Search PubMed⌕ Search

Biomedical subjects

R Zhang

Publications and source records attributed to R Zhang.

At least 469 records · Page 26Linked to original sources
PubMed1994

[Both 987P and F41 fimbriae produced by the same enterotoxigenic Escherichia coli strain isolated from a piglet with diarrhea].

An enterotoxigenic Escherichia coli strain isolated from a piglet with diarrhea was examined for the presence of fimbriea 987P and F41 by a direct agglutination (with MAbs), an indirect immunofluorescence technique (MAbs as first antibodies), SDS-PAGE and Western blots (antisera IgG as probes). Results of these techniques revealed that both 987P and F41 fimbrial adhesins were produced by the same strain, not by separate ones.

Adhesins, Escherichia coli↗
PubMed1993

Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver: population characteristics, newly identified deficient patients, and clinical implication in 5-fluorouracil chemotherapy.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (FUra), one of the most widely used anticancer drugs. Previous studies from our laboratory demonstrated the clinical importance of DPD in cancer patients (G. D. Heggie, J-P. Sommadossi, D. S. Cross, W. J. Huster, and R. B. Diasio. Cancer Res., 47: 2203-2206, 1987; B. E. Harris, R. Song, S-j. Soong, and R. B. Diasio. Cancer Res., 50: 197-201, 1990), particularly in those with DPD deficiency who experience severe FUra toxicity (including death) following FUra treatment [R. B. Diasio, T. L. Beavers, and J. T. Carpenter. J. Clin. Invest., 81: 47-51, 1988; B. E. Harris, J. T. Carpenter, and R. B. Diasio. Cancer (Phila.), 68: 499-501, 1991]. We now suggest that measurement of DPD activity may be useful in routine screening of cancer patients prior to FUra treatment. In this paper, we describe the following serial studies: (a) we developed a sensitive, accurate, and precise DPD assay and a storage method to stabilize DPD activity, permitting large scale DPD screening in cancer patients; (b) we demonstrated a normal distribution (Gaussian distribution) of human DPD activity from peripheral blood mononuclear cells (PBM-DPD) in a population study. Baselines for PBM-DPD with fresh and frozen samples were 0.425 +/- 0.124 (SD) and 0.189 +/- 0.064 nmol/min/mg protein, respectively. The 95% and 99% distribution ranges for both fresh and frozen samples were also determined, providing criteria for detection of DPD-deficient patients; (c) we identified nine new patients with profound or partial DPD deficiency; (d) we determined a baseline for human liver DPD activity, which was shown to be 0.360 +/- 0.182 nmol/min/mg protein (frozen samples); (e) we did a preliminary evaluation of liver DPD from deficient patients. Low liver DPD activity in two deficient patients correlated with low PBM-DPD activity. Using a polyclonal antibody raised against human liver DPD in our laboratory (Z. Lu, R. Zhang, and R. B. Diasio. J. Biol. Chem., 267: 17102-17109, 1992), Western blot analysis demonstrated decreased DPD protein in the liver cytosol from DPD-deficient patients compared to normal subjects. These results may be useful in improving the effectiveness and/or lessening the toxicity of FUra chemotherapy.

Adult↗
PubMed1993

Comparison of dihydropyrimidine dehydrogenase from human, rat, pig and cow liver. Biochemical and immunological properties.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines and has an important role in cancer chemotherapy with fluoropyrimidine drugs. Recently, we purified and characterized this enzyme from human liver and raised a rabbit polyclonal antibody against the purified human enzyme (Lu et al., J Biol Chem 267: 17102-17109, 1992). In the present study, using this purification procedure, DPD was purified to homogeneity from three other mammalian species, i.e. pig, rat, and cow. Comparison of the biochemical properties of these purified enzymes was conducted. Molecular masses of DPD from human, pig, rat, and cow liver were: 210, 204, 210, and 216 kDa, respectively. DPD from all four species appeared to be composed of two subunits. The apparent pI values were 4.6, 4.8, 4.85, and 5.25, respectively. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out with the purified DPD preparation, suggesting species differences in kinetic parameters. Amino acid composition of these purified enzymes also demonstrated slight species differences. In the present study, a rabbit polyclonal antibody against rat liver DPD was raised. Using polyclonal antibodies against human and rat liver DPD, immunoblotting demonstrated cross-reactivity among the four species. In summary, purification and comparison of DPD from different mammalian species will provide a basis for further biochemical and molecular studies of this enzyme.

