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Cross reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae venoms
9th World Congress
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skin when methanol and DMSO were the vehicles. HPLC analysis of the receptor fluid bathing human and guinea-pig skin indicated that more than 80% of the radioactivity was associated with PbTx-3.
Correlation between the enzymatic activity, anticoagulant and antiplatelet effects of phospholipase A 2 isoenzymes from Naja nigricollis crawshawii venom. R. MANJUNATHA Klrql and HERBERT J. EVANS (Department of Biochemistry, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA 23298, U.S.A.). THE THREE phospholipase A 2 isoenzymes from Naja nigricollis crawshawii snake venom inhibit both blood coagulation and platelet aggregation. To investigate the correlation between phospholipid splitting ability of these enzymes and their inhibitory activities, the effects of various preincubation times and the inclusion of EDTA were examined. Preincubation of plasma and thromboplastin with the phospholipase isoenzymes resulted in an increase in Ca2+-initiated clotting time with time of preincubation. Incubation of the isoenzymes with EDTA before their addition to the plasma - thromboplastin mixture reduced the anticoagulant effect. These results indicate that the catalytic activity contributes at least partially to the anticoagulant effect. However, inhibition of platelet aggregation appears to be independent of enzymatic activity since there is no increase in inhibition with time of preincubation of platelets and phospholipases, and inclusion of EDTA has no significant effect on inhibition or aggregation. All three enzymes appear to belong to class B of the platelet affector PLA 2 enzymes as determined by platelet effects, since they do not initiate platelet aggregation.
Secretion of paralytic shellfish toxins from scallop. MASAAKIKODAMA,l TAKEHIKO OGATA,1 SHIGERU SATO,1 NOBUYUKI INOGUCm,2 MIcmo SHIMIZU2 and HIDEO DAIDO2 (tLaboratory of Marine Biological Chemistry, School of Fisheries Sciences, Kitasato University, Iwate 022-01 and 2Iwate Prefectural Fisheries Experimental Station, Iwate 026, Japan). BIVALVESaccumulate paralytic shellfish toxins during the bloom of toxic dinoflagellates. The toxicity of bivalves decreases when the toxic dinoflagellates diminish from their environment. However, the decreasing rate of the toxicity is different from species of bivalves (OsmMA et al., 1982). Scallop, one of the economic species, is a hardly detoxified species. Its low rate of detoxification poses a severe problem to its culture industry. In this study, we describe the toxin secretion from toxic scallop and a device for detoxification of scallop. Specimens of toxic scallop were transplanted to the aquarium with filtered seawater and reared for several days at 10, 15 and 20°C. Aquarium water was replaced every day with freshly prepared filtered seawater. Replaced reared water was concentrated and tested for toxicity by mouse assay. All the samples showed toxicity. Analyses of partially purified toxin revealed the presence of paralytic shellfish toxins (PST), indicating that PST is secreted from scallop. The amounts of toxin secreted from scallop increased with increase of rearing temperature. When scallop were stimulated by instantaneous electric shock, immediate secretion of significant amounts of toxin was induced, showing that instantaneous electric shock accelerates the toxin secretion of scallop. These results suggest that instantaneous electric shock treatment under higher rearing temperature is effective for detoxification of toxic scallop. REFERENCE
OSHIMA,Y., YASUMOTO,T., KODAMA,M., OGATA,T., FUKUYO,Y. and MATSUURA,F. (1982) Bull. Jpn. Soc. Scient. Fish. 48, 525. Cross reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae venoms. F. KORNALIK,1 E. TAaORSKA,1 and D. MEnS2 (1Department of Pathophysiology, Prague CSSR, 2University of Frankfurt, F.R.G.). LETHAL, local and systemic effects of two African Viperidae and four Crotalidae were assayed on mice and rats and expressed as LDso, minimal hemorrhagic dose (MHD) and minimal defibrinating dose (MDD). Two commercial antivenoms: Behringwerke Nord Africa (BN) and Wyeth anticrotalidae (W) and gamma globulins prepared from hyperimmune rabbit serum were tested for their capacity to neutralize the toxic effects of the five venoms. Two LDsoS were mixed with 0.1 ml of various dilutions of antivenoms, injected i.v. to white mice and the death rate after 48 hr recorded. Cross reactivity (antiviperidae antivenom against Crotalidae venom and vice versa) was observed in nearly all cases. It was more evident with BN against Crotalidae than W against Viperidae. No cross reactivity could have been observed with monovalent antivenoms. Nearly a complete cross reactivity was observed with polyvalent as well as monovalent antivenoms in the neutralization of skin hemorrhages. The local effect was neutralized to 0 - 20% if antivenom was mixed with the venom. I.v. injection of any antivenom later than 20 min after the venom had no effect. Only Echis and Bothrops venoms cause defibrination in vivo. The capacity of all antivenoms to neutralize defibrination was very weak. 0.5 ml of antivenom neutralized I MDD only to 40 - 60% if injected
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9th World Congress
simultaneously, to less than 20% when injected 3 hr after the venom. Cross reactivity was more evident with W against Echis than BN against Bothrops.
