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Biomedical subjects

R D Sekura

Publications and source records attributed to R D Sekura.

At least 19 recordsLinked to original sources
PubMed1991

Safety and immunogenicity of hydrogen peroxide-inactivated pertussis toxoid in 18-month-old children.

The immunogenicity and adverse effects of an acellular pertussis vaccine consisting of a purified pertussis toxin inactivated with hydrogen peroxide (PTxd) was evaluated. Children aged 15 to 30 months were injected with 10 (n = 33) or 50 micrograms (n = 34) of PTxd or with diphtheria and tetanus toxoids and whole cell pertussis vaccine (DTP) (n = 34). All children had previously received three doses of DTP during infancy. Both dosages of PTxd induced higher IgG antibody (p less than 0.05 for 10 micrograms dose and p less than 0.01 for 50 micrograms dose) and pertussis antitoxin responses (p less than 0.01 for 50 micrograms dose) than DTP. The 50 micrograms dose gave slightly higher (though not significantly) antibody responses than the 10 micrograms dose of PTxd. None of the vaccines induced detectable IgM or IgA antibody responses to pertussis toxin. At 24 h, local reactions occurred in none of the children injected with 10 micrograms PTxd, 12% with 50 micrograms PTxd and 78% with DTP. Fever at 24 h occurred in 13% after 10 micrograms PTxd, in none after 50 micrograms PTxd and in 53% after DTP. Recipients of DTP, but not of PTxd, had significant increases in neutrophils and decreases in lymphocytes and haematocrit at 24 h (all p less than 0.05). None of the groups showed changes in blood glucose at 24 h. PTxd induced pertussis toxin antibody levels similar to those observed in patients convalescing from natural pertussis. This acellular pertussis vaccine deserves further evaluation for safety and immunogenicity in infants and for efficacy in preventing pertussis.

Antibodies, Viral↗
PubMed1991

Purification and characterization of the heat-labile toxin of Bordetella pertussis.

A procedure is described for purification of pertussis heat-labile toxin (PEHLT) from cells of Bordetella pertussis. The purification procedure, performed in the cold and in the presence of protease inhibitors, gives 1,350-fold purification with yields of about 60%. The toxin was shown to be a single-chain polypeptide of 140 kDa, pI 6.02. It was completely inactivated by heating at 56 degrees C for 60 min. Rabbit antiserum prepared against PEHLT neutralized the toxin and gave a single precipitin line on immunodiffusion. In immunodiffusion assays, this anti-PEHLT serum did not react with pertussis toxin, filamentous hemagglutinin, or preparations of pertussis adenylate cyclase. Purified PEHLT elicited dermonecrosis and atrophy of the spleen. PEHLT is extraordinarily active; 0.4 X 10(-12) g caused necrotic lesions in newborn mice, and with 18- to 20-g mice the 50% lethal dose was about 11 X 10(-9) g.

Adenylyl Cyclases↗
PubMed1988

Clinical, metabolic, and antibody responses of adult volunteers to an investigational vaccine composed of pertussis toxin inactivated by hydrogen peroxide.

A toxoid vaccine, composed of purified pertussis toxin inactivated with H2O2 (NICHD-Ptxd), was developed on the basis of evidence that serum neutralizing antibodies (antitoxin) would confer immunity to pertussis. In vivo and in vitro assays of NICHD-Ptxd showed only trace or nondetectable levels of pyrogenic, adenosine diphosphate-ribosyltransferase, binding and pharmacologic activities. Nevertheless, about 40% of the antigenicity of pertussis toxin was retained. Adult volunteers were injected, two times 6 weeks apart, with either 10 (n = 21), 50 (n = 25), or 75 (n = 30) micrograms/dose of one lot, Ptx-06, adsorbed onto AI(OH)3. Neither fever nor changes in the levels of leukocytes, lymphocytes, fasting blood glucose, or insulin were observed in the volunteers. The optimal immunizing dose, 50 micrograms, induced levels of antitoxin (geometric mean (GM) 302 U) comparable to those found in eight adults convalescent from pertussis (GM 269 U) and greater than those found in 18-month-old children after their fourth dose of diphtheria and tetanus toxoids and pertussis vaccine (GM 20.0 U, p less than 0.001). These data indicate that NICHD-Ptxd is safe and immunogenic in adults, and they justify its evaluation in infants and children.

Adolescent↗
PubMed1987

Guanosine 5'-O-(3-thiotriphosphate) reduces ADP-ribosylation of the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni) by pertussis toxin without causing dissociation of the subunits of Ni. Evidence of existence of heterotrimeric pt+ and pt- conformations of Ni.

