Biomedical subjects

W Wang

Publications and source records attributed to W Wang.

At least 937 records · Page 52Linked to original sources

PubMed1997

Kinetic mechanism of human inosine 5'-monophosphate dehydrogenase type II: random addition of substrates and ordered release of products.

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD to NADH. This reaction is the rate-limiting step of guanine nucleotide biosynthesis. IMPDH is a target of immunosuppressive, antiviral, anticancer, and antiparasitic chemotherapy. We have determined a minimal kinetic mechanism for human IMPDH type II using NAD analogs, isotope effects, hydride exchange, and presteady state kinetics. The values of kcat for the NAD analogs are similar despite a great variation in the structure and reactivity of the compounds. This observation suggests that a common step is rate-limiting, i.e., either hydrolysis of the E-XMP* intermediate or release of XMP. No Vm isotope effect is observed when 2-2H-IMP is the substrate, which indicates that hydride transfer is fast. This conclusion is confirmed by the observation of a burst of NADH production under presteady state conditions. These observations further suggest that either E-XMP* hydrolysis or XMP release is rate-limiting. V/Km deuterium isotope effects are observed for both substrates (1.9 for IMP and 2.5 for NAD), which indicates that substrate association is random. This result contradicts previous conclusions based on product inhibition studies. No NADH consumption is observed in the presence of XMP and IMPDH, which indicates that the overall reaction is irreversible. NADH consumption is observed in the presence of thio-NAD, IMP, and enzyme. These observations indicate that NADH traps the E-XMP* intermediate and demonstrates that hydride transfer is reversible. At infinite NADH, all of E-XMP* is trapped by NADH, as indicated by the equivalence of the rates of consumption of thio-NAD and NADH. Therefore the release of products is ordered, with NADH release preceding hydrolysis of E-XMP*.

Humans↗

PubMed1997

Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein.

The GM2 activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo function is to act as a substrate specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at neutral pH in vitro. Any other possible in vivo functions would require that some of the newly synthesized activator molecules not be targeted to the lysosome. The lysosomal targeting mechanism for the activator has not been conclusively identified. While earlier reports suggested that it is likely through the mannose-6-phosphate receptor, another more recent report demonstrated that deficient human cells could recapture nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mannose-6-phosphate pathway is likely the major intracellular, biosynthetic route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Additionally, we show that there exists a second lower affinity recapture pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphingolipids in the plasma membrane. Finally, we document that the pool of newly synthesized precursor activator protein contains a majority of molecules with a complex-type oligosaccharide, which cannot contain a functional mannose-6-phosphate targeting signal. These molecules makeup the secreted forms of the protein in normal human fibroblasts.

Amino Acid Sequence↗

PubMed1997

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase.

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.

Adenylosuccinate Synthase↗

PubMed1997

Neuroelectric correlates of response production and inhibition in individuals at risk to develop alcoholism.

P300 recordings were made from males at high risk (HR) for alcoholism and low-risk (LR) controls, participating in a visual go/no go reaction time paradigm. The go (button press) and no go (inhibit response) stimuli were large and small forms of the same letters. The LR group had significantly larger go than no go P300 amplitudes in the central, parietal, and temporal regions; the HR group manifested no response differences in any region. In the LR group compared to the HR group, both go and no go response amplitudes were larger over the entire head; no group differences in latencies were observed in any region. Surface energy magnitudes paralleled P300 amplitudes and were also larger in the LR group during both go and no go trials. Our findings indicate that HR individuals manifest widespread P300 amplitude deficits while performing a simple information-processing paradigm. These deficits, which may reflect genetic influences, preceded the onset of alcoholism and may function as a phenotypic marker for its development.

Adult↗

PubMed1997

Modulation of GABAA receptor function by tyrosine phosphorylation of beta subunits.

