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Biomedical subjects

R Zhang

Publications and source records attributed to R Zhang.

At least 397 records · Page 22Linked to original sources
PubMed1996

[Endothelin-1 receptors of the normal adrenal gland and adrenal tumors in human].

Endothelin-1 (ET-1) receptors of normal adrenal gland (ADR, 6 cases), aldosterone-producing adenoma (APA, 5 cases), idiopathic hyperaldosteronism (IHA, 4 cases) and pheochromocytoma (PHE, 6 cases) in human were measured by radioligand binding assay (RBA) of receptors. Binding studies using 125I-ET-1 as a radio ligand showed the presence of a single class of high-affinity binding sites for ET-1 in all of the above tissues. The values of dissociation constant (Kd) of ET-1 for its receptor were similar in ADR, APA and IHA (28.3 +/- 2.5, 27.9 +/- 6.1, 27.7 +/- 1.9 pmol/L, respectively), but the maximal binding capacity (Bmax) of ET receptor tended to be lower in APA tissue (107.2 +/- 13.2 fmol/mg protein) in comparison with ADR (P < 0.01) and IHA (P < 0.05, 274.9 +/- 40.8, 247.0 +/- 19.8 fmol/mg protein, respectively). Both the Kd (50.8 +/- 5.1 pmol/L) and Bmax (675.3 +/- 93.7 fmol/mg protein) in PHE were higher than those in ADR (P < 0.01), APA (P < 0.01) or IHA (P < 0.05 for Kd, P < 0.01 for Bmax). Our data may suggest that there is the down-regulation for ET-1 receptor in APA and support the concept of an important role of ET-1 in the paracrine-autocrine regulation of aldosterone and catecholamine secretion in the adrenal and adrenal tumors.

Adenoma↗
PubMed1996

[Cloning and identification of fliCi gene of Salmonella typhimurium].

Purified Salmonella typhimurium 8705 chromosomal DNA was digested with EcoRI. DNA fragments above 400 bp were obtained by Sepharcyl S-400 chromatography and cloned into vector pGEM-3Zf(-), then transformed into host cell LC2a (hag- is recA-). One out of 6013 transformants was found to be motile and this clone was named pGI4015. Miniprep proved that pGI4015 contained an inserted fragment about 15.3 kb. Motility/inhibition tests as well as Southern blot hybridization showed that pGI4015 bear the H-1i gene of S. typhimurium. With the aid of BamHI and SalI site, sequences unrelated to flagellin in the cloned DNA fragment was removed, and a 3.8kb fragment containing fliC gene was subcloned.

Cloning, Molecular↗
PubMed1996

[Chemical constituents of Epimedium wanshanense S. Z. He et Guo].

Five flavonoids were isolated from Epimedium wanshanense and identified as sagittatoside B, anhydroicaritin-3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-rhamnopyranoside, sagittatoside A, ikarisoside B and desmethylanhydroicaritin-3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L- rhamnopyranoside by means of IR, UV, 1HNMR, 13CNMR, MS and chemical evidence. They were obtained from this plant for the first time.

Drugs, Chinese Herbal↗
PubMed1996

Severe neurotoxicity following 5-fluorouracil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency.

Patients with decreased dihydropyrimidine dehydrogenase (DPD) activity are at increased risk for experiencing serious adverse reactions following 5-fluorouracil (5-FU)-based chemotherapy. Symptoms include severe and potentially life-threatening gastrointestinal toxicity, myelosuppression, and neurological toxicity. In the present study, we describe a 50-year-old Caucasian man who developed severe encephalopathy during his second cycle of 5-FU chemotherapy. The patient remained in a comatose state for 4 days but then showed dramatic improvement in his neurological status following continuous i.v. infusion of thymidine at 8 g/m2/day. Laboratory studies revealed the patient to be severely DPD deficient, as demonstrated by DPD enzyme activity from peripheral blood mononuclear cells being below the lower limit of the 95th percentile of a control population and by Western immunoblot analysis showing undetectable levels of DPD protein. Additional studies revealed a significant defect in pyrimidine catabolism with a 3.3- and 365-fold increase in the levels of uracil in plasma and urine, respectively, compared to normal subjects. Family studies suggest that the inheritance pattern of this syndrome is complex and most consistent with an autosomal recessive trait. This study demonstrates that cancer patients with DPD deficiency are at increased risk for developing severe neurological toxicity secondary to 5-FU chemotherapy, and that infusional thymidine should be considered as a potential rescue agent against this particular host toxicity.

