Biomedical subjects

V T Chow

Publications and source records attributed to V T Chow.

At least 37 records · Page 2Linked to original sources

PubMed1997

Amplification and sequencing of varicella-zoster virus (VZV) gene 4: point mutation in a VZV strain causing chickenpox during pregnancy.

The varicella-zoster virus (VZV) causes chickenpox (varicella) as the primary disease and shingles (zoster) as a recurrent manifestation of infection, both being generally benign and self-limiting. While these infections may be severe in adults and even life-threatening in immunosuppressed individuals, they may be amenable to effective antiviral drugs or varicella-zoster immune globulin, provided the treatment is administered early. The prompt diagnosis of VZV infections may be accelerated by rapid, sensitive and specific molecular techniques such as amplification by polymerase chain reaction (PCR) compared with slower and more cumbersome tissue culture and serological procedures. Based on the VZV gene 4 which encodes a transcriptional activator, primers were designed for use in PCR to amplify a target fragment of 381 bp. Distinct diagnostic bands were observed by agarose gel electrophoresis of PCR products of VZV strains isolated from 11 varicella and 7 zoster patients in Singapore, as well as of the Japanese vaccine Oka strain. The detection sensitivity of this PCR assay was determined to be 1 pg of purified VZV DNA equivalent to about 7,000 viral DNA copies. No target bands were amplified from negative control templates from five related human herpes-viruses and from human DNA. The specificity of the PCR products was ensured by direct cycle DNA sequencing, which revealed complete identity of the 18 VZV isolates with the published European Dumas strain. The strong sequence conservation of the target fragment renders this PCR assay highly reliable for detecting the VZV sequence. Only one VZV strain isolated from a patient with varicella during pregnancy exhibited a GGA to GAA point mutation at codon 46 of gene 4, culminating in the non-conservative substitution of Ser with Phe. The predicted secondary structure of the mutant polypeptide portrayed a radical alteration, which may influence its function in transcriptional activation.

Autoradiography↗

PubMed1996

HEP-COP, a novel human gene whose product is highly homologous to the alpha-subunit of the yeast coatomer protein complex.

A 4333-bp novel human cDNA sequence designated HEP-COP was isolated from the Hep3B hepatocellular carcinoma cell line by the RACE technique. Within HEP-COP was identified an ORF of 3672 bp encoding a deduced 1224-amino-acid (aa) sequence which exhibited striking homology with the 1201-aa sequence of RET1P, the alpha-subunit of the coatomer complex (alpha-COP) in Saccharomyces cerevisiae which participates in membrane transport between the endoplasmic reticulum and Golgi apparatus. The aa homology was highest in their N-terminal regions which each contained six WD-40 repeat motifs [Van der Voorn and Ploegh, FEBS Lett. 307 (1992) 131-134], and both proteins were predicted to be hydrophilic with similar estimated molecular masses of 138 324 and 135 599 Da, respectively. Northern blot hybridization demonstrated that HEP-COP was expressed in a wide range of human adult and fetal tissues. RT-PCR analysis revealed no differential expression of HEP-COP in 14 human cancer cell lines, as compared with normal control cells. Considering the close similarities between HEP-COP and yeast alpha-COP, and the ubiquitous expression of HEP-COP implying an essential cellular role, it is likely that HEP-COP is the human homologue of alpha-COP.

Amino Acid Sequence↗

PubMed1996

DENN, a novel human gene differentially expressed in normal and neoplastic cells.

