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The availability of complete genome sequences of many bacterial species is facilitating numerous computational approaches for understanding bacterial genomes. One of the major incentives behind the genome sequencing of many pathogenic bacteria is the desire to better understand their diversity and to develop new approaches for controlling human diseases caused by these microorganisms. This task has become even more urgent with the rapid evolution of antibiotic resistance among many bacterial pathogens. Novel drug targets are required in order to design new antimicrobials against antibiotic-resistant pathogens. The complete genome sequences of an ever increasing number of pathogenic microbes constitute an invaluable resource and provide lead information on potential drug targets. This review focuses on in silico analyses of microbial genomes, their host-specific adaptations, with specific reference to genome architecture, design, evolution, and trends in computational identification of microbial drug targets. These trends underscore the utility of genomic data for systematic in silico drug target identification in the post-genomic era.

Mucosal vaccines present several advantages over conventional parenteral vaccines including their ease of administration and low cost, both criteria being priorities for developing countries plagued by infectious diseases. A recombinant Lactococcus lactis strain producing the envelope domain III (EDIII) antigen from dengue virus serotype 2 was engineered, and the ability of the live recombinant bacteria to trigger a systemic anti-EDIII IgG antibody response upon nasal or oral administration to BALB/c and C57BL/6 mice was investigated. Results showed that the antibody response depended on the route of administration and on the mouse strain inoculated. Out of six, two and three C57BL/6 mice orally and nasally inoculated with the recombinant bacteria, respectively, displayed anti-EDIII antibody responses higher than that obtained in the mouse group intraperitoneally (i.p.) immunized with heat-inactivated dengue 2 virus. The protective potential of the immune sera was measured using the plaque reduction neutralizing test (PRNT) and results indicated that high anti-EDIII antibody levels did not correlate directly with high neutralizing activities. Immune sera from orally inoculated mice were found the most potent to neutralize in vitro dengue infection with neutralizing antibody activities in some cases higher than that obtained with the immune sera from mice i.p. injected with heat-inactivated virus.

The complete genome sequences of about 300 bacteria (mostly pathogenic) have been determined, and many more such projects are currently in progress. The detection of bacterial genes that are non-homologous to human genes and are essential for the survival of the pathogen represent a promising means of identifying novel drug targets. We present a subtractive genomics approach for the identification of putative drug targets in microbial genomes and demonstrate its execution using Pseudomonas aeruginosa as an example. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This strategy enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. It should be recognized that there are limitations to this computational approach for drug target identification. Distant gene relationships may be missed since the alignment scores are likely to have low statistical significance. In conclusion, the results of such a strategy underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1-VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral-host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.

The severe acute respiratory syndrome coronavirus (SARS-CoV) infects host cells with its surface glycosylated spike-protein (S-protein). Here we expressed the SARS-CoV S-protein to investigate its interactions with innate immune mechanisms in the lung. The purified S-protein was detected as a 210 kDa glycosylated protein. It was not secreted in the presence of tunicamycin and was detected as a 130 kDa protein in the cell lysate. The purified S-protein bound to Vero but not 293T cells and was itself recognized by lung surfactant protein D (SP-D), a collectin found in the lung alveoli. The binding required Ca(2+) and was inhibited by maltose. The serum collectin, mannan-binding lectin (MBL), exhibited no detectable binding to the purified S-protein. S-protein binds and activates macrophages but not dendritic cells (DCs). It suggests that SARS-CoV interacts with innate immune mechanisms in the lung through its S-protein and regulates pulmonary inflammation.

