Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 25 ABSTRACTS OF THE 33RD ANNUAL CONFERENCE OF THE MALAYSIAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY 27TH & 28TH AUGUST 2008, ISTANA HOTEL, KUALA LUMPUR
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 26Poster 1 AFFINITY CHROMATOGRAPHY TO STUDY INSECT GLUTATHIONE S-TRANSFERASES ZAZALI ALIAS1, ALAN CLARK, DING XUE, MILANA MALIUK AND RAMAVATI PAL 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia School of Biological Sciences, Victoria University of Wellington, Wellington, New ZealandIn 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 PASSAGE OF 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 1 Department of Physiology, Medical Centre, Universiti Kebangsaan Malaysia 2 Department of Obstetric & Gynecology, Medical Centre, Universiti Kebangsaan Malaysia, 3 Department of Pathology, Medical Centre, Universiti Kebangsaan Malaysia 4 Department of Human Anatomy, Faculty of Medicine and Allied Sciences, Universiti Putra MalaysiaHuman 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 FLUORESCENT INTENSITY 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 SembilanMammalian 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 27 Poster 4 THE RELEASE OF RECOMBINANT N PROTEIN OF NIPAH VIRUS FROM ESCHERICHIA COLI BY BEAD-MILLING SITI SARAH MOHD. NOOR1, WEN SIANG TAN2,3, TAU CHUAN LING3,2 AND BENG TI TEY1,3,* 1 Institute of Bioscience, 2Faculty of Biotechnology and Molecular Science, 3 Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, SelangorNipah 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 TEMPORARILY HYPERBILIRUBINEMIC 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 LumpurThe 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,* 1 Faculty of Engineering & Science, Universiti Tunku Abdul Rahman, Setapak, 53300 Kuala Lumpur, 2 Institute of Biological Sciences, Universiti Malaya, 50603 Kuala Lumpur * firstname.lastname@example.orgHydrocotyle 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 28Poster 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, ThailandHemocyanin (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 BY USING 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, MalaysiaRice 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 1 School of Biosciences & Biotechnology, Faculty of Science & Technology; 2 Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, National University of Malaysia, 43600 UKM Bangi, SelangorThe 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 29 Poster 11 QUANTITATIVE EXPRESSION OF IRON SUPEROXIDE DISMUTASE IN LEGIOENLLA PNEUMOPHILA USING REAL-TIME REVERSED TRANSCRIPTASE RT-PCR AUDREY KOW SIEW FOONG1, STACEY YONG FOONG YEE1 AND NG KIM YONG2 1 School of Science, Monash University Sunway Campus Selangor; 2Malaysian University of Science and Technology, SelangorLegionella 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 1 Department of Molecular Medicine and 2Department of Obstetrics and Gynaecology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaInfection 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-DEPENDENT APOPTOSIS IN HUMAN BREAST CANCER CELLS MCF-7 CHEAH YEW HOONG1, TEE THIAM TSUI1, NOOR RAIN ABDULLAH2 AND AZIMAHTOL HAWARIAH LOPE PIHIE11 School 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 LumpurThe 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 30Poster 14EFFECTS OF THE TREATMENT PERIOD WITH GLYCYRRHIZIC ACID ON 11β-HYDROXYSTEROID DEHYDROGEANSE TYPE 1 ACTIVITY IN RATS CHIA YOKE YIN1, TON SO HA1 AND KHALID BIN ABDUL KADIR2 1 School of Science, 2School of Medicine and Health Sciences, Monash University Sunway CampusGlycyrrhizic 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 EXCHANGE CHROMATOGRAPHY CHIN IUAN SHEAU1, 2, ABDUL MUNIR ABDUL MURAD1, 2, SHEILA NATHAN1,2, NOR MUHAMMAD MAHADI1, 2 AND FARAH DIBA ABU BAKAR1Faculty of Science and