Amino Acids↗
PubMed1993

Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10.

Interleukin 10 (IL-10), which was first discovered by its ability to inhibit the synthesis of various cytokines, most notably gamma interferon, from Th1 helper cells, displays pleiotropic immunoregulatory properties. Human and murine IL-10 have a high amino acid sequence identity (ca. 73%) which includes the conservation of all four cysteine residues in human IL-10 and the first four out of five cysteine residues for murine IL-10. Chemical analysis was used to determine that both recombinant human and recombinant murine IL-10 contain two disulfide bonds. The disulfide pairs for each were determined by mass spectrometric and reversed-phase HPLC analysis of trypsin-derived polypeptide fragments. The disulfide bond assignments for both species were similar in that the first cysteine residue in the sequence paired with the third and the second paired with the fourth. The fifth cysteine in murine IL-10 was determined by chemical modification to be unpaired. Far-UV circular dichroism analysis indicated that the secondary structure of recombinant human and murine IL-10 are composed of ca. 60% alpha-helix. Reduction of the disulfide bonds structurally destabilized the protein and led to a structure containing only 53% alpha-helix. The reduced protein displayed no in vitro biological activity in a mast cell proliferation assay. These studies indicate that IL-10 is a highly alpha-helical protein containing two disulfide bonds, either one or both of which are critical for its structure and function. In addition, these properties suggest that this interesting cytokine may belong to the alpha helical cytokine class of hematopoietic ligands.

Amino Acid Sequence↗
PubMed1993

Relationship between circadian-dependent toxicity of 5-fluorodeoxyuridine and circadian rhythms of pyrimidine enzymes: possible relevance to fluoropyrimidine chemotherapy.

Previous studies in experimental animals and patients have suggested a circadian variation in host toxicity following administration of 5-fluorodeoxyuridine (FdUrd) although the biochemical mechanisms are not fully understood. Thymidine kinase (TK; EC 2.7.1.21), the initial enzyme in the thymidine-phosphorylation pathway, is the first enzyme in the anabolism of FdUrd. Dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2), is the rate-limiting enzyme in the pyrimidine catabolic pathway and has been shown to be the key enzyme in FdUrd catabolism. The present study examined the relationship between the suggested circadian variation in FdUrd toxicity and potential circadian variations in the activity of these enzymes. Initial studies in Sprague-Dawley rats confirmed that the time of FdUrd administration affected death rate and other drug-related toxicities including loss of body weight, diarrhea, and bone marrow suppression, with the least toxicity and highest survival rate being observed in rats receiving FdUrd at 12:00 noon and 4:00 p.m. and the greatest toxicity and lowest survival rate at 12:00 midnight and 4:00 a.m. Statistical analysis revealed a circadian pattern in FdUrd toxicity (Cosinor analysis, P < 0.001). In subsequent studies with the same species, we simultaneously measured TK and DPD activities in several tissues at various times over 24 h. Under standardized light conditions (lights on, 6:00 a.m. to 6:00 p.m.; lights off, 6:00 p.m. to 6:00 a.m.), with sampling at 4-h intervals (4:00 and 8:00 a.m.; 12:00 noon; 4:00 and 8:00 p.m., and 12:00 midnight), a circadian variation in TK activity was observed (P < 0.0001, Cosinor analysis) in bone marrow, intestinal mucosa, liver, and spleen. In the same group of animals, a circadian pattern of DPD activity in liver and bone marrow was also observed (Cosinor analysis, P < 0.0001) that was inverse compared to the circadian variation in TK activity (Pearson correlation analysis, P < 0.05). Further statistical analysis indicated that the observed circadian variation in FdUrd toxicity was correlated with the circadian variation of TK activity and inversely correlated with DPD activity (Pearson correlation analysis, P < 0.05). Based on the above data, we conclude that the circadian pattern of TK and DPD activity may explain the observed circadian variation in toxicity as the time of FdUrd administration is varied. These results may be useful in the design of improved chemotherapeutic regimens using time-modified administration of FdUrd.

