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Biomedical subjects

W Wu

Publications and source records attributed to W Wu.

At least 163 records · Page 9Linked to original sources
PubMed1999

Genome screen for platelet monoamine oxidase (MAO) activity.

To identify loci involved in the control of platelet monoamine oxidase B (MAO-B) activity, a genomewide linkage screen was performed using 291 markers in 148 nuclear families containing a total of 1,008 nonindependent sib-pairs. Participants were genotyped and their platelet MAO-B activity levels were measured as part of the Collaborative Study on the Genetics of Alcoholism (COGA). Sib-pair analysis using Haseman-Elston regression was carried out with two programs. Two-point analysis on all pairs with SIBPAL indicated three markers with p-values below 0.01: D6S1018 (p = 0.0004), D2S1328 (p = 0.008), and D2S408 (p = 0.003). MAPMAKER/SIBS multipoint analyses using independent pairs(N = 409) gave maximal lod scores of 2. 0 on chromosome 6 and 1.1 and 1.4 for the two regions on chromosome 2. These results are consistent with linkage, but do not provide definitive evidence. We are currently creating a denser map in these regions and have begun genotyping a second sample in COGA.

Blood Platelets↗
PubMed1999

N-glycosylation contributes to the intracellular stability of prothrombin precursors in the endoplasmic reticulum.

Rat prothrombin (rFII) and human prothrombin (hFII) are processed differently during their biosynthesis in a manner dependent upon gamma-glutamyl carboxylation, a vitamin K-dependent posttranslational modification of prothrombin precursors. The role of N-glycosylation in the cellular processing of prothrombin was examined in hepatoma (H-35 and HepG2) and transformed kidney (HEK293) cell lines. Aglyco-rFII obtained by tunicamycin treatment was degraded in warfarin-treated H-35 cells, but not in vitamin K-treated cells. Fully glycosylated rFII is also selectively retained and degraded in warfarin-treated H-35 cells. When rFII and hFII were transiently expressed in tunicamycin-treated HEK293 cells, rFII but not hFII was degraded to generate a 48-KD species. This degradation was independent of gamma-carboxylation, indicating that the sensitivity of aglyco-rFII and aglyco-hFII toward this specific proteolysis differs in HEK293 cells. By expressing chimeric rFII/hFII constructs in tunicamycin-treated HEK293 cells, it was shown that the kringle 2 structure of prothrombin was responsible for this difference. The 48-KD species was not observed in tunicamycin-treated H-35 cells, suggesting that this specific proteolytic processing of aglyco-rFII is also cell-type specific. Expression of rFII in other tunicamycin-treated nonhepatic cells suggests that this cell-specific difference in processing might be determined by a difference between hepatic and nonhepatic cells. The site-directed mutagenesis utilized in these studies also establishes that the N-glycosylation sites of rFII are at residues Asn77, 101, 378, and 518.

Animals↗
PubMed1999

Parathyroid hormone-related protein is an independent prognostic factor for renal cell carcinoma.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has been shown to be the principal cause of humoral hypercalcemia associated with renal cell carcinoma (RCC). Recent studies have demonstrated that the amino-terminal region of PTHrP has growth factor-like activities, suggesting it may play a role in the development of RCC. In this study, expression of the carboxy-terminal region of PTHrP was assessed immunohistochemically and its significance in predicting the prognosis of RCC was studied. METHODS: Forty radical nephrectomy specimens were immunostained with a murine monoclonal antibody (9H7) against the carboxy-terminal region (amino acids 109-141) of PTHrP using the streptavidin-peroxidase enzyme conjugate method. Staining intensity was evaluated semiquantitatively and compared with clinicopathologic features of the corresponding RCC. RESULTS: Immunoreactivity to 9H7 was observed to be localized to the cytosol of tumor cells at various staining intensities. There were 30 cases (75.0%) with strong staining and 10 cases (25.0%) in which staining was weak or nonexistent. Staining intensity showed no significant correlation with gender, tumor greatest dimension, stage, or grade. Tumors of the clear cell type expressed PTHrP to a significantly greater extent than tumors of the granular cell type. Tumor recurrence was significantly greater in the weakly stained or unstained group compared with the strongly stained group (P = 0.035). Multivariate analysis indicated that PTHrP expression and tumor stage were equally significant prognostic indicators in RCCs measuring <10 cm in greatest dimension. CONCLUSIONS: Evident PTHrP(109-141) expression is present in the majority of RCCs. The results of the current study indicate PTHrP(109-141) may be a possible marker of cellular differentiation and may be useful for predicting recurrence free survival in RCC patients after radical nephrectomy.