We previously isolated and characterized the novel human gene MOST-1 (C8orf17) that is ubiquitously expressed in all cancer cell lines tested but differentially expressed in normal adult tissues. MOST-1 maps to chromosome region 8q24.2 whose amplification is frequently associated with breast and prostate cancers. RT-PCR analyses of breast and prostatic biopsies revealed MOST-1 overexpression and/or amplification in high-grade carcinomas. We raised and characterized a polyclonal antibody against a MOST-1-specific synthetic peptide. in vitro expression of MOST-1 protein revealed a tendency to exist as high molecular mass isoforms which are SDS-insoluble upon thermal stress. MOST-1 displayed cytoplasmic localization in four human cell lines (hTERT-HME1 normal mammary epithelial, MCF7 breast adenocarcinoma, PrEC normal prostate epithelial and DU145 prostate carcinoma), with polar expression during cell division. Knockdown of MOST-1 expression in DU145 cells resulted in reduced cell proliferation but enhanced apoptosis implying a putative mitogenic role of MOST-1. Yeast two-hybrid analyses demonstrated interaction with seven human proteins, most of which are overexpressed in tumors or involved in metabolic pathways. The interacting proteins were creatine kinase, Gardner feline sarcoma v-FGR oncogene product, telethonin, SNC73 protein, ferritin light chain, peripheral benzodiazepine receptor, and immunoglobulin C (mu) and C (delta) heavy chain. Co-immunoprecipitation assays validated the interactions of MOST-1 with the latter three proteins. Our results suggest that MOST-1 is associated with cell survival, proliferation and progression of cancer cells.

Laboratory testing for dengue virus is used to confirm the diagnosis of dengue virus infection and to differentiate dengue from other febrile tropical illnesses. There are few data on the clinical use of reverse transcription-polymerase chain reaction (RT-PCR) for diagnosis of dengue virus infection. We prospectively evaluated 121 consecutive patients with possible dengue who had samples submitted for RT-PCR, IgM serology, and virus culture. Results were compared with the final discharge diagnosis. Semi-nested RT-PCR was performed using genus- and serotype-specific NS3 consensus primers. Results of 112 patients were available for the final analysis. The RT-PCR was positive in 40 of 62 patients with dengue. Patients who were RT-PCR-positive alone showed a mean of 4.4 days to RT-PCR positivity compared with 5.9 days in patients who were RT-PCR-negative and IgM serology-positive (P = 0.03, Mann-Whitney U-test). The sensitivity, specificity, negative predictive value, and positive predictive value were 70, 100, 84, and 100%, respectively, for samples analyzed within 5 days of illness onset. The RT-PCR also provided epidemiological data regarding the prevailing dengue virus serotypes: 25 with Den-2, eight with Den-3, and seven with Den-1 infection. We propose an algorithm of dengue testing that uses RT-PCR within 5 days of illness onset, whereas IgM capture enzyme-linked immunosorbent assay is preferred for those presenting later.

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.

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/.

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., (Copyright 2006 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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/.

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.

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.

Niche dependent differential gene loss and overlapping genes have been proposed as means of achieving genome reduction by retaining indispensable genes and compressing maximum amount of information in available sequence space. Herein, we analyzed the differential gene loss and overlapping genes in bacterial genomes with different lifestyles. Our results clearly suggest that gene loss and overlapping genes could be a result of evolutionary pressure to minimize genome size. Comparative analysis of the genomes shows that the genomes display marked similarities in patterns of protein length and frequency. It is clear from our analysis that habitat is a major factor contributing to genome reduction. These comparisons increase our knowledge of the forces that drive the extreme specialization of the bacteria and its association to the host.

The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. E-rosetting assay is usually applied in the study of the modulation of CD2-CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 microM of BCECF-AM for 45 min at 37 degrees C before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF's excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2-CD58 interaction.