Animals↗
PubMed1993

A mathematical model of the kinetics and tissue distribution of 2-fluoro-beta-alanine, the major catabolite of 5-fluorouracil.

2-Fluoro-beta-alanine (FBAL) is the major metabolite of 5-fluorouracil (FUra), one of the most widely used anticancer drugs. It has been suggested previously that FBAL and/or its metabolites may have a role in the hepatotoxicity, neurotoxicity and cardiotoxicity resulting from FUra chemotherapy. Studies in patients and experimental animals have demonstrated that FBAL has a prolonged elimination compared with the parent drug, FUra. In the present manuscript, a mathematical model is developed for the kinetics and tissue distribution of FBAL. This model is based on recently published data from a study of the pharmacokinetics and disposition of FBAL in rats (Zhang et al., Drug Metab Dispos 20: 113-119, 1992). Satisfactory agreement was achieved between predicted and measured values, permitting an accurate evaluation of the kinetic and distribution parameters for FBAL. This model indicates that: (1) FBAL accumulates in several tissues including brain, heart, spleen, and enterohepatic system; and (2) enterohepatic circulation of FBAL and its bile acid conjugates has an important role in FBAL kinetics and distribution as demonstrated by a model in which enterohepatic circulation parameters were deleted.

Animals↗
PubMed1993

Functional properties of an H,K-ATPase/Na,K-ATPase chimera.

Cultured pig kidney epithelial cells were transfected with a chimeric P-type ATPase catalytic subunit composed of the NH2-terminal half of the rat gastric H,K-ATPase and the COOH-terminal half of the rat Na,K-ATPase (alpha 1 isoform). Low concentrations of ouabain (< or = 0.2 mM) were used to inhibit completely the endogenous pig Na,K-ATPase and high concentrations (5 nM) to test the sensitivity of the chimeric rodent pump. In the presence of a low concentration of ouabain, a small but significant inhibition of residual Rb+(K+) influx by 5 mM ouabain was observed in only the transfected cells. Conditions were found in which a similar component of Rb+ influx was inhibited by the gastric H,K-ATPase inhibitor SCH28080, consistent with SCH28080 binding to the extracellular H1-H2 loop of this enzyme. These experiments demonstrate that this chimera behaves as a functional ion pump and indicate that the protein domains involved in cardiac glycoside binding are not confined to the amino-terminal half of the Na,K-ATPase.

Animals↗
PubMed1993

A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k.

CHIP28k is an important water-transporting protein in the kidney proximal tubule and the thin descending limb of Henle [Zhang, Skach, Hasegawa, Van Hoek, & Verkman (1993) J. Cell Biol. 120, 359-369] that is homologous to human erythrocyte CHIP28 [Preston & Agre (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11110-11114]. Oligonucleotide-directed mutagenesis was used to identify the cysteine(s) involved in inhibition of the water-transporting function of CHIP28k by the mercurial HgCl2. Each of the four cysteines (at positions 87, 102, 152, and 189) were mutated to serine individually, or in combinations. In vitro transcribed cRNA was expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf) in the absence or presence of 0.3 mM HgCl2. Pf (in cm/s x 10(-4) measured at 10 degrees C) was 7 +/- 1 in water-injected oocytes. In wild-type CHIP28k, Pf was 58 +/- 7 (-HgCl2) and 12 +/- 1 (+HgCl2). Mutation of cysteine 87, 102, or 152, individually or in combinations, had little effect on oocyte Pf or on the inhibition by HgCl2. Mutation of cysteine 189 to serine or glycine gave similar Pf values of 49-56 (-HgCl2); however, Pf was not inhibited up to 1 mM HgCl2. Mutation of cysteine 189 to the larger amino acid tryptophan gave a low Pf of 9 +/- 1; coexpression with wild-type CHIP28k indicated that the tryptophan mutation was not dominant negative. Mutation of the asparagine 42 and 205 glycosylation sites to threonine had little effect on Pf.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Determination of total polyamines in tumor cells by high-performance capillary zone electrophoresis with indirect photometric detection.

