Biomedical subjects

H Zhang

Publications and source records attributed to H Zhang.

At least 523 records · Page 29Linked to original sources

PubMed2000

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts.

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.

Blood Proteins↗

PubMed2000

A carboxy-terminal alpha-helical segment in the rat skeletal muscle voltage-dependent Na+ channel is responsible for its interaction with the amino-terminus.

Cytoplasmic segments of the adult rat skeletal muscle sodium channel alpha-subunit (rSkM1) comprise a major portion (approximately 40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel's amino- and carboxy-termini and identify residues 1716-1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an alpha-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp1617.

Animals↗

PubMed2000

Expression of a TGF-beta superfamily protein, macrophage inhibitory cytokine-1, in the yeast Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-beta) superfamily cytokine. This is the first report of the expression of a correctly folded TGF-beta superfamily protein in a microbial organism. The protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which are significantly higher than we have been able to achieve using mammalian expression systems. Purification schemes are described, and the purified protein is immunologically identical to protein produced in a mammalian expression system. Protein expression was influenced by a number of factors, most significantly by the concentration of methanol used during the induction phase. However, with very high levels of MIC-1 induction, substantial amounts of MIC-1 monomer were also secreted.

Amino Acid Sequence↗

PubMed2000

Nociceptin inhibits capsaicin-induced bronchoconstriction in isolated guinea pig lung.

The isolated perfused guinea pig lung was used to investigate the effect of nociceptin against bronchoconstriction elicited by endogenous and exogenous tachykinins. The opioid receptor-like 1 (ORL1) receptor agonist, nociceptin/orphanin FQ (0.001-1 microM) produced a dose-related inhibition of the capsaicin-induced bronchoconstriction (10(-5)-10(3) microg) in isolated guinea pig lung (P<0.05), a response mediated by the release of endogenous tachykinins from lung sensory nerves. The new ORL1 receptor antagonist 1-[(3R, 4R)-1-Cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one (J-113397) (0.3 microM) significantly blocked the inhibitory effect of nociceptin/orphanin FQ (0.01 microM) on capsaicin-induced bronchoconstriction, whereas the non-selective opioid receptor antagonist naloxone (1 microM) had no effect. Nociceptin/orphanin FQ (1 microM) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist neurokinin A. In conclusion, the present data provide evidence that nociceptin inhibits capsaicin-evoked tachykinin release from sensory nerve terminals in guinea pig lung by a prejunctional mechanism. This inhibitory action occurs independently from activation of opioid receptors. The present study also indicates that J-113397 is a potent ORL1 receptor antagonist.

Animals↗

PubMed2000

Aggregation-based crystal growth and microstructure development in natural iron oxyhydroxide biomineralization products.

Crystals are generally considered to grow by attachment of ions to inorganic surfaces or organic templates. High-resolution transmission electron microscopy of biomineralization products of iron-oxidizing bacteria revealed an alternative coarsening mechanism in which adjacent 2- to 3-nanometer particles aggregate and rotate so their structures adopt parallel orientations in three dimensions. Crystal growth is accomplished by eliminating water molecules at interfaces and forming iron-oxygen bonds. Self-assembly occurs at multiple sites, leading to a coarser, polycrystalline material. Point defects (from surface-adsorbed impurities), dislocations, and slabs of structurally distinct material are created as a consequence of this growth mechanism and can dramatically impact subsequent reactivity.

Betaproteobacteria↗

PubMed2000

Effect of temperature on viscosity properties of some alpha-amino acids in aqueous urea solutions.

Viscosities for solutions of glycine, DL-alpha-alanine, DL-alpha-amino-n-butyric acid, DL-valine, DL-leucine and L-serine in 5 mol kg(-1) aqueous urea have been determined at 278.15, 288.15, 298.15 and 308.15 K. The viscosity B-coefficients for the amino acids in the aqueous urea solution have been calculated at different temperatures. The effect of temperature on the B-coefficients is discussed on the basis of the Feakins equation. The contribution of solute to the activation parameters (delta mu0*2, deltaH0*2, deltaS0*2) for viscous flow of the solution have been calculated, together with the Gibbs energy, enthalpy and entropy of transfer for the amino acids from the ground-state solvent to the hypothetical viscous transition state solvent. The contributions of the charged end group (NH3+, COO-) and CH2 groups of the amino acids to B-coefficient and delta mu0*2 have been also estimated using the linear correlations between B-coefficient or delta mu0*2 and the number of carbon atoms in the alkyl chains of the amino acids. All the activation parameters are discussed in terms of the solute-solvent interactions in the ground and transition states.

