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Biomedical subjects

P Zhang

Publications and source records attributed to P Zhang.

At least 163 records · Page 9Linked to original sources
PubMed2000

Cloning and characterization of human VPS35 and mouse Vps35 and mapping of VPS35 to human chromosome 16q13-q21.

Maintenance of different organelles in eukaryotic cells depends on sorting proteins, which ensure the proper delivery of organelle-specific proteins. The studies on yeast (Saccharomyces cerevisiae) VPS35, a hydrophilic membrane protein having a direct role in the retrieval of cargo proteins, suggest a mechanism underlying a possible lysosomal protein-sorting pathway in mammalian cells. Here, we report the isolation of human and mouse VPS35 cDNAs, which are 3208 and 3186 bp in length, respectively. The deduced proteins of the two cDNAs, which are both composed of 796 amino acids and share 99% identity, show homology to yeast VPS35 and other VPS35 homologues of various sources ranging from Schizosaccharomyces pombe to Drosophila melanogaster (31-56% identity and 49-71% similarity), especially in their amino- and carboxyl-termini. The conservation of VPS35 suggests that the function of this class of protein is important. The results of Northern hybridization of human VPS35 in 16 tissues showed that one transcript of 3.6 kb was highly expressed in brain, heart, testis, ovary, small intestine, spleen, skeletal muscle, and placenta and expressed at moderate or low levels in other tissues. Another transcript of 3.0 kb was also expressed with proportionally lower levels than the 3.6-kb transcript in all the tissues except that the 3.0-kb transcript was not detected in brain. Mouse Vps35 was widely expressed as a 3.4-kb transcript. In addition, human VPS35 was assigned to human chromosome 16q13-q21 by radiation hybrid mapping.

Amino Acid Sequence↗
PubMed2000

NMR structure of a DNA duplex containing nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and the structure of the unmodified control.

The three-dimensional structures of two DNA duplexes d(CATGAGTAC). d(GTACXCATG) (1) and d(CATGAGTAC).d(GTACTCATG) (2), where X represents 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, were solved using high resolution nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Good convergence was observed between final structures derived from A- and B-form starting geometries for both 1 and 2. Structures of 1 and 2 are right-handed duplexes within the B-form conformational regime. Furthermore, the structures of 1 and 2 are highly similar, with differences in the structures localized to the vicinity of residue 14 (X versus T). The pyrrole group of 1 is in the syn conformation and it is displaced towards the major groove. Furthermore, unlike T14 in 2, the base of X14 has reduced pi-pi stacking interactions with C13 and C15 and the nitro group of X14 is pointing out of the major groove. The structures presented here establish the basis of the thermal data of DNA duplexes containing X and should be informative during the design of improved wild card nucleobase analogs.

DNA↗
PubMed2000

Regulation of tumor necrosis factor cytotoxicity by calcineurin.

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.

Animals↗
PubMed2000

PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding.

The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1-estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1-mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein-protein interactions. These findings contribute to understanding how protein-protein interactions participate in hematopoietic differentiation and leukemogenesis. (Blood. 2000;96:2641-2648)

Biological Transport↗
PubMed2000

Production and evaluation of a panel of monoclonal antibodies against Haemophilus paragallinarum.

We report on the production and characterisation of monoclonal antibodies (MAbs) against Haemophilus paragallinarum, the causative agent of infectious coryza. A bank of 8 MAbs were produced by traditional techniques - four against the reference strain for Page serovar A (0083) and four against the reference strain for Page serovar C (Modesto). Seven of the eight MAbs were shown to be IgG(1) with one being nontypable. None of the MAbs had HI activity and none gave any detectable reaction when examined by Western blotting. None of the MAbs gave a positive reaction in the indirect ELISA with any of the eight type strains of Pasteurella species or sub-species. None of our 8 MAbs gave serovar specific reactions when used in an indirect ELISA format. There was a trend for the serovar A MAbs to give a higher titre with serovar A isolates/strains and a similar trend for the serovar C MAbs to give higher titres with the serovar C isolates/strains.

Antibodies, Monoclonal↗
PubMed2000

Effects of urea and sodium hydroxide on the molecular weight and conformation of alpha-(1-->3)-D-glucan from Lentinus edodes in aqueous solution.