Tumor cells contain much higher levels of common polyamines such as putrescine, spermidine, and spermine than normal cells. Our previous work demonstrated that high-performance capillary zone electrophoresis with indirect photometric detection was suitable for the determination of underivatized free polyamines in tumor cells. We now report improved sensitivity (7-fold), greater quantitative reproducibility, and quantitation of both bound and free, underivatized polyamines in PC 12 tumor cells. 1,7-Diaminoheptane was used as an internal standard for quantification. Femtomoles of three polyamines and simple cations such as K+ and Na+ were easily separated in less than 10 min. The quantitations for total spermidine and spermine (bound and free) were found to be 2-fold higher than the quantitation for free, unbound polyamines using our previous method. The signal response was linear over 2 orders of magnitude. The method described here provides a fast and simple technique for quantitation of bound, unbound, and total polyamines for use in the study of the biological role of polyamines.

Adrenal Gland Neoplasms↗
PubMed1993

Circadian rhythm of rat spleen cytoplasmic thymidine kinase.

The activity of thymidine kinase (TK, EC 2.7.1.21), the first enzyme of the thymidine phosphorylation pathway, was measured at various times over a 24-hr period in the spleens of Sprague-Dawley rats that had been housed under standardized conditions of light and dark for at least 4 weeks before the study. Spleen cytoplasmic TK activity was assayed with [2-14C]thymidine as substrate. Under "normal" light conditions (lights on 6:00 a.m.-6:00 p.m. and lights off 6:00 p.m.-6:00 a.m.), a circadian variation of TK activity was observed (P < 0.0001), Cosinor analysis) with peak activity (1.98 nmol product/hr/mg protein) at 1:00 a.m. (19 hr after light onset, HALO) and trough activity (0.40 nmol product/hr/mg protein) at 1:00 p.m. (7 HALO). Maximum enzyme activity exceeded minimum activity by approximately 5-fold. Reversing the light-dark cycle resulted in a corresponding shift in TK activity. Under these "reverse" conditions (lights on 6:00 p.m.-6:00 a.m. and lights off 6:00 a.m.-6:00 p.m.), a circadian variation in TK activity was also observed (P < 0.0001, Cosinor analysis) with peak activity (1.14 nmol product/hr/mg protein) at 12:00 noon (18 HALO) and trough activity (0.32 nmol/hr/mg protein) at 12:00 a.m. (6 HALO). Maximum enzyme activity exceeded minimum activity by approximately 4-fold. In summary, this study demonstrated for the first time that TK activity varies over a 24-hr period in association with the light-dark cycle.

Animals↗
PubMed1993

Structure and expression of the S locus-related genes of maize.

The extracellular of the putative receptor-like protein kinase, ZmPK1, is related to the self-incompatibility locus (S-locus) genes of Brassica. We have isolated and characterized a genomic DNA clone of ZmPK1 and three additional genes from maize that are highly related to ZmPK1. These three S-locus related genes do not appear to have the protein kinase catalytic domain that is found in ZmPK1. One or more of these genes are expressed specifically in the silks. This initial description of S-locus related genes in monocotyledonous plants suggests that the S-locus domain may be involved in several different cellular functions in a wide variety of plants.

Base Sequence↗
PubMed1993

Inducible removal of UV-induced pyrimidine dimers from transcriptionally active and inactive genes of Saccharomyces cerevisiae.

