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Inflammatory cell changes in peripheral blood and muscle during acute myonecrosis induced by subcutaneous injection of notechis II-5 (NII-5) from the venom of the Australian tiger snake, Notechis scutatus scutatus
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negative and 13 positive. Only 85% of the negative ST patients received antivenin (AV), while 38.5% of positive ST patients received AV. Of the ST negative patients receiving AV, 18 had acute reactions while all five positive ST patients who received AV developed acute reactions (;(2 p < 0.025). Of the ST negative patients suffering early reactions, seven of eight (87.5%) successfully received more AV. One of the ST positive patients reacting to initial AV treatment received readministration of AV, resulting in a second, more severe reaction. AV can be safely readministered to ST negative patients, but once an ST positive patient reacts to AV, successful AV administration is unlikely.
Cloning and sequence analysis of cDNAs coding for phospholipases A 2 from venom glands of sea- and land-snakes. FP~D~RIC DUCANCEL, GILBERTE GUIGNERY-FRELAT, JEAN-CLAUDE BOULAIN and ANDR~ Mf~NEZ (Service de Biochimie du Drpartement de Biologie, Centre d'Etudes Nucl~aires de Saclay, 91191 - Gif/Yvette, France). VENOMOUSsnakes belonging to Hydrophiidae, Elapidae and Viperidae (vipers and crotals), contain a variety of phospholipases A 2 (PLA2) , some of them being highly toxic. Poly ( A + ) mRNA have been isolated from the venom glands of a variety of sea-snakes and land-snakes. Corresponding cDNAs have been cloned and those encoding PLA 2 precursors have been selectively hybridized, using appropriate DNA probes. Nucleotide sequences have been elucidated. The deduced protein sequences of the PLA 2 precursors will be presented. Technical data associated with cloning experiments of cDNAs encoding snake toxins will be described in detail. REFERENCE GUIGNERY-FRELAT, G., DUCANCEL, F., Mf~NEZ,A. and BOULAIN,J. C. (1987) Nucl. Acid Res. 15, 5892.
Mechanism o f the local action o f the mammalian toxin isolated from a venom apparatus extract of the brown recluse spider (Loxosceles reclusa). MARK n . EDWARDS,i JOSEPH N. BEASLEY2 and COLLIS R. GERENi (Departments of 1Chemistry & Biochemistry and 2Animal Sciences, University of Arkansas, Fayetteville, AR 72701, U.S.A.). CHALLENGEof rabbits i.d. with the mammalian toxin isolated from a venom apparatus extract of brown recluse spiders produces a necrotic lesion which is very slow-healing. An aliquot of an extract of a rabbit skin lesion induced by toxin causes a lesion in a second rabbit. This confirms the report of ELGERT et al. (1974). An extract of that second lesion will induce a lesion in a third rabbit. The lesions induced by extracts decrease in intensity with increasing time betweeen toxin challenge and extract preparation. Tissue from a single rabbit skin lesion induced with toxin was extracted sequentially five times with buffered saline. The intensity of lesions produced by these extracts decreased only marginally between adjacent extracts. The ability of 24 hr lesion extracts to induce lesions in second rabbits is not neutralized by either goat or rabbit anti-brown recluse toxin. After partial purification of the lesion causing activity of these extracts, that activity was neutralized by the goat antiserum and partially neutralized by the rabbit antiserum. Antiserum injected directly into a challenge site at time zero does not eliminate lesion formation. ELISA of complete toxin challenge sites removed at zero time detect only 2% of the injected toxin. These findings suggest that the toxin is bound in vivo, thus becoming cryptic, and that the toxin does remain at the challenge site for a considerable time. This work is supported by NIH GM 33430 and BRSG 2 S07 RR 070101. REFERENCE ELGERT, K. D., ROSS, M. A., CAMPBELL,B. J. and BARRETT, J. T. (1974) Infect. Immun. 10, 1412.
