Preincubation Of Toxin Research Articles

Human α-Defensins Inhibit Clostridium difficile Toxin B

Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Inactivation of C difficile toxins by alpha-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine K(m) and median inhibitory concentration (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K(m) values for uridine 5'-diphosphate-glucose up to 10-fold. The IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.

G‐Protein Modulators Alter the Swimming Behavior and Calcium Influx of Paramecium tetraurelia

To assess the potential role of G-proteins in chemokinesis, Paramecium tetraurelia was pre-incubated with the G-protein modulator pertussis toxin. Pertussis toxin pretreatment significantly reduced Paramecium chemoattraction to sodium acetate and ammonium chloride in T-maze behavioral assays and depressed the frequency of avoidance reactions, indicating that heterotrimeric G-proteins may be involved with the motility response. To determine whether G-proteins exert their effect via the ciliary voltage-sensitive calcium channel, we examined responses of P. tetraurelia to the potent voltage-sensitive calcium channel agonist, deltamethrin. Pertussis toxin preincubation significantly reduced the toxic effects of deltamethrin exposure as determined by survival under depolarizing conditions and reduced the duration of backward swimming episodes in behavioral bioassays. Furthermore, non-hydrolyzable analogs of guanine nucleotides altered deltamethrin-stimulated calcium influx via calcium channels in isolated ciliary vesicles. Heterotrimeric G-protein subunits were subsequently detected in ciliary vesicles of P. tetraurelia by antibodies produced against Galpha and Gbeta subunits, and by 32P-ADP-ribosylation, indicating that proteins of the appropriate molecular weight are the target of pertussis toxin in these vesicles. These findings provide additional evidence that heterotrimeric G-proteins are associated with ciliary vesicles and that they play a role in the modulation of swimming behavior and the toxic action of deltamethrin in Paramecium.

G protein modulates thyroid hormone-induced Na(+) channel activation in ventricular myocytes.

To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T(3)) on Na(+) channel current (I(Na)) properties, I(Na) was recorded in adult guinea pig ventricular myocytes. T(3) (1 nM) acutely increased whole cell I(Na) and shifted the steady-state I(Na) inactivation curve dose dependently. When the pipette solution contained 100 microM GTP or GTPgammaS, the effect of T(3) on the whole cell I(Na) was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T(3) increased the open probability of single I(Na) by reducing the null probability. In the inside-out patch, T(3) effect was 10 times faster than that in whole cell and cell-attached patches while GTPgammaS was present and could be completely washed out. T(3) alone slightly increased the channel open probability by increasing the closed state to open state rate constant (k(CO)) and reducing the null probability. GTPgammaS exposure only increased the number of functional channels. T(3) and GTPgammaS synergistically enhanced the channel open probability 5.8 +/- 0.5-fold by increasing k(CO), decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T(3) acts on the cytosolic side of the membrane and acutely activates I(Na). Pertussis toxin-sensitive G protein modulation greatly magnifies the T(3) effects on the channel kinetics and null probability, thereby increasing the channel open probability.

Pertussis toxin converts hyperpolarizations caused by alpha(2)-adrenoceptor agonists containing an imidazoline moiety into depolarizations in MIN 6 cells.

Several alpha(2)-adrenoceptor agonists containing an imidazoline moiety inhibit insulin secretion when applied to beta cells. In the present study we investigated such imidazolines in regard to membrane potential effects in MIN 6 cells. We confirmed the inhibition of insulin release as reported in previous studies and showed an additional hyperpolarizing activity of the imidazolines using bisoxonol for membrane potential measurements. Pertussis toxin pre-incubation of MIN 6 cells converted the inhibitory imidazoline activity with regard to insulin release into a stimulatory effect. In addition, the marked hyperpolarization caused by the alpha-adrenoceptor agonists containing an imidazoline moiety was converted into a depolarizing effect after pertussis toxin pre-incubation. Adrenaline, an alpha-adrenoceptor agonist lacking an imidazoline moiety, also inhibited insulin release and hyperpolarized MIN 6 cells. Pertussis toxin pre-incubation led only to a loss of the inhibitory adrenaline effect and of the hyperpolarization but not to the stimulatory effects observed with the imidazolines. Thus a stimulatory effect of alpha-adrenoceptor agonists containing an imidazoline moiety was demasked by PTX in this study. It remains to be clarified to which extent a blockade of K(ATP) channels is responsible for this effect.