A novel 5843 bp human cDNA sequence was isolated from fetal liver cDNA using the RACE procedure. Within this cDNA was characterised an uninterrupted ORF of 3861 nt encoding a predicted hydrophilic protein of 1287 aa with a calculated molecular mass of 141790 Da and an isoelectric point of 5.41. An RGD cellular adhesion motif was identified within the putative serine- and leucine-rich protein. Northern blot hybridisation with a specific cDNA probe revealed differential levels of expression of 6.5 kb transcripts in 35 human tissues and cancer cell lines, with strongest signals observed in fetal brain and kidney; adult testis, ovary, brain and heart; and in the SW480, K-562, HeLa S3 and HL-60 cell lines. To reflect this feature, the novel gene was designated DENN for differentially expressed in normal and neoplastic cells. Furthermore, an alternative splicing event within DENN involving a 129 nt alternative exon encoding 43 aa was found in fetal liver and in several human cancer cell lines. Based on the predicted aa sequence, two peptides were designed and synthesised to raise rabbit polyclonal antisera which detected a distinct protein band of 140-145 kDa in Western blots of human cell lines. Immunofluorescent labelling of human cells with the same antibodies indicated predominant cell membrane localisation with some cytoplasmic staining.

Alternative Splicing↗

PubMed1995

Rapid, single-step RT-PCR typing of dengue viruses using five NS3 gene primers.

In order to detect and type dengue viruses in serum specimens, four type-specific downstream primers were designed for use with a consensus upstream primer in a reverse transcription and polymerase chain reaction (RT-PCR) assay. RT-PCR using these five primers amplified NS3 gene fragments of diagnostic sizes of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively, but not for Japanese encephalitis, Kunjin and yellow fever viruses. The conventional two-step RT-PCR procedure was simplified by combining RT and PCR in a single-step format with a "hot start". This RT-PCR protocol was applied successfully to dengue virus-spiked serum and dengue patient serum samples, and could detect as few as one PFU of dengue virus. This assay offers a rapid, specific and sensitive molecular technique for the simultaneous detection and typing of dengue viruses.

Base Sequence↗

PubMed1995

Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens.

BACKGROUND: More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed. OBJECTIVE: To develop a semi-nested polymerase chain reaction (PCR) assay based on primers within the NS3 gene for the simultaneous detection and typing of dengue viruses in human sera. STUDY DESIGN: A first round of single-step reverse transcription-polymerase chain reaction (RT-PCR) was carried out with a pair of consensus primers, followed by a second round of semi-nested amplification using the upstream consensus primer and four type-specific down-stream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID(50)-titrated virus-infected tissue culture fluid, and total RNA extracted from C6/36 cells infected with dengue and other flaviviruses, respectively. A retrospective study was performed on acute sera from thirteen patients with dengue (confirmed by virus isolation) employing semi-nested PCR in parallel with virus re-isolation and a single-step RT-PCR method for the typing of dengue viruses in human sera. RESULTS: The semi-nested PCR assay could detect up to 1 pfu of dengue virus, but not other flaviviruses. The semi-nested PCR and single-step RT-PCR assays correctly typed dengue viruses in twelve and five sera, respectively, whereas none of the sera was positive by virus re-isolation. CONCLUSIONS: This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.

Journal Article↗

PubMed1995

Differentiation of Neisseria gonorrhoeae IB-3 and IB-7 serovars by direct sequencing of protein IB gene and pulsed-field gel electrophoresis.

Serotyping of Neisseria gonorrhoeae based on the differential recognition by a panel of six monoclonal antibodies (MAbs) against Protein IB (PIB) is a valuable tool for studying the epidemiology of gonorrhoea. However, the predominance of certain serovars in specific geographic regions necessitates the development of new MAbs or new genotyping approaches. Nucleotide and amino-acid sequence analysis of PIB from strains within the IB-3 and IB-7 serovars revealed strain differences within the same serovar. Based on the generation of PIB nucleotide and deduced amino-acid sequences that centred on amino-acid residues 196 and 237, eight serovar IB-3 strains and nine serovar IB-7 strains were separately subdivided into five groups. Intra-serovar differences were also established by pulsed-field gel electrophoresis (PFGE) of macro-restriction fragments generated by SpeI- and NheI-cleavage of genomic DNA. There was good correlation between the results based on PIB gene PCR-sequencing and those based on PFGE analysis of macro-restriction fragment patterns. These data demonstrate the high precision of PFGE analysis and indicate that this approach can be used as a rapid epidemiological subtyping system for major serovars of N. gonorrhoeae.

