with sizes of 1-2kb, 2-4kb and >4kb, were then cloned into the pUC19plasmid vector and transformed into Escherichia coli. Analysis of the three libraries showed that the 1-2kb, 2-4kb and >4kb insert librariesare capable of producing a total of 960,000, 480,000 and 128,000 clones, respectively. Based on colour selection, the percentage ofrecombinant clones for the 1-2kb and 2-4kb insert libraries were estimated to be more than 98%, whereas for the >4kb insert library, onlyapproximately 25% of the clones were thought to contain inserts. Overall, these results indicated that the E. maxima small-insert genomiclibraries which have been constructed in this study will be a useful resource in genome studies of this species, particularly via the whole-genome shotgun approach.

Poster 31

SITE-DIRECTED MUTAGENESIS OF TYPE III POLYKETIDE SYNTHASE (PKS)FROM SARGASSUM BINDERI

HARIYANTI BAHARUM1, NG KIM YONG2, RAHA ABD RAHIM1 AND HO CHAI LING1*

1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor. 2Malaysia University of Science and

Technology, Unit GL33 (Ground Floor), Block C, Kelana Square, 17 Jalan SS7/26, 47301 Petaling Jaya, Selangor*Corresponding author: [email protected]

Type III polyketide synthases (PKSs) are involved in flower pigmentation, pathogen defence (phytoalexin), UV and visible light exposureresponse and symbiotic plant-pathogen interaction. Recent crystallographic and site-directed mutagenesis studies have revealed thestructural and functional details of type III PKSs in plants and bacteria that share almost similar three-dimensional overall fold andcommon active site architecture with an absolutely conserved Cys-His-Asn catalytic triad. In this study, we have cloned the cDNAencoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi. SbPKS was expressed in Escherichia coli strain BL21 (DE3)pLysS as a 61-kDa recombinant protein fused to His•Tag, Trx•Tag and S•Tag. Using the three-dimensional structure of type III PKS fromMycobacterium tuberculosis as a template, the model of SbPKS has been computed. Five mutants (H227G, H227G/L368V, H303Q/N336A, H227G/F93AV95A, and H227G/L368V/F93AV95A) were generated using the Stratagene QuikChange Site-directed MutagenesisKit to investigate the starter molecule selectivity and control of the final length of product of SbPKS. The resulting mutants will beassayed for their starter molecule specificities and selectivities.

Poster 32

INCREASED PERCENTAGE OF β-GALACTOSIDASE POSITIVE FIBROBLAST CELLS WITHEXPOSURE TO H2O2 IN STRESS-INDUCED PREMATURE SENESCENCE (SIPS) MODEL

HARYATI AHMAD HAIRI, GOON JO AAN, 1SUZANA MAKPOL, ROSLAN HARUN AND WAN ZURINAH WAN NGAH

Department Of Biochemistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

Oxidative stress caused by hydrogen peroxide (H2O2) can shorten the life span of cells in tissue culture. This phenomenon is termed stress-induced premature senescence (SIPS). The model of SIPS is reliable since reproducible results have been obtained from repeatedexperiment to monitor biomarkers of senescence. β-galactosidase (SA-β), a specific senescence-associated marker has widely been usedas a biomarker of cellular senescence in vivo and in vitro. The positive blue coloured cells of β-galactosidase at pH 6 has been reportedto be remarkably increase in senescence. In this research, human diploid fibroblasts (HDFs) at early passage were exposed with prolongeddose of 20 μM H2O2 for 2 weeks to represent subcytotoxic conditions that occur in aging. Our preliminary result shows that exposure ofHDFs to this low dose of H2O2 was able to increase β-gal staining by 21% at passage 10 which cells are considered senesce in a SIPSmodel. In contrary, control cells available in our laboratory which reached senescence at passage 30 was found to have increased β-galstaining by 65%. More research work are underway to determine if this is due to the difference in the source of HDFs.


Poster 33

ESTABLISHED METHOD OF TWO DIMENSIONAL ELECTROPHORESIS TECHNIQUE FORPROTEIN PATTERNS OF MICHELIA ALBA DC

HASLIZA HASSAN1, RADZALI MUSE1, MOHD. PUAD ABDULLAH2, JOHARI RAMLI1, MOHD ROSNI SULAIMAN3 ANDNOR ARIPIN SHAMAAN1

1Department of Biochemistry, 2Department of Molecular Cell Biology, Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 UPM, SERDANG, Selangor; Malaysia 3School of Food Science and Nutrition,

Universiti Malaysia Sabah, Locked Bag 2073, 88999 KOTA KINABALU, Sabah; Malaysia

An aromatic plant of Michelia alba DC. (Magnoliaceae) with very fine fragrant is well known to have high value of essential oils withvolatile monoterpenes as major compounds. The monoterpene synthesis enzymes commonly involved in biosynthesis of fragrancecompounds (monoterpenes) are well known as linalool synthase and cineole synthase. Though assays are available for several of theenzymatic steps of monoterpene biosynthesis, it would be quite hard to purify the enzymes for sequencing. Thus, proteomics seem to bepreferable to enzyme assaying in obtaining sequence information from all proteins connected with monoterpene biosynthesis. The aim ofthis study was to establish two dimensional electrophoresis techniques for Michelia alba DC., crude extracts. Two dimensional electrophoresistechniques according to O’Farrell, 1975 method were established in our laboratory using Bio-Rad Protean 2-D Cell. The results showedthat the 2D gel electrophoresis was reproducible and satisfactory using this technique. As conclusion, this protocol may applicable to otherhigher plant tissues such as fresh leaf and flower tissues.

Poster 34

AGROBACTERIUM-MEDIATED TRANSFORMATION OF PHB (POLY(R)-(-)-3-HYDROXYBUTIRATE)GENES INTO THE PLASTID OF ELAEIS GUINEENSIS JACQ.

HENG WEI SZE1, NIK MARZUKI SIDEK1 AND RUSLAN ABDULLAH2

1School of Bioscience and Biotechnology, Faculty of Science and Technology, UKM, 43600 Bangi, Selangor 2Sime Darby(Guthrie Research Chemara) Berhad, Jalan Sungai Ujong, 72000 Seremban, Negeri Sembilan

Oil palm is recognized as a socio-economically important edible crop cultivated in Malaysia. It has significant commercial values to thecountry as it comes only second to soy bean as far as providing vegetable oil for world wide consumption is concerned. Thus, it is crucialto not only genetically improve the crop qualitatively and quantitatively but also to engineer the plant to produce added-value products. Inthis study, the emphasis is more on integrating as well as cloning the PHB genes into the crop. PHB known as poly(R)-(-)-3-hydroxybutirate is an aliphatic polyester that possesses the thermoplastic entity and it is biodegradable either aerobically or anaerobically.Three expression vectors have been constructed namely pCAMBIA106, pCAMBIA107 and pCAMBIA108 with each vector harboringphaC, phaA and phaB respectively. These three PHB genes are controlled by Rubisco promoter. Besides the PHB genes and the promoter,each gene cassette also contained plastid sequence and a NOS terminator. Subsequently, these three expression vectors are transformedinto immature embryo of oil palm via Agrobacterium-mediated approach. Two strategies of transformation have been applied where in thefirst strategy; phaC, phaA and phaB have been transformed into three different individual immature embryos where each embryo carryingone gene respectively whilst in the second strategy; all of the PHB genes are transformed into a same immature embryo. The relevantbehind the first strategy is to study the levels of transformation, integration and expression of these three genes separately. While thesignificant of the second strategy is to examine whether a transgenic plant that own a thermoplastic property can be successfully produced.At the moment, the explants are generating and GUS assay has been performed and showed positive result.

Poster 36

OVER-EXPRESSION OF OIL PALM DIMINUTO/DWARF1 IN RICE

HUYNH KY1*, LE VINH THUC1, OOI SIEW ENG2, ZAMZURI ISHAK2

PARAMESWARI NAMASIVAYAM1Δ AND SUHAIMI NAPIS1Δ

1Cell and Molecular Biology Department, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400,UPM, Serdang Selangor, Malaysia 2Malaysia Oil Palm Board (MPOB), Bandar Baru Bangi, 43300, Kajang, Selangor, Malaysia

Many crop plants that have the mutants for genes that respond to plant hormones have been reported to show a dwarf phenotype. Inparticular, brassinosteroids are known as a regulator for plant growth and development. The mutant of brassinosteroid synthesis gene hasbeen identified in rice and was reported to have high homology to Arabidopsis thaliana DIMINUTO/DWARF1. Recently, cp 596 cDNAdesignated as EgDIM (Elaeis guineensis Jacq DIMINUTO) (EU805511) that encodes for a putative cell elongation protein was isolatedfrom the oil palm cell suspension culture. Sequence analysis of the full-length EgDIM cDNA revealed that the ORF is predicted to encodea 561 amino acid protein that has a high homology to a putative cell elongation protein DIMINUTO from Oryza sativa (82% identical,NP_921328). Expression analysis showed that EgDIM transcript abundantly found in female flowers. Over-expression of EgDIM drivenby double CaMV35S promoters revealed that the rice stem and the length of the rice seed were longer than wild type. These resultssuggest that EgDIM gene may play a critical role in cell elongation.


Poster 37

THE T102C POLYMORPHISM OF THE SEROTONIN 2A RECEPTOR GENE IN MALAYSIANPATIENTS WITH MAJOR DEPRESSIVE DISORDER

MOHD IZWAN ZAINOL1, VIJAYA L RAJ2, ELSA HANIFFAH MEJIA MOHAMED2, NOR ZURAIDA ZAINAL3 ANDZAHURIN MOHAMED2

Dept of Molecular Medicine1, Dept of Pharmacology2 and Department of Psychological Medicine3,Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Major depressive disorder (MDD) is a common mental disorder that manifests with several psychiatric symptoms. Recent studies suggestthat the brain dopaminergic and serotogenic systems are of major significance in the neuropathology and treatment of MDD. In the presentstudy, the relationship between MDD and the T102C polymorphism in serotonin 2A (5-HT2A) receptor gene was assessed in the Malaysianpopulation. The study involved 130 patients with MDD who were recruited from the University of Malaya Medical Centre (UMMC)Psychiatric Day Clinic, and 163 controls with no known personal and family history of MDD. The allelic and genotype frequencies of theT102C polymorphism of 5HT2A receptor gene between the MDD patients and the controls were then compared. The analysis of genepolymorphisms was performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP)techniques using MspI restriction enzyme. The RFLP results indicated that the uncut wild-type variant (TT) produced a single band (342bp), the heterozygous-type (TC) produced three bands (342 bp, 216 bp and 126 bp), while the mutant variant (CC) produced two bands(216 bp and 126 bp). The results of the study showed that the genotype frequencies of the MDD patients for 5-HT2A receptor gene were65%, 19% and 16% for the TT, TC and CC genotype respectively while the genotype frequencies in the control subjects were 43%, 50%and 7% for the TT, TC and CC genotype respectively. Allelic frequency of the T allele in the MDD patients was 0.742 and for the C allelethe frequency was 0.258. In the control subjects, the allelic frequency of T and C allele were 0.678 and 0.322 respectively. The allelic andgenotype frequencies were in Hardy-Weinberg Equilibrium. The results clearly showed that there were significant differences (ρ<0.02) inthe allelic and genotypic frequencies between MDD patients and control subjects in the Malaysian population for the T102C polymorphismof 5HT2A receptor gene, and hence this polymorphism may play a role in the aetiology of MDD.

Poster 38

TOXICITY ASSESSMENT OF MUNICIPAL LANDFILL LEACHATE IN MALAYSIA USINGFRESHWATER PRAWN (MACROBRACHIUM LANCHESTREI)

JAFFAR Y. M. ALKASSASBEH, *LEE YOOK HENG, M. SHUHAIMI OTHMAN AND SALMIJAH SURIF

School of Environmental and Natural Resource Sciences, *School of Chemical Sciences and Food Technology,Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

Landfill leachates have been implicated in environmental pollution, developmental anomalies, birth defect, and surface and groundwaterpollution worldwide. This study has been conducted to determine the toxicity of landfill leachate on adult freshwater prawn (Machrobrachiumlanchesteri) which for the first time, has been used as test organism for acute toxicity test of landfill leachate. Leachates were collectedfrom three landfill sites in Selangor, Malaysia; Air Hitam landfill (AHL), a sanitary landfill, Ampar Tenang (ATL), and Sungai Sedu (SSL)landfills, both are unregulated open landfills. The experiments were performed as three replicates using a total of 180 prawns. The statictest method of acute toxicity test was used. The temperature was maintained at 25±1°C, amount of dissolved oxygen was observed to benot less than 5 mg/l. The data obtained were statically evaluated by the use of the EPA computer program based on Finney’s ProbitAnalysis Method and a 96-h LC50 values of landfills leachate from the three landfills (AHL, ATL, and SSL) using M. lanchesteriindividuals with average weight of 0.3g and average length of 3.5cm were found to be 0.668, 2.08, and 3.195% respectively. The 95%confidence limits were (0.395-1.08%), (1.656-2.584), and (2.273-4.438) respectively. The results show that landfill leachate is highly toxicto freshwater prawn. Among the behavioral changes observed for the individual prawns at different leachate concentrations were; declinein general activity, loss of balance, swimming difficulties, breathing difficulties, lightening of skin color, and enlargement of the eyes.

Poster 39

BIOMASS NUTRIENT PROFILES OF THE MICROALGA CHLORELLA VULGARIS WITH DIFFERENTDRYING METHODS

JUNAIDA @ MAIMUNAH HASSAN BASARI1, NOR ASHIKEEN MUKTI1, WAN ZURINAH WAN NGAH1,RAZALI SABUDDIN2, A. RAZAK MUDA3 AND YASMIN ANUM MOHD YUSOF1

1Department of Biochemistry, Faculty of Medicine, 2Department of Management and Development, 3Department of Nutrition andDietetic, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur

Chlorella vulgaris (CV) is a unicellular green microalgae which grows well in tropical and subtropical climate. Biomass culture of CV isgaining popularity in many parts of the world for animal feeds as well as for human consumption as whole food. However, processing ofCV has been associated with loss of its nutrient composition. The aim of this study was to investigate the nutritional composition of CVwith various drying treatments. The data include the proximate composition (moisture, ash, protein, carbohydrates, fibre and lipids) andmineral elements (Na, K, Ca, Mg, Fe, Cu, Zn, Se). Chlorella vulgaris Beijerinck (strain 072, UMACC) was grown in a large tank under a12h light:12h dark regimen (pH 6.8 ; 25ºC in air containing 1% CO2) in Bold’s Basal Medium (BBM). The freeze-dried CV contained42.55% protein, 40.11% carbohydrate and 1% fibre whereas oven-dried CV contained 40.21% protein, 37.41% carbohydrate and 0.5%fibre. The contents of several minerals in 100g dry biomass were found higher in freeze-dried CV compared to oven dried CV: Ca (1654mg vs 1513 mg), Fe (2315 mg vs 1157 mg), K (831 mg vs 823 mg), Na (708 mg vs 248 mg), Cu (46 mg vs 42 mg) and Se (31 mg vs 16mg). In conclusion, the nutritional composition of CV is preserved better in freeze-dried compared to oven-dried method.


Poster 41

SYNTHESIS, CHARACTERIZATION OF BENZENESULFONOHYDRAZONES AND THEIRBIOLOGICAL ACTIVITIES

1LAILA MUSALAM, 1HAPIPAH MOHD. ALI, 1SHARIFUDIN M. ZAIN AND 2MAHMOOD AMEEN

1Chemistry Department, Faculty of Science, 2Molecular Medicine Department,Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

[email protected]

Benzenesulfanohydrazide and hydroxyacetophenone derivatives are able to form chelate complexes with transition metals .These ligandsand their complexes has active against certain bacteria. So it can be tested as further biological activities. Schiff bases of ( BSH) and(HAP) derivatives undergoes condensation reaction under reflux in acidic ethanol for 2 hours ,while their complexes are synthesizedunder reflux in basic ethanol for 5 hours. All the ligands and complexes characterized by IR, NMR, UV spectroscopy, TGA and xray(ifpossible) , before biological study is carried out The compounds are tested for anti-ulcerogenic activity. Sprague-Dawley rats werepretreated orally with different concentration of ligands before ethanol administration. The rats were sacrificed and their stomachs wereremoved for gastric lesions area measurement. The mucus was weighed and gastric juice was collected to determine the pH value. Acutetoxicity test has been carried out for each high (5g/kg) and low dose (2g/kg). Schiff bases of benzenesulfonohydrazone excellentlyinhibited gastric lesions on the glandular part of the stomach. It is believed that ulcer inhibition did not influence by production of mucusand gastric juice acidity. The hemorrhagic lesions were found to be prevented even though the mucus secreted is lower than that secretedby the positive control (cimetidine). This could explain other mechanism that may take place during the prevention. The biologicalactivities are run at Molecular Medicine Department, Faculty of Medicine, University of Malaya.

Poster 42

IS OIL PALM SOMATIC EMBRYOGENESIS RECEPTOR KINASE (EgSERK), A MARKER FOREMBRYOGENIC COMPETENCE?

LEE FONG CHIN1, PARAMESWARI NAMASIVAYAM1, MEILINA ONG ABDULLAH2 AND HO CHAI LING1

1Cell and Molecular Biology Department, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400,UPM, Serdang Selangor, Malaysia. 2Malaysia Oil Palm Board (MPOB), Bandar Baru Bangi, 43300, Kajang, Selangor, Malaysia

Somatic embryogenesis receptor-like kinase (SERK) is among the one that is claimed to confer embryogenic competence to somatic cellsof Daucus and Dactylis to form embryo later. SERK gene encodes a leucine-rich repeat (LRR) plant receptor-like kinase (RLK) and it hasbeen reported in a few plants including Arabidopsis, rice, maize, coconut and coco. We report the isolation of a full length oil palm SERKcDNA which is 2378bp in length and orthologous to Cocos nucifera SERK. The deduced amino acid sequence of oil palm SERK(EgSERK) cDNA (629 aa) has all the protein domains encoded by typical SERK proteins. These included signal peptide, leucine zipper,leucine rich repeats, proline rich region which serve as the unique features of SERK gene, transmembrane and kinase domain. EgSERKgene was then characterized by carrying out Southern analysis and real-time PCR. Based on Southern analysis, it was concluded that inthe oil palm genome, there might be more than one copy number of EgSERK gene. The expression profile of EgSERK was dissimilarfrom reported SERK gene of other plants whereby specific to embryogenic tissues. In oil palm, EgSERK gene was found expressed in allthe tested tissues including both embryogenic and non-embryogenic tissues. The expression level of EgSERK gene in mature vegetativetissues was higher than in reproductive tissues. The strongest gene expression was found in young leaves and the lowest in the root.However, in reproductive tissues, highest expression was in male flower. All these results suggest that EgSERK gene may have broaderrole in oil palm development rather than being specific to somatic embryogenesis.

Poster 43

COMPARISON OF GYNURA PROCUMBENS EXTRACT AND GLIBENCLAMIDE EFFECTS ONTRIGLYCERIDE LEVEL AND ADIPOSE TISSUE LIPOPROTEIN LIPASE ACTIVITY IN DIABETES

INDUCED RATS

LEE HUI WEN AND HALIMAH ABDULLAH SANI1

1Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor

Diabetes mellitus is a clinical condition characterized by elevated blood glucose level as a result of biochemical alterations in glucose andlipid metabolism due to the disturbance in insulin production, insulin action, or both. Diabetes mellitus affects lipid metabolism in fewways including the inhibition of lipoprotein lipase enzyme activity. Gynura procumbens, also known as “pokok sambung nyawa”, is aherbal shrub that has been used in Malaysia to treat various diseases. The aim of this research is to investigate the effects of Gynuraprocumbens leaf aqueous extract on blood triglyceride (TG) level and adipose tissue lipoprotein lipase (LPL) activity in diabetes-inducedrats, and comparing it with an anti-diabetic agent, Glibenclamide. Male Sprague Dawley rats (n = 30) were used and divided into 2 groups,which were normal (n = 15), and diabetic rats (n = 15). The rats were induced into diabetic with Streptozotocin (55 mg/kg) via intravenousinjection. Each group was subdivided into 3 groups with 5 rats each. The subgroups were control, Gynura procumbens leaf extract-treatedgroup (100 mg/kg) and Glibenclamide-treated group (5 mg/kg). Treatments were given daily for 14 days. On the 15th day, the rats weresacrificed and blood samples were taken from the aorta for determination of blood TG level, while epididymal adipose tissue sampleswere taken for LPL activity assay. The results obtained show that diabetes has increased blood TG level by 58.04 ± 2.09 mg/dl anddecreased adipose tissue LPL activity by 16.01 ± 3.79 U/mg in diabetic control rats as compared to normal rats. The treatment of Gynuraprocumbens leaf aqueous extract on diabetic rats was able to decrease blood TG level significantly (p<0.05) by 88.16 ± 2.15 mg/dl ascompared to diabetic control rats (391.34 mg/dl) and increased adipose tissue LPL activity significantly (p<0.05) by 5.22 ± 0.08 U/mg ascompared to diabetic control rats (4.85 U/mg). On the contrary, treatment of Glibenclamide on diabetic rats was only able to decreaseblood TG level by 36.09 ± 9.716 mg/dl and increased adipose tissue LPL activity by 2.86 ± 0.88 U/mg as compared to diabetic controlrats. Therefore, Gynura procumbens leaf aqueous extract has a better hypotriglyceridemic effect on diabetic rats compared to Glibenclamide.


Poster 44

MOLECULAR IDENTIFICATION AND CLONING OF STREPTOMONOSPORA SP. FROM MALAYSIAAND ANTARCTIC SOIL (BARRIENTOS ISLAND, ANTARCTIC)

LEE LEARN-HAN1, CHEAH YOKE-KQUEEN1*, HERNAN MOREANO ANDRADE2,MICHEAL CLEMENTE WONG VUI LING3, NURUL SYAKIMA AB MUTALIB1, ROSNIDA IDRIS1 AND SON RADU4

1Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor. 2Instituto Antartico Ecuatoriano,Guayaquil, Ecuador. 3Biotechnology Research Institute, Universiti Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah,Malaysia. 4Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia.

Soil antinomycetes have great potential in producing useful bioactive compounds. Halophiles like Streptomonospora occur as a newimportant resource as potential producers of novel bioactive compounds. Genus Streptomonospora formed a distinct branch in the 16SrRNAgene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, family Norcadiopsaceae. The use of genus specific primeroffers an effective approach for rapid identification of large number of strains and samples. In this study, 5 soil samples were gathered fromlocal soil comprises of different location. While 18 soil samples were collected from different locations throughout Barrientos Island,Antarctic to study the distribution of Streptomonospora. Furthermore molecular cloning was performed to make identical and multiple copiesof the target gene. For isolation of actinomycetes from soil, starch casein agar was used as selective media for actinomycetes growth. DNAwas extracted and subsequently 16S rRNA genes were amplified by specific-PCR yielded 1.5kb PCR product. The PCR product was used astemplate for nested PCR that target specific gene fragment of Streptomonospora that yielded 565 bp. PCR product was purified and proceedto molecular cloning using QIAGEN PCR cloning kit (Qiagen, Hilden, Germany). Through blue-white selection, insert was verified bycolony-PCR and colonies with transformations were preceded to plasmid DNA extraction (Eppendorf, Hamburg, Germany). Purified plasmidDNAs were served as templates for PCR to confirm the insertion of gene of interest. For results, 13 out of 143 isolates were positive forStreptomonospora which 2 isolates were isolated from local soil samples and 11 isolates were isolated from Barrientos Island soil samples.Results from the study demonstrated that nested PCR increases the specificity of DNA amplification by reducing background due to non-specific amplification. Sequencing result of purified plasmid DNA shown 96% homology with existing sequence of Streptomonospoara sp. ingene bank database, thus confirmed the identity of the specific band obtained. To conclude, nested-PCR enabled accurate, rapid and sensitiveapproach for identification of Streptomonospora from large numbers of different strains.

