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Y K Wang

Publications and source records attributed to Y K Wang.

At least 37 records · Page 2Linked to original sources
PubMed1997

Genetic analysis of the Rhizobium meliloti nifH promoter, using the P22 challenge phage system.

In several genera of bacteria, the sigma54-RNA polymerase holoenzyme (E sigma54) is a minor form of RNA polymerase that is responsible for transcribing genes whose products are involved in diverse metabolic processes. E sigma54 binds to the promoters of these genes to form a closed promoter complex. An activator protein is required for the transition of this closed promoter complex to an open complex that is transcriptionally competent. In this study, the P22-based challenge phage system was used to investigate interactions between E sigma54 and the Rhizobium meliloti nifH promoter. Challenge phages were constructed in which the R. meliloti nifH promoter replaced the binding site for the Mnt protein, a repressor of the phage P22 ant gene. When a Salmonella typhimurium strain that overexpressed sigma54 was infected with these challenge phages, E sigma54 bound to the nifH promoter and repressed transcription of the ant gene as seen by the increased frequency of lysogeny. Following mutagenesis of challenge phages that carried the R. meliloti nifH promoter, mutant phages that could form plaques on an S. typhimurium strain that overexpressed sigma54 were isolated. These phages had mutations within the nifH promoter that decreased the affinity of the promoter for E sigma54. The mutations were clustered in seven highly conserved residues within the -12 and -24 regions of the nifH promoter.

Bacteriophages↗
PubMed1995

Butyrate and TGF-beta downregulate Na,K-ATPase expression in cultured proximal tubule cells.

Attempts were made to examine the effect of growth inhibitors on regulation of Na,K-ATPase in primary cultures of renal proximal tubule cells. We observed that both TGF-beta and butyrate induced dose dependent decrease of Na,K-ATPase activity. The time course studies showed that the effect of TGF-beta preceded the effect of butyrate on inhibition of Na,K-ATPase activity. Both butyrate- and TGF-beta-induced inhibition of this enzyme were in general mediated by transcriptional decreases of alpha and beta mRNA and therefore also protein abundance. Like its effect on Na,K-ATPase activity, TGF-beta compared to butyrate induced much earlier decline in Na,K-ATPase alpha and beta mRNA and protein abundance. Butyrate did not affect gene expression of TGF-beta and anti-TGF-beta antibody had no effect on butyrate-induced inhibition of DNA synthesis. Obviously, both butyrate and TGF-beta can inhibit the expression of Na,K-ATPase alpha and beta mRNA, but TGF-beta does not mediate butyrate's effect.

Animals↗
PubMed1995

Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.

Automation↗
PubMed1995

Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.

Base Sequence↗
PubMed1995

Metabolism of cyclosporin G in the mouse, rat, and dog.

Cyclosporin G (CsG; Sandoz compound OG 37-325) is a cyclic undecapeptide with potent, immunosuppressive activity and is currently in clinical testing for prevention of transplanted solid organ rejection. Although structurally similar to cyclosporin A (CsA), results in animals suggest that CsG has a reduced potential for nephrotoxicity when compared with CsA, while retaining equivalent therapeutic efficacy. In the present study, the major metabolic pathways of CsG in the mouse, rat, and dog were investigated using radiolabeled drug substance to determine if interspecies differences in metabolism exist. The results indicated that the major metabolic pathways in these animal species are similar to those previously reported for CsA, including oxidative modifications at amino acids 1, 4, and 9, and concomitant cyclization of amino acid 1 in two of these metabolites. Moreover, the seven major CsG metabolites (designated GM19, GM1c9, GM4N9, GM1, GM9, GM1c, and GM4N) observed in animal excreta and/or blood were identical to those identified in humans. The major circulating metabolite in blood was GM9 (9-hydroxylated CsG) in all species. In addition, numerous unidentified minor metabolites were observed. Renal excretion was a minor elimination pathway, with the majority of drug-related material excreted via the fecal route. In conclusion, CsG was found to proceed through the same metabolic pathways in three animal species and humans, and that species differences in metabolism were primarily because of differences in the relative importance of the pathways observed.

