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B Wang

Publications and source records attributed to B Wang.

At least 361 records · Page 20Linked to original sources
PubMed1999

Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.

14-3-3 Proteins↗
PubMed1999

Immunoelectron microscopic study of glutamate inputs from the retrosplenial granular cortex to identified thalamocortical projection neurons in the anterior thalamus of the rat.

We have carried out an ultrastructural study to determine the characteristics and distribution of glutamate-containing constituents of the anterodorsal (AD) and anteroventral (AV) thalamic nuclei in adult rats. We used a polyclonal antibody to glutamate and a postembedding immunogold detection method in animals in which the neurons of AD/AV projecting to the cortex had been retrogradely labelled and the terminals of corticothalamic afferents anterogradely labelled by injection of cholera toxin-horseradish peroxidase (HRP) into the retrosplenial granular cortex. The heaviest immunogold labelling was over axon terminals 0.42 to 2.2 microm in diameter containing round synaptic vesicles and establishing Gray type 1 (asymmetric) synaptic contact (type 1 terminals) on HRP-labelled or non-labelled dendrites. Mean gold particle densities over such terminals were 3-4 times higher than the densities over the dendrites to which they were presynaptic and 5-6 times higher than over terminals establishing Gray type 2 (symmetric) synaptic contacts (type 2 terminals). Gold particle densities over neuronal cell bodies and dendrites and over a subpopulation of myelinated axons were intermediate between the densities over type 1 and type 2 terminals. In adjacent serial sections immunoreacted for gamma aminobutyric acid, type 2 terminals were heavily immunolabelled whereas type 1 terminals and other profiles with moderate gold particle densities after glutamate immunoreaction displayed very low labelling. A subpopulation of small type 1 axon terminals (up to 1 microm diameter) contained HRP reaction product identifying them as cortical in origin; they contacted small dendritic profiles (most <1 microm diameter) many of which also contained HRP reaction product. We conclude that terminals of the corticothalamic projection from retrosplenial granular cortex to AD/AV are glutamatergic and innervate predominantly distal dendrites of thalamocortical projection neurons.

Animals↗
PubMed1999

Energetic Iron(VI) chemistry: the super-iron battery

Higher capacity batteries based on an unusual stabilized iron(VI) chemistry are presented. The storage capacities of alkaline and metal hydride batteries are largely cathode limited, and both use a potassium hydroxide electrolyte. The new batteries are compatible with the alkaline and metal hydride battery anodes but have higher cathode capacity and are based on available, benign materials. Iron(VI/III) cathodes can use low-solubility K(2)FeO(4) and BaFeO(4) salts with respective capacities of 406 and 313 milliampere-hours per gram. Super-iron batteries have a 50 percent energy advantage compared to conventional alkaline batteries. A cell with an iron(VI) cathode and a metal hydride anode is significantly (75 percent) rechargeable.

Journal Article↗
PubMed1999

Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression.

Transcriptional activation of Hedgehog (Hh) target genes requires Cubitus interruptus, a 155 kDa cytoplasmic zinc finger protein (Ci155), which in the absence of Hh signaling is processed to form a nuclear repressor (Ci75). We show that Hh signaling reduces phosphorylation of Ci155, and this reduction in turn appears to decrease processing. Blocking processing with proteasome inhibitors or altered Ci proteins, however, is insufficient for activation of Hh targets. We find that Hh signaling increases the rate of Ci155 nuclear import, resulting in significant nuclear accumulation. Even in the absence of signaling, nuclear accumulation of Ci155 suffices for significant induction of Hh targets, and active nuclear export of Ci155 is an essential mechanism for maintenance of the unstimulated state.

Animals↗
PubMed1999

Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent.

Peptide-based immunotherapy is one strategy by which to selectively suppress the T cell-mediated destruction of beta cells and treat insulin-dependent diabetes mellitus (IDDM). Here, we investigated whether a panel of T cell epitopes derived from the beta cell autoantigen glutamic acid decarboxylase 65 (GAD65) differ in their capacity to induce Th2 cell function in nonobese diabetic (NOD) mice and in turn prevent overt IDDM at different preclinical stages of disease development. The panel consists of GAD65-specific peptides spanning aa 217-236 (p217), 247-265 (p247), 290-309 (p290), and 524-543 (p524). Our studies revealed that all of the peptides effectively prevented insulitis and diabetes when administered to NOD mice before the onset of insulitis. In contrast, only a mixture of p217 and p290 prevented progression of insulitis and overt IDDM in NOD mice exhibiting extensive beta cell autoimmunity. Immunization with the GAD65-specific peptides did not block IDDM development in NOD mice deficient in IL-4 expression. These findings demonstrate that GAD65-specific peptide immunotherapy effectively suppresses progression to overt IDDM, requires the production of IL-4, and is dependent on the epitope targeted and the extent of preexisting beta cell autoimmunity in the recipient.

Animals↗
PubMed1999

Bladder and urethral pressures evoked by microstimulation of the sacral spinal cord in cats.