Poster 45

PHARMACOLOGICAL PROPERTIES OF DIARYLPENTANOID DERIVATIVE ON CELLULARMODEL OF RHEUMATOID ARTHRITIS

KA-HENG LEE1, SYAHIDA AHMAD1,2, FARIDAH ABAS2,3, KHOZIRAH SHAARI2,4, DAUD AHMAD ISRAF ALI 2,5 ANDNORDIN HAJI LAJIS2,4

1Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Biosciences, 3Faculty of Food Science and Technology,4Faculty of Sciences, 5Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Rheumatoid arthritis is a chronic inflammatory disease characterized by abnormal immune phenomena involving macrophage (type-A synoviocyte)and synovial fibroblast (type-B synoviocytes) resulting in progressive joints destruction. In this study, the effects of diarylpentanoid derivative(MS71A), a new synthetic compound, was evaluated upon two cellular systems which are murine macrophage (RAW 264.7) and rabbit synovialfibroblast (HIG-82). MS71A inhibited main pro-inflammatory mediators and cytokines including nitric oxide (NO), tumor necrosis factor-alpha(TNF-α) and interleukin-1beta (IL-1β) production from lipopolysaccharide/interferon-gamma (LPS/IFN-γ) induced RAW 264.7, with IC50 valuesof 13.66 ± 0.61 μM, 9.40 ± 1.67 μM and 29.66 ± 0.72 μM respectively. In addition, MS71A significantly suppressed cartilage degradative matrixmetalloproteinase (MMP) including gelatinase (MMP-9) and collagenase activities from phorbol myristate acetate (PMA) induced HIG-82.These promising finding make MS71A as an alternative pharmacotherapy of rheumatoid arthritis treatment in future.

Poster 46

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA 2 EXPRESSION IN ORALLY-ADMINISTERED GLYCYRRHIZIC ACID-TREATED RATS

LIEW KAH LEONG1, TON SO HA1 AND KHALID BIN ABDUL KADIR2

1School of Science, 2School of Medicine and Health Sciences; Monash University Sunway Campus

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor with a pivotal role in adipocytedifferentiation and insulin sensitization. Synthetic ligands such as thiazolidinedione (TZD) were utilized to alleviate insulin resistanceamidst the presence of side effects. Studies on the ligand binding potential of glycyrrhizic acid (GA), a natural compound to PPARγdisplayed encouraging results in reducing blood glucose levels in diabetic KK-Ay mice. Therefore this study was performed to determinethe effects of GA on PPARγ2 expression levels in various tissues using real time PCR. The quantification was performed using a standardcurve of 8 log dynamic recombinant plasmids. Administration of GA did not display much difference in terms of PPARγ2 copy number inall treated tissues compared to controls (visceral adipose tissue – 1.52 x 1026: 1.09 x 1026; subcutaneous adipose tissue – 2.65 x 1027: 5.23x 1026; liver – 3.44 x 1021: 2.74 x 1021; kidney – 4.03 x 1021: 1.9 x 1021; quadriceps femoris – 3.83 x 1023: 7.5 x 1022; abdominal muscle –4.27 x 1024: 8.91 x 1023). Nevertheless, both the adipose tissues displayed the highest PPARγ2 expression, reiterating the importance ofPPARγ2 in adipogenesis. In conclusion, GA administration showed slight increase in PPARγ2 expression levels in all tissues.


Poster 47

CONSTRUCTION OF A HAPPY MAP FOR THE TWO LARGEST CONTIGS FROM THEEIMERIA TENELLA GENOME ASSEMBLY

LIK-SIN LIM1,2, PAUL H. DEAR3 AND KIEW-LIAN WAN1,2

1Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, 43600 UKM Bangi, Selangor DE;2School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,

43600 UKM Bangi, Selangor DE; 3MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Eimeria tenella is a major veterinary pathogen, particularly to the poultry industry. Sequencing of the E. tenella genome has been initiated in theeffort to enhance the development of more effective controls of the disease caused by the parasite. The current assembly of theE. tenella genome consists of slightly over 4700 contigs, and a HAPPY map for the whole E. tenella genome may help to translate this datasetinto a more valuable resource. In order to investigate the effectiveness of this approach, a HAPPY map was constructed for the two largest contigsfrom the assembly, namely contig_00031646 and contig_00031359 which are 515,133bp and 505,376bp in length, respectively. HAPPY mapmarkers were created for every ~20kb within each of the contigs. A total of 25 markers were designed for contig_00031646 and 23 markers weredesigned for contig_00031359. Results of the experiment showed that 19 of the 25 markers for contig_00031646 were successfully typed whileonly 14 out of the 23 markers for contig_00031359 were considered to be of a good quality. Analysis of the mapping data revealed that themarkers can be successfully arranged into two linkage groups, according to their respective contigs. Comparison between the position of themarkers on the map and in the genome sequence showed that the majority of the markers were co-linear. Small differences found between theorder of the markers on the map and in the genome sequence were thought to be due to the resolution of the HAPPY map. Overall, the results ofthis study showed that the HAPPY mapping approach will be useful in the generation of a framework for the assembly of the E. tenella genome.

Poster 48

LIPID PROFILE, SERUM FREE FATTY ACIDS AND LIPID DEPOSITION IN GLYCYRRHIZICACID-TREATED RATS

LIM WAI YEN ALFRED1, LIONG SHIH YEEN1, CHIA YOKE YIN1, TON SO HA1, KHALID BIN ABDUL KADIR2 ANDSHARIFAH NOOR AKMAL3

1School of Science, 2School of Medicine and Health Sciences, Monash University Sunway Campus,3Cytology Unit, Hospital Universiti Kebangsaan Malaysia

The metabolic syndrome is characterized by the co-existence of abdominal obesity, hyperglycaemia, hypertension, dyslipidaemia and insulinresistance. Increased activation of glucocorticoid receptors results in metabolic syndrome symptoms and the enhanced action of glucocorticoidshas therefore been speculated to play a causative role in the pathophysiology of the syndrome. Glycyrrhizic acid (GA), the primary bioactiveconstituent of licorice, is an inhibitor of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) that catalyzes the activation ofglucocorticoids. Oral administration of 50mg/kg of GA for a week showed non-significant but consistent improvement in the lipid profileparameters of treated rats relative to the control, with a decrease in serum triacylglycerol, total cholesterol and LDL cholesterol and elevationof HDL cholesterol (p>0.05). Serum free fatty acids also showed a similar trend of decrease (p>0.05). Histological analysis using Oil Red Ostaining showed significant decrease in the levels of lipid deposition in abdominal and quadriceps femoris muscle (p<0.05) but a non-significant decrease in the heart, kidney and liver (p>0.05). In conclusion, GA may potentially improve the symptoms of dyslipidaemia inmetabolic syndrome via a beneficial shift in lipid profile and serum free fatty acids and reduced tissue lipid accumulation.

Poster 49

OPTIMIZATION OF THE REAL TIME RT- PCR METHOD FOR ANTIOXIDANTASSOCIATED GENES OF HUMAN SKIN FIBROBLASTS

LINA WATI DURANI, SUZANA MAKPOL, 1CHUA KIEN HUI, YASMIN ANUM MOHD YUSOF, WAN ZURINAH WAN NGAH

Department Of Biochemistry and 1Physiology, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia

Antioxidant associated genes are involved in the scavenging of free radicals. The copper/zinc superoxide dismutase (SOD1) is present inthe cytosol, while manganese superoxide dismutase (SOD2) is present in the mitochondria. The primary function of SODs is to stop thesuperoxides anion from causing damage to the body. SODs convert the superoxide radical to hydrogen peroxide which is converted towater and oxygen by catalase (CAT) while glutathione peroxidase 1 (GPx1) is an efficient scavenger of hydrogen peroxide. In this study,we determined the specificity of the primers for antioxidant associated genes using quantitative real-time RT-PCR method which is ahighly sensitive technique for the detection and quantitation of mRNA. Primers for human GAPDH, SOD1, SOD2, CAT and GPx1 weredesigned with Primer 3 software and blasted with GeneBank database sequences in order to obtain primers with high specificity. GAPDHwas used as housekeeping gene. Real-time RT-PCR reaction was performed with RNA as templates, primer of the targeted genes andiScript One-Step RT-PCR kit with SYBR Green (Bio-Rad). Reactions were run using Bio-Rad iCycler with reaction profile of; cDNAsynthesis for 20 min at 50°C; pre-denaturation for 4 min at 95°C; PCR amplification for 38 cycles with 10 sec at 95°C and 30 sec at 61°C.These series of cycles were followed by a melt curve analysis to check the reaction specificity. Our results showed that all the primers usedwere specific to the target genes. Melting curves analysis showed a single amplified product for all genes and the melting temperatureswere in accordance ± 0.1. The PCR products which were checked on 2% agarose gel were corresponded to the expected size.


Poster 50

TRYPTOPHAN HYDROXYLASE (TPH) GENE POLYMORPHISMS AND ITS ASSOCIATION WITHMAJOR DEPRESSION IN THE MALAYSIAN POPULATION

LOKE AI CHIN1, NUR ZURAIDA ZAINAL2, LIAN LAY HOONG3, VIJAYA LECHIMI RAJ1,ELSA HANIFFA MEIJA MOHAMED1 AND ZAHURIN MOHAMED1

1Department of Pharmacology, 2Department of Psychological Medicine and3Department of Molecular Medicine, Faculty of Medicine, University of Malaya

Serotonin or 5-hydroxytryptamine (5-HT) is a neurotransmitter that is located in the brain-stem. A dysfunction of the 5-HT system has beenimplicated in the pathogenesis of psychiatric disease, including major depressive disorders (MDD). The tryptophan hydroxylase (TPH) is a rate-limiting enzyme which is involved in the biosynthesis of 5-HT. The TPH gene has been implicated as one of the major candidate genes for MDD.There are two isoforms of TPH, known as TPH-1 and TPH-2. TPH-1 gene is located at chromosome 11p15.3-p14, which is about 29 kilobaseslong. The aim of this study was to examine whether the polymorphism of A218C in intron 7 of the TPH-1 gene is associated with MDD in theMalaysian population. A total of 284 healthy volunteers and 142 patients with MDD were included in this study. Genotyping was carried outusing polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods, whereby the PCR products weredigested with BfaI enzyme. The RFLP results showed that the wild-type variant (CC) exhibited two bands (615bp, 245bp), the heterozygous-type(AC) had three bands (860bp, 615bp, 245bp) and the mutant variant (AA) remained uncut (860bp). The results of the study showed that thegenotype frequencies for MDD patients for the TPH A218C gene were 19.0%, 51.4% and 29.6% for the AA, AC and CC genotypes, respectively,while that of the healthy volunteers were 20.4%, 49.3% and 30.3% for the AA, AC and CC genotypes, respectively. No statistical significantdifferences were observed in the allelic and genotype frequencies between the MDD patients and control subjects [genotypes, χ2=0.194, degreesof freedom (d.f.)=2, P=0.907; alleles, χ2=0.008, d.f.=1, P=0.927]. There was no deviation from the Hardy-Weinberg equilibrium for thedistribution of the A218C polymorphism genotypes for both patient and control groups [MDD, χ2=0.225, d.f.=1, P=0.635; controls, χ2=0.006,d.f.=1, P=0.938]. These preliminary findings therefore indicate that the A218C polymorphism of the TPH gene may not play a major role in thepathogenesis in MDD. However, further studies need to be carried out using larger sample sizes to confirm the data obtained.

Poster 51

CHARACTERIZATION OF BURKHOLDERIA CEPACIA COMPLEX ENVIRONMENTAL ISOLATESFROM PENINSULAR MALAYSIA

LYE SIEW FEN1, NOR RASIMAH BT. MOHD NOOR1 AND SHEILA NATHAN1,2

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia &2Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, Bangi, Selangor

Burkholderia cepacia, a soil dwelling phytopathogen bacteria, is recognized as an important opportunistic pathogen among cystic fibrosispatients. Taxonomy studies have demonstrated that B. cepacia is made up of a cluster of at least nine closely related genomic species(genomovar) which are referred to as the B. cepacia complex (Bcc). In this study, we collected soil samples from selected states in PeninsularMalaysia to isolate environmental Bcc samples. The soil samples were prescreened on Burkholderia selective media and Bcc were confirmedusing the API 20NE kit. Biochemical assays were performed on the isolates in addition to killing assays to determine virulence. Four isolatesreferred to A, B, C and D were identified as Bcc members of B. cenocepacia, B. anthina and B. vietnamensis based on recA phylogenetic tree.Isolates B and D secreted beta-hemolysin while A and C were alpha-hemolysin. Protease activity was detected for isolates A and B based onthe production of a halo on skimmed milk agar. The nematode Caenorhabditis elegans (C. elegans), was fed on the bacteria and nematodeswere observed for 72 hours. With the exception of isolate D, none of the isolates achieved 50% lethality (LT50) of the nematode on nematodegrowth medium (NGM) and Pepton-Glucose-Sorbitol (PGS) medium throughtout the observation period of 72 hours. Thus, from this study,we have successfully isolated Bcc from the local soil environment but the majority of the isolates were avirulent towards C.elegans.

Poster 52

IDENTIFICATION OF FLAVONOIDS PRODUCTION FOR HYDROCOTYLE BONARIENSIS CELL CULTURE

MASOUMIAN M1. , MAZIAH M.2, ARBAKARIYA A.2 AND RADZALI M.2

1Institute of Bioscience, 2Faculty of Biotechnology and Biomolecular Sciences, Universitiy Putra Malaysia, 43400 UPM Serdang, Selangor

Plant flavonoids have been shown in recent years to be of vital significance to mankind as well as plants. The main objective in this study wasquantitative identification of flavonoids in leaf and callus culture of Hydrocotyle bonariensis a perennial prostrate herb and found mostly intropical and subtropical region of the world. The total flavonoid content has been carried out by colorimetric methods and the aglycones (acidhydrolysed) are quantified by HPLC analysis. Samples were oven-dried at 50 °C before been ground with mortar and pestle. A total of 0.25 gfinely dried powder was extracted with 20 ml of aqueous methanol containing 20 mM sodium diethyldithiocarbomate as an antioxidant. Fiveml 6 M HCL was added to each extraction to give a 25 ml solution. Extracts were refluxed at 90 °C for 2 h. Extract aliquots of 20 μl takenbefore and after hydrolysis, were filtred through a 0.45 μm filter and analyzed by reversed-phase HPLC on 150x3.9 mm I.D., 5μm particlediameter C18 symmetry Column;20 min gradient of acetonitrile in water (15-35%). Three replicated samples were analyzed similarly andfound to contain total flavonoids 18.08±1.24 mg g-1, 0.255±0.012 mg g-1 Quercetin and 0.089±.006 mg g-1 Kaempferol in dry mass of leaf. Indry mass of 16 days old callus total flavonoid 2.5±0.21 mg g-1, 0.098±0.007 mg g-1 Quercetin and 0.038±0.002 mg g-1 Kaempferol.


Poster 53

POLYMORPHISM OF IL-6 (-174G/C) PROMOTER GENE IN MALAYSIAN ASTHMATICS

MALYANAH SUPARMAN1, VIJAYA LECHIMI RAJ2, LIAM CHOMG KIN3, AMMU RADHAKRISHNAN4, RAKESH NAIDU5

AND MOHD RAIS MUSTAFA2

1Dept of Molecular Medicine, 2Dept of Pharmacology and 3Dept of Medicine, Faculty of Medicine, University of Malaya,Kuala Lumpur, 4Pathology Section, Faculty of Medicine, International Medical University, Kuala Lumpur,

5Monash University Malaysia, Selangor, Malaysia

Asthma is a chronic inflammatory disease characterized by lower airway hyperresponsiveness and by variable airflow limitation that canresolve spontaneously or through treatment. The inflammatory condition, including increase in the pro-inflammatory cytokine, IL-6, thatdetermines the superimposition of these inflammatory mechanisms on those involved in asthma. This increases the influence on airwaymuscle contractility [1]. The IL-6 gene has gained considerable interest because it has been associated with a variety of disease states andrecently, a G/C polymorphism has been reported at position -174 in the 5’ flanking region of the IL-6 promoter gene. The G allele isassociated with higher plasma concentrations of IL-6 [2]. In view of these findings, it was hypothesized that the IL-6 promoterpolymorphism at -174 could represent a genetic susceptibility factor for asthma. To assess the role of IL-6 polymorphism in asthma, the -174 G/C polymorphism of the IL-6 gene was analyzed in 94 patients with asthma and 114 healthy controls. Genotyping was performedwith polymerase chain reaction (PCR) and followed by restriction fragment length polymorphism (RFLP) method where the PCR productwas digested by the NlaIII restriction enzyme. The RFLP results showed that the wild type variant (GG) had two bands (167bp, 31bp), theheterozygous variant (GC) had four bands (167bp, 122bp, 45bp, 31bp) and the mutant variant had three bands (122bp, 45bp, 31bp). Theresults of the study showed that the genotype frequencies for asthmatic patients were 93.6%, 4.3 % and 2.1% for the GG, GC and CCgenotype respectively, while the control subjects had frequencies of 94.7%, 4.4% and 0.9% respectively for the above genotypes. Theallelic frequencies for asthmatic patients were 0.957 for G allele and 0.043 for C allele, while the allelic frequency for healthy controlsubjects were 0.969 for G allele and 0.031 for C allele. No significant difference in the distribution of IL-6 genotypes (P=0.718) and allelicfrequencies (P=0.519) was found. All allelic and genotype frequencies were in equilibrium with Hardy Weinberg. In conclusion, there isno significant association between the -174G/C polymorphism of IL-6 and the occurrence of asthma. This data does not support thehypothesis that IL-6 gene could represent a genetic susceptibility factor for asthma. However, additional variations in the IL-6 promoterregion together with the -174G/C polymorphism may define different haplotypes that show different levels of expression in the reportergene assays.

Poster 55

A SIMPLE MULTIPLEX PCR METHOD FOR FOUR Y-CHROMOSOME STRS:DYS388, DYS435, DYS437 AND DYS439

MIRSAED MIRINARGESI1, PATIMAH ISMAIL1, PARVIN PASALAR2,SIMA ATAOLLAHI ESHKOOR1, R.VASUDEVAN1 AND MOHAMED SALEEM1

1Molecular Biology Lab, Genetic Research Group, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,43400 UPM Serdang, Selangor, 2Faculty of Medicine, Tehran University of Health Sciences, Tehran, Iran

DYS (DNA Y-chromosome Segments) markers are Short Tandem Repeats (STRs) located at the non-recombining part of Y-chromosome.Thus, these markers are transmitted as haplotypes from fathers to sons throughout generations establishing patrilineages. These characteristicsshould render the Y-linked polymorphisms extremely useful as genetic tools for human evolution, forensic and other population geneticstudies. Since the Y-chromosome is passed down from generations without any recombination, individual markers cannot be combinedusing the product rule as it is commonly done with autosomal STRs occurring on different chromosomes. Ideally, a Y-chromosomehaplotype should include as many polymorphic loci as possible to improve the chance of separating any individual or male lineage. Inorder to obtain a high level of discrimination either a large number of Y-STRs can be run one at a time or combined into a multiplex. Thisinitiates us to do a simple multiplex PCR amplification for four polymorphic Y-chromosome-STRs: DYS388 (128-143 bp), DYS435 (210-228 bp), DYS437 (186-202 bp) and DYS439 (238-258 bp). Prior amplification, the sequences of DYS loci were obtained from HumanGenome Database and the primers were designed using Primer3 (v.0.4.0) online software and the accuracy was confirmed with BLASTsearch tool. Multiplex-PCR was optimized in a total volume of 25 μl includes 5 μl PCR Master Mix (iDNA), 150 nM DYS388 forwardand reverse primers, 100 nM of DYS435 forward and reverse primers, 50 nM of DYS437 forward and reverse primers, 50 nM of DYS439forward and reverse primers, and 0.05–2 ng of template DNA. The amplification was done under the one cycling conditions of 95 °C for12 min, 32 cycles at 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and then 65 °C for 30 min. In conclusion, multiplex-PCR methodis a simple and rapid way to amplify all the above STRs loci in a single reaction tube.


Poster 56

RELATIONSHIP BETWEEN T102C POLYMORPHISM OF THE 5HT2A RECEPTOR IN PATIENTSWITH SCHIZOPHRENIA

MOHD AIZAT MOHD ZAIN1, NOOR FADZLIN MAHALI1, TIONG CHEA PING2, NOR ZURAIDA ZAINAL2,ZAHURIN MOHAMED3 AND VIJAYA LECHIMI RAJ3

1Dept of Molecular Medicine, 2Dept of Psychological Medicine and3Dept of Pharmacology, Faculty of Medicine, University Malaya, 50603 Kuala Lumpur

Schizophrenia is a common and serious mental illness that negatively affects a person’s lifestyle. The aetiology of this disease is still amystery but there are many theories that indicate environment and genetic factors as possible factors. One of the genetic factors suggestedis the T102C polymorphism in the 5HT2A receptor gene. The T102C polymorphism is a silent polymorphism that does not change thesequence of amino acid of the protein that is yielded after translation of mRNA. Nevertheless, it is believed that T102C polymorphisminfluences the secondary structure and stability of mRNA1. Another possible explanation of association between the T102C polymorphismand schizophrenia is the linkage disequilibrium of the 5HT2A receptor genes or the adjacent genes that may be caused by thepolymorphism2. The aim of this study is to investigate the association between T102C polymorphism in 5HT2A receptor gene withschizophrenia in the Malaysian population. This study is important because there are contradictory results concerning the associationbetween schizophrenia and polymorphisms that may be explained by the different patterns of linkage disequilibrium across differentpopulations. In this study, the T102C polymorphism was analyzed by using a PCR-RFLP method and statistical analysis to determine thefrequency of this polymorphism in the Malaysian population. Subjects included 203 patients who fulfilled DSM-4 criteria for diagnosis ofschizophrenia, and 216 control individuals with no personal or family history of schizophrenia. Polymerase chain reaction (PCR) wasperformed with the appropriate primers. RFLP was performed by using MspI enzyme to digest the PCR product. The results of the RFLPshowed that the wild type variant had an uncut band of 342 bp, the heterozygous variant had 3 bands (342 bp, 216 bp and 126 bp) and themutant variant had 2 bands (216 bp and 126 bp). Allelic frequencies of the controls showed that the T and C frequencies were 0.722 and0.278 respectively, while that of the schizophrenia patients were 0.640 and 0.360 respectively. The allelic frequency between the twogroups was significantly different (p=0.014). The genotype frequencies for control were 49.5% (TT), 45.4% (TC) and 5.1% (CC) whilethat of the patients were 41.9% (TT), 44.3% (TC) and 13.8% (CC). The genotype frequencies between control and schizophrenia patientswere also significantly different (p=0.018). All allelic and genotype frequencies were in equilibrium with Hardy Weinberg. These resultsalso showed that the C allele had a 1.46 times higher risk (95% confidence interval 1.090 to 1.956) for schizophrenia. The findings of thisstudy indicate that the T102C polymorphism of 5HT2A is associated with risk of schizophrenia.