Isolation of a hemorrhagic fraction with a fibrinogenase activity from Cerastes vipera (Sahara Egyptian viper) venom. M. F. EL-ASMAR, H. NASSER, N. SWELAM, E. DAOUD, F. TASH and K. GHONEIM (Research Unit of Natural Toxins, Biochemistry Department Faculty of Medicine, Ain Shams University, Cairo, Egypt). A HEMORRHAGICfraction with a fibrinogenase activity was isolated from Cerastes vipera venom by Sephadex G100 and two cycles of DEAE-Sephadex A50 using different buffer molarities. SDS-PAGE showed one prominent band with a mol.wt of 105,000 and a faint one with a mol.wt of 85,000. The two bands appeared as one band by electrofocusing and showed an isoelectric point of 4. The preparation hydrolyzed fibrinogen, fibrin, albumin and casein. It had a hemorrhagic activity (MHD = 2pg) and an anti-coagulant activity. No phospholipase A2, phosphodiesterase or arginine ester hydrolase activities could be detected. Ca 2 + and Zn 2÷ were needed for its activity and was inhibited by EDTA. The preparation hydrolyzed etA chain of fibrinogen followed by fiB chain.
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differential leukocyte (WBC) count were made. The muscles were examined using histological, histochemical and immunocytochemical methods to define the nature of the invading WBC. In the peripheral blood, the hemoglobin concentration and platelet numbers remained unchanged but there was a marked fall in total WBC count most marked at 18 hr after injection. This reflected a striking drop in the numbers of circulating lymphocytes between 3 and 24 h after injection. There was an early, variable rise in neutrophils, no changes in monocytes were detected. In damaged muscle, neutrophils and macrophages had started to invade necrotic fibres by one hour after injection. Macrophages were the predominant cell type by 6 hr. Lymphocytes did not form a significant part of the infiltrate at any time point, therefore the fall in lymphocytes was not due to migration into the damaged tissue. Neither peripheral blood changes or local necrosis and infiltration with WBC was seen after injection with saline or notechis II-1 (an inactive homologue of NII-5). Lymphopenia has been documented after a tiger snake bite in man (GAYNOR, 1977). Our observations suggest that a falling lymphocyte count could be an early sign of significant envenomation in the clinical situation. REFERENCE GAYNOR, B. (1977) Med. d. Aust. 2, 191.
Similar peptide hepatoxins from four different cyanobacterial genera. JOHN E. ERIKSSON and JuSSl A.O. MERILUOTO (Department of Biology and Department of Biochemistry and Pharmacy Abo Akedemi, SF-20500 Turku, Finland). THE CYTOTOXICITY of peptide toxins isolated from the cyanobacteria Microcystis aeruginosa, Anabaena flosaquae, Oscillatoria agardhii and Nodularia spumigena (ERIKSSON et aL, 1988; ERIKSSON et al., in press; MERILUOTO and ERIKSSON, 1988) was studied with isolated rat hepatocytes. The toxins from all four species induced similar morphological alterations in hepatocytes. The formation of numerous surface swellings (blebs), as earlier reported for toxins from Microcystis (ERIKSSON et aL, 1987), could be observed with all four toxins. These alterations appeared already after a few min and totally disrupted the original cell morphology after 20 - 40 min. During this process no reduction in cell viability could be observed. The morphological alterations were followed by a marked redistribution of actin microfilaments, as demonstrated by immunofluorescence labelling of actin. Although only the cyclic peptide toxins from Microcystis has been characterized structurally in detail, our results indicate that all four toxins are toxicologically related. REFERENCES ERIKSSON, J. E., H.~GERSTRAND,H. and ISOMAA,B. (1987) Biochim. biophys. Acta 930, 304. ERIKSSON, J. E., MERILUOTO,J. A. O., KUJAR1, H. P., ~)STERLUND,K., {~ISTERLUND,K. and H.Z,LLBOM, L. (1988) Toxicon 26, 161. ER1KSSON, J. E., MER1LUOTO,J. A. O., KUJARI, H. P. and SKULBERG,O. M. Comp. Biochem. Physiol., in press. ERIKSSON, J. E., MERILUOTO, J. A. O., KUJARI, H. P., JAMEL AL-LAYL, K. and CODD, G. A. Toxicity Ass. in press. MERILUOTO, J. A. O. and ERIKSSON, J. E. (1988) J. Chromatogr. 438, 93.