Amino Acid Sequence↗

PubMed1995

Differentiation of Neisseria gonorrhoeae strains by polymerase chain reaction and restriction fragment length polymorphism of outer membrane protein IB genes.

OBJECTIVES: To employ polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for the rapid differentiation of Neisseria gonorrhoeae protein IB (PIB) isolates and to compare its usefulness with the widely accepted auxotype/serovar classification scheme. METHODS: The outer membrane protein IB genes of 47 gonococcal isolates belonging to 10 different serovars were amplified by PCR. The approximately 1 kb DNA products were then digested separately with restriction enzymes CfoI and MspA1I, and electrophoresed on agarose gels. RESULTS: Cleavage of PIB genes by MspA1I and CfoI differentiated all the N gonorrhoeae strains into five and six PCR-RFLP profiles, respectively. PCR-RFLP was more discriminatory than auxotyping, which classifies the strains into only two auxotypes. Some strains belonging to common serovars could be further differentiated. A combination of PCR-RFLP analysis, auxotyping and serotyping further increased the discrimination of the strains into 34 subtypes. The PCR-RFLP method was easy to perform, reliable, reproducible, and consistent with published nucleotide sequence data. CONCLUSION: The PCR-RFLP method can augment auxotyping and serotyping or be used as a preliminary screening tool to differentiate N gonorrhoeae strains in areas where serotyping reagents are not easily available.

Bacterial Outer Membrane Proteins↗

PubMed1994

Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from southeast Asia.

By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.

Amino Acid Sequence↗

PubMed1994

The c-erbA beta thyroid hormone receptor. Expression and cDNA sequence analysis of the hormone-binding domain in human cancer cell lines.

The human c-erbA beta protooncogene encodes a thyroid hormone receptor (comprising a hormone-binding domain and a DNA-binding domain) which modulates expression of specific genes, such as cell differentiation genes. Using the reverse transcription and polymerase chain reaction (RT-PCR) assay, significant expression of the c-erbA beta gene was detected in the SiHa, CaSki, HeLa cervical carcinoma; Hep3B, PLC/PRF/5, Mahlavu hepatocellular carcinoma; HT-1080 fibrosarcoma cell lines; as well as in normal MRC-5 embryo lung and FS-4 foreskin fibroblast cell lines. However, the Molt-4 leukaemia and Raji Burkitt's lymphoma cell lines exhibited very low levels of c-erbA beta expression. Single-strand conformation polymorphism analysis and direct sequencing of PCR products of the c-erbA beta hormone-binding domain cDNAs of these cell lines revealed identical sequences, but differed from the published human placental c-erbA beta sequence by five single base disparities. Sequencing of an aberrant fragment fortuitously amplified from the HT-1080 cDNA library demonstrated concordance with the cDNA of pregnancy-specific glycoprotein 4, which is related to the tumour marker, carcinoembryonic antigen.

Base Sequence↗

PubMed1993

Alternative splicing of the p53 tumor suppressor gene in the Molt-4 T-lymphoblastic leukemia cell line.

