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Cross-neutralization of coagulant enzymes in snake venoms by polyvalent antisera
9th World Congress
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The molecular mechanism of action of Clostridium perfringens type A enterotoxin. MORIHIRO MATSUDA,1 NAKABA SUGIMOTO,1 KUNIHIRO OZUTSUMI1 and MASAYUKI TAKAGI2 (1Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565; and 2College of General Education, 1-1 Machikaneyama, Toyonaka 560 ~, Osaka, Japan). AN ENTEROTOXIN ( M r 35,000) produced by C. perfringens type A causes diarrhea, disturbances of the secretory system in intoxicated animals and death, and shows cytotoxicity in sensitive tissue culture cells. We found that the toxin bound rapidly to HeLa and Vero cells in the absence of extracellular Ca 2 + and changed membrane permeability in the absence or presence of extracellular Ca 2 ÷ to the similar extent at 37°C and 4°C, and resulted in influx of Na t and efliux of K + and Mg 2+ and that if Ca 2÷ was present in the medium, Ca 2÷ influx resulted from the characteristic changes in membrane permeability caused at 37°C morphological alterations leading to cell death. In the presence of extracellular Ca 2 ÷, the enterotoxin induced massive release of noradrenaline from neurosecretory PC12 cells. Substances with molecular weights of over ca. 200 but not of less. than ca.200 protected the cells from the morphological alterations, showing that the 'holes' formed in the membrane are of a size that allows permeation of substances with mol.wts of less than ca.200. The enterotoxin was found, by patch clamp technique, to form ionic channels (40 - 450 pS) with occasionally long lasting ( ~ 10 sec) activities in artificial lipid bilayers.
Use of a synthetic peptide to induce cobrotoxin-neutralizing antisera. BRUCE D. MEADE1 and KEITH B. WARD 2 (iPathology Division, USAMRIID, Frederick, MD 21701, U.S.A.; and 2Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375, U.S.A.). COBROTOXIN, the post-synaptic neurotoxin from Naja naja atra, was used to study the feasibility of using
synthetic peptides as vaccines for snake neurotoxins. The two cobrotoxin peptides chosen for initial study were amino acids 18 - 34 [CB(l 8 -34); sequence: SGGETNCYKKRWRDHRG] and 25 - 41 [CB(25 - 41); sequence: YKKRWRDHRGYRTERGC]. The 10 amino acids common to these two peptides were predicted to be important immunologically because the region contains: (1) the hexapeptide in cobrotoxin with the highest hydrophilicity value, (2) the hexapeptide analogous to that in erabutoxin b with the highest solvent accessibility, (3) amino acids likely to contact the acetylcholine receptor, and (4) residues that are highly conserved among the short post-synaptic neurotoxins. Guinea pigs were immunized with either the free peptides or with the peptides coupled to ovalbumin via m-maleimidobenzoyl-N-hydroxysuccinimide.Free peptide CB(18 - 34) was poorly immunogenic, however, free peptide CB(25 - 41) induced antibodies that reacted at low titer in solid-phase enzyme immunoassay (EIA) with both CB(25 - 41) and purified cobrotoxin. Each conjugate induced antisera that reacted in EIA at moderate to high liter with CB(18 - 34), CB(25 - 41), and cobrotoxin. Antisera to an unrelated control peptide did not react with any of the antigens. In the mouse neutralization bioassay, antisera to CB(25 - 41)-ovalbumin neutralized 3 - 5/,g (3 - 5 LDso) of cobrotoxin per ml of serum. No other antiserum neutralized significantly. These data suggest that, with further optimization of sequence and presentation, synthetic peptides have potential as immunogens for post-synaptic neurotoxins.
Analysis of a toxic extract from the frog Atelopus oxyrhynchus. DIETRICH MEBS and KARL SCHMIDT(Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G.).
Atelopus oxyrhynchus (family: Bufonidae) is a brightly yellow colored frog which lives in mountain forest areas near Merida, Venezuela. Alcoholic extracts of several specimens proved to be extremely toxic producing paralytic symptoms when evaporated to dryness, dissolved in physiological saline and injected intraperitoneally into mice. The toxic component was isolated by gel filtration of the extract on Sephadex G-15. From the electrophoretic and thin-layer chromatographic behaviour as well as from gas chromatography-mass spectrometry studies it was concluded that the toxin involved is a tetrodotoxin-like substance.
