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Biomedical subjects

J Wu

Publications and source records attributed to J Wu.

At least 883 records · Page 49Linked to original sources
PubMed1997

Compromised allograft rejection response in transgenic mice expressing antisense sequences to retinoic acid receptor beta2.

Transgenic mice expressing antisense sequences to retinoic acid receptor beta2 (AS-RARbeta2 mice) were generated. The transgene was expressed in T cells, macrophages, and non-T spleen cells. These AS-RARbeta2 mice had four- to sevenfold higher endogenous RARbeta2 messages in their macrophages, comparing with their nontransgenic littermates, but the RARbeta protein level was significantly lower in these cells. This suggests that the antisense message interferes with RARbeta2 translation, and there is a feedback control of the RARbeta2 mRNA level in the macrophages. The endogenous RARbeta2 message was not detectable in T and B cells of either the transgenic or nontransgenic mice. These mice appeared to be healthy upon visual inspection. When transplanted with allogenic cardiac grafts heterotopically, the mice showed significantly compromised rejection response. These mice had a decrease of the macrophage population in spleen, using the nontransgenic littermates as references. The generation of alloantigen-specific T cell cytotoxicity was decreased in these AS-RARbeta2 mice. These results suggest that macrophage development and T cell function are affected by antisense expression of RARbeta2, and that anomaly in these cells might be contributing factors to the compromised immune response observed in the AS-RARbeta2 mice.

Animals↗
PubMed1997

Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking.

N and C-terminal halves of lactose permease, each with a single-Cys residue, were co-expressed, and crosslinking was studied. Iodine or N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), crosslinks Asn245 Cys (helix VII) and Ile52 --> Cys or Ser53 --> Cys (helix II). N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A) crosslinks the 245/53 Cys pair weakly, but does not crosslink 245/52, and 1,6-bis-maleimidohexane (BMH; flexible 16 A) crosslinks both pairs less effectively than o-PDM. Thus, 245 is almost equidistant from 52 and 53 by up to about 6 A. BMH or p-PDM crosslinks Gln242 --> Cys and Ser53 --> Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 A. Ligand binding increases crosslinking of 245/53 with p-PDM or BMH, has little effect with o-PDM and decreases iodine crosslinking. Similar effects are observed with 245/52. Ligand increases 242/53 crosslinking with p-PDM or BMH, but no crosslinking is observed with o-PDM. Therefore, ligand induces a translational or scissors-like displacement of the helices by 3-4 A. Crosslinking 245/53 inhibits transport indicating that conformational flexibility is important for function.

Amino Acid Sequence↗
PubMed1997

Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli.

By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles. In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment. In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect to the N-terminal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while ligand binding causes much greater increases in reactivity. The findings indicate that 4B1 binding induces a structural change in the permease that is much less widespread than that induced by ligand binding.

Antibodies, Monoclonal↗
PubMed1997

The Vav binding site (Y315) in ZAP-70 is critical for antigen receptor-mediated signal transduction.

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor-mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav- ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor-mediated signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Run-down of the GABAA response under experimental ischaemia in acutely dissociated CA1 pyramidal neurones of the rat.

1. The effect of experimental ischaemia on the response to gamma-aminobutyric acid (GABA) was assessed in acutely dissociated CA1 pyramidal neurones of rats, using the patch-clamp technique. 2. Rapid application of 3 x 10(-5) M GABA induced a bicuculline-sensitive inward Cl- current (IGABA) at a holding potential (Vh) of -44 mV. The peak amplitude of IGABA showed a time-dependent decrease (run-down) when it was recorded with the conventional whole-cell mode without internal ATP. The run-down was not observed when the intracellular ATP concentration ([ATP]i) was maintained by the nystatin-perforated recording with an intracellular Na+ concentration ([Na+]i) of 0 mM. 3. When [Na+]i was increased to more than 30 mM, the IGABA run-down was observed even with the nystatin-perforated recording. 4. The IGABA run-down observed at 60 mM [Na+]i with the nystatin method was further enhanced under experimental ischaemia without changes in the reversal potential of IGABA. The enhanced run-down was suppressed by application of the Na+,K(+)-ATPase inhibitors, ouabain and SPAI-1. 5. IGABA run-down during ischaemia was also accompanied by an outward holding current and a concomitant increase in intracellular free Ca2+ concentration ([Ca2+]i) in 48.5% of the neurones. The outward current was a Ca(2+)-activated K+ current, which was blocked by 3 x 10(-7) M charybdotoxin. 6. In the inside-out mode of the single-channel analysis, GABA activated three subconductance states with conductances of 33.4, 22.7 and 15.2 pS. Reduction of ATP concentration from 2 to 0 mM on the intracellular side suppressed the channel activities, while an increase in Ca2+ concentration from 0.7 x 10(-9) to 1.1 x 10(-6) M had no effect. 7. These results suggest that ischaemia induces the run-down of the postsynaptic GABA response at the GABAA receptor level, and that this run-down is triggered by a decrease in [ATP]i.