Amino Acids↗

PubMed2000

Insulin-like growth factor I-mediated degradation of insulin receptor substrate-1 is inhibited by epidermal growth factor in prostate epithelial cells.

We have sought to determine whether insulin-like growth factor I (IGF-I) regulates the levels of insulin receptor substrate-1 (IRS-1) in prostate epithelial cells. Exposure of prostate epithelial cells to IGF-I in the absence of other growth factors leads to a reduction in IRS-1 levels. Ubiquitin content of IRS-1 is increased in the presence of IGF-I, and inhibitors of the proteasome prevented the reduction of IRS-1 levels seen following IGF-I exposure. These results imply that IRS-1 is targeted to the proteasome upon exposure to IGF-I. The addition of epidermal growth factor (EGF) maintained IRS-1 levels even in the presence of IGF-I and inhibits IGF-I-dependent ubiquitination of IRS-1. Thus, these two growth factors, IGF-I and EGF, had antagonistic effects on IRS-1 protein levels in prostate epithelial cells. This regulation of IRS-1 reveals a novel level of cross-talk between the IGF-I and EGF signal pathways, which may have implications in tumors that harbor activating mutations in the EGF receptor.

Cells, Cultured↗

PubMed2000

Application of atmospheric pressure ionization time-of-flight mass spectrometry coupled with liquid chromatography for the characterization of in vitro drug metabolites.

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 microL/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic "A" ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed.

Animals↗

PubMed2000

Crystal structure of YbaK protein from Haemophilus influenzae (HI1434) at 1.8 A resolution: functional implications.

Structural genomics of proteins of unknown function most straightforwardly assists with assignment of biochemical activity when the new structure resembles that of proteins whose functions are known. When a new fold is revealed, the universe of known folds is enriched, and once the function is determined by other means, novel structure-function relationships are established. The previously unannotated protein HI1434 from H. influenzae provides a hybrid example of these two paradigms. It is a member of a microbial protein family, labeled in SwissProt as YbaK and ebsC. The crystal structure at 1.8 A resolution reported here reveals a fold that is only remotely related to the C-lectin fold, in particular to endostatin, and thus is not sufficiently similar to imply that YbaK proteins are saccharide binding proteins. However, a crevice that may accommodate a small ligand is evident. The putative binding site contains only one invariant residue, Lys46, which carries a functional group that could play a role in catalysis, indicating that YbaK is probably not an enzyme. Detailed sequence analysis, including a number of newly sequenced microbial organisms, highlights sequence homology to an insertion domain in prolyl-tRNA synthetases (proRS) from prokaryote, a domain whose function is unknown. A HI1434-based model of the insertion domain shows that it should also contain the putative binding site. Being part of a tRNA synthetases, the insertion domain is likely to be involved in oligonucleotide binding, with possible roles in recognition/discrimination or editing of prolyl-tRNA. By analogy, YbaK may also play a role in nucleotide or oligonucleotide binding, the nature of which is yet to be determined.

Amino Acid Sequence↗

PubMed2000

Molecular cloning, expression characterization, and mapping of a novel putative inhibitor of rho GTPase activity, RTKN, to D2S145-D2S286.

The Rho proteins are a class of small molecular GTPases that regulate multiple fundamental cellular processes by mediating the G-protein-coupled receptor signaling pathway. Rhotekin, which is one of the downstream target molecules of Rho with a Rho binding motif class I domain, can inhibit endogenous or RhoGAP-stimulating Rho GTPase activity to regulate the signaling pathway. Here, a novel human cDNA containing an intact open reading frame that encodes 544 amino acids has been identified. As this putative protein shares 84. 6% amino acid identity with mouse Rhotekin, and has a tandem Rho binding domain class 1 and Pleckstrin homology domain, it was regarded as a human homologue of the mouse Rhotekin and assigned a symbol of RTKN. With the human Rhotekin cDNA as a probe, Northern hybridization revealed that a 4.0-kb transcript was expressed at a high level in prostate and at a middle level in 13 of 16 tissues examined, but it cannot be detected in liver and lung tissues. Meanwhile, a 2.4-kb transcript was expressed at a middle level in prostate and another 3.0-kb transcript in kidney. In addition, the RTKN gene was localized to chromosome 2p13 between markers D2S145 at 6.94 cR (LOD > 12) and D2S286 at 8.12 cR (LOD > 9.7) by radiation hybrid panel mapping. Compared with BAC clone AC005041 sequence, there were 12 exons for the RTKN gene and it spanned a 16.5-kb genomic region.

