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Biomedical subjects

W B Wu

Publications and source records attributed to W B Wu.

At least 19 recordsLinked to original sources
PubMed2006

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81.

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.

Amino Acid Sequence↗
PubMed2006

Charge-orbital ordering and Verwey transition in magnetite measured by resonant soft X-ray scattering.

We report experimental evidence for the charge-orbital ordering in magnetite below the Verwey transition temperature T(V). Measurements of O K-edge resonant x-ray scattering on magnetite reveal that the O 2p states in the vicinity of the Fermi level exhibit a charge-orbital ordering along the c axis with a spatial periodicity of the doubled lattice parameter of the undistorted cubic phase. Such a charge-orbital ordering vanishes abruptly above T(V) and exhibits a thermal hysteresis, correlating closely with the Verwey transition in magnetite.

Journal Article↗
PubMed2005

Orbital symmetry and electron correlation in NaxCoO2.

Measurements of polarization-dependent soft x-ray absorption reveal that the electronic states determining the low-energy excitations of NaxCoO2 have predominantly a(1g) symmetry with significant O 2p character. In contrast to the prediction of band theory, doping-dependent O 1s x-ray absorption shows a large transfer of spectral weight, providing spectral evidence for strong electron correlations of the layered cobaltates. We also found that NaxCoO2 exhibits a charge-transfer electronic character rather than a Mott-Hubbard character.

Journal Article↗
PubMed2004

Spin and orbital magnetic moments of Fe3O4.

We present measurements of the spin and orbital magnetic moments of Fe3O4 by using SQUID and magnetic circular dichroism in soft x-ray absorption. The measurements show that Fe3O4 has a noninteger spin moment, in contrast to its predicted half-metallic feature. Fe3O4 also exhibits a large unquenched orbital moment. Calculations using the local density approximation including the Hubbard U method and the configuration interaction cluster-model suggest that strong correlations and spin-orbit interaction of the 3d electrons result in the noninteger spin and large orbital moments of Fe3O4.

Journal Article↗
PubMed2004

Orbital ordering in La0.5Sr1.5MnO4 studied by soft X-ray linear dichroism.

We found that the conventional model of orbital-ordering of 3x(2)-r(2)/3y(2)-r(2) type in the e(g) states of La0.5Sr1.5MnO4 is incompatible with measurements of linear dichroism in the Mn 2p-edge x-ray absorption, whereas these e(g) states exhibit predominantly cross-type orbital ordering of x(2)-z(2)/y(2)-z(2). LDA+U band-structure calculations reveal that such a cross-type orbital-ordering results from a combined effect of antiferromagnetic structure, Jahn-Teller distortion, and on-site Coulomb interactions.

Journal Article↗
PubMed2001

Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells.

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020 Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the alpha chain of fibrinogen preferentially and cleaved the beta chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.

Amino Acid Sequence↗
PubMed2001

Crotalin, a vWF and GP Ib cleaving metalloproteinase from venom of Crotalus atrox.

Binding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP lb antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP lb as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP lb antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin autoproteolytically degraded to approximately 14 and approximately 10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP lb interaction.

Amino Acid Sequence↗
PubMed2000

P2Y(2) receptor-stimulated phosphoinositide hydrolysis and Ca(2+) mobilization in tracheal epithelial cells.

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP(x)) accumulation and Ca(2+) mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [(3)H]InsP(x) accumulation and Ca(2+) mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y(1) receptors, N(6)-cyclopentyladenosine and AMP, alpha,beta-methylene-ATP and beta, gamma-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [(3)H]InsP(x) accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [(3)H]InsP(x) accumulation and Ca(2+) mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [(3)H]InsP(x) accumulation and Ca(2+) mobilization in canine TECs. P2Y(2) receptors may be predominantly mediating [(3)H]InsP(x) accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca(2+) mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.

Adenosine Diphosphate↗
PubMed1999

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells.

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.

Animals↗
PubMed1999

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells.

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.

Animals↗
PubMed1999

Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells.

1. Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). 2. BK and Lys-BK caused an initial transient peak of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. 3. Kinin B2 antagonists Hoe 140 (10 nM) and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (1 microM) had high affinity in antagonizing BK-induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. 4. Pretreatment of TECs with pertussis toxin (100 ng ml(-1)) or cholera toxin (10 microg ml(-1)) for 24 h did not affect the BK-induced IP accumulation and [Ca2+]i changes in TECs. 5. Removal of Ca2+ by the addition of EGTA or application of Ca2+-channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK-induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK-induced responses. 6. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+-free buffer and subsequently induced Ca2+ influx when Ca2+ was re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca2+]i, but had slight effect on BK-induced Ca2+ influx. 7. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor-gated Ca2+-channels and phospholipase C in TECs, respectively. 8. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C-mediated IP accumulation and Ca2+ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK-induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.