The weight-average molecular weight (Mw) and intrinsic viscosity ([eta]) of the alpha-(1-->3)-D-glucan (L-FV-II) from Lentinus edodes in 0.5 and 1.0 M NaOH aqueous solution containing urea, were studied by light scattering and viscometry. The Mw value of the glucan decreased with increase of the urea and NaOH concentration. A strong intermolecular hydrogen bonding confers water-insolubility on the glucan, but NaOH and especially urea, broke this hydrogen bonding leading to enhanced water-solubility. Use of 1.0 M urea-1.0 M NaOH as solvent broke not only intermolecular hydrogen bonds but also partial covalent bonds of the alpha-glucan in aqueous solution, resulting in a decrease of Mw and [eta]. The urea and NaOH concentrations, storage time with stirring, and mode of preparation of the polysaccharide in aqueous solution significantly affected the determination of Mw and [eta]. The dependences of specific rotation and fluorescence emission ratio of a probe on urea concentration showed that a change in the molecular conformation of the alpha-glucan in 0.5 M NaOH aqueous solution containing urea occurred in the range 0.4-0.6 M urea. The 0.5 M urea-0.5 M NaOH aqueous solution is a suitable solvent for the glucan, and the Mw and [eta] values obtained were 5.21 x 10(5) and 148 cm3 g(-1), respectively. Degradation of the glucan was obvious after storage for 15 months.

Dose-Response Relationship, Drug↗
PubMed2000

Regulation of interleukin-1beta -induced platelet-derived growth factor receptor-alpha expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase.

The potential role of p38 mitogen-activated protein (MAP) kinase in platelet-derived growth factor receptor-alpha (PDGF-Ralpha) gene expression was investigated using cultured rat pulmonary myofibroblasts. p38 MAP kinase was constitutively expressed in myofibroblasts and activated by interleukin (IL)-1beta. A pyridinylimidazole compound, SB203580, completely inhibited the ability of p38 MAP kinase activity to phosphorylate PHAS-1 substrate. SB203580 inhibited IL-1beta-induced up-regulation of PDGF-Ralpha mRNA and protein in a concentration-dependent manner. Other kinase inhibitors, including the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059, did not block up-regulation of PDGF-Ralpha. The IL-1beta-induced increase in the number of (125)I-PDGF-AA-binding sites at the cell surface was reduced >70% by pretreatment with SB203580. Accordingly, an enhancement of PDGF-AA-stimulated DNA synthesis following IL-1beta pretreatment was blocked >70% by SB203580. SB203580 did not affect IL-1beta-induced ERK activation, yet enhanced IL-1beta-induced JNK activation approximately 2-fold. Treatment of cells with SB203580 after inhibition of transcription by actinomycin D decreased the half-life of IL-1beta-induced PDGF-Ralpha mRNA from >4 to approximately 1.5 h. Moreover, pretreatment of cells with cycloheximide blocked induction of PDGF-Ralpha mRNA by IL-1beta, suggesting that de novo protein synthesis was required for PDGF-Ralpha mRNA stabilization. These data indicate that p38 MAP kinase regulates PDGF-Ralpha expression at the translational level by signaling the synthesis of an mRNA-stabilizing protein.

Animals↗
PubMed2000

Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK.

Activation of ERK-1 and -2 by H(2)O(2) in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO(-) in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using the phosphorylated heat- and acid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO(-) treatment and was maximally activated with 100 micrometer ONOO(-). Activation of ERK by ONOO(-) and H(2)O(2) was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H(2)O(2), but not by ONOO(-), demonstrating that the effect of ONOO(-) was not due to the generation of H(2)O(2). Both H(2)O(2) and ONOO(-) induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H(2)O(2), but not by ONOO(-). Both H(2)O(2) and ONOO(-) activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H(2)O(2), but not by ONOO(-). The MEK inhibitor PD98059 inhibited ERK activation by both H(2)O(2) and ONOO(-). Moreover, ONOO(-) or H(2)O(2) caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO(-) (but not H(2)O(2)) induced autophosphorylation and nitration of a glutathione S-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO(-) activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO(-)-induced ERK activation. However, MEK-1 activation is required for ONOO(-)-induced ERK activation in myofibroblasts. In contrast, H(2)O(2)-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.

Animals↗
PubMed2000

Rapid SLT gene detection on polyethylene-coacrylic acid film without molecular labels or surface-fouling agents.