Technology, Universiti Kebangsaan Malaysia1; UKM-MTDC Smart Technology Centre, Malaysia Genome Institute2Cutinase, 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 AND TYPE 2 ACTIVITIES CHOH LEANG CHUNG1, CHIA YOKE YIN1, TON SO HA1 AND KHALID BIN ABDUL KADIR2 1 School of Science, 2School of Medicine and Health Sciences, Monash University Sunway CampusElevated 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 31 Poster 17 EXPRESSION AND PURIFICATION OF EIMERIA TENELLA RECOMBINANT GPI-ANCHORED VARIANT SURFACE PROTEINS CHOW YOCK PING1, WAN KIEW LIAN1,2 AND SHEILA NATHAN1,2* 1 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia 2 Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, Bangi, SelangorEimeria 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 SEED EXTRACT AGAINST DAMAGES CAUSED BY NAJA NAJA SPUTATRIX VENOM N.H. TAN1, S.Y. FUNG1 AND S.M. SIM2 1 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaSeed 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 1 Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur 2 Institute of Medical Research, Jalan Pahang, 50588 Kuala LumpurThe 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 32Poster 20 DETECTION OF PHYTOCHELATIN SYNTHASE IN FUNGI ASSOCIATED WITH MANGROVE NOOR AFIZA BADALUDDIN, MARIAM TAIB, AZIZ AHMAD AND JAMILAH MOHD SALIM @ HALIMDepartment of Biological Sciences, Faculty of Science and Technology,Universiti Malaysia Terengganu, 21030 Kuala TerengganuPhytochelatins 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 AND BRAINS 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, SelangorWidespread 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 HEALTHY ADULT SUBJECTS ZAMRI CHIK1, ROMA C. BASU1, ROKIAH PENDEK2, LEE TOONG CHOW3 AND ZAHURIN MOHAMED1 1 Department of Pharmacology, 2Department of Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur 3Info Kinetic Sdn. Bhd., Kompleks Eureka, USAINS Holding, 11800 USM, PenangRifampicin 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 . 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 33 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 AND HAYATI ABDUL RAHMAN3 1 Department 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@example.comThe 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-STEROID HORMONE ASSOCIATED CANCERS? EMIDA MOHAMED1, PUTERI SHAFINAZ ABDUL-RAHMAN1, SITI ZAWIAH OMAR2 AND ONN HAJI HASHIM1 1 Department of Molecular Medicine and, 2Department of Obstetrics & Gynecology, Faculty of Medicine, University of Malaya, Kuala LumpurSex-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 AND ABU BAKAR SALLEH1 Enzyme and Microbial Technology Research, 1Faculty of Biotechnology and Biomolecular Sciences, 2 Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MalaysiaThe 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 34Poster 26 EFFECT OF TARRENA SP. UPON MUSHROOM TYROSINASE ENZYME ACTIVITYFARIDA HARYANI AB. AZIZ1, SYAHIDA AHMAD1,2, NORDIN HAJI LAJIS2,3, KHOZIRAH SHAARI2,3 AND FARIDAH ABAS2,4 1 Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Biosciences, 3Faculty of Sciences, 4 Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, SelangorTyrosinase 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 LEAVES DNA 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 1 Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, USM, 11800, PenangHerbal 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 CULTURE DURING THE INDIVIDUAL BTEX EXPOSURE FELLIE EDWIN AMIR1, WONG KOK KEE1, ABDUL JALIL ABDUL KADER2, OTHMAN OMAR1, BRID QUILTY3 AND SALMIJAH SURIF1 1 School 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, IrelandGroundwater 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 35 Poster 30CONSTRUCTION AND ANALYSIS OF SMALL-INSERT GENOMIC LIBRARIES OF EIMERIA MAXIMA HALIMAH ALIAS1 AND KIEW-LIAN WAN1,2 1 Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, 43600 UKM