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Effects of equinatoxin on single myelinated nerve fibres
40
First A s i a - Pacific Congress
to investigate an outbreak of disease in quail around Yogyakarta which had many characteristics of a mycotoxicosis, including reduction of disease after change of feed source. Clinically the quail exhibited opisthotonus and legs stretched fully backwards. One sample of feed was submitted at the time of the outbreak for analysis and found to contain 450 ppb aflatoxin BI, 15 ppb aflatoxin B2, 6000 ppb CPA and 500 ppb ochratoxin A. Zearalenone, T2 toxin and deoxynivalenol were not detected. We believe a diagnosis of CPA toxicosis may be warranted based upon the characteristic clinical signs, supported by histopathology and chemical analysis. The toxicological significance of CPA in poultry feeds and the interactive toxicity with afiatoxins should be further investigated.
Tick toxicoses and the causal toxins: tick paralysis. B. F. STONE and J. H. AYLWARD(CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia). PARALYISING ticks representing about 40 species occur in about 15 countries. As part of a study into the feasibility of producing vaccines to protect against paralysing toxicoses, notably the syndrome caused by the Australian paralysis tick, Ixodes holocyclus, the nature of the causal paralysing toxin ('holocyclotoxin') is being investigated. Extracts of whole ticks or of salivary glands have been subjected to chromatographic, electrophoretic and isoelectrofocussing separation methods to attempt isolation of holocyclotoxin. Salivary gland toxin, detoxified by glutaraldehyde, formed the active antigen in an experimental vaccine known from earlier work to stimulate immunity. A proteinaceous paralysing toxin, presumably identical to the original salivary gland toxin used in the successful vaccination trials, has been isolated in size-homogeneous form. Sequence data has also been obtained which should enable the construction of oligonucleotide probes, a copy DNA library from messenger RNA and finally expression of the protein by recombinant DNA techniques. This will enable the assessment of the antigenicity of any candidate protein(s) in detoxified form as a possible basis for a vaccine. An agreement has been negotiated with a manufacturer to produce a vaccine. Prospects for the production of this and other tick paralysis vaccines are reviewed.
Human toxic and allergic reactions due to the Australian paralysis tick Ixodes holocyclus. B. F. STONE,l M. GAUCIl and Y. H. THONG2 (tCSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia, and 2Immunobiology Laboratory, University Department of Child Health, Mater Children's Hospital, South Brisbane, Queensland 4101, Australia). OF APPROXIMATELY60 species of ticks in Australia, 13 species are known to attack humans. Five species may produce serious toxic or allergic effects and those due to the Australian paralysis tick, Ixodes holocyclus, are being studied most intensively. This tick may produce a lethal paralysis or potentially fatal anaphylaxis and as part of research into human reactions to this tick, hypersensitivity is being studied. Skin prick tests are being carried out with whole body extracts prior to similar tests with the active allergens to be separated by SDS acrylamide gel electrophoresis, transferred by Western blotting and identified by immunochemical methods. Radioimmunoassays (RIAs), radioallergosorbent assays (RASTs) and enzyme immunoassays (EIAs) are used to measure IgE levels in serum. IgE specifically released in response to tick allergens has been detected and quantitated most effectively in serum from reactive humans by means of an RIA. Some reactions, previously thought to be allergic, may well be due to vasoactive and chemotactic components of tick salivary secretions. The role of 'holocyclotoxin', the paralysing toxin from I. holocyclus and other bioactive salivary components appearing at the time of early tick attachment, when 'allergic-type' symptoms appear, is being investigated.
Effects o f equinatoxin on single myelinated nerve fibres. D. SUPUT(Institute of Pathophysiology, Faculty of Medicine, U.E.K., Zalo~ka 4, 61105 Ljubljana, Yugoslavia). THREE lethal and cytolytic proteins, named equinatoxins (EqT) I, II and III have been isolated and purified from the sea anemone Actinia equina L. It has been shown that EqT I and EqT II block sodium currents in skeletal muscle fibres of frog (Rana esculenta) in 10-100 pM concentrations, and that EqT III increases the leakage conductance so much that its effects on other membrane conductances cannot be measured reliably. As the mechanism of action of EqT on other excitable membranes is poorly understood, it seemed reasonable to investigate its action on single myelinated nerve fibres. Frog sensory fibres were dissected, mounted to the perspex cell and nodes of Ranvier were voltage clamped at 12°C. One to two minutes after application of either EqT I or EqT II in 10-100 nM concentration a complete block of potassium conductance developed, without noticable changes of the leakage conductance and sodium current amplitude. The block of potassium conductance was reversible, as the potassium current amplitude recovered completely in 2 - 5 min of washout of
First A s i a - Pacific Congress
41
the toxin with the control bathing solution. Effects of EqT III on the nodal membrane conductances were less coherent. Destruction of the nodal membrane, seen as a huge and irreversible increase of the leakage conductance after application of EqT III in 1 - 100 nM concentration was the most regular event. Compared to the frog skeletal muscle fibres, there was no noticahle decrease of the sodium current amplitude after the treatment of nerve fibres with EqT, which might imply that either the structure of the sodium channel or the structure of the membrane is different in nerve and muscle fibres. Higher concentrations of EqT needed to produce an effect on nerves are probably due to the 'buffering' capacity of the myelin sheath for EqT. Results allow the conclusion that EqT I and EqT II are potent potassium channel blockers in the node.