Amino Acid Sequence↗
PubMed1994

Expression and regulation of Na, K-ATPase in primary culture of proximal tubule cells.

We previously reported that butyrate slowed the downregulation of activities of differentiation marker enzymes for the proximal tubule during cellular proliferation. This work was designed to delineate whether butyrate also regulated activity of Na,K-ATPase, a basolateral membrane marker. We observed that Na,K-ATPase activity was decreased in cultured proximal tubule cells, which occurred before cell proliferation. When cultured proximal tubule cells approached confluency from day 4 to day 6, Na,K-ATPase activity was increased by 27%, but the increase was not seen in cultures under a lower plating density. Cultured proximal tubule cells under a large plating density also exhibited greater Na,K-ATPase activity than those under a small density. Na butyrate inhibited Na,K-ATPase activity throughout the course of primary culture and dependent on dose in the range 2-5 mM. At the confluent phase, 24-h treatment of butyrate (5mM) induced a 24% decrease in Na,K-ATPase activity, which is associated with coordinated decreases in both Na,K-ATPase alpha and beta subunit abundances and is mediated by coordinate decreases in both Na,K-ATPase alpha and beta mRNA levels. Moreover, Na butyrate, at a dose greater than 2 mM, inhibits proliferation of proximal tubular cells, but results in cell hypertrophy. Finally, the effect of butyrate on cell growth and Na,K-ATPase expression cannot be mimicked by other short chain fatty acids, such as acetate, hexanoate or octanoate.

Animals↗
PubMed1993

Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping.

A light microscope-based technique for rapidly constructing ordered physical maps of chromosomes has been developed. Restriction enzyme digestion of elongated individual DNA molecules (about 0.2 to 1.0 megabases in size) was imaged by fluorescence microscopy after fixation in agarose gel. The size of the resulting individual restriction fragments was determined by relative fluorescence intensity and apparent molecular contour length. Ordered restriction maps were then created from genomic DNA without reliance on cloned or amplified sequences for hybridization or analytical gel electrophoresis. Initial application of optical mapping is described for Saccharomyces cerevisiae chromosomes.

Chromosomes, Fungal↗
PubMed1993

Phorbol 12-myristate 13-acetate-induced phosphorylation of Op18 in Jurkat T cells. Identification of phosphorylation sites by matrix-assisted laser desorption ionization mass spectrometry.

Op18 is a widely expressed, cell cycle-regulated, phosphoprotein involved in signal transduction of a variety of stimuli. In actively proliferating Jurkat T cells which express Op18 at high level, phorbol 12-myristate 13-acetate (PMA) treatment induces a rapid increase in the level of several Op18 phosphorylated forms. To determine phosphorylation sites involved in the PMA effect, the major Op18 phosphorylated forms were resolved in Jurkat T cells, before and after treatment with PMA, using preparative immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. Tryptic fragments of phosphorylated Op18 were analyzed by two-dimensional thin layer peptide mapping and were resolved by reverse-phase high performance liquid chromatography prior to analysis by matrix-assisted laser desorption ionization mass spectrometry. Phosphorylation sites were identified by further treatment of the proteolytic fragments with different enzymes and determination of the mass shifts by matrix-assisted laser desorption ionization mass spectrometry. Two major phosphorylation sites were identified. Low constitutive levels of phosphorylation at Ser25 and Ser38 in Op18a and Op18b was demonstrated. Treatment with PMA resulted in enhanced phosphorylation of Ser25 in Op18a and of both Ser25 and Ser38 in Op18b. Taken together with prior studies of Op18 phosphorylation, the data suggest that Op18 phosphorylation occurs at identical sites in different tissues and organisms.

Amino Acid Sequence↗
PubMed1993

Maturation stage and proliferation-dependent expression of dUTPase in human T cells.