Cytolytic activity in the venom of the marine snail Conus californicus. ARNOLD S. KREGER (Department of Microbiology and Immunology, Bowman Gray School of Medicine, Winston-Salem, NC 27130, U.S.A.). EXTRACTS prepared from the venom duct of the predatory marine snail Conus californicus possessed cytolytic activity against erythrocytes obtained from twelve different animal species. Sheep, cow, chicken, and goat erythrocytes suspended in isotonic Tris-buffered saline (pH 7.4) were the most sensitive of the erythrocytes examined, and hemolytic activity was enhanced (ca. 33% increase) by the addition of Ca 2+ (10raM, final concentration) to the assay mixtures. The hemolytic activity was stable to lyophilization, was heat-labile (56°C, 30 min) and protease-sensitive, and was inhibited by a mixed bovine brain ganglioside preparation, cardiolipin, and sphingomyelin. Activity was not enhanced by chilling the erythrocytes after incubation with the extract at 37°C ('hot - cold lysis'), and activity was not affected by glycophorin, trypan blue, dithiothreitol, cholesterol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine. The cytolytic toxin(s) responsible for the observed hemolytic activity is antigenic (i.e. elicits the production of hemolysisneutralizing antibody in rabbits), has an apparent mol.wt of ca. 19,600, as estimated by gel filtration, and has isoelectric pH values of 6.0, 6.7 and 7.3.
Tetanus toxin: primary structure, disulfide bridges and proteolytic modifications of the extracellular form. KERSTIN KRIEGLSTEIN,1'2 AGNES HENSCHEN,2 ULRICH WELLER1 and ERNST HABERMANN1 (1Rudolf-Buchheim-Institute for Pharmacology, D-6300 Giessen, F.R.G.; 2Max-Planck-Institute for Biochemistry, D-8033 Martinsried, F.R.G.). A PREREQUISITEfor the understanding of structure-function relationships in tetanus toxin is the knowledge of amino acid sequence, disulfide bridges and proteolytic modifications. The toxin, a highly potent neurotoxin, is synthesized as a 150kDa single-chain protein, and a 1315 residue amino acid sequence has recently been predicted from its cDNA sequence. However, it has also been shown that its isolated extracellular form is composed of two disulfide-linked peptide chains, a 50 kDa and a 100 kDa chain. In the present work the differences between the true protein structure and the predicted structure were elucidated. Proteolytic modification was evaluated by N-terminal sequence analysis of the chains isolated after mercaptolysis and alkylation with vinylpyridine, and by analysis of C-terminal CNBr fragments of the chains. Cys residues with sulfhydryl groups were located by direct alkylation of the toxin with dimethylaminoazobenzeneiodoacetamide followed by CNBr cleavage and identification of labelled fragments. Disulfide-linked Cys residues were located by isolation and sequencing of CNBr fragments identified by their presence in chromatographies of alkylated and absence in those of mercaptolysed-alkylated samples. The positions of Cys residues connecting the chains were determined. For all separations reversed-phase HPLC was used. Posttranslational processing can obviously only be analysed on the proteinchemical level. The results are of importance for understanding the biological activity of tetanus toxin on the molecular level. REFERENCE EISEL, U., JARAUSCH, W., GORETZKI, K., HENSCHEN, A., ENGELS, J., WELLER, U., HUDEL, M., HABERMANN, E. and NIEMANN, H. (1986) EMBO J. 5, 2495.
