Biomedical subjects

W Wang

Publications and source records attributed to W Wang.

At least 19 recordsLinked to original sources

PubMed1992

Expression and regulation of cytochrome P-450I genes (CYP1A1 and CYP1A2) in the rat alimentary tract.

Cytochrome P-450 (P450) enzymes in the mucosa of the alimentary tract may be involved in the activation and/or inactivation of ingested xenobiotics and procarcinogens. Since the multiple P450 enzymes have overlapping substrate specificities and, in some cases, similar antigenic determinants, definitive identification of P450 genes that are expressed in various tissues requires molecular analysis. In this study, a sensitive and specific polymerase chain reaction assay along with hybridization analysis was used to examine the expression of the CYP1A gene family in the rat alimentary tract. CYP1A1 mRNA was expressed throughout the alimentary tract in untreated rats, as determined by the polymerase chain reaction. However, Northern blot analysis detected CYP1A1 mRNA and enzymatic activity only in small intestine and liver, with greater amounts in intestine. After treatment with beta-naphthoflavone, CYP1A1 mRNA and enzymatic activity was markedly induced in each alimentary tissue including esophagus, fore-stomach, glandular stomach, small intestine and colon. A single dose of inducer resulted in a rapid rise in CYP1A1 mRNA which was shown by nuclear run-on assays to be primarily due to an increase in transcription of the CYP1A1 gene. CYP1A2 mRNA was detected in significant amounts only in glandular stomach following induction although the polymerase chain reaction assay identified low levels of CYP1A2 mRNA in several other tissues. The definitive identification of the CYP1A genes that are expressed in alimentary tissue will allow the design of experiments to investigate the importance of these enzymes in the metabolism of carcinogens, and ultimately carcinogenesis, in the gastrointestinal tract. In addition, these data suggest that the aromatic hydrocarbon receptor, which mediates transcriptional induction of multiple genes by xenobiotics, is expressed through the alimentary tract.

Animals↗

PubMed1992

Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.

We have identified two 1.6 kb macronuclear DNA molecules from Euplotes crassus that hybridize to the alpha subunit of the Oxytricha telomere protein. We have shown that one of these molecules encodes the 51 kDa Euplotes telomere protein while the other appears to encode a homolog of the telomere protein. Although this homolog clearly differs in sequence from the Euplotes telomere protein, the two proteins share extensive amino acid sequence identity with each other and with the alpha subunit of the Oxytricha telomere protein. In all three proteins 35-36% of the amino acids are identical, while 54-56% are similar. The most extended regions of sequence conservation map within the N-terminal section; this section has been shown to comprise the DNA-binding domain in the Euplotes telomere protein. Our findings suggest that some of the conserved amino acids may be involved in DNA recognition and binding. The gene encoding the telomere protein homolog contains two introns; one of these introns is only 24 bp in length. This is the smallest mRNA intron reported to date.

Amino Acid Sequence↗

PubMed1992

Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation.

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.

Animals↗

PubMed1992

Laser photolysis of cytosine, cytidine and dCMP in aqueous solution.

Laser photolysis of cytosine and cytosine derivatives (cytidine and 2'-deoxycytidine-5'-monophosphate) using acetone as a sensitizer have been carried out in aqueous solution using a KrF laser. The absorption spectra of triplet states of cytosine and cytosine derivatives have been observed for the first time. From detailed kinetic analyses, the reaction mechanisms including triplet-triplet excitation transfer, and intramolecular excitation transfer within the exciplex arising from the interaction of triplet acetone with cytosine and cytosine derivatives, have been suggested. Accordingly, a series of kinetic parameters have also been obtained.

Cytidine↗

PubMed1992

Anti-HIV-I activity of linked lexitropsins.

Five groups of lexitropsin oligopeptides have been synthesized that are structurally related to the natural antiviral agents netropsin and distamycin and bearing two such moieties joined by flexible or rigid linkers. Inhibitory activity of these types of agents against murine leukemia retrovirus (MuLV) led to an evaluation of their inhibition of HIV-I in cell culture. The antiretroviral activity of the five different classes of lexitropsins is discussed in terms of their structural differences.

