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Biomedical subjects

J R White

Publications and source records attributed to J R White.

At least 19 recordsLinked to original sources
PubMed1992

Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Hypoglycemic drugs.

There are an estimated 14 million individuals in the United States with diabetes mellitus. These individuals suffer a very high incidence of podiatric complications. This article focuses on the pharmacologic agents used in the management of hyperglycemia in the patient with diabetes mellitus, including insulin and sulfonylureas.

Diabetes Mellitus, Type 1↗
PubMed1991

A bluetongue serogroup-reactive epitope in the amino terminal half of the major core protein VP7 is accessible on the surface of bluetongue virus particles.

Immunoelectron microscopy has been used to confirm that the core protein VP7 is accessible on the surface of bluetongue virus (BTV) particles. Monospecific antibodies generated to vaccinia virus-expressed VP7 and an anti-VP7 monoclonal antibody (MAb 20E9) bound to native virus particles and were localized by protein A-gold. In contrast, MAb 20E9 labeled directly with gold failed to gain access and bind, suggesting that VP7 is neither adventitiously adsorbed to the virion surface nor exposed in a manner such as protrusion through the outer capsid. Thus the surface layer of BTV may be considered as a net which only partially obscures the underlying core particle. Sequencing of VP7 revealed it to be an extremely hydrophobic protein, 350 amino acids in length with cysteine residues at positions 15, 65, and 154. Examination of VP7 in the cytosol of cells infected with either BTV or a vaccinia virus recombinant expressing VP7 indicated that the protein may exist as an oligomer, whose constituent monomers are not linked by intermolecular disulfide bonds. The cysteine residues in sodium dodecyl sulfate (SDS)-denatured, dithiothreitol (DTT)-treated VP7 were labeled with the fluorescent iodoacetamide AEDANS and the protein was cleaved by V8 protease. The size of the labeled peptides and knowledge of the location of potential V8 cleavage sites suggested that the enzyme cleaved VP7 at three locations (glutamic acid residues at positions 61, 104 (or 108), and 132 (or 134 or 135). Analysis of the fluorescent peptides generated by V8 protease cleavage of VP7 labeled with AEDANS in the absence of DTT (i.e., with any putative intramolecular disulfide bonds intact) suggested that the cysteine at position 154 was the only one accessible to AEDANS. The cysteines at positions 15 and 65 may therefore be linked via a disulfide bond. Denaturation of VP7 with SDS did not eliminate the capacity of the protein to bind MAb 20E9. However, the sensitivity of the epitope to reduction and acetylation and its resistance to either of these processes alone suggest that it may be located near a disulfide bond linking cysteines at positions 15 and 65. Confirmation that the epitope lay in the amino-terminal half of the VP7 came from immunoelectron microscopy experiments in which thin sections of bacteria expressing the complete VP7 and the amino-terminal half were probed with MAb 20E9 and protein A-gold.

Amino Acid Sequence↗
PubMed1991

A monoclonal antibody blocking ELISA detects antibodies specific for epizootic haemorrhagic disease virus.

The isolation of a monoclonal antibody (1G9/C9) with specificity for the epizootic haemorrhagic disease (EHD) serogroup has enabled the development of a highly sensitive and specific blocking ELISA (B-ELISA) for the detection of serum antibodies to EHD viruses. The assay was sensitive to blocking antibodies present in hyperimmune reference antisera to all six EHD serotypes tested but was unaffected by reference antisera to 19 South African and eight Australian serotypes of the related orbivirus bluetongue virus (BTV). The sensitivity of the EHD B-ELISA exceeded that of an indirect ELISA (I-ELISA) for EHD-specific antibody detection. Serum antibody titres to BTV and EHD in experimental and field sera, including a sentinel herd from which virus isolations were made, were examined in both the BTV and EHD B-ELISA tests. These results showed the B-ELISA was only sensitive to antibodies specific for the homologous serogroup in each case, even where sequential and mixed infections with each virus type occurred.

Animals↗
PubMed1991

Respiratory illness in nonsmokers chronically exposed to tobacco smoke in the work place.