Experiments were conducted to measure the bladder and urethral pressures evoked by intraspinal microstimulation of the sacral segments (S1-S2) in neurologically intact, chloralose anesthetized adult male cats. The bladder pressure was measured with a superpubic catheter and the urethral pressure was measured simultaneously at the level of the urethral sphincter and at the level of the penis using a two-element micromanometer. Intraspinal stimuli (typically 1 s, 20 Hz, 100 microA, 100 microseconds) were applied with activated iridium microwire electrodes in ipsilateral segments and intersegmental boundaries with a 250 micrometer mediolateral resolution and a 200 micrometer dorsoventral resolution. Increases in bladder pressures were generated by microstimulation in the intermediolateral region, in the lateral and ventrolateral ventral horn, and around the central canal. Simultaneous increases in urethral pressure were evoked by microstimulation in the ventrolateral ventral horn, but not at the other locations. Small reductions in urethral pressure (<10 cm H(2)O) were evoked at locations in the intermediate laminae and around the central canal. The magnitude of these pressure reductions was weakly dependent on the stimulus parameters. Stimulation around the central canal produced bladder contractions with either no change or a reduction in urethral pressure and voiding of small amounts of fluid. These results demonstrate that regions are present in the spinal intact anesthetized cat where microstimulation generates selective contraction of the bladder without increases in urethral pressure and that regions are present where microstimulation generates small reductions in urethral pressure.

Animals↗
PubMed1999

Transcriptional regulation of a receptor protein tyrosine phosphatase gene hPTP-J by PKC-mediated signaling pathways in Jurkat and Molt-4 T lymphoma cells.

The recently cloned type II receptor protein tyrosine phosphatase (RPTP) gene hPTP-J is a new member of the MAM (meprin, A5, PTPmicro) domain subfamily. We previously reported that hPTP-J mRNA was detected significantly in Jurkat T lymphoma cells and its expression was completely down-regulated by phorbol myristate acetate (PMA). In this study, we investigated what signaling pathways/molecules are involved in the transcriptional regulation of hPTP-J expression in Jurkat and Molt-4 T cell lines. The hPTP-J transcription was transiently up-regulated 20 min after the addition of PMA (20 ng/ml) to the Jurkat culture, followed by the complete down-regulation in 8 h after PMA addition. The transient up-regulation and the complete down-regulation induced by PMA was blocked by a PKC-specific inhibitor, GF109203X, suggesting that the regulatory effect of PMA on the hPTP-J transcription depends on protein kinase C activation. hPTP-J transcription was down-regulated not only by PMA but also by several signaling modulators including 1-oleoyl-2-acetylglycerol, forskolin, orthovanadate, manumycin and okadaic acid. Therefore, several signaling molecules such as protein tyrosine phosphatases, PP2A/CaMKIV and Ras are required for hPTP-J transcription in Jurkat and Molt-4 cells.

Cyclic AMP↗
PubMed1999

Substituted coumarins as esterase-sensitive prodrug moieties with improved release rates.

Our laboratory has recently reported a coumarin-based prodrug system for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. However, the release from this prodrug system was undesirably slow for some drug moieties. In this report, we describe the synthesis and evaluation of several substituted coumarin-based prodrugs of model amines with significantly increased release rates.

Animals↗
PubMed1999

Mice lacking NK cells develop an efficient Th1 response and control cutaneous Leishmania major infection.

NK cells are believed to play a critical role in the development of immunity against Leishmania major. We recently found that transplantation of wild-type bone marrow cells into neonatal tgepsilon 26 mice, which are deficient in T and NK cells, resulted in normal T cell development, but no or poor NK cell development. Using this novel model we analyzed the role of NK cells in the development of Th1 response and control of cutaneous L. major infection. Mice selectively lacking NK cells (NK-T+) developed an efficient Th1-like response, produced significant amounts of IL-12 and IFN-gamma, and controlled cutaneous L. major infection. Administration of neutralizing IL-12 Abs to NK-T+ mice during L. major infection resulted in exacerbation of the disease. These results demonstrate that NK cells are not critical for development of protective immunity against L. major. Furthermore, they indicate that IL-12 can induce development of Th1 response independent of NK cells in NK-T+ mice following L.major infection.

Animals↗
PubMed1999

Sol-gel thin-film immobilized soybean peroxidase biosensor for the amperometric determination of hydrogen peroxide in acid medium.

An acid-stable soybean-peroxidase biosensor was developed by immobilizing the enzyme in a sol-gel thin film. Methylene blue was used as a mediator because of its high electron-transfer efficiency. The sol-gel thin film and enzyme membrane were characterized by FT-IR, and the effects of pH, operating potential, and temperature were explored for optimum analytical performance by using the amperometric method. The H2O2 sensor exhibited a fast response (5 s), high sensitivity (27.5 microA/mM), as well as good thermostability and long-term stability. In addition, the performance of the biosensor was investigated using flow-injection analysis (FIA).

Enzymes, Immobilized↗
PubMed1999

Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo.

Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro.

Apoptosis↗
PubMed1999

Expression of CD1d2 on thymocytes is not sufficient for the development of NK T cells in CD1d1-deficient mice.

