Biomedical subjects

Z Wang

Publications and source records attributed to Z Wang.

At least 631 records · Page 35Linked to original sources

PubMed2000

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

Animals↗

PubMed2000

Tolerance to morphine at the mu-opioid receptor differentially induced by cAMP-dependent protein kinase activation and morphine.

Human neuroblastoma SH-SY5Y cells express endogenous mu-opioid receptor and develop cellular tolerance to morphine after prolonged (>/=4 h) treatment with morphine. Treatment with forskolin (25 microM, 12 h), an adenylyl cyclase activator, also desensitized mu-opioid receptor response to morphine (10 microM) by 38% (P<0. 001), which was reversed by the cyclic AMP (cAMP) dependent kinase inhibitor N-(2-aminoethyl)-5-isoquinolinesulfonamide (H8) (100 microM). Treatment with both morphine and forskolin appeared to cause an additive effect in desensitizing mu-opioid receptor. In mu-opioid receptor stably transfected human embryonic kidney 293 (HEK-mu) cells, morphine treatment produced cAMP upregulation, yet failed to induce mu-opioid receptor tolerance. However, treatment with forskolin (25 microM) or 8-bromo-cAMP (1mM) led to profound mu-opioid receptor tolerance, which was reversed by H8. These results demonstrate that cAMP-dependent kinase activation causes mu-opioid receptor tolerance. However, morphine-induced mu-opioid receptor tolerance in SH-SY5Y cells is not mediated by cAMP-dependent kinase activation. In addition, our results indicate that cAMP-upregulation does not necessarily lead to mu-opioid receptor tolerance.

Colforsin↗

PubMed2000

Metastable Equilibrium Solubility Behavior of Carbonated Apatites in the Presence of Solution Fluoride.

The aims of the present investigation were to assess the applicability of the metastable equilibrium solubility (MES) concept for the carbonated apatites (CAPs) over a range of pH and a wide range of solution fluoride concentrations and to examine the hypothesis that, in the presence of solution fluoride, a surface complex with the stoichiometry of fluorapatite (FAP) governs the MES behavior. Two CAP samples were prepared by precipitation from reaction media containing calcium nitrate (Ca(NO(3))(2).4H(2)O) and sodium phosphate (NaH(2)PO(4).H(2)O) at two different levels of sodium bicarbonate. The MES distributions of the two CAP preparations were determined by equilibrating approximately 10 mg of CAP powder in 2 L of 0.1 M acetate buffers (ionic strength=0.50 M) at pH 4.5 and 5.5 and at various levels of calcium, phosphate, and fluoride. The fluoride concentrations ranged from 0.03 to 12 ppm. From the compositions of the equilibrating buffer solutions, ion activity products based upon the stoichiometries of hydroxyapatite (HAP) and FAP were calculated in an attempt to determine the correct function governing the dissolution of the CAP preparations. The results of this study demonstrated that both CAP preparations exhibit the MES distribution phenomenon in solution media of varying pH and fluoride concentrations. Furthermore, the experimental MES data obtained with both CAP preparations at the lower pH (4.5) and at higher solution fluoride levels (>/=0.1 ppm) were essentially superimposable when plotted against the ion activity product based upon the stoichiometry of FAP, suggesting that in the presence of solution fluoride the MES governing surface complex may be an entity possessing a stoichiometry approximated by that of FAP. When the HAP stoichiometry was assumed to represent the surface complex, good superposition of the data was not possible. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Differential regulation and function of Fas expression on glial cells.

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.

Animals↗

PubMed2000

Regioselective synthesis of beta-N1- and beta-N3-alloxazine nucleosides

[structures: see text] A regio- and stereoselective glycosylation of ribose tetraester with persilylated alloxazine to give either beta-N1 or beta-N3 nucleosides is described. The N3 product is potentially of interest as a fluorescent nucleoside and is predicted to have the hydrogen-bonding characteristics of thymidine.

Journal Article↗

PubMed2000

The specific binding of small molecule isoprenoids to rhoGDP dissociation inhibitor (rhoGDI).