We evaluated CO levels as an index of cigarette smoke in the work place and analyzed diary entries on respiratory symptoms, eye irritation, chest colds and lost days from work due to respiratory illness in 40 passive smokers (nonsmokers chronically exposed to tobacco smoke in the work place) and 40 control subjects (nonsmokers not exposed to tobacco smoke in the work place) matched for age and gender. Passive smokers experienced greater CO levels during the workday. Also they reported significantly more cough, greater phlegm production, more shortness of breath, greater eye irritation, more chest colds and more days lost from work due to chest colds than control subjects. Nonsmoking workers and their employers are likely to incur significant financial loss because of missed workdays due to illnesses resulting from exposure to second-hand tobacco smoke.

Absenteeism↗
PubMed1990

Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a.

Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8R*,9S*,11S*)-(-)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2,3, 9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11-atrizadibenzo- [a,g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE (IC50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1-1000 nM) partially (less than 15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 micrograms/mL). Furthermore, prostaglandin E2 (PGE2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a (IC50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 +/- 12.6 ng/10(6) cells) and immunoreactive leukotriene C4 (LTC4) (27.0 +/- 2.6 ng/10(6) cells). Both histamine and LTC4 release was inhibited by staurosporine and K-252a with an IC50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.

Alkaloids↗
PubMed1990

Enhanced sexual behavior in exercising men.

The effects of 9 months of aerobic exercise on aerobic work capacity (physical fitness), coronary heart disease risk factors, and sexuality were studied in 78 sedentary but healthy men (mean age, 48 years). The men exercised in supervised groups 60 min per day, 3.5 days per week on average. Peak sustained exercise intensity was targeted at 75-80% of maximum aerobic working capacity. A control group of 17 men (mean age, 44 years) participated in organized walking at a moderate pace 60 min per day, 4.1 days per week on average. Each subject maintained a daily diary of exercise, diet, smoking, and sexuality during the first and last months of the program. Beneficial effects of chronic, vigorous exercise on fitness and coronary heart disease risk factors were obtained in close agreement with prior studies. Analysis of diary entries revealed significantly greater sexuality enhancements in the exercise group (frequency of various intimate activities, reliability of adequate functioning during sex, percentage of satisfying orgasms, etc.). Moreover, the degree of sexuality enhancement among exercisers was correlated with the degree of their individual improvement in fitness.

Anxiety↗
PubMed1990

Conformation of the VP2 protein of bluetongue virus (BTV) determines the involvement in virus neutralization of highly conserved epitopes within the BTV serogroup.

Seven neutralizing monoclonal antibodies (N-MAbs) were generated to an Australian isolate of bluetongue virus serotype 1 [BTV-1 (Aust)]. At least five of the N-MAbs were specific for epitopes on the outer coat protein VP2 and one was capable of binding SDS-treated protein in a Western blot. Six of the N-MAb panel bound and four of these neutralized BTV-1 (South Africa). None of the N-MAbs neutralized other Australian or South African BTV serotypes. However four of the N-MAb panel bound to a majority and two others bound with varying efficiency, to a more restricted but significant number of heterologous serotypes. Thus epitopes involved in the definition of one BTV serotype may be preserved on other serotypes but not be involved in their neutralization. To investigate the association between these epitopes and factors governing their expression, pools of neutralization-escape variants of BTV-1 (Aust), selected using each of six N-MAbs, were tested in virus neutralization and ELISA binding assays against the N-MAb panel. Each N-MAb displayed a unique reaction pattern with all 25 variants tested. All variants except one showed resistance to neutralization and/or reduced binding with at least three heterologous N-MAbs indicating the N-MAb-defined epitopes were mutually interactive. All 25 variants demonstrated increased resistance to neutralization by a bovine antiserum to BTV-1 (Aust). In total, the results from the variants revealed that the N-MAbs define seven distinct, interdependent neutralization epitopes which form at least part of a major neutralization domain on BTV. A majority of variants bound at least four and up to six N-MAbs, yet still resisted neutralization by them. This observation and the reaction of N-MAbs with BTV-1 (South Africa) and heterologous serotypes suggested that the conformation of the VP2 protein determines whether epitopes, conserved within the BTV serogroup, are involved in neutralization of individual serotypes of the virus.