Adult↗
PubMed1999

Eosinophil peroxidase nitrates protein tyrosyl residues. Implications for oxidative damage by nitrating intermediates in eosinophilic inflammatory disorders.

Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase have primarily focused on its unusual substrate preference for bromide (Br(-)) and the pseudohalide thiocyanate (SCN(-)) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO(2)(-)), a major end-product of nitric oxide ((.)NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically relevant concentrations of NO(2)(-). Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO(2)(-) with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and NO(2)(-). EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN(-) concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o'-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory conditions characterized by recruitment and activation of eosinophils.

Animals↗
PubMed1999

Reversible exposure of human platelet fibrinogen receptors by antiplatelet tetraspanin monoclonal antibodies via induction of a conformational change in membrane glycoprotein IIb/IIIa complex.

Antihuman platelet tetraspanin (CD9 antigen) monoclonal antibodies, HI117 and SJ9A4, can induce human platelet aggregation and secretion. As platelet aggregation is mediated by fibrinogen binding to its receptors exposed on platelet glycoprotein IIb/IIIa complex, we, therefore, investigated the induction of platelet fibrinogen receptors by HI117 and SJ9A4. It was found that HI117 and SJ9A4 induced specific fibrinogen binding to human platelets, suggesting that the two monoclonal antibodies evoked obvious exposure of fibrinogen receptors on human platelets. But in the absence of fibrinogen, the monoclonal antibody-exposed fibrinogen receptors gradually lost their capacity to bind fibrinogen and closed. Our results also showed that HI117 and SJ9A4, when activating platelets, caused a conformational change in glycoprotein IIb/IIIa complex, which must contribute to the exposure of functional fibrinogen receptors on this integrin. The effect of HI117 and SJ9A4 on glycoprotein IIb/IIIa complex seems, however, to be indirect, because the HI1117 and SJ9A4-induced fibrinogen binding was reduced by pretreatment of platelets with sphingosine, aspirin, apyrase, and/or PGI2. Taken together, we conclude that the antihuman platelet tetraspanin monoclonal antibodies, HI117 and SJ9A4, reversibly expose platelet fibrinogen receptors via inducing a conformational change in glycoprotein IIb/IlIa complex. Three signaling pathways, that is, thromboxane, secreted ADP, and cAMP pathways may be involved in this process, while protein kinase C activation seems to be the final common step of the three pathways.

Antibodies, Monoclonal↗
PubMed1999

Additive effect of NOS inhibitor and neurotrophic factors on the survival of injured Clarke's neurons.

The present study examined the effect of treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) together with peripheral nerve (PN) graft or brain-derived neurotrophic factor (BDNF) on the survival of CN neurons at the L1 level of the spinal cord following hemisection at T11. In control animals 41% of CN neurons survived 15 days after the hemisection, and 48% of these expressed NOS. Treatment with either PN graft implantation or continuous infusion of BDNF increased the survival rate of CN neurons to 70%; 70% of these expressed NOS. Combined L-NAME and PN graft or L-NAME and BDNF improved the rescue rate up to 94%, but only approximately 33% expressed NOS. Our results suggest that the expression of NOS might adversely influence the neuroprotective function of neurotrophic factors on injured CN neurons in the spinal cord.

Analysis of Variance↗
PubMed1999

Antibodies against human IFN-alpha and -beta recognized the immunosuppressive domain of HIV-1 gp41 and inhibit gp41-binding to the putative cellular receptor protein p45.