Pathogenesis o f tetanus, clinically relevant new concepts. K. TAKANO and F. KIRCHNER (Department of Physiology and Pathophysiology, University of GOttingen, G6ttingen, F.R.G.). THIS STUDY gives a review of the pathogenesis of the hyperactivity of the motor system during tetanus intoxication primarily of our own experiments on animals. Tetanus toxin at doses of 1 - 100,000 mouse MLD/kg was injected into the muscle or vein of the cat, rabbit or rat for experimental local or general tetanus. Tetanus toxin blocked not only the inhibitory synapses of the spinal motoneurones as had been believed, but also the excitatory synapses at lower doses of tetanus toxin. At the minimal dose which caused local tetanus the monosynaptic reflex was always depressed or blocked. Presynaptic inhibition could be observed in local tetanus during the period when the monosynaptic reflex was present. Many types of spinal inhibition were left intact in general tetanus. The rhythmic activity of alpha as well as gamma motoneurones in the spinal cord in general tetanus could not be observed distally to the level of spinal transection. We conclude that clinically relevant tetanus is most likely of central rather than of spinal origin.
Chemical modification o f phospholipase A2 from the venom o f king brown snake, Pseudechis australis. C. TAKASAKI, A. YANAGITA, A. SUGAMAand N. TAMIYA (Department of Chemistry, Tohoku University, Sendal, Japan). Pseudechis australis phospholipase A2, Pa-I l, (phospholipase activity, 3030 units/mg protein; LDs00.23 gg/g of mouse) is chemically modified and its enzymic and lethal activities are studied. (1) Carbamoylation of lysyl residues. The enzyme protein (with 14 lysyl residues) is carbamoylated with K'~CNO and a mixture of monocarbamoyl derivatives is separated by chromatography on a CM-52 column. Mono-carbamoyl derivatives on 58-, 63-, 81- and 85- lysine residues are isolated and other derivatives obtained in mixtures. 63-carbamoyl lysine Pa-11 shows one tenth of the enzymic and lethal activities as compared to the original protein. 58-carbamoyl lysine Pa-11 has 30°70 higher enzymic activity and slightly lower lethal activity. 81- and 85-carbamoyl derivatives have the same activity as the original protein. (2) Carhoxymethylation of methionine. Pa-ll is carboxymethylated at its only methionine-8 with ICH2COOH in 6 M guanidine at pH 3.0. The derivative shows 7070 and less than 4070 of the enzymic and lethal activities, respectively. (3) Nitrophenylsulphenylation of tryptophan. Pa-I ! is treated with nitrophenylsulphenyl chloride. The modification resulted in complete loss of both enzymic and lethal activities. These results suggest that the enzymic activity is indispensable for the lethal activity, although there is no proportional relationship between the two activities.
Anticholinergic action o f a pedicellarial glycoprotein from the sea urchin Toxopneustes pileolus. M. TAKEI,~ K. ENDOH,l H. NAKAGAWA2 and A. KIMURA2 (~Department of Pharmacology, School of Pharmacy, Tokushima University of Arts and Science, and 2Department of Health Science, Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770, Japan). A PEDICELLARIALglycoprotein (con A fraction) from the sea urchin Toxopneustespileolus has been fractionated by a combination of gel chromatography, ion-exchange chromatography and lectin affinity chromatography. The purpose of this study was to characterize the pharmacological properties of con A fraction in the isolated skeletal muscle preparation. We have found that the pedicellarial extract and gel chromatographic fraction antagonize acetylcholine-inducedcontraction of frog rectus ahdominis. Con A fraction also had the same effect as these fractions. The inhibitory effect of con A fraction was approximately 10 times higher than that of gel chromatographic fraction. In the high K ÷-induced contraction, however, no appreciable effect of con A fraction was observed. Likwise, in the presence of AChE inhibitor, the effect of con A fraction was completely blocked. The present data suggest that the inhibitory action of con A fraction are mediated through the cholinergic receptor sites.
First A s i a - Pacific Congress
to investigate an outbreak of disease in quail around Yogyakarta which had many characteristics of a mycotoxicosis, including reduction of disease after change of feed source. Clinically the quail exhibited opisthotonus and legs stretched fully backwards. One sample of feed was submitted at the time of the outbreak for analysis and found to contain 450 ppb aflatoxin BI, 15 ppb aflatoxin B2, 6000 ppb CPA and 500 ppb ochratoxin A. Zearalenone, T2 toxin and deoxynivalenol were not detected. We believe a diagnosis of CPA toxicosis may be warranted based upon the characteristic clinical signs, supported by histopathology and chemical analysis. The toxicological significance of CPA in poultry feeds and the interactive toxicity with afiatoxins should be further investigated.