The activities of small G-proteins are in part regulated by their interactions with GDI proteins. This binding is thought to be dependent on the C-terminal isoprenoid modification (geranylgeranyl or farnesyl) of these proteins. G-proteins are generally isoprenylated/methylated at their C-terminal cysteine residues. A quantitative fluorescence assay is reported here to evaluate the specificity of binding of rhoGDI. A rhodamine-labeled geranylgeranylated/methylated cysteine derivative is used to measure its binding to rhoGDI. Saturable binding in the low micromolar range is found with various geranylgeranylated/farnesylated analogues. Interestingly, the carboxymethylated derivatives bound significantly better than their free acid counterparts, suggesting that the state of methylation of the analogues is important for binding. The binding is also selective with respect to isoprenoid. Analogues containing hydrophobic modifications other than geranylgeranyl or farnesyl do not bind with significant affinities. These data demonstrate a substantial degree of specificity in the binding of isoprenoids to a protein important in signal transduction.

Anisotropy↗

PubMed2000

Gene expression, localization, and pharmacological characterization of endothelin receptors in diabetic rat bladder dome.

As there are significant amounts of functional endothelin receptors in the mammalian urinary tract, we examined the effect of experimental diabetes on the expression of endothelin receptors and their mRNAs in the rat bladder dome. The density of endothelin receptors in the rat bladder dome was higher (8 and 16 weeks following the onset of diabetes) than in age-matched controls. Insulin treatment, started 8 weeks after the induction of diabetes, partially reversed the endothelin receptor alterations. The pharmacological profile of the endothelin receptors in the bladder dome was similar in all groups and was consistent with the predominance of the endothelin ET(A) receptor subtype (ET(A):ET(B)=approximately 4:1). Autoradiographic studies demonstrated that the endothelin receptors were located in all tissue components of the bladder, including epithelial and muscular layers. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) data indicated that diabetes increased the expression level of gene transcripts for both endothelin receptor subtypes and that insulin treatment reversed the mRNA upregulation.

Animals↗

PubMed2000

Identification of an erythroid-enriched endoribonuclease activity involved in specific mRNA cleavage.

Stability of the human alpha-globin mRNA is conferred by a ribonucleoprotein complex termed the alpha-complex, which acts by impeding deadenylation. Using our recently devised in vitro decay assay, we demonstrate that the alpha-complex also functions by protecting the 3'-untranslated region (3'-UTR) from an erythroid-enriched, sequence-specific endoribonuclease activity. The cleavage site was mapped to a region protected by the alpha-complex and is regulated by the presence of the alpha-complex. Similar endoribonuclease cleavage products were also detected in erythroid cells expressing an exogenous alpha-globin gene. Nucleotide substitution of the target sequence renders the RNA refractory to the endoribonuclease activity. Insertion of the target sequence onto a heterologous RNA confers sequence-specific cleavage on the chimeric RNA, demonstrating the sequence specificity of this activity. We conclude that the alpha-complex stabilizes the alpha-globin mRNA in erythroid cells by a multifaceted approach, one aspect of which is to protect the 3'-UTR from specific endoribonuclease cleavage.

3' Untranslated Regions↗

PubMed2000

Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity.

Degenerate primers were designed for PCR amplification of unknown mouse immunoglobulin (Ig) light (L) and heavy (H) chain variable (V) genes. Each subgroup of mouse Ig gene sequences [Kabat, E.A., Wu, T. T., Perry, H.H., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Interest, 5th edn. US Department of Health and Human Services, Public Health Service, NIH.] was analyzed, and highly degenerate primers in the framework one (FR1) region were designed. A single highly degenerate FR1 primer sufficed for the amplification of light chains; for heavy chains, a series of FR1 primers was used. At the same time, we assessed the effect of 3' to 5' exonuclease activity of DNA polymerase on the utilization of these degenerate primers. Using Taq polymerase, which lacks 3' to 5' exonuclease activity, we successfully amplified the Ig VL and VH genes expressed in more than a hundred monoclonal hybridoma cell lines reactive against a phosphonamidate hapten. Sequence analysis of the cloned VL and VH genes, 52 of each, showed that they are derived from multiple germline families (10 of the 17 VL families and 9 of the 14 VH families) as recently defined [Martinez, C., Lefranc, M., 1998. The mouse (Mus musculus) immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp. Clin. Immunogenet. 15, 184.]. The universality of our primers was also demonstrated by successful amplification of other mouse hybridoma cell lines that are specific to different antigens.

