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Molecular pharmacology of clostridial toxins: a short overview
Tenth World Congress
485
resistance factors. In the initial set of experiments 82 and 108 strains produced enterotoxin and beta-haemolysin, respectively, regardless of their species designation and source of isolation. Enterotoxic activity was independent of the type of haemolysin production; however, the beta-haemolytic strains caused significantly more (P < 0.05) toxigenic response than the alpha- and non-haemolytic isolates. Aeromenas hydrophila and A. sobria elaborated more (P < 0.05) exterotoxin and beta-haemolysin than A. caviae. The Aeromonas strains of the three species that included all haemolytic types and failed to elaborate enterotoxin in the initial experiments did so after 1-3 passages through rabbit gut. The alpha- and non-haemolytic strains switched over to production of betahaemolysin once they showed positive ileal loop reaction; however, on repeated subcultures or on storage all of them reverted back to their original baemolytic characters. All the Aeromonas strains tested adhered to intestinal epithelial cells irrespective of species differences and source of isolation and caused agglutination of human, O, erythrocytes. A number of clinical and environmental strains invaded Hela ceils. Resistance to normal human serum was common amongst Aeromonas species. The data indicate that Aeromonas strains possess a number of virulence factors independent of their species designation and sources of isolation.
Nerve growth factors from snake venoms. Comparative aspects. J. SIIGUR, U. ARUMt~E,E. SI1GUR,V. PAALME,T. NEUMANand M. SAARMA(Institute of Chemical Physics and Biophysics, Estonian Academy of Sciences, Tallinn, Estonia). NERVE growth factor (NGF) is a neurotrophic protein with well-established survival effects, differentiation and maintenance function of sympathetic and embryonic sensory neurons. Vipera lebetina snake venom NGF was purified to homogeneity as judged by SDS electrophoresis. The purified NGF was used for producing monoclonal antibodies (MAbs). MAbs were purified by affinity chromatography using V. lebetina venom NGF-agarose and used for characterizing NGFs from snake venoms. Two isolated MAbs define two partially overlapping epitopes of the V. lebetina venom NGF which are not involved in the biological activity of the molecule. Both epitopes are also present on the/~NGF from the mouse salivary gland and on the NGFs from ten snake venoms but not on the bovine seminal plasma NGF. MAb cross-reacting with ten snake venom NGFs was linked to BrCN-activated agarose, and this immunoaffinity material was used for purifying NGF from V. lebetina, V. berus berus, V. rusellii, N. naja oxiana, N. naja, N. naja atra, Bungarus caeruleus and Agkistrodon halys venom. With this MAb NGF was also detected in the homogenates of salivary and venom glands of V. lebetina snake and salivary glands of non-poisonous snakes Elaphe dione and Natrix tessellata. The activities in the salivary glands of V. lebetina and E. dione were also revealed in bioassay with sensory ganglia of 9-day-old chick embryos and on primed rat pheochromocytoma PC 12 cells. The mol.wts of the NGF in nine snake venoms and in salivary glands of V. lebetina and non-poisonous snake E. dione were determined by Western immunoblot analysis with MAbs. These results show that NGF in all snake glands studied is synthesized in large precursor form which is cleaved into smaller intermediates. Using our results and literature data the classification of snake venom NGF is proposed.
Molecular pharmacology of clostridial toxins: a short overview. L.L. SIMPSON (Division of Environmental Medicine and Toxicology, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107, U.S.A.). CLOSTRIDIA produce numerous toxins that act on membranes and in the cytosol of eucaryotic cells. Experiments will be described dealing with the structure and biological activity of two groups of clostridial toxins, the neurotoxins and the binary toxins. Comparison data will also be presented on neurotoxins of snake venom origin. The identity of receptor(s) for clostridial neurotoxins remains unknown. However, certain lectins antagonize the binding of all neurotoxins, both peripherally and centrally. Clostridial neurotoxins are internalized by endocytosis and released into the cytosol via a pH-dependent step. Reductions in pH produce conformational changes in the holotoxins and the isolated chains, and they promote transitions from an aqueous to an organic phase. The intracellular poisoning effect of the neurotoxins remains to be determined, but in some cells it is coincident with changes in protein kinase C activity. Work has been done to compare the neurotoxins with the binary toxins, as well as with snake toxins. The binary toxins are ADP-ribosyltransferases that modify G-actin. The target for/n vivo pathophysiology appears to be endothelial cells of resistance vessels, but many other cells are also vulnerable. Whereas the neurotoxins always block mediator release, the binary toxins have variable effects on mediator release. Certain snake neurotoxins bind to ion channels, express phospholipase A2 activity, and block transmitter release. Clostridial neurotoxins do not bind to the same sites on ion channels, do not express phospholipase A 2 activity, and have a fundamentally different mechanism for blocking transmitter release.