Adenosine Triphosphate↗
PubMed1997

Involvement of cGMP in nociceptive processing by and sensitization of spinothalamic neurons in primates.

Central sensitization of spinothalamic tract (STT) neurons in anesthetized monkeys after intradermal injection of capsaicin depends in part on disinhibition. Protein kinase C is suggested to participate in this process. The present study shows that the nitric oxide-cGMP (NO-cGMP) signal transduction system also contributes to sensitization of wide dynamic range (WDR) STT neurons located in the deep dorsal horn. The NO-cGMP system was activated by microdialysis administration into the dorsal horn of 8-bromo-cGMP, an analog of cGMP. Sensitization of STT cells by 8-bromo-cGMP increased the responses of deep WDR STT cells to both weak and strong mechanical stimulation of the skin and simultaneously attenuated the inhibition of the same neurons produced by stimulation in the periaqueductal gray (PAG). In contrast, WDR STT cells in the superficial dorsal horn and high-threshold (HT) STT cells in superficial or deep layers showed reduced responses to mechanical stimulation of the skin after infusion of 8-bromo-cGMP, and PAG inhibition of these neurons was unaffected. Sensitization of STT cells and the attenuation of PAG inhibition induced by intradermal injection of capsaicin were prevented by preteatment of the dorsal horn with a guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The results support the hypothesis that activation of the NO-cGMP signal transduction system contributes to the sensitization of WDR STT neurons in the deep dorsal horn and helps explain why intradermal capsaicin injections often fail to sensitize superficial and HT STT cells. The results also support the idea that sensitization of STT cells is produced in part by disinhibition.

Animals↗
PubMed1997

Ordered YAC clone contigs assigned to rice chromosomes 3 and 11.

Yeast artificial chromosome (YAC) clones were arranged on the positions of restriction fragment length polymorphism (RFLP) and sequence-tagged site (STS) markers already mapped on the high-resolution genetic maps of rice chromosomes 3 and 11. From a total of 416 and 242 YAC clones selected by colony/Southern hybridization and polymerase chain reaction (PCR) analysis, 238 and 135 YAC clones were located on chromosomes 3 and 11, respectively. For chromosomes 3 and 11, 24 YAC contigs and islands with total coverage of about 46% and 12 contigs and islands with coverage of about 40%, respectively, were assigned. Although many DNA fragments of multiple copy marker sequences could not be mapped to their original locations on the genetic map by Southern hybridization because of a lack of RFLP, the physical mapping of YAC clones could often assign specific locations of such multiple copy sequences on the genome. The information provided here on contig formation and similar sequence distribution revealed by ordering YAC clones will help to unravel the genome organization of rice as well as being useful in isolation of genes by map-based cloning.

Blotting, Southern↗
PubMed1997

Carbocyclic oxetanocins lacking the C-3' methylene.

Using the observation that the side effects of aristeromycin (carbocyclic adenosine) were reduced by removing the methylene at the center in aristeromycin where phosphorylation occurs, derivatives of carbocyclic oxetanocin A (4a), oxetanocin G (4b), and 2-aminooxetanocin A (16) lacking the 3'-methylene have been prepared in racemic form. The only viruses for which an appreciable inhibitory effect of the compounds (minimum inhibitory concentration ranging from 1 to 40 microg/mL) was noted were herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). However, when directly compared for their antiviral potency against HSV-1 with their parents oxetanocin A and oxetanocin G, compounds 4a and 4b proved clearly less active.

Adenine↗
PubMed1997

Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling.

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

3T3 Cells↗
PubMed1997

Hyaluronan fragments induce nitric-oxide synthase in murine macrophages through a nuclear factor kappaB-dependent mechanism.

