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Biomedical subjects

S Chong

Publications and source records attributed to S Chong.

At least 55 records · Page 3Linked to original sources
PubMed1999

The antigen-presenting environment in normal and human papillomavirus (HPV)-related premalignant cervical epithelium.

The activation of HPV-specific T cells within the cervical microenvironment is likely to play an important part in the natural history of cervical intraepithelial neoplasia (CIN). The extent and the type of T cell activation will depend critically on the expression of MHC, costimulatory cell surface molecules and cytokines by keratinocytes and Langerhans cells within the cervical lesion. Expression of MHC class II (HLA-A-DR and -DQ), costimulatory/adhesion molecules (CD11a/18, CD50, CD54, CD58 and CD86) and cytokines (tumour necrosis factor-alpha (TNF-alpha) and IL-10) was therefore investigated by immunohistochemistry in normal squamous epithelium (n = 12), low-grade (n = 23) and high-grade (n = 18) squamous intraepithelial lesions of the cervix. CIN progression was associated with de novo expression of HLA-DR and CD54, and increased expression of CD58 by keratinocytes. However, significantly, there was no expression of any adhesion/costimulation molecule by epithelial Langerhans cells in any cervical biopsy studied. Furthermore, TNF-alpha, a potent activator of Langerhans cells, was expressed constitutively by basal keratinocytes in normal cervix (12+/12). but expression of this cytokine was absent in a number of CIN samples (20+/23 for low-grade, 12+/18 for high-grade CIN). Conversely, the suppressive cytokine IL-10 was absent in normal epithelium (0+/12), but was up-regulated in a number of CIN lesions (12+/23 for low-grade; 8+/18 for high-grade CIN). The restricted expression of costimulation/adhesion molecules and the nature of the cytokine microenvironment within the epithelium may act to limit effective immune responses in some CIN lesions.

Antigen Presentation↗
PubMed1999

Ability of the digene hybrid capture II test to identify Chlamydia trachomatis and Neisseria gonorrhoeae in cervical specimens.

The Digene Hybrid Capture II (HCII CT/GC) test is a combination test designed to detect Chlamydia trachomatis and Neisseria gonorrhoeae in a single specimen. It is a nucleic acid hybridization test which uses signal amplification to increase sensitivity. We compared its performance to that of culture on cervical specimens from 1,370 women. Direct fluorescent-antibody assay was used to resolve discrepant results for C. trachomatis. Samples were collected with a proprietary cervical brush or with endocervical swabs. The HCII CT/GC test proved to be sensitive and specific in detecting these organisms. Compared to N. gonorrhoeae culture, it had a sensitivity of 93% (87/94) and a specificity of 98.5% (1,244/1,263). Compared to C. trachomatis culture, the sensitivity was 97.7% (129/132) and specificity was 98.2% (1,216/1,238). Testing of some specimens with discrepant results by PCR suggested that the test would actually prove to be even more specific if it were compared to a nucleic acid amplification test (NAAT). The sensitivity of C. trachomatis culture was somewhat less, at 88.6% (117/132). The endocervical brush appeared to be better than Dacron swabs for collecting specimens. The HCII CT/GC test offers an attractive format that allows simultaneous detection of C. trachomatis and N. gonorrhoeae with a single specimen. An initial positive result is followed by repeat tests with probes to identify chlamydiae or gonococci. This test is more sensitive than C. trachomatis culture and is at least as sensitive as culture for gonococci. It deserves further evaluation and comparison with NAATs and may well offer an attractive alternative for diagnosis and screening of these infections.

Bacteriological Techniques↗
PubMed1999

Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax(1) is a 40-kDa phosphoprotein, predominantly localized in the nucleus of the host cell, which functions to transactivate both viral and cellular promoters. It seems likely that HTLV-1, through expression of the viral regulatory protein Tax(1), provides some initial alteration in cell metabolism predisposing the development of ATL. Here, we demonstrate that HTLV-1 infection in T-cell lines and patient samples causes overexpression of an early G(1) cyclin, cyclin D2. The transcriptional up-regulation of the cyclin D2 gene is due to activation of Tax on the cyclin D2 gene. More important, we find that overexpression of cyclin D2 is accompanied by acquisition of new partners such as cyclin-dependent kinase 2 (cdk2), cdk4, and cdk6 in infected cells. This is in contrast to uninfected T cells, where cyclin D2 associates only with cdk6. Functional effects of these cyclin-cdk complexes in infected cells are shown by hyperphosphorylation of Rb and histone H1, indicators of active progression into S phase as well as changes in cellular chromatin and transcription machinery. These studies link HTLV-1 infection with changes of cellular cyclin gene expression, hence providing clues to development of T-cell leukemia.

Animals↗
PubMed1999

Insulin-like growth factor I stimulates cardiac myosin heavy chain and actin synthesis in the awake rat.

