Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A genetically engineered gene fusion was constructed which encoded a nontoxic derivative of the A fragment of diphtheria toxin joined to the C180 peptide of the S1 subunit of pertussis toxin. The product of this gene fusion, termed the DTA-C180 protein, was purified from the periplasm of Escherichia coli to approximately 80% purity. The DTA-C180 protein possessed an apparent molecular weight of 43,000 by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DTA-C180 protein was cleaved into two tryptic peptides, which migrated with apparent molecular weights of approximately 22,000. One tryptic peptide reacted with diphtheria antitoxin, while the other tryptic peptide reacted with anti-C180 peptide immunoglobulin G. The DTA-C180 protein did not inhibit protein synthesis or stimulate clustering morphology in Chinese hamster ovary cells. The DTA-C180 protein elicited an immune response, in guinea pigs, against both the DTA and C180 peptide components of the fusion protein, with alum being a more efficient adjuvant than Freund's adjuvant for eliciting neutralization titers. Neutralization titers elicited by DTA-C180 protein were weaker than those elicited by diphtheria toxoid and pertussis toxin 9K/129G, a genetically engineered double mutant of pertussis toxin. Three doses of DTA-C180 protein yielded a neutralization titer of 1/750 against pertussis toxin in Chinese hamster ovary cells and a neutralization titer of 1/50 against diphtheria toxin in Vero cells. This is the first report of a protein derived from a recombinant S1 subunit that elicits a neutralizing titer against pertussis toxin.
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PMID:Construction of a diphtheria toxin A fragment-C180 peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin. 145 39

The purpose of this study was to identify and characterize functional G proteins that couple regulatory peptides with lacrimal secretory functions. Membranes were prepared from isolated rat exorbital lacrimal gland acini, and guanosine 5'-triphosphate (GTP)-dependence of adenylyl cyclase activity, known to be coupled with regulation of secretion, was measured. The guanine nucleotide GTP produced a biphasic response in the activity of membrane-bound adenylyl cyclase during a 10 min incubation with a maximum stimulation at 10(-5) mol/l GTP. Significant inhibition occurred at a dose of 10(-3) mol/l GTP, with cyclic adenosine monophosphate (cAMP) production reduced to less than basal levels. The effect of ADP-ribosylation of membrane proteins by the toxins produced by Vibrio cholera or Bordetella pertussis on lacrimal adenylyl cyclase was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and laser densitometry. Cholera toxin treatment of membranes resulted in dose-(0.5-100 micrograms/ml) and time-dependent (0-45 min) adenosine diphosphate (ADP)-ribosylation of two membrane proteins with M(r) values of 42,000 and 45,000. Pertussis toxin treatment resulted in the specific ADP-ribosylation of a single protein that migrates with an M(r) value of 41,000. This also was dose (0.5-25 micrograms/ml) and time dependent (0-30 min). Incorporation of 32P into the 45,000 M(r) and 42,000 M(r) proteins in the presence of 50 micrograms/ml cholera toxin was guanine nucleotide dependent, with a two- to threefold increase in labeling when the membranes were incubated with 1 or 0.5 mmol/l GTP. This effect was enhanced in the presence of the nonhydrolyzable GTP analog GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Guanine nucleotide binding proteins in the dual regulation of lacrimal function. 146 5