Activated macrophages play a critical role in controlling chronic tissue inflammation through the release of a variety of mediators including cytokines, chemokines, growth factors, active lipids, reactive oxygen, and nitrogen species. The mechanisms that regulate macrophage activation in chronic inflammation are poorly understood. A hallmark of chronic inflammation is the turnover of extracellular matrix components, and recent work has suggested that interactions with the extracellular matrix can exert important influences on macrophage effector functions. We have examined the effect of low molecular weight fragments of the extracellular matrix glycosaminoglycan hyaluronan (HA) on the induction of nitric-oxide synthase (iNOS) in macrophages. We found that HA fragments induce iNOS mRNA, protein and activity alone, and markedly synergize with interferon-gamma to induce iNOS gene expression in murine macrophages. In addition, we found that resident tissue alveolar macrophages respond minimally, but inflammatory alveolar macrophages exhibit a marked induction in iNOS expression in response to HA fragments. Finally, we demonstrate that the mechanism of HA fragment-induced expression of iNOS requires activation of the transcriptional regulator nuclear factor kappaB. These data support the hypothesis that HA may be an important regulator of macrophage activation at sites of chronic tissue inflammation.

Animals↗
PubMed1997

Tetrahydroberberine inhibits acetylcholine-induced K+ current in acutely dissociated rat hippocampal CA1 pyramidal neurons.

The effects of a novel chemical type of dopamine receptor antagonist, the tetrahydroprotoberberine analogs (THPBs), on acetylcholine (ACh)-induced current were studied in freshly dissociated pyramidal neurons from rat hippocampal CA1 area using the nystatin perforated patch-clamp recording technique. Under voltage clamp conditions, the ACh-induced outward current (IACh) is sensitive to the muscarinic receptor antagonist, atropine and the K+ channel blocker, TEA. The reversal potential of IACh (-84.1 +/- 0.8 mV) is close to the K+ equilibrium potential, indicating that the IACh is mediated by a muscarinic receptor, and is carried mainly by K+. Tetrahydroberberine (THB) markedly reduced the IACh while its chemical analogs, l-stepholidine (l-SPD) or l-tetrahydropalmatine (l-THP), had little effect on the IACh. The half-maximal inhibitory concentration (IC50) of THB was 1.3 x 10(-5) M for a 10(-5) M ACh-induced IACh. THB suppressed the maximum of the ACh concentration-response curve without shifting the Hill coefficient, indicating a non-competitive inhibition. It is concluded that THB non-competitively inhibits the ACh-induced K+ current in a concentration-dependent manner, and that this inhibitory effect provides further evidence that THB plays its pharmacological roles in the central nervous system by effects other than through blockade of dopamine receptors.

Acetylcholine↗
PubMed1997

A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry.

A highly activated ester containing a fixed positive charge, S-pentafluorophenyl [tris(2,4,6-trimethoxyphenyl)phosphonium]acetate bromide (TMPP-AcSC6F5 bromide), has been synthesized as a reagent for N-terminal modification of peptides. Stable in aqueous acetonitrile solution during extended storage, TMPP-AcSC6F5 bromide reacts with unprotected peptides through p-(dimethylamino)pyridine (DMAP)-promoted amidation in aqueous acetonitrile (15 min, ambient temperature) to form N-TMPP-Ac derivatives of peptides. These peptide derivatives are readily amenable to analysis by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Greater than 90% conversion has been observed in transforming low-nanomole quantities of analyte using molar ratios of 1:5:10 (peptide/reagent/ DMAP). For reactions at the picomole level a slightly modified stoichiometry, with molar ratios of 1:10:500, is employed. Owing to the high reaction efficiency and the tolerance to moderate excess reagent and base during analysis by FAB- and MALDI-MS, the reaction mixture containing the modified peptides can be analyzed directly in most cases, without sample cleanup. Examples of the preparation and analysis of a variety of N-TMPP-acetyl-peptides (TMPP-Ac-peptides) ranging from hexamers to 15-mers are given. Collisionally activated dissociation tandem mass spectrometry of TMPP-Ac-derivatives showed dominant a-type ions, accompanied by d- and c-type ions in some cases, allowing sequence determination to be made in a straightforward manner.

Amino Acid Sequence↗
PubMed1997

Extracellular particles of polymeric material formed in n-hexadecane fermentation by Pseudomonas aeruginosa.