The expression of the p53 tumor suppressor gene in ten human cell lines (nine cancers and one normal) was studied using reverse transcription, polymerase chain reaction (PCR) and direct sequencing. Using P53U and P53D primers for amplifying a 371-base pair (bp) target fragment spanning exons 7-10 of p53 cDNA, normal-sized PCR products were amplified from 9 cell lines but not from the Hep3B hepatocellular carcinoma (HCC) cell line. An additional larger band (504 bp) was observed for the Molt-4 T-lymphoblastic leukemia cell line. Employing P531 and P53D primers which flank a 76-bp p53 cDNA fragment, 76 bp as well as 209 bp products were generated by PCR of Molt-4 cDNA. Direct sequencing of the 504 bp and 209 bp bands confirmed the presence of a 133 bp insertion between exons 9 and 10 in the aberrant transcript. This insertion was homologous to a 130-bp sequence within the wild-type p53 intron 9, except for 2 point mutations and 3 base insertions. Sequencing of P53U/P53D PCR products of Molt-4 genomic DNA revealed an 8 bp deletion just downstream to the 133 bp insertion, creating a novel donor splicing site within intron 9. This site, coupled with an inherent acceptor splicing site just upstream to the 133 bp insertion, suggests that the 133 bp stretch represents an alternative exon. The occurrence of a termination signal within this alternative transcript is predicted to culminate in a truncated p53 translational product. The sequences of the 371 bp PCR products of Molt-4, HT-1080, SiHa, CaSki, HeLa and MRC-5 cell lines corresponded with the wild-type p53 cDNA. G-->T transversions at the third base of codon 249 of p53 were detected in Mahlavu and PLC/PRF/5 HCC lines, while a TAC to CAC mutation at codon 234 was observed in an allele of the Raji Burkitt lymphoma line.

Alternative Splicing↗

PubMed1993

Comparative analysis of the restriction endonuclease profiles of the Dumas and Singapore strains of varicella-zoster virus.

The incidence of varicella in Singapore has been increasing since 1984. In 1991, 17,930 cases were reported in a population of about 3 million. A serological survey completed in 1990 demonstrated that only 43% of the cohort had antibodies to varicella-zoster virus (VZV), indicating inadequate herd immunity. To exclude novel VZV strains, representative VZV isolates from 9 chicken pox and 4 zoster patients were characterised by restriction endonuclease analysis. DNAs were extracted from viral isolates propagated in MRC5 human embryo lung cells and were digested separately with BglII, EcoRI, PstI, SalI, and XbaI enzymes. The cleavage profiles of these VZV strains derived from both chicken pox and zoster lesions revealed no distinct differences. This observation implies that the current upsurge of chicken pox most likely stems from closely related VZV genotypes infecting a susceptible population with insufficient herd immunity. Comparison of the restriction fragments of the Singapore and the Dumas strains revealed polymorphisms of the SalI-D, SalI-E, and XbaI-I fragment lengths, which correlated with variable regions I, II, and III of the VZV genome, thereby representing geographically distinct genotypic variants of VZV.

Chickenpox↗

PubMed1993

Polymerase chain reaction and direct sequencing of Neisseria gonorrhoeae protein IB gene: partial nucleotide and amino acid sequence analysis of strains S4, S11, S48 (serovar IB4) and S34 (serovar IB5).

A pair of primers were designed for the polymerase chain reaction (PCR) to amplify a 341-base pair fragment of the gene encoding the outer membrane protein IB (PIB) of Neisseria gonorrhoeae. This PCR technique is specific and sensitive, being able to detect gonococcal strains belonging to ten different PIB serovars, but not PIA gonococcus nor other negative control bacteria. PCR products of four representative PIB strains were directly sequenced. Of the three strains belonging to serovar IB4, two (S11 and S48) shared identical nucleotide and amino acid sequences in the PIB region examined. The third IB4 strain (S4) revealed sequences identical to the published IB26 strain (P9). The sequences of strains P9, S4, S11 and S48 were found to differ from those of strain S34 (serovar IB5). The PCR sequencing technique can further differentiate strains belonging to a common serovar and establish clonal relationships among strains. As a molecular epidemiological tool, the PCR-sequencing strategy can augment existing typing methods including serotyping.

Amino Acid Sequence↗

PubMed1993

Use of NS3 consensus primers for the polymerase chain reaction amplification and sequencing of dengue viruses and other flaviviruses.