The ADP-ribosyl moiety of NAD was transferred to a 40-kDa protein when rat liver nuclei were incubated with pertussis toxin. The 40-kDa substrate in the nuclei displayed unique properties as follows, some of which were apparently distinct from those observed with the toxin-substrate GTP-binding protein (Gi) in the liver plasma membranes. 1) The nuclear 40-kDa protein was recognized with antibodies reacting with the alpha-subunits (alpha i-1 and alpha i-2) of Gi, but not with anti-Go-alpha-subunit antibody. 2) The nuclear protein had a higher mobility than alpha-subunit of the plasma membrane-bound Gi upon electrophoresis with a urea/sodium dodecyl sulfate-containing polyacrylamide gel. 3) The nuclear protein was not extracted from the nuclei with 1% Triton X-100, whereas Gi was easily solubilized from the plasma membranes. 4) There was a beta gamma-subunit-like activity in the nuclei, which was assayed by an ability to support pertussis toxin-catalyzed ADP-ribosylation of a purified alpha-subunit of Gi. Moreover, a 36-kDa protein in the nuclei was recognized with antibody raised against purified beta-subunits of Gi. 5) Pertussis toxin-induced ADP-ribosylation of the nuclear protein was selectively inhibited by the addition of a nonhydrolyzable GTP analogue, and its inhibitory action was competitively blocked by the simultaneous addition of GDP or its analogues, as had been observed with plasma membrane-bound Gi. It thus appeared that a novel form of alpha beta gamma-trimeric GTP-binding protein serving as the substrate of pertussis toxin was present in rat liver nuclei. In order to examine a possible role of the nuclear GTP-binding protein, rats were injected with carbon tetrachloride, a necrosis inducer of hepatocytes. There was a marked increase in the nuclear substrate activity from 3-6 days after the injection, without a significant change in the activity of Gi in the plasma membranes. The time course of the increase corresponded with a recovering stage from the hepatocyte necrosis. These results suggested that the nuclear GTP-binding protein found in the present study might be involved at some stages in the hepatocyte growth.
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PMID:A GTP-binding protein in rat liver nuclei serving as the specific substrate of pertussis toxin-catalyzed ADP-ribosylation. 154 91

The expression of many of the known virulence determinants of Bordetella pertussis is coordinately regulated by the vir regulatory locus and reduced in response to environmental signals called modulators. We have previously identified eight TnphoA gene fusions in B. pertussis in which the expression of alkaline phosphatase was maximal in the absence of the modulators nicotinic acid and MgSO4. We have termed the genes identified by these fusions vir-activated genes. Here we report the characterization of these TnphoA mutant strains. Four fusion strains were defective in known virulence determinants. For one of these, fusion strain SK39, Southern blot hybridization demonstrated that TnphoA was inserted in the S1 subunit gene of pertussis toxin. Hemagglutination assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblots identified three fusions strains, SK16, SK75, and SK91, that were defective in filamentous hemagglutinin. Whereas all three filamentous hemagglutinin-defective mutants showed either normal or enhanced colonization, the pertussis toxin-defective mutant showed a marked defect in pulmonary persistence. Of the four other fusion strains, two were deficient in outer membrane proteins. One of these, strain SK8, was defective in a major outer membrane protein of 95 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This strain colonized mouse lungs less well and did not induce lymphocytosis after aerosol challenge. The other strain, SK34, was defective in four outer membrane proteins, three of which were detectable only on a Western blot with polyclonal sera against B. pertussis. Two of our gene fusion strains did not show any defect in identifiable vir-regulated proteins.
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PMID:Characterization of vir-activated TnphoA gene fusions in Bordetella pertussis. 165 62

We have previously demonstrated that human bronchial smooth muscle cells possess a single class of high-affinity binding sites for endothelin 1. In this study, we further characterized the receptor for endothelin 1 and evaluated the signal transduction mechanisms of this peptide. Stimulation of cultured human bronchial smooth muscle cells with endothelin 1 induced mobilization of Ca2+ from both intracellular and extracellular pools with a biphasic increase in cytoplasmic free Ca2+ concentration. Endothelin 1 increased cellular levels of inositol phosphates and diacylglycerol, indicating activation of phospholipase C, but induced production of inositol phosphates in smooth muscle cell membranes only in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Treatment of smooth muscle cells with pertussis toxin failed to block the endothelin 1-induced increase in inositol phosphate production and Ca2+ mobilization. These results suggest that the receptor for endothelin 1 in bronchial smooth muscle is coupled to phospholipase C through a pertussis toxin-insensitive G protein. Affinity crosslinking experiments identified the endothelin 1 receptor as a single band with an apparent molecular weight of approximately 70,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, further supporting the functional evidence that endothelin 1 receptor belongs to the G protein-linked rhodopsin type of receptor superfamily.
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PMID:Mechanisms of calcium mobilization and phosphoinositide hydrolysis in human bronchial smooth muscle cells by endothelin 1. 165 61