The effect of the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the GTP analog which activates the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni), on the pertussis toxin-mediated ADP-ribosylation reaction was studied in detail. Two effects were discerned: a stimulation of the ADP-ribosyltransferase activity of the toxin, akin to what was described for ATP and GDP in a previous report (Mattera, R., Codina, J., Sekura, R., and Birnbaumer, L. (1986) J. Biol. Chem. 261, 11173-11179), and a decrease in the ability of Ni to be a substrate for the activated toxin. Both effects were time-dependent with activation of the toxin being somewhat faster than inactivation of Ni. The effect of the addition of GTP gamma S on Ni was readily reversed by excess GDP and attenuated by increasing EDTA in the medium from 0.35 to 10 mM, suggesting dependence on trace concentrations of a divalent cation. It is suggested that this cation is Mg2+ on the basis that low (5-10 nM) concentrations of Mg2+ are needed for the endogenous GTPase activity of Ni (Sunyer, T., Codina, J., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 15447-15451). Sucrose density gradient analysis of the Ni X GTP gamma S complexes with decreased susceptibility to ADP-ribosylation by pertussis toxin showed the same sedimentation parameters as Ni or Ni X GDP complexes, indicating that the molecule of Ni with GTP gamma S bound is heterotrimetric as opposed to dissociated into alpha i X GTP gamma S plus beta X gamma. Thus, these experiments define two conformations of heterotrimeric Ni: one -pt+, ADP-ribosylated by pertussis toxin, and the other pt-, poorly or not ADP-ribosylated by pertussis toxin. This latter, hitherto unrecognized conformation, is stabilized by the addition of strongly activating guanine nucleotides such as GTP gamma S and guanyl-5'-yl imidodiphosphate and should be important in the train of events that lead from an inactive heterotrimeric Ni to a fully active and dissociated Ni.

ADP Ribose Transferases↗
PubMed1987

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin.

Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.

Adenosine Diphosphate Ribose↗
PubMed1987

Pertussis toxin blocks melatonin-induced pigment aggregation in Xenopus dermal melanophores.

The molecular mechanism of action for the pineal hormone melatonin was explored by testing melatonin interaction with the components of the hormone-sensitive adenylate cyclase complex in a Xenopus dermal melanophore bioassay. Forskolin was employed to stimulate melanosome dispersion. The ability of melatonin to reverse forskolin-stimulated pigment dispersion was assessed, as was the effect of pertussis toxin on the ability of melatonin to aggregate dispersed pigment. Forskolin elicited dispersal of melanosomes in a dose dependent manner (EC50 = 12 nM) in meninges from stage 52-56 tadpoles of Xenopus laevis. Maximal pigment dispersion was obtained with 100 nM forskolin. Melatonin reversed this effect of forskolin (EC50 = 1.5 nM), causing pigment aggregation. Pertussis toxin blocked the melatonin-induced aggregation (EC50 = 358 ng/ml). Prior treatment of the melanophore containing meningeal explants with pertussis toxin results in blockade of melatonin induced pigment aggregation. A 41 kDa pertussis toxin substrate is found in explant homogenates treated with 32P-NAD and pertussis toxin. The availability of this substrate is reduced by prior treatment of intact explants with pertussis toxin and depletion of melatonin responsiveness corresponds to depletion of the 41 kDa substrate. Together, these data suggest that melatonin action upon amphibian dermal melanosomes is mediated by a system requiring a protein similar to the regulatory protein Ni used by mammalian cells to mediate the action of hormones which inhibit adenylate cyclase through a cell surface receptor.

Adenylate Cyclase Toxin↗
PubMed1987

Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin.

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contributes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti-immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lymphocytes.

Animals↗
PubMed1987

Control of aldosterone production by angiotensin II is mediated by two guanine nucleotide regulatory proteins.

The involvement of guanine nucleotide regulatory proteins in the steroidogenic response of the adrenal glomerulosa to angiotensin II (AII) was investigated by analyzing the effects of Bordetella pertussis toxin (PT) on several aspects of AII action. These included receptor binding, stimulation of aldosterone production and GTPase activity, inhibition of cAMP production, and attenuation of the aldosterone response at high angiotensin concentrations. Pretreatment of glomerulosa cells with PT abolished the inhibitory effects of both AII and somatostatin (SRIF) on ACTH-stimulated cAMP production. Under the same incubation conditions, the stimulation of aldosterone secretion by submaximal and maximal steroidogenic concentrations of AII was completely unaffected by the toxin. However, the attenuation of steroid responses seen with supramaximal concentrations of AII was abolished. In addition, the ability of SRIF to inhibit AII-stimulated steroid production was markedly reduced by PT treatment. The binding of [125I]AII to high affinity sites in intact cells and particulate fractions, and modulation of the binding by guanine nucleotides, were unaffected by toxin pretreatment, even under conditions where a 40-41K protein was completely ADP ribosylated. In contrast, the toxin substantially diminished the binding of [125I]Tyr0-SRIF to SRIF receptors in glomerulosa cells (by 50% after 5 h and by 90% after 20 h). These results indicate that Ni or a similar protein probably mediates the inhibition of cAMP formation by AII and the attenuation of the steroid response by high concentrations of AII as well as the inhibitory actions of SRIF in the adrenal glomerulosa cell. Furthermore, the lack of effect of PT on AII binding and stimulation of GTPase activity suggests the existence of an additional pertussis-insensitive guanine nucleotide-regulatory protein that is activated by lower concentrations of AII and mediates the stimulation of aldosterone production.