Antiviral Agents↗

PubMed1992

ATP is a coupling modulator of parallel Na,K-ATPase-K-channel activity in the renal proximal tubule.

A fundamental and essential property of nearly all salt-transporting epithelia is the tight parallel coupling between the magnitude of the K-conductive pathway at the basolateral membrane and the activity of the Na,K-dependent ATPase (Na,K-ATPase). In the present study, we demonstrate that the coupling response in the renal proximal tubule is governed, at least in part, through the interaction between ATP-sensitive K channels and Na,K-ATPase-mediated changes in intracellular ATP levels. First, we identified a K-selective channel at the basolateral membrane, which is inhibited by the cytosolic addition of ATP. Second, conventional microelectrode analysis in the isolated perfused proximal straight tubule revealed that these channels are the major determinant of the macroscopic K conductance so that ATP-mediated changes in the open probability of the K channel could alter the extent of K recycling. Indeed, the increase in the macroscopic K conductance upon stimulation of transcellular Na transport and pump activity was found to be paralleled by a decrease in intracellular ATP. Finally, a causal link between parallel Na,K-ATPase-K-channel activity and ATP was established by the finding that intracellular ATP loading uncoupled the response. With our recent observations that similar ATP-sensitive K channels are expressed abundantly in other epithelia, we postulate that ATP may act as a universal coupling modulator of parallel Na,K-ATPase-K-channel activity.

Adenosine Triphosphate↗

PubMed1992

Antibodies directed against synthetic peptides distinguish between the catalytic subunits of protein phosphatases 1 and 2A.

Two antipeptide antibodies (designated type 1 antibody and type 2A antibody) were raised against synthetic peptides, Cys-Thr-Pro-Pro-Arg-Asn-Ser-Ala-Lys-Ala-Lys-Lys and Cys-Val-Thr-Arg-Arg-Thr-Pro-Asp-Try-Phe-Leu, corresponding to the carboxyl termini of the catalytic subunits of protein phosphatase 1 and phosphatase 2A (Cys was added for specific coupling to carrier protein). These antipeptide antibodies were highly specific and were useful in discriminating between protein phosphatase 1 and phosphatase 2A in crude extracts or purified preparations. Type 2A antibody reacted with both native and denatured protein phosphatase 2A whereas under the same condition type 1 antibody reacted only with denatured protein phosphatase 1.

Amino Acid Sequence↗

PubMed1992

Effect of NADPH oxidase inhibition on endothelial cell ELAM-1 mRNA expression.

Neutrophil adherence to endothelium is partially mediated by the expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells activated by agents such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). To elucidate molecular mechanisms involved in the induction of ELAM-1 on endothelial cells, we investigated the effect of the NADPH oxidase inhibitor, apocynin (4-hydroxy-3-methoxyacetophenone), on ELAM-1 mRNA expression in human umbilical vein endothelial cells (HUVEC) by Northern blot analysis. Apocynin downregulated both LPS- and PMA-induced ELAM-1 mRNA expression in a dose-dependent manner. Our results suggest NADPH oxidase might play a key role in ELAM-1 mRNA expression in HUVEC.

Acetophenones↗

PubMed1992

An improved ELISA with linear sweep voltammetry detection.

An improved ELISA combined with linear sweep voltammetry detection of p-nitrophenol generated by an enzyme has been investigated in this study. p-nitrophenol, produced from alkaline phosphatase catalysing p-nitrophenyl phosphate, yielded an oxidative peak at 1.06 V (vs. Ag/AgCl) with a wax-impregnated tubular graphite anode. Without separation, the small three-electrode system was directly inserted in the well of an ELISA plate for detection. The detection limit for p-nitrophenol was 1 x 10(-6) M, lower than that obtained by measuring the absorbance of p-nitrophenol. The feasibility of utilizing linear sweep voltammetry as a detection scheme was demonstrated by determining metallothionein, granulocyte-colony stimulating factor and Xenopus laevis keratin using the above new system. The method was simple, reproducible and much more sensitive than traditional spectrophotometry.

