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Biomedical subjects

R Wang

Publications and source records attributed to R Wang.

At least 271 records · Page 15Linked to original sources
PubMed1999

Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells.

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.

Adolescent↗
PubMed1999

Mechanism of the cleavage specificity of Alzheimer's disease gamma-secretase identified by phenylalanine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein.

Proteolytic processing of the amyloid precursor protein by beta-secretase yields A4CT (C99), which is cleaved further by the as yet unknown gamma-secretase, yielding the beta-amyloid (Abeta) peptide with 40 (Abeta40) or 42 residues (Abeta42). Because the position of gamma-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the Abeta domain with phenylalanine, stably transfected the constructs in COS7 cells, and determined the effect of these mutations on the cleavage specificity of gamma-secretase (Abeta42/Abeta40 ratio). Compared with wild-type A4CT, mutations at Val-44, Ile-47, and Val-50 led to decreased Abeta42/Abeta40 ratios, whereas mutations at Thr-43, Ile-45, Val-46, Leu-49, and Met-51 led to increased Abeta42/Abeta40 ratios. A massive effect was observed for I45F (34-fold increase) making this construct important for the generation of animal models for Alzheimer's disease. Unlike the other mutations, A4CT-V44F was processed mainly to Abeta38, as determined by mass spectrometry. Our data provide a detailed model for the active site of gamma-secretase: gamma-secretase interacts with A4CT by binding to one side of the alpha-helical transmembrane domain of A4CT. Mutations in the transmembrane domain of A4CT interfere with the interaction between gamma-secretase and A4CT and, thus, alter the cleavage specificity of gamma-secretase.

Alzheimer Disease↗
PubMed1999

Implications of presenilin 1 mutations in Alzheimer's disease.

Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are the most common genetic factors underlying the development of early-onset familial Alzheimer's disease (FAD). To investigate the pathogenic mechanism of PS1 mutations linked to FAD, we established inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1(M146L or deltaexon 10) under the control of the Lac repressor. Using this inducible PS1 system, the influence of PS1 mutations on the generation of endogenous murine Abeta species was assessed using a highly sensitive immunoblotting technique. The induction of mutated PS1 resulted in an increase in the extra- and intracellular levels of two distinct Abeta species ending at residue 42, Abeta1-42 and its N-terminally truncated variant(s), Abetax-42. In addition, the intracellular generation of these Abeta42 species was completely blocked by brefeldin A. In contrast, it exhibited differential sensitivities to monensin such that there was an increased accumulation of intracellular Abetax-42 but an inhibition of intracellular Abeta1-42 generation. These data strongly suggest that Abetax-42 is generated in a proximal Golgi compartment, whereas Abeta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage which occurs (i) in the ER or the early Golgi apparatus prior to gamma-secretase cleavage, or (ii) in the distinct sites where Abetax-42 and Abeta1-42 are generated. To date, the site of Abeta42 generation has not been firmly established. Our data provide new information regarding the site of Abeta42 generation mediated by the FAD-linked mutant PS1.

Alzheimer Disease↗
PubMed1999

Pan DR binding sequence provides T-cell help for induction of protective antibodies against Plasmodium yoelii sporozoites.

Pan-DR epitope (PADRE) peptides have demonstrated the capacity to deliver help for antibody responses in vivo. They were also found, fortuitously, to be able to provide significant helper T-cell activity in vivo. This suggested that linear constructs, containing the PADRE epitope, might be as efficient at generating an immune response as large multivalent antigens. Plasmodium falciparum and P. yoelii PADRE constructs were capable of inducing a high titre IgG antibody response that recognized intact sporozoites. We now report that these antibodies can inhibit sporozoite invasion of hepatocytes in vitro and that mice immunized with the PyCSP-PADRE linear construct were protected when challenged with P. yoelii sporozoites.

Amino Acid Sequence↗
PubMed1999

The crayfish plasma clotting protein: a vitellogenin-related protein responsible for clot formation in crustacean blood.

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.

Amino Acid Sequence↗
PubMed1999

Three-dimensional placement of the conserved 530 loop of 16 S rRNA and of its neighboring components in the 30 S subunit.

Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved. Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding. Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits. The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base. The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure.