Antibodies, Monoclonal↗
PubMed1990

Effects of pentoxifylline on proteinuria in normotensive patients with diabetes mellitus.

In 1985, investigators reported that four months of pentoxifylline therapy resulted in a significant decrease in proteinuria (46 percent reduction) and plasma fibrinogen concentrations (18 percent reduction) in patients with diabetes. Our study evaluated four normotensive patients with diabetes and documented proteinuria who were treated with pentoxifylline 400 mg tid for four months. A consistent decrease in proteinuria (as measured by the urine protein/creatinine ratio) and plasma fibrinogen concentrations was not observed. Therapy with pentoxifylline was discontinued after four months.

Adult↗
PubMed1989

Differentiation of second messenger systems in mast cell activation.

Pretreatment of rat peritoneal mast cells with either Staurosporine or an analog K-252a, lead to a dose-related inhibition of histamine release when stimulated with Anti-IgE (IC50: Staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two PKC inhibitors (1-1000 nM) failed to inhibit histamine release induced by compound 48/80 (0.5-1 micrograms/ml). Exposure of Anti-Asc-IgE sensitized mouse bone marrow derived mast cells to Asc-BSA lead to the release of both histamine (510 ng +/- 12.6 ng/10(6) cells) and immunoreactive Leukotriene C4 (27.0 +/- 12.6 ng/10(6) cells). LTC4 release was inhibited by Staurosporine and K-252a with an IC50 of 75 nM for both compounds. Pretreatment of rat peritoneal mast cells with PMA 100 nM lead to a small but significant release of histamine (18.3 +/- 3.6%). Pretreatment of these cells with K-252a or Staurosporine lead to a dose related inhibition of histamine release with an ED50 of 10 nM for Staurosporine and 60 nM for K-252a. Treatment of rat peritoneal mast cells with the calcium ionophore A23187 lead to a significant release of histamine which was not inhibited by either of the two kinase inhibitors (0.1-1000 nM). The two kinase inhibitors also inhibited mouse bone marrow derived mast cell proliferation in response to IL-3 with IC50 of 80 nM for Staurosporine and 270 nM for K-252a.

Alkaloids↗
PubMed1988

Localization of the nonstructural protein NS1 in bluetongue virus-infected cells and its presence in virus particles.

Seven monoclonal antibodies to the nonstructural protein NS1 of an Australian isolate of bluetongue virus (BTV) have been used in immunofluoresence and immunogold procedures to locate NS1 in virus-infected cells and cytoskeletons. The antibodies fall into three groups indicating that NS1 contains at least three antigenic sites. One group consists of four antibodies which react solely with cytoskeleton-associated virus-specific tubules. A second group contains one antibody which reacts with cytoskeleton-associated virus particles, released viruses, and purified virus and core particles. Two antibodies constituting a third group react with both tubules and cytoskeleton-associated and released virus particles. NS1 was found in [35S]methionine-labeled, purified virus and core particles. Immunofluorescence tests reveal that those antibodies which react with virus particles also bind to cytoskeleton-associated virus inclusion bodies (VIB). The nature of this association was examined by probing cytoskeletons of BTV-infected cells with antibodies to NS1 and protein A-gold. VIB observed in thin sections were not uniformly labeled. Gold was associated with fibrillar arrays found around virus particles either leaving or in close proximity to the VIB. Fibrillar material was not found in association with all virus particles elsewhere in the cell and this suggests that fibril-virus complexes may be intermediate in virus morphogenesis.

Animals↗
PubMed1988

Antibody competition studies with gold-labelling immunoelectron microscopy.

Competitive studies, involving neutralizing monoclonal antibodies (NTmAbs) to outercoat proteins of Akabane virus were performed by immunoelectron microscopy. The experiments were designed to determine whether the NTmAbs were directed against the same or spatially different epitopes. Characteristics of NTmAbs in direct and indirect gold-labelling studies were determined. It was found that the protein A method gave cross-contamination of the immuno-gold complexes whereas direct conjugation of the NTmAbs to gold probes gave clean, specific and intense labelling. Analysis of dilution curves confirmed that saturation of antigenic sites did not occur and secondly determined the optimum working dilutions for the conjugated probes. The data generated in the preliminary studies enabled reliable results to be obtained from the double-labelling competitive experiment. We found that the 2 NTmAbs were directed to either the same epitope or to 2 separate but neighbouring epitopes where the binding of one NTmAb inhibited the binding of the second. The results demonstrate that if reliable data is to be obtained in double-labelling immunoelectron microscopical studies then experiments must be meticulously designed.