Poster 57

LACTOBACILLUS PLANTARUM STRAINS ISOLATED FROM MALAYSIAN FERMENTED FOODSHARBOR TWO DIFFETENT CLASSES OF STRUCTRUAL BACTERIOCIN GENES

MORTEZA SHOJAEI MOGHADAM1, FOO HOOI LING1,2*, RAHA ABDUL RAHIM1,2 AND ADAM LEOW THEAN CHOR1

1Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.2Institue of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Bacteriocins comprise a large and diverse group of antimicrobial proteins or peptides. Although bacteriocins can be found in numerousGram-positive and Gram-negative bacteria, those produced by Lactic acid bacteria (LAB) have received special attention since last decadedue to their potential application in food industry as natural biopreservatives. Different bacteriocins have been reported for different strainsof Lactobacillus plantarum, such as plantaricin N and W, plantaricin EF, JK, S and NC8. In this study, seven pairs of bacteriocin gene-specific primers were employed to study the bacteriocin structural genes that present in six strains of L. plantarum isolated fromMalaysian fermented foods. Interestingly, two structural bacteriocin genes encoding plantaricin W (class I bacteriocin) and plantaricin EF(class IIb bacteriocin) were amplified using total DNA as template. The other bacteriocin genes were shown to be absent since no PCRproduct was obtained at lower annealing temperatures. Nevertheless, this is the first report pertaining to the existence of two classes ofbacteriocin genes from a single strain of L. plantarum. Simultaneous existing of two bacteriocin genes belong to two different classeswould contribute to the broad inhibitory spectrum of the producer cell. The result obtained in this study support further our previous resultfor the broad antagonistic spectrum of producer cells. However, the origin of both classes of bacteriocin structural genes, eitherchromosomally or plasmid encoded, could not be verified since total DNA was used as template in this study. In addition, multipleplasmid bands were obtained from plasmid profiling study, indicating the strains harbor a few plasmids. Hence, bacteriocin that producedby the six strains of L. plantarum could be plasmid encoded since certain bacteriocins of LAB have been reported to be plasmid encoded.


Poster 58

ASSOCIATION ANALYSIS OF THE SEROTONIN 5-HT2C RECEPTOR AND DOPAMINE D3RECEPTOR GENE POLYMORPHISMS WITH SCHIZOPHRENIA IN THREE ETHNIC GROUPS

ZAHURIN MOHAMED1, VIJAYA L RAJ1, NOOR FADZLIN MAHALI2,SITI ZALEHA SUKI1, EHM MOHAMED1 AND NOR ZURAIDA ZAINAL3

1Dept of Pharmacology, 2Dept of Molecular Medicine, and 3Dept of Psychological Medicine, Faculty of Medicine,University of Malaya, Kuala Lumpur, Malaysia

Schizophrenia is a common psychiatric disease which affects about one percent of people world-wide. Recent studies suggest that the interactionbetween brain dopaminergic and serotonergic systems is of major significance in the neuropathology and treatment of schizophrenia. Thepharmacogenetics of schizophrenia has thus focused on studying dopamine and serotonin-related genes. In the present study, the relationshipbetween schizophrenia with two candidate genes were assessed in three ethnic populations namely that of Malays, Chinese and Indian-origin.Specifically, the allelic and genotype frequencies of -759C/T polymorphism of the 5-HT2C receptor gene and Ser9Gly polymorphism of theDRD3 receptor gene between schizophrenic patients and controls in the Malaysian population were compared. The allelic and genotypefrequencies of -759C/T polymorphism of the 5-HT2C receptor gene and Ser9Gly polymorphism of the DRD3 receptor gene between the 3 ethnicgroups were then compared. An attempt was then made to determine the genotype association between the two polymorphisms in schizophrenia.The study comprised of 62 schizophrenic patients and 64 healthy unrelated controls. The analysis of the gene polymorphisms was performedusing polymerase chain reaction and restriction length polymorphisms techniques. The results of the study showed that the genotype frequenciesfor schizophrenia for the 5-HT2C receptor gene were 73%, 3% and 23% for the CC, CT and TT genotype respectively, while the control subjectshad frequencies of 69%, 11% and 20% for the CC, CT and TT genotype respectively. For the DRD3 polymorphism, the genotype frequencieswere 50%, 42% and 8% for the AA, AG and GG genotype respectively in the schizophrenia patients, while the control subjects had frequenciesof 55%, 37% and 8% respectively for the above genotypes. No significant difference was found to exist in the frequencies of allele and genotypebetween schizophrenic patients and control subjects (p>0.05) for both of the genes studied in all the three ethnic groups. In term of genotypeassociation, there was no association between the polymorphism of 5-HT2C and DRD3 in all of the nine possible combinations (p value =0.499). It was interesting however, that the CT/AG and CT/GG combination genotypes were only present in schizophrenia patients but furtherconclusion cannot be made due to the small sample size. Our early findings therefore indicate that the 5-HT2C receptor gene and the dopamineD3 receptor gene may not play a substantial role in schizophrenia nor help evaluate susceptibility to schizophrenia.

Poster 59

ISOLATION OF STEAROYL-ACP DESATURASE (SAD) cDNA FROM JESSENIA BATAUA

MUHAMAD SYAFIQ SHARIAN, PARAMESWARI NAMASIVAYAM1, HO CHAI LING1, AND UMI SALAMAH RAMLI

Advanced Biotechnology & Breeding Center (ABBC), Malaysian Palm Oil Board (MPOB), Bandar Baru Bangi, Selangor,Malaysia, 1Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Jessenia bataua, a potential new oil crops is one of the MPOB’s germplasm collections planted in different parts of the country under the oil palmbreeding programme with the aim to identify and disseminate good germplasm materials for joint trials and development by the industry. Jesseniaoil is very similar composition to olive oil. The mesocarp is also an excellent animal feed stuff. The oil contains about 78% oleic acid and 13%palmitic acid. This paper reports our early effort to study fatty acid biosynthetic genes from Jessenia by molecular approach. Our first target is toisolate and characterize fatty acid biosynthetic genes from Jessenia including stearoyl-ACP desaturase (SAD). SAD is a plastidial soluble enzymethat catalyzes the conversion of stearoyl-ACP to oleoyl-ACP by primary introduction of cis double bond between carbon position 9 and 10. Usingdegenerates primers designed based on SAD sequence of other plants available in the gene bank, partial cDNA sequences of SAD have beenamplified from Jessenia by rapid amplification of cDNA ends (RACE) PCR. The RACE products were used to amplify the full length SADcDNA with size of about ~1.6 kb. The nucleotide sequence revealed an open reading frame of 1182 bp encoding 393 amino acids. The predictedprotein has a molecular weight of 45.02 KDa and contains 3 different domains; Acyl-ACP Desaturase, Ferritin-like and Fatty Acid Desaturase 2domains. BLAST search analysis revealed a very high similarity to SAD from other crops with highest homology (96%) was to the oil palm.Currently, work is being carried out to clone the ORF into expression vector prior to expression studies in bacteria and plant systems.

Poster 60

MAPK SIGNALING INVOLVEMENT IN CAENORHABDITIS ELEGANS DEFENSE AGAINSTBURKHOLDERIA PSEUDOMALLEI INFECTION

MUHAMMAD AZFAR ABDULLAH1 AND SHEILA NATHAN1,2

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor& 2Malaysia Genome Institute, UKM-MTDC Smart Technology Centre

The nematode Caenorhabditis elegans has been used as a model to study host-pathogen interaction in particular the innate immune response. Themitogen-activated protein kinase (MAPK) is an integral part of a cell’s response to physiological stimuli. MAPK signaling is found to beevolutionarily-conserved and has been described to be involved in C. elegans defense against bacterial pathogen. In this study, we attempted toidentify components of the MAPK signaling pathway involved in combating the Gram-negative bacteria Burkholderia pseudomallei infection.Age-matched MAPK-mutant strains of C. elegans (MAPKKK homolog nsy-1, MAPKK mek-1, MAPK pmk-1 and MKP vhp-1) were exposed toa B. pseudomallei clinical (H10) and animal (Sheep4523) isolate. The infected nematode was then scored for live and dead worms every 4 hoursuntil complete lethality was observed. Wild-type N2 Bristol worms were exposed to the same pathogen as a positive control. Worm strains fedwith E. coli OP50 functioned as the negative control. A Kaplan-Meier statistical analysis was performed to calculate the time taken for 50% of theinfected worms to die (LT50). Based on the statistical analysis, we demonstrate that the mutant worms MAPKKK nsy-1, MAPKK mek-1 as wellMAPK pmk-1 died significantly faster compared to the wild-type N2 worms. This implies that these components of the MAPK signalingpathway contribute a significant role to the immune response of C. elegans towards the pathogen B. pseudomallei.


Poster 61

IDENTIFYING DIFFERENTIALLY EXPRESSED PROTEINS IN CELL LINES INFECTED WITH THEH5N1 VIRUS BY PROTEOME ANALYSIS

MUHAMMAD MUZHAFAR MOHAMAD NOOR1,2, SAIFUL ANUAR KARSANI1, IEKHSAN OTHMAN2,SHARIFAH SYED HASSAN2 AND ABDUL RAHMAN OMAR3

Faculty of Science, University of Malaya, Kuala Lumpur1, School of Medical & Health Sciences, Monash University Malaysia2

and Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor3

Highly pathogenic H5N1 (HPAI H5N1) virus has persisted in Asia at least since 1997. It is established in domestic poultry populations inAsia (primarily chickens and domestic ducks) and has subsequently spread to parts of Europe and Africa. In 1997, a human death resultingfrom HPAI H5N1 virus infection in Hong Kong was reported; since then there have been nearly 200 human cases with 109 fatalities. H5N1,a subtype of the Influenza A virus can cause illness (as well as mortality) in humans and many other animal species. The term bird flu is anonscientific term that has been coined to describe the HPAI H5N1 viruses. The presence of the virus may cause alterations as well as specificchanges in the body metabolism which, in turn may lead to abnormalities in life processes. These may be caused either by the absence orpresence of some factors, co-factors, enzymes as well as proteins. In this study, we have used two-dimensional gel electrophoresis to identifythese possible changes in selected cell lines (Madin-Darby canine kidney [MDCK] & chicken embryonic lung cell lines) following infectionwith the H5N1 virus. A number of protein spots were found to have altered expression dynamics. The determination of the identity of theseproteins by mass spectrometry will allow us to propose their possible roles in virus-host interaction and disease progression.

Poster 62

2-DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF COLORECTAL CANCER SERUM

MUNIRAH MIHAT1, SAIFUL ANUAR KARSANI2, ROHANA YUSOF3, COLIN NG4, SANJIV MAHADEVA1,APRIL CARMILLA ROSLANI4 AND ROSMAWATI MOHAMED1

Department of Medicine1, Department of Biomolecules3, Department of Surgery4, Faculty of Medicine,Institute of Biological Sciences2, Faculty of Science, University of Malaya

Colorectal cancer (CRC) is one of the most common cancers in the world, ranking as the third cancer-caused death in western countries such asthe United States and Europe. CRC is also the third most common cancer reported in the Malaysian population. It is a cancer that develops in thecolon and rectum. Most CRC cases occur in individuals aged 50 and above. It usually does not cause any symptoms until the cancer reaches anadvanced stage. CRC develops from a series of genetic and epigenetic events, causing changes in epithelia cells transforming it to carcinomacells. In western countries, it has been reported that hyperplastic polyps usually precedes colorectal cancer, however, this is not a common case inMalaysia. In an effort to understand and elucidate the mechanisms that may be involved in the progression and manifestation of the disease, weused two-dimensional gel electrophoresis to identify proteins that are differentially expressed in the serum of patients with CRC. Using thisapproach, we have identified at least 30 individual protein spots as being differentially expressed in the serum of CRC patients. The identificationof these spots by mass spectrometry may provide insights on the role(s) played by these proteins in the development and progression and CRC.

Poster 63

LIPOPROTEIN LIPASE GENE EXPRESSION AND TRIACYLGLYCERIDE LEVELS IN RATS GIVENGLYCYRRHIZIC ACID AND FED ON COMBINATION OF NORMAL AND HIGH FAT DIET

NG SHING WEI1, TON SO HA1 AND KHALID ABDUL KADIR2

1School of Arts and Sciences, 2School of Medicine and Health Sciences, Monash University, Sunway Campus, Malaysia

Visceral obesity is the major underlying cause of metabolic syndrome (MS) and associated with insulin resistance (IR). High-fat dietarypatterns and sedentary lifestyles have shown to be the pivotal factors contributing to the high prevalence of visceral obesity. The developmentof visceral obesity is associated with how our body regulates the fat consumed. Lipoprotein lipase (LPL) plays a pivitol role in regulating lipidmetabolism and the enzyme is thought to have significant effects on obesity and IR which could lead to Type 2 diabetes mellitus (T2DM) andMS. Glycyrrhizic acid (GA), a constituent of licorice from the dried root of Glycyrrhiza glabra have shown to improve glucose tolerance andinsulin sensitivity. However, GA has not been studied for its effect on lipid metabolism. Therefore, this project was undertaken to study theeffect of GA on serum lipid levels and LPL gene expression in rats fed a combination of normal diet (ND) and high fat diet (HFD), simulatinghuman dietary patterns. Data showed all the rats were obese but no significant differences in weight gain were seen after 4 weeks.Normoglycemia were seen in rats given GA supplementation but were found to be hyperinsulimic and had decreased insulin sensitivity. Ratsgiven a ND the first fortnight with GA supplementation followed by a HFD on the second fortnight with water (Group C) and with the samediet given in Group C but with 4 weeks of GA supplementation (Group D) had normal triacylglyceride (TAG) levels. However, rats on 4weeks of HFD without GA supplementation (Groups A) and with GA supplementation on the second fortnight (Group B) werehypertriglyeridemic. Lipoprotein lipase (LPL) gene expression in the rats given GA for any period showed similar restricted tissue expressionprofile: high in subcutaneous adipose tissues (SAT), visceral adipose tissues (VAT) and heart, while low in abdominal muscle (AM),quadriceps femoris skeletal muscle (QFSM), kidney and liver. Results showed LPL gene expression in VAT and liver in rats from Group Bwere significantly lower compared to rats from Group A (p=0.000). The AM showed significant increased LPL gene expression (p=0.001).While rats from Group D showed significant higher LPL gene expression in AM (p=0.000), kidney (p=0.002) and liver (p=0.010) but lower inheart (p=0.033) than rats from Group C. In conclusion, it was found that with a HFD and GA supplementation at any period, hypretriglyceridemiacould be prevented at an early stage. However, high-fat feeding showed early signs of metabolic syndrome such as obesity, insulin resistanceand hypertriglyceridemia despite GA supplementation for any period of time. Moreover, GA seems to play a role in lipid metabolism,affecting the regulation of LPL gene expression in various tissues with respond to dietary patterns.


Poster 64

RADICAL SCAVENGING AND ANTIOXIDANT PROPERTIES OF THE EXTRACTS OFANACARDIUM OCCIDENTALE AND PIPER BETLE

NOOR NAZIRAHANIE ABRAHIM, M.S KANTHIMATHI AND AZLINA ABDUL AZIZ

Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur

In recent years, a number of studies have been carried out in order to determine the abilities of plants and herbs to scavenge free radicalattack. Malaysia is rich with its flora and fauna and there is great potential to discover novel compounds. The leaves of Anacardiumoccidentale and Piper betle have been selected to screen for their radical scavenging and reducing properties. Anacardium occidentale isalso known as ‘gajus’ and is eaten as ‘ulam’ among Malaysian. Meanwhile Piper betle or ‘sirih’ is popular among the Malay and Indianfolks. Both plants were extracted with hexane, ethyl acetate, methanol and water through a sequential extraction technique. The Folin-Ciocalteu Assay was used to determine the total phenolic content of each extract and results were expressed in mg Gallic Acid Equivalents(GAE)/g. The radical scavenging and antioxidant ability of the extracts were tested using the DPPH, FRAP and Superoxide Anion assays.Through the Folin-Ciocalteu Assay, the ethyl acetate extract of Piper betle showed the highest phenolic content with 852.3 ± 4.71 mgGAE/g. Followed by A.occidentale (methanol) (346.8 ± 4.84 mg GAE/g), P.betle (hexane) (266.9 ± 6.05 mg GAE/g), A.occidentale (ethylacetate) (112.9 ± 1.08 mg GAE/g), A.occidentale (water) (61.6 ± 3.49 mg GAE/g), P.betle (methanol) (52.3 ± 5.5 mg GAE/g), P.betle(water) (47.7 ± 5.38 mg GAE/g) and lastly A.occidentale (hexane) (5.4 ± 3.61 mg GAE/g). The FRAP value of each extract were in theorder of P.betle (ethyl acetate) (6.05 ± 0.1 mmole/g) > A.occidentale (methanol) (3.68 ± 0.14 mmole/g) > A.occidentale (ethyl acetate)(1.08 ± 0.02 mmole/g) > P.betle (hexane) (0.9 ± 0.006 mmole/g) > A.occidentale (water) (0.81 ± 0.02) > P.betle (methanol) (0.48 ± 0.01mmole/g) > P.betle (water) (0.35 ± 0.01 mmole/g) > A.occidentale (hexane) (0.02 ± 0.01 mmole/g). The ability of the extracts to scavengethe DPPH radical was determined through its IC50 value which was expressed in ug/ml. The IC50 values of each of the extracts were asfollows, P.betle (ethyl acetate) (29 ug/ml) < A.occidentale (methanol) (34.43 ug/ml) < A.occidentale (ethyl acetate) (131.65 ug/ml) <P.betle (hexane) (151.19 ug/ml) < A.occidentale (water) (192.83 ug/ml) < P.betle (methanol) (339.6 ug/ml). Results for the SuperoxideAnion Assay were expressed as IC50 value. Piper betle (ethyl acetate) showed the lowest IC50 value with 52.84 ug/ml while A.occidentale(methanol) with 63.22 ug/ml. The results demonstrated that the extracts of P.betle (ethyl acetate) and A.occidentale (methanol) have potentradical scavenging and antioxidant properties.

Poster 65

COMPARISON OF BIOACTIVE COMPOUNDS IN OF MEDICINE PLANT EXTRACTS

NORZAINAH AHMAD, NORDIANA AHMAD MAREKAN AND ISHAK MAT

Unit Kanser MAKNA-USM, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang

Catharanthus roseus (Cr), Pereskia bleo (Pb), Pereskia grandifolia (Pg) and Lingzhi spp have been used as traditional medicine bydifferent societies. Some of the effects may be associated with the presence of natural phytocompounds such as alkaloid or flavonoid. Thepotential health effects of flavonoid, alkaloid, and glycoprotein demand that methods be developed for their determination and quantificationin plant extracts. Cr and Lingzhi were selected since these species have also been commonly used in traditional medicine for theirpurported anti cancer and anti-inflammatory. The plants were extracted with water and ethanol. Flavonoid and alkaloid were analyzedwith HPTLC. Carbohydrates were extracted from Pereskia with 80% ethanol extract, heat and precipitate with ethanol. Alkaloidsseparated well in HPTLC analysis (solvent system chloroform-ethyl acetate) with vinblastine and vincristine as reference standards.Ethanol extracts of Pb contain high flavonoid; Cr extracts contain flavanol at Rf 0.56 with HPTLC (solvent system ethyl acetate: formicacid: glaciar acetic acid: water). Protein contents from Cr, Pb and Yellow Lingzhi in range 15 to 35 %. Hydrolysed polysaccharides fromPb and Pg analysed with HPLC [Refractive Index detector, Microsorb 100 C8 column (250x4.6 mm), mobile phase- water and flow rate0.5 ml/min] showed chromatogram at retention time in range from 5.5 to 5.6 mins with mannose, rhamnose, glucosamine and galactose asreference standards. Our investigation showed that Pb, Cr and Lingzhi spp contain beneficial flavonoids, in addition to the carbohydratesand other protein contents. Therefore this may contribute to the healths that are often traditionally associated with consumption ofproducts from these plant species.

Poster 66

SCREENING OF FRUCTOOLIGOSACCHARIDES (FOS) PRODUCING MICRO FUNGI USINGSUBMERGED CULTURES

NORAZIAH, A.Y.A, MASHITAH M.D.A*, AND NOORLIDAH, A.B

aSchool of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal, Penang,Malaysia, bInstitute of Biological Sciences, Faculty of Sciences, University of Malaya, 50603 Kuala Lumpur

Eleven isolates of micro fungi from the family of trichocomaceae, hypocreaceae, and moniliaceae were screened for fructooligosaccharides(FOS) yield using sugar cane syrups as a substrate. The best isolate Penicillium islandicum produced 65% (w/w) of FOS yield from 20%(v/v) of sugar cane syrups after 24 hours of reaction time. The extracellular fructosyltransferase (FTase) activity and biomass productionwere 5.54x102 IU/ml and 3.33x10-3 g/L, respectively. Then, this strain was cultivated batch-wise at variable concentrations of sucrose inorder to improve FOS yield. Under the best fermentation condition, FOS yield reached 97.8% (w/w) of sucrose conversion.


Poster 67

SCREENING OF ANGIOTENSIN CONVERTING ENZYME 1 (ACE) INHIBITORY ACTIVITY INSEARCH OF POTENTIAL ANTI-HYPERTENSIVE MUSHROOMS

NORHUDA MOHD ANSOR, NOORLIDAH ABDULLAH AND NORHANIZA AMINUDIN

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Lembah Pantai, Kuala Lumpur

In the old days, other than useful as food, mushrooms were also known to be useful in treating hyperglycemia, lowering blood pressure orhypertension and other illnesses. In this study, our aim was to investigate the anti-hypertensive potential of selected mushroom species.Mushroom fruiting bodies were extracted with hot water and the freeze-dried crude extracts were later subjected to AngiotensinConverting Enzyme (ACE) inhibitory activity assay. Substrates used were Hippuryl-Histidyl-Leucine (HHL) and N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly (FAPGG). IC50 values for respective mushroom species were 54.4 μg/ml (Pleurotus cystidiosus), 50.3 μg/ml (Pleurotusflorida), 66.8 μg/ml (Pleurotus eryngii), 56.2 μg/ml (Pleurotus sajor-caju), 57.6 μg/ml (Pleurotus flabellatus), 43.2 μg/ml (Agaricusbisporus) and 44.4 μg/ml (Ganoderma tsugae) with the presence of HHL as substrate. In the case of FAPGG, IC50 values were notdetermined, however, the pattern of inhibition percentage were similar to HHL. This shows that HHL is a more suitable substrate for theevaluation of ACE inhibitory activity for mushrooms. The IC50 results suggest that A. bisporus and G. tsugae are potentially goodcandidates to be developed further as anti-hypertensive agents. Studies are currently ongoing in order to determine the type of inhibitionand also to identify the bioactive compound(s).

Poster 68

NUTRIGENOMIC ANALYSIS OF CRUDE EXTRACTS OF TAMARINDUS INDICA

NURAHANANI RAZALI, AZLINA ABDUL AZIZ AND SARNI MAT JUNIT

Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia

Tamarindus indica L. or locally known as asam jawa grows naturally in many tropical regions. Various parts of the plant are used as foodcomponent and in herbal medicine. Studies have demonstrated the beneficial effect of the fruit of T. indica in reducing serum levels ofcholesterol and triglycerides when fed to animals. However, the mechanisms for the lipid lowering action of this plant have not beenelucidated. The aim of the study was to investigate the molecular mechanisms underlying the effects of the leaves and fruits of T. Indicaon lipid metabolism and oxidative stress in liver HepG2 cell lines using GeneChip microarray technique. HepG2 cells were treated withthe leave and fruit extracts of T. Indica. The experiments were carried out in triplicates. Gene expression microarray studies wereperformed on total RNA isolated from control and T.indica-treated HepG2 cells using Affymetrix Human Genome 1.0 S.T arrays. The datagenerated in this study were pre-analysed using the NetAffx Analysis Centre which correlates the GeneChip® array results with arraydesign and annotation information and finally refines the results by determining certain annotations of normalization control. The PartekGenomics Suite software was used to further analyse the data. A threshold of 1.5 was used to limit the data set to genes that areupregulated or downregulated by 1.5-fold or greater in order to identify differentially expressed genes. Comparisons between control andHep G2 cells treated with the leaves revealed approximately 988 upregulated and 1128 downregulated genes. On the other hand, in controland Hep G2 cells treated with the fruits, 590 upregulated and 656 downregulated genes were identified. Amongst the down-regulatedgenes are those involved in cholesterol synthesis and lipoprotein metabolism.

