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Vincent T K Chow

Publications and source records attributed to Vincent T K Chow.

At least 19 recordsLinked to original sources
PubMed2006

In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. METHODS: Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. RESULTS: The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 microg/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05-10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. CONCLUSION: This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei.

Animals↗
PubMed2006

Apoptosis Gene Information System--AGIS.

Genes implicated in apoptosis have great relevance to biology, medicine and oncology. Here, we describe a unique resource, Apoptosis Gene Information System (AGIS) that provides data for over 2400 genes involved directly or indirectly, in apoptotic pathways of more than 350 different organisms. The organization of this information system is based on the principle of one-gene, one record. AGIS will be updated on a six monthly basis as new information becomes available. AGIS can be accessed at: http://www.cellfate.org/AGIS/.

Amino Acid Sequence↗
PubMed2006

Gene fusion in Helicobacter pylori: making the ends meet.

Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era.

Amino Acid Sequence↗
PubMed2006

Functional and evolutionary analyses on expressed intronless genes in the mouse genome.

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life--bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.

Animals↗
PubMed2006

The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells.

Deregulation of the cell cycle is a common strategy employed by many DNA and RNA viruses to trap and exploit the host cell machinery toward their own benefit. In many coronaviruses, the nucleocapsid protein (N protein) has been shown to inhibit cell cycle progression although the mechanism behind this is poorly understood. The N protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) bears signature motifs for binding to cyclin and phosphorylation by cyclin-dependent kinase (CDK) and has recently been reported by us to get phosphorylated by the cyclin-CDK complex (Surjit, M., Kumar, R., Mishra, R. N., Reddy, M. K., Chow, V. T., and Lal, S. K. (2005) J. Virol. 79, 11476-11486). In the present study, we prove that the N protein of SARS-CoV can inhibit S phase progression in mammalian cell lines. N protein expression was found to directly inhibit the activity of the cyclin-CDK complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant down-regulation in E2F1-mediated transactivation. Coexpression of E2F1 under such conditions could restore the expression of S phase genes. Analysis of RXL and CDK phosphorylation mutant N protein identified the mechanism of inhibition of CDK4 and CDK2 activity to be different. Whereas N protein could directly bind to cyclin D and inhibit the activity of CDK4-cyclin D complex; inhibition of CDK2 activity appeared to be achieved in two different ways: indirectly by down-regulation of protein levels of CDK2, cyclin E, and cyclin A and by direct binding of N protein to CDK2-cyclin complex. Down-regulation of E2F1 targets was also observed in SARS-CoV-infected VeroE6 cells. These data suggest that the S phase inhibitory activity of the N protein may have major significance during viral pathogenesis.

Animals↗
PubMed2006

The VP1 protein of human enterovirus 71 self-associates via an interaction domain spanning amino acids 66-297.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease (HFMD). Several recent outbreaks of HFMD in East Asia were associated with neurological complications and numerous deaths. In 2000, an outbreak in Singapore afflicted thousands of children, resulting in four fatal cases from whom EV71 was isolated. The virus possesses four structural proteins VP1, VP2, VP3, and VP4, each of which is involved in forming the pentameric icosahedral structure of the virus. Here we report that the full-length VP1 structural protein of EV71 is capable of self-association. Dimerization of VP1 was tested using the yeast two-hybrid system, fluorescence resonance energy transfer (FRET) analysis, in vitro coupled transcription-translation binding assays, and mammalian cell transcription-translation experiments. Dimerization of various truncated versions of the VP1 protein was also studied by mutational analysis. Systematic deletions of parts of VP1 revealed that the region spanning amino acids 66-132 of VP1 contains the major dimerization domain. However, the region between amino acids 132 and 297 was indispensable, and contributed largely to increasing the strength of the interaction. This ability of EV71 VP1 to self-associate and to participate significantly in forming the characteristic icosahedral capsid strongly enhances the pathogenicity and stability of the virus to withstand the environment of the gastrointestinal tract.

Amino Acids↗
PubMed2006

Differential dengue cross-reactive and neutralizing antibody responses in BALB/c and Swiss albino mice induced by immunization with flaviviral vaccines and by infection with homotypic dengue-2 virus strains.