Animals↗

PubMed2000

Isolation and characterization of monoclonal antibodies directed against subunits of human RNA polymerases I, II, and III.

Human nuclei contain three different RNA polymerases: polymerases I, II, and III. Each polymerase is a multi-subunit enzyme with 12-17 subunits. The localization of these subunits is limited by the paucity of antibodies suitable for immunofluorescence. We now describe eight different monoclonal antibodies that react specifically with RPB6 (also known as RPA20, RPB14.4, or RPC20), RPB8 (RPA18, RPB17, or RPC18), RPC32, or RPC39 and which are suitable for such studies. Each antibody detects one specific band in immunoblots of nuclear extracts; each also immunoprecipitates large complexes containing many other subunits. When used for immunofluorescence, antibodies against the subunits shared by all three polymerases (i.e., RPB6, RPB8) gave a few bright foci in nucleoli and nucleoplasm, as well as many fainter nucleoplasmic foci; all the bright foci were generally distinct from speckles containing Sm antigen. Antibodies against the two subunits found only in polymerase III (i.e., RPC32, RPC39) gave a few bright and many faint nucleoplasmic foci, but no nucleolar foci. Growth in two transcriptional inhibitors-5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole and actinomycin D-led to the redistribution of each subunit in a characteristic manner.

Animals↗

PubMed2000

Isolation of genes from the rhabdoid tumor deletion region in chromosome band 22q11.2.

We employed exon trapping and large-scale genomic sequence analysis of two bacterial artificial chromosome clones to isolate genes from the region between the IGLC and BCR in chromosome 22q11.2. At the time these studies were initiated, one previously identified gene, GNAZ, was known to map to this region. Two genes, RTDR1 and RAB36, were cloned from this portion of 22q11, which is heterozygously or homozygously deleted in pediatric rhabdoid tumors of the brain, kidney and soft tissues. RTDR1 is a novel gene with a slight homology to a yeast vacuolar protein. RAB36 is a member of the Rab family of proteins. A series of primary rhabdoid tumors with chromosome 22q11 deletions were screened for mutations in the coding sequences of RTDR1, GNAZ and RAB36, but did not demonstrate any disease-specific alterations. Recently, INI1, which maps to the distal portion of the deletion region in 22q11, was identified as the candidate rhabdoid tumor suppressor gene. Further studies of RTDR1 and RAB36 are required to determine whether their absence contributes to the progression of rhabdoid tumors. Alternatively, these genes may be candidates for other diseases that map to human chromosome 22.

Amino Acid Sequence↗

PubMed2000

A new zinc ribbon gene (ZNRD1) is cloned from the human MHC class I region.

Eleven unique cDNA fragments were identified from YAC B30H3, which spans 330 kb in the human major histocompatibility complex class I region. One fragment (CAT80) was mapped 80 kb telomeric to the HLA-A locus. Using this cDNA fragment as probe, Northern analysis reveals a ubiquitously expressed transcript of about 850 nt in all 16 tissues tested. Based on the cDNA fragment sequence, a full-length cDNA of 858 bp that contains an open reading frame of 378 bp was cloned. Within the putative polypeptide of 126 amino acids, two zinc-ribbon domains were identified: Cx2Cx15Cx2C at the N-terminal and Cx2Cx24Cx2C at the C-terminal. The C-terminal domain is well conserved throughout evolution, including archaea, yeast, Drosophila, nematodes, amphibians, and mammals. The conserved amino acid sequence, CxRCx6Yx3QxRSADEx2TxFxCx2C, is highly homologous to the yeast RNA polymerase A subunit 9 and transcription-associated proteins. Alignment with genomic DNA demonstrates that this gene spans 3.6 kb and consists of four exons and three introns. Cross-species Northern analysis reveals a mouse homolog of a similar size and with an expression profile similar to those of the human gene. We have named this gene ZNRD1 for zinc ribbon domain-containing 1 protein.

