Student Projects

Biological Sciences Graduate Congress 2005

The Biological Sciences Graduate Congress is organised by the Department of Biological Sciences, Faculty of Science, National University of Singapore.

Functional Genomics and Structural Biology (Oral)

I Prize:

IDENTIFICATION OF THE HUMAN SWEAT PROTEINS

Sravanthy Gopinath , Zhang Xin, Bi Xuezhi, Chew Fook Tim
Department of Biological Sciences, National University of Singapore, Singapore 117543
E-mail (presenting author): sravanthy@nus.edu.sg

The human sweat has been shown to contain diverse proteins, in addition to salts and urea. Several of these may have specific anti-microbial and other immune functions. To identify and analyze the putative role of sweat proteins we first obtained a 2-dimensional liquid chromatography tandem mass spectrometric (2D-LC-MS-MS) profile and 2D-gel electrophoresis (2DGE) proteome map of human sweat. Eccrine sweat from several individuals were collected, pooled and processed by exhaustive dialysis, lyophilized and precipitated. A proteome map was then obtained between pH 4-7. Subsequent in-gel digestion and tandem mass spectrometric analysis lead to the identification of each of these proteins. Via 2D-LC-MS-MS, we identified more than 300 components with two or more peptides matches. These included immunoglobulins (IgG; IgE), dermcidin precursor and lysozymes. Via 2DGE, multiple isoforms of major sweat proteins such as zinc alpha glycoprotein (ZAG), fatty acid binding protein-5, lactotransferrin, prolactin-induced protein, cystatin, proteinase inhibitor, clusterin and psoriasin, were identified. We have thus identified many human sweat components. Qualitative and quantitative variations of these proteins in pathologic conditions may lead to the detection of new biomarkers for specific diseases. Evaluation of their specific functions may also provide insights into sweatrelated disorders, auto-antigens and possible immuno-modulatory and anti-microbial molecules.

II Prize:

NANODROPLET MICROARRAYS AS HIGH-THROUGHPUT ENZYMATIC SCREENING TOOLS

Mahesh Uttamchandani ¹ , Yao Shao Qin ^1,2
1 Department of Biological Sciences, National University of Singapore, Singapore 11754
² Department of Chemistry, National University of Singapore, Singapore 117543
E-mail (presenting author): mahesh@nus.edu.sg

The ability to rapidly screen huge libraries of compounds represents a major bottleneck in the discovery of biologically relevant molecules and drug leads. In response to this pressing need, we have developed novel microarray platforms that enable the large scale assessment of enzymes, both in activity-based profiling and in inhibitor discovery. One such platform – Nanodroplet microarrays– uses robotic printing to produce spatially addressed droplets on glass slides precoated with fluorogenic substrates.We tested our system using 37 representative enzymes and successfully obtained fluorescence readouts across temporal and stochiometric dimensions concurrently, on a single microarray substrate. We further exploited this strategy in the quick andcost-effective identification of potent molecules, which perturb target proteins meaningfully in an activity dependent manner.Specifically, a 400-member library of putative metalloprotease inhibitors was synthesized combinatorially and designed with a potent zinc-binding group to target these compounds to the active site of this important class of enzymes for thermolysin. Overall, this strategy offers not only a rapid method for functional profiling and enzyme characterization, but also a viable format for identifying bioactive compounds in drug discovery.

III Prize:

STRUCTURAL STUDIES OF TYPE THREE SECRETION VIRULENCE FACTOR FROM THE PATHOGENIC BACTERIUM AEROMONAS HYDROPHILA

Yvonne Tan Yih Wan, Yu Hongbing, Leung Ka Yin, Jayaraman Sivaraman, Mok Yu-Keung Henry
Department of Biological Sciences, National University of Singapore, Singapore 117543
E-mail (presenting author): g0306264@nus.edu.sg

Aeromonas hydrophila is an opportunistic bacterial pathogen that infects a variety of aquatic and terrestrial animals including humans. Like many other gram negative pathogenic bacteria, Aeromonas hydrophila relies on type three secretion systems to translocate virulence factors into the host cell. A series of proteins from the TTSS were selected for structural studies, among them were chaperone AcrH, translocators AopB and AopD and AscE of unknown function. AscE was expressed, purified and crystallized. The structure of AscE will be solved using SAD experiment. We suspect that AscE functions as a chaperone for two proteins, AscG, which is functionally unknown and AscF which is a syringe forming protein. AscF has a tendency to self polymerize, however this formation is not seen when it is attached to AscE. AscG is expressed as an insoluble protein but is soluble when attached to AscE.

