Biomedical subjects

P Wang

Publications and source records attributed to P Wang.

At least 19 recordsLinked to original sources

PubMed2000

Determination of the binding specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through the screening of a combinatorial phosphotyrosyl peptide library.

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.

Amino Acid Sequence↗

PubMed2000

Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates.

Platelet-derived growth factor receptor beta (PDGFRbeta) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown. We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners. The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways.

Caveolae↗

PubMed2000

Cloning, characterization, and tissue distribution of mouse phosphodiesterase 7A1.

We have cloned a cDNA representing mouse phosphodiesterases (PDE) 7A1. The open reading frame encodes a protein of 482 amino acids with a predicted molecular mass of 55417. Like human PDE7A variants, mouse PDE7A1 and A2 are 5' splice variants from a common gene. The distinct N-terminal sequence of mouse PDE7A1 is highly homologous to the corresponding sequence of human PDE7A1 with a similarity of 98% but not to that of mouse PDE7A2 (with a similarity of 12%), and is more hydrophilic than that of mouse PDE7A2. Mouse PDE7A1 expressed in SF9 cells has been compared with human PDE7A1 under identical conditions. Mouse PDE7A1 has a Km for cAMP of 0.2 microM, an optimal pH of 7.5, an IC(50) value of 14 microM for 3-isobutyl-1-methylxanthine (IBMX), and is dependent on Mg(2+) for activity. All these characteristics are very similar to those of human PDE7A1. In mice, PDE7A1 is expressed in tissues of the immune system (lymph node, thymus, spleen, and blood leukocyte), testis, brain, kidney and lung but not in skeletal muscle, heart, embryo, or liver, while PDE7A2 is expressed in skeletal muscle, heart, embryo, and kidney, but not in the other tissues. This tissue distribution profile is very similar to that in humans, and hence suggests that PDE7A1 and 7A2 might play a similar role in different species.

1-Methyl-3-isobutylxanthine↗

PubMed2000

II. Structure and specificity of the interaction between the FHA2 domain of Rad53 and phosphotyrosyl peptides.

The forkhead-associated (FHA) domain is a protein module found in many proteins involved in cell signaling in response to DNA damage. It has been suggested to bind to pThr sites of its target protein. Recently we have determined the first structure of an FHA domain, FHA2 from the yeast protein Rad53, and demonstrated that FHA2 binds to a pTyr-containing peptide (826)EDI(pY)YLD(832) from Rad9, with a moderate affinity (K(d) ca. 100 microM). We now report the solution structure of the complex of FHA2 bound with this pTyr peptide. The structure shows that the phosphate group of pTyr interacts directly with three arginine residues (605, 617, and 620), and that the leucine residue at the +2 position from the pTyr interacts with a hydrophobic surface on FHA2. The sequence specificity of FHA2 was determined by screening a combinatorial pTyr library. The results clearly show that FHA2 recognizes specific sequences C-terminal to pTyr with the following consensus: XX(pY)N(1)N(2)N(3), where N(1)=Leu, Met, Phe, or Ile, N(2)=Tyr, Phe, Leu, or Met, and N(3)=Phe, Leu, or Met. Two of the selected peptides, GF(pY)LYFIR and DV(pY)FYMIR, bind FHA2 with K(d) values of 1.1 and 5.0 microM, respectively. The results, along with other recent reports, demonstrate that the FHA domain is a new class of phosphoprotein-binding domain, capable of binding both pTyr and pThr sequences.

Amino Acid Sequence↗

PubMed2000

Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.

Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer.

Amino Acid Sequence↗

PubMed2000

Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb.