CD1 is an MHC class I-like molecule that has been conserved throughout mammalian evolution. Unlike MHC class I molecules, CD1 can present unique nonprotein antigens to T cells. The murine CD1 locus contains two highly homologous genes, CD1d1 and CD1d2. CD1d1 is essential for the development of a major subset of NK T cells that promptly secrete IL-4 following activation. However, the function of CD1d2 has not yet been demonstrated. In the present study, we examined the expression of CD1d2 in CD1d1-deficient (CD1d1 degrees) mice with the anti-CD1 Ab 3H3. Unlike CD1d1, which is expressed by all lymphocytes, CD1d2 can be detected only on the surface of thymocytes. To determine whether CD1d2 can select a unique subset of NK T cells, we compared the remnant population of NK T cells in CD1d1 degrees and CD1d1, CD1d2-double deficient (CD1d1 degrees CD1d2 degrees) mice. No significant difference in the number of NK T cells and cytokine secretion capacity can be detected between CD1d1 degrees and CD1d1 degrees CD1d2 degrees mice, indicating that CD1d2 cannot substitute for CD1d1 in NK T cell development. The inability of CD1d2 to select NK T cells is not due to the structural constraints of CD1d2 since CD1d2-transfected cells can be recognized by both NK T cell hybridomas and freshly isolated NK T cells. Given the structural similarities, it is possible that the low levels of surface expression and limited tissue distribution of CD1d2 may prevent it from functioning in the selection and expansion of NK T cells.

Animals↗
PubMed1999

Multiple developmental roles for CRAC, a cytosolic regulator of adenylyl cyclase.

Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DeltaPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DeltaPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-DeltaPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway.

Adenylyl Cyclases↗
PubMed1999

The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association.

Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition.

Adenosine Triphosphate↗
PubMed1999

Effective treatment of autoimmune disease and progressive renal disease by mixed bone-marrow transplantation that establishes a stable mixed chimerism in BXSB recipient mice.

Male BXSB mice spontaneously develop autoimmune disease with features similar to systemic lupus erythematosus. To determine whether this autoimmune disease can be treated as well as prevented by bone-marrow transplantation (BMT) and, at the same time, whether the immunity functions of lethally irradiated recipients can be reconstituted fully, male BXSB mice were engrafted with mixed T cell-depleted marrow (TCDM) both from fully allogeneic autoimmune-resistant BALB/c mice and from syngeneic autoimmune-prone BXSB mice, after the onset of autoimmune disease in the recipient mice. BMT with mixed TCDM from both resistant and susceptible strains of mice (mixed BMT) established stable mixed chimerism, prolonged the median life span, and arrested development of glomerulonephritis in BXSB mice. BMT with mixed TCDM also reduced the formation of anti-DNA antibodies that are observed typically in male mice of this strain. Furthermore, mixed BMT reconstituted the primary antibody production in BXSB recipients impressively. These findings indicate that transplantation of allogeneic autoimmune-resistant TCDM plus syngeneic autoimmune-prone TCDM into lethally irradiated BXSB mice can be used to treat autoimmune and renal disease in this strain of mice. In addition, this dual bone-marrow transplantation reconstitutes the immunity functions and avoids the immunodeficiencies that occur regularly in fully allogeneic chimeras after total body irradiation. This report describes an effective treatment of progressive renal disease and autoimmunity by establishing a stable mixed chimerism of TCDM transplantation from allogeneic autoimmune-resistant BALB/c mice plus syngeneic autoimmune-prone BXSB mice into BXSB mice.

Animals↗
PubMed1999

Familial aggregation of blood pressure in a rural Chinese community.

This study investigated blood pressure in 1,183 Chinese nuclear families (mother, father, and first two children) via a cross-sectional 1994-1997 survey. The mother's, the father's, and the first sibling's blood pressures were each significantly and independently related to the second sibling's blood pressure after adjustment for sex, age, height, weight, education, smoking, and alcohol consumption. The association was consistent across the four age strata (6-10, 11-14, 15-19, and > or = 20 years). The rate of high systolic blood pressure in the second sibling was lowest (2.3%) when both parents and the first sibling were in the low blood pressure tertile (low-low group) and highest (26.0%) when these family members were in the high blood pressure tertile (high-high group). The rate was intermediate if only the parents (10.7%, high-low group) or the first sibling (8.4%, low-high group) was in the high blood pressure tertile. As compared with the low-low group, the odds ratios for the high-high, high-low, and low-high groups were 14.3 (95% confidence interval 4.3-48.2), 4.3 (95% confidence interval 1.2-15.6), and 3.9 (95% confidence interval 1.1-14.4), respectively. A similar pattern was found for diastolic blood pressure. The data indicate a strong familial aggregation of blood pressure in this population and show that such a familial influence on blood pressure can be detected from early childhood onward.

Adolescent↗
PubMed1999

Human single-chain Fv immunoconjugates targeted to a melanoma-associated chondroitin sulfate proteoglycan mediate specific lysis of human melanoma cells by natural killer cells and complement.

Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.

Animals↗