Functional Genomics and Structural Biology Posters

I Prize:

CBASS: A CURATED DATABASE OF ASIAN SEA BASS SEQUENCES

Chee Choong Hoh , Nurilda Hayati Misnan, Nurul Yuziana Mohd
Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia 43600
E-mail (presenting author): ivan.hoh@gmail.com

Random sequencing from cDNA and genomic libraries, known as Expressed Sequence Tag (EST) and Genome Survey Sequence (GSS) respectively, provides a useful source of information for studies such as gene discovery, comparative genomics, gene expression profiling and genome annotation. By adopting both the EST and GSS approaches, we have initiated an effort to study the genome of the economically important Asian sea bass, Lates calcarifer . A total of 14,869 high quality EST and 7,288 GSS sequences were generated respectively from nonnormalized cDNA (of spleen, liver, brain and muscle tissues) and genomic libraries from the Asian sea bass. Even though EST and GSS are documented to be excellent approaches in gene discovery and genome studies, the sheer volume of data involved are often overwhelming. In addition, these sequence data are normally redundant and without sufficient curation. In addressing these issues, we have developed CBASS (a Curated dataBase of Asian Sea bass Sequences), a novel database which consists of annotated non-redundant EST and GSS sequences. Raw sequence data generated were subjected to stringent clustering protocols and subsequently 10^th assembled into a non-redundant dataset of clusters known as Unique Transcripts (UTs). These novel UTs were then curated manually based on information inferred using a repertoire of bioinformatics tools. Annotated features include gene expression profile, putative functions and domain structures, conserved motifs, putative homology to model organisms, and association with Gene Ontology functional groups. In addition to being an efficient data storage and retrieval system, CBASS provides a comprehensive and an in-depth curation of the plethora of sequences from the Asian sea bass.

II Prize:

CRYSTAL STRUCTURE OF DENMOTOXIN, A SPECIES-SPECIFIC POSTSYNAPTIC NEUROTOXIN FROM THE VENOM OF COLUBRID SNAKE BOIGA DENDROPHILA

Joanna Pawlak¹, Enrico Stura², Madhav Bhatia³, André Ménez² , R Manjunatha Kini^1
1 Department of Biological Sciences, National University of Singapore, Singapore 117543
² Département d’Ingénierie et d’Etudes des Protéines, Commissariat à l’Energie Atomique, Gif-sur-Yvette Cedex, France
³ Department of Pharmacology, National University of Singapore, Singapore 117543
E-mail (presenting author): scip1336@nus.edu.sg

Crystal structure of Denmotoxin, isolated from colubrid snake Boiga dendrophila (Mangrove catsnake) was solved at 1.9Å by molecular replacement method and refined to an R-factor of 20.2%. Denmotoxin polypeptide chain is organized in three finger-like β stranded loops emerging form the globular core, stabilized by four conserved disulphide bridges. The first loop possesses an additional (fifth) disulphide bond; inferring Denmotoxin to be a member of non-conventional toxin class. Competitive binding experiments showed Denmotoxin’s ability to bind mice muscular-type α1βγδ and chimeric neuronal α7 acetylcholine receptors (AChRs). In the organ bath experiments, Denmotoxin displayed postsynaptic neuromuscular activity by irreversible inhibition of indirectly stimulated twitches of chick biventer cervicis nerve-muscle preparations. However, it induced significantly smaller and readily reversible inhibition effect on electrically evoked twitches of the mouse hemi-diaphragm nerve-muscle preparations. This data indicates species specific differences of acetylcholine receptors in susceptibility to this postsynaptic neurotoxin with chick α1βγδ muscle AChR being up to 100-fold more sensitive as compared to that of mice. Interestingly, Boiga dendrophila being an arboreal inhabitant of the lowland rainforests and mangrove swamps preys mostly on birds suggesting the correlation between toxin specificity with target prey population forming the snake’s diet.

III Prize:

PROTEOMIC ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN LITOPENAEUS VANNAMEI STOMACH CELLS AFTER WHITE SPOT SYNDROME VIRUS INFECTION

Hao-Ching Wang¹, Han-Ching Wang², Shao-En Peng², Chu-Fang Lo², Shyh-Horng Chiou^1
1 Institute of Zoology, National Taiwan University, Taipei, Taiwan
² Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
E-mail (presenting author): d92b46001@ntu.edu.tw

To better understand the pathogenesis of white spot syndrome virus (WSSV) and to determine which cell pathways might be affected after WSSV infection, two-dimensional gel electrophoresis (2-DE) was used to produce protein expression profiles from samples taken at 48 hpi from the stomachs of Litopenaeus vannamei that were either specific pathogen free or else infected with WSSV. Seventy-five protein spots that consistently showed either a marked change (>50%) in accumulated levels or else were highly expressed throughout the course of WSSV infection were identified by in-gel trypsin digestion followed by LC-nanoESI-MS/MS and a search of bioinformatics databases. A total of 72 proteins were positively identified. Many of these proteins have roles in cellular responses such as energy production (WSSV infection induced up-regulation of fructosebiphosphate aldolase, glycoaldehyde-3-phosphate dehydrogenase, enolase, cytochrome oxidase polypeptide VIb, and the alpha and beta subunits of mitochondrial ATP synthase) and calcium homeostasis (up-regulation of sarco/endoplasmic reticulum-type calcium-transporting ATPase and calreticulin, down-regulation of a calcium binding protein of the invertebrate sarcoplasmic calcium-binding protein family). This study is the first global analysis of differentially expressed proteins in WSSV-infected shrimp and is an important first step in investigating the molecular pathogenesis of this virus-associated shrimp disease.