The commercially available copolymer of 10 mol% acrylic acid and polyethylene is easily formed into a nonfluorescing, non-polynucleotide-adsorbing film. The film has surface carboxylate functions whose concentration can be increased by heating to 80 degrees C in 30% NaOH. The carboxylate groups will react at pH approximately 7 with commercially available, oligo-DNA, 2-8 ng/microl, that has been synthesized with a C(12)-alkylamino tail on the 5'-end. The reaction is mediated with water-soluble carbodiimide reagent and is assumed to result in a primary amide bond between the polymer film and the modified oligo-DNA. The tethered oligo-DNA retains its hybridization activity, and its surface concentration is sufficient to permit qualitative, labelless detection of hybridized target by fluorescence after brief staining with ethidium bromide. The film is used to detect Shiga-like toxin gene II (SLT-II) from Escherichia coli O157:H7 after asymmetric, capillary, PCR amplification, and a 4-h hybridization. Captured target may be removed from the film using distilled water, after which the film can be used again without noticeable loss of activity. The method provides relatively rapid detection of PCR amplimers without having to use molecular labels, or surface-fouling agents.

Acrylates↗
PubMed2000

Characterization of the active site of group B streptococcal hyaluronan lyase.

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.

Calcium↗
PubMed2000

DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins.

DNA vaccination was employed to study immune responses to hepatitis C virus (HCV) proteins. As an immunizing strategy, we studied immune responses of BALB/c (H-2d) and C57BL/6 mice (H-2b) to HCV genes delivered intramuscularly as a polycistronic construct capsid/E1/E2/NS2/NS3 (pRC/C-NS3) encoding 5 structural and nonstructural proteins. We also evaluated canarypox virus containing the same HCV genes as a means for potentiating immune responses to naked DNA. Our results indicate that mice that received a polycistronic pRC/C-NS3 with canarypox booster had enhanced antibody and cellular responses to HCV proteins. Immunodominant CD8(+) T cell responses to several HCV structural and nonstructural proteins, characterized by cytotoxicity and interferon (IFN)-gamma production or IFN-gamma production without significant cytotoxicity, were observed in both strains of mice. The combination of naked DNA with a nonreplicating canarypox booster encoding HCV polycistronic pRC/C-NS3 genes appears to diversify and enhance T cell responses to HCV proteins.

Animals↗
PubMed2000

China's forest policy for the 21st century.

A half-century policy of forest exploitation and monoculture in China has led to disastrous consequences, including degradation of forests and landscapes, loss of biodiversity, unacceptable levels of soil erosion, and catastrophic flooding. A new forest policy had been adopted in China called the Natural Forest conservation Program (NFCP), which emphasizes expansion of natural forests and increasing the productivity of forest plantations. Through locally focused management strategies, biodiversity and forest resources will be sustained, and downstream regions will be better protected from flooding. This new policy is being implemented with a new combination of policy tools, including technical training and education, land management planning, mandatory conversion of marginal farmlands to forest, resettlement and retaining of forest dwellers, share in private ownership, and expanded research. These policy tools may have wider relevance for other countries, particularly developing countries.

Agriculture↗
PubMed2000

Beta-carotene and protein oxidation: effects of ascorbic acid and alpha-tocopherol.

The effect of beta-carotene on protein oxidation was examined under different oxygen (O(2)) tensions and with other antioxidants: alpha-tocopherol, ascorbic acid, and mixtures of antioxidants. Human serum albumin (HSA) was incubated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) to induce protein oxidation (carbonyl formation), under 15, 150, and 760 torr of O(2) tension. Antioxidant activity was related to O(2) tension, antioxidant concentrations and interaction between mixtures of antioxidants: (1) Under 15 torr of O(2), incubating HSA with AAPH, 1. 6 microM beta-carotene, 80 microM alpha-tocopherol, 160 microM ascorbic acid, and mixtures (0.1 microM beta-carotene, 5.0 microM alpha-tocopherol and 10 microM ascorbic acid) resulted in 24, 29, 39, and 44% reduction of carbonyl formation, respectively. (2) Under 150 torr of O(2) tension, the antioxidant effect of beta-carotene was decreased by 4% but increasing O(2) tension did not diminish the antioxidant effects of alpha-tocopherol, ascorbic acid, or antioxidant mixtures. (3). Under 760 torr of O(2) tension, adding 1. 6 microM beta-carotene resulted in 26% more carbonyl formation. (4) Under 760 torr of O(2) tension, the antioxidant effect of ascorbic acid was decreased 32% compared to what was observed at 150 torr of O(2) tension. Changes in O(2) tension had no effect on the antioxidant effect of alpha-tocopherol. The mixture of antioxidants inhibited carbonyl formation by 37% and was 7% less effective than that of 15 and 150 torr of O(2) tension. High concentration of beta-carotene produces more protein oxidation in the presence of high O(2) tension by a prooxidant mechanism. Mixtures of beta-carotene, alpha-tocopherol, and ascorbic acid provided better protective effects on protein oxidation than any single compound.