Protein tyrosine phosphorylation is a key event in diverse intracellular signaling pathways and has been implicated in modification of neuronal functioning. We investigated the role of tyrosine phosphorylation in regulating type A GABA (GABAA) receptors in cultured CNS neurons. Extracellular application of genistein (50 microM), a membrane-permeable inhibitor of protein tyrosine kinases (PTKs), produced a reversible reduction in the amplitude of GABAA receptor-mediated whole-cell currents, and this effect was not reproduced by daidzein (50 microM), an inactive analog of genistein. In contrast, intracellular application of the PTK pp60(c-src) (30 U/ml) resulted in a progressive increase in current amplitude, and this potentiation was prevented by pretreatment of the neurons with genistein. Immunoprecipitation and immunoblotting of cultured neuronal homogenates indicated that the beta2/beta3 subunit(s) of the GABAA receptor are tyrosine phosphorylated in situ. Moreover, genistein (50 microM) was found to be capable of decreasing GABAA currents in human embryonic kidney 293 cells transiently expressing functional GABAA receptors containing the beta2 subunit. Thus, the present work provides the first evidence that native GABAA receptors are phosphorylated and modulated in situ by endogenous PTKs in cultured CNS neurons and that phosphorylation of the beta subunits may be sufficient to support such a modulation. Given the prominent role of GABAA receptors in mediating many brain functions and dysfunctions, modulation of these receptors by PTKs may be important in a wide range of physiological and pathological processes in the CNS.

Animals↗

PubMed1997

Life on biomembranes viewed with the atomic force microscope.

Since its invention in 1986, the atomic force microscope (AFM) has become one of the most widely used near-field microscopes. Surfaces of hard samples are imaged almost routinely with atomic resolution. Soft biological surfaces, however, are still challenging. In this brief review, the AFM technique is introduced to the experimental biologist. We discuss recent data on imaging molecular structures of biomembranes, and give detailed information on the application of the AFM with three representative examples. One is imaging plasma membrane turnover of transformed renal epithelial cells during migration in vivo, another is visualizing a cloned and isolated potassium channel usually located in kidney, and a third is imaging macromolecular pore complexes of the nuclear envelope of aldosterone-sensitive kidney cells and of Xenopus laevis oocytes. The review ends with the conclusion that nuclear pores can serve as birthday candles on a Guglhupf.

Animals↗

PubMed1997

A naturally processed peptide presented by HLA-A*0201 is expressed at low abundance and recognized by an alloreactive CD8+ cytotoxic T cell with apparent high affinity.

In contrast to T cells that respond to peptides presented by self MHC molecules, alloreactive T cells recognize determinants expressed on nonself MHC molecules. Because current positive selection models suggest that T cell affinity toward a nonself MHC molecule would be lower than that toward a self MHC molecule, we previously proposed that vigorous alloreactive responses would be generated preferentially toward those antigenic peptide complexes presented at the highest density on the cell surface. The high abundance of two class I MHC-associated peptides that have been identified as allo- or xenoantigens is consistent with this hypothesis. We report here the identification of a naturally processed peptide YLDPAQQNL that is presented by HLA-A*0201 and recognized by an alloreactive T cell clone. This peptide appears to originate from an unknown member of the zinc finger proteins. Quantitation by mass spectrometry indicates that this peptide is present on the surface at 85 to 125 copies per cell, comparable with the density of several other epitopes presented by HLA-A*0201 to self MHC-restricted T cells. In addition, based on the affinity of the peptide for HLA-A*0201 and the half-maximal peptide concentration required for T cell sensitization, this alloreactive T cell appears to have an affinity similar to or higher than that of many self MHC-restricted T cells. These data suggest that allogeneic responses can be directed against antigenic determinants of low abundance and that recognition of alloreactive peptides is not limited by a lower affinity of T cells for nonself MHC molecules.

Clone Cells↗

PubMed1997

Characterization of the affinity of the G(M2) activator protein for glycolipids by a fluorescence dequenching assay.

The G(M2) activator protein is a substrate specific cofactor for degradation of G(M2) ganglioside by lysosomal beta-hexosaminidase A. Mutations in the gene encoding the activator result in the AB-variant form of G(M2) gangliosidosis. The activator protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide binding site(s), and an area that interacts with hexosaminidase A. In this report a fluorescence dequenching assay specific for only the hydrophobic binding pocket is evaluated and optimized. It is shown that various glycolipids inhibit the transport between liposomes of a self-quenching fluorescent lipid probe, octadecylrhodamine, by the activator protein. The level of inhibition produced by each glycolipid is then used to characterize the oligosaccharide-binding specificity of the activator. The fluorescence dequenching assay is also used to evaluate the functionality of a truncated form of the activator protein. Our results indicate that this simple assay can be used to determine structure-function relationships within the normal or mutant forms of the activator. The data suggest that the C-terminus of the activator is required to produce a functional hydrophobic binding pocket.