Animals↗
PubMed1998

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells.

1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, betaI, betaII, gamma, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, betaI, betaII, gamma, delta, epsilon and theta from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 microM PMA for either 30 min or 6 h did not significantly change the KD, and Bmax receptor for BK binding (control: KD=1.7+/-0.3 nM; Bmax=50.5+/-4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC-alpha, betaI, betaII, delta, epsilon, gamma, and theta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Animals↗
PubMed1997

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells.

Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPgammaS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPgammaS > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, alpha,beta-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPgammaS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPgammaS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.

Adenosine Triphosphate↗
PubMed1997

Biodegradation of benzene, toluene, and other aromatic compounds by Pseudmonas sp. D8.

Pseudomonas sp. D8 strain, which has the potential to utilize toluene as a sole carbon source, was isolated. At a concentration of 100 mg/l, this strain was found to efficiently degrade toluene and benzene (both individually and in mixture) in culture medium at 30 degrees C and pH7. Following a two-hour lag phase, complete biodegradation of 100 mg/l toluene or benzene occurred within 6 to 8 hours. The addition of nitrate, phosphate, or sulfate at various concentrations were found to have significant influence on both toluene and benzene degradation. In addition, results show that the D8 strain has the ability to degrade monochlorophenols, nitrophenols, and phenol, but not aliphatic compounds. Inoculation of groundwater samples containing 100 mg/l toluene or benzene with Pseudmonas sp. D8 resulted in rapid degradation within 24-33 hours.

Benzene↗
PubMed1996

Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells.

1. A direct [3H]-bradykinin ([3H]-BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial cells (TECs). Based on receptor binding assay, TECs have specific, saturable, high-affinity binding sites for [3H]-BK. 2. The specific [3H]-BK binding was time- and temperature-dependent. Equilibrium of association of [3H]-BK with the BK receptors was attained within 30 min at room temperature and 1 h at 4 degrees C, respectively. 3. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 1.5 +/- 0.2 nM and a maximum receptor density (Bmax) of 53.2 +/- 5.2 fmol mg-1 protein. The Hill coefficient for [3H]-BK binding was 1.00 +/- 0.02. The association (K1) and dissociation (K-1) rate constants were (7.6 +/- 1.1) x 10(6) M-1 min-1 and (9.2 +/- 1.5) x 10 M-3 min-1, respectively. KD, calculated from the ratio of K-1 and K1, was 1.2 +/- 0.3 nM, a value close to that calculated from Scatchard plots of binding isotherms. 4. Neither a B1 receptor selective agonist (des-Arg9-BK, 0.1 nM - 10 microM) nor antagonist ([Leu8, des-Arg9]-BK, 0.1 nM - 10 microM) significantly inhibited [3H]-BK binding to TECs, which excludes the presence of B1 receptors in canine TECs. 5. The specific binding of [3H]-BK to canine TECs was inhibited by the B2 receptor selective antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (Hoe 140, 0.1 nM-10 microM) and [D-Arg0, Hyp3, Thi5.8, D-Phe7]-BK, 0.1 nM - 10 microM) and agonists (BK and kallidin, 0.1 nM-10 microM) with a best fit by a one-binding site model. The order of potency for the inhibition of [3H]-BK binding was kallidin = BK = Hoe 140 > [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK. 6. BK and kallidin significantly induced concentration-dependent accumulation of IPs with a half-maximal response (EC50) at 17.6 +/- 3.5 and 26.6 +/- 5.3 nM, respectively, while the B1-selective agonist, des-Arg9-BK did not stimulate IPs accumulation and the B1-selective antagonist [Leu8, des-Arg9]-BK did not inhibit BK-induced IPs accumulation. Two B2-selective antagonists, Hoe 140 and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK, inhibited BK-stimulated IPs accumulation with apparent pKB values of 8.8 +/- 0.3 and 7.0 +/- 0.3, respectively. 7. It is concluded that the pharmacological characteristics of the BK receptors in canine cultured TECs are primarily of the B2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.

Animals↗