485
resistance factors. In the initial set of experiments 82 and 108 strains produced enterotoxin and beta-haemolysin, respectively, regardless of their species designation and source of isolation. Enterotoxic activity was independent of the type of haemolysin production; however, the beta-haemolytic strains caused significantly more (P < 0.05) toxigenic response than the alpha- and non-haemolytic isolates. Aeromenas hydrophila and A. sobria elaborated more (P < 0.05) exterotoxin and beta-haemolysin than A. caviae. The Aeromonas strains of the three species that included all haemolytic types and failed to elaborate enterotoxin in the initial experiments did so after 1-3 passages through rabbit gut. The alpha- and non-haemolytic strains switched over to production of betahaemolysin once they showed positive ileal loop reaction; however, on repeated subcultures or on storage all of them reverted back to their original baemolytic characters. All the Aeromonas strains tested adhered to intestinal epithelial cells irrespective of species differences and source of isolation and caused agglutination of human, O, erythrocytes. A number of clinical and environmental strains invaded Hela ceils. Resistance to normal human serum was common amongst Aeromonas species. The data indicate that Aeromonas strains possess a number of virulence factors independent of their species designation and sources of isolation.
Nerve growth factors from snake venoms. Comparative aspects. J. SIIGUR, U. ARUMt~E,E. SI1GUR,V. PAALME,T. NEUMANand M. SAARMA(Institute of Chemical Physics and Biophysics, Estonian Academy of Sciences, Tallinn, Estonia). NERVE growth factor (NGF) is a neurotrophic protein with well-established survival effects, differentiation and maintenance function of sympathetic and embryonic sensory neurons. Vipera lebetina snake venom NGF was purified to homogeneity as judged by SDS electrophoresis. The purified NGF was used for producing monoclonal antibodies (MAbs). MAbs were purified by affinity chromatography using V. lebetina venom NGF-agarose and used for characterizing NGFs from snake venoms. Two isolated MAbs define two partially overlapping epitopes of the V. lebetina venom NGF which are not involved in the biological activity of the molecule. Both epitopes are also present on the/~NGF from the mouse salivary gland and on the NGFs from ten snake venoms but not on the bovine seminal plasma NGF. MAb cross-reacting with ten snake venom NGFs was linked to BrCN-activated agarose, and this immunoaffinity material was used for purifying NGF from V. lebetina, V. berus berus, V. rusellii, N. naja oxiana, N. naja, N. naja atra, Bungarus caeruleus and Agkistrodon halys venom. With this MAb NGF was also detected in the homogenates of salivary and venom glands of V. lebetina snake and salivary glands of non-poisonous snakes Elaphe dione and Natrix tessellata. The activities in the salivary glands of V. lebetina and E. dione were also revealed in bioassay with sensory ganglia of 9-day-old chick embryos and on primed rat pheochromocytoma PC 12 cells. The mol.wts of the NGF in nine snake venoms and in salivary glands of V. lebetina and non-poisonous snake E. dione were determined by Western immunoblot analysis with MAbs. These results show that NGF in all snake glands studied is synthesized in large precursor form which is cleaved into smaller intermediates. Using our results and literature data the classification of snake venom NGF is proposed.
Molecular pharmacology of clostridial toxins: a short overview. L.L. SIMPSON (Division of Environmental Medicine and Toxicology, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107, U.S.A.). CLOSTRIDIA produce numerous toxins that act on membranes and in the cytosol of eucaryotic cells. Experiments will be described dealing with the structure and biological activity of two groups of clostridial toxins, the neurotoxins and the binary toxins. Comparison data will also be presented on neurotoxins of snake venom origin. The identity of receptor(s) for clostridial neurotoxins remains unknown. However, certain lectins antagonize the binding of all neurotoxins, both peripherally and centrally. Clostridial neurotoxins are internalized by endocytosis and released into the cytosol via a pH-dependent step. Reductions in pH produce conformational changes in the holotoxins and the isolated chains, and they promote transitions from an aqueous to an organic phase. The intracellular poisoning effect of the neurotoxins remains to be determined, but in some cells it is coincident with changes in protein kinase C activity. Work has been done to compare the neurotoxins with the binary toxins, as well as with snake toxins. The binary toxins are ADP-ribosyltransferases that modify G-actin. The target for/n vivo pathophysiology appears to be endothelial cells of resistance vessels, but many other cells are also vulnerable. Whereas the neurotoxins always block mediator release, the binary toxins have variable effects on mediator release. Certain snake neurotoxins bind to ion channels, express phospholipase A2 activity, and block transmitter release. Clostridial neurotoxins do not bind to the same sites on ion channels, do not express phospholipase A 2 activity, and have a fundamentally different mechanism for blocking transmitter release.