Binding Sites↗

PubMed1997

Expression patterns and transcript processing of ftt-1 and ftt-2, two C. elegans 14-3-3 homologues.

A wide diversity of biological functions have been attributed to the highly conserved and ubiquitous 14-3-3 protein family. Yet how much of this diversity is inherent in the basic structure of 14-3-3 and how much is due to isoform specific functions is not yet fully understood. Here, two Caenorhabditis elegans 14-3-3 isoforms whose protein sequences are 90% similar were found to differ significantly in both their genomic structure and expression patterns. The two genes, ftt-1 (IV) (fourteen-three-three) and ftt-2 (X), differ in both the position and sequence of their introns. Since the various intron/exon boundaries respect neither functional nor structural protein motifs, the introns appear to be relatively recent evolutionary additions. ftt-1(IV) encodes three germline enhanced transcripts, two of which are related through the differential use of alternative poly(A) addition sites. RNA in situ hybridization studies reveal high levels of ftt-1 throughout the gonad with particularly high levels in the distal arm. In contrast, ftt-2 (X) encodes a single transcript which is expressed somatically. In embryos, high levels of ftt-1 transcripts appear to be maternally supplied, whereas ftt-2 is expressed as an early zygotic transcript whose expression pattern later localizes to the posterior region of post-proliferative embryos. These expression pattern differences between ftt-1 and ftt-2 suggest that these two 14-3-3 isoforms perform distinct biological roles within the worm.

14-3-3 Proteins↗

PubMed1997

Subtyping of D20S85 STR alleles by single-strand conformation polymorphism (SSCP) analysis.

During a population study of STR locus D20S85, we discovered two types of sequence variations by direct sequencing of the alleles: two transitions each of G to A and A to G occur in the 5' flanking region in the individuals possessing allele 6 and some of those possessing allele 7 [1]. Using single-strand conformation polymorphism (SSCP) analysis, we were able to distinguish two subtypes of allele 7 from each other. This analysis method enables rapid screening for STR alleles of the same length with different sequences, and should find application to other complex STR loci because of the practical advantage of simplicity in comparison to sequencing.

Forensic Medicine↗

PubMed1997

Interactions between conserved residues in transmembrane helices 1, 2, and 7 of the thyrotropin-releasing hormone receptor.

The roles of conserved residues in transmembrane helices (TMs) of G protein-coupled receptors have not been well established. A computer-generated model of the thyrotropin-releasing hormone receptor (TRH-R) indicated that conserved Asp-71 (TM-2) could interact with conserved asparagines 316 (TM-7) and 43 (TM-1). To test this model, we constructed mutant TRH-Rs containing polar or alanine substitutions of these residues. The maximal activities of N43A and N316A TRH-Rs were diminished, whereas D71A (Perlman, J. H., Nussenzveig, D. R., Osman, R., and Gershengorn, M. C. (1992) J. Biol. Chem. 267, 24413-24417) and N43A/N316A TRH-Rs were inactive. Computer models of D71A and N43A/N316A TRH-Rs show similar changes from native TRH-R in their TM bundle conformations. The inactivity and the similarity of the computer models of D71A and N43A/N316A TRH-Rs are consistent with the idea that Asp-71 bridges Asn-43 and Asn-316 and suggest that activity is critically dependent on these interactions. The conservation of these residues suggests these specific interactions involving TMs 1, 2, and 7 may be structurally important for all members of the rhodopsin/beta-adrenergic receptor subfamily of G protein-coupled receptors.

Amino Acid Sequence↗

PubMed1997

Does elevated nitric oxide production enhance the release of prostacyclin from shear stressed aortic endothelial cells?