Tandem mass spectrometric investigations on cyclic peptides. THAIYA KRISHNAMURTHY,1 MARQUERITE E. BROOKS,1 DONALD F. HUNT,2 JEFFREY SHABANOWITZ,2 WAYNE W. CARMICHAEL3 and K. m. MEREISH4 (1U.S. Army Chemical Research, Development, and Engineering Center, Aberdeen Proving Ground, MD 21010-5423; 2University of Virginia, Charlottesville, VA 20903; 3Wright State University, Dayton, OH 45435; 4U.S. Army USAMRIID, Ft Detrick, Frederick, MD 21701, U.S.A.). THE CYCLIC peptides produced by some blue-green algae, termed microcystins, have been found to be hepatotoxic. Cattle loss and human health hazards due to these toxins have been reported throughout the world. Accurate information on their structures and properties, etc., were not known. These are required for their specific detection in environmental samples and subsequent decontamination of the sources. Hence, we have elucidated the structures of several blue-green algal toxic peptides using nanomole quantities of the intact peptides by utilizing the state-of-the-art fast atom bombardment tandem mass spectrometry (FAB-MS/MS) techniques in combination with high performance liquid chromatography"thermospraymass spectrometric (LC/MS) methods. This innovative approach could also be applied for investigating other biologically active cyclic peptides and depsipeptides. Rapid, sensitive and specific LC/MS methods of analysis were developed and applied for the trace analysis of microcystins in toxic blooms. Approaches undertaken for the structural elucidation and selective detection of these cyclic peptides will be discussed during this presentation.
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skin when methanol and DMSO were the vehicles. HPLC analysis of the receptor fluid bathing human and guinea-pig skin indicated that more than 80% of the radioactivity was associated with PbTx-3.
Correlation between the enzymatic activity, anticoagulant and antiplatelet effects of phospholipase A 2 isoenzymes from Naja nigricollis crawshawii venom. R. MANJUNATHA Klrql and HERBERT J. EVANS (Department of Biochemistry, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA 23298, U.S.A.). THE THREE phospholipase A 2 isoenzymes from Naja nigricollis crawshawii snake venom inhibit both blood coagulation and platelet aggregation. To investigate the correlation between phospholipid splitting ability of these enzymes and their inhibitory activities, the effects of various preincubation times and the inclusion of EDTA were examined. Preincubation of plasma and thromboplastin with the phospholipase isoenzymes resulted in an increase in Ca2+-initiated clotting time with time of preincubation. Incubation of the isoenzymes with EDTA before their addition to the plasma - thromboplastin mixture reduced the anticoagulant effect. These results indicate that the catalytic activity contributes at least partially to the anticoagulant effect. However, inhibition of platelet aggregation appears to be independent of enzymatic activity since there is no increase in inhibition with time of preincubation of platelets and phospholipases, and inclusion of EDTA has no significant effect on inhibition or aggregation. All three enzymes appear to belong to class B of the platelet affector PLA 2 enzymes as determined by platelet effects, since they do not initiate platelet aggregation.