Cross-neutralization of coagulant enzymes in snake venoms by polyvalent antisera. DIETRICH MEBSand INA CLAUS (Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G.). POLYVALENT antivenoms (Behringwerke North Africa, Central Africa and Orient; South African Institute of Medical Research (SAIMR); Wyeth anticrotalidae antivenom) were tested for their ability to inhibit (neutralize) the coagulant (fibrinogen) activity of the following snake venoms: Agkistrodon acutus, Bitis gabonica, Bothrops atrox and Crotalus adamanteus. Remarkable cross-neutralization was observed; for instance, the activity of the Asian Agkistrodon acutus venom is inhibited by SAIMR as well as Behringwerke Orient antivenom, that of Bothrops atrox venom by SAIMR and Wyeth antivenom. By affinity chromatography using a fibrinogen-coagulase partially purified from Bitis gabonica venom as ligand, specific antibodies were isolated from Behringwerke Orient antivenom which inhibited the activity of the enzymes in Agkistrodon acutus and
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9th World Congress
Bothrops atrox, but not in Crotalus adamanteus venom. This supports the assumption that fibrinogen-coagulants from the venom of different snake species may share common antigenic properties.
Cyanogenic ornamental plants of Oklahoma. F. N. MGnEMEand G. E. BURROWS(Department of Zoology and Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). INTOXICATION due to ingestion of cyanogenic plants is not a common problem but it is a serious one because of the resulting high case fatality rate. Numerous plants, especially those of the family Rosaceae, are considered hazardous because of their cyanide content. In most plants the cyanide is bound in an inactive form as a glycoside, such as dhurrin and amygdalin. In some species or under certain conditions the concentrations of these glycosides may become sufficiently high as to represent a hazard when the plant is eaten. Because of the widespread usage of purported cyanogenic plants as ornamentals and the possible ingestion of such plants by livestock, pets and children, the present study was undertaken to identify the most hazardous plant species. Plants were collected in the Stillwater, Oklahoma, area and from the extensive horticulture collection at Oklahoma State University. The evaluation was based on the picrate paper test (BURNSIDE, 1954) which, although only semi-quantitative at best, is very adaptable to large numbers of samples. The tests were carried out over a 2 yr period with sampling at two different times of the year to compensate for possible seasonal variations in cyanide content. Based on these data, plants could be characterized as consistently highly cyanogenic (Nandina domestica), cyanogenic (Plantanus occidentalis) or non-cyanogenic (Pyracantha coccinea). Seasonal variations were not a significant factor. REFERENCE BURNSIDE, J. E. (1954) Vet. Med. 49, 136-146.
Immunological relationships between snake phospholipase .4 2 neurotoxins. JOHN L. MIDDLEBROOK1 and IVAN I. KAtSER~ (1pathology Division, U.S. Army Med. Res. Inst. for Infect. Dis., Frederick, MD 21701; 2Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, U.S.A.). WE HAVEpurified l0 phospholipase A 2 neurotoxins found in elapid and viperid snake venoms and immunized rabbits with each toxin. Antisera were obtained to the following toxins: ammodytoxin, beta-bungarotoxin, caudoxin, concolor toxin, crotoxin, Mojave toxin, notexin, pseudexin, taipoxin and textilotoxin. To determine the immunological relatedness of the toxins, ELISA assays were run wherein serial dilutions of homologous or heterologous antisera were incubated on plastic plates previously coated with neurotoxins. The amount of rabbit antibody binding to the toxins was determined by using a second antibody conjugated with peroxidase. Extensive cross reactions were observed and it was possible to classify toxins into three moderately to strongly cross-reacting serogroups. One group was the elapid-derived toxins: taipoxin, notexin, pseudexin and textilotoxin. Also falling into this serogroup were the acidic phospholipase A 2 from Naja naja atra and porcine pancreatic phospholipase A 2. A second serogroup was composed of the crotalid and viperid neurotoxins: crotoxin, concolor toxin, Mojave toxin, caudoxin and ammodytoxin. The third serogroup had only one member, beta-bungarotoxin. Antisera to several other toxins, such as botulinum neurotoxins A and B and cobrotoxin, did not cross react with any of the phospholipase A 2 neurotoxins, confirming the specificities cited above. In all cases, the homologous antisera were effective at protecting mice from the lethal effects of the toxins. In certain instances, heterologous antisera could also protect. For example, antisera to N. n. atra phospholipase A 2 (LDso 5000/zg/kg) and notexin (17/~g/kg) were effective at protecting from taipoxin (2/zg/kg). These data suggest that the snake phospholipase A 2 neurotoxins have common epitopes and, in some instances, common neutralizing epitopes.