Brain Diseases↗
PubMed1995

Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C.

The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D. (1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.

Amino Acid Sequence↗
PubMed1995

Functional role of endothelin ETA and ETB receptors in venous and arterial smooth muscle.

The functional importance of endothelin ETA and ETB receptors in selected arterial and venous smooth muscle preparations was characterized. Endothelin-1 induced force in the saphenous and jugular veins is normally mediated by endothelin ETB-like receptors. However, desensitization or pharmacological block of these receptors reveals an endothelin ETA receptor population that is of sufficient size to mediate full endothelin-1-evoked force. Block of either endothelin ETA or endothelin ETB receptors alone is insufficient to antagonize endothelin-1-evoked force in saphenous vein. Endothelin-1-induced force in hamster aorta may also be mediated by activation of both endothelin ETA and ETB receptors. However, activation of endothelin ETB-like receptors alone is insufficient to generate a full endothelin-1 response. Sarafotoxin S6c treatment, to desensitize endothelin ETB receptors, failed to affect the responses of rat aorta and rabbit carotid artery to endothelin-1 or endothelin ETA receptor antagonists. These findings indicate that selective endothelin receptor antagonists will vary enormously in their efficacy against endothelin-induced force in different vascular beds.

Amino Acid Sequence↗
PubMed1995

Functional glycosylation sites of the rat luteinizing hormone receptor required for ligand binding.

The contribution of N-linked glycosylation to the ligand binding activity of the rat luteinizing hormone receptor (LHR) was studied in wild-type and mutant LHR expressed in mammalian (COS1) cells and overexpressed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane domain were equivalent in both cell lines. Tunicamycin-treated transfected Sf9 cells expressed a carbohydrate-free LH receptor that lacked hormone binding activity. Functional carbohydrate chains essential for binding activity were localized to glycosylation sites at Asn-173 and Asn-152. Glycosidase treatment of the double mutant N173Q/N152Q revealed the presence of at least one additional carbohydrate chain at Asn-269, Asn-277, or Asn-291 that does not contribute to hormone binding. Asn-77 was not glycosylated, but its mutation to Gln reduced hormone binding. LHR expressed in insect cells contained only high mannose carbohydrate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl chains indicated that only the proximal N-acetylglucosamine residue, which is common to high mannose and complex carbohydrate forms, is necessary for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolecular folding of the nascent receptor rather than in its interaction with the hormone.

Alternative Splicing↗
PubMed1995

Scintillation proximity assay to measure binding of soluble fibronectin to antibody-captured alpha 5 beta 1 integrin.

A scintillation proximity assay (SPA) has been developed to measure binding of alpha 5 beta 1 integrin, a heterodimeric cell-surface adhesion receptor, to fibronectin. This assay utilizes an anti-beta 1 integrin monoclonal antibody to simultaneously capture alpha 5 beta 1 from a cellular lysate and couple the integrin to anti-mouse IgG antibody-coated SPA beads for detection of 125I-fibronectin binding. The assay does not require prior purification of alpha 5 beta 1 nor physical separation of bound and free 125I-fibronectin. Chinese hamster ovary cells that stably overexpress human alpha 5 integrin (CHO#7 cells) were used as a source of alpha 5 beta 1 fibronectin receptor. Using the anti-hamster beta 1 monoclonal antibody 7E2 to capture alpha 5 beta 1 from a CHO#7 cell lysate, this SPA assay allowed measurement of specific 125I-fibronectin binding as defined by displacement by the Arg-Gly-Asp containing peptide GRGDSP or the anti-human alpha 5 antibody P1D6. IC50 values for displacement of 125I-fibronectin binding by GRGDSP and the novel cyclic peptides cRGDGF, cRGEGF, and cRRETAWA were 2.6, 0.045, 3.2, and 37 microM, respectively. Specific 125I-fibronectin binding to alpha 5 beta 1 from C8161 human melanoma cells was also measured using anti-human beta 1 antibodies. This method should be generally useful to measure cell-free ligand binding to receptors that are difficult to purify.