Flaviviral infections produce a distinct array of virus-induced intracellular membrane alterations that are associated with the flaviviral replication machinery. Currently, it is still unknown which flaviviral protein(s) is/are responsible for this induction. Using yeast two-hybrid and co-immunoprecipitation analyses, we demonstrated that the NS3 protein of dengue virus type 2 interacted specifically with nuclear receptor binding protein (NRBP), a host cellular protein that influences trafficking between the endoplasmic reticulum (ER) and Golgi, and that interacts with Rac3, a member of the Rho-GTPase family. Co-expression of NS3 and NRBP in baby hamster kidney cells exhibited significant subcellular co-localization, and revealed the redistribution of NRBP from the cytoplasm to the perinuclear region. Furthermore, a set of membrane structures affiliated with the rough ER at the perinuclear region was induced in cells transfected with NS3. These structures are reminiscent of the virus-induced convoluted membranes previously observed in flavivirus-infected cells. This interaction between dengue viral and host cell proteins as well as the formation of the NS3-induced membrane structures suggest that NS3 may subvert the role of NRBP in ER-Golgi trafficking.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The virus is a novel coronavirus isolated from patients exhibiting atypical pneumonia and may have originated from wild animals such as civet cats in southern China. The genome of SARS-CoV is a positive-sense, single-stranded RNA whose sequence is distantly related to all known coronaviruses that infect humans and animals. Like other known coronaviruses, SARS-CoV is an enveloped virus containing three outer structural proteins, namely the membrane (M), envelope (E), and spike (S) proteins. The nucleocapsid (N) protein together with the viral RNA genome presumably form a helical core located within the viral envelope. The SARS-CoV nucleocapsid (N) protein is a 423 amino-acid, predicted phospho-protein of 46 kDa that shares little homology with other members of the coronavirus family. A short serine-rich stretch, and a putative bipartite nuclear localization signal are unique to it, thus suggesting its involvement in many important functions during the viral life cycle. In this report we have cloned the N gene of the SARS coronavirus, and studied its property of self-association to form dimers. We expressed the N protein as a fusion protein in the yeast two-hybrid system to demonstrate self-association and confirmed dimerization of the N protein from mammalian cell lysates by coimmunoprecipitation. Furthermore, via deletion analysis, we have shown that the C-terminal 209 amino-acid region constitutes the interaction domain responsible for self-association of the N protein to form dimers.

Motivation: The past decade has seen extension in the methods of sequence analysis from single gene based to analyzing multiple genes and proteins simultaneously. Consequently, there is a need for software tools that will allow mining of these enormous datasets at genome level effectively. A key challenge is to make them user-friendly, available to a larger community and integrate with public domain software without much hassle., Results: A web-based interactive computational tool is described for visualization and comparison of gene order from prokaryotic and selected viral genome data. Many intriguing similarities and differences in gene order of multiple genomes can be compared and revealed. The interface facilitates easy extraction of the nucleotide sequence of the gene of interest and BLAST analysis against GenBank at NCBI to provide insights into gene functions and orthologs of the gene in other species.

Dendritic cells (DCs) populate atherosclerotic lesions and might be involved in the regulation of immune reactions in atherosclerosis. The present work was undertaken to examine a possible association of DCs with Chlamydophila pneumoniae in human atherosclerotic plaques obtained by endarterectomy. C. pneumoniae was identified in 17 of 60 (28%) atherosclerotic plaques by a combination of immunohistochemistry and polymerase chain reaction (PCR). Double immunohistochemistry identified the presence of C. pneumoniae within S100(+) DCs that were localised predominantly in the deep layer of the intima under the necrotic core. Quantitative analysis showed that there were no differences in the numbers of DCs between C. pneumoniae(+) and C. pneumoniae(-) groups of atherosclerotic specimens. There were also no differences in the expression of Lag-antigen and HLA-DR by DCs between the groups of specimens. Markers of DC activation CD80 and CD86 were absent from both groups of specimens. Flow cytometry analysis of the effects of C. pneumoniae infection on immature monocyte-derived DCs in vitro showed no changes in the expression of CD1a, MHC class II, CD80 and CD86. The results of this study demonstrate that C. pneumoniae might infect DCs within the atherosclerotic intima but whether the presence of C. pneumoniae in DCs affects the intensity of immune reactions in atherosclerosis needs further clarification.