We have developed a database of lymphoid polypeptides detected by two-dimensional polyacrylamide gel electrophoresis to aid in studies of leukemogenesis and of mutation affecting protein structure. In prior studies, we observed a 19-kDa phosphopolypeptide which was induced with proliferation in mature T cells and constitutively expressed in immature thymocytes. In this report we describe the identification of this polypeptide as the phosphorylated form of dUTPase (EC 3.6.1.23), following cDNA cloning of the gene, based on a partial amino acid sequence of the phosphopolypeptide. Studies of the expression and phosphorylation of dUTPase in human T cells indicate that accumulation and phosphorylation of dUTPase in mature T cells occur in a cell cycle-dependent manner. Interestingly, noncycling immature thymocytes express constitutively high levels of phosphorylated and unphosphorylated dUTPase. These results suggest an important role for dUTPase in immature thymocytes that is independent of proliferation.

Amino Acid Sequence↗
PubMed1993

Hepatitis C virus infection: its role in chronic liver diseases.

In order to find out the infectious rate of hepatitis C virus (HCV) among chronic liver diseases, we investigated antibodies against hepatitis C virus and HCVRNA by method of ELISA, RIBA and RT-PCR in 410 patients with chronic liver disease. The prevalence of HCV infection was found to be 4%. Whereas the positive rate of HBsAg and HBV-DNA of these cases was 69% and 58%, respectively. There is no statistical significance between HCV infectious rate of patients with positive and negative HBsAg. The relative low infectious rate of HCV infection among chronic hepatic diseases indicates that HBV infection plays a more important role in causing chronic hepatitis than that of HCV. Thus, special emphasis should be paid to the preventive and therapeutic measures against hepatitis B in China.

China↗
PubMed1993

Establishment and application of SPA-co-operated ELISA for detection of anti-HCV-IgM.

A staphylococcus aureus protein A co-operated ELISA (SPA-ELISA) for the detection of anti-HCV-IgM has been established using HCV antigenic polypeptide, SPA-bearing germs and horseradish peroxidase labelled anti-human IgM. The specificity of SPA-ELISA has been confirmed by some substitution tests, blocking tests and destroying test with 2-mercaptoethanol. The results showed that the rate of anti-HCV-IgG in a group of patients with acute hepatitis and there were significant difference in anti-HCV-IgM was higher than that of anti-HCV-IgM detected rates between patients with acute hepatitis and those with chronic hepatitis (32.26%, P < 0.01). On the other hand, the positive rates of anti-HCV-IgM were 53.66% and 63.41% in transfusion associated hepatitis, 38.10% and 42.86% in sporadic hepatitis, 6.11% and 16.33% in people who have had active social activities, 40.00% and 10.00% in a group of blood donors respectively. Furthermore, taking into account the characteristics of HCV polypeptide used, its easiness of manipulation, and elimination of the interference of anti-HCV-IgG in sera, the new SPA-ELISA is believed to be of practical value in clinical and epidemiological studies of hepatitis C.

Enzyme-Linked Immunosorbent Assay↗
PubMed1993

[Evaluation of DNA content in glandular expansion and adenocarcinoma of gastric mucosa].

Gastric mucosa biopsies from 61 patients with gastric adenocarcinoma and glandular expansion of different nature were used to study DNA content with photo-cytometry technique. It was found that the number of diploid and near diploid cells in simple glandular expansion and dysplastic glandular expansion of gastric mucosa gradually decreased and finally disappeared in gastric adenocarcinoma. The proliferating diploid cells increased with the seriousness of the dysplastic hyperplasia and the DNA ploidy patterns illustrated in histogram became widened with a right shift of the highest peak with a significant increase of the DNA content, moreover, aneuploid cells could also be found. The DNA content of cell nucleus and the ploidy histogram of the grade III dysplastic glandular expansion approached those of tubular adenocarcinoma.

Adenocarcinoma↗
PubMed1992

[Synthesis cloning and expressions in E coli of human insulin A and B chain genes].