Amino Acid Sequence↗
PubMed1995

In vivo stability, disposition and metabolism of a "hybrid" oligonucleotide phosphorothioate in rats.

Oligodeoxynucleotide phosphorothioates containing segments of 2'-O-methyloligoribonucleotide phosphorothioates at both 3'- and 5'-ends (hybrid oligonucleotide) have been shown to be potent antisense agents. In the present study, in vivo biostability, disposition, and excretion of a 25-mer hybrid oligonucleotide were determined in rats after i.v. bolus administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. The plasma disappearance curve for the hybrid oligonucleotide could be described by a two-compartmental model, with half-lives of 0.34 and 52.02 hr, respectively. The majority of the radioactivity in plasma was associated with the intact hybrid oligonucleotide. Urinary excretion represented the major pathway of elimination, with 21.98 +/- 3.21% (mean +/- SD) of the administered dose excreted within 24 hr and 38.13 +/- 2.99% over 240 hr post-dosing. The majority of the radioactivity in urine was associated with the degradative products with lower molecular weights, but the intact form was also detected by HPLC analysis. Fecal excretion was a minor pathway of elimination with 2.34 +/- 0.13% of the administered dose excreted over 24 hr and 6.74 +/- 0.40% over 240 hr post-dosing. A wide tissue distribution of hybrid oligonucleotide was observed based on radioactivity levels, and analysis by HPLC showed that the majority of the radioactivity in tissues was associated with the intact hybrid oligonucleotide. Further analyses of the experimental data provided a comprehensive pharmacokinetic analysis of hybrid oligonucleotide in each tissue. Compared with a previously examined oligodeoxynucleotide phosphorothioate (GEM 91) that has a similar nucleotide sequence, the hybrid oligonucleotide had a shorter distribution half-life and a longer elimination half-life, based on the quantitation of radioactivity in plasma. Although it had a similar tissue distribution pattern compared with other oligonucleotide phosphorothioates such as GEM 91, the hybrid oligonucleotide was more stable in vivo, which may be important in the development of antisense oligonucleotides as therapeutic agents.

Animals↗
PubMed1995

Mutation of arginine 276 to methionine changes Mg2+ cooperativity and the kinetic mechanism of fructose-1,6-bisphosphatase.

Arginine 276 of porcine liver fructose-1,6-bisphosphatase (FBPase) was mutated to methionine by site-directed mutagenesis on the basis of the crystal structure of the enzyme [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., & Lipscomb, W.N. (1993) Biochemistry 32, 1844-1857]. The mutant and wild-type forms of the enzyme were purified to homogeneity and characterized by circular dichroism spectrometry (CD) and initial-rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzymes on the basis of the CD data. Replacement of Arg 276 with methionine caused a significant decrease in the enzyme's activity. The kcat for the mutant enzyme was only about 0.67% of that of the wild-type enzyme. Most importantly, the mutation caused the total loss of cooperativity for Mg2+ and changed the kinetic mechanism to one in which the substrate adds to FBPase before Mg2+ and in which all steps equilibrate rapidly relative to the conversion of the ternary complex of enzyme, substrate, and Mg2+ to products. The Ka for Mg2+ increased by only about 5-fold relative to that of the wild-type enzyme. The mutation did not change the Ki for AMP or the Hill coefficient of this allosteric inhibitor. The Ki for fructose 2,6-bisphosphate was increased by 16-fold compared with that of the wild-type enzyme. The Km for fructose 1,6-bisphosphate was similar to that of the wild-type enzyme. It is concluded that Arg 276 is critical for activity and Mg2+ cooperativity with FBPase and it determines the enzyme's kinetic mechanism.

