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Does the specific crotoxin inhibitor from Crotalus durissus terrificus serum behave as a soluble crotoxin acceptor?
720
Report
and Abstracts
Myoglobinuria was observed as early as 30 min and up to 48 hr. Light and electron microscopic studies were done on soleus and gastrocnemius muscles at various time intervals of envenomation. Light microscopic study showed disruption of muscle fibres with hypercontraction, vacuolation, and delta lesions from as early as 5 min. Myonecrosis was severe by 1-7 hr. Peak infiltration by macrophages occurred between 24 and 48 hr. By 5-10 days regeneration was maximal with many myotubes with clear central row of nuclei. Regeneration was almost complete by 3 weeks. Electron microscopic study showed dissolution and degeneration of Z band as early as 5 min to 1 hr. By 24 hr numerous macrophages were seen among the necrotic muscle. The sacrolemmal membrane was disrupted but the basal lamina was intact. By 5 days a clear central row of nuclei was seen; satellite cells were also seen. Regeneration was complete by 3 weeks except for in a few areas. Electron microscopic study of nerve fibres showed degenerative changes by 24 hr onwards with vacuolation of Schwann cell cytoplasm and the axoplasm of the nerve with myehn splitting. Thus the myotoxic PLA, which cause extensive myolysis also have a neurotoxic effect on the nerves. Does the speciJic croroxin inhibitor from Crotalus durissus terrificus serum behave as a soluble crotoxin acceptor? J. Perales,’ C. Villela,’ G. Domont,’ V. Choumet? B. Saliou, * H. Moussatche,’ C. Bon* and G. Faure’ (’ Departamento Fisiologia e Farmacodinamica, Instituto Oswald0 Cruz, Rio de Janeiro, Brazil; and *Unite des Venins, Institut Pasteur, Paris, France). An antivenom protein component that specifically neutralizes crotoxin, the main lethal component of rattlesnake venom, has been identified in the blood of Crotalus durissus tertificus snake. The crotoxin inhibitor has been purified by gel filtration on Sephadex G-200 and Superose 12 columns, and by ion-exchange chromatography on DEAE-Sephacel. It is a glycoprotein of 130 kDa made of the non-covalent association of two polypeptides of 23 and 25 kDa. These two homologous peptides are characterized by the absence of a methionine residue and a high content of acidic, hydrophobic and cystein residues. The neutralizing effect of the purified crotoxin inhibitor has been demonstrated in mice by lethality assays. Biochemical investigations performed in virro indicate that the serum inhibitor (1) efficiently inhibits the phospholipase activity of crotoxin and of its isolated subunit CB; (2) specifically interacts with crotoxin and causes its dissociation; and (3) binds to the phospholipase subunit CB, but does not associate with the acidic chaperon subunit CA. The molecular mechanism of the interaction between the serum inhibitor and crotoxin therefore seems to be very similar to that of crotoxin with its membrane acceptor. It is therefore tempting to suggest that the serum inhibitor acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, preventing its action on the neuromuscular system. Mechanism of action of Lophozozymus (Department of Biochemistry, Faculty
pictor toxin. C. 0. Lau, Q. T. Li, H. E. Khoo, R. Yuen and C. H. Tan of Medicine, National University of Singapore, Singapore).