Sequence-comparison indicates existing sequence-similarity between receptor-binding regions of human type 1 IFNs (IFN-alpha, -beta and -omega) and HIV-1 gp41. Previous findings had suggested that the increased levels of antibodies against human IFN-alpha and -beta in HIV-1-infected individuals are associated with a common epitope on gp41, IFN-alpha and -beta. To clarify the relationship between human type I interferon and HIV-1 gp41 and the protective mechanism of an IFN-alpha-vaccine, we prepared antisera against human IFN-alpha, -beta and HIV-1 gp41, and examined crossreaction of these antisera and their inhibition of gp41 binding to its binding protein p45. Mouse antisera against IFN-alpha and -beta could recognize HIV-1 recombinant soluble (aa539-684) and gp41 immunosuppressive peptide (ISP, aa583-599), while normal mouse sera (pre-immune sera) did not. Mouse antisera to rsgp41 crossreacted with IFN-alpha and -beta. Besides, mouse antisera to IFN-alpha and beta, like mouse anti-rsgp41 antiserum, could inhibit gp41-binding to its putative cellular receptor protein p45, while normal mouse serum did not. These results indicate that antibodies crossreacting with gp41 ISP, IFN-alpha and -beta, could be induced by this common immunological epitope in vivo.

Amino Acid Sequence↗
PubMed1999

Directional guidance of neuronal migration in the olfactory system by the protein Slit.

Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration have remained unclear. Here we report that the secreted protein Slit repels neuronal precursors migrating from the anterior subventricular zone in the telencephalon to the olfactory bulb. Our results provide a direct demonstration of a molecular cue whose concentration gradient guides the direction of migrating neurons. They also support a common guidance mechanism for axon projection and neuronal migration and suggest that Slit may provide a molecular tool with potential therapeutic applications in controlling and directing cell migration.

Animals↗
PubMed1999

Genetic variability at the amyloid-beta precursor protein locus may contribute to the risk of late-onset Alzheimer's disease.

In a series of sibpairs with late onset Alzheimer's disease, we have examined the segregation of the loci involved in the early onset, autosomal dominant form of the disorder by using flanking microsatellite repeat markers: thus we have used APP-PCR3 and D21S210 to examine the segregation of the amyloid-beta precursor protein (APP) gene, the markers DI 4S77 and D14S284 to examine the segregation of the presenilin 1 (PSI) gene and the markers D1S227, D1S249 and D1S419 to examine the segregation of presenilin 2 (PS2). We carried out our analyses on the whole dataset of 291 affected sibpairs, and on subsets comprising those sibpairs in which neither had an apolipoprotein E4 allele (65 affected sibpairs) and those in which both had an apolipoprotein E4 allele (165 affected sibpairs). We used the programs SPLINK to generate allele frequencies and MAPMAKER/SIBS to analyze our results. We examined the segregation of the markers D19S908 and D19S918 that are close to the apolipoprotein E (ApoE) gene as a positive control to assess whether the methods we are employing have the capability to identify known loci. The sibpair approach to the identification of genetic risk loci is relatively insensitive as indicated by the failure of the ApoE locus to reach statistical significance (P = 0.06). Nevertheless, these data suggest that neither the PS1 nor the PS2 gene is a major locus for late-onset AD, but that the APP gene cannot be ruled out as a risk locus in those sibships without an E4 allele (P = 0.014). The possibility that APP is indeed a locus for late onset disease will need confirmation in other series of familial cases.

Age of Onset↗
PubMed1999

The C domain of HIV-1 gp41 binds the putative cellular receptor protein P62.

OBJECTIVE: To characterize the binding of gp41 with the putative receptor protein P62. DESIGN: HIV-1 gp41 binds several cellular proteins by two binding sites, one of which has been shown to bind to a putative receptor protein P45 (45 kDa). Based on this, an attempt was made to determine the relationship between the two binding sites and P62 (62 kDa). METHODS: Using surface plasmon resonance (SPR) measurement, the interaction was measured between recombinant soluble gp41 (rsgp41, Env aa539-684) and protein P62. Inhibition of this interaction was attempted by the use of synthetic peptides (P1, aa583-599; P2, aa646-674) corresponding to the two binding sites in gp41. In addition, the direct binding of P62 to peptide P2 was examined in an enzyme-linked immunosorbent assay. RESULTS: Using SPR measurement, the interaction between P62 and rsgp41 was confirmed, and the interaction was found to be inhibited by only the synthetic peptide P2 sequence that corresponds to the C domain of gp41; neither P1 nor a control peptide inhibited the interaction. Moreover, like rsgp41, P2 was able to bind P62 whereas P1 and another recombinant gp41 (aa567-648 that does not include the C domain) were not. CONCLUSIONS: P62 bound rsgp41 and the synthetic peptide P2. This interaction could be inhibited only by P2. These results indicate that the C domain of HIV-1 gp41 binds P62.