Animals↗

PubMed1992

Isolation and characterization of the human sucrase-isomaltase gene and demonstration of intestine-specific transcriptional elements.

The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.

Adenocarcinoma↗

PubMed1992

Polymerase II promoter activation: closed complex formation and ATP-driven start site opening.

Studies on bacterial RNA polymerases have divided the initiation pathway into three steps, namely (i) promoter binding to form the closed complex; (ii) DNA melting to form an open complex, and (iii) messenger RNA initiation. Potassium permanganate was used to detect DNA melting by mammalian RNA polymerase II in vitro. Closed complexes formed in a rate-limiting step that was stimulated by the activator GAL4-VP16. Adenosine triphosphate was then hydrolyzed to rapidly melt the DNA within the closed complex to form an open complex. Addition of nucleoside triphosphates resulted in the melted bubble moving away from the start site, completing initiation.

Adenosine Triphosphate↗

PubMed1992

Dose-response implications of the transient nature of electromagnetic-field-induced bioeffects: theoretical hypotheses and predictions.

Data in the literature imply that the relationship between exposure and bio-effect involves more than a simple time integral of the field strength to which the living system has been subjected. Windows--ranges in which the system exhibits enhanced sensitivity--have been reported for power (or field strength), frequency, and the duration of the exposure. In this paper we show that such isolated window effects can be accounted for by recognizing the transient character of the response of the biological system. The principal assumption here is that the direct effect of the field is to increase the rates of production and degradation of mRNA or proteins. In this paper we review and extend the mathematical model that quantifies this. The model predicts that, for a given field strength, certain optimum relatively short duration exposures cause significantly larger bio-effects than exposure for much longer or much shorter times. The thinking embodied in the model should provide a framework for obtaining a meaningful working definition of "effective dose" and for predicting the response of subjects to environmental electromagnetic fields. It should help in deciding the relevant variables in the design and analysis of epidemiological studies.

Animals↗

PubMed1992

Comparative molecular modeling of the active subunit of human kininase I.

The structure of the enzymatically active subunit of human plasma carboxypeptidase N was determined by computer aided model building by homology using the structural coordinates from carboxypeptidase A. The active site of carboxypeptidase N has been well conserved in comparison with carboxypeptidase A. Differences in substrate specificity can be explained by the comparison of energetically favorable binding sites for different atomic probe groups.

Amino Acid Sequence↗

PubMed1992

Human serum carboxypeptidase U: a new kininase?

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its instability at room temperature and at 37 degrees C, we named it "unstable carboxypeptidase" (carboxypeptidase U, CPU). The enzymatically active subunit of carboxypeptidase U was purified and exhibits a single band at 53 kDa on SDS-PAGE.

Carboxypeptidases↗

PubMed1992

Conjunctival penetration of insulin and peptide drugs in the albino rabbit.

An in vitro model was used to evaluate the conjunctival penetration of three peptides, [D-ala2]metenkephalinamide (YAGFM, MW 647), substance P (MW 1348), and insulin (MW 5778), in comparison with two nonpeptides, atenolol (MW 266) and timolol (MW 433). All three peptides were hydrolyzed to varying extents during penetration across the conjunctiva. The permeability coefficient for intact YAGFM and insulin was 4.5 +/- 0.3 and 4.6 +/- 0.7 microns sec-1, respectively. These values were about two to five times lower than those for atenolol and timolol. No permeability coefficient could be calculated for substance P, since its transconjunctival flux never reached steady state. The conjunctival penetration of YAGFM and insulin was improved by about two and three times, respectively, with the addition of 1% Na glycocholate. Increasing the Na glycocholate concentration was more effective than changing the type of bile salt in improving the conjunctival penetration of insulin. The maximum factor of improvement was 12, as the Na glycocholate concentration was raised to 4%. The way in which Na deoxycholate, glycocholate, and taurocholate affected the conjunctival penetration of atenolol, timolol, and insulin suggests that these three bile salts improved mainly the transcellular penetration of the compounds studied.