The goals of the work reported here were (i) to identify distinct GABA(A)/benzodiazepine receptors in the rat hippocampus and olfactory bulb using receptor binding assays, and (ii) to determine the affinities and selectivities of benzodiazepine receptor ligands from structurally diverse chemical families at each site identified. These studies were aided by the use of software AFFINITY ANALYSIS SYSTEM, developed in our laboratory for analysis of receptor binding data that allows the determination of receptor heterogeneity using non-selective radioligands. Saturation binding assays using [3H]RO15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a]-[1,4]benzodiazepine-3-carboxylate) revealed two binding sites in each of these two tissues. The higher affinity site corresponds to alpha(5) subunit-containing GABA(A) receptor and the lower affinity site to a combination of alpha(1), alpha(2), and alpha(3) subunit-containing receptors. These results should be useful in the challenging task of identifying the various functional GABA(A) receptors in the central nervous system, and in providing a link between receptor affinities and in vivo activities of the GABA(A)/benzodiazepine receptor ligands studied.

Animals↗

PubMed2000

Characterization of the protein phosphatase 1 catalytic subunit in endothelium: involvement in contractile responses.

We have previously demonstrated the direct involvement of a type 1 Ser/Thr phosphatase (PPase 1) in endothelial cell (EC) barrier regulation [Am. J. Physiol. 269:L99-L108, 1995]. To further extend this observation, we microinjected either the Ser/Thr PPase inhibitor, calyculin, or the PPase 1 inhibitory protein, I-2 into bovine pulmonary artery EC and demonstrated both an increase in F-actin stress fibers and a shift from a regular polygonal shape to a spindle shape with gaps apparent at the cell borders. Northern blot analysis with specific cDNA probes revealed the presence of three major PPase 1 catalytic subunit (CS1) isoforms (alpha, delta, and gamma) in human and bovine EC. To characterize the myosin-associated EC CS1 isoform, myosin-enriched bovine EC fraction was screened with anti-CS1alpha and anti-CS1delta antibodies The anti-CS1delta antiserum, but not anti-CS1alpha antiserum cross reacts with the CS1 isoform present in myosin-enriched fraction and CS1delta was found in stable association with EC myosin/myosin light chain kinase (MLCK) complex in MLCK immunoprecipitates under nondenaturing conditions. Consistent with these data, overexpression of CS1delta-GFP construct in bovine endothelium followed by immunoprecipitation of CS1 with anti-GFP antibody revealed the stable association of CS1delta with actomyosin complex. Finally, screening of a human EC oligo(dT)-primed cDNA library with a probe encoding a rat CS1delta cDNA segment yielding several positive clones that encoded the entire CS1delta open reading frame and partially noncoding regions. Sequence analysis determined a high homology ( approximately 99%) with human CS1delta derived from a teratocarcinoma cell line. Together, these data suggest that CS1delta is the major of PPase 1 isoform specifically associated with EC actomyosin complex and which participates in EC barrier regulation.

Amino Acid Sequence↗

PubMed2000

Substituted oxazole benzenesulfonamides as potent human beta3 adrenergic receptor agonists.

As a part of our investigation into the development of orally bioavailable beta3 adrenergic receptor agonists, we have identified a series of substituted oxazole derivatives that are potent beta3 agonists with excellent selectivity against other beta receptors. Several of these compounds showed excellent oral bioavailability in dogs. One example, cyclopentylethyloxazole 5f is a potent beta3 agonist (EC50 = 14 nM, 84% activation) with 340-fold and 160-fold selectivity over beta1 and beta2 receptors, respectively, and has 38% oral bioavailability in dogs.

Adrenergic beta-3 Receptor Agonists↗

PubMed2000

Lysophosphatidylcholine activates p38 and p42/44 mitogen-activated protein kinases in monocytic THP-1 cells, but only p38 activation is involved in its stimulated chemotaxis.