Analyses of the Pore Forming Ability of Bacillus thuringiensis Cry1A Mutant Toxins Using a Light-Scattering Technique

Abstract The pore formation properties of Bacillus thuringiensis Cry1 wild type and domain I and II mutant toxins were studied on Manduca sexta brush border membrane vesicles (BBMV) using a light-scattering technique. Wild type Cry1Ac, Cry1Ab, and Cry1Ba; Cry1Ab mutant toxins A92E, Y153D, R368A/R369A, and F371A; and Cry1Ac mutant toxin A92D were analyzed. In a direct mixing assay the mutant toxins Y153D, R368A/R369A, F371A, and Cry1Ba did not elicit a response in a 1-min signal-monitoring period. Mutant toxins A92D and A92E elicited slight responses. After preincubation of toxin with BBMV, the signal recovery response increased for all toxins. The signal recoveries caused by A92D and A92E were greater than Y153D-, F371A-, and R368A/R369A-induced signal recoveries, which were slightly greater than Cry1Ba-induced recoveries. By increasing the monitoring period to 3 min in direct mixing experiments, we observed greater pore formation by A92D. A92E had a response similar to, but lower than that of A92D. The response induced by both wild type and mutant toxins decreased when the hyperosmotic solution was changed from KCl to sucrose. However, in the presence of sucrose the responses induced by A92D and A92E were substantially reduced relative to KCl.

Activation of A(3) adenosine receptor induces calcium entry and chloride secretion in A(6) cells.

We have previously demonstrated that in A(6) renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A(1) and A(2A) receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A(3) receptor-selective agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca(2+) influx. The agonist-induced rise in [Ca(2+)](i) was significantly inhibited by the selective adenosine A(3) receptor antagonists, 2,3-diethyl-4, 5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A(1) or A(2) receptors supporting the hypothesis that Cl-IB-MECA increases [Ca(2+)](i) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elicited Ca(2+) entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca(2+)](i) response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data suggest that in A(6) cells a G(s)/protein kinase A pathway is involved in the A(3) receptor-dependent increase in calcium entry.

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans.

Pertussis toxin sensitive endothelin-1 coupling to inositol phosphate formation via a GTP-binding protein: comparison in SHR and WKY cultured aortic smooth muscle cells.

The effects of pertussis toxin on endothelin-1 and noradrenaline coupling to inositol phosphate (IP) formation was investigated in cultured aortic smooth muscle cells from 14 week SHR and WKY rats. Endothelin-1 (10(-6) M) stimulated IP formation was decreased in cells from SHR compared to WKY (WKY 1117 +/- 157, SHR 668 +/- 85% of basal). Pre-incubation with pertussis toxin produced a significant and similar reduction in endothelin stimulated IP production in both SHR (54% reduction) and WKY (55%). However, the observed reduction in endothelin-1 stimulated IP accumulation was still apparent in SHR when compared to WKY. Pertussis toxin preincubation followed by removal of extracellular calcium reduced further the endothelin responses by similar amounts in SHR and WKY cells, but SHR stimulated IP formation remained significantly decreased compared to WKY. The extent of pertussis toxin ADP-ribosylation of Gi alpha was similar in both SHR and WKY cells. Endothelin-1 produced a reduction in the extent of ADP-ribosylation of Gi alpha and this was of similar magnitude in both SHR and WKY cell membranes. In contrast, noradrenaline stimulated IP formation was unaffected by pertussis toxin pre-incubation. It was concluded that SHR cells do not appear to have an alteration in endothelin-1 activated, pertussis toxin sensitive G-protein coupling to IP formation or in the dependence of inositol phosphate formation on extracellular calcium.

The somatostatin receptors SSTR1 and SSTR2 are coupled to inhibition of adenylyl cyclase in Chinese hamster ovary cells via pertussis toxin-sensitive pathways.

Somatostatin exerts multiple effects throughout the body by binding to specific somatostatin receptors. Two classes of somatostatin receptors, SRIF1 and SRIF2, have been distinguished biochemically and pharmacologically. Two cDNAs have been recently isolated that encode somatostatin receptors 1 and 2 (SSTR1 and SSTR2, respectively). The pharmacological characteristics of receptors expressing these cDNAs resemble those of the SRIF2 and SRIF1 classes of somatostatin receptors, respectively. We stably expressed the rat homologs of both receptors in Chinese hamster ovary (CHO) cells (type K1). These transfected cell lines recognized the endogenous ligands SS14 and SS28 with high affinity, whereas the synthetic analog MK678 identified only SSTR2. In preparations of CHO-SSTR1 or CHO-SSTR2 cells, SS14 and SS28 inhibited forskolin-stimulated adenylyl cyclase activity by approximately 35%, with ED50 values in the nanomolar range. The adenylyl cyclase inhibition was dependent upon the guanine nucleotide GTP and could be ablated with pertussis toxin preincubation. The present data indicate that SSTR1 and SSTR2 are coupled to inhibition of adenylyl cyclase via pertussis toxin- sensitive G-proteins.