Cross-Linking Reagents↗
PubMed1999

Ser-534 in the hinge 1 region of Arabidopsis nitrate reductase is conditionally required for binding of 14-3-3 proteins and in vitro inhibition.

14-3-3 proteins bind to the hinge 1 region of nitrate reductase (NR) and inhibit its activity. To determine which residues of NR are required for 14-3-3-inhibitory interactions, wild-type and mutant forms of Arabidopsis NR were examined in the yeast two-hybrid system and in vitro inhibition assays. NR fragments with or without hinge 1 were introduced into yeast with one of seven Arabidopsis 14-3-3 isoforms (called GF14s). NR fragments (residues 1-562 or 487-562) containing hinge 1 interacted with all GF-14s tested; an NR fragment (residues 1-487) lacking hinge 1 did not. GF14 binding to NR fragments was dependent on Ser-534, since Asp or Ala substitutions at this site blocked the interaction. Revertants with second site substitutions restoring interaction between GF14omega and the Ala- or Asp-substituted NR fragments were identified. One isolate had a Lys to Glu substitution at position 531, which is in hinge 1, and six isolates had Ile to Leu or Phe substitutions at 561 in the heme binding region. Double mutant forms of holo-NR (S534D plus K531E, I561F, or I561L) were constructed and found to be partially inhibited by protein extracts from Arabidopsis containing 14-3-3 proteins. Wild-type NR is phosphorylated and inhibited by these extracts, but S534D single mutant forms are not. These results show that inhibitory NR/14-3-3 interactions are dependent on Ser-534 but only in the context of the wild-type sequence, since substitutions at second sites render 14-3-3 binding and in vitro NR inhibition independent of Ser-534.

14-3-3 Proteins↗
PubMed1999

[Zinc deficiency inhibited the proliferation and differentiation of rat osteoblast in vitro].

The effect of zinc deficiency on proliferation and differentiation of rat osteoblast was studied in vitro. The osteoblasts were isolated from the rat calvaria, and were subcultured in DMEM medium. Zinc deficiency model in cell culture was made by chelating zinc from the medium with a special chelator TPEN. Incorporation of 3H-thymidine was measured to show the DNA synthesis at different time point, and cell cycle was analyzed by flow cytometry. A histochemistry method was used to detect changes of osteoblast cytoskeleton. Incorporation of 3H-proline was measured to show the synthesis of collagen. Alkaline phosphatase activity (AKP) and osteocalcin content were measured by enzyme dynamics method and radioimmunoassay respectively. The results showed that the incorporation of 3H-thymidine in the zinc deficient group was significantly less than that in the control group at different time points. The cells were blocked at G2/M phase of cell cycle, which is related to the impairment of cytoskeleton. Collagen synthesis, osteocalcin content and AKP activity in the zinc deficient group were reduced significantly as compared with those in the control group. But no differences in all the measurements were observed between the control group and the group of zinc deficiency supplemented with zinc. Therefore, it was concluded that zinc deficiency inhibited the proliferation and differentiation of rat osteoblast in vitro.

Alkaline Phosphatase↗
PubMed1999

Separation of promethazine and thioridazine using capillary electrophoresis with end-column amperometric detection.

Promethazine and thioridazine were separated and detected by capillary electrophoresis with end-column amperometric detection. The influence of pH value on oxidation potential, the peak current and the resolution were studied and the following conditions was selected: 0.03 M Na2HPO4 and 0.015 M citric acid at pH 3.0, detection potential at 1.10 V. The detection limits of these two substances were in the range of 10(-8) mol/l. The linear range spanned two to three orders of magnitude. This method was applied to the detection of promethazine and thioridazine spiked in urine.

Antipsychotic Agents↗
PubMed1999

Effects of nicotine on K+ channel currents in vascular smooth muscle cells from rat tail arteries.