Consensus primers for the polymerase chain reaction were designed based on conserved motifs within the serine protease and RNA helicase domains encoded by the NS 3 genes of dengue and other flaviviruses. Target fragments of 470 bp were amplified on cDNA templates synthesized from RNAs of dengue types 1, 2, 3, and 4, Japanese encephalitis, Kunjin, and yellow fever viruses using random or specific downstream primers. PCR of oligo(dT)-primed cDNAs from Japanese encephalitis and Kunjin viral RNAs did not yield target bands. As few as 10(3) copies of dengue viral RNA could be detected. Direct DNA sequencing of PCR products of reference strains of dengue 2 (NGC), Kunjin (MRM 61C) and yellow fever (17 D) viruses demonstrated complete concurrence with published data. However, 2 nucleotide differences were observed between our data for dengue 3 H87 strain and the published sequence, resulting in a single amino acid disparity. Differences at 21, 16, and 11 nucleotide positions were noted between dengue 1 Hawaii and S 275/90; dengue 4 H 241 and 814669; Japanese encephalitis Nakayama and JaOArS 982 viral strains, culminating in only 4, 1 and 1 amino acid residue differences, respectively. These amino acid disparities occurred outside putative active sites of the enzymatic domains, emphasizing the important role of the NS3 protein in flaviviral replication. This RNA-PCR consensus primer strategy coupled with DNA sequencing represents a valuable tool for the molecular diagnosis and epidemiology of dengue and other flaviviral infections.

Aedes↗

PubMed1993

Cancer and viruses.

Viruses implicated in the development of human cancers include hepatitis B (and C) viruses in hepatocellular carcinoma; human papillomaviruses in anogenital cancers; Epstein-Barr virus in nasopharyngeal carcinoma and Burkitt's lymphoma; human T-cell leukaemia/lymphoma viruses in adult T-cell leukaemia/lymphoma; and indirectly, human immunodeficiency viruses in Kaposi's sarcoma and B-cell lymphoma. Together, they contribute significantly to the cancer statistics in the Southeast Asian region. Neoplastic proliferation may be instigated by the presence and expression of viral oncogenes which may be integrated into the host genome and/or exist in episomal molecules. Critical viral genes may also interfere with host genes, resulting in the activation of cellular proto-oncogenes and/or the inactivation of anti-oncogenes and their products. The molecular pathogenesis of virally-induced cancers has led to major breakthroughs in the understanding of carcinogenesis at a molecular level. The occurrence of some of these viruses in a significant proportion of normal individuals suggests long latency periods necessitating multi-step co-operating events arising from multi-factorial agents such as host genetic susceptibility, immunological and hormonal status, as well as chemical and physical cocarcinogens in the environment. Successful intervention achieved with effective vaccines such as the hepatitis B vaccine and measures to severe the chain of viral transmission culminating in reduced incidence of the corresponding cancer will provide conclusive evidence for the virus-cancer relationship.

Anus Neoplasms↗

PubMed1992

Patterns and implications of subclinical vulval human papillomavirus infection: the impact of PCR analysis.

This study was performed to elucidate the presence of human papillomavirus (HPV) infection of the vulva by colposcopy, histology and the polymerase chain reaction (PCR). Colposcopy defined 5 patterns of vulval epithelial lesions inconspicuous to the naked eye. Of these 75 subclinical vulval lesions, HPV infection was diagnosed by histology in 20.0% of minor epithelial changes with faint acetowhitening, 52.2% of conspicuous acetowhite lesions, 63.0% of acetowhite areas with satellite lesions, 84.6% of villous lesions, and 85.7% of villous lesions with surrounding acetowhitening. The corresponding HPV DNA positivity rates by PCR were 60.0%, 73.9%, 70.4%, 84.6% and 100% respectively. The oncogenic HPV type 16 was detected by PCR in 37.3% of the samples. These results provide firm evidence for the prevalent existence of subclinical HPV lesions of the vulva. Some of these infections may not produce significant morphologic changes detectable by colposcopy or histology. Subclinical vulval lesions are common and may constitute a reservoir for repeated cervical HPV infections, as well as a source of contamination of cervical samples for HPV DNA detection by sensitive molecular techniques.