Differential display (DD)-RT-PCR was employed to analyze mRNAs from ECV304 human endothelial-like cells undergoing apoptosis following infection with the virulent New Guinea C strain of dengue virus type 2 in order to elucidate the cellular gene responses to dengue viral infection at the transcriptional level. We isolated, sequenced, and identified 203 differentially expressed and overlapping cDNA fragments, all of which were of human origin except 1 that was of viral origin. Out of these, 78 were individual distinct clones comprising 46 and 32 expressed sequence tags (ESTs) that exhibited upregulated and downregulated trends, respectively. Of the 78 differentially expressed mRNAs, 16 did not match any characterized genes or ESTs. The remaining 62 mRNAs modified by dengue virus infection matched known genes, including those encoding components of the cell cycle (Anillin, CDC27), cytoskeleton (epsilon-tubulin), signal transduction (OPHN1, PPP2R2A, TIRAP), protein translation and modification (EIF3S10, IF2, TMEM1), transcriptional regulation (alpha-NAC, C20orf104, EGR1, ELP2), apoptotic cell death (RICK), membrane (BPAG1), and mitochondrial-related proteins. Semiquantitative RT-PCR and real-time RT-PCR authenticated further the altered expression patterns of selected genes of interest. These data demonstrate the feasibility of mRNA DD in providing insights into the complex responses of the transcriptional machinery of permissive and apoptotic human endothelial-like cells in the pathogenesis of dengue and/or its complications induced by the virulent dengue virus type 2., (Copyright 2004 Wiley-Liss, Inc.)

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We have used three-way genome comparisons to identify essential genes from Pseudomonas aeruginosa. Our approach identified 306 essential genes that may be considered as potential drug targets. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This approach enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. These results underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.

The human genome is revisited using exon and intron distribution profiles. The 26,564 annotated genes in the human genome (build October, 2003) contain 233,785 exons and 207,344 introns. On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length. These results suggest constraints on the splicing machinery to splice out very long or very short introns and provide insight to optimal intron length selection. Interestingly, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the determined chromosome size with a coefficient of correlation r = 0.95 and r = 0.97, respectively. These results suggest their implication in genome design.

There is still substantial uncertainty concerning the association between Chlamydia pneumoniae and ischemic heart disease. This may partly be explained by the adjustment for potential confounders in different population studies. This is the first study in Singapore to look at the association of C. pneumoniae seropositivity with ischemic heart disease in a multivariate analysis. A random sample of 714 persons aged between 35 and 69 years was selected from the participants of the Singapore National Health Survey conducted in 1998. Data on clinical measurements and conditions were collected using biochemical tests and interviewer-based questionnaires. Ischemic heart disease was defined by the Rose questionnaire and included history suggestive of angina and/or myocardial infarction. Immunoglobulin G antibodies for C. pneumoniae were detected using an indirect microimmunofluorescence test, and seropositivity was defined as IgG titers > or = 1:16. There were no statistically significant differences in the prevalence rates of seropositivity to C. pneumoniae among the three ethnic groups, that is, Chinese (80.4%), Malays (74.0%), and Asian Indians (73.2%). There was no association between seropositivity and ischemic heart disease after adjustment for age alone (OR 1.00, 95% CI 0.54-1.83) or for age, sex, and other risk factors of atherosclerosis (OR 0.99, 95% CI 0.53-1.84). C. pneumoniae Immunoglobulin G seropositivity was not associated with an increased risk of ischemic heart disease as defined by the Rose angina questionnaire in Singapore.

During 1999-2000, a sustained mumps outbreak in the highly vaccinated population in Singapore was attributed to vaccine failure associated with the Rubini vaccine strain. To explain this phenomenon, the complete nucleotide and amino acid sequences of the hemagglutinin-neuraminidase (HN) gene of eight mumps virus isolates from patients with parotitis in Singapore were determined and compared with those of known vaccine strains. Phylogenetic trees constructed on the basis of HN nucleotide and amino acid sequences showed that the Singapore mumps virus isolates were more closely related to the Urabe strain and belonged to a different cluster from the Rubini and Jeryl-Lynn strains. The Rubini vaccine showed only approximately 93% nucleotide and approximately 96% amino acid sequence similarity to Urabe and Singapore isolates. Compared with the vaccine strains, six of the eight isolates lacked the extracellular glycosylation site at residues 400-402. Other significant amino acid disparities (e.g., at residue 354) may also affect the antigenic properties of the HN protein. These findings suggest that the evolution and adaptation of the currently circulating mumps virus strains in the community has led to the emergence of genetically distinct viral strains. The low vaccine efficacy of the Rubini strain represents a major reason for the recent mumps resurgence and failure of mumps immunization in Singapore., (Copyright 2003 Wiley-Liss, Inc.)