Administration, Topical↗

PubMed1992

H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis.

H36 is a 120,000-D membrane glycoprotein that is expressed during the differentiation of skeletal muscle. H36 cDNA clones were isolated from a lambda UniZapXR rat myotube cDNA library and sequenced. The deduced amino acid sequence demonstrates that H36 is a novel integrin alpha chain that shares extensive homology with other alpha integrins that includes: (a) the GFFKR sequence found in all alpha integrins; (b) a single membrane spanning region; (c) conservation of 18 of 22 cysteines; and (d) a protease cleavage site found in the non-I region integrin alpha chains. The cytoplasmic domain of H36 is unique and additional regions of nonhomology further indicate H36 is distinct from all other alpha chains. In keeping with current nomenclature we designate this alpha chain alpha 7. Northern blots demonstrate that expression of H36-alpha 7 mRNA is regulated both early in the development of the myogenic lineage and later, during terminal differentiation. Detection of H36-alpha 7 mRNA coincides with conversion of H36- myogenic precursor cells to H36+ cells. H36-alpha 7 mRNA is present in replicating myoblasts: expression increases upon terminal differentiation and is markedly reduced in developmentally defective myoblasts. In addition, H36-alpha 7 mRNA is not detected in C3H10T1/2 cells. It is in myotubes derived from myoblasts obtained by treatment of 10T1/2 cells with azacytidine or transfection with MRF4. Immunoblots and immunofluorescence demonstrate that the H36-alpha 7 chain is associated with integrin beta 1. Affinity chromatography demonstrates that H36-alpha 7 beta 1 selectively binds to laminin. The expression of H36-alpha 7 on secondary myoblasts during the development of the limb in vivo corresponds with the appearance of laminin in the limb, with the responsiveness of secondary myoblast proliferation to laminin, and with the onset of increased muscle mass, suggesting that H36-alpha 7 modulates this stage in limb development. We conclude that H36-alpha 7 is a novel alpha integrin laminin binding protein whose expression is developmentally regulated during skeletal myogenesis.

Amino Acid Sequence↗

PubMed1992

Reversal of the malignant phenotype by an anti-ras ribozyme.

In this study a ribozyme (catalytic RNA) was designed to site specifically cleave the mRNA of the activated H-ras gene expressed in human bladder carcinoma EJ cells. The optimal conditions for catalytic cleavage by the ribozyme were demonstrated in vitro. A synthetic DNA encoding the ribozyme was cloned into a mammalian expression vector (pH beta APr-1) and transfected into EJ cells. The expressed ribozyme significantly altered the morphology and suppressed the growth of EJ cells in vitro. These cell lines were examined for their malignant potential in athymic (nude) mice by an orthotopic (transurethral) implantation model, which recapitulates the invasive potential of various bladder carcinomas. EJ tumors expressing the H-ras ribozyme were characterized by a marked reduction in tumor take and invasion compared to those formed by control EJ cells. These differences resulted in almost a twofold increase in survival of mice implanted with ribozyme-containing EJ cells. These results further elucidate the role of ras genes in tumorigenicity and invasion, as well as introduce ribozymes as a new class of anticancer agents.

Animals↗

PubMed1992

The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA.

The assembly of activated RNA polymerase II (pol II) transcription complexes has been investigated by assaying whether pre-assembly of intermediate complexes reduces the extended time required for start-site melting. The results show that a closed complex requiring factors IIA, IID, and the acidic activator GAL4-AH forms in a rate-limiting step. This directs the templates into a productive assembly pathway. Factor TFIIB is then added rapidly, affording further protection against diversion into nonproductive pathways. These events are followed by a series of rapid steps in which the remaining general factors are assembled onto the template, which is then melted using the energy of ATP hydrolysis.

Base Sequence↗