Preparative HPLC separation of maitotoxin from crude extracts of Gambierdiscus toxicus. DONALDM. MILLER,1 DONALD R. TINDALL2 and MARK JACYNO1 (Departments of 1Physiology and 2Botany, Southern Illinois University, Carbondale, IL 62901-6512, U.S.A.). MAITOTOX1N is one of several toxins involved in the ciguatera syndrome. Ciguatera is a disease in humans caused by the ingestion of fish which have acquired toxicity from dinoflagellates in their diet. Gambierdiscus toxicus, a dinoflagellate known to produce maitotoxin was grown in large scale culture and harvested. Cells were extracted in methanol and toxic fractions were partitioned in ether/water. The water soluble extract was taken up in methanol and fractionated on a Waters Delta Prep-3000 preparative HPLC system. Thirty-five milligrams were loaded on a 15 micron C-18 column (5 cm x 28 cm) at 4 ml/min. The sample was run at a flow rate of 20 ml/min, with 100% methanol as the mobile phase for 65 min. The detector system utilized was a Lambda-Max, Model 481 set at 210 nm. Eluent was collected in six l0 min fractions. These fractions were evaporated to dryness, weighed on a Cahn microbalance and resuspended in 1 ml of methanol. Maitotoxin,
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The molecular mechanism of action of Clostridium perfringens type A enterotoxin. MORIHIRO MATSUDA,1 NAKABA SUGIMOTO,1 KUNIHIRO OZUTSUMI1 and MASAYUKI TAKAGI2 (1Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565; and 2College of General Education, 1-1 Machikaneyama, Toyonaka 560 ~, Osaka, Japan). AN ENTEROTOXIN ( M r 35,000) produced by C. perfringens type A causes diarrhea, disturbances of the secretory system in intoxicated animals and death, and shows cytotoxicity in sensitive tissue culture cells. We found that the toxin bound rapidly to HeLa and Vero cells in the absence of extracellular Ca 2 + and changed membrane permeability in the absence or presence of extracellular Ca 2 ÷ to the similar extent at 37°C and 4°C, and resulted in influx of Na t and efliux of K + and Mg 2+ and that if Ca 2÷ was present in the medium, Ca 2÷ influx resulted from the characteristic changes in membrane permeability caused at 37°C morphological alterations leading to cell death. In the presence of extracellular Ca 2 ÷, the enterotoxin induced massive release of noradrenaline from neurosecretory PC12 cells. Substances with molecular weights of over ca. 200 but not of less. than ca.200 protected the cells from the morphological alterations, showing that the 'holes' formed in the membrane are of a size that allows permeation of substances with mol.wts of less than ca.200. The enterotoxin was found, by patch clamp technique, to form ionic channels (40 - 450 pS) with occasionally long lasting ( ~ 10 sec) activities in artificial lipid bilayers.