Intake of nicotine has been related in many cases to acute or chronic hypertension. Using the patch-clamp technique the effect of nicotine on voltage-dependent K+ channel currents in rat tail artery smooth muscle cells was studied. Nicotine at concentrations of 1-100 microM or 0.3-3 mM increased or decreased, respectively, the amplitude of the tetraethylammonium-sensitive K+ currents. Pretreatment of cells with 10 microM dihydro-beta-erythroidine hydrobromide, a nicotinic receptor antagonist, abolished the excitatory effect (n=6), but not the inhibitory effect (n=10), of nicotine on K+ channel currents. The activation of nicotinic receptors with 100 microM 1,1-dimethyl-4-phenylpiperazinium iodide increased K+ channel currents by 27.4+/-3.8% (n=13, P < 0.01). Our results indicate that the excitatory and inhibitory effects of nicotine on K+ channels are respectively mediated by a nicotinic receptor-dependent mechanism and by a direct interaction of nicotine with K+ channels.

4-Aminopyridine↗
PubMed1999

Integrating qualitative and quantitative liquid chromatography/tandem mass spectrometric analysis to support drug discovery.

During the early phase of a drug discovery process, in order to facilitate the selection of drug candidates from a discovery research program, a liquid chromatography tandem mass spectrometry (LC/MS/MS) strategy has been developed to assess the preliminary pharmacokinetics and metabolism of new drug entities. For pharmacokinetic studies using multiple reaction monitoring (MRM), the parent drug concentration and its 'suspected' (predictable) metabolites were monitored in the biological samples simultaneously. For metabolic identification, the general methodologies most frequently used to search for metabolites include full scan, precursor-ion scan, product-ion scan and neutral-loss scan. However, when the metabolites do not produce intense signals for tandem mass spectrometry, a more sensitive and selective assay has to be developed, and MRM would be the method of choice. Likewise, an intelligent guess as to which MRM transitions ought to be used for the metabolites will be considered, based on the product ion mass spectrum of the parent drug.

Acetonitriles↗
PubMed1999

Amyloid phenotype characterization of transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes.

Doubly transgenic mice (PSAPP) overexpressing mutant APP and PS1 transgenes were examined using antibodies to Abeta subtypes and glial fibrillary acidic protein (GFAP). Visible Abeta deposition began primarily in the cingulate cortex of PSAPP mice at approximately 10 weeks of age. By 6 months, the mice had extensive amyloid deposition throughout the hippocampus and cortex as well as other regions of the brain. Highly congophilic deposits consisting of N-terminal normal and modified forms of Abeta were identified, reminiscent of those found in human AD brain. Both immunohistochemistry and mass spectrometry showed that Abeta42 forms were underrepresented relative to Abeta40, and Abeta43 was undetectable. Deposits were associated with prominent gliosis which increased with age, but in 14-month-old PSAPP mice, GFAP immunoreactivity in the vicinity of amyloid deposits was substantially reduced compared to APP littermates. These mice have considerable utility in the study of the amyloid phenotype of AD.

Age Factors↗
PubMed1999

Feline penile erection induced by transurethral administration of sodium nitroprusside.

Nitric oxide (NO) is an important mediator in the relaxation of cavernosal smooth muscle. The aim of this study was to investigate the in vivo feline erectile response after transurethral administration of sodium nitroprusside (SNP), a NO donor drug. Erectile responses after administration of transurethral SNP were compared with those elicited by an intracavernosal control triple-drug combination (1.65 mg papaverine, 25 microg phentolamine, and 0.5 microg prostaglandin E1). SNP was administered via a 20-gauge Jelco intravenous catheter in a volume of 200 microl and changes in intracavernosal pressure, penile length, and systemic blood pressure were monitored. The control triple-drug combination was administered via a 30-gauge needle at the end of each experiment to serve as a control reference. Transurethral administration of SNP (1-4 mg) induced penile erection in a dose-dependent manner with minimal changes in systemic blood pressure. The maximum increase in intracavernosal pressure and penile length after transurethral administration of SNP (4 mg) was significantly less than after the intracavernosal injection of the control triple-drug combination (P<0.01). These data suggest that transurethral administration of SNP can induce an erectile response in cats with minimal side effects.

Alprostadil↗
PubMed1999

Corticotropin-releasing hormone-immunopositive nerve elements in apposition to chicken gonadotropin-releasing hormone I-containing perikarya in Japanese quail (Coturnix coturnix japonica) brain.