Adaptor Proteins, Signal Transducing↗
PubMed1999

No metal cofactor in orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) is an important enzyme that catalyzes the final step of de novo pyrimidine nucleotide biosynthesis. The mechanism of this unique enzyme and whether metal ions play any role in catalysis have been topics of intense research interest. In this report, the role of Zn in ODCase was reexamined. Atomic absorption (AA) and X-ray absorption (XAS) spectroscopic studies did not detect zinc in active enzyme samples at high concentration. The XAS results also indicated the absence of other transition metal ions in ODCase.

Catalysis↗
PubMed1999

Expression of DMBT1, a candidate tumor suppressor gene, is frequently lost in lung cancer.

DMBT1 is a candidate tumor suppressor gene located at 10q25.3-26.1. Homozygous deletion of the gene was found in a subset of medulloblastoma and glioblastoma multiforme; lack of expression was noted in the majority of these tumors. In adult tissues, DMBT1 is highly expressed only in lung and small intestine tissues, indicating its important role in these organs. By analyzing lung cancer cell lines and primary lung tumors using reverse transcription-PCR, we found that 100% (20 of 20) of small cell lung cancer (SCLC) cell lines and 43% (6 of 14) of non-small cell lung cancer (NSCLC) cell lines lacked DMBT1 expression. Furthermore, 45% (9 of 20) of the primary NSCLCs exhibited a markedly low level of gene expression compared with corresponding normal lung tissues, indicating that lack of gene expression also occurs in primary lung cancers. To determine the potential mechanisms for lack of DMBT1 expression in lung cancer, we analyzed tumor cell lines for potential intragenic homozygous deletions of the gene and found such homozygous deletions in 10% (4 of 40) of SCLC cell lines but in none of 14 NSCLC cell lines. Moreover, the loss of expression could not be rescued by treatment with a demethylation agent (5-azacytidine) in two NSCLC cell lines lacking DMBT1 expression, suggesting that de novo methylation of the promoter region of the gene is unlikely to play a role in inactivation of the gene. We then sequenced the whole coding region of DMBT1 in 8 NSCLC cell lines that expressed DMBT1 and 20 primary NSCLCs. A potential point mutation at codon 52 was detected in a NSCLC cell line and resulted in an amino acid change from serine to tryptophan. Three common polymorphisms were also detected in tissues analyzed. Our data demonstrate that DMBT1 expression is frequently lost in lung cancer due to gene deletion and to other not yet identified mechanisms, suggesting that inactivation of DMBT1 may play an important role in lung tumorigenesis.

Agglutinins↗
PubMed1999

3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for eosinophil-dependent tissue injury in vivo.