Biochemistry, Physiology & Biotechnology (Oral)

I Prize:

IDENTIFICATION OF PEPTIDES SPECIFIC FOR DEXTRAN BINDING AND THEIR USE AS AFFINITY TAG FOR PURIFICATION ONTO SEPHADEX® GELS

Surisa Suwannarangsee ¹ , Magali Remaud-Simeon ² , Warawut Chulalaksananukul 3
¹ Biological Science Program, Chulalongkorn University, Bangkok, Thailand 10330
² Laboratoire de Biotechnologies - Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, 135 avenue de Rangueil, 31077 Toulouse Cedex 4, France
³ Department of Botany, Chulalongkorn University, Bangkok, Thailand 10330
E-mail (presenting author): surisa_b@yahoo.com

Principally Leuconostoc mesenteroides and oral Streptococcus sp. produce glucansucrases. They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. All of glucansucrases possess the same organizations which compose of 4 domains. One interesting domain is a domain often referred as C-terminal glucan binding domain (GBD). The function of C-terminal domain has not been totally elucidated yet. The objectives of our study are to investigate the precise role of GBD in glucan binding and to generate shortest fragment that could be employed as a tag for affinity chromatography onto available Sephadex® supports. To that aim, the C-terminal domain of dextransucrase from Leuconostoc mesenteroides NRRL B512-F (DSR-S) was cloned and used as template to generate the truncated forms of GBD. Fourteen GBD variants were constructed and then estimated the dextran binding properties. We found that the DSR-S determinants responsible for glucan binding a re localized at the two third C-terminal end of GBD. This result was confirmed by affinity electrophoresis against a soluble dextran. Moreover, efficiency of the shortest GBD tag onto Sephadex® column was determined using thioredoxin as a model of study. Overall of results revealed that high levels of protein purity were obtained after simple run chromatographic purification. So, this GBD derivative tag considered as the appropriate and convenient tool for recombinant protein purification in Sephadex® gel column.

II Prize:

IN SILICO AND IN VITRO CEL48A ENGINEER IN CLOSTRIDIUM ACETOBUTYLICUM ATCC824

Virunanon C ¹ , Soucaille P ² , Chulalaksananukul W ^3
1 Biological Sciences Program, Chulalongkorn University, Bangkok, Thailand 10330
² Centre de Bioingénierie Gilbert Durand, Départment de Génie Biochimique et Alimentaire, INSA, UMR CNRS 5504. UMR. INRA. 792, 135, avenue de Rangueil, 31077 Toulouse, Cedex4, France
³ Department of Botany, Chulalongkorn University, Bangkok, Thailand 10330
E-mail (presenting author): knutz2@hotmail.com

The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulolytic substrates and associated plant cell wall. To date, seven genes coding for the components of Clostridium cellulolyticum cellulosome have been identified. It has recently been reported that celF of cellulase gene of Clostridium cellulolyticum is active on degraded swallen avicel more efficiently than substitued soluble CMcellulose or crystalline avicel (Reverbel-Leroy et al., 1996). These models of mesophilic clostridial cellulosomes have a large cluster which is very similar to that of Clostridium acetobutylicum ATCC824. In contrast, C. acetobutylicum is unable to grow on cellulolytic substrate even though it can produce cellulosomal complex (Sabathe et al., 2002). They concluded that C. acetobutylicum can produce cellulosome but inactive. For this reason, we tried to modify cel48A by direct mutagenesis method to restore the activity of Cel48A. We constructed new hybrid molecules of cel48A dockerin domain and cel48F catalytic domain. The results show that the purified hybrid Cel48A-Cel48F has proficient activity on all cellulolytic substrates that we inspected. Interestingly, the hybrid protein shows activity on crystalline cellulose higher than efficient native Cel48F for 3 fold (31.512 and 13.4 IU/µmol respectively). Overall results revealed that the chimera protein exhibited enhanced synergistic action on crystalline cellulose than in native catalytic domain of cellulase.

