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Biomedical subjects

T Nguyen

Publications and source records attributed to T Nguyen.

At least 19 recordsLinked to original sources
PubMed1992

Isolation and characterization of the human glutathione S-transferase A2 subunit gene.

We have isolated and characterized a second human liver glutathione S-transferase (GST) subunit gene. The nucleotide sequence of this gene indicates that it encodes the alpha class subunit A2, with a coding region of about 13 kb. Using reverse transcription assays it could be shown that the A2 subunit gene is expressed in human liver and HepG2 cells. The transcription initiation site has been determined by primer extension analysis. A "TATA"-sequence was found 26 nucleotides upstream from the transcription start site. A comparison of the structure of the A2 subunit gene with that of the A1 subunit gene shows significant sequence identity between the two genes. Southern blot analysis of restriction endonuclease digests of human DNA indicates that there may be several more human alpha class GST genes.

Amino Acid Sequence↗
PubMed1992

Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease.

Structural and nonstructural regions of the HCV-encoded polyprotein have been expressed in recombinant yeast, bacteria, or insect cells and used to capture and measure reactive antibodies circulating in different individuals. The putative nucleocapsid protein (C) and nonstructural proteins 3-5 (NS3-NS5) were found to contain the most immunodominant epitopes. The NS3, NS4, and C regions were expressed in yeast in the form of a fused, chimeric polyprotein (C25) and a capture assay for reactive antibody was developed. This anti-C25 assay detects all previously identified HCV-seropositive cases and provides a substantially more sensitive diagnostic for both acute and chronic HCV infections than the current anti-C100-3 (NS4) assay. Anti-C25 was detected more frequently than anti-C100-3 in chronic, transfusion-associated non-A, non-B hepatitis patients from the United States (95% vs. 71%) and Japan (98% vs. 82%), in cryptogenic cirrhosis patients from the United States (62% vs. 28%), and in hepatitis B surface antigen-negative cases of hepatocellular carcinoma from Japan (83% vs. 63%). These data indicate that HCV has a greater role in these liver diseases than was previously thought. In volunteer United States blood donors sampled following the introduction of anti-C100-3 screening, the prevalence of anti-C25 and anti-C100-3 was 0.5% and 0.08%, respectively.

Antigens, Viral↗
PubMed1992

Effects of sedatives on GABA-mediated chloride flux into cerebral cortical microsacs prepared from emotional and non-emotional mice.

Some strains of rats and mice express increased momentary fear or emotionality when exposed to a novel environment. Previous studies have found significantly fewer diazepam binding sites in the brains of Balb/cJ mice compared to C57BL and AKR/J mice and this has been suggested to contribute to the increased emotionally of the 'nervous' Balb strain. The influx of 36Cl into cerebral cortical microsacs was used to functionally assess the effects of GABA, diazepam and pentobarbital in the Balb mice compared to nonemotional animals (C57 and ICR mice). Slight differences in the ability of GABA to increase chloride influx were found among the three strains. Pentobarbital potentiation of GABA-mediated chloride flux was slightly higher in the ICR mice compared to Balb and C57. Diazepam potentiation of the effects of GABA, however, was significantly decreased in the Balb mice, strengthening the hypothesis that the benzodiazepine receptor is involved in mediating animal emotionality.

Analysis of Variance↗
PubMed1992

Regulation of rat glutathione S-transferase Ya subunit gene expression. DNA-protein interaction at the antioxidant responsive element.

We have characterized the interaction of the antioxidant responsive element (ARE) in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene with its trans-acting factor. The ARE core sequence, 5'-ggTGACaaaGC-3', previously identified as the cis-acting element required for activation of the Ya subunit gene by planar aromatic compounds and phenolic antioxidants, is shown to be the high affinity recognition motif for a trans-acting factor(s) as determined by gel mobility shift assays as well as methylation interference and protection studies. The DNA-protein interaction appears to occur in the major groove and involves the GpG dinucleotide preceding and the G residue within the TGAC tetramer on the coding strand of the core sequence. In addition, DNase I protection analysis maps an extended region 5' from the core recognition motif, which was shown previously to be essential for basal activity of the ARE. The trans-acting factor is present in nuclear extracts from untreated and tert-butylhydroquinone-treated cells as determined by photochemical cross-linking experiments. The cross-linked protein appears to be a heterodimer with subunit molecular weights of approximately 28,000 and approximately 45,000.

Animals↗
PubMed1992

Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide.

Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to the constitutively expressed rat liver Yc1 subunit. The significance of the fact that both mouse Yc and rat Yc2 exhibit high catalytic activity towards AFB1-8,9-epoxide, whereas rat Yc1 possesses little activity towards this compound, is discussed in terms of structure/function.

Aflatoxin B1↗
PubMed1992

3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.