Poster 69

EXPRESSION AND CHARACTERIZATION OF SELECTED ESSENTIAL GENES ENCODED BYBURKHOLDERIA PSEUDOMALLEI (EXPRESSION AND CHARACTERIZATION OF SMPB IN

BURKHOLDERIA PSEUDOMALLEI)

NUR AAINA MOHD MOHAYADI1, AMIR RABU2 AND RAHMAH MOHAMED3

1School of Biosciences & Biotechnology, Faculty of Science & Technology; 2Malaysia Genome Institute,UKM-MTDC Smart Technology Centre, National University of Malaysia, 43600 UKM Bangi, Selangor

Burkholderia pseudomallei is a causative agent of meliodosis. B. pseudomallei K96243 had been completely sequenced but till now notmuch information is known about its protein and its interaction. It also have a high survivality rate in a stress condition but the mechanisminvolve in its fatal infection and how it remain quiescent in host for 62 years is still remain unknown. Thus, there is an urgent need tocreate an efficient vaccine against this diseases started by developing a research in order to understand its protein. This study involvecloning and expression of the essential gene of B. pseudomallei which are non-homolog to human based on the previous In silico analysisby Chong et al. which had identified 312 essential genes for Burkholderia pseudomallei survivality which may also become as a potentialdrug targets. One of the listed essential gene is SmpB. SmpB codes for Small protein B which is a unique protein that bind to tmRNA andform tmRNA-SmpB complex. TmRNA-SmpB complex released stalled ribosomes causes by lack of in frame stop codon in mRNA henceact as a quality control for translation process in cell. In this project, the gene had been amplified followed by cloning into cloning vector,TOPO TA and then expression vector, pET-28b. The recombinant plasmid was transformed into BL21 Star™(DE3) as an expression host.The expressed protein will be analyzed through SDS and Western Blot and the expression condition will be optimized and proteinpurification will be carried out.


Poster 70

EFFECT OF SITE-DIRECTED MUTAGENESIS ON PUTATIVE LIPASE LID

NUR HANA MD. JELAS1, RAJA NOOR ZALIHA RAJA ABD RAHMAN1, MAHIRAN BASRI2 AND ABU BAKAR SALLEH3

Department of Microbiology, 3Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,2Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Thermostable L2 lipase gene from Bacillus sp. L2 was mutated at its early N-terminal region. Changes of one amino acid producednoticeable difference in its total activity as in previous study, it showed that the lipase activity was increased in response to the mutation.Due to this, some changes in substrate selectivity of the mutated lipase were also expected as N-terminal region of a lipase protein wasassociated with the structure of its lid. However, the results revealed that the lipase retains its preference for hydrolysis of long chain fattyacids which save its properties of a true lipase with a random regio-selectivity. This shows that the amino acid where the point mutationhappened was not associated with the lid structure that determines the enzyme’s substrate selectivity.

Poster 71

CLONING AND EXPRESSION OF HEAT SHOCK SIGMA FACTORS FROMBURKHOLDERIA PSEUDOMALLEI

NUR HAZWANI HARMAN SHAH1, FARAH LIYANA ABD AZIZ1, RAHMAH MOHAMED1,2 AND AMIR RABU1

1Faculty of Biotechnology and Biosciences, Universiti Kebangsaan Malaysia,43600 UKM Bangi, Selangor, 2Malaysia Genome Institute

Corresponding author: [email protected]

Burkholderia pseudomallei is generally considered a saprophyte Gram-negative bacteria, causes the infectious disease melioidosis that ismostly restricted to Southeast Asia and Australia. From in silico analysis, rpoE and rpoH have identified as essential genes inB. pseudomallei and also non homologous to human. These two genes encoded alternative sigma factor, σE and σ32 respectively which areinvolved in transcription of heat shock genes and extracytoplasmic function. The rpoH and rpoE were amplified from B. pseudomalleistrain D286 and cloned into cloning vector (TOPO TA) and later subcloned into expression vector (pET28b). The genes were expressedfor 1 hours (rpoH) and 4 hours (rpoE) in Escherichia coli BL21 (DE3) Star after induction with 1mM IPTG at 37°C. Expression yieldsproteins with size of 34.4 and 22.5 kDa for rpoH and rpoE respectively which can be visualized by Coomassie staining. Next, rpoH andrpoE proteins were detected on nitrocellulose membrane during Western Blot after 2 hours incubation with monoclonal anti-polyHistidine-Alkaline Phosphatase which specifically bind to 6X His-Tag on target protein The protein will be undergoing purification using Ni-NTAaffinity column chromatography.

Poster 72

IMMUNOLOGICAL ASSESSMENT OF DNA VACCINE AND RECOMBINANT BCG (rBCG) VACCINEAGAINST ENTEROVIRUS 71 INFECTION

NUR AYUNI KADIR1, NURULHASANAH OTHMAN2, MUSTAFFA MUSA3 AND ZAINUL F. ZAINUDDIN4

1Faculty of Medicine & Health Sciences, Universiti Darul Iman Malaysia, Kota Campus, 20400 Kuala Terengganu, Terengganu.2Institute for Research in Molecular Medicine (INFORMM), 11800 Univesiti Sains Malaysia, Pulau Pinang.

3School of Medical Sciences & 4School of Health Sciences, Health Campus, Universiti Sains Malaysia,16150 Kubang Kerian, Kelantan

Enterovirus 71 (EV71) is a highly infectious causative agent of hand, foot and mouth disease (HFMD) in children and could lead to severeneurological complications. In Malaysia, the first epidemic occurred in 1997 in Sarawak and caused 34 deaths due to severe neurologicalsyndrome. There is currently no vaccine available against EV71. Therefore, vaccination is considered the most effective means to controldisease outbreaks. Our group successfully constructed two candidate vaccines in two different formats which are DNA vaccine (namelypVaxUbVP1) and recombinant BCG vaccine (namely rBCGV1). Synthetic gene encoding the VP1 gene of EV71 fused to ubiquitincomplex (UbGR) was constructed using assembly PCR technique. UbGR-VP1 synthetic gene in DNA vaccine system was codonoptimized for expression in E. coli and Salmonella whereas rBCG1 was codon optimized for expression in mycobacterium. Immunogenicityof both candidate vaccines were performed in BALB/c (H-2d) mice. The results indicate that IgG2a subclass antibody was significantlyhigher compared to IgG1 in both candidate vaccines when tested against purified UbGR-VP1 fusion protein. Splenocytes obtained frompVaxUbVP1 immunized mice showed higher level of lymphocyte proliferation compared to splenocytes from rBCGV1 immunized mice.Analyses of intracellular cytokines show that CD4+ and CD8+ T cells from pVaxUbVP1 and rBCGV1 immunized mice were stimulated byUbGR-VP1 protein to express significant levels of IFN- γ, IL-2 and IL-4 when compared to control. Data from this study suggested thatboth candidate vaccines enhanced the stimulation of immune system towards the T helper 1 (Th1) pathway. In conclusion, both candidatevaccines are immunogenic in mice and further study should be carried out in order to evaluate the efficacy of these candidate vaccines.pVaxUbVP1 and rBCGV1 show potential for further development as a vaccine against EV71.


Poster 73

EXPRESSION OF AN OIL PALM FATB THIOESTERASE GENE PROMOTER IN ARABIDOPSIS THALIANA

NURHAFIZAH R., ABRIZAH O., MOHAMAD ARIF A.M. AND MAHADZIR J.

Malaysian Palm Oil Board, P.O Box 10620, 50720 Kuala Lumpur, Malaysia

Acyl-acyl carrier protein (ACP) thioesterase catalyses the hydrolysis of the acyl-ACP thioester to free fatty acid and ACP commonly from a growingacyl chain of 16 or 18 carbons. Work on an oil palm FatB type acyl-ACP thioesterase gene, designated EgFatB, yielded information on its promoterregion. The thioesterase promoter has great potential significance in the expression of desirable foreign genes in the developing seed tissue of oilpalm. In order to investigate the expression of the cloned thioesterase gene, the upstream region of the gene, which estimated to contain all regulatoryelements, was fused to a β-glucuronidase (GUS) reporter gene. The resulting construct was designated pCB2TP and introduced into Arabidopsisthaliana via floral dip transformation. Transgenic plants containing pCB2TP construct were selected by spraying a solution of Basta (glufosinate)herbicide. Twelve individual Basta resistant plants from T3 homozygous lines were obtained. Seeds of these plants were analysed for fatty acidcomposition by gas chromatography. The palmitate (C16:0) level was increased compared with the wild type A. thaliana.

Poster 74

P53 CODON 72 POLYMORPHISMS OF NON-MELANOMA SKIN CANCER FROM THE ARCHIVALFORMALIN-FIXED PARAFFIN-EMBEDDED TISSUES AMONG THREE MAIN RACES IN MALAYSIA

NURUL SYAKIMA AB MUTALIB1, CHEAH YOKE-KQUEEN1*, SHIRAN MOHD SIDIK2, LEE LEARN-HAN1,TAN GEOK-CHIN3 AND HAYATI ABDUL RAHMAN3

1Department of Biomedical Sciences, 2Department of Pathology, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor. 3Department of Pathology, Faculty of Medicine,

Hospital Universiti Kebangsaan Malaysia, Jalan Ya’acob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur

Non-melanoma skin cancer (NMSC) is classified among the ten most frequent cancers in Malaysia according to National Cancer Registry 2003and it is divided into two histologic types; basal cell carcinoma (BCC) which is the most frequent and squamous cell carcinoma (SCC). Acommon polymorphism at codon 72 located at exon 4 of p53 tumor suppressor gene has been reported to be associated with increasedsusceptibility to several cancers and its influence on cancer risk has been studied for different types of cancer with mixed and inconsistent results.With respect to sporadic NMSC, there are few studies, with small sample sizes, and no publication on Malaysian population so far. In this presentstudy, the frequency of p53 codon 72 polymorphism in 60 patients with NMSC was investigated from the archival formalin-fixed paraffin-embedded (FFPE) tissues. FFPE tissues of NMSC were obtained from the period of 2003 to 2006 from HUKM. An optimized extraction methodwas developed for the DNA isolation from the archival FFPE tissue. Touchdown PCR was performed to detect desired polymorphisms. Seventypercents of NMSC FFPE possesses Arg/Arg, twenty percents with Pro/Pro and ten percents with Arg/Pro. In total, there was no significantdifference in the p53 codon 72 genotypes between BCC and SCC when tested with Chi-square test (SPSS 14.0, P=0.05). Besides that, among theChinese, 72.2% possessed Arg/Arg, 19.4% with Pro/Pro and 8.3% possessed Arg/Pro were observed. On the other hand, Malay exhibited 61%with Arg/Arg, 27.8% for Pro/Pro and the rest 11.1% were Arg/Pro. Among Indian, eighty percents were Arg/Arg, 20% were Arg/Pro and nonewas Pro/Pro. No significant difference in the p53 codon 72 genotypes in regard of gender (P=0.219), race (P=0.771), tumor location (P=0.490)and age group (P=0.235). However, there was an apparent age-associated increase in the Arg/Arg genotype but statistically not significant. Inaddition, Random Amplified Polymorphic DNA analysis with three selected random primers (i.e. OPO-09, OPO-12 and OPO-20) were appliedto this study to further clustered the samples for preliminary biomarker search. In conclusion, NMSC histologic types and demographiccharacteristics did not affect genotypes distribution but BCC and SCC distributions were influenced by age group, race and tumor location.

Poster 75

HUMANIZATION OF MURINE MONOCLONAL ANTI-CEA(ANTI-CARCINO EMBRYONIC ANTIGEN) ANTIBODY

NURULFARHANA HUSSIN, HEILLY CHONG, NURUL ATIQAH ALI AKHBAR, KHAIRUL EZANI, JONG HANG SIONGAND ZULKEFLIE ZAMROD

Department of Protein Science, Inno Biologics Sdn Bhd. Lot 1, Persiaran Negeri BBN, Putra Nilai, 71800 Nilai, Negeri Sembilan

The potential of using monoclonal antibodies (mAbs) that specifically target tumor antigens for in vivo diagnosis of various cancers is beingsteadily realized. These mAbs could be conjugated to radio-nuclides making it possible to image the tumor as well as other metastasizingtissue or cells. mAbs have also been found to be useful in experimental therapies such as treatment of malignancies and immmunosuppression.Majority of the mAbs used in these classes involved the use of rodent antibodies. However, there’s one common issue arises from this modeli.e. the mAbs is limited by themselves as a result of becoming a target of the host’s immune system (human/patient) by provoking an anti-globulin response or commonly known as HAMA (human anti-mouse antibody) response. These antibodies that triggered the HAMAresponse will be immediately cleared from the host’s system. The success of using antibodies for in vivo tumor-targeting and therapy isdependent upon the uptake of the antibody molecule (penetrability) by the tumor tissue and the rate of clearance of the antibody by the bodyimmune system. Therefore, in order for the mAb to be efficient, it must minimize its HAMA response and stays in the host’s body longenough for it to be useful. With the advances of recombinant technology, it is now possible to generate rodent antibody and “humanized”them for use in human. These, “humanized antibodies” retain the specificity of the rodent antibodies, posses a human constant regions whichmay interact with human effector mechanism more effectively and in addition present less of a HAMA response from the patient. In thisstudy, we will be describing the humanization of a murine anti-CEA (anti-carcinoembryonic antigen) antibody through a string of molecularbiology and bioinformatics process. The end product is a humanized anti-CEA antibody which could be useful in the in vivo imaging ofcolorectal cancer. This R&D program proposes to make available to Malaysians a diagnostic technology for imaging cancer foci using one ormore tumor antigen-binding proteins labeled with a radio-nuclide or with other energy-emitting immunoconjugates or immunotoxins (fortherapy). The project will be a concerted effort by researchers from 3 organizations, viz., Inno Bio, Agensi Nuklear Malaysia (previouslyknown as MINT) and UMBI (HUKM) encompasses immunologists, molecular biologists, radio-pharmacists, nuclear medicine physicians,clinical oncologists and process development and bio-manufacturing engineers. The project also covers technology development for thegeneration of labeled antibodies for in vivo diagnosis and monitoring of cancerous cells and tissue. This is a primer to the larger scope ofbuilding local capability in commercial development of antibodies for therapy of cancer and autoimmune disease in humans.


Poster 76

CYTOTOXICITY EFFECT OF PURE COMPOUNDS ON HSC-2, K-562, MCF7 AND NCI-H23 CANCERCELL LINES

ONG WEI TEING1, LIM CHAN KIANG1 AND LIM YANG MOOI1*

1Department of Bioscience and Chemistry, Faculty of Engineering and Science,Universiti Tunku Abdul Rahman, Jln. Genting Klang, Setapak, 53300, Kuala Lumpur, Malaysia

*Corresponding author: [email protected]

Contrary to accepted belief, cancer is not a new disease, which has appeared with modern civilization. Cancer statistics are extremelyshocking nowadays and affects one in three people. Current treatment of cancer botched to fully deal with the nuisance of cancer, and thisinstigate the enthusiasm to discover anti-cancer drugs with better efficacy. In this study, the cytotoxicity activity of pure and partiallypurified compounds isolated from local plants were evaluated against various cancer cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. The cancer cell lines used were HSC-2, K-562, MCF7 and NCI-H23 cell lines. The purecompounds used included friedelin, friedelan-1,3-dione, fraxidin, catechin, dimethylmangostin, 5’-demethoxycadensin G, rubraxanthoneand pinocembrin. Compounds were tested at the concentrations of 50μg/ml, 25μg/ml, 12.5μg/ml, 6.25μg/ml, 3.13μg/ml, 1.56μg/ml, and0.78μg/ml via seven-point serial dilution of MTT assay, in order to ascertain the IC50 value of each compound which exhibited significantcytotoxic effects. From the results obtained, rubraxanthone suggested to reveal promising cytotoxic effect against NCI-H23 cancer cellline with recorded IC50 value of 4.25μg/ml. In addition, catechin and pinocembrin also showed strong and selective cytotoxicity effects.The results obtained suggest the potency of compounds isolated from local plants as anticancer agents.

Poster 77

ISOLATION OF DIFFERENTIALLY EXPRESSED GENES IN PINEAPPLES USING DIFFERENTIALDISPLAY REVERSE TRANSCRIPTION –POLYMERASE CHAIN REACTION

ONG WEN DEE AND VIJAY KUMAR

Biotechnology Research Institute, Universiti Malaysia Sabah, Locked Bag No. 2073, 88999 Kota Kinabalu, Sabah

The mechanism involved in gene expression by mRNA Differential Display RT-PCR has been extensively used for the isolation ofdifferentially expressed genes between RNA populations. This study describes the use of Differential Display Reverse Transcription-PCR(DDRT-PCR) to screen for differently expressed genes in ripe and unripe pineapple fruits. Total RNA was isolated using phenol/chloroform method, purified and reverse transcribed using three different two-base anchored oligo dTs primers (dT12GG, dT12GC, dT12GT)to bind to the mRNA poly A tail. The resulting cDNAs was then used as templates for DD-PCR with the combination of oligo dTs andthree different 10-mer arbitrary primers (5’-CAGCCCAGAG-3’, 5’-TGAGCGGAGA-3’, 5’-GGGTAACGCC-3’). DDRT-PCR productswere then separated on a 6% non-denaturing polyacrylamide gel. Differentially expressed genes in the ripe and unripe pineapple fruit wereidentified based on the banding profiles. The differently expressed bands will be excised, re-amplified, sequenced and screened forfunctional properties using bioinformatics tools.

Poster 78

LETHAL MOUSE MODEL EVALUATION OF PROTECTION AGAINST CONGENITALTOXOPLASMOSIS WITH RECOMBINANT SAG2 PROTEIN IN GUINEA PIGS

WAN OMAR, A., NGAH ZASMY, U., HAIRUL BAZLI, H, MALINA, O, RUKMAN, AH, *WAN ZAIDAH, A.

Medical Parasitology Unit, Department of Microbiology and Parasitology, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, Serdang 43400, Selangor Darul Ehsan; *Department of Haematology, School of Medical Sciences,

Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan

Primary infection with Toxoplasma gondii during pregnancy can induce fetal pathology and abortion both in human and animals. Thepresent study describes the development of an experimental model of congenital toxoplasmosis in guinea pig. We have recently cloned,expressed and purified a recombinant protein SAG2 in Pichia pastoris. SAG2 of T. gondii is a major surface protein known as anattachment ligand that also has good antigenicity and immunogenicity. The infectious status of pups delivered from guinea pigs wereevaluated in a lethal mouse model. Guinea pigs were immunized subcutaneously with recombinant SAG2 in alum. As a negative control,alum alone was injected. Mean titers of antibodies at the moment of delivery, correspond respectively to 9 to 10 weeks after the thirdSAG2 injection and 2 to 3 weeks after parasite infection. Anti-T. gondii antibody titers increased markedly in both the vaccinated andinfected groups. A greater mortality was observed in litters from adjuvant immunized mothers (n= 10) whereby 21 pups were born, ofwhich 9 were retrieved from dead mothers and 15 were stillborn. In contrast, out of 24 pups derived from SAG2 vaccinated group (n=10),only 3 and 6, respectively, were retrieved from dead mothers or stillborn. More than eighty percent (83.3%) of the stillborn pups from thenon-vaccinated group were infected with tachyzoites whereas the incidence of T. gondii infection among still born pups from the SAG2-vaccinated group reached only 22.2 % (Fisher’s test, P < 0.01). In both the groups, none of the pups retrieved from dead mothers wereinfected. In summary, this work demonstrates that the guinea pig model is suitable for studying congenital toxoplasmosis and for testingprotective effect of vaccine candidates against maternofetal transmission.


Poster 79

CONSTRUCTION OF A PLANT-BASED VACCINE DELIVERY VECTOR FOR CANDIDATE GENES OFAVIAN INFLUENZA VIRUS, STRAIN H5N1

PUA TEEN LEE1, LOH HWEI SAN1, FESTO MASSAWE1, TAN CHON SENG2 AND ABDUL RAHMAN OMAR3

1School of Biosciences, Faculty of Science, The University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor.2Biotechnology Research Center, Malaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor.

3Institute of Bioscience, University Putra Malaysia, 43400 UPM Serdang, Selangor

Avian Influenza (AI) virus, strain H5N1, causes a serious infectious disease that affects the respiratory, digestive and nervous system of birdsresulting into a high degree of morbidity and mortality. The H5N1 strain is highly virulent and considered as a zoonotic pathogen. Accordingto WHO (2008), the disease caused by this virus has resulted in millions of dead animals, including 243 human deaths globally. There istherefore an urgent need to develop a strategy to control the AI due to the grave difficulty in managing the pandemic potential and social-economic impacts, especially in the largest outbreak area of Southeast Asian countries. Vaccination is the best strategy for managing thedisease and its eventual eradication. This research seeks to use plants as a biofactory to develop a delivery system of vaccine for a locallyisolated strain of AI virus. The local isolate of AI virus and the other isolates found in Southeast Asia, including those reported in Hong Kongand Thailand, have nearly identical protein sequences. It is therefore hypothesized that the local strain will potentially provide higher level ofimmunoprotectivity against the Southeast Asian AI disease. Plant systems have been used to produce several recombinant proteins due to theirgreat advantages over other competing systems such as microbial fermentation and mammalian cell culture. The plant system has lowproduction cost, it is easy to scale-up, and has low risk of contamination with animal pathogens. In the present study, we describe theconstruction of AI genes, namely the haemagglutinin type 5 (HA) and neuraminidase type 1 (NA) into Potato Virus X (PVX)-basedexpression binary vector, pGR107, which is driven by a 35S promoter and terminator with a right and left border. A full-length of HA and NAgenes were firstly identified and separately cloned into the pGR107 vector through ClaI and SalI restriction cutting sites. Polyhistidine-tag(His-tag) was incorporated into C-terminal of both genes to ease purification of expressed recombinant proteins. The constructed vectors wereverified and transformed into Agrobacterium tumefaciens LBA 4404. In this study, tobacco leaves were infiltrated with Agrobacteriumharbouring the constructed vectors carrying the individual HA and NA genes. Once the successful expression of recombinant proteins intobacco is confirmed, further optimization for higher level of expression will be pursued. This study provides great opportunity for theexploration of plant-manufactured AI viral proteins which could potentially be pursued for the production of edible vaccine.

Poster 80

THE EFFECT OF CATHARANTHUS ROSEUS EXTRACTS ON THE PROTEIN PROFILE ANDCYTOTOXICITY ACTIVITY OF HUMAN COLORECTAL CANCER CELLS HT-29

RAFEDAH ABAS, ADILAH ABDUL KHALIL, NORHIMAN AHMAD AND ISHAK MAT

Advanced Medical and Dental Institute, EUREKA complex, Universiti Sains Malaysia, 11800 USM, Penang

Colorectal cancer is the third commonest cause of cancer in both males and females in Malaysia. There are many plant derived products, such astaxol, vincristine and vinblastine that have been used as chemotherapeutic agents to inhibit or kill the cancer cells. A number of techniques, suchas flow cytometry, HPLC or mass spectrometry, have been used to assess the proteomic changes of the target cells for these products. In thisstudy, we have used Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique in combination with densitometry toassess the preliminary changes in protein expression of a colon carcinoma cell line, HT-29, treated with extracts of C. roseus. For the study,different preparations of the leaves were processed using the standard extraction procedures. The cytotoxic effect of extracts were tested on theHT-29 cells using MTS assay while the SDS-PAGE in combination with densitometry technique were used to assess changes in protein profile.Ethyl acetate and n-butanol extracts showed moderate cytotoxicity effect towards HT-29 cells with IC50 values of 48.2 μg/mL and 67.6 μg/mL,respectively. The HT-29 cells treated with C. roseus extracts were lysed and proteins were extracted and subjected to SDS-PAGE. The similaritiesand differences of the protein profiles were analysed by bioimaging system using GeneTool analysis software. The results from the densitometricanalysis showed that most of the protein levels of HT-29 cells treated with ethyl acetate extract were reduced when compared to cell extracts fromuntreated cells. In conclusion, the use of the SDS-PADE/densitomeric technique is able to detect variation in protein levels in the target HT-29cells in response to C. roseus extracts. However, further studies need to be carried out using 2D polyacrylamide gel electrophoresis to obtain fullproteome of HT-29 cells in order to study the differential expression of protein following treatment of C. roseus extracts.