Oxidized LDLs (OxLDLs) have been shown to be involved in recruitment of blood monocytes into the arterial subendothelial space, which is the earliest step in atherogenesis, but the underlying molecular mechanisms are poorly understood. The present study demonstrated that lysophosphatidylcholine (LPC), a major phospholipid component of OxLDL, strongly evoked phosphorylation and activation of p38 and p42/44 mitogen-activated protein kinases in monocytic cells. The stimulation of p38 and p42/44 occurred in a dose- and time-dependent manner, reaching the maximal activation at 25 microg/mL LPC within 5 minutes. Interestingly, inhibition of p38 activation by OxLDL or LPC, using its selective inhibitors (SB203580 and SKF86002), completely blocked OxLDL- or LPC-stimulated chemotaxis of THP-1 cells, which was measured in a transwell chemotaxis assay. In contrast, inhibition of p42/44 activation by its potent inhibitor (PD98059) did not block OxLDL- or LPC-stimulated chemotaxis. Moreover, expression of a p38 dominant-negative mutant (p38AF) reduced cell chemotaxis significantly. In addition, activation of p38 by LPC was apparently mediated neither by scavenger receptors nor by tyrosine kinase receptors. It was, however, effectively blocked by pertussis toxin and substantially reduced by phospholipase C inhibitor (U73122) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002). LPC also inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner, indicating that Gi/Go proteins likely mediated the effects of LPC. Our results suggested that OxLDL/LPC efficiently activated both p38 and p42/44, but only the activation of p38 was functionally associated with OxLDL-/LPC-induced chemotaxis in THP-1 cells.

Cells, Cultured↗

PubMed2000

Transition of the blastomere cell cycle from cell size-independent to size-dependent control at the midblastula stage in Xenopus laevis.

Dissociated animal cap blastomeres of Xenopus laevis blastulae were cultured at a low Ca level (1 microM) from 9th to 18th cell cycle at 22 +/- 1 degrees C and observed by a time-lapse video recorder. Blastomeres cleaved unequally to increase variability in cell size as cell cycles progressed, but synchronously at a constant cell cycle time of about 30 min up to the 12th cleavage in diploid cells, and up to the 13th cleavage in haploid cells, regardless of their cell sizes. Thereafter, blastomeres cleaved asynchronously at varying cell cycle times in proportion to the inverse square of their radii. The transition from the cell size-independent to -dependent cell cycles occurred at the critical cell radius, 37.5 microm for the diploid and 27.9 microm for the haploid. While the protein synthesis inhibitor, cycloheximide (CHX) lengthened cell cycle times two- to six-fold, epidermal growth factor (EGF) had no significant effect on the cell cycle. CHX-treated blastomeres synchronously cleaved at a constant cell cycle time of 60 min up to the 12th cleavage. Thereafter, cell cycle times became variable in proportion to the inverse square of radii in the presence of CHX at 0.10-0.14 microg/ml, but to the inverse cube of radii at 0.18 microg/ml. The critical cell size of CHX-treated blastomeres for the transition from cell size-independent to -dependent cell cycles remained the same as that of untreated blastomeres. Frequency distributions of cell cycle times of synchronous cell cycles were monomodal with the peak at 30 min, except for CHX-treated blastomeres with the peak at 60 min. In contrast, frequency distributions of asynchronous cell cycles were polymodal with peaks at multiples of a unit time of 30-35 min. To explain these results, we propose that blastomere cytoplasm has 30-min cycles that repeatedly produce mitosis promoting factor (MPF) in a quantity proportional to the cell surface area. MPF is neutralized when it titrates a nuclear inhibitor present in a quantity proportional to the genome size, and sequestered in the nucleus. When the total amount of MPF produced exceeds the threshold required to titrate all of the inhibitor, mitosis is initiated.

Animals↗

PubMed2000

Identification of alternative splicing variants of the beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II with different activities.

The beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II (beta CaMKII) was identified by searching through an expressed sequence tag database and rapid amplification of cDNA 5'-ends and was assigned to chromosome 7. Reverse transcription-polymerase chain reaction and sequencing analysis identified at least five alternative splicing variants of beta CaMKII (beta, beta6, betae, beta'e, and beta7) in brain and two of them (beta6 and beta7) were first detected in any species. When expressed in HEK 293 cells, the Ca(2+)/calmodulin-dependent kinase activity of beta7, the shortest variant, was much lower than that of either beta (the longest one) or betae (the medium one), suggesting possible regulation of beta CaMKII activity by alternative splicing.