Activation of human platelets induced by cerastobin, a thrombin-like enzyme,from the Egyptian Sahara snake viper (Cerastes vipera) venom. TAHER M. FARID,1 ANTHONY T. Tu 1 and M. FARID EL-ASMAR2 (1Biochemistry Department, Colorado State University, Fort Collins, CO 80523, U.S.A.; 2Biochemistry Department, Faculty, of Medicine, Ain Shams University, Abassia, Cairo, Egypt).
CERASTOBIN, a thrombin-like enzyme with arginine esterase activity, was purified from crude Cerastes vipera venom. Unlike thrombin-like enzymes isolated from other snake venoms, cerastobin had a potent platelet aggregatory effect. The activation of human platelets was not related to ADP release and/or prostagiandins synthesis. Cerastobin showed proteolytic activity on towards protein constituents of the platelet's cytoskeleton. It hydrolyzed actin, actin-binding protein, and P23s- This may explain at least, part of the aggregatory mechanism(s) of cerastobin. Electron microscopic studies of the stimulated platelets revealed changes in their morphology including the appearance of pseudopodia, dilatation of the canalicular system with the formation of peripheral balloons, as well as centeralization of the platelet organelles. Some inhibitors of the esteratic activity of cerastobin, also inhibited its ability to aggregate platelets. Characterization of crotoxin isoforms, comparison of their molecular structure and of their biological activities. GRAZYNA FAURE and CASSIANBON (Laboratoire des Venins, Unit6 associre Pasteur/INSERM No. 285 Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France).
CROTOXIN, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus presents an extensive heterogeneity. Fractionation of the venom by high performance liquid chromatography (FPLC), in
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negative and 13 positive. Only 85% of the negative ST patients received antivenin (AV), while 38.5% of positive ST patients received AV. Of the ST negative patients receiving AV, 18 had acute reactions while all five positive ST patients who received AV developed acute reactions (;(2 p < 0.025). Of the ST negative patients suffering early reactions, seven of eight (87.5%) successfully received more AV. One of the ST positive patients reacting to initial AV treatment received readministration of AV, resulting in a second, more severe reaction. AV can be safely readministered to ST negative patients, but once an ST positive patient reacts to AV, successful AV administration is unlikely.
Cloning and sequence analysis of cDNAs coding for phospholipases A 2 from venom glands of sea- and land-snakes. FP~D~RIC DUCANCEL, GILBERTE GUIGNERY-FRELAT, JEAN-CLAUDE BOULAIN and ANDR~ Mf~NEZ (Service de Biochimie du Drpartement de Biologie, Centre d'Etudes Nucl~aires de Saclay, 91191 - Gif/Yvette, France). VENOMOUSsnakes belonging to Hydrophiidae, Elapidae and Viperidae (vipers and crotals), contain a variety of phospholipases A 2 (PLA2) , some of them being highly toxic. Poly ( A + ) mRNA have been isolated from the venom glands of a variety of sea-snakes and land-snakes. Corresponding cDNAs have been cloned and those encoding PLA 2 precursors have been selectively hybridized, using appropriate DNA probes. Nucleotide sequences have been elucidated. The deduced protein sequences of the PLA 2 precursors will be presented. Technical data associated with cloning experiments of cDNAs encoding snake toxins will be described in detail. REFERENCE GUIGNERY-FRELAT, G., DUCANCEL, F., Mf~NEZ,A. and BOULAIN,J. C. (1987) Nucl. Acid Res. 15, 5892.