Poster 81

THE EFFECT OF MITRAGYNA SPECIOSA IN CARRAGEENAN-INDUCED INFLAMMATORY PAINTEST IN RATS

RAJA ELINA RAJA AZIDDIN, MUSTAFA ALI MOHD, ZAHURIN MOHAMED AND MOHD RAIS MUSTAFA

Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur

The species Mitragyna speciosa is endemic to Southeast Asia. In Malaysia it grows in the northern states of Kedah and Perlis as well as in theEast coast states and is commonly known as Ketum or Biak by the local Malays. The interest to study the analgesic property of the Mitragynaspeciosa from Malaysia is based on previous scientific research data which have shown promising evidence of the analgesic properties of theThai species. The effect of Mitragyna speciosa extracts on persistent pain was studied using an animal model of inflammatory hyperalgesia.Carrageenan-induced inflammatory pain test in rats is a well-known pain test for assessing the thermal hyperalgesia after carrageenan-inducedinflammation. Injection of Carrageenan into rat hind paws display a spontaneous pain behavior. In response to a noxious thermal stimulus, thewithdrawal latency of the carrageenan-injected, inflamed hindpaw is significantly reduced compared with the contralateral, saline treatedpaws which remains unchanged and is used as a control. This is consistent with the induction of thermal hyperalgesia by carrageenan. Thismodel mimics more closely the time course of postoperative pain or other types of persistent injury. Mitragyna speciosa alkaloids at 30 mg/kgwere able to relieve thermal hyperalgesia similar to Aspirin at 20 mg/kg. The effect was significant by 1 hour and reached a maximum by 3hours. After 4 hours, its effect decreased and was shown as an increase in paw withdrawal latency.


Poster 83

TWO-DIMENSIONAL GEL ELECTROPHORETIC PROFILING OF LOW ABUNDANCE SERUM PROTEINS OF PATIENTSWITH NASOPHARYNGEAL CARCINOMA WHO TYPES 2 AND 3

RAMARAO SERIRAMALU1, PUTERI SHAFINAZ ABDUL RAHMAN1, ANITA ZARINA BUSTAM2,ALAN KHOO3 AND ONN HAJI HASHIM1

1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur.2Clinical Oncology Unit, University of Malaya Medical Centre, 50603 Kuala Lumpur.

3Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur

Nasopharyngeal carcinoma (NPC) is a disease not easily detected at early stages and not often treated by surgery. The search for reliablebiomarkers to detect NPC is ongoing. Using gel-based proteomic studies, we have previously profiled the high abundance proteins inserum samples of patients with WHO type 3 NPC and shown the up-regulated expression of ceruloplasmin compared to those obtainedfrom normal healthy individuals. In the present study, we have analysed the expression of serum proteins of lower abundance bysubjecting serum samples to albumin and IgG depletion, prior to analysis by 2-dimensional gel electrophoresis (2-DE). The 2-DEprofiling studies were performed on three cohorts of serum samples, i.e., those obtained from patients with WHO type 3 NPC, patientswith WHO type 2 NPC and age-matched normal healthy individuals. Our data demonstrated that the three cohorts of serum samplesstudied demonstrated different altered expression of several low abundance proteins.

Poster 84

EFFECT OF 1-METHYLCYCLOPROPENE (1-MCP)TREATMENT ON EKSOTIKA PAPAYA FRUIT SOFTENING

RAZMAN KOSNIN, ZAINON MOHD. ALI AND ROOHAIDA OTHMAN

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,43600 UKM Bangi, Selangor

Applications of 1-methylcyclopropene (1-MCP), an ethylene antagonist, have been used on Eksotika papaya (Carica papaya L. cv.Eksotika), a climacteric fruit, to study the softening related changes during ripening at ambient temperatures. Preliminary experimentsshowed that 1-MCP treatment at 90ppb on 5% yellow(Y) was more promising than the 25%Y fruit in extending the fruit ripening periodand maintaining the fruit quality and firmness until 100%Y stage. 1-MCP treatment delayed skin color development of 5%Y fruit by about5 days and slowed down the fruit softening. Effect of the treatment on three of the dominant softening enzymes activities ie.α-galactosidase, β-galactosidase and pectin methylesterase were also studied. 1-MCP treatment significantly delayed the increase in thesethree enzymes activities and appeared to correlate positively with the decreasing in tissue firmness particularly the β-galactosidase activity.Results of this study suggest that 1-MCP treatment delays Eksotika papaya fruit softening by suppressing the cell wall degrading activitiesthat contributes in maintaining the fruit quality.

Poster 85

IDENTIFICATION OF CHEMICAL COMPOUNDS IN THE CATHARANTUS ROSEUS LEAVES BYHIGH PERFORMANCE LIQUID CHROMATOGRAPHY COUPLE WITH TIME OF FLIGHT MASS

SPECTROMETRY

ROHANIZAH ABDUL RAHIM1, RAFEDAH ABAS1, SYAZWANI ISMAIL1, NORHISHAM SHUKUR1,NORHIMAN AHMAD1 AND ISHAK MAT1

1Unit Kanser MAKNA-USM, Advanced Medical & Dental Institute, Suite 121 & 141,EUREKA Complex, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

Natural products plants have been widely used as folkloric medicines in different societies. Many efforts have been used to developspecific analytical methods for identifying those chemical compounds. Catharanthus roseus became one of the best studied medicinalplant because it contains two alkaloids, vincristine and vinblastine, that have found wide application as anti-cancer compounds. In thisstudy we have used HPLC technique to fractionate the first four fractions of the aqueous extracts of the Catharanthus leaves. Thesefractions were subsequently subjected to LCtof/MS analysis to obtain the molecular formula of the extracts and the identification of thepossibilities of the structure of the chemical compounds was done by chemspider software based on the monoisotopic mass and calculatedmass. One of the fraction was found to have at least six compounds. Subsequently, this fraction will be analysed by NMR and CHNanalysis in order to confirm the structure of the compounds.


Poster 86

LEUCAENA LEUCOPHALA AS A POTENTIAL ANTI DIABETIC FUTURE DRUG

ROSMIYANI SAHDOM1, FUZIAH LATIF2, CHEE BENG GIN3,AND SYAKIRA M HUSSEIN1

1Faculty of Health and Life Sciences, Management and Science University, 40100 Shah Alam, Selangor,2Hospital Tapah, Ipoh, 3Forest Research Institute(FRIM)

Leucaena leucocephala seeds (petai belalang) has been used by a portion of people in the world as a folks remedy for a personal antidiabetic control, regardless of its use as a coffee substitute in certain regional areas. This tropical fodder species are easily found growingin secondary forests, cleared lands and also bushes, mostly as cattle feeds. It has been found that it is quite well known with 72% ofMalaysian are aware of this seeds and are also consuming them as a blood sugar control. These days, more consumers are consideringalternative and natural product medicine. It showed that consumption of this seeds is relatively the same in rural and urban areas.Ironically, the seeds are mostly grilled, 50%, and others are either taken fresh or boiled. Many agree that it has an acceptable and bettertaste in comparison to other bitter taste of anti diabetic herbs, with a slightly sweet taste and pungent smell. Survey shows that nearly100% people whom have consumed this seeds reported no side effects with a majority intake at a frequency of once a month. More than5% feels that the seed has the same function as a diabetic drug and may be used as an alternative in future. It is assumed that takingleucocephala at an early age could prevent diabetes during middle ages due to its blood glucose lowering properties. However, researcheswith a high interest on these potential seeds are encouraged to comprehend a scientific testing involving efficacy and toxicity. This is dueto the presence of mimosine toxicity, a substance causing hair loss and stomach problems with high intake of the seeds.

Poster 87

PROBING UREA AND GUANIDINE HYDROCHLORIDE DENATURATION OF BOVINE SERUMALBUMIN WITH ERYTHROSIN B BINDING

ADYANI AZIZAH ABDUL HALIM, HABSAH ABDUL KADIR AND SAAD TAYYAB

Biomolecular Research Group, Institute of Biological Sciences, Faculty of Science,University of Malaya, 50603 Kuala Lumpur

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were studied using erythrosin B (ErB) bindingas a probe. A significant red shift of 12 nm along with increase in absorbance at λmax were noted in the absorption spectrum of ErB(10 μM) upon addition of 10 μM BSA. The absorption difference spectrum, produced in the wavelength range, 450-600 nm wascharacterized by the presence of a maximum at 545 nm and two minima around 448 and 522 nm. Addition of urea/GdnHCl to ErB-BSA(1:1) mixture produced a change in the absorption difference spectrum. The magnitude of absorption difference (ΔAbs.) at 545 nm showeda progressive decrease on increasing denaturant (urea/GdnHCl) concentrations and followed the denaturation curve. The urea denaturationwas found to be a three-state, two-step transition with the accumulation of an intermediate around 4.7-5.0 M urea concentrations.Similarly, GdnHCl denaturation also showed the presence of an intermediate around 2.25-2.6 M GdnHCl concentrations. However, both(urea and GdnHCl) transition curves were found to be different in terms of start-, mid- and end-points of transitions. The value of freeenergy of stabilization, GD

H2O as determined from urea/GdnHCl denaturation curves was similar to those reported for a number ofproteins. Taken together, these results suggest that ErB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.

Poster 88

PHENOTYPIC EFFECT OF AN OIL PALM STEAROYL-ACP DESATURASE INARABIDOPSIS THALIANA

SAFIZA, M.1, ABRIZAH, O.1, SITI NOR AKMAR, A.2 AND MOHAMAD ARIF A. M.1

1Malaysian Palm Oil Board, P.O. Box 10620, 50720 Kuala Lumpur, Malaysia. 2Department of Agriculture Technology, Faculty ofAgriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Analysis of transgene expression in transgenic plants is essential for evaluating the efficiency of a transformation system and functionalityof the transgene expression in the plant cells. Our effort was to study the over-expression of oil palm stearoyl-ACP desaturase (SAD1)gene in Arabidopsis thaliana var. Columbia by phenotypic analysis. These transgenic Arabidopsis plants were transformed with geneexpression vectors containing cDNA encoding SAD1 (with either plastid targeting sequence or mature protein) and phosphinothrycinacetyl transferase (bar) gene as a selectable marker. The stability of the inserted SAD1 gene in transgenic Arabidopsis was confirmed byPolymerase Chain Reaction (PCR) using gene specific primers. Lipids from green leaf of wild-type and T3 transgenic Arabidopsis wereanalyzed via fatty acid analysis using Gas Chromatography. Growth development analysis was conducted through quantitative analysis onroot growth and hypocotyl length. The PCR amplified fragments were identical in size to SAD1 gene fragments. Over-expression effectswere detected in the transgenic Arabidopsis plants with plastid targeting sequence (either from vector or SAD1 gene) by its fatty acidcomposition. However, the over-expression does not affect the growth development of the transgenic Arabidopsis plants.


Poster 89

CONSTRUCTION OF A BURKHOLDERIA PSEUDOMALLEI MUTANT AUXOTROPH FOR THESHIKIMATE PATHWAY

SAM KIN KIT1, RAHMAH MOHAMED1, 2 AND SHEILA NATHAN1,2

1Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, 2Malaysia Genome Institute, Heliks EmasBlock, UKM-MTDC Smart Technology Centre, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia

Burkholderia pseudomallei is a Gram negative bacterium. This bacterium is the causative agent for melioidosis, an endemic disease inSouth East Asia and Northern Australia. To date, there is no vaccine for melioidosis and B. pseudomallei is resistant to most antibiotics. Asthis pathway is absent in humans, this makes it an interesting target for both vaccine and drug development. Numerous studies otherbacteria have suggested that the shikimate pathway has high potential as a vaccine target. The objective of this project was to create aB. pseudomallei mutant auxotrophic for the shikimate pathway. The gene encoding the final enzyme from the Shikimate pathway, aroCwas cloned in two fragments into the suicide plasmid pSM430. The two fragments were cloned flanking two reporter gene, lacZ and CatR.The clone pSM_aroC was electroporated into B. pseudomallei clinical isolate R15/05. Transformants were prepared for homologousrecombination and mutants were selected on agar with chloramphenicol (150 μg/ml), sucrose (15%) and X-gal supplemented with thethree aromatic amino acid phenylalanine, tyrosine and tryptophan. Further analysis on selected colonies is underway to confirm the loss ofability to complete the shikimate pathway.

Poster 90

HETEROLOGOUS EXPRESSION OF TRIOSE PHOSPHATE ISOMERASE ENZYME FROM ANANTARCTIC BACTERIUM

LIEW YU CHENG, SEE TOO WEI CUN, AND FEW LING LING

School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.Email: [email protected]

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesizecold-adapted enzymes. Psychrophilic enzymes produced by the cold-adapted microorganisms usually display higher catalytic efficiencyand thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. Our collection of cold-tolerantmicroorganisms isolated from the Antarctic region has offered a potential source for psychrophilic enzymes. Previously our group hadsuccessfully cloned the open reading frame for triose phosphate isomerase (TIM) gene from an Antarctical bacterium known as phi9. TheORF was cloned into a pET-14b plasmid. The full length TIM protein was subsequently expressed in E. coli BL21(DE3) strain, purified asHis-tag protein and confirmed to be catalytically active. Results showed that induction with 0.25 mM of IPTG yielded more solublefraction of TIM. Under this concentration of IPTG induction, roughly 90% of soluble TIM was produced while 3 hours of induction timeat room temperature (28°C) was the best condition for the expression and solubility of this protein. This work laid the foundation forfurther biochemical and structural characterizations of TIM from a psychrophilic bacterium by providing a highly purified recombinantprotein sample.

Poster 91

VARIATIONS IN THE QUANTIFICATION OF GENISTEIN DURING WAVELENGTH SHIFT

SENTHILKUMAR SUBRAMANIAN, NURHANANI RAZALI,AZLINA ABDUL AZIZ AND SARNI MAT JUNIT

Department of Molecular Medicine, Faculty of Medicine, University of Malaya,Kuala Lumpur 50603, Malaysia

Genistein is an important isoflavone aglycone, naturally occurring plant substance, which has high potential in the prevention ofatherosclerosis, and cardiovascular diseases. Despite these beneficial effects, adverse effects of genistein were also observed. Thyroidenlargement in rats and humans, especially children and women, fed with soybeans (that are known to have high levels of genistein), hadbeen reported. A wide range of analytical techniques has been applied for the determination of genistein in foods and biological materials.Selection of optimum wavelength (λmax) is an important factor during the quantification of the compounds. Deviation from the λmax, affectsthe quantification of the compound. The present study is designed to document the percentage variation in the exact quantity of thegenistein, when deviating from the λmax. Genistein (≥98% purity, Sigma) was quantified through HPLC (NovaPak reversed phase C18

column; acetonitrile and water with trifluoroacetic acid, pH 2.5; 20 - 40% acetonitrile in 20 mins; 0.5 ml/min), at various wavelengths,between 240 & 280 nm. Genistein showed maximum peak height at 260nm and decreased on either side of the wavelength. Also,quantification through peak area showed its maximum at 260nm, which dropped on both sides. This confirmed that genistein has λmax at260nm and the quantification was affected by deviating from 260nm.


Poster 92

THE EFFECT OF BERBERINE ON PLASMODIUM LACTATE DEHYDROGENASE (pLDH) ACTIVITYIN PLASMODIUM BERGHEI-INFECTED ERYTHROCYTES

SHAFARIATUL AKMAR ISHAK, LIANA MARHALIM, TAN TEE PING AND HASIDAH MOHD SIDEK

School of Biosciences & Biotechnology, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor

Plasmodium LDH (pLDH), an antimalarial target enzyme is involved in glycolysis and ATP production in the malarial parasites. The presentstudy aims to investigate the effect of berberine (Aldrich Chemical Company Inc), a plant alkaloid with reported antimalarial properties, onpLDH. Chloroquine was used as a reference drug in this study. A total of fifty mice were divided into two groups; a group infected withPlasmodium berghei (n=40), and another as non-infected controls (n=10). For the group of infected mice, twenty were treated with 0.001-10μg/ml chloroquine while the other twenty were treated with 0.001-100 μg/ml berberine. Blood samples were collected from the study animalswhen parasitemia levels reached 1-2% for the in vitro pLDH analysis. Prior to analysis, the blood samples were incubated (24 hours at 37°Cin a candle jar), lysed using HEPES buffer, centrifuged at 10 000 xg to obtain supernatant samples containing pLDH. The supernatants werethen transferred into 96-well microtiter plates containing Malstat reagent (20 mg of sodium L-lactate. 5.5 mg of basic Tris buffer (Tris-Cl) and3.7 mg of 3-acetyl pyridine adenine dinucleotide (APAD) dissolved in 1 ml of distilled water) followed by addition NBT-PES mixture (1.6mg of nitroblue tetrazolium (NBT) and 0.1 mg of phenazine ethosulphate (PES) dissolved in 1 ml of distilled water). Absorbance wasmeasured at 650 nm using an ELISA plate reader. Percentage of inhibition of pLDH activities in samples from berberine- and chloroquine-treated animals were determined using readings obtained from control non-infected blood samples. Chloroquine was found to exert stronginhibition of pLDH activity with IC50 value of 0.01-0.1μg/ml (~ 0.05 μg/ml). Berberine, also showed strong inhibition of pLDH activities withan IC50 of 1-10 μg/ml (~7 μg/ml). In addition, a total of thirty mice were used to study the effect of 10 mg/kg chloroquine or berberine on theprogression of parasitemia and survival in P.berghei-infected mice. Intraperitoneal administration of 10 mg/kg chloroquine or berberine intoinfected mice for four consecutive days suppressed development of parasitemia and prolonged survival of the mice.

Poster 93

ANTIOXIDANT ACTIVITIES OF THE EXTRACT OF CASHEW SHOOTS

SHAGHAYEGH KHALEGHI , AZLINA ABDUL AZIZ AND SARNI MAT JUNIT

Department of Molecular Medicine, Faculty of Medicine , University of Malaya, 50603, Kuala Lumpur

The cashew plant (Anacardium occidentale) is a member of the family Anacardiaceae. This plant has been used for years as food and a sourceof traditional medicine to treat various diseases including malaria and yellow fever. In Malaysia the bark decoction of this plant is used fordiarrhea. A.occidentale is also a perfect fountain for phytochemicals which contain potent antioxidant activities. An antioxidant is a moleculecapable of showing or preventing the oxidation of other molecules. Oxidation reaction can produce free radicals, which start chain reactionsthat damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions bybeing oxidized themselves. The aim of our study was to determine the antioxidative potential of the shoots of A.occidentale and to determineits effect on lipoprotein metabolism using cell culture. Here we reported the results of our first objective. The total phenolic content andantioxidant activities of methanol, ethyl acetate, hexane and water extracts of the shoots of A. occidentale were measured. The total phenoliccontent was evaluated using the Folin-Ciocateu assay. The antioxidant capacity of the samples was measured using FRAP assay, which isbased on the ferric reducing properties of antioxidants. Results showed that the methanol extract of A. occidentale contained substances thatwas potent reducing agents compared to the other extracts. The order of antioxidant potency of the plant extract is methanol (3.479 +0.0069)>water (1.655 + 0.0018)>ethyl acetate (0.257 + 0.0010)>hexane (0.081 + 0.0012) mmole/g dry weight. Since the methanol extractcontained the highest antioxidant activity, we can conclude that most of the antioxidants present in the shoots of this plant are polar in nature.

Poster 94

MORPHOLOGICAL ANALYSIS OF THE TRUNCATED LEAVES SYNDROME SEEDLINGS IN OIL PALM

HABIB S.H.1, NAMASIVAYAM P.1*, HO C.-L.1 AND SHARIFAH SHARUL RABIAH SYED ALWEE2

1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,43400 Serdang, Selangor, Malaysia. 2Felda Agricultural Research Sdn. Bhd.

*Corresponding author: [email protected] Phone: (60)03-89468344 Fax: (60)03-89467510

Tissue culture of oil palm is important for rapid multiplication of uniform planting materials with desired characteristics such as yield,slow vertical growth and disease resistance. However, some tissue culture derived seedlings often lead to an abnormality termed “selfpruning” or truncated leaf syndrome (TLS). These plants possess leaves that look like grasshopper damage, have stunted growth withonly a stub as remains. The plants with severe symptoms will be eventually culled off. To date, no extensive research has been done on theTLS seedlings. Therefore, an attempt has been taken to investigate the morphological and anatomical differences of TLS seedlings incomparison to the wild type seedlings using light and scanning electron microscopy studies. On the basis of the percentage of TLSoccurence in a clone, three categories have been created and they are high (70-100%), moderate (20-69%), and mild (less than 20%).Normal and abnormal seedlings differ in terms of growth and vigority, leaf size and shape, root number, volume as well as length, and sizeof shoot apical meristem. Depressed and wavy leaf surface was observed by light microscopy in TLS seedlings. Size and shape of thestomata was also different in TLS leaf compared to wild type leaf. TLS leaves had larger mesophyll cells compared to wild type.


Poster 95

MOLECULAR IDENTIFICATION OF ISOLATED ENDOPHYTIC FUNGI WITH POTENTIALANTIBACTERIAL PROPERTIES ISOLATED FROM GARCINIA MANGOSTANA

AND GARCINIA PARVIFOLIA

SIM JIUN-HORNG1, CHEAH YOKE-KQUEEN1*, KHOO CHAI-HOON1 AND SON RADU2

1Department of Biomedical Science, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan

2Department of Food Science, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan

*Correspondence: [email protected]

Estimated there may be as many as 1 million different Fungi species in our planet. Only 0.1 million fungi had been describe and named,endophytic fungi could be part of the million undiscovered fungi species. Endophytic fungi are endosymbiont that lives within a plant forat least part of its life without causing any apparent disease. Endophytic fungi are ubiquitous and have been found in all the species ofplants studied to date. Although abundant, the extent of their contribution to fungal biodiversity remains unclear. Since endophytic fungiare poorly known, especially in the tropics, current estimates of fungal species are probably conservative. Besides that, fungi which livewithin plant also represent an excellent source of secondary metabolites such as antibiotics, bio-insecticides, fine chemicals and enzymesdue to their special host interaction. The wide ranges of compounds produced by endophytic fungi have been shown to combat pathogensand even cancers in animals including humans. Two local fruit plants namely mangosteen (Garcinia mangostana) and brunei cherry(Garcinia parvifolia) obtained from fruit farm, Johor were used in this study. Different part of the fruits sample such as leaf, stem, seed,flower and root were subjected for the isolation process. A total of 37 endophytic fungi were successfully recovered using Potato DextroseAgar. Endophytic fungi were further tested with fourteen different pathogens for the antibacterial bioactive compound screening. Only onedistinguish isolate exhibited potential bioactive compound towards 6 pathogens (Staphylococcus aures, Listeria monocytogenes, Aeromonashydrophilla, Bacillus megaterium, Bacillus subtilis and Streptococcus pyogenes) using well diffusion assay. The isolate was furtheridentified by molecular approach. On the other hand, genetic relationships among the isolated endophytic fungi were accomplished byRAPD with three arbitrary primers (OPO 6, OPO10 and OPO16).