Use of a synthetic peptide to induce cobrotoxin-neutralizing antisera. BRUCE D. MEADE1 and KEITH B. WARD 2 (iPathology Division, USAMRIID, Frederick, MD 21701, U.S.A.; and 2Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375, U.S.A.). COBROTOXIN, the post-synaptic neurotoxin from Naja naja atra, was used to study the feasibility of using
synthetic peptides as vaccines for snake neurotoxins. The two cobrotoxin peptides chosen for initial study were amino acids 18 - 34 [CB(l 8 -34); sequence: SGGETNCYKKRWRDHRG] and 25 - 41 [CB(25 - 41); sequence: YKKRWRDHRGYRTERGC]. The 10 amino acids common to these two peptides were predicted to be important immunologically because the region contains: (1) the hexapeptide in cobrotoxin with the highest hydrophilicity value, (2) the hexapeptide analogous to that in erabutoxin b with the highest solvent accessibility, (3) amino acids likely to contact the acetylcholine receptor, and (4) residues that are highly conserved among the short post-synaptic neurotoxins. Guinea pigs were immunized with either the free peptides or with the peptides coupled to ovalbumin via m-maleimidobenzoyl-N-hydroxysuccinimide.Free peptide CB(18 - 34) was poorly immunogenic, however, free peptide CB(25 - 41) induced antibodies that reacted at low titer in solid-phase enzyme immunoassay (EIA) with both CB(25 - 41) and purified cobrotoxin. Each conjugate induced antisera that reacted in EIA at moderate to high liter with CB(18 - 34), CB(25 - 41), and cobrotoxin. Antisera to an unrelated control peptide did not react with any of the antigens. In the mouse neutralization bioassay, antisera to CB(25 - 41)-ovalbumin neutralized 3 - 5/,g (3 - 5 LDso) of cobrotoxin per ml of serum. No other antiserum neutralized significantly. These data suggest that, with further optimization of sequence and presentation, synthetic peptides have potential as immunogens for post-synaptic neurotoxins.
Analysis of a toxic extract from the frog Atelopus oxyrhynchus. DIETRICH MEBS and KARL SCHMIDT(Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G.).
Atelopus oxyrhynchus (family: Bufonidae) is a brightly yellow colored frog which lives in mountain forest areas near Merida, Venezuela. Alcoholic extracts of several specimens proved to be extremely toxic producing paralytic symptoms when evaporated to dryness, dissolved in physiological saline and injected intraperitoneally into mice. The toxic component was isolated by gel filtration of the extract on Sephadex G-15. From the electrophoretic and thin-layer chromatographic behaviour as well as from gas chromatography-mass spectrometry studies it was concluded that the toxin involved is a tetrodotoxin-like substance.
Cross-neutralization of coagulant enzymes in snake venoms by polyvalent antisera. DIETRICH MEBSand INA CLAUS (Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G.). POLYVALENT antivenoms (Behringwerke North Africa, Central Africa and Orient; South African Institute of Medical Research (SAIMR); Wyeth anticrotalidae antivenom) were tested for their ability to inhibit (neutralize) the coagulant (fibrinogen) activity of the following snake venoms: Agkistrodon acutus, Bitis gabonica, Bothrops atrox and Crotalus adamanteus. Remarkable cross-neutralization was observed; for instance, the activity of the Asian Agkistrodon acutus venom is inhibited by SAIMR as well as Behringwerke Orient antivenom, that of Bothrops atrox venom by SAIMR and Wyeth antivenom. By affinity chromatography using a fibrinogen-coagulase partially purified from Bitis gabonica venom as ligand, specific antibodies were isolated from Behringwerke Orient antivenom which inhibited the activity of the enzymes in Agkistrodon acutus and
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9th World Congress
Bothrops atrox, but not in Crotalus adamanteus venom. This supports the assumption that fibrinogen-coagulants from the venom of different snake species may share common antigenic properties.