Alternative Splicing↗

PubMed2000

The dissociation between upregulated endothelins and hemodynamic responses during polymicrobial sepsis.

Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although studies have suggested that endothelins (ETs) contribute to the development of shock after a bolus injection of endotoxin, little is known about the role of ETs in the transition from the hyperdynamic phase to the hypodynamic phase of sepsis. To study this, male adult rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. Plasma levels of ET-1 and ET-2 were measured by radioimmunoassay at 2, 5, 10 h (i.e. the early stage of sepsis), and 20 h (late stage) following CLP or sham operation. Tissue levels of ET-1 and ET-2 were determined in the heart, lungs, small intestine, and spleen at 5 h after CLP or sham operation. In addition, preproendothelin-1 (precursor of ET-1) gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) at 5 h in the heart, lungs, small intestine, spleen, and liver. The results indicate that plasma levels of ET-1 and ET-2 were not different from values of sham groups at 2 and 20 h, but were significantly higher than the sham values at 5 and 10 h after CLP. While there were no significant increases in tissue levels of ET-1 and ET-2 at 5 h post-CLP, RT-PCR analysis indicates a significant upregulation of preproendothelin-1 gene expression in the heart, spleen, and liver (but not in the lungs or small intestine) at 5 h after the onset of sepsis. These results indicate that the heart, spleen, and liver appear to be important ET-producing organs during the early stage of sepsis. The lack of significant increases in tissue ET levels could be due to the possibility that the newly converted peptide is quickly transferred to the bloodstream. Since the hyperdynamic phase of sepsis occurs at 2-10 h and the hypodynamic phase occurs at 20 h after CLP, the increased plasma levels of ET at 5 and 10 h suggest that mediators other than ETs (such as adrenomedullin) are responsible for producing the biphasic hemodynamic responses during the progression of polymicrobial sepsis.

Animals↗

PubMed2000

Mechanism of the beneficial effects of pentoxifylline during sepsis: maintenance of adrenomedullin responsiveness and downregulation of proinflammatory cytokines.

BACKGROUND: Although it is known that pentoxifylline (PTX) produces various beneficial effects during sepsis, it remains unknown whether this agent has any salutary effects on the depressed vascular responsiveness to adrenomedullin (ADM), a novel potent vasodilatory peptide, under such conditions. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). One hour after CLP, PTX (50 mg/kg body wt) or vehicle (normal saline) was infused intravenously over 90 min. Twenty hours after CLP (i.e., the late, hypodynamic stage of sepsis), the thoracic aorta and small intestine were isolated and preconstricted by norepinephrine. Rat ADM (10(-7) M) was applied, and the percentage of ADM-induced relaxation in the aortic rings and resistance vessels in the small intestine was determined. In addition, plasma ADM was determined by radioimmunoassay and tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 levels were measured by enzyme-linked immunosorbent assay. RESULTS: The percentage of ADM-induced vascular relaxation in the aortic rings and resistance vessels of the isolated gut was significantly reduced 20 h after CLP. Administration of PTX early after the onset of sepsis, however, prevented the decrease in vascular ADM responsiveness at the macro- and microcirculatory levels. Plasma ADM levels increased after CLP, irrespective of PTX infusion, indicating that the effect of PTX was not mediated by altering ADM release. The upregulated TNF-alpha, IL-1beta, and IL-6 during late sepsis were, however, attenuated by PTX administration, suggesting that maintenance of ADM responsiveness by this agent appears to be due to downregulation of these cytokines. CONCLUSIONS: Since early administration of PTX maintains vascular ADM responsiveness even during the late stage of sepsis, this agent appears to be a useful adjunct in preventing the deterioration in hemodynamics and cardiovascular function during the progression of polymicrobial sepsis.

Adrenomedullin↗

PubMed2000

Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells.