To determine the effect of insulin-like growth factor I (IGF-I) on cardiac contractile protein synthesis in vivo, we measured L-[ring-2, 6-3H]phenylalanine incorporation into myosin heavy chain and actin during intravenous infusions (4 h) of either saline or IGF-I (1 microgram. kg-1. min-1) in awake rats. After an overnight fast, IGF-I increased myosin synthesis by 29% compared with saline (11.5 +/- 0.8 vs. 8.9 +/- 0.6%/day, P < 0.01) and actin synthesis by 26% (7.2 +/- 0.3 vs. 5.7 +/- 0.3%/day, P < 0.01), with similar effects in left and right ventricles and a comparable effect on mixed cardiac protein. When amino acids were infused with IGF-I, a further increase in myosin synthesis was observed (P < 0.01). In fed rats, despite higher baseline synthesis rates than in fasted rats (P < 0. 01), IGF-I also increased the synthesis of myosin (12.3 +/- 0.5 vs. 9.9 +/- 0.5%/day, P < 0.01) and actin (8.8 +/- 0.3 vs. 7.5 +/- 0. 2%/day, P < 0.01) compared with saline. IGF-I infusion had no hypoglycemic effect and did not change heart rate or blood pressure. Thus relatively low-dose IGF-I has a direct action in vivo to acutely increase heart contractile protein synthesis in both fasted and fed awake rats.

Actins↗
PubMed1998

Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.

Affinity Labels↗
PubMed1998

Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein.

Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.

Amino Acid Substitution↗
PubMed1998

Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity.

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and -70 degreesC overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or -70 degreesC and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.

Adolescent↗
PubMed1998

Hepatitis G virus encodes protease activities which can effect processing of the virus putative nonstructural proteins.

The genome of a recently identified virus, hepatitis G virus (HGV), shows considerable homology to hepatitis C virus (HCV). Two HGV proteases similar to nonstructural proteins NS2 and NS3 of HCV were identified, and their cleavage site specificity was investigated. Amino acids essential for the protease activities were determined by mutation analysis. NS4A of HGV was demonstrated to be a cofactor for NS3-mediated proteolysis, with a region critical for activity residing between Leu1561, and Ala1598.

Amino Acid Sequence↗
PubMed1997

Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs.

The protein splicing element (intein) of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae catalyzes both protein splicing and site-specific DNA cleavage. It has been demonstrated that the conserved splice junction residues are directly involved in protein splicing and the central dodecapeptide motifs are required for DNA cleavage. To examine whether the splicing activity of the intein can be structurally separated from the endonuclease motifs, we made large in-frame deletions at the central region of the intein. We demonstrate for the first time that protein splicing can proceed efficiently after the removal of the central region of the intein including the endonuclease motifs. Our results suggest that the N- and C-terminal regions of the Sce VMA intein may form a separate domain that is not only catalytically sufficient for protein splicing but also structurally independent from the endonuclease domain.

Carrier Proteins↗
PubMed1997

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Affinity Labels↗
PubMed1997

Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women.

In a comparison of commercial ligase chain reaction (LCR; Abbott) and polymerase chain reaction (PCR; Roche) assays, measuring plasmid genes of Chlamydia trachomatis, some specimens were found to be negative by either or both assays but positive in traditional culture or antigen detection tests. Of 767 women, 35 were found to be infected by cervical or urine testing. Twenty three specimens from 16 women may have contained inhibitors in six cervical swabs (CS) and 15 first void urines (FVU). By performing dilution and 'spiking' experiments on five FVU, inhibitors of PCR, LCR or both, which disappeared by dilution, were demonstrated. Confirmatory assays were used which amplified segments of the major outer membrane gene by PCR or LCR. When comparisons of assays were made on a single specimen type, the sensitivities of the amplification assays, compared to an expanded reference standard, were as follows: on CS, PCR was 93.8% (30/32) and LCR was 96.9% (31/32); on FVU, PCR was 76.6% (23/30) and LCR was 93.3% (28/30). When a combined calculation was made to determine the ability of the assays to detect patients infected in the cervix or urethra by testing FVU, the sensitivities dropped to 71.4% (25/35) for PCR and 80.0% (28/35) for LCR: CS sensitivity was 88.6% (31/35) for both amplified tests. There were two CS and five FVU false-positives by PCR which reduced to one CS and three FVU in the combined analysis. There were no false-positives by LCR. Inhibitors and low levels of chlamydial plasmid nucleic acids may have contributed to lower than expected sensitivities, suggesting a possible need for internal positive controls, especially for PCR, when testing urine. More studies with multiple sampling and more than one amplification assay are needed to confirm these findings and to identify and remove inhibitors of amplification assays.

Cervix Uteri↗
PubMed1997

Cysteine 265 is in the active site of, but is not essential for catalysis by tRNA-guanine transglycosylase (TGT) from Escherichia coli.