Amino Acid Sequence↗
PubMed1992

DNA damage and mutation in human cells exposed to nitric oxide in vitro.

Nitric oxide (NO.) is a physiological messenger formed by several cell types. Reaction with O2 forms oxides that nitrosate amines at pH values near 7. We now report experiments in which NO. was added to intact human cells and to aerobic solutions of DNA, RNA, guanine, or adenine. TK6 human lymphoblastoid cells were mutated 15- to 18-fold above background levels at both the HPRT and TK gene loci. Xanthine and hypoxanthine, from deamination of guanine and adenine, respectively, were formed in all cases. NO. induced dose-responsive DNA strand breakage. Yields of xanthine ranged from nearly equal to about 80-fold higher than those of hypoxanthine. Yields of xanthine and hypoxanthine from nucleic acids were higher than those from free guanine and adenine. This was most pronounced for xanthine; 0.3 nmol/mg was formed from free guanine vs. 550 nmol/mg from calf thymus RNA. Nitric oxide added to TK6 cells produced a 40- to 50-fold increase in hypoxanthine and xanthine in cellular DNA. We believe that these results, plus the expected deaminations of cytosine to uracil and 5-methylcytosine to thymine, account for the mutagenicity of nitric oxide toward bacteria and mammalian cells.

Cell Survival↗
PubMed1992

Isolation and characterization of a human glutathione S-transferase Ha1 subunit gene.

We have isolated and characterized a human liver glutathione S-transferase Ha1 subunit gene. The gene spans approximately 12 kilobases and comprises seven exons separated by six introns. The transcription initiation site has been determined by primer extension analysis. A TATA box is located 26 nucleotides upstream from the transcription initiation site, an adenine residue. RNA blot analysis reveals that the gene is expressed at significantly higher levels in human liver than in HepG2 cells. The isolation and characterization of a human glutathione S-transferase Ha1 subunit gene should facilitate a detailed analysis of its transcriptional regulation.

Amino Acid Sequence↗
PubMed1992

Assessment of spinal and femoral bone density by dual X-ray absorptiometry: comparison of lunar and hologic instruments.

Clinical application of techniques for assessing bone mineral density (BMD) requires accurate and precise measurements that can be related to clearly defined normal ranges. In this study we investigated the clinical interpretation of BMD values in a group of individuals measured on the same day with two different dual-energy x-ray densitometers (Lunar DPX and Hologic QDR 1000). The BMD results were analyzed as absolute values in g/cm2 and with respect to young and age-specific normals as defined by each manufacturer. Absolute BMD values measured by the two instruments were highly correlated (lumbar spine r = 0.98, femoral neck r = 0.95; p less than 0.0001). In the lumbar spine, the two instruments assigned almost identical values when expressed as a percentage of age-matched values and as a percentage of young normals, despite a small but systematic difference between the values assigned for the latter index. In the femoral neck, however, there were significant differences in assignments between instruments, expressed both as a percentage of young normal (mean difference 6.2%) and with respect to age-matched values (mean difference 3.3%). In particular, in premenopausal subjects femoral neck values with the Hologic instrument were assigned significantly lower values. This study shows effective comparability between these two instruments for absolute and relative values for the lumbar spine, as well as for absolute values at the femoral neck, but important differences for normality assignments at the femoral neck. These latter differences may produce bias in the "diagnosis" of femoral neck osteoporosis and may have important implications for clinical decision making.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMed1992

Estimating the fraction of invariable codons with a capture-recapture method.

A codon-based approach to estimating the number of variable sites in a protein is presented. When first and second positions of codons are assumed to be replacement positions, a capture-recapture model can be used to estimate the number of variable codons from every pair of homologous and aligned sequences. The capture-recapture estimate is compared to a maximum likelihood estimate of the number of variable codons and to previous approaches that estimate the number of variable sites (not codons) in a sequence. Computer simulations are presented that show under which circumstances the capture-recapture estimate can be used to correct biases in distance matrices. Analysis of published sequences of two genes, calmodulin and serum albumin, shows that distance corrections that employ a capture-recapture estimate of the number of variable sites may be considerably different from corrections that assume that the number of variable sites is equal to the total number of positions in the sequence.

Biological Evolution↗
PubMed1992

Hemihypertrophy as possible sign of renal cell carcinoma.

In infants and children there is a well-recognized association of hemihypertrophy with visceral malignancies, including those of the kidney as well as benign renal disorders. As a result of our report on an adult with segmental hemihypertrophy and renal cell carcinoma, we suggest that if this combination represents an association similar to that found in children, a radiographic-urologic evaluation is needed to diagnose renal neoplasms at an early stage, and to disclose the presence of treatable benign renal disease.

Carcinoma, Renal Cell↗
PubMed1992

Tomographic myocardial perfusion imaging with technetium-99m teboroxime during adenosine-induced coronary hyperemia: correlation with thallium-201 imaging.

