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W C Thompson

Publications and source records attributed to W C Thompson.

At least 19 recordsLinked to original sources
PubMed2006

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells.

Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298.

An endothelial nitric oxide synthase (eNOS) polymorphism in exon 7 (894 G/T) resulting in glutamate or aspartate, respectively, at position 298 on the protein is correlated with severity of cardiopulmonary diseases. Because glutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. We now show in transfected cells, primary human endothelial cells, and human hearts, that eNOS with aspartate, but not glutamate, at position 298 is cleaved, resulting in the generation of 100-kDa and 35-kDa products. Recombinant or native eNOS was examined by immunoblotting either in lysates (COS7) or after partial purification over 2',5'-ADP-Sepharose and calmodulin-Sepharose. Immunoblotting after SDS/PAGE with a carboxyl-terminal antibody showed a single major protein band in the predicted position for eNOS at 135 kDa. An additional band at approximately 100 kDa was present only in the recombinant 298Asp eNOS and in the eNOS synthesized by primary cells and heart tissue with a G/T genotype. Using an eNOS amino-terminal-specific antibody, an immunoreactive band at approximately 35 kDa, corresponding to the residual N-terminal cleavage fragment, was observed in those cells with a T genotype. Thus, eNOS with aspartate but not glutamate at position 298 is cleaved, resulting in the generation of N-terminal 35-kDa and C-terminal 100-kDa fragments. Thus, the eNOS gene with polymorphisms at nucleotide 894 generates protein products with differing susceptibility to cleavage, suggesting that, in contrast to prior predictions, this polymorphism has a functional effect on the eNOS protein.

Animals↗
PubMed1999

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.

ADP Ribose Transferases↗
PubMed1999

Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl2. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.

Animals↗
PubMed1999

A study of various morphologic variables and troponin I in pericardial fluid as possible discriminators of sudden cardiac death.

Pathologists frequently examine victims of sudden cardiac death. In some cases, a firm diagnosis of cardiac-related death can be made based on conclusive gross and histologic findings. In many other cases, we find evidence supportive of, but not diagnostic of, cardiac death (e.g., atherosclerotic coronary artery disease, cardiomegaly, myocardial scarring). A final cohort consists of cases of sudden death with minimal to mild cardiac disease, no other significant pathology, and negative toxicologic studies. This prospective study compared 38 cardiac-related deaths with 52 control cases with respect to concentrations of pericardial cardiac troponin I (cTnI), heart weight, evidence of old and/or recent myocardial injury, and presence of significant coronary artery disease. The influence of documented chest trauma and/or perimortem cardiopulmonary resuscitation (CPR) on levels of cTnI was also analyzed. Even though median cTnI levels were significantly higher in cardiac deaths than in controls (p = .003), cTnI was not found to be a significant predictor of cardiac deaths, as determined by discriminant analysis (p = .52). Heart weight >500 g, evidence of old and recent myocardial injury, and significant coronary artery disease were seen statistically more often in cardiac deaths than in controls (p < or = .005 in each case), and median age was significantly higher in cardiac deaths than in controls (p = .001). Based on a stepwise logistic regression model, significant coronary artery disease, old and recent myocardial injury, and heart weight >500 g were found to contribute significantly to the prediction of cardiac death. Finally, neither chest injury nor CPR significantly affected concentrations of cTnI in pericardial fluid. These data confirm that the presence of acute and remote myocardial injury, significant coronary artery disease, and cardiomegaly (heart weight >500 g) strongly supports the diagnosis of a cardiac-related death. In contrast to a recently published report, we do not find that elevated concentrations of cTnI in pericardial fluid are strong indicators of cardiac-related deaths using our methodology.

Adult↗
PubMed1998

Interaction of integrin alpha 7 beta 1 in C2C12 myotubes and in solution with laminin.

The dimer of integrin alpha 7 and beta 1 is a major laminin-binding receptor in skeletal muscle. We studied interactions of integrin alpha 7 beta 1 with the extracellular matrix protein laminin in solution and in intact cells. Integrin alpha 7 beta 1 bound to EHS laminin (laminin-1, composed of alpha 1, beta 1, and gamma 1 chains), but not to endogenous laminin expressed in C2C12 myotubes. Northern blot analysis demonstrated that C2C12 myotubes synthesized laminin-1 alpha, beta, and gamma subunits mRNAs. C2C12 laminin was, however, immunologically distinct from EHS laminin; it was not recognized by 5D3 anti-laminin-1 monoclonal antibody, whereas 5A2 and LT3 antibodies reacted equally well with C2C12 and EHS laminins. Following deglycosylation of EHS laminin, separation of the subunits by SDS-PAGE, Western blotting, and partial amino acid sequencing of the protein bands, the epitope recognized by 5D3 antibody was localized to the gamma 1 laminin chain. Following binding in vitro, the complex of EHS laminin and integrin alpha 7 beta 1 was subject to chemical cross-linking. The two proteins did not undergo cross-linking at the cell surface, consistent with the fact that in intact, resting myotubes integrin alpha 7 beta 1 interacted poorly with EHS laminin, which may reflect a limited accessibility of integrin alpha 7 beta 1 in the membrane to laminin or an inactive state of the integrin.