Poster 96

ASSOCIATION BETWEEN FAAH P129T POLYMORPHISM ANDADDICTION TO STIMULANTS IN THE MALAYSIAN POPULATION

M.S. SIM1, V.L. RAJ1, A.H.SULAIMAN2 AND Z.MOHAMED1

Department of Pharmacology1 and Department of Psychological Medicine2,Faculty of Medicine, University of Malaya

Drug abuse is a major problem in Malaysia and its impact on the society is increasing. Although environmental factors contribute to drugaddiction, genetic factors also play a significant role estimated at 40–60% of the total risk. Fatty acid amide hydrolase (FAAH) is amammalian integral membrane enzyme that terminates the activity of a large class of endogenous signaling lipids termed fatty acidamides. Recent studies suggest that the FAAH P129T mutation is a common mutation in the FAAH gene and it is significantly associatedwith drug addictive traits. The single nucleotide polymorphism results in a missense mutation (385C→A) in the FAAH gene that convertsa conserved proline residue to threonine (Pro129Thr), thereby producing a functionally altered enzyme that displays normal catalyticproperties but has an enhanced sensitivity to proteolytic degradation. This gene encodes the endocannabinoid-inactivating enzyme, fattyacid amide hydrolase (FAAH) that in homozygous form, is strongly associated with the abuse of stimulant drugs which includeamphetamine and ‘Ecstasy’. The objective of this study was to determine whether any association exists between the Pro129Thr variant ofthe FAAH gene with dependence to stimulants in the Malaysian population. The study comprised of 105 males who are known to beaddicted to stimulant drugs and are rehabilitated at the Drug Rehabilitation Centre, Papar, Sabah, and 108 healthy unrelated controls. Theanalysis of the gene polymorphisms was performed using polymerase chain reaction (PCR) followed by restriction fragment lengthpolymorphism (RFLP) technique The results of the study showed that the genotype frequencies of the addicts for the FAAH gene P129Twere 64.8%, 27.6% and 7.6% for the CC, CA and AA genotype respectively, while the control subjects had frequencies of 75.9%, 20.4%and 3.7%. Genetic mutation in the Pro129Thr variant of the FAAH gene showed a chi-square value of 3.669 (P=0.055) and the odds ratiovalue of 1.691 with 95% confidence interval of 1.018 – 2.808. Preliminary results suggest that there is a difference in genotypefrequencies in the FAAH P129T mutation between those who are addicted to stimulants and controls even though the difference was notsignificant. These results also seem to suggest that there is a 1.691 increase in the risk for addiction for the A allele. An increase in samplesize in the continuing study will reveal whether this mutation may constitute an important risk factor for stimulant drug dependence in theMalaysian population.


Poster 97

MOLECULAR CLONING AND RECOMBINANT PROTEIN PRODUCTION OF GDP-MANNOSEPYROPHOSPHORYLASE, GDP-MANNOSE-3’, 5’-EPIMERASE AND GALACTOSE-1-PHOSPHATE

URIDYLYLTRANSFERASE FROM GRACILARIA CHANGII IN ESCHERICHIA COLI

SIOW ROUH SAN, TEOH SEDDON, HO WAN YONG AND HO CHAI LING*

Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor.


GDP-mannose pyrophosphorylase (GMP) is an essential enzyme catalyzing the formation of GDP-mannose which serves as mannosyldonor for the biosynthesis of glycolipids, lipopolysaccharides and polysaccharides, from mannose-1-phosphate and GTP. GDP-mannose-3', 5’-epimerase (GME) catalyzes the reversible epimerization of GDP-D-mannose to yield GDP-ß-L-galactose and GDP-L-gulose. On theother hand, galactose-1-phosphate uridylyltransferase is one of the four key enzymes involved in the Leloir pathway of galactosemetabolism. GALT converts UDP-glucose and galactose-1-phosphate to UDP-galactose and glucose-1-phosphate. The objective of thisstudy is to clone the open reading frames of GMP, GME and GALT from a red seaweed, Gracilaria changii, for recombinant proteinproduction in Escherichia coli. The open reading frame of GMP, GME and GALT were cloned using rapid amplification of 5'complementary DNA ends (5' RACE) PCR or EST approach. Primers with appropriate restrictions sites were designed to amplify the openreading frames of GMP, GME and GALT by PCR to facilitate directional cloning of the cDNA into pET32a in frame with the fusion tagsavailable. GMP, GME and GALT were expressed in E. coli strain BL21 (DE3) pLysS as recombinant proteins fused to Trx•TagTM,His•TagTM and S•TagTM. The expression conditions of these recombinant proteins were optimized, respectively; to produce solublerecombinant proteins. The activities of the recombinant proteins will be assayed and their roles in producing the precursors of agar will beinvestigated.

Poster 98

CONSTRUCTION OF A VACCINE CANDIDATE FOR HIGHLY VIRULENT INFECTIOUS BURSALDISEASE VIRUS USING PLANT VIRUS-BASED VECTOR

SITI HASMAH MOHTAR1, LOH HWEI SAN1, FESTO MASSAWE1 AND ABDUL RAHMAN OMAR2

1School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor.2Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Plants have now become an alternative source for production of immunotherapeutic molecules including vaccines. Plants expressingrecombinant antigens can be used directly as an oral vaccine, which can be fed raw without further cumbersome purification. Furthermore,plant system has the capability of producing vaccine in large amounts at low production cost and with high stability over other expressionsystems. Transformation of a recombinant vector transiently in plants allows the expression of proteins in a short period over theagrobacterium transformation system. Plant virus based-vectors have been widely used as transient expression vectors to facilitate genedelivery and protein expression in plant systems. Several plant RNA viruses such as tobacco mosaic virus (TMV) and potato virus X(PVX) have been constructed as viral based-vectors that employ different viral genome strategies. The present study is aimed atdeveloping a vaccine candidate for highly virulent infectious bursal disease virus (hvIBDV) by constructing a chimeric plant virus capableof expressing the VP2 gene of hvIBDV in plant systems. This paper describes the construction of a recombinant plant virus-based vector,namely the potato virus X-VP2 (PVX/VP2), comprising of the full length coding region for the VP2 gene of hvIBDV. The hvIBDV is ahighly virulent strain of the infectious bursal disease virus (IBDV) which causes contagious disease in young chickens. The viral genomecontains two segments of double stranded RNA genomes which consist of different functional viral proteins. The VP2 gene has beenidentified as the main host protective antigen of IBDV and it has the ability to elicit neutralizing antibodies. Therefore, the VP2 gene hasbeen chosen as the target protein for the development of subunit vaccine. In this study we first identified and isolated the VP2 gene fromhighly virulent strain of IBDV. The primers flanking VP2 gene region were incorporated with EcoRV and SalI restriction recognitionsequences at both 5’ and 3’ sites to facilitate the cloning of VP2 gene onto PVX vector. The VP2 gene was ligated in between the triplegene block and the coat protein coding regions of the PVX vector. The recombinant PVX/VP2 vector was cloned into E. coli and thepositive clones were further analyzed by restriction enzyme and PCR analyses. Once identified, the correct orientation of recombinantPVX/VP2 vector will be transiently expressed in plants such as alfalfa (Medicago sativa). Current preliminary findings demonstrate thepotential of PVX as an expression vector for the presentation of the hvIBDV VP2 gene in the plant viral coat protein and delivery into aplant system.


Poster 99

TWO DIMENSIONAL GEL ELECTROPHORETIC ANALYSIS OF URINARY PROTEINS OF PATIENTSWITH OVARIAN AND CERVICAL CANCERS

SITI SUHANA ABD. SOHEIMI1, LIM BOON KIONG2, ONN HAJI HASHIM3 AND ADAWIYAH SURIZA SHUIB1

1Institute of Science and Biology, Faculty of Science, University Malaya, Kuala Lumpur. 2Department of Obstetrics andGynaecology, 3Department of Molecular Medicine, Faculty of Medicine, University Malaya, Kuala Lumpur

In Malaysia, cervical carcinoma is the second most common cause of mortality due to cancer among women age 15-69, followed byovarian carcinoma (age 15-69) (Second Report of The National Cancer Registry Cancer Incidence In Malaysia 2003). Recently, urinaryproteomic is becoming a new appealing study in discovering potential biomarkers in human diseases, especially in cancer. Earlierdetection of the disease can be made in general population due to the non-invasive nature of getting samples. The aim of current work wasto develop a typical urinary proteome map of normal individuals and compare it with those obtained from patients with ovarian andcervical cancers. Urine collected from normal individuals and cancer patients were centrifuged, dialyzed and freeze-dried. Three hundredmicrogram of the urinary protein was then subjected to two dimensional gel electrophoresis. The proteins were first separated based ontheir charge (pI) using Immobiline Dry Strip pH 3-10L and followed by second separation based on the molecular weight using 12.5%SDS-PAGE gel. The protein spots were detected using silver staining. Screenings for abnormally expressed proteins were made bycomparing the proteome maps of normal individuals and cancer patients using Image Master 2D Platinum Software 6.0. The urinaryproteome maps obtained from patients with ovarian carcinoma and cervical cancer were generally comparable but different from thoseobtained from normal individuals. Further analysis will be done to identify the resolved proteins detected in the urinary proteome maps.

Poster 100

CHARACTERIZATION OF BACTERIOCIN GENES IN LACTOBACILLUS PLANTARUM I-UL4

TAI HUI FONG1, MORTEZA SHOJAEI MOGHADAM1, RAHA ABDUL RAHIM1,2 AND FOO HOOI LING1,2

1Faculty of Biotechnology and Biomolecular Sciences,2Institute of Biosciences, University Putra Malaysia, 43400 UPM Serdang, Selangor

Bacteriocins, a group of proteinaceous antimicrobial peptides, have received vast attention in the past few years. Bacteriocins produced byGenerally Regarded as Safe (GRAS) organisms like Lactic acid bacteria (LAB) are of particular interest due to their potential applicationsin food industry. The genetic determinants of bacteriocins in LAB encompass several genes that are organized into operons and functionsin immunity, processing, externalization and regulation. The present study revealed the bacteriocin genes involved in the bacteriocinproduction by Lactobacillus plantarum I-UL4, a probiotic LAB that was isolated from Malaysian food. PCR amplification of bacteriocingenes was accomplished by using thirteen bacteriocin gene-specific primers designed for the detection of structural, immunity, ABCtransporter and regulatory genes of bacteriocin. Six amplified fragments were purified and analyzed for their DNA sequences. The DNAsequences of the fragments amplified by primer Pln EF, Pln I, Pln G, Pln D and Pln W, were highly homologous to Plantaricin EF,Plantaricin I, Plantaricin G, Plantaricin D and Plantaricin W respectively. The DNA sequence analysis of the amplified fragments showedsignificant homology (95-100 %) to bacteriocin genes namely Plantaricin EF, Plantaricin I, Plantaricin G, Plantaricin D, and PlantaricinW, which were encoded for structural, immunity, ABC transporter, regulatory, and structural genes, respectively.

Poster 101

GENE TRANSFER TO HEPG2 CELLS USING T7 BACTERIOPHAGES

TANG KIE HIE1, KHATIJAH MOHD. YUSOFF1,2 AND TAN WEN SIANG1, 2

1Faculty of Biotechnology and Biomolecular Sciences,2Institute of Biosciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Hepatitis B is a major public health problem worldwide that may lead to chronic liver diseases, cirrhosis and hepatocellular carcinoma. Itis caused by hepatitis B virus (HBV) which is responsible for 500,000 deaths annually. During the initial step of HBV infection, the PreSdomain of HBV particularly amino acids 21-47, is believed to be involved in the attachment to hepatocyte. In order to study virus-cellinteractions, the genetically modified T7 phages displaying regions of HBV surface antigen were used to transfect human hepatocarcinomacell line HepG2. The DNA of phage could be recovered from cell lysate and confirmed by PCR whereas infectious form of internalizedphage was measured by plaque forming assay. The bacteriophage-cell interactions were further confirmed by immunofluorescencetechnique. Under fluorescence microscope, internalized phage exhibited the appearance as fluorescent dots. At high MOI (≈ 1.25 x 105),unmodified T7 phages were also capable of transfecting HepG2 at low efficiency and were thought to be taken up by nonspecific uptake.However, surface modification of T7 phage particles particularly by displaying the PreS regions enhanced phage uptake resulted in moreefficient in vitro gene transfer. The ability of recombinant T7 phages to transfect HepG2 cells demonstrates the potential of phagedisplayed system as gene therapy for liver cancer.


Poster 102

ETHNOMEDICINAL APPROACH IN SCREENING OF CYTOTOXICITY AND DPPH RADICALSCAVENGING ACTIVITIES OF LOCAL MEDICINAL PLANTS

TAN HOR YUE1, TOI LEE ROU1, LEE CHIN YOONG2, ONG HEAN CHOOI3,ANTHONY HO SIONG HOCK1 AND LIM YANG MOOI1*

1Department of Bioscience and Chemistry, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman,Jln. Genting Klang, Setapak, 53300 Kuala Lumpur, Malaysia. 3Institute of Biological Sciences, Faculty of Science,

University Malaya, Malaysia. 2PK Herbal Research Centre, 15-2, Jalan Kledang Timur Satu, Bandar Baru Menglembu,31450 Menglembu, Ipoh, Perak Malaysia.


Despite all the advancement in current cancer therapy, many cancers are still ineffectively treated due to the side effects. The finding of theanticancer agent from the natural products has raised the great interest nowadays due to the better therapeutic performance. The aim of thisstudy is to screen the selected local medicinal plants on the cytotoxicity and the antioxidant activities with the hope to assess its potentialin cancer treatment. The selection of the plants was through the ethnomedicinal approach in which the plants were collected based on thelocal medicinal use in cancer treatment. The cytotoxicity of total 102 ethanolic crude extract from different part of 48 local medicinalplants were evaluated by using MTT assay tested on HL-60 cell line. The result showed 44% of the extracts exhibit strong cytotoxicactivities. The three with most potent cytotoxicity activity extracts were further investigated to determine IC50 by serial dilution which isthe leaf of Calotropis gigantean, fruit and the leaf of Melia azedarach. Due to the strong cytotoxicity exhibited by the leaves extract thatderived from Calotropis gigantean, the IC50 was not able to determine therefore IC20 was recorded which is 6μg/ml. IC50 for fruit and theleaf of Melia azedarach were 14μg/ml, 18μg/ml, respectivel. The antioxidant activity of 102 crude extract were then accessed through theDPPH assay. The leaf of Rodgersia sambucifolia and the leaf of Leea indica showed the strongest antioxidant capacity with the percentageinhibition of 85.57±4.23 and 80.97±5.54 respectively. The result of preliminary screening revealed a high potential in the local medicinalplants mentioned above. This encourages the further work in isolating the pure compounds which may help in cancer treatment.

Poster 103

BIOASSAY-GUIDED ISOLATION OF CYTOTOXIC COMPOUNDS FROM AMARANTHUS SPINOSUSAND CLEOMEl GYNANDRA

TAN SU YIN1, LIM CHAN KIANG1 AND LIM YANG MOOI1*

1Department of Bioscience and Chemistry, Faculty of Engineering and Science,Universiti Tunku Abdul Rahman, Jln. Genting Klang, Setapak, 53300, Kuala Lumpur, Malaysia.


Nowadays, discovery of novel anticancer drugs, which selectively eliminate neoplastic cell phenotype is crucial in the treatment of cancer.In this study, cytotoxic compounds from Amaranthus spinosus and Cleome gynandra leaves extracts were isolated using bioassay-directedisolation techniques. The isolation of active cytotoxic compounds was carried out in three steps: sequential extraction, fractionation usingcolumn chromatography and compound determination using thin layer chromatography and analytical high performance liquidchromatography. Cytotoxicity preliminary screening and IC50 values of fractions were evaluated by using MTT assay. Pre-screening resultshowed that the magnitude of cytotoxicity was predominant in A. spinosus methanol extract and C. gynandra ethyl acetate extract.Fraction AS2b isolated from A. spinosus possessed notably high cytotoxic effect against HSC-3 and HSC-4 cell lines with IC50 values of7.00 μg/ml and 10.50 μg/ml, respectively. Fraction CG4c from C. gynandra exhibited potent cytotoxicity against K-562 cells (IC50 = 10.50μg/ml). Present study confirms the cytotoxic properties of A. spinosus and C. gynandra.

Poster 105

EURYCOMANOL FROM EURYCOMA LONGIFOLIA JACK MEDIATED APOPTOSIS IN HUMANBREAST CANCER CELLS MCF-7

TEE THIAM TSUI1, CHEAH YEW HOONG1, MEENAKSHII NALLAPPAN2, AZIMAHTOL HAWARIAH LOPE PIHIE1

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKMBangi, Selangor 2Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

The present study investigates the antiproliferative effect, mode of cell death and the mechanism of action of eurycomanol, a quassinoidfrom the root of Eurycoma longifolia Jack on the human breast cancer cell line MCF-7. Eurycomanol exhibites cytotoxic activity towardsMCF-7 (IC50=15.23±0.66 μg/ml) and is less sensitive against a normal breast cell line MCF-10A (IC50=66.31±0.47 μg/ml). Based on theIC50 values obtained, eurycomanol displayed a certain extent of cytoselectivity towards normal breast cells. The antiproliferative activity ofeurycomanol was due to apoptosis induced in MCF-7 cells and not necrosis. This was demonstrated by the Hoechst 33258 nuclear stainingassay showing typical apoptotic morphology such as DNA fragmentation, cell shrinkage, and nuclear condensation. The apoptosistriggered by eurycomanol in MCF-7 cells was associated with the down-regulation of the antiapoptotic BCL-2 protein expression inducingdisruption of the mitochondrial membrane potential thus favouring apoptosis. Next, the processing of the 35 kDa executioner procaspase-7 was detected. Active caspase-7 then cleaved and inactivated poly (ADP-ribose) polymerase (PARP-1) resulting in nuclear fragmentation.These results, therefore, suggest eurycomanol exert antiproliferative effects on MCF-7 cells by inducing apoptosis through the down-regulation of BCL-2 protein levels, activation of caspase-7 and inactivation of PARP-1.


Poster 106

EXPRESSION AND CHARACTERIZATION OF YjeE- HOMOLOGUE PROTEIN INBURKHOLDERIA PSEUDOMALLEI

TENGKU YASMIN PUTRI T MOHD YUSOF1, RAHMAH MOHAMED1,2 AND AMIR RABU1*

1Faculty of Biotechnology and Biosciences, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,2MALAYSIA GENOME INSTITUTE

* Corresponding author: [email protected]

The Gram-negative pathogen Burkholderia pseudomallei are the causative agent of melioidosis, a serious human and animal disease. Aputative YjeE-homologue protein is a protein of unknown function, encoded by the BPSL 0860 gene of B. pseudomallei. This protein wasselected as part of a structural genomics project for its characterization. The protein is considered essential to bacteria because the gene ispresent in most bacterial genome but not in eukaryote. Amino acids sequences show homology to other proteins only for the presence ofthe Walker A motif G-X-X-X-X-G-K-T that indicates the possibility of a nucleotide-binding protein. The closest protein similarity is 42%to known protein structure of putative enzyme YjeE from Haemophilus influenzea.. The BPSL 0860 were amplified from B. pseudomalleiD286 and cloned into cloning vector (TOPO TA) and then subcloned into expression vector (pET28b). The gene was expressed for 3 hoursin Escherichia coli BL21 (DE3) by using IPTG (1 mM) as inducer. From the expression experiment, the protein was successfully obtainedwith size of 19.6 kDa. Further experiment shows it is soluble and can be detected by using anti his antibody. The protein was eluted by30% of imidazole and purified by Nickel-Nitrilotriacetic metal affinity chromatography. The protein will be undergoing biochemical andbiophysical characterization.

Poster 108

EXPRESSION OF PROLIFERATOR-ACTIVATED RECEPTOR GAMMA 2IN GLYCYRRHIZIC ACID TREATED RATS

TIU KHAI MING1, TON SO HA1 AND KHALID BIN ABDUL KADIR2

1School of Science, 2School of Medicine and Health Sciences; Monash University Malaysia

Peroxisome proliferator-activated receptor gamma isoform 2 (PPARγ2) has been implicated as the nodal transcriptional regulation point inexpression of fat-specific genes. There are increasing evidence that suggest the association of pathogenesis of insulin resistance in relationto transcriptional regulation by PPARγ2. Glycyrrhizic acid (GA), a potential agonist of PPARγ2, has been reported ameliorate metabolicdisturbance associated with insulin resistance, which is one of the common risk factors for metabolic syndrome and type 2 diabetesmellitus. This study aimed at determining the effect of GA on insulin resistance and expression of PPARγ2 in rats fed with GA (100mg/kg) for 24 hours. Expression of PPARγ2 in various rats tissues were quantified using Real Time PCR based on a standard curve of 8 logdynamic of recombinant PPARγ2 plasmid. GA-administered rats recorded similar copy number expression of PPARγ2 compared tocontrol rats (p>0.05) in liver (1.31. x 1012:1.06 x 1012), abdominal muscle (5.01 x 1011:3.07 x 1010), quadriceps fermoris muscle (1.48 x1011:1.1 x 1011) and subcutaneous adipose (2.08 x 1027:8.12 x 1026) tissues. Noticeable increment of PPARγ2 levels were observed invisceral adipose (3.75 x 1016:2.76 x 1010). GA administration induced significant reduction in blood glucose level and HOMA-IR index(p<0.05). In conclusion, these results suggest that GA might act as ligand of PPARγ2 in visceral adipose tissues.

Poster 109

HOMEOSTASIS MODEL OF ASSESSMENT – INSULIN RESISTANCE (HOMA-IR) ANDHISTOLOGICAL ANALYSIS ON TISSUES OF RATS ADMINISTERED WITH GLYCYRRHIZIC ACID

CHIA YOKE YIN1, TON SO HA1 AND KHALID BIN ABDUL KADIR2

1School of Science, 2School of Medicine and Health Sciences; Monash University Sunway Campus

Insulin resistance is the key factor underlying the pathogenesis of metabolic syndrome and Type II diabetes. Previous studies had broadlyshown that glycyrrhizic acid (GA) inhibits the postprandial blood glucose rise in genetically diabetic KK-Ay mice. Therefore this studywas conducted to determine the effects of GA on homeostasis model assessment – insulin resistance (HOMA-IR) and histological analysison tissues using rat as the animal model. 40 male rats, Rattus norvegicus (Sprague Dawley strain) were used in this study. The rats weresubjected to 12-, 24- and 48-hours treatment and fed ad libitum. The treated and control groups were administered intraperitoneally withGA (50mg/kg) and saline respectively. Blood glucose and serum insulin levels were measured and HOMA-IR was calculated. Histologicalanalysis was done using Haematoxylin-Eosin (H&E) and Periodic Acid Schiff (PAS) Stain on the tissues studied (e.g. subcutaneous andvisceral adipose tissue and liver). It was found that GA-administered rats for 12, 24 and 48 hours had lower blood glucose but only the24-hour GA administered rats showed significant decrease (p<0.05). There was no significant difference in the serum insulin levelsbetween the control and GA-administered rats at 12-, 24- and 48-hour (p>0.05). The homeostasis model assessment (HOMA-IR) in GA-administered rats was significantly lowered at 12 and 48 hours (p<0.05) while a slight increase was seen at 24 hours (p>0.05). Histologicalanalysis on subcutaneous adipose tissue showed increase in the number of small-size adipocytes while visceral adipose tissue showedshrinkage after GA administration. Increased glycogen deposition in the liver was observed in the GA-administered rats in both the 12-and24-hours. The data signified that GA could increase insulin sensitivity by reducing blood glucose and insulin levels. This study maybeuseful for further studies on Type II diabetes.