The prior UV irradiation of alpha haploid Saccharomyces cerevisiae with a UV dose of 25 J/m2 substantially increases the repairability of damage subsequently induced by a UV dose of 70 J/m2 given 1 h after the first irradiation. This enhancement of repair is seen at both the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. The presence in the medium of the protein synthesis inhibitor, cycloheximide in the period between the two irradiations eliminated this effect. Enhanced repair still occurred if cycloheximide was present only after the final UV irradiation. This indicated that the first result is not due to cycloheximide merely blocking the synthesis of repair enzymes associated with a hypothetical rapid turnover of such molecules. The enhanced repairability is not the result of changes in chromatin accessibility without protein synthesis, merely caused by the repair of the damage induced by the prior irradiation. The data clearly show that a UV-inducible removal of pyrimidine dimers has occurred which involves the synthesis of new proteins. The genes known to possess inducible promoters, and which are involved in excision are RAD2, RAD7, RAD16 and RAD23. Studies with the rad7 and rad16 mutants which are defective in the ability to repair HML alpha and proficient in the repair of MAT alpha showed that in rad7, preirradiation enhanced the repair at MAT alpha, whereas in rad16 this increased repair of MAT alpha was absent. The preirradiation did not modify the inability to repair HML alpha in either strain. Thus RAD16 has a role in this inducible repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1993

Nucleotide sequence relationships of double-stranded RNAs in flax rust, Melampsora lini.

Flax rust, Melampsora lini strain SP6, contains 11 double-stranded (ds) RNA molecules with a total length of about 25 kbp. The dsRNAs are inherited in three genetic units: the L unit comprising a single 5.2 kbp dsRNA and contained within a 40-nm virus-like particle, and the A and B units each consisting of five dsRNAs (A1-A5, and B1-B5) ranging in size from 1.2 to 2.7 kbp. This paper reports the isolation of a cDNA library representing 10 of the 11 dsRNAs. By nucleic-acid hybridization techniques it has been shown that all ten sequences are unique showing no detectable cross-hybridization with any other dsRNA present in the rust. A near full-length sequence of 1932 bp of the B3 dsRNA is reported and contains several open reading frames, the largest of which comprises most of the molecule.

Amino Acid Sequence↗
PubMed1993

Acquired amegakaryocytic thrombocytopenic purpura with a Philadelphia chromosome.

A 30-year-old Chinese man with acquired amegakaryocytic thrombocytopenic purpura (AATP) and a Ph chromosome is reported. At presentation, he had severe thrombocytopenia resulting in epistaxis, gingival bleeding, and ecchymoses, while other hematologic values were within the normal range. Bone marrow aspiration showed no megakaryocytes, with a normal appearance of erythroblastic and granulopoietic series. He failed to respond to prednisone treatment, and underwent a progress from isolated thrombocytopenia to full pancytopenia. At last he died of spontaneous intracranial hemorrhage. An in vitro culture for granulocyte-macrophage precursors showed very few colonies. Karyotypic analysis revealed a standard Ph chromosome translocation, t(9;22)(q34;q11), in the majority of bone marrow cells. Southern blot analysis using a 3' bcr-HE probe didn't detect a rearrangement within the bcr DNA sequence. This patient, in fact, was a myelodysplastic disorder, initially presenting as AATP. The diagnosis of chronic myelogenous leukemia was excluded on the basis of clinical and hematologic findings. The heterogeneity of Ph chromosome in myelodysplastic syndrome is discussed.

Adult↗
PubMed1993

Measurement of chain tilt angle in fully hydrated bilayers of gel phase lecithins.

