Bioproduction of rubratoxin in a glucose-mineral salts broth with nutritional supplements and metabolic inhibitors.

A sterile glucose-salts broth fortified with various metabolic inhibitors and nutritional supplements was inoculated with conidia of Penicillium rubrum P3290, and incubated quiescently at 28 degrees C for 14 days. Potassium sulfite and sodium metabisulfite at all test concentrations caused moderate reduction in rubratoxin formation; at high concentrations (greater than or equal to 2.7 X 10(-2)M) accumulation of fungal tissue was also retarded. Production of rubratoxin and cell mass was inhibited by p-aminobenzoic acid; syntheses of toxin were completely blocked by 7.5 X 10(-2)M of the vitamin. Effects of sodium fluoride on P. rubrum cultures grown on inorganic nitrogen sources varied from inhibition of mold growth and (or) rubratoxin A production to reduction in formation of rubratoxin B. With organic nitrogen sources, fluoride caused a 30 and 60% reduction in synthesis of rubratoxins A and B, respectively. Sodium acetate at all test concentrations enhanced formation of rubratoxin; mold growth was enhanced when acetate concentration was larger than or equal to 6.0 X 10(-2)M. A moderate reduction in mold growth was caused by lower acetate concentrations (1.2 X 10(-2)M or 2.4 X 10(-2)M). Sodium arsenite and iodoacetate at test concentrations blocked mold growth and toxin formation; sodium azide and 2,4-dinitrophenol caused a marked reduction in mold growth but inhibited toxin formation completely. However, sodium azide permitted slight growth and toxin formation when mold cultures were incubated for 28 days.     

Alteration of Trichostrongylus colubriformis egg permeability by Bacillus thuringiensis israelensis toxin

A toxin from the bacterium Bacillus thuringiensis israelensis is lethal to nematode eggs. Exposure of eggs of the ruminant nematode Trichostrongylus colubriformis to the toxin significantly increased the eggs' permeability to radiolabeled phenylalanine within 2 hr. Calcium chloride inhibited the toxin-induced change in egg permeability. Iodine staining of eggs that were exposed to the microbial toxin revealed that egg permeability was altered within 5 min and was dependent on the dose of toxin. Addition of 34 mM sucrose, 17 mM sodium chloride, or 17 mM potassium chloride to the eggs' medium increased the toxin's lethality. Exopeptidase activity in eggs of T. colubriformis was reduced significantly after exposure to the B. t. israelensis toxin. Tetrodotoxin, tetraethylammonium chloride, ouabain, 4-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid (SITS), 4,4'-diisothiocyano-2,2'disulfonic acid stilbene (DIDS), valinomycin, and sodium vanadate, which affect membrane transport, had no significant effect on the activity of B. t. israelensis toxin for eggs. Likewise, a series of nucleotides and their derivatives had no effect on the toxin's activity. Ovicidal activity of the microbial toxin was increased by 4-aminopyridine (4.4 X), but was decreased by furosemide (97 X), nigericin (263 X), or monensin (125 X). Microscopic measurement of T. colubriformis eggs after treatment with the microbial toxin revealed no significant size change.     

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannosides of the side chains also failed to bind toxin, indicating that the main mannoside chains were essential for the receptor activity. The reversible adsorption of killer toxin to mannoprotein was demonstrated by linking it covalently to Sepharose and using this material for affinity chromatography. A 90-fold increase in the specific activity of a preparation of killer toxin KT 28 was achieved in this way.     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Targeting of mycotoxins to reticuloendothelial system of mice with carrier erythrocytes

The tricothecene mycotoxin, T-2 toxin, was encapsulated in bovine erythrocytes for in vivo delivery of T-2 toxin to macrophages. Intraperitoneal injection of bovine carrier erythrocytes (5 X 10(8) cells) containing T-2 toxin saturated mouse liver uptake of erythrocytes by 6 h postinjection. At 24 h postinjection, 20% of the injected carrier cells containing toxin were localized in the liver of mice. Saturation of the liver uptake of bovine carrier cells was independent of encapsulated or free T-2 toxin. A dose of T-2 toxin sufficient to inhibit 50% of the macrophage protein synthesis was targeted to the liver via the carrier erythrocytes. A methodology for delivery of highly toxic molecules to liver macrophages is described.     