The xanthid crab Lophozozymus pictor contains a potent water-soluble toxin (LPTX), which is fatal upon ingestion. Besides exhibiting neurotoxic and haemolytic properties, it is also cytotoxic and causes the release of K+ from cells. The toxin was also found to inhibit the uptake of 2-[‘4C]deoxy-o-ghicose into HeLa cells. To obtain an insight into the mechanism of toxin action of LPTX, its effects on mitochondria and hposomes were investigated. LPTX did not cause passive osmotic swelling of mitochondria as measured by light scattering experiments and therefore appeared to lack intrinsic ionophoretic activity. Using fluorescence spectroscopy, it was also found that LPTX did not interact with liposomes containing phosphatidylcholine (PC), phosphatidylserine, PC: sphingomyehn (1: 1)and PC: phosphatidylethanolamine (1: 1).However, ouabain was able to prevent its toxic effects. This antagonistic action of ouabain (a selective inhibitor of the plasma membrane Na+/K+-ATPase) on LPTX action suggests that the toxic activity of LPTX might be mediated via its effect on the membrane ion pump but not through interaction with membrane lipids. Inhibition of synaptosomal uptake of choline and GABA by sea anemone cytolysins. H. E. Khoo, J. P. C. Lim and C. H. Tan (Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore 051 I). Magnificalysin (HMg) I and II and equinatoxin (EqTx) II are cytolytic toxins extracted from the sea anemones Hereractis magnr$ca and Actinia equina, respectively. The toxins inhibited choline and GABA uptake by synaptosomes in a dose-dependent manner but did not cause synaptosomal lysis. The inhibition of both choline and GABA uptake could be overcome by the addition of exogenous sphingomyehn suggesting that there might be interaction between these cytolysins and the phospholipid. In conclusion, although the precise mechanisms involved in cytolysis and inhibition of the neurotransmitter uptake are unknown, they appeared to be different because the inhibitory effect was seen in the absence of lytic activity. Pharmacological activity of four fraciions isolated from the venom of the scorpion Buthus martensi karsch. J. P. Gong,’ R. M. Kini, M. C. E. Gwee: and P. Gopalakrishnakone’ (Venom and Toxin Research Group, Departments of ‘Pharmacology, and *Anatomy, Faculty of Medicine and Bioscience Cent&, Faculty of Science, National University of Singapore, Singapore). Venoms from scorpions are generally complex mixtures of pharmacologically active polypeptides, and several have been documented which cause nerve depolarization with enhancement of adenergic transmission. Four fractions (MK: 1. 2, 3, 4) isolated from the crude venom (MKV) of Burhus martensi karsch by gel filtration
Report
and Abstracts
Myoglobinuria was observed as early as 30 min and up to 48 hr. Light and electron microscopic studies were done on soleus and gastrocnemius muscles at various time intervals of envenomation. Light microscopic study showed disruption of muscle fibres with hypercontraction, vacuolation, and delta lesions from as early as 5 min. Myonecrosis was severe by 1-7 hr. Peak infiltration by macrophages occurred between 24 and 48 hr. By 5-10 days regeneration was maximal with many myotubes with clear central row of nuclei. Regeneration was almost complete by 3 weeks. Electron microscopic study showed dissolution and degeneration of Z band as early as 5 min to 1 hr. By 24 hr numerous macrophages were seen among the necrotic muscle. The sacrolemmal membrane was disrupted but the basal lamina was intact. By 5 days a clear central row of nuclei was seen; satellite cells were also seen. Regeneration was complete by 3 weeks except for in a few areas. Electron microscopic study of nerve fibres showed degenerative changes by 24 hr onwards with vacuolation of Schwann cell cytoplasm and the axoplasm of the nerve with myehn splitting. Thus the myotoxic PLA, which cause extensive myolysis also have a neurotoxic effect on the nerves. Does the speciJic croroxin inhibitor from Crotalus durissus terrificus serum behave as a soluble crotoxin acceptor? J. Perales,’ C. Villela,’ G. Domont,’ V. Choumet? B. Saliou, * H. Moussatche,’ C. Bon* and G. Faure’ (’ Departamento Fisiologia e Farmacodinamica, Instituto Oswald0 Cruz, Rio de Janeiro, Brazil; and *Unite des Venins, Institut Pasteur, Paris, France). An