Amino Acid Sequence↗
PubMed1995

Differential effects of interleukin-12 treatment on gene expression by allostimulated T cells from young and aged mice.

Alloantigen stimulation was used to examine the effect of interleukin (IL-12) treatment of stimulated cells from young and aged mice on the expression of mRNAs for perforin and granzyme B, two proteins known to be intimately involved in an important lytic pathway used by CTL, and mRNA for interferon (IFN)-gamma, production of which is highly stimulated by IL-12 As reported previously, IL-12 augmented the lytic activity by cells from both young and aged mice, although the relative increase was greater for the latter. The mRNAs encoding perforin and granzyme B were both marginally enhanced at early time points (for cells from young mice) or throughout the stimulation (for cells from aged mice) following allo-stimulation in the presence of IL-12. The levels of augmentation of these mRNAs was consistent with the augmentation of lytic activity. In contrast, mRNA encoding IFN-gamma was markedly enhanced throughout stimulation in cells from animals of both age groups, corresponding to the more substantial increase in interferon protein in response to IL-12.

Aging↗
PubMed1995

Activated mast cells produce interleukin 13.

When mast cells are activated through their immunoglobulin (Ig)E receptors, release of low molecular weight mediators like histamine is followed by secretion of multiple cytokines, including interleukin (IL)-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor. Here we report that stimulated mast cells also synthesize IL-13 mRNA and protein; secretion of this cytokine may be of particular importance because of its ability to stimulate IgE expression. IL-13 transcripts detected by a semiquantitative reverse transcriptase-mediated polymerase chain reaction assay were induced within 30 min after stimulation of mast cells by dinitrophenyl plus monoclonal IgE anti-dinitrophenyl, and peaked at about 1 h. Within 3 h of IgE stimulation, secreted IL-13 bioactivity, estimated by proliferation of an IL-13-dependent cell line, reached levels equivalent to 1-2 ng/ml of IL-13. When added to human B lymphocytes, the mast cell-derived IL-13 activity (like bone fide IL-13) induced Ig C epsilon transcripts, DNA recombination characteristic of the isotype switch to C epsilon, and the secretion of IgE protein. These results suggest a model of local positive feedback interactions between mast cells and B cells, which could play a role in the pathogenesis of atopy.

Animals↗
PubMed1995

Subjective interpretation, laboratory error and the value of forensic DNA evidence: three case studies.

This article discusses two factors that may profoundly affect the value of DNA evidence for proving that two samples have a common source: uncertainty about the interpretation of test results and the possibility of laboratory error. Three case studies are presented to illustrate the importance of the analyst's subjective judgments in interpreting some RFLP-based forensic DNA tests. In each case, the likelihood ratio describing the value of DNA evidence is shown to be dramatically reduced by uncertainty about the scoring of bands and the possibility of laboratory error. The article concludes that statistical estimates of the frequency of matching genotypes can be a misleading index of the value of DNA evidence, and that more adequate indices are needed. It also argues that forensic laboratories should comply with the National Research Council's recommendation that forensic test results be scored in a blind or objective manner.

DNA↗
PubMed1995

Confirmation and quantitation of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene by gas chromatography/mass spectrometry. Use of microwave irradiation for rapid preparation of trimethylsilyl and T-butyldimethylsilyl derivatives.

The authors developed a technique by which cocaine metabolites as well as cocaethylene can be identified and quantitated using gas chromatography/mass spectrometry (GC/MS), and deuterated standards of benzoylecgonine, cocaine, ecgonine methyl ester, and cocaethylene as internal standards. The authors used a solid phase extraction (Clean Screen, Worldwide Monitoring Corporation, (Horsham, PA)) technique, and rapid derivatization of metabolites using microwave irradiation before GC/MS analysis. The t-butyldimethylsilyl derivatives (prepared by microwave irradiation in 2.5 minutes, 640 Watts [W]) offered better baseline separation and chromatography than conventional trimethylsilyl derivatives. The assay was linear between 0.87-17.3 mumol/L of benzoylecgonine (250-5,000 ng/mL) concentration, 0.83-16.6 mumol/L of cocaine (250-5,000 ng/mL), 0.79-15.8 mumol/L of cocaethylene (250-5,000 ng/mL) and 1.25-25.1 mumol/L of ecgonine methyl ester (250-5,000 ng/mL). The yields are comparable between the microwave technique and conventional heating.