III Prize:

EFFECTS OF POLYMER SIZE AND CONCENTRATION OF CHITOSAN ON GROWTH AND PRODUCTION OF OKRA [ ABELMOSCHUS ESCULENTUS (L.) MOENCH ], INFECTION OF OKRAYELLOW VEIN MOSAIC VIRUS, AND FEEDING OF BEET ARMYWORM ( SPODOPTERA EXIGUA HÜBNER , 1808)

Wongchai C ¹ , Lotrakul P ¹ , Chadchawan S ¹ , Pichyangkura R ^2
1 Environment and Plant Physiology Research Unit, Department of Botany, Chulalongkorn University, Bangkok, Thailand 10330
² Department of Biochemistry, Chulalongkorn University, Bangkok, Thailand 10330
E-mail (presenting author): wongchaimee@hotmail.com

The application of chitosan for agricultural propose in Thailand has received much interest in recent years because of its potential as an agent for biological control and plant growth promoter. In this study, polymeric and oligomeric 80% DD chitosan (P80 and O80) and an uncharacterized commercial chitosan (UCC) were used at different concentrations (25, 50, and 100 ppm) as foliar spray on okra ( Abelmoschus esculentus (L.) Monech) cv. India 9701 and Yamato Green. The effects of chitosan on growth and production, infection of Okra yellow vein mosaic virus and feeding of beet armyworm ( Spodoptera exigua Hübner, 1808) were detected. It was found that P80 at 100 ppm and O80 at 25 ppm affected plant growth and production. Interestingly, O80 at 50 ppm slightly reduced the percent virus infection in cv. Yamato Green. Application of chitosan did not clearly affect the beet armyworm feeding. It was also found that at 50 ppm, UCC50 could significantly enhance the foliar proteinase inhibitor (PI) level of Yamato Green cultivar within 24 hour after treatment. These results clearly indicated that polymer sizes and concentrations of chitosan used differently affected growth and production of okra, viral infection and feeding of beet armyworm.

Biochemistry, Physiology & Biotechnology Posters

I Prize:

HEMEXTIN- A SNAKE VENOM PROTEIN COMPLEX THAT INHIBITS THE TISSUE FACTOR-FACTOR VIIA ACTIVITY

Yajnavalka Banerjee ¹ , Jun Mizuguchi ² , Sadaaki Iwanaga ² , R Manjunatha Kini ^1
1 Department of Biological Sciences, National University of Singapore, Singapore 117543,
² Blood Products Research Department, Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan 8691298
E-mail (presenting author): yajnavalka@gmail.com

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) present in the blood with freshly exposed tissue factor (TF) to form TF-FVIIa complex. Unwanted clot formation, however, can lead to death and debilitation due to vascular occlusion and hence anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences indicate that they belong to the three-finger toxin family. Hemextin A but not hemextin B exhibit mild anticoagulant activity. However, hemextin B forms a complex (hemextin) with hemextin A and enhances its anticoagulant potency. With the help of CD, ITC, MS, DLS and size-exclusion chromatography we have shown the formation of 1:1 complex between these two proteins. Studies with “dissection approach” and reconstituted extrinsic tenase showed that hemextin A and hemextin prolong clotting by inhibiting TF-FVIIa activity. Kinetic studies showed that hemextin inhibits the activity of FVIIa-sTF noncompetitively with a Ki of 50 nM. Hemextin is the first reported inhibitor of FVIIa from snake venom. Molecular interactions of hemextin with FVIIa/TF-FVIIa provide a new paradigm in the search for anticoagulants.

II Prize:

ISOLATION AND DETECTION OF A NOVEL SECOND MESSENGER CYCLICDI-GMP FROM DIVERSE BACTERIAL SPECIES

Li Fangyan , Weng-Keong Choy, Sanjay Swarup
Department of Biological Sciences, National University of Singapore, Singapore 117543
E-mail (presenting author): lifangyan@nus.edu.sg

MorA is a newly discovered GGDEF and EAL domain-containing regulator, which affects flagellar development and biofilm formation in various Pseudomonas species. However, the function and regulatory pathway of MorA are still unknown. The two domains have been shown in several studies to be associated respectively with cyclic-di-GMP cyclase and phosphdiesterase activities that regulate the level of cyclic-di- GMP. We hypothesize that a fine balance in the two activities and thus the level of cyclic-di-GMP affect the signaling pathways to control the motility and biofilm pathways. Therefore, the establishment of an isolation and detection method of cyclic-di-GMP is important for elucidating the regulatory cascade of MorA. We have initiated a study to isolate this second messenger from two bacterial species, Pseudomonas and Gluconacetobacter. We use G.xylinus, in which the first diguanylate cyclase was identified, as a model to establish a method of extracting cyclic-di-GMP using perchloric acid, and subsequent isolation and detection by HPLC and MALDI-TOF mass spectrometry.