We investigated whether cross-reactive and/or cross-protective antibodies against dengue virus could be generated in 6-week-old BALB/c mice by immunization with currently approved flaviviral vaccines, i.e., Japanese encephalitis (JE) BIKEN and yellow fever (YF) 17D. Cross-reactivity with dengue antigens was apparent in at least one-third each of JE-vaccinated mouse sera and of JE/YF-vaccinated mouse sera by dengue enzyme immunoassay, but was not detected in sera of mice immunized with YF vaccine alone. All the immunized BALB/c mice failed to generate neutralizing antibodies against the New Guinea C laboratory (NGC-lab) strain of dengue virus type 2. In addition, we determined the specificity of neutralizing antibodies elicited in 3-week-old Swiss albino mice against two homotypic dengue-2 strains, i.e., NGC-lab and Singapore 1999 (SING/99). Although sera from virus-inoculated mice displayed better neutralization against the corresponding strain, antibodies elicited by NGC-lab exhibited a significantly poorer neutralizing response against the SING/99 strain compared to antibodies elicited by SING/99 against NGC-lab. The differences may be related to sequence variations of approximately 3% between the envelope proteins of both strains. Amino acid disparities at positions 71 (Glu --> Ala), 112 (Ser --> Gly) and 124 (Ile --> Asn), which are found in dengue-2 neutralization escape mutants, were also found in the SING/99 strain. The envelope sequence differences may explain diminished binding of NGC-lab-induced neutralizing antibodies to neutralizing epitopes within the envelope of the SING/99 strain, resulting in a lower titer of neutralizing antibodies against another strain of the same serotype.

Albinism↗
PubMed2006

Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection.

Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.

Cell Line, Tumor↗
PubMed2005

Gene expression profiles of U937 human macrophages exposed to Chlamydophila pneumoniae and/or low density lipoprotein in five study models using differential display and real-time RT-PCR.

Many studies on atherosclerosis have indicated an association with Chlamydophila pneumoniae, a respiratory intracellular pathogen that can infect bronchoalveolar macrophages, and be transported to sites of vascular injury. Furthermore, the connection between low density lipoprotein (LDL), macrophages, foam cell formation, and atherosclerosis is well established. Five study models were designed to mimic the early events in plaque formation, i.e. untreated U937 human macrophages as control; macrophages treated with LDL only; macrophages infected with the AR-39 strain of C. pneumoniae only; and the last two models in which macrophages were exposed to both C. pneumoniae and LDL but in different order. Differential display RT-PCR was employed to analyze mRNAs from the five models to elucidate the transcriptional responses arising from macrophage interactions with C. pneumoniae and/or LDL. Out of 235 expressed sequence tags, the altered expression patterns of 29 selected genes at various time-points were authenticated by semi-quantitative and real-time RT-PCR. In relation to atherosclerosis, some interesting modified transcripts are involved in cell proliferation (CDC2, CDC27, CDC42EP3, GBDR1, GPR6, RPL27A, Similar to COX2), apoptosis (BCL2A1), ubiquitination (CTSH, HSPC150, NUC2), and structural integrity (ACTG1, FLOT1). Other genes exhibiting altered expression include those that modulate inflammatory and immune responses or signal transduction linking infection to host immunity (ARPP-19, BLAME, HCA58, IK cytokine, IRS2, STAT1P1). The transcriptional alterations of critical human genes in response to C. pneumoniae infection and foam cell development provide insights into certain key molecular pathophysiological mechanisms of atherosclerosis.

Cell Proliferation↗
PubMed2005

Structure-activity studies of peptides from the "hot-spot" region of human CD2 protein: development of peptides for immunomodulation.

CD2 is a cell surface protein belonging to the immunoglobulin superfamily (IgSF) that plays a key role in mediating adhesion between human T-lymphocytes and target cells. The interaction between cell-adhesion molecules CD2 and CD58 is critical for immune response. Modulation or inhibition of these interactions has been shown to be therapeutically useful. Synthetic 12-mer linear and cyclic peptides and cyclic hexapeptides from the beta-turn and beta-strand region (hot spot) of human CD2 protein were designed to modulate CD2-CD58 interaction. The 12-amino acid synthetic cyclic peptides effectively blocked the interaction between CD2 and CD58 proteins as demonstrated by E-rosetting and heterotypic adhesion assays. NMR and molecular modeling studies indicated that these cyclic peptides exhibit beta-turn structure in solution and closely mimic the beta-turn structure of the surface epitopes of CD2 protein. The designed cyclic peptides with beta-turn structure have the ability to modulate CD2-CD58 interaction.