Mechanism o f the local action o f the mammalian toxin isolated from a venom apparatus extract of the brown recluse spider (Loxosceles reclusa). MARK n . EDWARDS,i JOSEPH N. BEASLEY2 and COLLIS R. GERENi (Departments of 1Chemistry & Biochemistry and 2Animal Sciences, University of Arkansas, Fayetteville, AR 72701, U.S.A.). CHALLENGEof rabbits i.d. with the mammalian toxin isolated from a venom apparatus extract of brown recluse spiders produces a necrotic lesion which is very slow-healing. An aliquot of an extract of a rabbit skin lesion induced by toxin causes a lesion in a second rabbit. This confirms the report of ELGERT et al. (1974). An extract of that second lesion will induce a lesion in a third rabbit. The lesions induced by extracts decrease in intensity with increasing time betweeen toxin challenge and extract preparation. Tissue from a single rabbit skin lesion induced with toxin was extracted sequentially five times with buffered saline. The intensity of lesions produced by these extracts decreased only marginally between adjacent extracts. The ability of 24 hr lesion extracts to induce lesions in second rabbits is not neutralized by either goat or rabbit anti-brown recluse toxin. After partial purification of the lesion causing activity of these extracts, that activity was neutralized by the goat antiserum and partially neutralized by the rabbit antiserum. Antiserum injected directly into a challenge site at time zero does not eliminate lesion formation. ELISA of complete toxin challenge sites removed at zero time detect only 2% of the injected toxin. These findings suggest that the toxin is bound in vivo, thus becoming cryptic, and that the toxin does remain at the challenge site for a considerable time. This work is supported by NIH GM 33430 and BRSG 2 S07 RR 070101. REFERENCE ELGERT, K. D., ROSS, M. A., CAMPBELL,B. J. and BARRETT, J. T. (1974) Infect. Immun. 10, 1412.
Isolation of a hemorrhagic fraction with a fibrinogenase activity from Cerastes vipera (Sahara Egyptian viper) venom. M. F. EL-ASMAR, H. NASSER, N. SWELAM, E. DAOUD, F. TASH and K. GHONEIM (Research Unit of Natural Toxins, Biochemistry Department Faculty of Medicine, Ain Shams University, Cairo, Egypt). A HEMORRHAGICfraction with a fibrinogenase activity was isolated from Cerastes vipera venom by Sephadex G100 and two cycles of DEAE-Sephadex A50 using different buffer molarities. SDS-PAGE showed one prominent band with a mol.wt of 105,000 and a faint one with a mol.wt of 85,000. The two bands appeared as one band by electrofocusing and showed an isoelectric point of 4. The preparation hydrolyzed fibrinogen, fibrin, albumin and casein. It had a hemorrhagic activity (MHD = 2pg) and an anti-coagulant activity. No phospholipase A2, phosphodiesterase or arginine ester hydrolase activities could be detected. Ca 2 + and Zn 2÷ were needed for its activity and was inhibited by EDTA. The preparation hydrolyzed etA chain of fibrinogen followed by fiB chain.
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differential leukocyte (WBC) count were made. The muscles were examined using histological, histochemical and immunocytochemical methods to define the nature of the invading WBC. In the peripheral blood, the hemoglobin concentration and platelet numbers remained unchanged but there was a marked fall in total WBC count most marked at 18 hr after injection. This reflected a striking drop in the numbers of circulating lymphocytes between 3 and 24 h after injection. There was an early, variable rise in neutrophils, no changes in monocytes were detected. In damaged muscle, neutrophils and macrophages had started to invade necrotic fibres by one hour after injection. Macrophages were the predominant cell type by 6 hr. Lymphocytes did not form a significant part of the infiltrate at any time point, therefore the fall in lymphocytes was not due to migration into the damaged tissue. Neither peripheral blood changes or local necrosis and infiltration with WBC was seen after injection with saline or notechis II-1 (an inactive homologue of NII-5). Lymphopenia has been documented after a tiger snake bite in man (GAYNOR, 1977). Our observations suggest that a falling lymphocyte count could be an early sign of significant envenomation in the clinical situation. REFERENCE GAYNOR, B. (1977) Med. d. Aust. 2, 191.