III Prize:

ISOLATION AND SCREENING OF LACTIC ACID PRODUCING BACTERIA FROM LOCAL FOOD SOURCES

Siew-Chen Tan, Irene KP Tan
Institute of Biological Sciences, University of Malaya, Malaysia
E-mail (presenting author): s_chen1380@yahoo.com

The objective of this study was to isolate lactic acid producing bacteria from some local food. ”Tairu” (fermented yogurt), ”tempe” (fermented soya bean cake) and salad (mixture of green vegetables) were separately blended in sterile 0.85% saline solution, and 1% w/v was used to inoculate MRS medium containing 2% w/v of either glucose, lactose, sorbitol, sucrose or galactose. The cultures were incubated in 37 ºC, 100 rpm shaking for one week. Aliquots of the cultures were dilution-spreaded on agar plates to obtain single colonies. Out of 48 isolates screened, 21 were gram-positive, catalase-negative, non gas-forming in the gel plug test, produced clear zones on GYP-CaCO₃ agar, and produced blue to light green colonies on HHD medium, indicating that they are homofermentative lactic acid producing bacteria. 10 of the homofermentative isolates were then tested in a range of temperatures (15 ºC, 37 ºC, 45 ºC), pH (4.5, 7.0, 9.0) and NaCl concentrations (5%, 7.5%, 10%). These serve as a rapid discriminatory test to select for isolates with desirable traits e.g. tolerance to high temperature, low pH and high NaCl concentrations. Isolates having the latter traits and which produce high amounts of lactic acid (comparatively) will be selected to test their ability to utilize effluents from palm oil mill, rubber mill and food processing plants as fermentation substrates.

Cell and Molecular Biology (Oral)

I Prize:

GENE STRUCTURES OF TWO FUNCTIONALLY DIVERSE PROTHROMBIN ACTIVATORS, TROCARIN D AND COAGULATION FACTOR X, IN TROPIDECHIS CARINATUS SNAKE

Reza MA ¹ , Swarup S ¹ , Kini RM ^1,2
1 Department of Biological Sciences, National University of Singapore, Singapore 117543
² Virginia Commonwealth University, Richmond, Virginia, USA
E-mail (presenting author): reza@nus.edu.sg

Recently we have shown that Australian rough scaled snake, Tropidechis carinatus possesses two parallel prothrombin activator systems. Trocarin D, a venom prothrombin activator plays an offensive role as toxin, whereas factor X (FX) plays role in hemostatic function. These two proteins are structurally similar and have identical domain architecture. But, their functional difference mandates a highly tissue-specific expression; trocarin D is expressed ~1150 times higher in the venom gland compared to FX expression in liver. Moreover, the expression of FX is constitutive, whereas that of trocarin D is inducible. Therefore, it is interesting to study the gene structure and regulation of expression of these two closely related proteins with divergent functional roles in snake. Here we present the complete gene structure of trocarin D and FX from T. carinatus . Both of the genes have 8 exons and all the exon-intron boundaries are almost at the same position. Introns of these two genes show high identity (>85%), indicating a recent gene duplication event. Interestingly, the promoter of trocarin D has a big insertion of 264 bp (-29 to -293). This region of trocarin D promoter may be responsible for high level of tissue-specific expression.

II Prize:

FUNCTIONAL ANALYSIS OF AGL6 AND AGL13 - ARE THEY INVOLVED IN THE FLOWERING PROCESS OF ARABIDOPSIS THALIANA ?

Teo Lai Lai ¹ , Lee Candy LY ¹ , Tan Hugh TW ¹ , Kumar Prakash P ¹ , Yu Hao ^1,2
1 Department of Biological Sciences, National University of Singapore, Singapore 117543
² Temasek Life Sciences Laboratory, 1- Research Link, NUS, Singapore 117604
E-mail (presenting author): g0202193@nus.edu.sg

The flowering process of plants involves the transition from vegetative phase to reproductive phase. Many of the regulators involved in this process are genes that belong to the MADS-box superfamily, all having a onserved domain, namely the MADS-box. AGL6 and AGL13 are two MADS-box genes with unknown function. However, it has been thought that these two genes may be involved in the flowering of Arabidopsis thaliana . Our preliminary results indicated that the mRNA transcripts of both AGL6 and AGL13 have been up-regulated during floral transition period in Arabidopsis thaliana . Overexpression of these genes in wild-type background resulted in early flowering phenotype, whereas knock-down of both genes produced late flowering phenotypes. These results strongly indicated that AGL6 and AGL13 are promoters of flowering. The ongoing works include identifying the genetic pathway(s) in which these genes act in, as well as finding the interacting protein partners and the direct downstream genes.