Adjuvants, Immunologic↗
PubMed2005

Microarray and real-time RT-PCR analyses of a novel set of differentially expressed human genes in ECV304 endothelial-like cells infected with dengue virus type 2.

The cellular and molecular pathways of dengue infection have not been as intensively studied compared to the host immunological responses. Changes in mRNA expression levels of ECV304 human endothelial-like cells following infection with the virulent New Guinea C strain of dengue virus type 2 were analyzed by a microarray system comprising 7600 oligonucleotide cDNAs. After normalization against the uninfected control using two independent software programs, 111 genes exhibited at least a 1.5-fold difference in expression level. Out of these, 21 mRNAs were upregulated while 90mRNAs were downregulated. Quantitative real-time RT-PCR was then performed to determine the expression patterns of 15 selected genes of interest involved in the cell cycle (MAD3), apoptosis (RIPK3, PDCD8), cellular receptors (H963, CCR7, KLRC3), transcriptional regulation (RUNX3, HNF4G, MIZ1), signal transduction (HSP27, TRIP, MAP4K4), enzymes (angiotensinogen), protein transport (AP4M1), and cytoskeleton (ACTA2). Dengue virus infection resulted in the downregulation of the C-terminal alternatively spliced p53 variant, the pro-apoptotic IG20 and IG20-SV2 isoforms, and the Fas apoptosis inhibitory molecule (FAIM). Most of the real-time RT-PCR data showed concordance with the normalized microarray data. Hence, real-time RT-PCR validation of high-throughput gene microarray screening is important and necessary before further conclusions on gene expression can be drawn. This study elucidated novel information on the complex responses at the transcriptional level in susceptible human endothelial-like cells induced by a virulent dengue virus strain implicated in the pathogenesis of dengue and/or its complications.

Apoptosis↗
PubMed2005

Insights to metabolic network evolution by fusion proteins.

Human fusion proteins consisting of two or more fusion partners of prokaryotic origin exhibit accreted function. Recent studies have elucidated the importance of fusion proteins in complex regulatory networks. The significance of fusion proteins in cellular networks and their evolutionary mechanism is largely unknown. Here, we discuss the association of six fusion proteins with the citric acid cycle. We define possible gene fusion scenarios and show that they produce metabolites with high connectivity for complex networking. Complex networking of metabolites requires proteins with incremental structural architectures and functional capabilities. Such higher order functionality is frequently provided by fusion proteins. Therefore, evolution of fusion proteins capable of producing metabolites with greater connectivity for enhanced cross-talk between pathways is critical for the selection of multiple trajectories in maintaining a stoichiometric balance during regulation. The association of six fusion proteins with the citric acid cycle and their capability to produce metabolites with high connectivity index is intriguing. This suggests that fusion gene products and their evolution have had a key role in the selection of complex multifaceted networks. In addition, we propose that fusion proteins have gained additive biochemical function for a balanced regulation of metabolic networks.

Biological Evolution↗
PubMed2005

Computational prediction of SEG (single exon gene) function in humans.

Human genes are often interrupted by non-coding, intragenic sequences called introns. Hence, the gene sequence is divided into exons (coding segments) and introns (non-coding segments). Consequently, a majority of them are multi exon genes (MEG). However, a considerable amount of single exon genes (SEG) are present in the human genome (approximately 12%). This amount is sizeable and it is important to probe their molecular function and cellular role. Hence, we performed a genome wide functional assignment to 3750 SEG sequences using PFAM (protein family database), PROSITE (database of biologically meaningful signatures or motifs) and SUPERFAMILY (a library covering all proteins of known 3 dimensional structure). PFAM assigned 13% SEG to trans-membrane receptor genes of the G-protein coupled receptor (GPCR) family and showed that a majority of SEG proteins have DNA binding function. PROSITE identified 336 unique motif types in them and this accounts for 25% of all known patterns, with a majority having PHOSPHORYLATION and ACETYLATION signals. SUPERFAMILY assigned 33% SEG to the membrane all alpha (proteins containing alpha helix structural elements according to SCOP (structural classification of proteins) definition). Functional assignment of SEG proteins at multiple levels (sequence signals, sequence families, 3D structures) using PFAM, PROSITE and SUPERFAMILY is envisioned to suggest their selective and predominant molecular function in cellular systems. Their function as DNA binding, phosphorylating, acetylating and house-keeping agents is intriguing. The analysis also showed evidence of SEG expression and retro-transposition. However, this information is inadequate to draw concerted conclusion on the prevalent role played by these proteins in cellular biology. A complete understanding of SEG function will help to explore their role in cellular environment. The derived datasets from these analyses are available at http://sege.ntu.edu.sg/wester/intronless/human/.