Secretion of paralytic shellfish toxins from scallop. MASAAKIKODAMA,l TAKEHIKO OGATA,1 SHIGERU SATO,1 NOBUYUKI INOGUCm,2 MIcmo SHIMIZU2 and HIDEO DAIDO2 (tLaboratory of Marine Biological Chemistry, School of Fisheries Sciences, Kitasato University, Iwate 022-01 and 2Iwate Prefectural Fisheries Experimental Station, Iwate 026, Japan). BIVALVESaccumulate paralytic shellfish toxins during the bloom of toxic dinoflagellates. The toxicity of bivalves decreases when the toxic dinoflagellates diminish from their environment. However, the decreasing rate of the toxicity is different from species of bivalves (OsmMA et al., 1982). Scallop, one of the economic species, is a hardly detoxified species. Its low rate of detoxification poses a severe problem to its culture industry. In this study, we describe the toxin secretion from toxic scallop and a device for detoxification of scallop. Specimens of toxic scallop were transplanted to the aquarium with filtered seawater and reared for several days at 10, 15 and 20°C. Aquarium water was replaced every day with freshly prepared filtered seawater. Replaced reared water was concentrated and tested for toxicity by mouse assay. All the samples showed toxicity. Analyses of partially purified toxin revealed the presence of paralytic shellfish toxins (PST), indicating that PST is secreted from scallop. The amounts of toxin secreted from scallop increased with increase of rearing temperature. When scallop were stimulated by instantaneous electric shock, immediate secretion of significant amounts of toxin was induced, showing that instantaneous electric shock accelerates the toxin secretion of scallop. These results suggest that instantaneous electric shock treatment under higher rearing temperature is effective for detoxification of toxic scallop. REFERENCE
OSHIMA,Y., YASUMOTO,T., KODAMA,M., OGATA,T., FUKUYO,Y. and MATSUURA,F. (1982) Bull. Jpn. Soc. Scient. Fish. 48, 525. Cross reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae venoms. F. KORNALIK,1 E. TAaORSKA,1 and D. MEnS2 (1Department of Pathophysiology, Prague CSSR, 2University of Frankfurt, F.R.G.). LETHAL, local and systemic effects of two African Viperidae and four Crotalidae were assayed on mice and rats and expressed as LDso, minimal hemorrhagic dose (MHD) and minimal defibrinating dose (MDD). Two commercial antivenoms: Behringwerke Nord Africa (BN) and Wyeth anticrotalidae (W) and gamma globulins prepared from hyperimmune rabbit serum were tested for their capacity to neutralize the toxic effects of the five venoms. Two LDsoS were mixed with 0.1 ml of various dilutions of antivenoms, injected i.v. to white mice and the death rate after 48 hr recorded. Cross reactivity (antiviperidae antivenom against Crotalidae venom and vice versa) was observed in nearly all cases. It was more evident with BN against Crotalidae than W against Viperidae. No cross reactivity could have been observed with monovalent antivenoms. Nearly a complete cross reactivity was observed with polyvalent as well as monovalent antivenoms in the neutralization of skin hemorrhages. The local effect was neutralized to 0 - 20% if antivenom was mixed with the venom. I.v. injection of any antivenom later than 20 min after the venom had no effect. Only Echis and Bothrops venoms cause defibrination in vivo. The capacity of all antivenoms to neutralize defibrination was very weak. 0.5 ml of antivenom neutralized I MDD only to 40 - 60% if injected
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9th World Congress
simultaneously, to less than 20% when injected 3 hr after the venom. Cross reactivity was more evident with W against Echis than BN against Bothrops.
Cytolytic activity in the venom of the marine snail Conus californicus. ARNOLD S. KREGER (Department of Microbiology and Immunology, Bowman Gray School of Medicine, Winston-Salem, NC 27130, U.S.A.). EXTRACTS prepared from the venom duct of the predatory marine snail Conus californicus possessed cytolytic activity against erythrocytes obtained from twelve different animal species. Sheep, cow, chicken, and goat erythrocytes suspended in isotonic Tris-buffered saline (pH 7.4) were the most sensitive of the erythrocytes examined, and hemolytic activity was enhanced (ca. 33% increase) by the addition of Ca 2+ (10raM, final concentration) to the assay mixtures. The hemolytic activity was stable to lyophilization, was heat-labile (56°C, 30 min) and protease-sensitive, and was inhibited by a mixed bovine brain ganglioside preparation, cardiolipin, and sphingomyelin. Activity was not enhanced by chilling the erythrocytes after incubation with the extract at 37°C ('hot - cold lysis'), and activity was not affected by glycophorin, trypan blue, dithiothreitol, cholesterol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine. The cytolytic toxin(s) responsible for the observed hemolytic activity is antigenic (i.e. elicits the production of hemolysisneutralizing antibody in rabbits), has an apparent mol.wt of ca. 19,600, as estimated by gel filtration, and has isoelectric pH values of 6.0, 6.7 and 7.3.