The neuroanatomic basis of how stress inhibits reproduction in birds is not understood. To address this question we used double-label immunofluorescence histochemistry to determine whether corticotropin-releasing hormone (CRH)-immunoreactive (ir) neuronal elements contact chicken gonadotropin-releasing hormone I (cGnRH I)-ir somata in brains of Japanese quail. The double-label system used a sheep anti-cGnRH I primary antibody with a secondary antibody conjugated to dichlorotriazinylaminofluorescein dihydrochloride for green fluorescence and a rabbit anti-CRH antibody with a secondary conjugated to Texas Red for red fluorescence. Immunohistochemical (IHC) distribution of both peptides resembled that in previous reports using single-label IHC. In four areas of the quail brain in which CRH nerve fibers and cGnRH I somata co-occurred (bed nucleus commissural pallii, nucleus preopticus medialis, nucleus septalis lateralis and nucleus accumbens), numerous instances were found of CRH-ir nerve fibers or terminals in apposition to cGnRH I cell bodies. These interactions provide a potential neuroanatomic route by which CRH may directly inhibit the activity of cGnRH-I-containing neurons, thereby inhibiting gonadotropin output and halting or slowing the progression of reproductive cycles. It remains to be demonstrated by electron microscopy whether these interactions, which appear abundant by IHC, represent instances of synaptic contact, as has been demonstrated to occur in analogous areas in mammalian species.

Animals↗
PubMed1999

Selective neointimal gene transfer in an avian model of vascular injury.

Avian models of atherosclerosis helped pioneer the study of vascular biology, and offer economic and technical advantages over mammalian models. As an initial step towards investigating important molecular pathways involved in avian atherogenesis and restenosis, we developed a recombinant adenovirus (Ad) which expresses the reporter gene beta-galactosidase (beta-gal), and applied it to cultured chicken vascular smooth muscle cells (SMCs) and a rooster model of acute vascular injury. In cultured chicken SMCs, recombinant gene expression increased as a function of multiplicity of infection (MOI) and incubation time. Maximal expression occurred at an MOI of 10(4) plaque-forming units (pfu)/cell with approximately 50% of quiescent and non-quiescent chicken SMCs expressing beta-gal. Human aorta SMCs had two- to four-fold increased beta-gal expression compared with chicken SMCs at all MOI and incubation times. In vivo instillation of recombinant Ad into uninjured rooster femoral artery segments revealed low efficiency endothelial cell expression of the reporter gene. In contrast, recombinant Ad infection of rooster femoral artery segments 3-21 days after balloon injury revealed up to 60% of luminal surface beta-gal expression, confined predominantly to the neointimal layer. Peak reporter gene expression efficiencies occurred in arterial segments infected 3 days after balloon injury. Uninfected and control Ad infected arteries had no detectable beta-gal expression. Rooster neointimal cells targeted by the recombinant Ad were identified as alpha-smooth muscle actin containing cells by immunohistochemistry. We conclude that Ad-mediated gene transfer is efficient and selective for the neointima in the rooster acute vascular injury model, and offers the potential to efficiently introduce exogenous genes that may impact on the injury response. This model of acute vascular injury may also be manipulated into more established avian models of atherosclerosis, permitting the investigation of acute injury progression to chronic injury.

Adenoviridae↗
PubMed1999

Preoperative auricular wax pattern duplication for surgical template fabrication.

Craniofacial implants have been shown to provide excellent stability and retention for auricular prosthodontic rehabilitation. The locations of implant placement are critical to achieve optimal prosthetic results. This article describes a procedure for fabricating a surgical template from a diagnostic wax-up of an ear without destroying the wax pattern. A full-contour template can assist in proper positioning of craniofacial implants, which in turn can complement the prosthetic result.

Acrylic Resins↗
PubMed1999

Presurgical confirmation of craniofacial implant locations in children requiring implant-retained auricular prosthesis.

Early prosthetic treatment is believed to be psychologically beneficial to children who have an ear defect resulting from congenital malformation. Lack of soft tissue landmarks and minimal bone thickness in deformed auricular areas are sometimes problematic for craniofacial implant placement in these children. This article describes procedures to confirm locations of craniofacial auricular implants by using computed tomographic scan information and a surgical stent. The procedures can also be applied to children and adults to identify locations for auricular implant placement. This method ensures precise locations for implant placement and it also provides a better prosthodontic prognosis for auricular defect patients.

Child↗