Interaction of cholera toxin with ganglioside GM1 receptors in supported lipid monolayers

Lipid monolayers formed at the air-water interface containing the ganglioside GM1 in egg yolk phosphatidylcholine have been transferred according to the Langmuir-Blodgett technique to glass cover slips coated with octadecyl- or hexadecyltrichlorosilane and carbon-coated electron microscope grids. Monolayer transfer has been demonstrated with fluorescence microscopy, by the transfer of a fluorescent phospholipid analogue, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine or Lucifer yellow labeled GM1 (LY-GM1), incorporated into the lipid monolayer. Incubation of supported monolayers with solutions of fluorescein-labeled cholera toxin (FITC cholera toxin) resulted in specific binding of the toxin to monolayers containing GM1, as revealed by fluorescence microscopy. Lateral diffusion coefficients were measured for both the receptor (LY-GM1) [(3.9 +/- 2.1) X 10(-8) cm2/s] and the receptor-ligand complex (GM1-FITC cholera toxin) [(8.9 +/- 3.2) X 10(-9) cm2/s] according to the technique of fluorescence recovery after photobleaching. In separate studies, GM1-containing monolayers transferred to electron microscope grids were incubated with solutions containing unlabeled cholera toxin, followed by negative staining with uranyl acetate. Electron microscopy revealed patches of stained cholera toxin molecules (diameter approximately 70 A) in crystalline, two-dimensional hexagonal arrays. Optical diffraction and image reconstruction showed the arrangement of the cholera toxin molecules in a planar hexagonal cell, a = 81 A. These initial reconstructions give structural information to a resolution of approximately 30 A and indicate a doughnut-shaped molecule with a central aqueous channel.     

Interconversions of aglycone and host-selective toxin from Helminthosporium sacchari

Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[³H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.     

Diphtheria toxin-receptor interaction: A polyphosphate-insensitive diphtheria toxin-binding domain

Inositol hexaphosphate, and other polyphosphates, inhibit diphtheria toxin-mediated cytotoxicity by binding to the toxin at a highly cationic site called the P site and preventing toxin binding to cell surface receptors. The binding of diphtheria toxin to a solubilized cell surface glycoprotein (150,000 daltons) is also inhibited by these polyphosphates. Treatment of this 150,000 dalton diphtheria toxin-binding cell surface glycoprotein with papain yielded an 88,000 dalton and a 74,000 dalton diphtheria toxin-binding glycoprotein whose binding to toxin was no longer inhibited by inositol hexaphosphate. This result suggests a model of diphtheria toxin-receptor interaction in which the toxin receptor possesses one binding site which interacts with the P site of the toxin in a $\text{polyphosphate-sensitive}$ fashion, and another binding site (located within the papain-derived 74,000–88,000 dalton glycoproteins) which can interact with the toxin at a site distinct from the P site (the X site) in a $\text{polyphosphate-insensitive}$ fashion. This X site-receptor interaction may be involved in the binding of CRM proteins that bind to the toxin receptor but that do not bind polyphosphates, or it may be involved in the entry process of the toxin.     

Biochemical effects of PR toxin on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Metabolic responses of Sertoli cells in culture to various concentrations of follicle stimulating hormone and cholera toxin.

The concentration of cholera toxin required for half-maximal stimulation of cAMP production by Sertoli cell enriched cultures (4.48 X 10(2) microgram/ml) is greater than that required for half-maximal stimulation of 17beta-estradiol synthesis from testosterone (2.34 X 10(-4) microgram/ml), [3H]thymidine incorporation into DNA (1.48 X 10(-5) microgram/ml), or androgen binding protein production (2.43 X 10(-6) microgram/ml). The same relative dose response hierarchy was obtained with respect to stimulation of Sertoli cells with follicle stimulating hormone (FSH) preparations. Again, highest concentrations were required to elicit maximal cAMP production. The data are discussed in relation to an apparent paradox: If cAMP is the mediating 'second messenger' following stimulation by FSH or cholera toxin, why should highest concentrations of these agents be required to elicit 50% of maximal cAMP levels?     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom

A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl-L-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and beta-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant (Km), for benzoyl-L-arginine ethyl ester, and inhibition constant (Ki), for DFP, were found to be 6.9 X 10(-3) and 1.93 X 10(-4) M, respectively.     

Developmental Appearance of Sodium Channel Subtypes in Rat Skeletal Muscle Cultures

22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one- or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive (K = 0.6-5.0 X 10(-8) M) and an insensitive population (Ki = 3.3-4.9 X 10(-6) M). In primary rat muscle cultures, the sensitivity of the toxin-stimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki = 2.4 X 10(-7)M). A single population was also found in 6-day-old cultures (Ki = 5.3 X 10(-7) M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high- and low-affinity sites for the toxin (Ki = 1.3 X 10(-9) M and 9.6 X 10(-7) M). We conclude that a single, toxin-activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high-affinity population evolves with time in primary rat muscle cultures. The expression of a high-affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.     