III Prize:

FUNCTIONAL ANALYSIS OF A GENE FOR CYTOKININ BINDING PROTEIN

Mandar Godge , Kumar PP
Department of Biological Sciences, National University of Singapore, Singapore 117543
E-mail (presenting author): mandar.godge@nus.edu.sg

Plant hormones play a central role in development, growth and adaptation of a plant. Cytokinins are a group of plant hormones and they include N6-substituted adenine derivatives. Hormone signal transduction is important in proper interactions among various hormones necessary to regulate development. Beyond the phosphorelay signaling elements, the cytokinin signaling pathway remains obscure. The present study is a step towards elucidation of the underlying mechanism of cytokinin signal transduction pathway. We have identified a cDNA encoding putative cytokinin binding protein (CBP) from Petunia. Also, we have isolated its homologs from Arabidopsis thaliana , Brassica alboglabra , Amaranth, Chrysanthemum, Rice and Sugarcane. This gene encodes for a protein that has high similarity to S-adenosyl L-homocysteine hydrolase, which regulates transmethylation reactions. To investigate the involvement of CBP in plant morphogenesis, we are generating transgenic plants overexpressing the gene and also inhibiting it by dsRNA interference and antisense gene technology. Also, we investigated the role of CBP in plant development by Virus Induced Gene Silencing. Further, we are attempting to isolate the proteins interacting with CBP with the help of Tandem Affinity Purification

Cell and Molecular Biology Posters

I Prize:

STRESS-INDUCED ALTERATIONS IN PARKIN SOLUBILITY PROMOTE PARKIN AGGREGATION AND COMPROMISE PARKIN'S PROTECTIVE FUNCTION

Wang Cheng ^1,2 , Ko Hanseok ³ , Thomas Bobby ³ , Tsang Fai ⁴ , Tay Shiam-Peng ^2
1 Department of Biological Sciences, National University of Singapore, Singapore 117543
² National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433
³ Institute for Cell Engineering, Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA
⁴ Department of Physiology, 2 Medical Drive, National University of Singapore, Singapore 117597
E-mail (presenting author): g0203585@nus.edu.sg

Mutations in parkin are currently recognized as the most common cause of familial Parkinsonism. Emerging evidence also suggest that parkin expression variability may confer a risk for the development of the more common, sporadic form, of Parkinson’s disease (PD). Supporting this, we have recently demonstrated that parkin solubility in the human brain becomes altered with age. Since parkin apparently functions as a broadspectrum neuroprotectant, the resulting decrease in the availability of soluble parkin with age may underlie the progressive susceptibility of the brain to stress. Interestingly, we also observed that many familial-PD mutations of parkin alter its solubility in a manner that is highly reminiscent of our observations with the aged brain. The converging effects on parkin brought about by aging and PD-causing mutations are probably not trivial, and suggest that environmental modulators affecting parkin solubility would increase an individual’s risk of developing PD. Using both cell culture and in vivo models, we demonstrate here that several PD-linked stressors, including neurotoxins (MPTP, Rotenone, 6-OHDA), paraquat, NO, dopamine and iron, induce alterations in parkin solubility and result in its intracellular aggregation. Furthermore, the depletion of soluble, functional forms of parkin is associated with reduced proteasomal activities and increased cell death. Our results suggest that exogenously introduced stress as well as endogenous dopamine could affect the native structure of parkin, promote its misfolding, and concomitantly compromise its protective functions. Mechanistically, our results provide a link between the influence of environmental (and intrinsic) factors and genetic susceptibilities in PD pathogenesis.

II Prize:

ENDOCRINE-DIRECTED EXPRESSION OF THE GONADOTROPIN _ -SUBUNIT GENES TARGETS THE DISRUPTION OF DISTINCT HISTONE DEACETYLASE COMPLEXES

Stefan Lim ¹ , Mingshi Koh ² , Mohammed Nizam bin Abdul Kadir ² , Meng Yang ² , Zhiyong Ye ² , Philippa Melamed ^2
1 Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117597
² Department of Biological Sciences, National University of Singapore, Singapore 117543
E-mail (presenting author): g0301469@nus.edu.sg

The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced by gonadotropes within the anterior pituitary. Their production as separate and distinct α and β subunits is tightlyregulated by GnRH from the hypothalamus. Using immature gonadotropic αT3-1 cells, we show that the β subunit genes are normally repressed by unique complexes containing histone deacetylases (HDACs), which either hypoacetylate the chromatin or inhibit transcriptional activators, together with other co-repressors. Treatment with GnRH then restores β subunit expression. Through confocal microscopy, we demonstrate that GnRH causes the cytoplasmic localization of pan-cellular HDACs 4 and 7. It also causes nuclear export of HDAC 5, which is abolished when we mutate two key serine residues within its N-terminal extension. Research has shown that all such class IIa HDACs possess these serine residues that are variously phosphorylated by ERK1/2 and CaMK, both known to be activated by GnRH. Furthermore, 14-3-3 chaperone proteins have been found to recognize these phosphorylated sites and bind them to mediate HDAC nuclear export. It is thus possible that GnRH relieves the repression of the β subunit genes by disrupting the HDAC-containing complexes, through the export of any nuclear HDAC 4, 5 or 7, which is likely 14-3-3-dependent.