Single-photon emission computed tomographic imaging with technetium-99m teboroxime during exercise has been found to be feasible and the results correlate with those obtained with thallium-201. This study examined the feasibility of this technique and compared tomographic imaging with technetium-99m teboroxime during adenosine-induced coronary hyperemia with thallium-201 imaging. With the patient positioned on the imaging table, adenosine was infused at a rate of 140 micrograms/kg per min for 6 min. At 4 min, 20 to 25 mCi (740 to 925 MBq) of technetium-99m teboroxime was injected intravenously and imaging was started as soon as the infusion was completed with use of a 180 degrees anterior arc and 32 stops at 10 s/stop (total imaging time 7.8 min). Rest images were obtained 60 to 90 min later with use of a similar dose of technetium-99m teboroxime. Exercise tomographic thallium images were obtained within 2 weeks of the teboroxime studies. In the 20 patients studied, the teboroxime images were normal in 2 (50%) of 4 normal subjects and abnormal in 15 (94%) of 16 patients with coronary artery disease; 4 of the 15 had a fixed defect and 11 a reversible defect. There was agreement between teboroxime and thallium studies in 16 patients (80%), in 319 (80%) of 400 segments and in 50 (83%) of 60 vascular segments (p less than 0.05). In two normal subjects, an apparent fixed defect involving the inferior wall was seen on the teboroxime but not the thallium images and was thought to be due to an attenuation artifact secondary to extracardiac activity in the left lobe of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1992

Curdlan sulfate and HIV-1: II. In vitro long-term treatment of HIV-1 infection with curdlan sulfate.

Short-term (1 h) treatment with a newly synthesized sulfated polysaccharide, curdlan sulfate (CRDS), showed relatively weak blocking effects on the binding of human immunodeficiency virus type 1 (HIV-1) to the surface of H9 cells. To investigate whether long-term treatment with CRDS could strengthen this effect, CRDS in various doses (0.1, 1, 10, and 100 micrograms/ml) was used in 2-week treatment periods in four separate protocols or "Procedures." SF titers and p24 antigen levels were partially suppressed during long-term CRDS treatment but returned to control levels after the treatment was terminated. In addition, no direct cytotoxicity of CRDS to H9 cells or H9/HIV-1 cells was observed in vitro in the course of continuous exposure to 100 micrograms/ml CRDS for 2 weeks. These results demonstrate the effectiveness of long-term treatment of cells infected with HIV-1 in inhibiting virus expression. The most dramatic inhibition results were obtained when the compound was present both at the time of exposure of cells to virus and during a long-term follow-up treatment. These results show that CRDS inhibits both the cell-free and cell-associated transmission of HIV-1 to host cells and interferes with early events in virus infection. In contrast, CRDS exhibits no significant virucidal activity and has little effect on already infected cells.

Antiviral Agents↗
PubMed1992

Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury.

Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions > 1:500 but < 1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10(-7)-10(-6) M) or recombinant human interleukin-8 (10(-9)-10(-8) M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3 +/- 4.6, mean +/- SEM; n = 7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6 +/- 17.7; p < 0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1 +/- 22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

[Lymph node metastasis of melanoma of unknown primary tumor].

Sixteen patients with clinical stage II melanoma of unknown primary site (regional lymph node metastasis) were followed up between 1978 and 1988. The 5-year survival rate was 47%. These results seem to parallel those for stage II disease with known primary tumor sites, as has been observed in other data from the literature. The hypotheses put forward to explain such lesions are the spontaneous regression of the primary tumor, and the onset of primary malignant melanoma in the lymph nodes.

Adult↗
PubMed1991

Human dopamine D5 receptor pseudogenes.

Molecular cloning studies have now identified five structurally homologous genes encoding the biosynthesis of the human dopamine receptors, DRD1, DRD2, DRD3, DRD4, and DRD5. Two of these dopamine receptors (DRD1 and DRD5) are encoded by intronless genes. To ascertain whether there are other intronless genes that share identity with the gene (DRD5) encoding the DRD5 receptor, we used a cloning method based on the polymerase chain reaction (PCR). Human genomic DNA was amplified by PCR with oligodeoxyribonucleotides (oligos) based on the DRD5 nucleotide (nt) sequence. Amplification of nt sequences between these oligos allowed the isolation of two independent intronless genes that share identity with DRD5. The full-length clones have also been isolated by screening human genomic libraries. The deduced amino acid sequences for these genes, PG-1 and PG-2, share 91% and 92% identity to DRD5, respectively. However, each of the genes contains differences in the coding regions that would render these genes incapable of encoding functional receptors. Thus, the human genome contains at least two DRD5 pseudogenes, consistent with in situ human chromosomal hybridization analysis which reveals the presence of two pseudogenes.

Amino Acid Sequence↗