Poster 110

ROOTS, STEMS AND LEAVES OF HYPTIS SUAVEOLENS EXERT INHIBITORY ACTIVITY ON THEPROLIFERATION OF BREAST CANCER CELL LINES

UMA MAGESWARY THLIAMPLAM1, M.S.KANTHIMATHI1, UMAH RANI KUPPUSAMY1 AND CHRISTOPHE WIART2

1Department of Molecular Medicine, 2Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur

Medicinal plants have been used for centuries to treat and cure different kinds of ailments including cancer. These plants have been screenedfor their biological activites all around the world. Hyptis suaveolens, (Lamiaceae), commonly known as Lerkuing or Selaseh hutan, is a strongscented herb used traditionally in the treatment of diarrhoea and as an insect repellent. It has been found to possess antimicrobial activity.However the sole effect of this plant on cancer has not been reported. Cancer is a group of diseases in which cells grow and divide withoutrespect to normal limits, invade and destroy adjacent tissues (invasive) and sometimes spread to other locations of the body (metastatic).Breast cancer is the most prevalent type of cancer among women and the incidence of this cancer has increased significantly around theworld. In the present study, aqueous extracts of H. suaveolens leaves and methanolic crude extracts of its roots and stems were tested onbreast cancer cell lines (MCF-7 and MDA-MB-231) for anti-proliferative effect. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay was used to quantitate the viable cells. The aqueous extract of H. suaveolens leaves and the methanolic crude extract of theroots and stems exerted dose-dependent inhibition (dose range of 100-500 μg/ml) on both cell lines. The root extract exerted more potentinhibitory effect on both the MDA-MB-231 (estrogen-unresponsive) and MCF-7 (estrogen-responsive) cell line, compared with the methanolicextract of the stem and the aqueous extract of the leaves. The concentration of root extract that caused 50% inhibition (IC50) of MDA-MB-231and MCF-7 was 150 μg/ml and 100 μg/ml, respectively. The concentration of leaves and stem that caused 50% inhibition (IC50) of MDA-MB-231 was 100 μg/ml. The IC50 for the concentration of leaves and stem of the MCF-7 could not be determined. Methanolic crude extracts of theleaves, roots and stems were then fractioned by solvent-solvent partitioning to yield 4 fractions: hexane, butanol, ethyl acetate and water. Allthese fractions were assayed for growth inhibitory effect on the MDA-MB-231 and MCF-7 cell lines. The hexane root fraction, once againexerted more potent inhibitory effect on both the cells lines with an IC50 of less than 100 μg/ml.

Poster 111

SCREENING FOR ASSOCIATION OF NOTCH4 GENE POLYMORPHISMSIN MALAYSIANS WITH SCHIZOPHRENIA

WAN CHING LEE1, NUR ZURAIDA ZAINAL2, LIAN LAY HOONG3, VIJAYA LECHIMI RAJ1,ELSA HANIFFA MEIJA MOHAMED1 AND ZAHURIN MOHAMED1

1Department of Pharmacology, 2Department of Psychological Medicine and3Department of Molecular Medicine, Faculty of Medicine, University of Malaya

The NOTCH pathway is an evolutionarily conserved cell-cell signaling mechanism, and its one key role of which is to decide the cell’sfate, especially during the neural developmental process. The NOTCH4 gene is located at 6p21.3, and this gene encodes a member of theNOTCH family which share structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like (EGF) repeats, and an intracellular domain consisting of multiple, different domain types. The NOTCH4 protein functions as areceptor for membrane bound ligands, and may play a role in vascular, renal and hepatic development. A recent study reported that asingle nucleotide polymorphism SNP1 in the 5’ flanking region of the NOTCH4 gene is highly associated with schizophrenia in theBritish population. To investigate the genetic association between the NOTCH4 polymorphism and schizophrenia in the Malaysianpopulation, 202 patients with schizophrenia and 389 normal controls were included in this study. Genotyping of the SNP1 was determinedby Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assays. PCR products were digested with MspIenzyme resulting in wild type variants (TT) with one band (382bp), heterozygous types (TC) exhibiting three bands (382bp, 268bp and114bp) and mutant variants (CC) with two bands each (268bp and 114bp). The results of the study showed that the genotype frequenciesfor schizophrenia were 27.5%, 47.3%, and 25.2% for the TT, TC, and CC genotypes, respectively, while the control subjects hadfrequencies of 19.8%, 50.5% and 29.7% for the TT, TC and CC genotypes, respectively. The odds ratio value among the patients againstthe controls was 0.99, indicating that the group of patients is slightly less likely to have an SNP1 mutated if compared to the control group.The results of this study showed that the genotypic distribution did not show a significant [χ2= 4.465, degree of freedom (d.f)=2, P=0.107]and allelic distribution of SNP1 show a significant [χ2= 3.968, d.f=1, P=0.046] association with schizophrenia in the Malaysianpopulation. The genotype distribution of NOTCH4 SNP1 polymorphism was in Hardy-Weinberg Equilibrium (HWE) for both controlsand patients [controls, χ2= 0.003, d.f=1, P=0.959; patients, χ2= 0.001, d.f=1, P=0.973]. The preliminary results of the present study, doesnot support the original finding that the above polymorphism of the NOTCH4 gene is associated with schizophrenia.

Poster 112

RTPCR AMPLIFICATION AND CLONING OF PARTIAL DNA SEQUENCE CODING FOR OIL PALM(E. GUINEENSIS) PHYTOENE SYNTHASE

WAN NUR SYUHADA WAN SULAIMAN1, OMAR ABD RASID1, AHMAD PARVEEZ GHULAM KADIR1, ANDZULQARNAIN MOHAMED2

1Advanced Biotechnology and Breeding Center, Malaysian Palm Oil Board. No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000Kajang Selangor, Malaysia, 2Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur

Carotenoids with provitamin A activity are essential components of the human diet, and there is considerable evidence that many carotenoidshave anti-cancer activity. Oil palm, being naturally rich in the carotenoids and other minor components, offers great potential to be exploited forcommercial development of value-added products. To date, for the carotenoids pathway, especially in oil palm, there is incomplete knowledge ofthe genes involved. This work was aimed at the isolation of cDNA clone coding for phytoene synthase from E. guineensis. In this work, we havesuccessfully obtained a 863 bp fragment that codes for a partial length (289 amino acids) through RTPCR using hybrid primers. The fragment(pEPSY89) was cloned into a PCRII-TOPO vector and its complete DNA sequence was determined. DNA sequence analysis showed that theclone is highly identical to phytoene synthase of other species at about 81%. Moreover, the E. guineensis psy sequence relates closely withE. oleifera psy at about 95% which was previously isolated. Further analysis showed the presence of a conserved aspartate-rich catalytic domainwhich was essential for catalytic activity. This domain is present in the partial sequence of E.oleifera psy.


Poster 113

IMPROVEMENT OF GENE TRANSFER EFFICIENCY FOR THE TRANSFORMATION OF FLOWERCOLOUR GENE IN TIBOUCHINA SEMIDECANDRA COGN.

WILSON THAU LYM YONG1, JANNA ONG ABDULLAH2 AND MAZIAH MAHMOOD3

1Biotechnology Research Institute, Universiti Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia.2Department of Microbiology, 3Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti

Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

Genetic engineering of a wide variety of plant species has led to the improvement of plant traits. In this study, genetic transformation ofTibouchina semidecandra Cogn. with sense and antisense dihydroflavonol-4-reductase (DFR) genes using the Agrobacterium-mediatedmethod was carried out. D-galactose, tyrosine, aluminium chloride and ascorbic acid were used to enhance the transformation efficiency.Plasmids pBETD10 and pBETD11, each harbouring the DFR gene at different orientations (sense and antisense) and the selectable markernptII for kanamycin resistance, were transformed into the shoot and nodal explants of T. semidecandra using an improved transformationprotocol. Putative transformants were selected in the presence of kanamycin at their respective optimized concentration. The resultsshowed that approximately 5.3% of pBETD10-transformed shoots and 9.3% of the nodes regenerated whereas only 4.7% (shoots) and8.3% (nodes) regenerated with pBETD11 transformation. The presence and integration of the sense and antisense DFR genes into thegenome of T. semidecandra were verified by polymerase chain reaction (PCR) and nucleotide sequence alignment and confirmed bysouthern analysis. The regenerated putative transformants were acclimatized to glasshouse conditions. Approximately 69.4% of thepBETD10-transformed and 57.4% pBETD11-transformed T. semidecandra survived after transfer to the glasshouse. The colour changescaused by the transformation event were observed at the budding stage of the putative transformants where greenish buds were producedby both T. semidecandra plants harbouring the sense and antisense DFR transgenes.

Poster 114

PLANTLET P(AA)RODUCTION THROUGH DEVELOPMENT OF ACTIVE MERISTEM FROM MALEINFLORESCENCE OF BANANA, MUSA ACUMINTA CV. ‘PISANG MAS’ (AA)

S. WIRAKARNAIN * M.T. ROSNA AND S.CHANDRAN

Institute of Biological Sciences, Faculty of Science, University Malaya, 50603,Kuala Lumpur, Malaysia(*Co-responding author E-mail: [email protected] Phone No: +603-7957 3336 / 03-7955 1116 Fax: +603-7967 4178 )

We have developed a protocol for establishing active meristem of banana, Musa acuminta cv. ‘Pisang Mas’ (AA ) derived from maleinflorescence. The white floral meristem appeared after 21 days in medium supplemented with 10μM of N6-benzylaminopurine (BA) and1μm indole -3-acetic acid (IAA). White floral meristem were optimized in medium supplemented with 100μM (BA) and 1μM (IAA) untila high amount of compact floral meristem were obtained. These compact floral meristems were induced to compact active green meristemafter 21 days in medium supplemented with 21.5 μM NAA(Napthaleneacetic acid) and 50 μM of BA. MS medium supplemented with10μM of N6-benzylaminopurine (BA) and 1μm indole -3-acetic acid (IAA) were suitable for shoot production. The regenerated shootswere rooted after being transferred onto MS medium supplemented with 1% (w/v) of activated charcoal for 14 days. This study hasdemonstrated that green active meristem derived from male inflorescence can produce plantlet and could be an alternative material formass micropropagation of bananas.

Poster 115

QUANTITATIVE DETERMINATION OF EXOGENOUS GANGLIOSIDES EXPRESSED BY YAC-1MURINE CANCER CELL LINE

WONG CHIN PIOW1 AND LIM YANG MOOI1*

1Department of Bioscience and Chemistry, Faculty of Engineering and Science,Universiti Tunku Abdul Rahman, Jln. Genting Klang, Setapak, 53300, Kuala Lumpur.


Gangliosides play important cellular roles which includes cell surface recognition and regulator of cell growth. Overexpression ofgangliosides is a common feature of numerous types of tumours, which includes neuroblastoma, lymphoma, melanoma, and leukemia.Overexpression of gangliosides influences the progression, proliferation, invasion, angiogenesis and immunosuppression of humancancers. The acidic ninhydrin assay is a common method used to determine the amount of sialic acid. However, this method is notoriginally conceived to determine exogenous gangliosides expressed by cancer cells. Therefore, the acidic ninhydrin assay is evaluated andoptimized to measure exogenous gangliosides expressed by YAC-1 murine cancer cell line. Results obtained demonstrated optimal ratio ofchloroform: methanol: cell supernatant used to extract gangliosides from cell supernatant was at 8:4:3. Cell seeding density at 4 x 105

cells/ml was required to give the highest detectable amount of gangliosides after three days incubation. Commercially availablemonosialogangliosides, GM1 was used as standard to validate the assay. Result obtained showed distinct differences in the optical density(OD) of positive control containing monosialogangliosides, GM1 (OD = 2.11) from negative control without monosialogangliosides, GM1

(OD = 0.70) with optical density differences at 1.41 OD. In conclusion, the gangliosides extraction technique and cell seeding densitywere optimized, and acidic ninhydrin assay is able to determine exogenous gangliosides expressed by YAC-1 murine cancer cell line.


Poster 116

IDENTIFICATION OF IMPORTANT RESIDUES FOR CATALYSIS AND SUBSTRATE SPECIFICITY INHUMAN CHOLINE KINASE BY SITE-DIRECTED MUTAGENESIS

WONG MUN TENG, FEW LING LING AND SEE TOO WEI CUN

School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan

Phosphatidylcholine (PtdCho) is a prominent constituent of eukaryotic and some prokaryotic membranes. The biosynthesis of PtdCho isaccomplished by three pathways, which are CDP-choline pathway, methylation pathway and phosphatidylcholine synthase pathway.Choline kinase (CK) is the initial enzyme of the CDP-choline pathway and localized in the supernatant fraction of cells. CK catalyzes bothcholine and ethanolamine by ATP in the presence of magnesium ion. Recently, the high activity of CK in transformed cells and tumors hasled to increasing interest in CK inhibition as possible anti-tumor therapy. Alignment of derived protein sequence of CK and ethanolaminekinase (EK) from human, Drosophila and yeast revealed a number of highly conserved residues, which include the choline kinase motifand Brenner’s phosphotransferase motif. Brenner’s phosphotransferase motif appeared to be specific in CK/EK family and possiblyimportant for binding of choline. To identify the amino acid residue of human CK (hCK) that is important for catalysis, a conservedaspartate at position 342 in hCKα2 was mutated to asparagine. The mutant construct was successfully cloned into pET-14b vector andoverexpressed in E. coli BL21(DE3). The mutant protein D342NhCKα2 showed dramatic loss of activity with only 12.46% of wild typeprotein activity remained. The result indicated that precise position of carboxylate group and its negative charge is essential for catalysis.Both hCKα2 and D342NhCKα2 were able to phosphorylate choline and ethanolamine. Choline is the preferred substrate for D342NhCKα2,its Km for choline is lower than ethanolamine. The apparent Km for choline (0.13 mM) is 1.18 times higher than the wild type protein, butthe Vmax (7.70 U/mg) was about eight times lower than the wild type. Apparent Km for ethanolamine for D342NhCKα2 is 29.94 mM,which is about 598 times higher compared to wild type hCKα2. The Km and Vmax for ATP of mutant protein increased by 3 and 4 timesrespectively compared to the wild type. The results indicate that the carboxylate group of aspartate may be required for binding withsubstrate choline, ethanolamine or ATP. Besides, the precise location of negative carboxylate group might be critical for catalysis bycoordinating the magnesium ion. Previous study on mutation of aspartate 320 (corresponding to aspartate 342 in hCKα2) to alanine shownto be responsible for activity inhibition or disruption of homo-dimer complex in mouse CKα1. Therefore, mutation of aspartate 342 in thiswork might also cause the activity inhibition or disruption of homo-dimer complex in hCKα2.

Poster 117

SEQUENCE COMPARISON BETWEEN TWO EIMERIA SPECIES

YEA-LING TAY1,2 AND KIEW-LIAN WAN1,2

1School of Biosciences and Biotechnology, Faculty of Science and Technology,2Malaysian Genome Institute, UKM-MTDC Smart Technology Centre Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor

Eimeria tenella and E.maxima are two of the seven Eimeria species that are known to infect chicken and cause the intestinal diseaseknown as coccidiosis. As an initial effort to compare the genome of the two species, three bacterial artificial chromosome (BAC) clonesfrom an E.maxima genome library were sequenced and analyzed. Clone sizes were estimated based on restriction digestion and pulsed-field gel electrophoresis. The BAC clones were then fragmented nebulization. Fragments with sizes between 3.0 kb and 5.0 kb werepurified by low-melting point agarose electrophoresis and cloned into the pCR®4Blunt-TOPO® plasmid vector. Subsequently, clones wererandomly selected and sequenced from both ends to at least 10 times coverage. The sequence reads generated were pre-processed and thenassembled using the Staden Package. Analysis with Tandem Repeat Finder revealed that, similar with E.tenella, the trinucleotide CAG andthe heptanucleotide AGGGTTT, were found to be abundant in the E.maxima genome sequence. Sequence comparison between the twoEimeria species showed that regions with high similarity could be found. These include regions containing genes such as glucose-6-phosphate isomerase (GPI), phosphatidylinositol-4-phosphate 5-kinase (PIP5K), AAA family ATPase and conserved hypothetical proteins.

Poster 118

BIOASSAY GUIDED ISOLATION OF POTENTIAL CANCER CHEMOPREVENTIVE CONSTITUENTSFROM KYLLINGA BREVIFOLIA AND HOMALOCLADIUM PLATYCLADUM

YEOH SZE MUN1, LIM CHAN KIANG1 AND LIM YANG MOOI1*

1Department of Bioscience and Chemistry, Faculty of Engineering and Science,Universiti Tunku Abdul Rahman, Jln. Genting Klang, Setapak, 53300, Kuala Lumpur, Malaysia.


The classical approach for the diagnosis of cancers is generally absent of significant decrease in cancer mortality. Thus, searching andisolating potential chemoprevention agents to interfere with process of carcinogenesis had driven the attention of public. Leaves ofKyllinga brevifolia and stems of Homalocladium platycladum were subjected for bioassay-guided isolation and were evaluated via shortterm in vitro anti-tumour promoting assay with the induced Raji cells by phorbol 12-myristate 13-acetate (PMA) and sodium n-butyrate(SnB). Methanolic extracts for both plant exhibit strong inhibitory effects of Epstein-Barr virus early antigen (EBV-EA) induction andpreserved a high viability (CV > 80%) of raji cells which the inhibition rate were 52.59% and 62.51% respectively. Hence, bothmethanolic extracts were further purified by chromatography technique and 13 fractions being isolated from Kyllinga brevifolia methanolextract while 9 fractions were obtained from methanol extract of Homalocladium platycladum. Five out of thirteen fractions which are K1,K2, K3, K4, and K6 from Kyllinga brevifolia methanol extract display remarkable inhibitory effects on the EBV-EA induction. Whereas,just one fraction, H4 was being isolated from methanol extract of Homalocladium platycladum give promising anti-tumour promotingeffect with the inhibition rate of 72.12% and high cell viability of 88.10%. Further isolation and purification need to carry out for thebioactive pure compounds for both plants so that potential chemopreventive agents can be identify.


Poster 119

ISOLATION AND CLONING OF MICRORNAS IN PINEAPPLES

YEW CHEE WEI AND VIJAY KUMAR

Biotechnology Research Institute, University Malaysia Sabah, Locked Bag No. 2073, 88999 Kota Kinabalu, Sabah

Plant microRNAs are around 21-nucleotide, endogenous non-coding small RNAs, that play an important role in regulating geneexpression at the post-transcriptional level. It does this by complementary binding to specific mRNA targets and induces gene silencingeither by translational repression or mRNA degradation. As there is lack of information in the understanding of fruit ripening processmediated by microRNAs, this study aims to isolate and characterize microRNA expression profiles particularly for the tropical non-climacteric fruit, pineapple. Total RNA was extracted from fruit tissues using conventional phenol/chloroform method Approximately 50μg of total RNA was then fractionated on a 15% denaturing polyacrylamide gel. Small RNAs within the size range of 17-25bp, containingthe microRNAs were then excised and gel-purified. Next, 3’-linker (5’-rAppCTGTAGGCACCATCAAT-NH2-3’) and 5’-linker(5’-ATCGTrArGrGrCrArCrCrUrGrArArA-3’) were ligated sequentially to the 3’ and 5’-end respectively, enabling amplification of theligated RNAs through RT-PCR. After that, the amplified small RNAs were concatemerized through the use of BanI and T4 DNA ligase.Concatemers with a range of 300-800bp were subsequently cloned and sequenced. The putative microRNAs will be verified usingbioinformatics tools and further comparison with public miRNA databases will be conducted.

Poster 120

AN EVALUATION ON FRIDELAN-1,3-DIONE, CATECHIN, 5’-DEMETHOXYCADENSIN G, ANDPINOCEMBRIN AS POTENTIAL CANCER CHEMOPREVENTIVE AGENTS

YIP SHIAU CHOOI1, LIM CHAN KIANG1, PAUL LIM VEY HONG2 AND LIM YANG MOOI1*

1Department of Bioscience and Chemistry, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Jln. GentingKlang, Setapak, 53300, Kuala Lumpur, Malaysia. 2Tung Shin Hospital, Jalan Pudu, 55100, Kuala Lumpur, Malaysia.


Multistage carcinogenesis commonly has a latency of long time period and tumour promotion is the only reversible stage that can besuppressed by chemopreventive agents. Chemopreventive agents are the naturally-derived substances with the ability to inhibit or reversecarcinogenesis. In this study, pinocembrin from Xanthophyllum brevipes, 5’-demethoxycadensin G from Cratoxylum glaucum, friedelan-1,3-dione from Mesua daphnifolia, and catechin from Garcinia penangiana were evaluated for anti-tumour promoting activity, DPPH freeradical scavenging activity, and xanthine/xanthine oxidase inhibition activity. Five-point serial dilutions with the concentrations rangingfrom 10.00 μg/ml to 0.31 μg/ml for anti-tumour promoting assay, and 30.00 μg/ml to 0.94 μg/ml for DPPH free radical scavenging assaywere conducted. Pinocembrin and 5’-demethoxycadensin G showed strong anti-tumour promoting activity at the IC50 value of 0.80 μg/mland 2.20 μg/ml, respectively. Friedelan-1,3-dione showed moderate activity at the IC50 value of 9.70 μg/ml. Catechin exhibited very mildactivity and the IC50 was unable to be determined. All compounds did not show active activity in scavenging free radical, except catechinwith the IC50 value of 6.50 μg/ml. In the xanthine/xanthine oxidase inhibitory assay, only 5’-demethoxycadensin G and catechindemonstrated active superoxide anion scavenging activity at 20.00 μg/ml, with the inhibition rate of 67.27 % ± 6.11 % and 70.69 % ± 3.19 %,respectively. The strong anti-tumour activity and superoxide anion scavenging activity exhibited by 5’-demethoxycadensin G indicate thatthere is a correlation between antioxidant activity and anti-tumour promoting activity. Preliminary results suggest that more biologicalassays are needed to further evaluate the chemopreventive properties of pinocembrin, 5’-demethoxycadensin G, and catechin.

Poster 121

INSULIN-LIKE GLUCOSE UPTAKE ACTIVITY OF FICUS DELTOIDEA METHANOL EXTRACT ININSULIN RESPONSIVE CELL LINE

ZAINAH ADAM1, BEH JOO EE2, MUHAJIR HAMID2, AMIN ISMAIL3 AND SHAFII KHAMIS1

1Medical Technology Division, Malaysian Nuclear Agency, Bangi 43000 Kajang.2Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang.

3Department of Nutrition and Dietetic, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang

Ficus deltoidea, which is locally known as ‘mas cotek’ is one of the common medicinal plants used in Malaysia. This epiphytic plant, fromthe family of moraceae has been claimed to have antidiabetic property. However, there is still no scientific evidence to confirm its antidiabeticproperty. Antidiabetic agents can exert beneficial effects in the diabetic environment by enhancing insulin secretion, improving or mimickinginsulin action, reducing glucose production from liver and inhibiting glucose absorption from small intestine. This study was designed toevaluate the effect of Ficus deltoidea methanol extract on glucose uptake activity in insulin responsive cells, 3T3F442A adipocytes and L6myotubes using radiotracer assay. In L6 myotubes, Ficus deltoidea methanol extract significantly enhance basal glucose uptake by 1.5-fold atall concentration investigated. Under insulin-mediated state, the uptake was significantly enhanced by 1.36-fold at concentration of 500μg/ml.In 3T3F442A adipocytes, basal glucose uptake was stimulated by 1.69-fold at concentration of 50 μg/ml whereas under insulin-mediatedstate, no stimulation was observed. This observation suggests that the methanol extract of Ficus deltoidea has insulin-like glucose uptakestimulatory acivity in 3T3F442A adipocytes and L6 myotubes which partly evidenced the antidiabetic property of this plant. Therefore Ficusdeltoidea represents a possible antidiabetic dietary adjunct and has the potential to be developed as future oral antidiabetic agent.


Poster 123

SCREENING FOR THE PRODUCTION OF FRUCTOSYLTRANSFERASE (FTase) BY FUNGI STRAINSAND EFFECT OF AGITATION ON ENZYMATIC RATE

MASHITAH M.D.1, SITI HATIJAH M.1, NOORLIDAH A.2 AND SALMIAH U.3

1School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Seri Ampangan, 14300 Nibong Tebal,Penang, Malaysia 2Institute of Biological Sciences, Faculty of Sciences, University of Malaya, 50603 Kuala Lumpur.