Computational Biology↗
PubMed2005

Intermedilysin release by Streptococcus intermedius: effects of various antibacterial drugs at sub-MIC levels.

Intermedilysin is a cytolytic toxin produced by Streptococcus intermedius, a pathogen of humans. In vitro studies showed that exposure of S. intermedius to sub-minimum inhibitory concentration (MIC) levels (1/2 MIC) of protein-inhibiting antibiotics and nucleic acid-inhibiting antibiotics decreased intermedilysin release by S. intermedius. The most potent antibiotic was clindamycin. On the other hand, exposure to cell wall-inhibiting antibiotics generally showed insignificant changes in intermedilysin release at sub-MIC concentrations. Investigations into possible mechanisms underlying this sub-MIC effect with clindamycin showed that there was selective decrease in biosynthesis and release of toxin after exposure to 1/2 MIC condition. However, no significant differences in the mRNA levels of the intermedilysin gene were observed.

Anti-Bacterial Agents↗
PubMed2005

Microarray and real-time RT-PCR analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (SARS) coronavirus infection of Vero cells.

Vero E6 African green monkey kidney cells are highly susceptible to infection with the newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV), and they are permissive for rapid viral replication, with resultant cytopathic effects. We employed cDNA microarray analysis to characterize the cellular transcriptional responses of homologous human genes at 12 h post-infection. Seventy mRNA transcripts belonging to various functional classes exhibited significant alterations in gene expression. There was considerable induction of heat shock proteins that are crucial to the immune response mechanism. Modified levels of several transcripts involved in pro-inflammatory and anti-inflammatory processes exemplified the balance between opposing forces during SARS pathogenesis. Other genes displaying altered transcription included those associated with host translation, cellular metabolism, cell cycle, signal transduction, transcriptional regulation, protein trafficking, protein modulators, and cytoskeletal proteins. Alterations in the levels of several novel transcripts encoding hypothetical proteins and expressed sequence tags were also identified. In addition, transcription of apoptosis-related genes DENN and hIAP1 was upregulated in contrast to FAIM. Elevated Mx1 expression signified a strong host response to mediate antiviral resistance. Also expressed in infected cells was the C-terminal alternative splice variant of the p53 tumor suppressor gene encoding a modified truncated protein that can influence the activity of wild-type p53. We observed the interplay between various mechanisms to favor virus multiplication before full-blown apoptosis and the triggering of several pathways in host cells in an attempt to eliminate the pathogen. Microarray analysis identifies the critical host-pathogen interactions during SARS-CoV infection and provides new insights into the pathophysiology of SARS.

Animals↗
PubMed2005

Comparative study of overlapping genes in bacteria, with special reference to Rickettsia prowazekii and Rickettsia conorii.

Overlapping genes have been proposed as a means of achieving genome reduction by compressing the maximum amount of information in limited sequence space. In this report, comparative analyses of the overlapping genes of genomes of nine bacteria with different lifestyles were performed. The results clearly suggest that overlapping genes may be a result of evolutionary pressure to minimize genome size. The genomes of two closely related obligatory intracellular parasites - Rickettsia prowazekii and Rickettsia conorii - were investigated further. Detailed analyses of these two genomes revealed that mutations at the ends of coding regions and elimination of intergenic DNA are the main forces that determine overlapping of genes.

Biological Evolution↗
PubMed2005

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation.

The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.

14-3-3 Proteins↗