Human insulin A and B chain genes were designed and synthesized by using a rapid and simple method. The synthesized A and B chain genes were cloned separately. The expression (plasmids) pWR 590-HIA and pWR 590-HIB were constructed, and the two plasmids can direct the synthesis of the approximately 590 amino acid-long truncated beta-galactosidases fused to human insulin A or B chains. The fused A or B chain proteins were isolated from the fermented cells and cleaved with BrCN. The resulting mixtures were sulfonated and the sulfonated A and B chains were purified. Human insulin was obtained by using an A and B chain combination method.

Amino Acid Sequence↗
PubMed1992

[Studies on the function of Ser579 and Arg580 in beta-subunit of penicillin G acylase with the method of site-specific mutagenesis].

According to the comparison of amino acid sequence between PGA (Penicillin G Acylase) and PBPs (Penicillin Binding Protein), We suggest that No. 565-595 peptide fragment in beta-subunit of PGA may be a substrate-binding site of enzyme. Plasmid pTZGA was constructed by cloning the 2.6 kb PGA gene of pWGA into phagemid pTZ18U The technique of site-specific mutagenesis was used to study the role of residue No. 579 (Ser) and No. 580 (Arg) of PGA. Four kinds of mutants were obtained (Ser579-->Gly579, Arg580-->Gly580, Arg580-->Glu580, Arg580-->Lys580), both Glu580 and Gly580 mutants showed no activity of enzyme and Lys580 mutant remained 30% and Gly579 mutant kept 70% activity of wilde type. The same protein expression of four mutants according to the results of ELISA indicate that mutation does not affect the expression of PGA, but Arg580 residue may be essential for substrate-binding or catalysis of PGA.

Amino Acid Sequence↗
PubMed1991

[Radiotherapy of cancer of the gastric cardia--report on 40 patients].

Fourty patients with cancer of the gastric cardia as proven by pathology or cytology were treated by radiation therapy from January 1985 to October 1986. All patients except four who refused operation had lesions too advanced for surgical intervention. Telecobalt was used to deliver 60-70 Gy/6-7 wk through 3 or 4 portals. In some patients, 45-60 Gy was also given to the supraclavicular area. 90% of patients had symptomatic improvement and 80% showed objective improvement on X ray barium meal. The 1-, 2- and 3-year survival rates were 83%, 43% and 23%. Ten percent of patients survived the third year without evidence of disease. The influence of sex, age, siege of lesion and X ray findings at the conclusion of treatment were analyzed. Female patients and those whose lesions completely resolved at the completion of radiotherapy had more favorable outcome than the others (P less than 0.01, P less than 0.05). It is suggested that radiation therapy may be an effective means in the combination treatment or palliative treatment of cancer of the gastric cardia.

Adenocarcinoma↗
PubMed1990

The cytoplasmic HBVDNA implication: HBV replication or non-replicative status.

Intrahepatocellular HBVDNA was investigated in patients with chronic hepatitis B by in situ hybridization assay, employing biotin-labelled HBVDNA as the specific probe. It was found that HBVDNA was predominantly present in the cytoplasm of hepatocytes. In general the cases with cytoplasmic HBVDNA could be grouped as follows: 1) cytoplasmic HBVDNA coinciding with intrahepatic envelope protein and nucleocapsid component expression, this pattern of the cytoplasmic HBVDNA would be replicative; 2) cytoplasmic HBVDNA accompanied by intrahepatic single HBsAg expression, common to HBVDNA integrated into host genome with only HBsAg being found frequently in liver; 3) with only cytoplasmic HBVDNA, with neither HBsAg nor HBcAg expression, representative for non-replicative status. It is speculated that the reason for the occurrence of the cytoplasmic hybridization signal in cases with non-replicative HBVDNA may be due to DNA-RNA transcript hybrid. The prudent way to evaluate the implication of the cytoplasmic HBVDNA is to consider the combination of HBVDNA location, intrahepatic gene product expression, and HBV markers in the serum.

Cytoplasm↗