Cholera and pertussis toxin-mediated ADP-ribosylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical 32P-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the alpha-subunits of Gs. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from Gs alpha. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endogenous ADP-ribosylation in brain: initial characterization of substrate proteins. 168 21

A rapid two-step purification to homogeneity of the calmodulin-activated adenylyl cyclase from urea extracts of Bordetella pertussis organisms (strain 114) is described. Catalytic and invasive activities are purified 30- and 177-fold, respectively, and virtually no degraded forms are found. Specific activities are 0.4 mmol/min/mg and 0.5 mumol/mg of enzyme protein/mg of cell protein/min for catalytic and invasive activities, respectively. The 15 amino-terminal amino acids agree with those deduced from the DNA sequence, as does the molecular mass of 175 kDa (guanidine) or 177 kDa (urea) obtained by equilibrium sedimentation. The larger apparent molecular mass seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be ascribed to anomalous migration. Half-maximal cyclase activation occurs at 3-4 X 10(-10) M calmodulin in the presence of Ca2+ and at 2 X 10(-8) M calmodulin in its absence. Ca2+ activation is maximal at 60-100 microM free CaCl2 (at low calmodulin concentrations), and free Ca2+ concentrations above approximately 125 microM are inhibitory at any calmodulin concentration. Extracellular Ca2+ is essential for intoxication. In Chinese hamster ovary cells, exogenous calmodulin does not inhibit penetration of the cyclase.
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PMID:Invasive adenylyl cyclase of Bordetella pertussis. Physical, catalytic, and toxic properties. 169 22

The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.
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PMID:A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes. 169 70

The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals. Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature. We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci. Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events. The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B. pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions. The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature. Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active. In B. pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects. In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations.
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PMID:Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene. 173 30

Systemic infusion of angiotensin II, a potent agonist, using doses that are initially subpressor, eventually produces sustained blood pressure elevation and reductions in glomerular capillary ultrafiltration coefficient characterized by enhanced signal transduction to angiotensin II and other agonists. In this setting, there is a significant increased affinity of angiotensin II binding to smooth muscle and glomerular mesangial receptors and enhanced sensitivity and magnitude of angiotensin II-induced decrements in cyclic AMP. Since G proteins are important modulators of binding and signal transduction, the present studies were designed to test the hypothesis that differences in the relative amounts of G proteins may be present and have accounted for differences observed. G proteins were identified and quantitated by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radiolabeling in the presence of activated toxins with [gamma-32P]NAD+, immunoprecipitation, and immunoblotting. A 168% and 465% increase in pertussis toxin-catalyzed ADP ribosylation of alpha 40-41 was found in angiotensin II-treated groups over control groups for glomerular and mesenteric membranes, respectively. Immunoblotting revealed a 250% and 35% increase in the levels of the Gi isoforms alpha i-2 and alpha i-3, respectively, and a decrease of 53% in alpha i-1 from the angiotensin II-treated group. No differences were observed in cholera toxin labeling or immunoblotting of Gs. These results demonstrate multiple mechanisms whereby angiotensin-induced signal transduction can be modulated involving both the receptors and G proteins. These observed differences in G proteins in systemic and renal vasculature accompanying angiotensin II infusion suggest the possibility of a regulatory role in the pathophysiology of angiotensin II-induced hypertension and renal disease.
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PMID:Angiotensin II-induced changes in guanine nucleotide binding and regulatory proteins. 173 48


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