3Wood Construction & Protection Programme, Product Development Division, Forest Research Institute Malaysia (FRIM),52109 Kepong, Selangor, Malaysia

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their ability for the production fructosyltransferase(FTase). Penicillium islandicum showed the highest production with FTase activity of 506 IU/ml after 48 hour fermentation of the culture.The agitation effects on FTase activity were studied at 100 to 300 rpm in shake flask culture. The results showed that FTase activities were64.47, 101.06, 49.31, 34.09 and 29.71 IU/ml at the agitation rate of 100, 150, 200, 250 and 300 rpm, respectively. Thus, showing that theagitation rate of 150 rpm was suitable for FTase production by Penicillium islandicum using commercial sugar as a carbon source.

Poster 124

MOLECULAR ASSESSMENT OF HERBICIDE RESISTANCE IN VARIOUS FOOD SAMPLES

KHOO SIEW PING1, CHEAH YOKE KQUEEN1*, AND SON RADU2

1Faculty of Medicine and Health Sciences, 2Faculty of Food Science and Technology,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia


The employment of genetically modified organisms (GMOs) is the fastest growing trend in the history of agriculture, with an annual growthexceeding 10% every year for the last decade. The global planted area has increased from 1.66 million ha in 1996 to 102 million ha in 2006.GMOs are products of gene technology characterizes by improved functional properties. This is achieved by taking copies of genes from the cellsof a plant, animal or microbe and inserting them into another cell to get a desired characteristic. This production of GM foods might poseunknown risks in either short or long terms. The recent occurrence of several food crises has emphasized food safety and protection ofconsumer’s health as main objectives for the labeling legislation. Therefore, in more than 15 countries, compulsory labeling is required forproducts that contain GMOs or derived product above a certain threshold. The information given to consumers is also essential for them choosingcertain foods over others. The legislation has necessitated development of detection methods for GMO. In the present study, the PolymeraseChain Reaction (PCR) was used to detect 187 samples comprises of certified reference materials (soybean and maize powder), raw seeds(soybean and maize), processed food and animal feed. The amplification products are analyzed by agarose gel electrophoresis and ethidiumbromide staining of the separated DNA fragment. However, there is a risk that an artefact having same size of the target sequence may have beenamplified and the most reliable way to confirm the amplified PCR product is sequencing. As a result, the amplified PCR product was cloned andthe plasmid was extracted and sent to sequencing. However, the lack of quantitative information obtained with the end point PCR, which isimposed by the legislation requirements, the real-time PCR strategies is developed for the quantitative detection in GM foods.

Poster 125

ANTIMICROBIAL ACTIVITY OF DUABANGA GRANDIFLORA

M OTHMAN1, H S LOH2, C WIART1, T J KHOO1 AND K N TING1

1School of Pharmacy, 2School of Bioscience, University of Nottingham Malaysia Campus, Semenyih, Selangor

The search for new anti-infection agents is becoming increasingly important with the emerging problems of resistance to many of the currentavailable antibiotics. There are evidences that higher plants exhibit promising and to some extent potent antimicrobial activity. In this study,we have investigated a rainforest plant named Duabanga Grandiflora (Sonneriataceae) for its potential antimicrobial activity. The extractionof the plants was carried out as described previously using hexane (H), ethyl acetate (EA) and ethanol (E). Crude extracts from these solventswere then tested for their potential antimicrobial activity using two different methods: (1) pour plate disc diffusion (PPDD)3 and (2)turbidometric (TB). The bacterial strains tested included; gram negative Escherichia coli and gram positive Staphylococcus aureus.Streptomycin and ampicillin were used as positive controls. Results obtained from the PPDD method showed that both the EA and E extractsinhibited the growth of both bacteria in a dose-dependent manner. E. coli treated with 3, 1 and 0.3mg of EA extracts produced 18.3, 14.3 and11.3mm inhibiton diameter (ID) respectively. Whilst Staph. aureus incubated with same doses of the EA extract exhibited 18, 13 and 9.7mmID respectively. Ethanol crude extract of Duabanga grandiflora tested on E. coli at 3, 1, 0.3 and 0.1mg dose produced ID of 18.3, 14.7, 11.7and 8.7mm respectively. The same extract at the same doses were tested on Staph. aureus and the ID obtained were 18.4, 16, 12.3 and 9.3mm.A much lower dose of E crude extract at 0.03mg also inhibited growth of Staph. aureus with ID of 7mm. E. coli and Staph. aureus incubatedwith streptomycin at 30, 10, 3 and 1μg produced respective ID of 21.4, 20, 16 and 12 for E. coli and 30.4, 27.6, 22.7 and 21 for Staph. aureus.Ampicillin at the same doses exerted ID of 19, 13.7, 8.3 and 0 for E. coli and 17.7, 15.4, 14 and 0 for Staph. aureus. In the TB assay, crudeextracts obtained from all 3 solvents exhibited antimicrobial activities in a concentration-dependent manner against both types of bacteriaafter a 24-h incubation period. Survival index (SI) was calculated from the TB graphs and showed that crude extracts from H, EA and E at aconcentration of 3mg/ml inhibited growth of E. coli with SI of 80, 2.8 and 7.6 respectively. SI for Staph. aureus treated with same extracts at3mg/ml were 8, -0.2 and -8.3. Streptomycin at 10μg/ml gave a SI of 1.1 for E. coli and 1 for Staph. Aureus. Ampicillin at 10μg/ml on the otherhand, gave a SI of 2.1 and 1.5 for E.coli and Staph. Aureus, respectively. In conclusion, crude extracts from Duabanga grandiflora possessedunequivocal dose-dependent antimicrobial actions with increasing activity observed in the following fractions: ethanol > ethyl acetate >hexane. Further fractionation and separation of these crude extracts is warranted to elucidate the chemical entities of these antibacterial agents.


Poster 126

AUGMENTATION OF ADIPONECTIN (ACRP30) SECRETION IN 3T3-F442A ADIPOSE CELLS BYETHANOL EXTRACTS FROM SCOPARIA DULCIS LINN.

MUHAJIR HAMID1*, JALIFAH LATIP2, MOHD PUAD ABDULLAH3, ZAINAH ADAM4 AND BEH JOO EE1

1Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, UPM, 43400 UPM Serdang, Selangor.2School of Chemical Science and Food Technology, UKM, 43600 UKM Bangi, Selangor. 3Department of Cell and Molecular

Biology, Faculty of Biotechnology and Biomolecular Sciences, UPM, 43400, Serdang, Selangor. 4Radiation processingtechnology, Malaysian Nuclear Agency (Nuclear Malaysia) Bangi, 43000 Kajang, Selangor.

* Corresponding author: 03-89466707

Adiponectin (Acrp 30) is a crucial hormone which is secreted by adipocytes, plays a pivotal role in regulating cholesterol synthesis, lipidoxidation and glucose uptake activity epidemiologically ameliorates cardiovascular diseases, obesity and Type 2 diabetes. In order toinvestigate whether Scoparia dulcis Linn could promote adiponectin secretion from fully differentiated adipocytes, 3T3-F442A adiposecells were treated with five concentrations (0.05, 0.1, 0.2, 0.4 and 0.6 mg/ml) of ethanol extracts of Scoparia dulcis leaves and roots and incombination with either 50 nM or 100 nM insulin using Enzyme-Linked Immunosorbent Assay (ELISA). It was found that the treatmentcombination of 0.2 mg/ml ethanol extract of Scoparia dulcis roots with insulin (50 nM and 100 nM) significantly (p< 0.05) increasedadiponectin secretion (≥6 folds) compared to control. Amazingly, this augmentation of Acrp30 secretion by 0.2 mg/ml ethanol extract ofScoparia dulcis roots is in an insulin-independent manner. These data suggest that ethanol-soluble active compound(s) in Scoparia dulcisroot enhance Acrp30 secretion and potentially improve metabolic syndromes in body.

Poster 127

ANTIOXIDANT PROPERTIES OF GNETUM GNEMON L., (GNETACEAE) STICK CRUDE EXTRACTS

DAYANA W., RADZALI M., JOHARI R., MAZIAH M. AND SYAHIDA A.

Natural Product Biochemistry Labarotary, Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences.Universiti Putra Malaysia, 43400UPM Serdang, Selangor; Malaysia.

Fax no:603-89430913 & E-mail:radzali @ biotech. upm.edu.my.

Gnetum gnemon is a family of Gnetaceae and is locally known as ‘melinjo or belinjo’. It has been grown well several years ago in the SouthEast Asia countries such as some parts of Indonesia, Malaysia, Thailand and Philippines for small scale food industries such as simple foods,biscuits or crispy chips. This plant species from mature fruits to young leaves or shoots is also widely found having as anti-asthmaticproperties, anti-inflammatory properties and reduce constipation. The antioxidant properties of G. gnemon,.L, stick were biochemicallystudied using DPPH scavenging assay using different organic solvents. The stick of G.gnemon were biochemically extracted using methanol,ethanol, water, hexane and chloroform solvent. The highest antioxidant activity was in methanol extract (28.35%) and the lowest was inhexane extract (17.37%) at concentration of 300ug/ml. The result showed stick of G.gnemon have ability to scavenge free radical. From thisstudy, we expect local G.gnetum could be another potential source of natural dietary antioxidant to replace several synthesized antioxidantthat might have carcinogenic effects and also in an application as a new natural skin-whitening agent.

Poster 128

ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS EXTRACTED FROM FRESH FLOWER ANDLEAF TISSUES OF CANANGA ODORATA (Hook. F. & Thomson)

NURAZAH Z., RADZALI M., JOHARI R. AND SYAHIDA A.

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,University of Putra Malaysia, 43400 UPM Serdang, Selangor.

E-mail: [email protected]

Cananga odorata (Hook. F. & Thomson) from Annonaceae family which is locally known as ‘kenanga’ or ‘ylang-ylang’ is widely used foraromatherapy and in perfumery industries. The high value of essential/volatile oils (fragrance) from C. odorata, which is commonlyknown as ylang-ylang oil is one of good natural sources that shows many biological properties since ancient times. The essential oils fromfresh flower and leaf of C. odorata were extracted by hydro-distillation process using simultaneous distillation extraction (SDE) modifiedwith Likens-Nickerson apparatus. The yield of essential oil extraction with n-pentane solvent from fresh flower and leaf tissues were 0.92± 0.31 and 0.23 ± 0.11% respectively. The antioxidant properties were evaluated through three antioxidant assays; 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, ferric reducing antioxidant power (FRAP) assay and beta carotene bleaching assay. The flowerand leaf essential oil showed moderate antioxidant activity in DPPH scavenging assay with 40.57 ± 2.72 and 40.88 ± 3.07% of antioxidantactivity respectively, each at concentration of 100μg/ml. At the same concentration, flower essential oil showed only some antioxidantactivity in FRAP assay with 21.04 ± 1.34 and 22.50± 1.48% of antioxidant activity from leaf essential oil. In beta carotene bleachingassay, antioxidant activities for both essential oils were quite comparable to the positive standards (BHT, linalool and eugenol) that hadbeen used. The antioxidant activities in beta carotene bleaching assay were 61.83 ± 0.53 and 57.20 ± 1.04% from flower and leaf essentialoils, each at concentration of 100μg/ml, respectively. From this study, volatile oils (essential oils) from C. odorata fresh flower and leaftissues possessed some potent antioxidant properties.


Poster 129

ANTIOXIDANT PROPERTIES OF THE EXTRACTS FROM DIFFERENT AERIAL PARTS OFBARRINGTONIA RACEMOSA L. (PUTAT KAMPUNG)

NURUL MARIAM H., RADZALI M., JOHARI R. AND SYAHIDA A.

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra malaysia, 43400 UPM,SERDANG, Selangor, Malaysia. E-mail : [email protected]

The antioxidant properties of extracts from leaf, stick and bark tissues of Barringtonia racemosa L. were studied. The antioxidantproperties of boiling water, methanol and ethanol extracts from this plant were screened for their 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical scavenging capacity, reducing power and β-carotene linoleate model system including total phenolics, flavonoids and carotenoids.Methanolic extracts of all aerial parts contained relatively higher level of total phenolics than other extracts (leaf:16.2 ± 0.02 mg GAE/gFDW tissue, stick:29.9 ± 0.02 mg GAE/g FDW tissue, bark:21.78 ± 0.20 mg GAE/g FDW tissue) and ethanolic extracts in all aerial partsgave higher level of total flavonoids (leaf: 60.57 ± 4.31 mg Quercetin/g FDW tissue, stick: 59.04 ± 4.99mg Quercetin/g FDW tissue, bark:96.61 ± 1.73 mg Quercetin/g FDW tissue). The amount of β-carotene was found higher in methanolic and ethanolic extracts of leaf part(342.2 ± 8.79 μg β-carotene/g FDW tissue; 356.9 ± 0.93 μg β-carotene/g FDW tissue) respectively. The methanolic and ethanolic extractsin all aerial parts exhibited very strong antioxidant properties when compared to standard antioxidants such as butylated hydroxyl toluene(BHT), ascorbic acid (Vitamin C) and α-tocopherol in the DPPH radical scavenging, reducing power and β-carotene linoleate assays atconcentration of 80μg/ml. These results demonstrate that methanolic and ethanolic extracts of different aerial parts of B.racemosa mayhave the potential to be used as antioxidant agents compared to the boiling water extracts which showed the lowest antioxidant activity.

Poster 130

MOLECULAR MODELLING OF UTP-GLUCOSE-1-PHOPSHATE URIDYLYLTRANSFERASE OFEUCHEUMA DENTICULATUM

1,2MOHD NORHISAMo HAMID, 1,2ZETI AZURA MOHAMED HUSSEIN AND 1,2ROOHAIDA OTHMAN1School of Biosciences & Biotechnology, Faculty of Science & Technology, Universiti Kebangsaan Malaysia 43600 UKM Bangi.

2Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia 43600 UKM Bangi

UGPase is a key enzyme that catalyzes the formation of UDP-glucose, which is a precursor in the synthesis of polysaccharide. This enzymecatalyzes the reversible formation of UDP-glucose and pyrophosphate from glucose-1-phosphate and UTP in an Mg2+-dependent reaction.Therefore, with the objective of deducing comparative structural models, we have described a molecular model emphasizing the uniqueness ofUGPase from Eucheuma denticulatum that should generate insights into the structural distinctiveness of this protein as compared to structurallyresolved UGPase from other species. Sequence and structure analyses were conducted to provide detailed information of the protein and toidentify important residues that involve in the biological activity of UGPase. X-ray resolved structures of UGPase from Arabidopsis thaliana areavailable from Protein Data Bank (PDB). Hence, we modeled UGPase from Eucheuma denticulatum using comparative modeling strategy. Sincethe protein structures used for the alignment and modeling have well conserved structural motifs and regions, and functional information is alsoavailable, the problem of low sequence identity was overcome. The quality of the sequence alignment and the optimization of gaps were verifiedwith the secondary structure corresponding positions. The templates were aligned and were used as a profile for aligning the target sequence andto obtain an alignment, which was then used for modeling UGPase. Three-dimensional structure of this protein was generated usingSWISSModel, with 2ICY, 2ICX and 1Z90 as the templates. Ramachandaran plots were generated of every model produced. The protein structuregenerated from the first approach mode has the best quality with 87.2% residues in most favored region and 12.0% residues in additional allowedregions. Verify3D was then applied and showed that the protein structure built from the first approach has a good scoring average and stable thanthe other protein structures. Two important domains were identified i.e. catalytic centre domain and sugar binding domain, which play animportant role in the function of this protein. The catalytic centre domain functions as a positively charged active site, which interacts with thenegative charges of the UDP-glucose. From the multiple sequence alignment, a conserved region was identified, i.e. nucleotide binding loopregion which is crucial in binding with the nucleotide. Moreover, some important residues were recognized in this study (Lys130, His225, Lys289and Lys397) and these residues are responsible in interacting with the charges in the substrate.

Poster 131

DENGUE VIRUS UP-REGULATES THE PROMOTER ACTIVITY OF THE MHC CLASS I GENE

SHATRAH OTHMAN1, NOORSAADAH ABDUL RAHMAN2, ROHANA YUSOF1

1Department of Molecular Medicine, Faculty of Medicine, 2Department of Chemistry, Faculty of Science, University of Malaya,50603 Kuala Lumpur, Malaysia.

Mammalian cells express surface major histocompatibility complex class I (MHC-I) molecules, which is crucial for immune recognitionand MHC-restricted cytolysis as part of the adaptive immune system. In contrast to most viruses that have little or no effect on the level ofMHC expression and a few that decrease the MHC expression or antigen processing and presentation, infections by Flaviviruses such asWest Nile Virus, have been associated with a virus-specific increase of major histocompatibility complex class I (MHC-I) molecules onthe cell surface. In dengue virus infection, the mechanism by which the virus regulates the MHC-I remains elusive. We have previouslyreported the development of a system that employs the dual luciferase assay to analyze the modulation of MHC-I gene transcription, themajor determinant of the level of MHC molecule synthesis and expression. Here, pHLA firefly luciferase reporter construct containingfull-length MHC-I promoter were transiently transfected into the HepG2 human liver cells, together with a second reporter vectorexpressing the Renilla luciferase (pRen) which is used for normalization purposes. The cells were then treated and incubated with eithermedium alone (mock-infection) or with one of the four serotypes of dengue virus. Following incubation, the cells were lysed and the ratioof the firefly:renilla luciferase activities were determined. In this study, we have demonstrated that all serotypes of dengue virus produceda higher firefly:renilla luciferase ratio when compared to the mock-infected cells. This indicates an up-regulation of the pHLA luciferaseactivity that is due to an induction on the MHC-I promoter. The level of induction, however, varies among the serotypes. Increase in thepromoter activity results in the up-regulation of transcription and translation of the MHC-I gene. It is postulated that upon infection bydengue virus, there is increased DNA-binding protein which may be viral or non-viral of origin that targets for certain transcriptionregulatory elements within the -427bp to -1 of the MHC-I promoter. Several aspects of the promoter induction will be discussed. Furtherinvestigation is also currently undertaken to identify the regulatory element and the DNA-binding proteins involved.


Poster 132

DNA FINGERPRINTING OF MARINE BACTERIA FROM SPONGES ATKARAH ISLAND, TERENGGANU

NORAZNAWATI I ., SHAZANI S., MOHD-NUR-FIRDAUS A. L., CHEW M. Y. AND LUKMAN-HAKIM M. D.

Biological Sciences, Department, Faculty of Science and Technology,Universiti Malaysia Terengganu, 21300 Kuala Terengganu, Malaysia.

[[email protected]]

The bacteria associated with sponges have attracted great interest worldwide. In order to understand the association of marine bacteria withsponges, molecular tools such as Random Amplified Polymorphic DNA (RAPD) were used to investigate the bacterial community. Applicationof DNA fingerprinting was used to identify DNA-based genetic polymorphism for determining the genetic variability and degree of polymorphismof marine bacteria from sponges. In this study, sponges from Karah Island, Terengganu had been focused at depths of 5 to 10 meters. Some of theRAPD profiles show mono and polymorphic patterns for each primers screened, OPA-07, -08, -10, OPB-05, -06, -07 and OPC-02 that producedclear and reproductive bands. There are 51 polymorphic RAPD profiles from the selected primers. The overall polymorphism among spongeswas 60.18% and the productive RAPD marker was in the range of 100-3200 bp. The mean similarity index of the marine bacteria associated withsponges was 0.43 ± 0.11 and genetic distance obtained was 0.33 – 0.67. Genetic variety was high as no same genotypes present. Further studiesshould ideally be integrated with more sponges species and primers to obtain precise results of the degree of polymorphism.

Poster 133

CHANGES IN LIPID PROFILE, SERUM LEPTIN AND ANTHROPOMETRY MEASUREMENTSAMONG OBESE ADULTS IN A WEIGHT LOSS PROGRAM

MAT LUDIN CHE MAT1, WAN MANAN WAN MUDA2, HASENAN NORDIN3, ROHANA JALIL3 AND WAN SURIATI WAN NIK2

1School of Dental Sciences, USM Health Campus, Kubang Kerian Kelantan 2School of Medical Sciences, USM Health Campus,Kubang Kerian Kelantan, 3School of Health Sciences, USM Health Campus, Kubang Kerian Kelantan

A low fat diet and low calorie intake together with a structured exercise are believed able to reduce weight and lower the lipid profile whichis one of the contributing factor to heart disease among obese adults. However, the effectiveness of this kind of structured exercise to thepeople in Asia, including Malaysia is not clear. It may be due to the varied food preferences and way of life and habits of the Asians which arequite different with those in the West, thus their fluctuating body weight. This research was carried out to assess the lipid profile, serum leptinlevels and anthropometric measurements before and after a 12 week weight loss program among obese adults using a selective diet andexercise approach without the use of any medications and drugs. 67 participants that fulfilled the required criteria (adult 18 years and above,BMI 28.0 kg/m2, female, not pregnant, not on any medication, supplements or drugs and certified fit by a doctor to participate in the 12 weeksweight loss program) were selected from HUSM and Pasir Mas in Kelantan. All participants agreed to abide to the rule and regulations as perthe programmed procedures. During the intervention period, participants were required to perform 30 minutes aerobic exercises in themorning and jogging for 2 kilometer or brisk walking in the evening. Participants were monitored and advised to take a low fat and low sugardiets during the entire 12 week program. Anthropometric measurements, lipid profile, serum leptin and blood sugar samples were taken fromthe participants before and after the 12 week intervention periods and analyzed. The investigation showed that Total serum cholesterol andtriglyceride levels were significantly reduced, whereas HDL level and cholesterol ratio to HDL LDL which is the indicator for CVD, wereincreased among the obese participants whose body weight had reduced during the 12 weeks intervention but the control participants showedno significant changes. Body weight, BMI and body fat percentage of participants were also reduced significantly as compared to the controlgroup. The serum leptin level of the participants showed a reduction with the lowering of the body weight. Based on all measured parameterswe found that participants from Pasir Mas area have shown atypically significant changes from participants in HUSM. Thus, a weight lossprogram using a selective diet and exercise approach has certainly shown a significant impact to the changes of lipid profile levels, serumleptin, anthropometry measurements and reduction of serum glucose among obese adults.

Poster 134

INTERACTION OF THE CHAMPEDAK GALACTOSE BINDING LECTIN WITH O-LINKEDOLIGOSACCHARIDES OF HUMAN CHORIONIC GONADOTROPIN

SITI SHAHIRAH CHE MURAT1, ONN HAJI HASHIM2, SOFIAH SULAIMAN3 AND ADAWIYAH SURIZA SHUIB1

1Institute of Biological Sciences, Faculty of Science, 2Department of Molecular Medicine, Faculty of Medicine and3Department of Obstetrics and Gynecology, Faculty of Medicine, University Malaya, Kuala Lumpur

Human chorionic gonadotropin (hCG) is a glycoprotein hormone containing two non-covalently-linked polypeptides. Both the α and β subunitsof hCG contain two N-lnked oligosaccharides, while the β-subunit has four extra O-linked oligasaaccharides. In this study, SDS-PAGE, 2DE andWestern blotting were done to determine the binding specificity of the champedak galactose binding lectin (CGB) towards the O-linkedoligosaccharides of urinary hCG. The CGB lectin was purified from the seeds of Atocarpus integer using galactose affinity chromatography.Urine samples were obtained from women who were pregnant and those with molar pregnancy. Purified hCG were subjected to separation by2DE and CGB lectin blotting. Image Master 2D Platinum Software 6.0 was used for analysis of the 2DE gels. Different profiles were observedwhen purified hCG samples, urine of normal unpregnant and pregnant women, and those with molar pregnancy were analysed. With exceptionof normal urine samples, all other samples demonstrated three protein bands. Urine of normal pregnant women and those with molar pregnancydemonstrated two extra bands. When analysed by 2DE and lectin blotting, only two clusters of protein spots appeared. These clusters were mostprobably the β subunits or their fragments. Futher analysis will be done to identify the isoforms of hCG which can be recognized by the CGBlectin before the latter can be used as probe to detect differences in urine hCG isoforms.