The somatostatin receptors SSTR1 and SSTR2 are coupled to inhibition of adenylyl cyclase in Chinese hamster ovary cells via pertussis toxin- sensitive pathways

Somatostatin exerts multiple effects throughout the body by binding to specific somatostatin receptors. Two classes of somatostatin receptors, SRIF1 and SRIF2, have been distinguished biochemically and pharmacologically. Two cDNAs have been recently isolated that encode somatostatin receptors 1 and 2 (SSTR1 and SSTR2, respectively). The pharmacological characteristics of receptors expressing these cDNAs resemble those of the SRIF2 and SRIF1 classes of somatostatin receptors, respectively. We stably expressed the rat homologs of both receptors in Chinese hamster ovary (CHO) cells (type K1). These transfected cell lines recognized the endogenous ligands SS14 and SS28 with high affinity, whereas the synthetic analog MK678 identified only SSTR2. In preparations of CHO-SSTR1 or CHO-SSTR2 cells, SS14 and SS28 inhibited forskolin-stimulated adenylyl cyclase activity by approximately 35%, with ED50 values in the nanomolar range. The adenylyl cyclase inhibition was dependent upon the guanine nucleotide GTP and could be ablated with pertussis toxin preincubation. The present data indicate that SSTR1 and SSTR2 are coupled to inhibition of adenylyl cyclase via pertussis toxin- sensitive G-proteins.

Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin

B. G. Stiles, B. C. Lidgerding, F. W. Sexton and S. B. Guest. Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin. Toxicon 29, 1195–1204, 1991.—Twelve monoclonal antibodies against cobrotoxin from Naja naja atra venom were tested for cross-reactivity with eight different snake toxins, binding to linear epitopes, prevention of cobrotoxin binding to acetylcholine receptor (AchR) in vitro, and protection in mice concomitantly given a lethal dose of cobrotoxin. The antibodies were highly specific, as evidenced by little reactivity with other snake toxins. None of the monoclonal antibodies bound to reduced cobrotoxin or synthesized 8-mer regions spanning the whole molecule, thus suggesting the recognition of conformational epitopes. The in vitro binding of toxin to AchR was competitively inhibited (23–79%) with a 1.66 : 1 mole ratio of antibody : AchR. Preincubation of monoclonal antibody with toxin before adding AchR (3 : 1 mole ratio of AchR : antibody) inhibited the in vitro binding of toxin to AchR by 20–80%. Monoclonal antibodies added after the preincubation of toxin with AchR did not dissociate the toxin-AchR complex. An antibody : toxin mole ratio of 2.5 : 1, with 6 μg of cobrotoxin, delayed the time to death of mice 3.7–23.8-fold over control mice. The monoclonal antibodies that most effectively prevented in vitro binding of toxin to AchR also provided the longest delay in time to death in mice.

Effects of epidermal growth factor on signal transduction in rabbit parietal cells

The aim of this study was to elucidate the cellular mechanisms of action of epidermal growth factor (EGF) inhibition of parietal cell secretion. EGF effects on histamine- and carbachol-stimulated [14C]aminopyrine (AP) uptake and intrinsic factor (IF) secretion were evaluated in isolated rabbit parietal cells. EGF inhibited histamine-stimulated [14C]AP uptake and IF secretion through a reduction in stimulated adenosine 3',5'-cyclic monophosphate (cAMP) levels. EGF decreased the phosphorylation of a cytosolic 30-kDa, histamine-stimulated, cAMP-dependent protein kinase substrate. These effects on histamine-stimulated activation were reversed by pertussis toxin preincubation. EGF inhibited carbachol-stimulated [14C]AP uptake and IF secretion, but did not alter the carbachol-stimulated Ca2+ transient. These results indicate that EGF inhibits histamine-stimulated secretion through the inhibitory Gi guanosine 5'-triphosphate-binding protein and carbachol-stimulated secretion through a mechanism independent of the activation of an increase in intracellular Ca2+.

Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils.

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.