antivenom protein component that specifically neutralizes crotoxin, the main lethal component of rattlesnake venom, has been identified in the blood of Crotalus durissus tertificus snake. The crotoxin inhibitor has been purified by gel filtration on Sephadex G-200 and Superose 12 columns, and by ion-exchange chromatography on DEAE-Sephacel. It is a glycoprotein of 130 kDa made of the non-covalent association of two polypeptides of 23 and 25 kDa. These two homologous peptides are characterized by the absence of a methionine residue and a high content of acidic, hydrophobic and cystein residues. The neutralizing effect of the purified crotoxin inhibitor has been demonstrated in mice by lethality assays. Biochemical investigations performed in virro indicate that the serum inhibitor (1) efficiently inhibits the phospholipase activity of crotoxin and of its isolated subunit CB; (2) specifically interacts with crotoxin and causes its dissociation; and (3) binds to the phospholipase subunit CB, but does not associate with the acidic chaperon subunit CA. The molecular mechanism of the interaction between the serum inhibitor and crotoxin therefore seems to be very similar to that of crotoxin with its membrane acceptor. It is therefore tempting to suggest that the serum inhibitor acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, preventing its action on the neuromuscular system. Mechanism of action of Lophozozymus (Department of Biochemistry, Faculty
pictor toxin. C. 0. Lau, Q. T. Li, H. E. Khoo, R. Yuen and C. H. Tan of Medicine, National University of Singapore, Singapore).
The xanthid crab Lophozozymus pictor contains a potent water-soluble toxin (LPTX), which is fatal upon ingestion. Besides exhibiting neurotoxic and haemolytic properties, it is also cytotoxic and causes the release of K+ from cells. The toxin was also found to inhibit the uptake of 2-[‘4C]deoxy-o-ghicose into HeLa cells. To obtain an insight into the mechanism of toxin action of LPTX, its effects on mitochondria and hposomes were investigated. LPTX did not cause passive osmotic swelling of mitochondria as measured by light scattering experiments and therefore appeared to lack intrinsic ionophoretic activity. Using fluorescence spectroscopy, it was also found that LPTX did not interact with liposomes containing phosphatidylcholine (PC), phosphatidylserine, PC: sphingomyehn (1: 1)and PC: phosphatidylethanolamine (1: 1).However, ouabain was able to prevent its toxic effects. This antagonistic action of ouabain (a selective inhibitor of the plasma membrane Na+/K+-ATPase) on LPTX action suggests that the toxic activity of LPTX might be mediated via its effect on the membrane ion pump but not through interaction with membrane lipids. Inhibition of synaptosomal uptake of choline and GABA by sea anemone cytolysins. H. E. Khoo, J. P. C. Lim and C. H. Tan (Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore 051 I). Magnificalysin (HMg) I and II and equinatoxin (EqTx) II are cytolytic toxins extracted from the sea anemones Hereractis magnr$ca and Actinia equina, respectively. The toxins inhibited choline and GABA uptake by synaptosomes in a dose-dependent manner but did not cause synaptosomal lysis. The inhibition of both choline and GABA uptake could be overcome by the addition of exogenous sphingomyehn suggesting that there might be interaction between these cytolysins and the phospholipid. In conclusion, although the precise mechanisms involved in cytolysis and inhibition of the neurotransmitter uptake are unknown, they appeared to be different because the inhibitory effect was seen in the absence of lytic activity. Pharmacological activity of four fraciions isolated from the venom of the scorpion Buthus martensi karsch. J. P. Gong,’ R. M. Kini, M. C. E. Gwee: and P. Gopalakrishnakone’ (Venom and Toxin Research Group, Departments of ‘Pharmacology, and *Anatomy, Faculty of Medicine and Bioscience Cent&, Faculty of Science, National University of Singapore, Singapore). Venoms from scorpions are generally complex mixtures of pharmacologically active polypeptides, and several have been documented which cause nerve depolarization with enhancement of adenergic transmission. Four fractions (MK: 1. 2, 3, 4) isolated from the crude venom (MKV) of Burhus martensi karsch by gel filtration