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Biomedical subjects

S Chong

Publications and source records attributed to S Chong.

At least 19 recordsLinked to original sources
PubMed1999

Single-shot echo-planar imaging of multiple sclerosis: effects of varying echo time.

Our aim was to determine the relative merits of short and long echo times (TE) with single-shot echo-planar imaging for imaging cerebral lesions such as multiple sclerosis. We examined seven patients with clinically definite multiple sclerosis were imaged at 1.5 T. Patients were scanned with spinecho, single-shot echo-planar imaging, using TEs of 45, 75, 105, and 135 ms. Region of interest (ROI) measurements were performed on 36 lesions at or above the level of the corona radiata. The mean image contrast (IC) was highest (231.1) for a TE of 45 ms, followed by 75 ms (218.9), 105 ms (217.9), and 135 ms (191.6). When mean contrast-to-noise ratios (C/N) were compared, the value was again highest (29.7) for TE 45 ms, followed by 75 ms (28.9), 105 ms (28.5), and 135 ms (26.3). In a lesion-by-lesion comparison, TE 45 ms had the highest IC and C/N in the largest number of cases (50 % and 47.2 %, respectively). IC and C/N for TE 45 ms were superior to those of 75 ms in 64% and 58%, respectively. These results support the use of relatively short TEs for single-shot echo-planar imaging in the setting of cerebral lesions such as multiple sclerosis.

Cerebral Cortex↗
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

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

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↗
PubMed1996

X-ray absorption spectroscopy of the zinc site in tRNA-guanine transglycosylase from Escherichia coli.

A key step in the post-transcriptional modification of tRNA with queuine in Escherichia coli is the exchange of the queuine precursor, preQ1 into tRNA. This reaction is catalyzed by tRNA-guanine transglycosylase (TGT). We have previously shown that the E. coli TGT is a zinc metalloprotein [Chong et al. (1995) Biochemistry 34, 3694-3701]. Site-directed mutagenesis studies indicated that cysteines 302, 304, 307 and histidine 317 constitute the four ligands to the zinc. The involvement of histidine 317 is somewhat confounded by the presence of histidine 316. We have examined the zinc site in TGT (wt) and TGT (H317C) by X-ray absorption spectroscopy. The TGT (wt) data are most consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. Interestingly, the data for TGT (H317C) are also consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. The outer shell imidazole scattering for TGT (H317C) appears to be somewhat more ordered than that for TGT (wt), consistent with our previous suggestion that the wild-type enzyme may exist in two conformations the predominant one involving histidine 317 liganding to the zinc and the minor conformer involving histidine 316 liganding to the zinc. The minor conformer, with histidine 316 coordinating the zinc, appears to have an overall conformation that is subtly different from that of the wild-type enzyme. While TGT (H317C) has kinetic parameters very similar to the wild-type, it does not form the homotrimer quaternary structure of the wild-type. TGT (H317A) has previously [Chong et al. (1995) Biochemistry 34, 3694-3701] been found to contain a significant amount of zinc, but is essentially inactive. This suggests that careful analysis of EXAFS data can reveal subtle conformational changes in metal binding sites that are not observed in more common probes of protein conformation such as CD spectroscopy.

Absorptiometry, Photon↗
PubMed1996

Methylation sensitive protein binding to an intragenic active X-specific methylated region in the M. robustus Hprt gene.

An intragenic region of the wallaroo (Macropus robustus) hypoxanthine phosphoribosyltransferase gene which contains three active X-specific methylated cytosines was examined for protein(s) binding. In vitro DNase I footprinting of unmethylated DNA identified three footprints, one of which (footprint I) contained two of the known differentially methylated sites (a HpaII and a HhaI site). Methylation of the footprint I HpaII site only, abolished the formation of several, specific DNA-protein complexes in mobility shift assays. UV cross-linking experiments indicated that polypeptides involved in the methylation sensitive interactions with footprint I had molecular weights ranging from 72 to 48 kDa. Analogous results were obtained with nuclear extracts from both eutherian and metatherian cells, indicating that these proteins are conserved. We suggest that the binding of these proteins to the inactive X may play some role in gene silencing, with the active gene being protected from this effect by methylation of the binding site.

Amino Acid Sequence↗