Amino Acid Sequence↗

PubMed2000

Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry.

Studying the permeability of compounds across a Caco-2 cell monolayer is an established in vitro model to screen for oral absorption and to evaluate the mechanism of transport. This assay can also be used to evaluate compounds as potential P-glycoprotein substrates and/or inhibitors. The traditional methods of sample analysis (high-performance liquid chromatography (HPLC) with a UV or fluorescence detector) limit the throughput and sensitivity of this assay. Data are presented here describing the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the analysis of samples derived from the Caco-2 cell studies. During the analysis an automatic switching valve was used to divert the flow from the HPLC column to waste for the first minute, preventing the early eluting salts from entering and contaminating the LC/MS interface. This approach allows the rapid and accurate determination of drug transport across the Caco-2 cell monolayer. The high sensitivity and specificity of LC/MS/MS make this technique an ideal candidate for the low concentration and high throughput routine analysis of Caco-2 cell solutions, especially if multiple compounds are administered and analyzed simultaneously. Thus, the use of LC/MS/MS will increase the value of the Caco-2 cell assay as an in vitro screening tool.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Involvement of hepatocyte epidermal growth factor receptor mediated activation of mitogen-activated protein kinase signaling pathways in response to growth inhibition by a novel K vitamin.

Compound 5 (Cpd 5), a synthetic K vitamin analogue, or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is a potent inhibitor of epidermal growth factor (EGF)-induced rat hepatocyte DNA synthesis and induces EGF receptor (EGFR) tyrosine phosphorylation. To understand the cellular responses to Cpd 5, its effects on the EGF signal transduction pathway were examined and compared to those of the stimulant, EGF. Cpd 5 induced a cellular response program that included the induction of EGFR tyrosine phosphorylation and the activation of the mitogen-activated protein kinase (MAPK) cascade. EGFR tyrosine phosphorylation was induced by Cpd 5 in a time- and dose-dependent manner. Coimmunoprecipitation studies demonstrated that both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR was associated with increased amounts of adapter proteins Shc and Grb2, and the Ras GTP-GDP exchange protein Sos, indicating the formation of functional EGFR complexes. Although EGFR phosphorylation was induced both by the stimulant EGF and the inhibitor Cpd 5, the timing and intensity of activation by EGF and Cpd 5 were different. EGF activated EGFR transiently, whereas Cpd 5 induced an intense and sustained activation. Cpd 5-altered cells had a decreased ability to dephosphorylate tyrosine phosphorylated EGFR, providing evidence for an inhibition of tyrosine phosphatase activity. Both EGF and Cpd 5 caused an induction of phospho-extracellular response kinase (ERK), which was also more sustained with Cpd 5. Moreover, whereas Cpd 5 induced a striking translocation of phosphorylated ERK from cytosol to the nucleus, no significant nuclear translocation occurred after stimulation with EGF. The data suggest that this novel compound causes growth inhibition through antagonism of EGFR phosphatases and consequent induction of EGFR and ERK phosphorylation. This is supported by experiments with PD 153035 and PD 098059, antagonists of phosphorylation of EGFR and MAP kinase kinase (MEK), respectively, which both antagonized Cpd 5-induced phosphorylation and the inhibition of DNA synthesis. These results imply a mechanism of cell growth inhibition associated with intense and prolonged protein tyrosine phosphorylation. Protein tyrosine phosphatases may thus be a novel target for drugs designed to inhibit cell growth.

Animals↗

PubMed2000

A method for fast multislice T1 measurement: feasibility studies on phantoms, young children, and children with Canavan's disease.