Base Sequence↗

PubMed1991

Diagnostic sensitivity of polymerase chain reaction and Southern blot hybridization for the detection of human papillomavirus DNA in biopsy specimens from cervical lesions.

Human papillomaviruses (HPVs) are associated with benign and malignant neoplasms of the cervix. One of the criteria for their etiologic role requires an assessment of whether virtually all or only a small fraction of lesions contain viral genomes. DNA preparations from colposcopically directed punch biopsies of cervical lesions were analyzed by Southern blot hybridization and the polymerase chain reaction (PCR) for the presence of HPV DNA. The biopsy specimens represented different pathologic entities (koilocytosis, condyloma, cervical intraepithelial neoplasia, and invasive carcinoma). In Southern blot hybridization with radioactive probes for HPV 11, 16, 18, 31, and 33, HPV DNA was detected in 74% of the biopsy specimens (42 of 57 cases), with the predominant types being HPV 16 and HPV 18. In contrast, after PCR amplification with primers yielding fragments of characteristic size for HPV 11, 16, and 18, the analysis of the same 57 biopsy specimens revealed that all samples were positive for at least one HPV type. To exclude false-positive PCR results, controls without HPV DNA were interspersed at regular intervals, and results were evaluated only if these controls remained HPV negative. To exclude false-negative results due to failure of the reaction, a target sequence within the c-Ha-ras-1 gene was used as an internal control. All HPV typing results obtained by Southern blot hybridization were in agreement with HPV typing by PCR. The higher number of positive samples in the latter analysis stems from the increased sensitivity of PCR, which was which was effective in identifying as few as 10-100 HPV DNA molecules; in contrast, the sensitivity of Southern blot hybridization was 1 pg, or approximately 10(5) molecules of HPV DNA. The authors conclude that, with sufficiently sensitive diagnostic methods, HPV DNA can be detected in most, if not all, neoplastic cervical lesions.

Base Sequence↗

PubMed1990

Thermus aquaticus DNA polymerase-catalysed chain reaction for the detection of human papillomaviruses.

To delineate the conditions for the polymerase chain reaction (PCR) using primers specific for human papillomavirus (HPV) types 6b, 16 and 18, a number of important technical features were analysed. Buffer, concentrations of magnesium, Taq polymerase, primers and DNA templates, annealing temperature, and extension time were studied by a combination of gel electrophoresis, Southern and slot-blot hybridization. Amplification of E6 gene fragments of HPV-16 and HPV-18 generated bands of 110 bp and 154 bp respectively, as predicted. However, amplification of a segment within the long control region of HPV 6b yielded an unexpected size of 340 bp. Different conditions were found for each HPV type-specific primer pair. These results, and the applications of PCR in HPV research and in an increasingly wide range of fields in medical virology are discussed.

Base Sequence↗

PubMed1990

Genital human papillomavirus infection among women from major ethnic groups in Singapore.

The close epidemiological relationships between specific genital human papillomavirus (HPV) types and neoplasia of the cervix uteri have been extensively documented worldwide, including Singapore. Cervical cancer incidence rates in Singapore show variations between the major ethnic groups. To ascertain the corresponding HPV infection rates among the various races in Singapore, we analysed the cervical smears of 225 women by filter in situ DNA hybridization, and compared the data with a previous similar study. Fourteen (6.2%) individuals were HPV-positive, with HPV 16 and HPV 31 being the commonest types. No significant difference between HPV positivity rates in Chinese (5.0%) and in Malays (6.7%) was found, even though Chinese have a higher cervical cancer incidence than Malays. Furthermore, the cervical HPV carriage rate among women with normal cytology was 5.9%. In the light of reports of high genital HPV prevalence rates detected by DNA amplification, these data support the notion that HPV infection is commonly latent and requires the cooperation of other factors for cervical carcinogenesis.

Adult↗