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.

Amino Acid Substitution↗

PubMed2000

Drosophila beta spectrin functions independently of alpha spectrin to polarize the Na,K ATPase in epithelial cells.

Spectrin has been proposed to function as a sorting machine that concentrates interacting proteins such as the Na,K ATPase within specialized plasma membrane domains of polarized cells. However, little direct evidence to support this model has been obtained. Here we used a genetic approach to directly test the requirement for the beta subunit of the alphabeta spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. beta Spectrin mutations were lethal during late embryonic/early larval development and they produced subtle defects in midgut morphology and stomach acid secretion. The polarized distributions of alphabeta(H) spectrin and ankyrin were not significantly altered in beta spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independently of one another, and that ankyrin is upstream of alphabeta spectrin in the spectrin assembly pathway. In contrast, beta spectrin mutations had a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for beta spectrin in determining the subcellular distribution of the Na, K ATPase and, unexpectedly, this role is independent of alpha spectrin.

Animals↗

PubMed2000

Self-compression of high-intensity femtosecond optical pulses and spatiotemporal soliton generation

Self-compression of high-intensity femtosecond pulses has been observed in a number of atomic and molecular gases and solid bulk material. The evolution of the femtosecond pulse parameters during the self-compression has been studied under a variety of experimental conditions. Generation of spatiotemporal solitons has been achieved by the combined action of self-compression and self-focusing.

Journal Article↗

PubMed2000

Cytokine-induced Src homology 2 protein (CIS) promotes T cell receptor-mediated proliferation and prolongs survival of activated T cells.

Members of the suppressor of cytokine signaling (SOCS) family were discovered as negative regulators of cytokine signaling by inhibition of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway. Among them, cytokine-induced Src homology 2 (SH2) protein (CIS) was found to inhibit the interleukin 3- and erythropietin-mediated STAT5 signaling pathway. However, involvement of SOCS proteins in other signaling pathways is still unknown. This study shows that the expression of CIS is selectively induced in T cells after T cell receptor (TCR) stimulation. In transgenic mice, with selective expression of CIS in CD4 T cells, elevated CIS strongly promotes TCR-mediated proliferation and cytokine production in vitro, and superantigen-induced T cell activation in vivo. Forced expression of CIS also prolongs survival of CD4 T cells after TCR activation. Molecular events immediately downstream from the TCR are not changed in CIS-expressing CD4 T cells, but activation of mitogen-activated protein (MAP) kinase pathways by TCR stimulation is significantly enhanced. Together with the increased MAP kinase activation, a direct interaction of CIS and protein kinase Ctheta was also demonstrated. These results suggest that CIS is one of the important regulators of TCR-mediated T cell activation. The functions of CIS, enhancing TCR signaling and inhibiting cytokine signaling, may be important in the regulation of immune response and homeostasis.

Animals↗

PubMed2000

An LC-MS-MS method for the comprehensive analysis of cocaine and cocaine metabolites in meconium.

A sensitive, precise, and accurate liquid chromatography-mass spectrometry (LC-MS-MS) method was developed to quantitate cocaine and cocaine metabolites, which were simultaneously extracted from suspected drug-positive meconium samples using solid-phase extraction. The ability to analyze cocaine and multiple cocaine metabolites in meconium makes this method a powerful tool for the study of cocaine exposure and metabolism in neonates. Of 22 samples, only 1 did not show the presence of cocaine or any metabolite of cocaine. The identified metabolites varied both qualitatively and quantitatively between samples. Ecgonine appears to hold the most promise as a diagnostic marker compound for neonatal cocaine exposure as this metabolite was present in 21 of 21 of the positive samples tested, and at a relatively high median concentration. However, a core group of eight metabolites (present in at least 20 of 21 positive samples) was identified that appears to possess the greatest utility for determining cocaine exposure. Finally, the use of this method for assessment of the magnitude of fetal cocaine exposure was demonstrated.

Chromatography, Liquid↗