Until 1998, varicella-zoster virus (VZV) was generally considered sufficiently stable to allow the use of a single sequenced virus (VZV-Dumas) as a consensual representation of the world VZV genotype. But recent investigations have uncovered a gE mutant virus called VZV-MSP with a second genotype and a distinguishable accelerated cell spread phenotype. A subsequent study suggested that single nucleotide polymorphisms (SNPs) could be applied toward the genetic analysis of the VZV genome. To further assess the scope of genetic variation in the VZV genome on a worldwide basis, we carried out an extensive SNP analysis of structural glycoprotein genes gB, gE, gH, gI, gL, as well as the IE62 regulatory gene in viruses collected from Western Europe, North America and Asia, including the VZV vaccine strain. The SNP data showed segregation of viral isolates of Asian origin from those of Western ancestry into distinct phylogenetic clades. Unexpectedly, however, VZV from Thailand segregated with VZV from Iceland and the United States, i.e. it was more Western than Asian in nature. Further, SNP analysis disclosed strikingly unusual genotypes, e.g. gH genes with up to five missense mutations and gL genes with insertions of an in-frame methionine codon. In summary, these VZV genomic analyses have shown that individual VZV strains, like closely related human beings, have distinctive SNP profiles containing private alleles within just five VZV genes (gB, gH, gE, gL and IE62) that provide a fingerprint to localize ancestry of the viral strain., (Copyright 2002 Elsevier Science Ltd.)

We have previously isolated and characterized a novel human gene HUEL (C4orf1) that is ubiquitously expressed in a wide range of human fetal, adult tissues and cancer cell lines. HUEL maps to region 4p12-p13 within the short arm of chromosome 4 whose deletion is frequently associated with bladder and other carcinomas. Here we present the genomic organization, sizes and boundaries of exons and introns of HUEL. The GC-rich upstream genomic region and 5' untranslated region (UTR) together constitute a CpG island, a hallmark of housekeeping genes. The 3250 bp HUEL cDNA incorporates a 1704 bp ORF that translates into a hydrophilic protein of 568-amino acids (aa), detected as a band of approximately 70 kDa by Western blotting. We have isolated the murine homolog of HUEL which exhibits 89% nucleotide and 94% amino acid identity to its human counterpart. The HUEL protein shares significant homology with the minimal DNA-binding domain (DNA-BD) of the DNA repair protein encoded by the xeroderma pigmentosum group A (XPA) gene. Other notable features within HUEL include the putative nuclear receptor interaction motif, nuclear localization and export signals, zinc finger, leucine zipper and acidic domains. Mimosine-mediated cell cycle synchronization of PLC/PRF/5 liver cancer cells clearly portrayed translocation of HUEL into the nucleus specifically during the S phase of the cell cycle. Yeast two-hybrid experiments revealed interactions of HUEL with two partner proteins (designated HIPC and HIPB) bearing similarity to a mitotically phosphorylated protein and to reverse transcriptase. Co-immunoprecipitation assays validated the interaction between HUEL and HIPC proteins in mammalian cells. HUEL is likely to be an evolutionarily conserved, housekeeping gene that plays a role intimately linked with cellular replication, DNA synthesis and/or transcriptional regulation.

Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT-PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.

Enterovirus 71 (EV71) is the most common aetiological agent detected in cases of hand, foot and mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. A comparison of the capsid proteins implicated in the pathogenicity of the fatal and non-fatal strains of EV71, reveals a high degree of homology (93%-100% identity). To facilitate diagnostic immunoassays and vaccine development, a consensus structural model for the EV71 coat protein has been developed based primarily on the homologous structure of the bovine enterovirus. The overall architecture of the virion closely resembles those of related icosahedral picornaviruses. Detailed atomic modelling of the fatal 5865/SIN/00009 strain has been carried out, and the functional regions (known and predicted) from closely related viruses mapped onto the surface of the predicted structure. From the model, we have identified two putative immunogenic regions, one of which is unique to EV71. The hydrophobic pocket within VP1, found in bovine enterovirus, poliovirus and rhinovirus, is also conserved in EV71.