Quantification of Clostridium botulinum type A toxin and organisms in the feces of a case of infant botulism and examination of other related specimens

Bacteriological examinations were performed on the first case of infant botulism in Japan (an infant boy aged 79 days at onset of illness). Clostridium botulinum type A toxin and organisms were detected continually in the stools of the infant for at least 31 days and 39 days, respectively. The highest levels of the toxin and of the population of the organisms, 7.8 X 10(4) LD50/g and 1.3 X 10(6) colony forming units (cfu)/g, were detected in the stool specimen taken on the 20th day of illness. Type A organisms were detected also in the honey fed to the infant before onset of illness, teats of his feeding bottle, soil specimens taken at the house entry and the vacuum-cleaner dust. Fecal excretion of the toxin and organisms was no longer detected from the 68th day of illness and he recovered.     

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.     

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.     

SDF-1-induced activation of ERK enhances HIV-1 expression

Chemokine receptors are not only able to bind chemokines but, together with CD4, they serve as an entry door for the human immunodeficiency virus type 1 (HIV-1). The signalling capacity of chemokine receptors, which is of fundamental importance for chemokine-induced chemotaxis, is not used by HIV-1 to enter a target cell, nor by chemokines or chemokine-derived ligands to inhibit viral entry. In addition, an ill-defined signal triggered by chemokines can, under some circumstances, lead to an increase in HIV-1 expression. We show here that, in infected cells, exposure to SDF-1 leads to an increased expression of a X4 strain of HIV-1. A similar increase can be induced by an N-terminal peptide of SDF-1 which had previously been shown to elicit an intracellular calcium response and to inhibit the entry of X4 strains of HIV-1. We demonstrate the involvement of extracellular signal-regulated kinases (ERK) in this phenomenon. SDF-1 activates ERK-1 and ERK-2 in Jurkat cells. In HeLa cells, ERK-2 only is activated by SDF-1 or by a SDF-derived peptide. This ERK activation can be blocked by pertussis toxin and by the MEK inhibitor U0126. Most importantly, SDF-1-dependent HIV-1 expression is abolished by pretreating the cells with pertussis toxin or with U0126. The consequences of this SDF-1-induced, ERK-dependent modulation of HIV-1 expression in infected cells may have a clinical relevance for eradicating latent viruses.     

Acetylcholine inhibition in rabbit sinoatrial node is prevented by pertussis toxin

The effects of acetylcholine (ACh) were examined on the naturally occurring slow action potentials (APs) of the isolated, organ-cultured, spontaneously beating sinoatrial (SA) node of the rabbit, in the presence or absence of pertussis toxin. The sensitivity of the SA-node preparations to ACh was not altered after 24 h incubation in organ culture medium. Activation of the muscarinic receptor hyperpolarized the cells and reduced the frequency of spontaneous activity at low concentrations (1 X 10(-6) and 3 X 10(-6) M), and completely abolished automaticity at higher concentrations (1 X 10(-5) M). However, stimulated activity was maintained. Increased concentrations (1 X 10(-4) M) of ACh completely abolished excitability. When the SA-node preparations were cultured in the presence of 0.5 micrograms/mL pertussis toxin, concentrations of ACh as high as 1 X 10(-4) M had no effect on the AP parameters and frequency of spontaneous activity. The results indicate that inactivation of G proteins by pertussis toxin caused inhibition of the ACh effects on the automaticity of the SA node. In addition, the blocking effect of ACh to the naturally occurring slow APs was also inhibited by pertussis toxin. We conclude that in the rabbit SA node, the effects of ACh on automaticity and on the slow channels are mediated by G protein.     

Genomic Relatedness of Clostridium difficile strains from different toxinotypes and serogroups

Clostridium difficile strains can be divided into sixteen toxinotypes (0 and I to XV) according to changes in their toxin genes. To determine the genomic similarity between toxinotypes, two molecular typing techniques were used, AP-PCR and PFGE. Strains were selected from five serogroups (A1, A15, E, F, X) and represented non-toxinogenic isolates, strains with toxin genes identical to the reference C. difficile strain, VPI 10463 (toxinotype 0), and strains with variant toxin genes from toxinotypes III, IV, V, VI, VII, VIII, IX, and XI. The strains studied formed three main clusters, which correlated well with serogroups: in the first were strains from serogroup A15 and E; in the second, serogroup A1 strains; and in the third, strains from serogroups F and X. Within these three clusters strains of a single toxinotype were grouped together. Toxinotypes III, IV and VIII were more similar to strains with ordinary toxin genes or non-toxinogenic isolates within the same serogroup than to other toxinotypes. Toxinotypes V, VI, VII, and XI, which exhibit similar changes in their toxin genes, seem to be more closely related one to another than to other toxinotypes. It can be concluded that variant Clostridium difficile strains do not have a common ancestor and that groups of different toxinotypes arose independently from strains with ordinary toxin genes.