We have developed a multislice protocol for quantitative T1 measurements in which the processing time and the acquisition time are under 2 minutes each for a complete brain study of 15 slices. An echoplanar, inversion-recovery image sequence is designed to collect data suitable for analysis using a linear regression algorithm. The precision is approximately twice the noise to signal ratio of the images. The accuracy of the protocol is better than 1% for T1 in the range 0-2 seconds and deviates slightly for longer T1 values. The protocol is insensitive to B1 field values. If needed, the data can be postprocessed using a slow, nonlinear algorithm to give an accuracy of less than 1% and a precision of approximately the noise to signal ratio throughout a range of T1 values from 0 to 4 seconds.

Algorithms↗

PubMed2000

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection.

Sixteen peptides (trimers to hexamers) were designed for use as a set of pI markers for capillary isoelectric focusing (CIEF). Each peptide contains one tryptophan residue for detection by UV absorption and other amino acid residues having ionic side chains, which are responsible for focusing to its pI. The pIs of these peptides were determined by slab-gel IEF using commercial carrier ampholytes. The focused peptides in the gel were detected by absorption measurement at 280 nm using a scanning densitometer and the pH gradient was determined by measuring the pH of the gel using an oxidized metal membrane electrode. The pI values of the peptides ranged from 3.38 to 10.17. The obtained values agreed well with the predicted ones, which were calculated based on amino acid compositions, with root mean square differences of 0.15 pH unit. The peptides were detected at 280 nm as very sharp peaks when separated by CIEF. The pI values of some standard proteins were redetermined by CIEF by using this set of peptide pI markers and the values agreed closely with those reported previously. The sharp focusing, stability, high purity and high solubility of these synthetic pI markers should facilitate the profiling of a pH gradient in a capillary and the determination of the pI values of proteins.

Hydrogen-Ion Concentration↗

PubMed2000

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis.

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis (CE) were exemplified with p-allylcalix[4]arene (pACX4) which was immobilized to the fused silica surface using gamma-methacryloxypropyl-trimethoxysilane (gamma-MAPS) as linking agent. Successful gamma-ACX4 coating was suggested by the greatly decreased electroosmotic flow (EOF), due to the introduction of phenolic hydroxyl groups on the inner surface of the capillaries. A slight slope of EOF versus pH at pH <8 would help make the separation reproducible. The coated columns also featured a low ultraviolet (UV) absorption background and long lifetime (> 6 months at 4<pH<9). Moreover, their special selectivity on phenolic compounds indicated a certain extent of selective interactions between solutes and the calixarene coating, which effected separations partially based on an electrochromatographic mechanism.

Calixarenes↗

PubMed2000

Prevalence and phylogenetic characterisation of TT-virus in the blood donor population of Auckland, New Zealand.

TT-virus (TTV, patient initials: T.T.), a novel DNA virus, was first isolated in Japan in 1997 from serum of a patient with post-transfusion hepatitis of unknown aetiology. To date, the contribution of TTV to liver disease remains doubtful. The potential for transmission via blood and blood products makes it essential to establish the prevalence of TTV viraemia in the blood donor population. 413 blood donor serum samples were chosen randomly, the DNA was extracted and TTV-specific DNA amplified by nested polymerase chain reaction (PCR). TTV infection was present in 13 out of 413 (3.15%) blood donors in the Auckland region of New Zealand using a set of primers targeting open reading frame (ORF) 1. These 13 amplification products (264 bp) were sequenced and TTV genotypes determined. Alignment with published TTV sequences showed that seven (53.8%) of the thirteen positive serum samples belonged to genotype 1, five (38.5%) belonged to genotype 2 and one (7.7%) could not be classified as either genotype 1 or 2. One hundred twenty-seven blood donor serum samples were retested with a second set of primers targeting the 5' region of the TTV genome in a single round PCR. Forty-three samples were positive for TTV DNA with these primers resulting in a prevalence of 37%. The data demonstrate that TTV is present among New Zealand blood donors and support the need for further investigation into the natural history of TTV infection.

Blood Donors↗