Similar peptide hepatoxins from four different cyanobacterial genera. JOHN E. ERIKSSON and JuSSl A.O. MERILUOTO (Department of Biology and Department of Biochemistry and Pharmacy Abo Akedemi, SF-20500 Turku, Finland). THE CYTOTOXICITY of peptide toxins isolated from the cyanobacteria Microcystis aeruginosa, Anabaena flosaquae, Oscillatoria agardhii and Nodularia spumigena (ERIKSSON et aL, 1988; ERIKSSON et al., in press; MERILUOTO and ERIKSSON, 1988) was studied with isolated rat hepatocytes. The toxins from all four species induced similar morphological alterations in hepatocytes. The formation of numerous surface swellings (blebs), as earlier reported for toxins from Microcystis (ERIKSSON et aL, 1987), could be observed with all four toxins. These alterations appeared already after a few min and totally disrupted the original cell morphology after 20 - 40 min. During this process no reduction in cell viability could be observed. The morphological alterations were followed by a marked redistribution of actin microfilaments, as demonstrated by immunofluorescence labelling of actin. Although only the cyclic peptide toxins from Microcystis has been characterized structurally in detail, our results indicate that all four toxins are toxicologically related. REFERENCES ERIKSSON, J. E., H.~GERSTRAND,H. and ISOMAA,B. (1987) Biochim. biophys. Acta 930, 304. ERIKSSON, J. E., MERILUOTO,J. A. O., KUJAR1, H. P., ~)STERLUND,K., {~ISTERLUND,K. and H.Z,LLBOM, L. (1988) Toxicon 26, 161. ER1KSSON, J. E., MER1LUOTO,J. A. O., KUJARI, H. P. and SKULBERG,O. M. Comp. Biochem. Physiol., in press. ERIKSSON, J. E., MERILUOTO, J. A. O., KUJARI, H. P., JAMEL AL-LAYL, K. and CODD, G. A. Toxicity Ass. in press. MERILUOTO, J. A. O. and ERIKSSON, J. E. (1988) J. Chromatogr. 438, 93.
Activation of human platelets induced by cerastobin, a thrombin-like enzyme,from the Egyptian Sahara snake viper (Cerastes vipera) venom. TAHER M. FARID,1 ANTHONY T. Tu 1 and M. FARID EL-ASMAR2 (1Biochemistry Department, Colorado State University, Fort Collins, CO 80523, U.S.A.; 2Biochemistry Department, Faculty, of Medicine, Ain Shams University, Abassia, Cairo, Egypt).
CERASTOBIN, a thrombin-like enzyme with arginine esterase activity, was purified from crude Cerastes vipera venom. Unlike thrombin-like enzymes isolated from other snake venoms, cerastobin had a potent platelet aggregatory effect. The activation of human platelets was not related to ADP release and/or prostagiandins synthesis. Cerastobin showed proteolytic activity on towards protein constituents of the platelet's cytoskeleton. It hydrolyzed actin, actin-binding protein, and P23s- This may explain at least, part of the aggregatory mechanism(s) of cerastobin. Electron microscopic studies of the stimulated platelets revealed changes in their morphology including the appearance of pseudopodia, dilatation of the canalicular system with the formation of peripheral balloons, as well as centeralization of the platelet organelles. Some inhibitors of the esteratic activity of cerastobin, also inhibited its ability to aggregate platelets. Characterization of crotoxin isoforms, comparison of their molecular structure and of their biological activities. GRAZYNA FAURE and CASSIANBON (Laboratoire des Venins, Unit6 associre Pasteur/INSERM No. 285 Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France).
CROTOXIN, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus presents an extensive heterogeneity. Fractionation of the venom by high performance liquid chromatography (FPLC), in