Bangi, Selangor DE; 2 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor DEEimeria 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* 1 Department 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: firstname.lastname@example.orgType 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 WITH EXPOSURE 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, MalaysiaOxidative 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 36Poster 33 ESTABLISHED METHOD OF TWO DIMENSIONAL ELECTROPHORESIS TECHNIQUE FOR PROTEIN PATTERNS OF MICHELIA ALBA DC HASLIZA HASSAN1, RADZALI MUSE1, MOHD. PUAD ABDULLAH2, JOHARI RAMLI1, MOHD ROSNI SULAIMAN3 AND NOR ARIPIN SHAMAAN1 1 Department 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; MalaysiaAn 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 1 School 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 SembilanOil 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 NAPIS1Δ1 Cell 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, MalaysiaMany 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 37 Poster 37 THE T102C POLYMORPHISM OF THE SEROTONIN 2A RECEPTOR GENE IN MALAYSIAN PATIENTS WITH MAJOR DEPRESSIVE DISORDER MOHD IZWAN ZAINOL1, VIJAYA L RAJ2, ELSA HANIFFAH MEJIA MOHAMED2, NOR ZURAIDA ZAINAL3 AND ZAHURIN MOHAMED2 Dept of Molecular Medicine1, Dept of Pharmacology2 and Department of Psychological Medicine3, Faculty of Medicine, University of Malaya, Kuala Lumpur, MalaysiaMajor 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 USING FRESHWATER 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, MalaysiaLandfill 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 39BIOMASS NUTRIENT PROFILES OF THE MICROALGA CHLORELLA VULGARIS WITH DIFFERENT DRYING METHODS JUNAIDA @ MAIMUNAH HASSAN BASARI1, NOR ASHIKEEN MUKTI1, WAN ZURINAH WAN NGAH1, RAZALI SABUDDIN2, A. RAZAK MUDA3 AND YASMIN ANUM MOHD YUSOF11 Department of Biochemistry, Faculty of Medicine, 2Department of Management and Development, 3Department of Nutrition and Dietetic, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala LumpurChlorella 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 38Poster 41 SYNTHESIS, CHARACTERIZATION OF BENZENESULFONOHYDRAZONES AND THEIR BIOLOGICAL ACTIVITIES 1 LAILA MUSALAM, 1HAPIPAH MOHD. ALI, 1SHARIFUDIN M. ZAIN AND 2MAHMOOD AMEEN 1 Chemistry Department, Faculty of Science, 2Molecular Medicine Department, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. email@example.comBenzenesulfanohydrazide 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 FOR EMBRYOGENIC COMPETENCE? LEE FONG CHIN1, PARAMESWARI NAMASIVAYAM1, MEILINA ONG ABDULLAH2 AND HO CHAI LING11 Cell 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, MalaysiaSomatic 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 ON TRIGLYCERIDE LEVEL AND ADIPOSE TISSUE LIPOPROTEIN LIPASE ACTIVITY IN DIABETES INDUCED RATS LEE HUI WEN AND HALIMAH ABDULLAH SANI1 1 Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, SelangorDiabetes 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.
Malaysian Journal of Biochemistry and Molecular Biology (2009) 17(1) 39 Poster 44 MOLECULAR IDENTIFICATION AND CLONING OF STREPTOMONOSPORA SP. FROM MALAYSIA AND 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 RADU41Faculty 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 CELLULAR MODEL OF RHEUMATOID ARTHRITIS KA-HENG LEE1, SYAHIDA AHMAD1,2, FARIDAH ABAS2,3, KHOZIRAH SHAARI2,4, DAUD AHMAD ISRAF ALI 2,5 AND NORDIN HAJI LAJIS2,4 1 Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Biosciences, 3Faculty of Food Science and Technology,4 Faculty of Sciences, 5Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MalaysiaRheumatoid 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 1 School of Science, 2School of Medicine and Health Sciences; Monash University Sunway CampusPeroxisome 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.