Nitric oxide (NO) enhances prostacyclin (PGI2) production in agonist-stimulated endothelial cells, while peroxynitrite formed from NO and superoxide anion has been shown to activate cyclooxygenase. Using cultured bovine aortic endothelial cells (BAEC) exposed to arterial levels of laminar shear stress of 25 dynes/ cm2, we tested the hypothesis that NO mediated the elevated synthesis of PGI2 by shear stressed endothelium. Shear stress caused a large and rapid burst and sustained release of NO and PGI2 with the cumulative production at 1 hr enhanced 9.96-fold (n = 4, p < 0.005) and 9.16-fold (n = 3, p < 0.005), respectively, over stationary control production of 0.0257 nmol-NO/cm2-BAEC and 0.0193 ng-PGI2/cm2-BAEC. The NO synthase inhibitors, N(G)-nitro-L-arginine methyl ester (100 microM, LNAME) and N(G)-nitro-L-arginine (10 microM, LNA), caused 87.5 and 65% reductions (n = 3, p < 0.02) of cumulative NO release at 1 hr, respectively, and 45 and 55% reductions (n = 3, p = 0.025) of PGI2 release, respectively. About half of the elevated production of PGI2 in shear stressed cells was due to NO-dependent signaling, indicating that hemodynamic control of these two dilatory molecules is partially coupled.

Animals↗

PubMed1997

A study of Escherichia coli adenylosuccinate synthetase association states and the interface residues of the homodimer.

The state of aggregation of adenylosuccinate synthetase from Escherichia coli is a point of controversy, with crystal structures indicating a dimer and some solution studies indicating a monomer. Crystal structures implicate Arg143 and Asp231 in stabilizing the dimer, with Arg143 interacting directly with bound IMP of the 2-fold related subunit. Residue Arg143 was changed to Lys and Leu, and residue Asp231 was changed to Ala. Matrix-assisted laser desorption ionization mass spectroscopy and analytical ultracentrifugation of the wild-type and the mutant enzymes indicate a mixture of monomers and dimers, with a majority of the enzyme in the monomeric state. In the presence of active site ligands, the wild-type enzyme exists almost exclusively as a dimer, whereas the mutant enzymes show only slightly decreased dissociation constants for the dimerization. Initial rate kinetic studies of the wild-type and mutant enzymes show similar kcat and Km values for aspartate. However, increases in the Km values of GTP and IMP are observed for the mutant. Changes in dissociation constants for IMP are comparable with changes in Km values. Our results suggest that IMP binding induces enzyme dimerization and that two residues in the interface region, Arg143 and Asp231, play significant roles in IMP and GTP binding.

Adenylosuccinate Synthase↗

PubMed1997

A new class of membrane-bound chemokine with a CX3C motif.

Chemokines direct the trafficking of white blood cells in immune surveillance, playing a key role in inflammatory and infectious diseases such as AIDS. All chemokines studied so far are secreted proteins of relative molecular mass approximately 7K-15K and fall into three families that are defined by a cysteine signature motif: CXC, CC and C (refs 3, 6, 7), where C is a cysteine and X any amino-acid residue. We report here the identification and characterization of a fourth human chemokine type, derived from non-haemopoietic cells and bearing a new CX3C fingerprint. Unlike other chemokine types, the polypeptide chain of the human CX3C chemokine is predicted to be part of a 373-amino-acid protein that carries the chemokine domain on top of an extended mucin-like stalk. This molecule can exist in two forms: either membrane-anchored or as a shed 95K glycoprotein. The soluble CX3C chemokine has potent chemoattractant activity for T cells and monocytes, and the cell-surface-bound protein, which is induced on activated primary endothelial cells, promotes strong adhesion of those leukocytes. The structure, biochemical features, tissue distribution and chromosomal localization of CX3C chemokine all indicate that it represents a unique class of chemokine that may constitute part of the molecular control of leukocyte traffic at the endothelium.

Amino Acid Sequence↗

PubMed1997

Differential regulation of neuronal proteoglycans by activation of excitatory amino acid receptors.