Amino Acid Motifs↗

PubMed2000

Model for the analysis of binary time series of respiratory symptoms.

Environmental epidemiologic research on respiratory symptoms presents unique types of data, typically requiring simultaneous analysis of both time- and person-varying factors. In this paper, the authors propose a new, simple model that incorporates such factors and controls for each person's prior history of symptoms. The Yale Mother and Infant Health Study was undertaken to investigate the effects of ambient pollutant concentrations, meteorologic changes, and demographic variables on daily respiratory symptoms in both mothers and infants. This analysis was restricted to 673 mothers followed in southwestern Virginia from June 10 to August 31, 1995. Of the person-varying factors, husband's level of education, nested within marital status, and having pets in the home were related to an increased likelihood of new episodes; however, neither was related to duration of symptoms. Interestingly, women who were unmarried were least likely to have new episodes of respiratory symptoms, while those with the most highly educated spouses were most likely to have new episodes. Having pets in the home increased the likelihood of a new episode. Having a history of allergies and having children in day care were found to be related to the symptom of a runny or stuffy nose, in terms of both incidence and duration. The level of coarse particles was related to the incidence of new episodes of runny or stuffy nose, and a higher level prolonged the duration of symptoms. Sulfate level was not related to the incidence of new episodes but was associated with the duration of the episodes.

Adult↗

PubMed2000

Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1.

High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1-specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a K(d) = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (k(on) and k(off), respectively) were determined, and they yielded a dissociation constant (K(d)) of 3.2x10(-9) mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1-mediated cell-extracellular matrix and cell-cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury. (Blood. 2000;95:3788-3795)

Cell Adhesion↗

PubMed2000

[Effect of smoking on the guanylin in the injured lung of rats].

In order to survey the influence of smoking on the level of guanylin in lung tissue in rats, a method of passive smoking was used to set up chronic bronchitis model in rats. The content of guanylin was measured by radioimmunoassay. In the smoking group, remarkable pathological changes in the lung tissue were found. There was no significant difference in the contents of guanylin in the plasma of experimental rats, compared with the control(P < 0.05). But in the lung tissue, the difference was significant(P < 0.01).

Animals↗

PubMed2000

Synthesis and characterization of the dimethyl-substituted bisbenzimidazole ligand and its manganese complex.

Macrocycles with unique properties provide new avenues for the design of novel catalysts and materials. Here, we report, for the first time, the synthesis and characterization of the dimethyl-substituted bisbenzimidazole ligand (Me2BBZ) and its manganese complex (Mn-Me2BBZ). The Me2BBZ ligand is similar to porphyrin and phthalocyanine macrocycles in terms of its cavity size and metal-binding mode, but owing to electronic and charge differences, it exhibits properties that make it distinct from its structural counterparts. For instance, the optical spectra of bisbenzimidazoles lack transitions in the 500-900 nm region. Perhaps the most significant feature of the Me2BBZ ligand, however, is its inherent nonplanarity. Geometric restraints within this nonplanar ligand give rise to two atropisomers, which, when separated, could have potential in chiral catalysis and recognition. In addition, here we show that this nonplanarity can help to promote unusual crystal-packing interactions. Within the structure of the Mn-Me2BBZ complex, intermolecular pi-stacking interactions of the phenyl and benzimidazole groups lead to the formation of a distinct two-dimensional "staircase" lattice comprised of alternating Mn-Me2BBZ atropisomers. The potential significance of this structural arrangement is revealed by temperature-dependent magnetic studies that indicate weak antiferromagnetic coupling between the metal ions in the crystal. Fine-tuning of these long-range electronic and magnetic interactions could be useful for the design of novel molecular materials.

Journal Article↗

PubMed2000

Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors.

Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal-regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal-regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH(2)-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by alpha(1)-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.

Animals↗

PubMed2000

Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis.

Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild-type enzyme. The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I.

Amino Acid Substitution↗

PubMed2000

Modulation of HIV-1 replication by a novel RhoA effector activity.

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Galpha13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity.

Amino Acid Sequence↗

PubMed2000

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical.

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.

Animals↗