Detection of specific reaction products is a powerful approach for dissecting out pathways that mediate oxidative damage in vivo. Eosinophil peroxidase (EPO), an abundant protein secreted from activated eosinophils, has been implicated in promoting oxidative tissue injury in conditions such as asthma, allergic inflammatory disorders, cancer, and helminthic infections. This unique heme protein amplifies the oxidizing potential of H2O2 by utilizing plasma levels of Br- as a cosubstrate to form potent brominating agents. Brominated products might thus serve as powerful tools for identifying sites of eosinophil-mediated tissue injury in vivo; however, structural identification and characterization of specific brominated products formed during EPO-catalyzed oxidation have not yet been reported. Here we explore the role of EPO and myeloperoxidase (MPO), a related leukocyte protein, in promoting protein oxidative damage through bromination and demonstrate that protein tyrosine residues serve as endogenous traps of reactive brominating species forming stable ring-brominated adducts. Exposure of the amino acid L-tyrosine to EPO, H2O2, and physiological concentrations of halides (100 mM Cl-, </=100 microM Br-) produced two new major products with distinct retention times on reverse phase HPLC. The products were identified as 3-bromotyrosine and 3, 5-dibromotyrosine by electrospray ionization mass spectrometry and multinuclear (1H and 15N) NMR spectroscopy. Formation of the ring-brominated forms of the amino acid occurred readily at neutral pH with the enzymatic system and a variety of reactive brominating species, including HOBr/OBr-, N-bromoamines, and N,N-dibromoamines. Addition of primary amines (e.g., Nalpha-acetyllysine and taurine) to L-tyrosine exposed to either HOBr/OBr- or the EPO-H2O2-Br- system enhanced phenolic ring bromination, suggesting N-bromoamines are preferred brominating intermediates in these reactions. Reduction of N-bromoamines (e.g., Nalpha-acetyl,Nepsilon-bromolysine) by L-tyrosine was shown to result in the loss of reactive halogen with a near stoichiometric increase in the level of tyrosine ring bromination (i.e., carbon-bromine bonds). Although both EPO and MPO could use Br- to halogenate protein tyrosine residues in vitro, only EPO effectively brominated the aromatic amino acid at physiological levels of halides and H2O2. Collectively, these results suggest that 3-bromotyrosine and 3,5-dibromotyrosine are attractive candidates for serving as molecular markers for oxidative damage of proteins by reactive brominating species in vivo. They also suggest that in biological mixtures where amine groups are abundant, the trapping of EPO-generated HOBr/OBr- as N-bromoamines will serve to effectively "funnel" reactive brominating equivalents to stable ring-brominated forms of tyrosine.

Animals↗
PubMed1999

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons.

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.

Animals↗
PubMed1999

No association between the alpha-2 macroglobulin I1000V polymorphism and Alzheimer's disease.

Recent reports have suggested that variability in the alpha2-macroglobulin gene is a genetic risk factor for Alzheimer's disease. Here we have both tested a common polymorphism in the gene (I1000V) for association with the disease in a four-site case control study design, and tested the locus for linkage in a large series of sibpairs afflicted with late onset disease. Our results fail to show an association between this polymorphism and disease.

Age of Onset↗
PubMed1999

Determination of interfacial tension from crystallization and dissolution data: a comparison with other methods.

Methods for the determination of interfacial tension between a solid and a liquid are reviewed including solubility/particle size, crystallization and dissolution kinetics. The use of solubility as a function of particle size, originally put forward by Ostwald and later corrected by Freundlich, may be unjustified for determining interfacial tension at solid-liquid interfaces. The interfacial tension values between solutions and sparingly soluble minerals such as hydroxyapatite, fluorapatite, brushite, octacalcium phosphate, calcium oxalate monohydrate, barium sulfate, calcium sulfate, calcite, and divalent metal fluorides are discussed. A comparison of these results is made with contact angle or wetting measurements. The interfacial tension values obtained from constant composition reaction kinetics are of the same order of magnitude as those determined using a contact angle method involving thin layer wicking techniques.

Chemistry Techniques, Analytical↗
PubMed1999

Peripheral nerve graft and neurotrophic factors enhance neuronal survival and expression of nitric oxide synthase in Clarke's nucleus after hemisection of the spinal cord in adult rat.

The present study examined the effects of peripheral nerve (PN) graft and neurotrophic factors on the expression of nitric oxide synthase (NOS) and the survival of Clarke's nucleus (CN) neurons at the first lumbar spinal segment (L1) 15 days after hemisection of the spinal cord at T11. Normal intact CN neurons did not express NOS. Forty-one percent of the ipsilateral CN neurons survived after hemisection at T11, and 48% of the surviving neurons expressed NOS. Transplantation of PN graft at the lesion site promoted the survival of CN neurons to 71% and increased the expression of NOS to 70%. Continuous infusion of brain-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophic-3, but not glial cell-derived neurotrophic factor, at the lesion site enhanced the survival of CN neurons to about 65%. Among the surviving neurons about 70% were NOS-positive. These results indicated that transplantation of autologous PN graft or continuous infusion of neurotrophic factors could enhance the survival of axotomized CN neurons. In addition, the survival-promoting function of the neurotrophic agents was coincided with the upregulation of the expression of NOS. However, whether the upregulation of NOS expression in injured CN neurons is related to the rescue function or is a side effect of the neurotrophic factors is not clear and needed further investigation.

Age Factors↗