Adenosine Monophosphate↗
PubMed1995

Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription in endothelial cells.

To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS) gene expression, we have cloned the eNOS promoter and fused it to a luciferase reporter gene to map regions of the promoter important for basal transcription in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1 luciferase (LUC) (-1600 to +22 nucleotides) yielded a 35-fold increase in promoter. Progressive deletion from -1600 to -1033 (F2 and F3 LUC) did not significantly influence eNOS promoter activity. Further deletion from -1033 to -779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter activity, and still further deletion from -779 to -494 (F5 LUC) did not markedly influence activity. Deletion from -494 to -166 (F6 LUC) reduced eNOS promoter activity by 40-50%. Specific mutation of the consensus GATA site (-230) in the F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of GATA-2 to the oligonucleotide sequence containing the -230 GATA site. Next, we mutated the Sp1 site (-103) in the F3 and F6 LUC constructs and in the F3 GATA mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90% reduction in normalized luciferase activity. Gel shift and antibody supershift analysis using a BAEC nuclear extracts demonstrated four specific, DNA-protein complexes binding to the eNOS Sp-1 site, with the slowest migrating form composed of Sp1 and another nuclear protein. These data demonstrate that the Sp1 site is an important cis-element in the core eNOS promoter.

Amino Acid Oxidoreductases↗
PubMed1995

Site-directed mutagenesis of the substrate binding site of porcine fructose-1,6-bisphosphatase.

Asn 212, Arg 243, Tyr 244, Tyr 264, and Lys 274, which are conserved in all known primary sequences of fructose-1, 6-bisphosphatase, are located in the substrate binding domain on the basis of the crystal structure of the enzyme. Mutations of the five residues of porcine liver fructose-1,6-bisphosphatase (Asn212Ala, Arg243-Met, Tyr244Phe, Tyr264Phe, and Lys274Leu) were carried out by site-directed mutagenesis. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism spectrometry. The mutants exhibited kcat values that are similar to those of the wild-type enzyme. The Km values for fructose 1,6-bisphosphate of the mutants are 6- to 44-fold higher than that of the wild-type enzyme. The Ki values for fructose 2,6-bisphosphate and AMP of the mutants increased from 56- to 1950-fold and 12- to 27-fold, respectively, relative to the wild-type enzyme. The alteration of inhibition constants for both inhibitors suggest that these five active site residues are involved in the inhibition by fructose 2,6-bisphosphate and AMP. No apparent differences in secondary structure of the wild-type and mutant forms of fructose-1,6-bisphosphatase were observed as measured by circular dichroism spectrometry. This report demonstrates that Asn 212, Arg 243, Tyr 244, Tyr 264, and Lys 274 not only are the sites for substrate binding, but also play an important role in the binding affinity of inhibitors fructose 2,6-bisphosphate and AMP.

Adenosine Monophosphate↗
PubMed1995

Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?

The aim of the research was to study the role played by extracellular O2-radicals, which are implicated in cardiac cell damage and the protective effect by cell-permeable, nitroxide, superoxide dismutase-mimics. Cardiomyocytes cultures from 1-day-old rats served as the test-system. Experiments were performed since 5th day in culture when > 80% of the cells were beating myocardial cells. Oxidative damage was induced by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM glucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate damages were spontaneous beating, lactate dehydrogenase release and ATP level. The rhythmic pulsation was followed microscopically. To determine the kinetics of cytosolic enzyme release from the cells, media samples were collected at various points of time and assayed for enzyme activity. To determine the cellular ATP, cells were washed with sodium phosphate buffer, scraped off and boiled for 3 min with sodium phosphate buffer. Following centrifugation the supernatant was collected and ATP was determined by the chemiluminogenic assay using firefly tails. The present results indicate that nitroxide stable free radicals in the millimolar concentration range, provide full protection without toxic side-effect. Unlike exogenously added SOD that failed to protect, exogenous catalase provided almost full protection. In addition, the metal-chelating agent dipyridyl, but not diethylene-triamine-pentaacetate or desferrioxamine, protected the cultured cells. The present results suggest that H2O2 is the predominant toxic species mediating the oxidative damage whereas extracellular superoxide radical does not contribute to cultured cardiomyocyte damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Region-related Fos expression in hypothalamic dopaminergic neurons of rats by intraperitoneal administration of an endogenous satiety substance, 2-buten-4-olide.