Cell surface proteoglycans (PGs) have been implicated in neuronal growth, migration and differentiation and can play pivotal roles both in cell-cell and cell-substrate interactions during the development of the nervous system. We have previously shown that glutamate activation of excitatory amino acid receptors induces the synthesis and release of PGs with neurite-promoting activity from hippocampal neurones. In this study, we have investigated the activity-dependent regulation of mRNA expression of two PGs in fetal hippocampal neurones using a competitive reverse transcriptase-polymerase chain reaction and correlated this expression with neuronal growth. Both cerebroglycan (CBG), a glycosylphatidylinositol-anchored heparan sulphate PG, and neurocan, a developmentally regulated chondroitin sulphate PG, are expressed in hippocampal neurones. Exposure of hippocampal neurones to 100 microM glutamate for 5 min resulted in an increase in CBG mRNA levels and an increase in axonal and dendritic length. The increase in CBG mRNA levels following glutamate exposure was mediated via both N-methyl-D-aspartate and metabotropic receptor activation.

Animals↗

PubMed1997

Cleavage of CCK 33 by recombinant PC2 in vitro.

The specificity and kinetic properties of CCK 33 cleavage by recombinant prohormone convertase 2 (PC2) was investigated using a combination of Sephadex G-50 chromatography, HLPC and RIA methods. It is shown that CCK 33 can be cleaved effectively by PC2 to form CCK 8, the reaction of which has a K(m) of 104.8 microM. No CCK 22 or other products were detected and the reaction is completely inhibited by the PC2 inhibitor, p-CMS (p-chloromercuriphenylsulfonic acid), suggesting that the cleavage is PC2 specific. The time course of this reaction shows an initial lag phase followed by a rapid increase in velocity consistent with a previously reported spontaneous transformation from a 71 kDa relatively inactive form into a more active form of 62 kDa (1). PC2 did not cleave recombinant pro CCK or CCK 1-21. These results demonstrate that PC2, an enzyme usually associated with dibasic cleavages, can cleave easily at single basic residues.

4-Chloromercuribenzenesulfonate↗

PubMed1997

Brain-derived neurotrophic factor spares choline acetyltransferase mRNA following axotomy of motor neurons in vivo.

Choline acetyltransferase (ChAT) is a functional and specific marker gene for neurons such as primary motor neurons that synthesize and release acetylcholine as a neurotransmitter. In adult mammals, transection of the peripheral nerve results in a loss of immunoreactivity for ChAT in the injured motor neurons without affecting their cell number. Using a quantitative RNase protection assay, we have investigated dynamic changes in ChAT mRNA levels following axotomy of motor neurons in the brainstem of adult rats. One week after transection of the left hypoglossal nerve, levels of ChAT mRNA in the ipsilateral side of the hypoglossal motor nucleus decreased dramatically to around 10% when compared to the uninjured contralateral side. When cut axons were chronically exposed to brain-derived neurotrophic factor (BDNF) for 1 week, ChAT mRNA levels were maintained at 63% of control levels. Thus, BDNF can abrogate the injury-induced loss of ChAT mRNA in mature motor neurons in vivo. In contrast, neither neurotrophin 4/5 nor nerve growth factor could prevent the decrease in message. This effect of BDNF on ChAT mRNA levels following peripheral injury to motor neurons demonstrates the existence of regulatory pathways responsive to neurotrophic factors that can "rescue" or "protect" cholinergic gene expression.

Animals↗

PubMed1997

Osteoclasts are capable of particle phagocytosis and bone resorption.

Osteoclasts are multinucleated cells specialized for the function of lacunar bone resorption. Although they are known to be capable of phagocytosis of inert particles, it is not known whether this abolishes their ability to respond to hormones or to form resorption lacunae. Human and rat osteoclasts were isolated from giant cell tumours of bone and rat long bones, respectively, and cultured on coverslips and cortical bone slices, both in the presence and in the absence of particles of latex (1 micron diameter) and polymethylmethacrylate (PMMA) (< 50 microns). By light microscopy, it was evident that osteoclasts which had phagocytosed both latex and PMMA particles remained responsive to calcitonin. Osteoclast phagocytosis of particles was also evident on scanning electron microscopy, where it could also be seen that these cells were associated with the formation of resorption lacunae. These findings underline the fact that the osteoclast is a true member of the mononuclear phagocyte system and that phagocytosis does not abrogate either its hormonal response to calcitonin or its highly specialized function of bone resorption. That osteoclasts which have phagocytosed biomaterial particles such as PMMA are still able to carry out lacunar bone resorption is of interest in clinical conditions such as aseptic loosening, where a heavy foreign body particle load is often associated with extensive bone resorption.

Animals↗