Adenosine Diphosphate Ribose↗
PubMed1987

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes.

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Adenosine diphosphate ribosylation of G proteins by pertussis and cholera toxin in isolated membranes. Different requirements for and effects of guanine nucleotides and Mg2+.

ADP ribosylation of membranes by pertussis toxin (PT) and cholera toxin (CT) was studied as a function of addition of ATP, various guanine nucleotides, Mg2+, and inorganic phosphate (Pi). ADP ribosylation of a 40 kilodalton (kDa) band by PT is markedly enhanced by ATP and GTP and is strongly inhibited by Pi or Mg2+. GTP analogs (GTP gamma S and GMP-adenyl-5'-yl imidodiphosphate) were less effective. In contrast, ADP ribosylation of two substrates for CT (of 42 and 50 kDa) is stimulated by Pi, Mg2+, and GTP or GTP analogs such as GTP gamma S, but is unaffected by ATP. These stimulatory conditions correlate well with GTP-mediated activation of stimulated nucleotide-binding regulatory component of adenyl cyclase. Optimal conditions for ADP ribosylation by PT do not correlate simply with conditions thought to lead to stabilization of an inactive form of inhibitory nucleotide-binding regulatory component of adenyl cyclase (Gi) or Gi-like protein; rather, the data suggest the involvement of both a stimulatory nucleotide site on PT (positively affected by either ATP or GTP) and a stabilizing site on the PT substrate (affected by GDP, GDP beta S, or GTP). Treatment of membranes with Lubrol PX increased ADP ribosylation by PT by as much as 25- to 30-fold, but inhibited the action of CT. Using defined conditions for ADP ribosylation by PT and CT, distinct labeling patterns were observed in thyroid, brain, corpus luteum, liver, heart, and erythrocytes membranes. All membranes were more intensely labeled by PT rather than CT.

Adenosine Diphosphate Ribose↗
PubMed1986

The interaction of nucleotides with pertussis toxin. Direct evidence for a nucleotide binding site on the toxin regulating the rate of ADP-ribosylation of Ni, the inhibitory regulatory component of adenylyl cyclase.

The interaction of nucleotides with pertussis toxin (PT), and their effects on the ability of the toxin to ADP-ribosylate pure Ni, were evaluated. [32P]ATP (10 nM) bound directly to dithiothreitol-activated PT. This binding was competitively inhibited by nucleotides and anions with the following IC50 concentrations in order of decreasing potency: ATP = ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)) = 0.2-0.3 microM, GDP beta S (guanosine-5'-O-(2-thiodiphosphate)) = 2-3 microM, GTP gamma S (guanosine-5'-O-(3-thiotriphosphate)) = 10-15 microM, ADP = 20-25 microM, GTP = 30-40 microM, GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) = 100-150 microM, GDP = 150-200 microM, Pi = SO4(2-) = 20 mM and Cl- = acetate = 30-35 mM. Treatment of PT with ATP, AMP-P(NH)P, GTP, GDP, or GDP beta S, resulted in a stimulated state of NAD+-Ni ADP-ribosyltransferase activity. Addition of ATP, AMP-P(NH)P (adenyl-5'-yl imidodiphosphate), GTP, GDP, and GDP beta S to the ADP-ribosylation reactions resulted in increased rates of ADP-ribosyl-Ni formation. It is concluded that these effects on the nucleotides are due to their action to stimulate the activity of PT. At concentrations of PT between 0.04 and 0.4 microgram/ml, the stimulation of ADP-ribosylation of Ni effected by nucleotides was hysteretic in nature, exhibiting an approximately 25-min long lag when GDP was used as the activating nucleotide. These lags decreased with increasing concentrations of PT, and were abolished by pretreatment of the toxin with GDP or ATP. Preliminary incubation of Ni with GDP had no effect on the lag in its ADP-ribosylation by non-nucleotide treated PT. Addition of divalent cations (Mg2+, Mn2+, and Ca2+) inhibited formation of ADP-ribosyl-Ni, possibly by causing aggregation and denaturation of Ni. This is the first demonstration that both adenine and guanine nucleotides interact directly with PT and act to stimulate its activity to ADP-ribosylate Ni, and that guanine nucleotides do so regardless of whether they are nucleoside di- or triphosphates.