Amidines↗
PubMed2000

Activity of p44/42 MAP kinase in the caudal subnucleus of trigeminal spinal nucleus is increased following perioral noxious stimulation in the mouse.

The extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2), also referred to as the p44/42 mitogen-activated protein kinase (p44/42 MAP kinase), plays an essential role in neuronal signal transduction, but its function involved in nociceptive response has not been deeply studied yet. Here we report immunohistochemical evidence that p44/42 MAPK might be critical in nociceptive response. We found that after formalin was injected into the perioral skin of the upper lip of mice, the number of activated p44/42 MAPK-like immunoreactive neurons was significantly increased in the laminae I and II of the caudal subnucleus of the trigeminal spinal nucleus (Sp5C). The positive neurons and fibers were mostly concentrated in the middle portion of Sp5C dorsoventrally, where the afferent fibers innervating the skin of the upper lip are terminated. The reactive products were localized in perikarya, dendrites, nuclei, and diffusely in the neuropil. The present result suggests that p44/42 MAPK may be important in the transmission and modulation of noxious information in Sp5C.

Animals↗
PubMed2000

Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.

A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome.

Amino Acid Sequence↗
PubMed2000

Detection of hypoxia in human squamous cell carcinoma by EF5 binding.

Localization and quantitation of 2-nitroimidazole drug binding in low pO2 tumors is a technique that can allow the assessment of hypoxia as a predictive assay. EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] is such a drug, and it has been shown to be predictive of radiation response in rodent tumors. Using fluorescence immunohistochemical techniques, we provide data on the presence, distribution, and levels of EF5 binding as a surrogate for hypoxia in human head and neck and uterine cervix squamous cell cancers (SCCs). Six patients with SCC were studied. Four patients had head and neck tumors, and two had uterine cervix cancers. The incubation of fresh tissue cubes in EF3 under hypoxic conditions ("reference binding") demonstrated that all tumors were capable of binding drug, and that this binding varied by a factor of 2.9-fold (174.5-516.1) on an absolute fluorescence scale. In the five patients treated at the lowest drug doses (9 mg/kg), in situ binding was quantitatable. For all six patients, the maximum rate of in situ binding varied by a factor of 6.7 between the lowest and highest binding tumor (24.8-160.3) on an absolute fluorescence scale. In tumors with high binding regions, intratumoral heterogeneity was large, extending from minimal fluorescence (<1%) up to 88.6% of reference binding. In tumors with minimal binding, there was little intratumoral heterogeneity. These studies demonstrate substantial heterogeneity of in situ binding between and within individual squamous cell tumors.

Adult↗
PubMed2000

Direct observation of single-molecule generation at a solid-liquid interface

Direct observation of single-molecule generation from a chemical reaction was achieved at a solid-liquid interface. The reaction between fluorescamine and immobilized N'-(3-trimethoxysilylpropyl)diethylenetriamine (DETA) was studied at the single-molecule level. Time-lapse fluorescence images of single-molecule products, excited by the evanescent field generated at a quartz-liquid interface, were recorded to follow the chemical reaction to its completion. The reactions were restricted to the approximately 1 nm thick layer nearest to the interface. Analysis of the photoelectron intensity of the fluorescent product of the reaction and its distribution shows that the reaction kinetics goes through a transition from zeroth-order to first-order as the reaction proceeds. This approach offered a novel means to study single-molecule reactions at the solid-liquid interface. It also enabled the investigation of reaction kinetics and chemical mapping of surface heterogeneity at the single-molecule level.

Journal Article↗