III Prize:

RHO MEDIATES CYTOKINESIS AND EPIBOLY VIA ROCK IN ZEBRAFISH

Shih-Lei Lai ¹ , Ching-Nung Chang ¹ , Pei-Jen Wang ² , Shyh-Jye Lee ^1
1 Institute of Zoology, National Taiwan University, Roosevelt Road, Taipei 106, Taiwan
² Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan
E-mail (presenting author): F90243012@ntu.edu.tw

To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3- xoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3- induced defects. Distributions of actin and the cleavage furrow associated _ -catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.

Biodiversity, Conservation & Ecology (Oral)

I Prize:

PREY PREFERENCE AND DIET OF PARACYRBA WANLESSI , A MOSQUITOEATING JUMPING SPIDER

Jeremy Woon, Chin Yujia, Li Daiqin
Department of Biological Sciences, National University of Singapore, Singapore 117543
E-mail (presenting author): g0306239@nus.edu.sg

Spiders are commonly envisaged as generalist predators, but recent studies have shown that certain species of spiders demonstrate a pronounced prey preference. Some salticid species have even been shown to grow faster and larger when fed with their preferred prey. Paracyrba wanlessi is known to feed on a large proportion of mosquitoes in nature. However, it is unclear if the observed proportion of prey taken in nature is indicative of a prey preference or if it is due to the relative abundance of different prey. We performed prey choice tests to examine if the spiders show a preference for mosquitoes, and also if hunger levels affect this preference. Our results demonstrated that P. wanlessi juveniles show a distinct preference for mosquitoes over both fruit flies and crickets. Experiments were also conducted to investigate whether the growth rate and development of P. wanlessi were influenced if the spiders were fed on strict diets of either Drosophila melanogaster or Aedes lbopictus . There appears to be a trend towards slower growth, later maturity and higher mortality for spiders reared on a diet of D. melanogaster as compared to those reared on diets of blood fed or non-blood fed mosquitoes or a mixed diet.

II Prize:

PARASITIZING QUEENLESS RED DWARF HONEY BEE COLONIES

Piyamas Nanork ^1,2,3 , Jürgen Paar ² , Nadine C Chapman ² , Siriwat Wongsiri ¹ , Benjamin P Oldroyd ^2
1 Department of Biology, Chulalongkorn University, Bangkok, Thailand 10330
² School of Biological Sciences A12, University of Sydney, New South Wales 2006, Australia
³ Department of Biology, Faculty of Science, Mahasarakham University, Khamriang, Kantarawichai, Mahasarakham , Thailand 44150
E-mail (presenting author): pnanork@yahoo.com

In honeybee colonies with a queen-worker dimorphism, workers mostly refrain from activating their ovaries, and suppress reproduction of worker nestmates by worker policing, in which worker-laid eggs are eaten. However, if a colony becomes queenless, workers eventually begin laying eggs, and rear a last batch of males before the colony finally dies. Breakdown of worker policing in queenless colonies makes them vulnerable to parasitism by the eggs of workers from other colonies. We will show that in the red dwarf honeybee, Apis florea , this window of opportunity is actively exploited, and up to 30% of males are sons of parasitizing workers.

III Prize:

IMPORTANCE OF RESERVOIRS FOR THE CONSERVATION OF FRESHWATER MOLLUSCS IN A TROPICAL URBAN LANDSCAPE

Reuben Clements ¹ , Lian Pin Koh ² , Tien Ming Lee ³ , Rudolf Meier ¹ , Daiqin Li ^1
1 Department of Biological Sciences, National University of Singapore, Singapore 117543
² Department of Ecology and. Evolutionary Biology, Princeton University, Princeton, NJ, USA
³ University of California, San Diego, USA
E-mail (presenting author): g0403015@nus.edu.sg

Freshwater species and ecosystems are gravely imperiled, particularly within urban landscapes of tropical Asia. In Singapore, we determined: (1) the importance of six different habitats (i.e. catchment reservoirs, estuarine reservoirs, forest streams, rural streams, ponds and monsoon canals) for conserving freshwater molluscan diversity; (2) key environmental factors (e.g., pH) affecting molluscan distribution; (3) important biogeographical determinants (e.g., area) of molluscan richness within each habitat; and (4) the habitat affinities of introduced species. High sampling saturation was achieved at most study habitats with minimal sampling effort, suggesting that the utilization of molluscs as bio-indicators can expedite freshwater conservation initiatives. Estuarine reservoirs had the highest molluscan richness (6.0 ± 2.0), while both reservoir types possessed species compositions distinct from other habitats and contained majority (76%) of the sampled species. Reservoirs, therefore, serve to conserve the bulk of local freshwater malacofauna, especially if they are maintained at nearneutral pH levels (i.e., ~ 7.3) and contain large substrates (i.e., rocks). Area was the best predictor of molluscan richness across all habitats, implying that larger freshwater habitats require higher conservation priorities than smaller ones. Introduced species (e.g., Pomacea canaliculata ) had high affinities for reservoirs, which are in need of monitoring to curb population expansions.