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694-3701; Garcia et al. (1966), Biochemistry 35, 3133-3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850-2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.

Binding Sites↗
PubMed1997

Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, and Mycoplasma genitalium in First-void Urine Specimens by Multiplex Polymerase Chain Reaction.

Background: Sexually transmitted diseases are often caused by one or more microorganisms, and asymptomatic carriage and transmission may be of significance. Testing for more than one organism in a single assay could be a useful approach to laboratory diagnosis. Methods and Results: A multiplex polymerase chain reaction (PCR) assay was developed that employed specific primers targeted to the 7.5-kb cryptic plasmid of Chlamydia trachomatis, the cppB gene of the 4.2-kb cryptic plasmid of Neisseria gonorrhoeae, the 140-kd major adhesion protein gene of Mycoplasma genitlium, and the urease gene of Ureaplasma urealyticum. All four polymerase chain reaction products were detectable by agarose gel electorphoresis and were confirmed by Southern hybridization using fluorescein isothiocyanate-labeled oligonucleotide probes and enhanced chemiluminescent detection. Using purified DNA preparations, multiplex PCR had a reproducible detection limit of 1 fg of C. trachomatis DNA, 100 fg of N. gonorrhoeae DNA, and 10 fg U. urealyticum DNA and M. genitalium DNA, which converts to 1-2 genomic equivalents (ge) of C. trachomatis and N. gonorrhoeae, 4 ge of M. genitalium, and 10 ge U. urealyticum. Multiplex PCR was compared with individual uniplex polymerase chian reaction PCR assays by testing 117 first-void urine samples (91 men, 26 women) from Canadian or Kenyan patients. Multiplex PCR detected 45 of 46 (97.8%) urines with C. trachomatis DNA, 42 of 42 (100%) urines with N. gonorrhoeae DNA, 17 of 17 (100%) urines with U. urealyticum DNA, 4 of 4 (100%) urines with M. genitalium DNA, 12 of 12 urines that had DNA from two bacteria, and 2 of 2 urines with DNA from three bacteria. Multiplex PCR correctly identified bacteria in 92 of 93 urines for an overall sensitivity of 98.9%. Specificity calculations were 100% for C. trachomatis (71/71), N. gonorhoeae (75/75), U. urealyticum (100/100), and M. genitalium (113/113). Conclusions: Multiplex PCR provided a single sensitive and specific test for the detection of four bacteria in first-void urine samples. Testing of first-void urine samples by multiplex PCR could facilitate studies aimed at improving our understanding of the epidemiology of these important sexually transmitted diseases.

Journal Article↗
PubMed1997

Sequence variation and phylogenetic analysis of envelope glycoprotein of hepatitis G virus.

A transfusion-transmissible agent provisionally designated hepatitis G virus (HGV) was recently identified. In this study, we examined the variability of the HGV genome by analysing sequences in the putative envelope region from 72 isolates obtained from diverse geographical sources. The 1561 nucleotide sequence of the E1/E2/NS2a region of HGV was determined from 12 isolates, and compared with three published sequences. The most variability was observed in 400 nucleotides at the N terminus of E2. We next analysed this 400 nucleotide envelope variable region (EV) from an additional 60 HGV isolates. This sequence varied considerably among the 75 isolates, with overall identity ranging from 79.3% to 99.5% at the nucleotide level, and from 83.5% to 100% at the amino acid level. However, hypervariable regions were not identified. Phylogenetic analyses indicated that the 75 HGV isolates belong to a single genotype. A single-tier distribution of evolutionary distances was observed among the 15 E1/E2/NS2a sequences and the 75 EV sequences. In contrast, 11 isolates of HCV were analysed and showed a three-tiered distribution, representing genotypes, subtypes, and isolates. The 75 isolates of HGV fell into four clusters on the phylogenetic tree. Tight geographical clustering was observed among the HGV isolates from Japan and Korea.

Amino Acid Sequence↗
PubMed1997

Ability of commercial ligase chain reaction and PCR assays to diagnose Chlamydia trachomatis infections in men by testing first-void urine.

A total of 287 men (37.6% with symptoms of urethritis) attending a hospital-based sexually transmitted disease clinic had urethral swabs tested by culture and by direct fluorescent-antibody assay. First-void urine (FVU) was tested for Chlamydia trachomatis by commercially available ligase chain reaction (LCR) and PCR assays. By using an expanded reference standard, 35 men (12.2%) were found to be positive. By performing LCR and PCR, the infection prevalence was found to be approximately twice (11.5 and 12.2%, respectively) that determined swab testing. The sensitivity values were 94.3% for LCR and 100% for PCR. One of the two positive specimens missed by LCR contained inhibitors. PCR produced five false-positive results and LCR produced one.

Chlamydia Infections↗
PubMed1996

Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.

Amino Acid Sequence↗