Tetanus toxin: primary structure, disulfide bridges and proteolytic modifications of the extracellular form. KERSTIN KRIEGLSTEIN,1'2 AGNES HENSCHEN,2 ULRICH WELLER1 and ERNST HABERMANN1 (1Rudolf-Buchheim-Institute for Pharmacology, D-6300 Giessen, F.R.G.; 2Max-Planck-Institute for Biochemistry, D-8033 Martinsried, F.R.G.). A PREREQUISITEfor the understanding of structure-function relationships in tetanus toxin is the knowledge of amino acid sequence, disulfide bridges and proteolytic modifications. The toxin, a highly potent neurotoxin, is synthesized as a 150kDa single-chain protein, and a 1315 residue amino acid sequence has recently been predicted from its cDNA sequence. However, it has also been shown that its isolated extracellular form is composed of two disulfide-linked peptide chains, a 50 kDa and a 100 kDa chain. In the present work the differences between the true protein structure and the predicted structure were elucidated. Proteolytic modification was evaluated by N-terminal sequence analysis of the chains isolated after mercaptolysis and alkylation with vinylpyridine, and by analysis of C-terminal CNBr fragments of the chains. Cys residues with sulfhydryl groups were located by direct alkylation of the toxin with dimethylaminoazobenzeneiodoacetamide followed by CNBr cleavage and identification of labelled fragments. Disulfide-linked Cys residues were located by isolation and sequencing of CNBr fragments identified by their presence in chromatographies of alkylated and absence in those of mercaptolysed-alkylated samples. The positions of Cys residues connecting the chains were determined. For all separations reversed-phase HPLC was used. Posttranslational processing can obviously only be analysed on the proteinchemical level. The results are of importance for understanding the biological activity of tetanus toxin on the molecular level. REFERENCE EISEL, U., JARAUSCH, W., GORETZKI, K., HENSCHEN, A., ENGELS, J., WELLER, U., HUDEL, M., HABERMANN, E. and NIEMANN, H. (1986) EMBO J. 5, 2495.
Tandem mass spectrometric investigations on cyclic peptides. THAIYA KRISHNAMURTHY,1 MARQUERITE E. BROOKS,1 DONALD F. HUNT,2 JEFFREY SHABANOWITZ,2 WAYNE W. CARMICHAEL3 and K. m. MEREISH4 (1U.S. Army Chemical Research, Development, and Engineering Center, Aberdeen Proving Ground, MD 21010-5423; 2University of Virginia, Charlottesville, VA 20903; 3Wright State University, Dayton, OH 45435; 4U.S. Army USAMRIID, Ft Detrick, Frederick, MD 21701, U.S.A.). THE CYCLIC peptides produced by some blue-green algae, termed microcystins, have been found to be hepatotoxic. Cattle loss and human health hazards due to these toxins have been reported throughout the world. Accurate information on their structures and properties, etc., were not known. These are required for their specific detection in environmental samples and subsequent decontamination of the sources. Hence, we have elucidated the structures of several blue-green algal toxic peptides using nanomole quantities of the intact peptides by utilizing the state-of-the-art fast atom bombardment tandem mass spectrometry (FAB-MS/MS) techniques in combination with high performance liquid chromatography"thermospraymass spectrometric (LC/MS) methods. This innovative approach could also be applied for investigating other biologically active cyclic peptides and depsipeptides. Rapid, sensitive and specific LC/MS methods of analysis were developed and applied for the trace analysis of microcystins in toxic blooms. Approaches undertaken for the structural elucidation and selective detection of these cyclic peptides will be discussed during this presentation.