Adenosine Diphosphate Ribose↗
PubMed1986

Coupling of the thyrotropin-releasing hormone receptor to phospholipase C by a GTP-binding protein distinct from the inhibitory or stimulatory GTP-binding protein.

Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.

Adenosine Diphosphate Ribose↗
PubMed1986

D-2 dopamine receptor-mediated inhibition of pro-opiomelanocortin synthesis in rat intermediate lobe. Abolition by pertussis toxin or activators of adenylate cyclase.

Stimulation of the D-2 dopamine receptor inhibits pro-opiomelanocortin (POMC) synthesis in isolated rat intermediate lobe tissue. Intermediate lobe tissue was incubated in the absence or presence of various dopaminergic compounds, and then its capacity to incorporate [3H]tyrosine into POMC was tested. D-2 dopaminergic agonists caused a dose-dependent inhibition of POMC synthesis; the maximal inhibitory effect was approximately a 50% reduction in the amount of POMC synthesized. D-2 dopaminergic antagonists blocked the inhibitory effect of each agonist. Pretreatment of the tissue with pertussis toxin abolished the D-2 dopaminergic inhibition of POMC synthesis. The potency of pertussis toxin in abolishing the dopaminergic inhibition of POMC synthesis corresponded to its potency in abolishing the D-2 dopaminergic inhibition of adenylate cyclase activity. Cholera toxin, forskolin, and 8-bromo-cAMP, compounds that activate the cAMP pathway, enhanced the capacity of intermediate lobe tissue to synthesize POMC and counteracted the dopaminergic inhibition of POMC synthesis. Incubation of intermediate lobe tissue for 24 h with bromocriptine, a D-2 dopaminergic agonist, decreased the POMC mRNA content by 46% as determined by hybridization of RNA to a 32P-labeled probe. Incubation of intermediate lobe tissue with forskolin increased the level of POMC mRNA; incubation of the tissue with a combination of bromocriptine and forskolin also resulted in an increase in the level of POMC mRNA. It is proposed that Ni, the inhibitory guanyl nucleotide binding protein, and possibly adenylate cyclase mediate the dopaminergic inhibition of POMC synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Dual effects of pertussis toxin on lymphoid cells in culture.

Pertussis toxin (Ptx), a component of Bordetella pertussis, is responsible for many of the biological activities of this bacterium, including its potent adjuvant capacity. In attempt to better understand the Ptx activity on the immune response in vivo, we have examined the effect of Ptx on certain lymphoid cell responses in vitro which could be targets for the adjuvant activity of this molecule. Ptx was found to stimulate a variety of cell responses which include (a) increased production and release of interleukin-1 (IL-1) by human monocytes and murine macrophages; (b) co-mitogenesis, in combination with IL-1, in cultures of murine thymocytes; (c) mitogenesis in cultures of various peripheral lymphocytes; (d) increased production of IL-2 in cultures of human blood lymphocytes and rodent splenocytes; and (e) elevated release of IL-3 in cultures of murine spleen cells. In addition to its stimulatory effects, however, Ptx was found to inhibit responses of both mononuclear phagocytes and lymphocytes to other stimuli. Most activities of Ptx in vitro were achieved at the optimal concentration range of 1-10 micrograms/ml, which is 100-1000 times higher than that showing adjuvant effects in vivo. Possible explanations for the dual effect of Ptx and for the discrepancy in doses optimal for the effects in vivo and in vitro are discussed.

Animals↗
PubMed1985

Adenine nucleotides directly stimulate pertussis toxin.

Both cholera toxin and pertussis toxin catalyzed ADP-ribosylation of purified bovine brain tubulin. The effect of cholera toxin was evident in the absence or presence of nucleotides. In contrast, pertussis toxin required adenine nucleotides for its ADP-ribosylating activity. ATP, ATP gamma S, App(NH)p, deoxy-ATP, and ADP all supported pertussis toxin-catalyzed ADP-ribosylations in the absence or presence of EDTA, suggesting that nucleotide hydrolysis was not involved. Adenine nucleotides also promoted pertussis toxin-catalyzed ADP-ribosylation of heat-treated bovine serum albumin. This result suggests that adenine nucleotides directly affect pertussis toxin. ATP stimulation of pertussis toxin-catalyzed hydrolysis of NAD to ADP-ribose supports this hypothesis.

Adenine Nucleotides↗