An effect of 2-buten-4-olide (2-B4O), an endogenous satiety substance, on hypothalamic dopamine neurons of rats was studied by double-label immunohistochemistry for c-Fos and tyrosine hydroxylase (TH). Intraperitoneal administration of 2-B4O induced Fos-like immunoreactivity in TH-immunoreactive neurons in the periventricular area and paraventricular nucleus but not in the arcuate nucleus. Thus, the present results suggest that tuberohypophysial dopamine neurons are regulated under the influence of endogenous 2-B4O.

4-Butyrolactone↗
PubMed1995

Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1.

An antisense oligodeoxynucleotide phosphorothioate, namely gene expression modulator 91 (GEM 91), has been demonstrated to have significant anti-human immunodeficiency virus activity in various tissue culture models. The present study was undertaken to determine the pharmacokinetics and tissue distribution of GEM 91 in rats following i.v. bolus administration of 35S-radiolabeled GEM 91. Plasma disappearance curves for GEM 91-derived radioactivity could be described by the sum of two exponentials, with half-lives (mean +/- SEM) of 0.95 (+/- 0.07) and 47.57 (+/- 14.48) hr. Urinary excretion represented the major pathway of elimination of GEM 91, with 26.67 +/- 6.46% (mean +/- SD) of the administered dose excreted within 24 hr and 58.12 +/- 4.36% over 240 hr after GEM 91 administration. Fecal excretion was a minor pathway of elimination of GEM 91 with 1.4 +/- 0.62% (mean +/- SD) of the administered dose excreted over 24 hr and 8.54 +/- 0.64% over 240 hr. A wide tissue distribution of GEM 91 was observed. During the initial 30 min, the highest levels of tissue radioactivity were found in the kidney, liver, spleen, lungs, and heart. Radioactivity was retained over longer time periods in the kidneys, liver, heart, and intestine. Analyses of the extracted radioactivities from plasma, kidney, and liver by gel electrophoresis showed the presence of both intact GEM 91 and degradative products with smaller molecular weights. Radioactivity in urine was found to be degradative metabolites of GEM 91. Based on the experimental data, pharmacokinetic parameters for GEM 91 in each tissue and biological fluids were calculated using computer-based two-compartmental i.v. bolus or absorption models. This study is important not only in providing the basis for future studies of GEM 91 in humans, but also in understanding the pharmacology and toxicology of antisense oligodeoxynucleotide phosphorothioates, in general.

Animals↗
PubMed1995

Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene.

The product of the vav proto-oncogene, p95vav or Vav, is tyrosine phosphorylated upon stimulation of T and B cells by antigen and other receptors, and contains motifs associated with signal transduction. To determine its role in vivo, we used vav-gene-targeted embryonic stem cells and RAG-2-/- blastocyst complementation. The vav(-/-)-RAG-2-/- chimaeras displayed thymic atrophy with reduced numbers of peripheral T cells. Whereas the total number of B cells was normal, the subset of peritoneal B-1 (CD5+) cells was missing. The vav-/- T and B cells were hyporeactive when stimulated through antigen receptors, but vav-/- T cells proliferated on exposure to phorbol ester and calcium ionophore, whereas B cells responded normally to bacterial mitogen, lipopolysaccharide or the CD40 ligand. Thus, we have established here a functional role for vav in the control of T- and B-cell development and activation.

Animals↗