Antibodies, Monoclonal↗
PubMed1988

Studies with enzyme-linked immunosorbent assays for the serodiagnosis of bluetongue and epizootic haemorrhagic disease of deer.

An ELISA for the detection of serum antibody in sheep, cattle and goats to the viruses of bluetongue (BTV) and epizootic haemorrhagic disease of deer (EHDV) has been developed. Two methods of antigen preparation were analysed for efficacy in the ELISA and inter-group seroreactivity. A freeze-thaw (F/T) antigen appeared to have a narrower specificity than a cytoskeletal preparation from infected cells (P200) which contained all viral proteins. A higher background reactivity was seen when using the P200 antigen, suggesting that a F/T antigen, perhaps as a composite of serotypes, would be of greater value in an ELISA to replace current methods for antibody screening. The effect of multiple infections with unrelated orbiviruses was found to have no effect on the detection of antibody to BTV and EHDV by ELISA. The ELISA was able to demonstrate development and persistence of antibody to BTV in cattle over the course of 120 days.

Animals↗
PubMed1988

Evaluation of a monoclonal antibody blocking ELISA for the detection of group-specific antibodies to bluetongue virus in experimental and field sera.

In order to overcome serological cross-reactions among orbivirus serogroups, which can hinder the accurate diagnosis of bluetongue virus (BTV) infection of livestock, a blocking ELISA (B-ELISA) incorporating a monoclonal antibody (20E9B7G2) with specificity for the BTV serogroup was developed. Experimental antisera raised to South African BTV serotypes 1 to 19 were tested in the B-ELISA and all blocked the binding of 20E9B7G2 to BTV antigen. The sensitivity and specificity of the assay was evaluated with a range of experimental and field sera and compared to a sensitive indirect ELISA (I-ELISA) for the detection of BTV-specific antibodies. The specificity of the B-ELISA was absolute for antibodies to BTV, showing no cross-reaction with experimental antisera to serotypes of the closely related orbivirus causing epizootic haemorrhagic disease of deer. The sensitivity of the B-ELISA exceeded that of the I-ELISA. In particular, the B-ELISA detected a BTV-specific antibody response much earlier after infection that the I-ELISA, while still exhibiting full sensitivity to BTV antibody titres several months after infection.

Animals↗
PubMed1988

Bacterial lipopolysaccharide reduces macrophage lipoprotein lipase levels: an effect that is independent of tumor necrosis factor.

Human monocyte-derived macrophages secrete lipoprotein lipase (LPL) in culture. The regulation of human macrophage LPL production is poorly understood. Since bacterial lipopolysaccharide (LPS) alters production of several macrophage secretory products, its effect on human monocyte-derived macrophage LPL was tested. LPS treatment produced a dramatic dose-dependent decrease in LPL activity in macrophage-conditioned media. At 100 ng/ml LPS, medium LPL activity dropped by 60%. The effect of LPS on macrophage LPL activity was rapid, was blocked by polymixin B, and was not due to cytotoxicity. LPS lowers (by about 60%) the steady state level of LPL mRNA, suggesting that its effect is exerted at the level of mRNA metabolism. Since LPS stimulates macrophage production of cachectin/tumor necrosis factor (TNF), a potent inhibitor of LPL production by the 3T3-L1 adipocyte-like cell line, it was determined whether TNF reduces macrophage LPL levels. Treatment of human macrophages with up to 1000 U/ml of recombinant human TNF had no effect on macrophage LPL activity. When TNF was added in combination with LPS, no additional effect on LPL activity was observed over that seen with LPS alone. Furthermore, the LPS effect was not blocked by a monoclonal anti-TNF antibody. Thus, bacterial LPS potently decreases macrophage LPL activity and mass independent of an autocrine effect of TNF.

Arteriosclerosis↗