Cyanogenic ornamental plants of Oklahoma. F. N. MGnEMEand G. E. BURROWS(Department of Zoology and Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). INTOXICATION due to ingestion of cyanogenic plants is not a common problem but it is a serious one because of the resulting high case fatality rate. Numerous plants, especially those of the family Rosaceae, are considered hazardous because of their cyanide content. In most plants the cyanide is bound in an inactive form as a glycoside, such as dhurrin and amygdalin. In some species or under certain conditions the concentrations of these glycosides may become sufficiently high as to represent a hazard when the plant is eaten. Because of the widespread usage of purported cyanogenic plants as ornamentals and the possible ingestion of such plants by livestock, pets and children, the present study was undertaken to identify the most hazardous plant species. Plants were collected in the Stillwater, Oklahoma, area and from the extensive horticulture collection at Oklahoma State University. The evaluation was based on the picrate paper test (BURNSIDE, 1954) which, although only semi-quantitative at best, is very adaptable to large numbers of samples. The tests were carried out over a 2 yr period with sampling at two different times of the year to compensate for possible seasonal variations in cyanide content. Based on these data, plants could be characterized as consistently highly cyanogenic (Nandina domestica), cyanogenic (Plantanus occidentalis) or non-cyanogenic (Pyracantha coccinea). Seasonal variations were not a significant factor. REFERENCE BURNSIDE, J. E. (1954) Vet. Med. 49, 136-146.
Immunological relationships between snake phospholipase .4 2 neurotoxins. JOHN L. MIDDLEBROOK1 and IVAN I. KAtSER~ (1pathology Division, U.S. Army Med. Res. Inst. for Infect. Dis., Frederick, MD 21701; 2Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, U.S.A.). WE HAVEpurified l0 phospholipase A 2 neurotoxins found in elapid and viperid snake venoms and immunized rabbits with each toxin. Antisera were obtained to the following toxins: ammodytoxin, beta-bungarotoxin, caudoxin, concolor toxin, crotoxin, Mojave toxin, notexin, pseudexin, taipoxin and textilotoxin. To determine the immunological relatedness of the toxins, ELISA assays were run wherein serial dilutions of homologous or heterologous antisera were incubated on plastic plates previously coated with neurotoxins. The amount of rabbit antibody binding to the toxins was determined by using a second antibody conjugated with peroxidase. Extensive cross reactions were observed and it was possible to classify toxins into three moderately to strongly cross-reacting serogroups. One group was the elapid-derived toxins: taipoxin, notexin, pseudexin and textilotoxin. Also falling into this serogroup were the acidic phospholipase A 2 from Naja naja atra and porcine pancreatic phospholipase A 2. A second serogroup was composed of the crotalid and viperid neurotoxins: crotoxin, concolor toxin, Mojave toxin, caudoxin and ammodytoxin. The third serogroup had only one member, beta-bungarotoxin. Antisera to several other toxins, such as botulinum neurotoxins A and B and cobrotoxin, did not cross react with any of the phospholipase A 2 neurotoxins, confirming the specificities cited above. In all cases, the homologous antisera were effective at protecting mice from the lethal effects of the toxins. In certain instances, heterologous antisera could also protect. For example, antisera to N. n. atra phospholipase A 2 (LDso 5000/zg/kg) and notexin (17/~g/kg) were effective at protecting from taipoxin (2/zg/kg). These data suggest that the snake phospholipase A 2 neurotoxins have common epitopes and, in some instances, common neutralizing epitopes.
Preparative HPLC separation of maitotoxin from crude extracts of Gambierdiscus toxicus. DONALDM. MILLER,1 DONALD R. TINDALL2 and MARK JACYNO1 (Departments of 1Physiology and 2Botany, Southern Illinois University, Carbondale, IL 62901-6512, U.S.A.). MAITOTOX1N is one of several toxins involved in the ciguatera syndrome. Ciguatera is a disease in humans caused by the ingestion of fish which have acquired toxicity from dinoflagellates in their diet. Gambierdiscus toxicus, a dinoflagellate known to produce maitotoxin was grown in large scale culture and harvested. Cells were extracted in methanol and toxic fractions were partitioned in ether/water. The water soluble extract was taken up in methanol and fractionated on a Waters Delta Prep-3000 preparative HPLC system. Thirty-five milligrams were loaded on a 15 micron C-18 column (5 cm x 28 cm) at 4 ml/min. The sample was run at a flow rate of 20 ml/min, with 100% methanol as the mobile phase for 65 min. The detector system utilized was a Lambda-Max, Model 481 set at 210 nm. Eluent was collected in six l0 min fractions. These fractions were evaporated to dryness, weighed on a Cahn microbalance and resuspended in 1 ml of methanol. Maitotoxin,