In n-hexadecane fermentation by Pseudomonas aeruginosa, the production of rhamnolipids (biosurfactants) caused by nitrogen source limitation was observed during the stationary phase. The rhamnolipids caused severe foaming in the process, particularly at higher pH (ca. > 6.8). To reduce the foaming, several runs were made at a lower pH, i.e. 6.5 +/- 0.1. Some, however, behaved anomalously different. The rhamnolipid synthesis was very low. Instead, a large quantity of waxy particles with sizes up to 3-5 mm were formed. The waxy material was practically insoluble in all of the organic solvents tested, suggesting the cross-linked polymeric structure. A white, brittle solid was obtained after the material was thoroughly washed (with 0.05 M sodium bicarbonate, distilled water, and hexane) and vacuum-dried. Characterization of the washed material was made by Fourier-transformed infrared (FTIR) spectroscopy, thin-layer chromatography (TLC), and wet chemical analyses. It was found to contain less than 2.5% (w/w) proteins and less than 9% carbohydrates, of which only one sugar component was observed and tentatively identified as glucose by the TLC analysis. The material was, therefore, predominantly hydrocarbon-based, with oxidized functional groups such as esters, alcohols, and carboxylic acids being identified by FTIR. The lipase-catalyzed esterification of fatty acids and alcohols, within the oil droplets or at the oil/water interface of low local water activity, is postulated to play an important role in the waxy particle formation.

Alkanes↗
PubMed1997

Effect of sertraline on regional metabolic rate in patients with affective disorder.

Seventeen patients with major affective disorder completed a 10-week, placebo-controlled, randomized trial of the serotonin reuptake inhibitor sertraline. Patients underwent positron emission tomography with 18F-deoxyglucose and were assessed with the Hamilton Depression Rating Scale at baseline and 10 weeks after treatment with sertraline or placebo. The middle frontal gyrus, an area previously characterized by decreased metabolic activity in depressive patients, showed relatively increased activity on both sides after sertraline when contrasted with temporal and some occipital areas. Sertraline was associated with a significantly increased relative metabolic rate in right parietal lobe and in left occipital area 19, and a decreased metabolic rate in right occipital area 18. Other areas that differed between controls and a larger cohort of 39 depressive patients--including medial frontal lobe, cingulate gyrus, and thalamus--also showed a normalization of metabolic rate after sertraline.

1-Naphthylamine↗
PubMed1997

Rapamycin inhibits proteasome activator expression and proteasome activity.

Rapamycin (RAPA) is a potent immunosuppressive drug, and certain of its direct or indirect targets might be of vital importance to the regulation of an immune response. In this study, we used differential hybridization to search for human genes whose expression was sensitive to RAPA. Seven RAPA-sensitive genes were found and one of them encoded a protein with high homology to the alpha subunit of a proteasome activator (PA28 alpha). This gene was later found to code for the beta subunit of the proteasome activator (PA28 beta). Activated T and B cells had up-regulated PA28 beta expression at the mRNA level. Such up-regulation could be suppressed by RAPA, FK506, and cyclosporin A. RAPA and FK506 also repressed the up-regulated PA28 alpha messages in phytohemagglutinin (PHA)-stimulated T cells. At the protein level, RAPA inhibited PA28 alpha and PA28 beta in the activated T cells according to immunoblotting and confocal microscopy. Probably as a consequence, there was a fourfold increase of proteasome activities in the peripheral blood mononuclear cell lysate after the PHA activation. RAPA could inhibit the enhanced part of the proteasome activity. Considering the critical role played by the proteasome in degrading regulatory proteins, our data suggest that the proteasome activator is a relevant and important downstream target of rapamycin, and that the immune response could be modulated through the activity of the proteasome.

Cysteine Endopeptidases↗
PubMed1997

Effect of a cola beverage on the bioavailability of itraconazole in the presence of H2 blockers.

Thirty-three healthy individuals participated in an open-label, randomized, three-way crossover study designed to compared the bioavailability of a single 200-mg oral dose of itraconazole when administered alone or after treatment with ranitidine, both with and without coadministration of a cola beverage. Each treatment phase was separated by a 2-week washout period. Participants pretreated with ranitidine were required to have a gastric pH of at least 6.0 before receiving itraconazole. An analysis of the area under the curve (AUC) and peak plasma concentration (Cmax) data indicated that the bioavailability of itraconazole was significantly reduced when the gastric pH was increased by pretreatment with ranitidine but showed that this effect was counteracted by the coadministration of an acidic solution (e.g., a cola beverage) that transiently reduced the gastric pH. These findings suggest that the coadministration of an acidic beverage with itraconazole may be an effective approach in improving the bioavailability of itraconazole in patients who are hypochlorhydric or who are taking gastric acid suppressants.

Adult↗