The tilt angle theta tilt of the hydrocarbon chains has been determined for fully hydrated gel phase of a series of saturated lecithins. Oriented samples were prepared on glass substrates and hydrated with supersaturated water vapor. Evidence for full hydration was the same intensity pattern of the low angle lamellar peaks and the same lamellar repeat D as unoriented multilamellar vesicles. Tilting the sample permitted observation of all the wide angle arcs necessary to verify the theoretical diffraction pattern corresponding to tilting of the chains towards nearest neighbors. The length of the scattering unit corresponds to two hydrocarbon chains, requiring each bilayer to scatter coherently rather than each monolayer. For DPPC, theta tilt was determined to be 32.0 +/- 0.5 degrees at 19 degrees C, slightly larger than previous direct determinations and considerably smaller than the value required by recent gravimetric measurements. This new value allows more accurate determinations of a variety of structural parameters, such as area per lipid molecule, A = 47.2 +/- 0.5 A2, and number of water molecules of hydration, nw = 11.8 +/- 0.7. As the chain length n of the lipids was increased from 16 to 20 carbons, the parameters A and nw remained constant, suggesting that the headgroup packing is at its excluded volume limit for this range. However, theta tilt increased by 3 degrees and the chain area Ac decreased by 0.5 A2. This behavior is explained in terms of a competition between a bulk free energy term and a finite or end effect term.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Analysis of lectin receptors in normal nasal mucosa, nasal polyp, inverted papilloma and papillary adenocarcinoma.

In order to investigate the changes in glycoprotein structure in the process of cellular differentiation of the nasal mucosa, formalin-fixed, paraffin-embedded biopsy specimens of normal nasal mucosae, nasal polyps, inverted papillomas and papillary adenocarcinomas were analysed by the Avidin Biotin-Peroxidase Complex technique for the demonstration of peanut agglutinin (PNA) receptors, concanavalin ensifomis agglutinin (ConA) receptors, ulex europeaus agglutinin (UEA-I) receptors, wheat germ agglutinin (WGA) receptors, carcino-embryonic antigen (CEA) and keratin. The quantity and distribution of PNA receptors, ConA receptors, UEA-I receptors and CEA were different, in relation to the varying pathological changes. The results suggest that the glycoprotein structure in the cells of the nasal mucosa will change following their differentiation and malignant transformation, which may be helpful in establishing the diagnosis.

Adenocarcinoma, Papillary↗
PubMed1993

Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28.

The localization and transporting properties of a kidney protein homologous to human erythrocyte protein CHIP28 was evaluated. The cDNA encoding rat kidney protein CHIP28k was isolated from a rat renal cortex cDNA library. A 2.8-kb cDNA was identified which contained an 807 bp open reading frame encoding a 28.8 kD protein with 94% amino acid identity to CHIP28. in vitro translation of CHIP28k cDNA in rabbit reticulocyte lysate generated a 28-kD protein; addition of ER-derived microsomes gave a 32-kD transmembrane glycoprotein. Translation of truncated RNA demonstrated glycosylation of residue Asn42 which is predicted to lie between the first and second transmembrane domains. Expression of in vitro transcribed mRNA encoding CHIP28k in Xenopus oocytes increased oocyte osmotic water permeability (Pf) from (4 +/- 1) x 10(-4) to (33 +/- 4) x 10(-4) cm/s at 10 degrees C; the increase in oocyte Pf was weakly temperature dependent and inhibited by HgCl2. Two-electrode voltage clamp measurements indicated that CHIP28k was not permeable to ions. Oocyte Pf also increased with expression of total mRNA from kidney cortex and papilla; the increase in Pf with mRNA from cortex, but not kidney papilla, was blocked by coinjection with excess antisense CHIP28k cRNA. In situ hybridization of a 150 base cRNA antisense probe to tissue sections from rat kidney showed selective CHIP28k localization to epithelial cells in proximal tubule and thin descending limb of Henle. Pf in purified apical membrane vesicles from rat and human proximal tubule, and in proteoliposomes reconstituted with purified protein, was very high and inhibited by HgCl2; stripping of apical vesicles with N-lauroylsarcosine enriched a 28-kD protein by 25-fold and yielded a vesicle population with high water, but low urea and proton permeabilities. CHIP28k identity was confirmed by NH2-terminus sequence analysis. These results indicate that CHIP28k is a major and highly selective water transporting protein in the kidney proximal tubule and thin descending limb of Henle, but not collecting duct.

Amino Acid Sequence↗