Biodiversity, Conservation & Ecology Posters

I Prize:

MOLECULAR PHYLOGENY AND EVOLUTION OF LARVAL FEEDING HABITS IN THE FAMILY SCATHOPHAGIDAE (DIPTERA)

Sujatha Narayanan Kutty ¹ , M.Bernasconi ² , Rudolf Meier ^1
1 Department of Biological Sciences, National University of Singapore, Singapore 117543
² University of Zurich-Irchel, Zurich, Switzerland
E-mail (presenting author): g0306071@nus.edu.sg

The relatively small family Scathophagidae (Diptera) displays with its 250 described species, a wide variety of breeding and feeding habits in their larval stage. These range from leaf-mining over dung feeding to predation. In order to trace and study the evolution of the diverse natural history across the family, a robust phylogenetic tree for the Scathophagidae is needed. A phylogenetic analysis based on COI and Cytb sequences by Bernasconi et al . identified major clades, but most of the nodes needed more support. Here, we present the results of a cladistic analysis on 63 species from 22 genera based on additional DNA sequences from the mitochondrial genes 12S , 16S , Cytb and the nuclear genes 28S , Ef1 - alpha and Pol lI . The results confirm the monophyly of the Scathophagidae. Based on our tree, phytophagy, or more particularly leaf mining, seems to be the ancestral larval feeding habit. The species in the phytophagous genera are generally host-specific for particular monocot and/or dicot host plants. From here shifts to saprophagy and predation appear to have occurred. Most saprophagous species are dung-feeding, with the larvae of a few species being specialized for feeding on decaying seaweed. Predation has evolved at least twice in the family.

II Prize:

SEX, FLIES, AND VIDEO TAPES: THE EVOLUTION OF MATING BEHAVIOUR IN SEPSIDAE

Nalini Puniamoorthy, Kathy Feng-Yi Su, Rudolf Meier
Department of Biological Sciences, National University of Singapore, Singapore 11754 3
E-mail (presenting author): sepsids@gmail.com

Sepsidae is a family of flies with worldwide distribution. Male sepsid flies possess extremely diverse foreleg and genital morphology, making them suitable as model organisms for the study of rapid speciation and the evolution of sexual dimorphism. In order to correlate morphological diversity with behaviour, we have studied the mating behaviour for 12 species. We find that the behaviour is as diverse as the morphology, with more than 30 different behavioural elements found across the 12 species. These courtship repertoires are very species specific and are often marked by species-specific behavioural elements. In order to reconstruct the evolution of behaviour in Sepsidae , we generated a robust phylogenetic tree for the family based on ten gene fragments totaling 7.2kbp per species. The cladistic analysis based on 70 species from 21 genera (out of 30 in the family) confirms the monophyly of the family and we find that most behavioural characters show high levels of homoplasy, thus indicating that courtship behaviour is evolutionarily labile. Our results demonstrate that studying behaviour based on a phylogenetic tree provides valuable insights into the evolution of mating behaviour.

III Prize:

CARBON FLUX THROUGH BACTERIA IN A EUTROPHIC TROPICAL ENVIRONMENT: PORT KLANG WATERS

Bong Chui Wei, Lee Choon Wen
Institute of Biological Sciences (Microbiology Unit), Faculty of Science, University of Malaya, Malaysia 50603
E-mail (presenting author): chuiweibong@yahoo.com

Carbon flux through bacteria was analyzed in Port Klang waters, a eutrophic tropical environment from September 2004 until February 2005. Water quality was poor due to low dissolved oxygen (DO) concentration (<200 µM), and high total suspended solids (TSS) (>260 mg L–1). TSS was mainly inorganic in nature, with particulate organic matter <5% of TSS. Two episodes of hypoxia (DO<125 µM) were observed in early December 2004 and February 2005. Based on marine water quality data collected by the Department of Environment of Malaysia, the water quality at and around Port Klang deteriorated from 1990 to 2003. Over 10 years (1994–2003), TSS increased 132 mg L–1, and DO decreased by 48 µM. The NO 3 :PO 4 ratio was low, ranging from 0.05 to 0.38, suggesting nitrogen limitation for the phytoplankton. Gross primary production (GPP) correlated significantly with NO 3 (R2=0.867, n=5, p<0.05). The low NO 3 concentration in February 2005 could have limited GPP, and indirectly triggered hypoxia. GPP correlated with community respiration (R2=0.956, n=5, p<0.01) except in February 2005 when there was uncoupling between primary production and heterotrophy. Heterotrophic metabolism was probably supported by other sources of allochthonous organic matter (e.g. the Klang River) during this period.  

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