Cocaine↗
PubMed1995

Carbamazepine-salicylate interaction in normal and uremic sera: reduced interaction in uremic sera.

Displacement of phenytoin and valproic acid by salicylate have been described. We studied carbamazepine-salicylate interactions in normal and uremic sera, which have not been studied. Salicylate caused significant displacement of carbamazepine from protein binding, leading to higher concentrations of free carbamazepine. The concentrations of free carbamazepine were always significantly higher in uremic sera than in normal sera. However, when uremic sera were supplemented with both carbamazepine and salicylate, we observed a much lower displacement of carbamazepine and only a slight increase in free carbamazepine concentration. Treatment of uremic sera with activated charcoal corrected the binding deficiency for carbamazepine. Known uremic compounds like hippuric acid and indoxyl sulphate can only partly explain the observed displacement of carbamazepine in uremic sera. We conclude that salicylate displaces carbamazepine from protein binding in normal sera, but this interaction is significantly reduced in uremic sera.

Carbamazepine↗
PubMed1994

Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: requirement for a second signaling pathway.

Thrombin mitogenesis in fibroblasts requires two distinguishable subsets of signals; one generated by proteolytic cleavage, the other by high-affinity cell surface binding. Characterizing two closely related mouse embryo (ME) cell lines with high numbers of thrombin binding sites, we found that one line, B11-A, responds mitogenically to thrombin, epidermal growth factor (EGF), and serum, whereas the B11-B cell line is responsive to EGF and serum, but not to thrombin. The B11-B defect responsible for loss of thrombin responsiveness is not due to differences in the number of high-affinity binding sites, the affinity of thrombin binding to these sites, or to differences in cell surface expression of proteolytically activated receptors for thrombin (PART). The defect is also not associated with an inability of thrombin to activate PART since thrombin stimulates the cleavage-dependent induction of the proto-oncogene c-fos in both B11-A and B11-B cells. Various combinations of thrombin, synthetic thrombin receptor peptide, TRP-14 (SFFLRNPGENTFEL), platelet-derived growth factor (PDGF), and phorbol 12-myristate 13-acetate (PMA) were used to better define the defect in thrombin-mediated mitogenesis in B11-B cells. Direct activation of protein kinase C with PMA in combination with thrombin did not overcome B11-B nonresponsiveness. However, mitogenic responsiveness was regained in B11-B cells by simultaneous addition of PDGF and either thrombin or TRP-14. Therefore, the B11-B defect may involve a set of signals initiated by nonproteolytic thrombin interactions distinct from those initiated by PART, but related to the downstream signals initiated by the tyrosine kinase-associated growth factors, EGF and PDGF.

Amino Acid Sequence↗
PubMed1994

Microwave-induced rapid preparation of fluoro-derivatives of amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine for GC-MS confirmation assays.

We prepared trifluoroacetyl, pentafluoropropyl, and heptafluorobutyl derivatives of amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) in 45 s, 1 min, and 6 min, respectively, by using microwave irradiation. Conventional techniques require heating the reaction mixture for 15 min at 40 degrees C for trifluoroacetyl derivatives, 15 min at 75 degrees C for pentafluoropropyl derivatives, and 40 min at 60 degrees C for heptafluorobutyl derivatives. The mass-spectral fragmentation patterns and the gas-chromatographic retention times of the derivatives obtained by both microwave irradiation and conventional heating were similar. Perfluorooctanoyl derivatives of amphetamine can be prepared quantitatively by either heating the reaction mixture for 30 min at 60 degrees C or by 1 min of microwave irradiation. Conversion of methamphetamine and MDMA to the corresponding perfluorooctanoyl derivatives was not quantitative by either technique, although the yield of the derivative in the conventional technique was much higher.

3,4-Methylenedioxyamphetamine↗
PubMed1993

Extracranial carotid artery aneurysm.

Although rare, extracranial carotid aneurysms are common enough to be considered in the differential diagnosis of a mass in the neck or posterior pharynx. The unexpected finding of an aneurysm of the carotid artery during the biopsy of a mass in the neck for histologic diagnosis could be catastrophic. The case presented demonstrates the importance of diagnostic imaging of the neck prior to biopsy of a clinically presumed solid mass.

Aneurysm↗
PubMed1992

The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice.

We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine.

Animals↗