Tick toxicoses and the causal toxins: tick paralysis. B. F. STONE and J. H. AYLWARD(CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia). PARALYISING ticks representing about 40 species occur in about 15 countries. As part of a study into the feasibility of producing vaccines to protect against paralysing toxicoses, notably the syndrome caused by the Australian paralysis tick, Ixodes holocyclus, the nature of the causal paralysing toxin ('holocyclotoxin') is being investigated. Extracts of whole ticks or of salivary glands have been subjected to chromatographic, electrophoretic and isoelectrofocussing separation methods to attempt isolation of holocyclotoxin. Salivary gland toxin, detoxified by glutaraldehyde, formed the active antigen in an experimental vaccine known from earlier work to stimulate immunity. A proteinaceous paralysing toxin, presumably identical to the original salivary gland toxin used in the successful vaccination trials, has been isolated in size-homogeneous form. Sequence data has also been obtained which should enable the construction of oligonucleotide probes, a copy DNA library from messenger RNA and finally expression of the protein by recombinant DNA techniques. This will enable the assessment of the antigenicity of any candidate protein(s) in detoxified form as a possible basis for a vaccine. An agreement has been negotiated with a manufacturer to produce a vaccine. Prospects for the production of this and other tick paralysis vaccines are reviewed.
Human toxic and allergic reactions due to the Australian paralysis tick Ixodes holocyclus. B. F. STONE,l M. GAUCIl and Y. H. THONG2 (tCSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia, and 2Immunobiology Laboratory, University Department of Child Health, Mater Children's Hospital, South Brisbane, Queensland 4101, Australia). OF APPROXIMATELY60 species of ticks in Australia, 13 species are known to attack humans. Five species may produce serious toxic or allergic effects and those due to the Australian paralysis tick, Ixodes holocyclus, are being studied most intensively. This tick may produce a lethal paralysis or potentially fatal anaphylaxis and as part of research into human reactions to this tick, hypersensitivity is being studied. Skin prick tests are being carried out with whole body extracts prior to similar tests with the active allergens to be separated by SDS acrylamide gel electrophoresis, transferred by Western blotting and identified by immunochemical methods. Radioimmunoassays (RIAs), radioallergosorbent assays (RASTs) and enzyme immunoassays (EIAs) are used to measure IgE levels in serum. IgE specifically released in response to tick allergens has been detected and quantitated most effectively in serum from reactive humans by means of an RIA. Some reactions, previously thought to be allergic, may well be due to vasoactive and chemotactic components of tick salivary secretions. The role of 'holocyclotoxin', the paralysing toxin from I. holocyclus and other bioactive salivary components appearing at the time of early tick attachment, when 'allergic-type' symptoms appear, is being investigated.
Effects o f equinatoxin on single myelinated nerve fibres. D. SUPUT(Institute of Pathophysiology, Faculty of Medicine, U.E.K., Zalo~ka 4, 61105 Ljubljana, Yugoslavia). THREE lethal and cytolytic proteins, named equinatoxins (EqT) I, II and III have been isolated and purified from the sea anemone Actinia equina L. It has been shown that EqT I and EqT II block sodium currents in skeletal muscle fibres of frog (Rana esculenta) in 10-100 pM concentrations, and that EqT III increases the leakage conductance so much that its effects on other membrane conductances cannot be measured reliably. As the mechanism of action of EqT on other excitable membranes is poorly understood, it seemed reasonable to investigate its action on single myelinated nerve fibres. Frog sensory fibres were dissected, mounted to the perspex cell and nodes of Ranvier were voltage clamped at 12°C. One to two minutes after application of either EqT I or EqT II in 10-100 nM concentration a complete block of potassium conductance developed, without noticable changes of the leakage conductance and sodium current amplitude. The block of potassium conductance was reversible, as the potassium current amplitude recovered completely in 2 - 5 min of washout of
First A s i a - Pacific Congress
41
the toxin with the control bathing solution. Effects of EqT III on the nodal membrane conductances were less coherent. Destruction of the nodal membrane, seen as a huge and irreversible increase of the leakage conductance after application of EqT III in 1 - 100 nM concentration was the most regular event. Compared to the frog skeletal muscle fibres, there was no noticahle decrease of the sodium current amplitude after the treatment of nerve fibres with EqT, which might imply that either the structure of the sodium channel or the structure of the membrane is different in nerve and muscle fibres. Higher concentrations of EqT needed to produce an effect on nerves are probably due to the 'buffering' capacity of the myelin sheath for EqT. Results allow the conclusion that EqT I and EqT II are potent potassium channel blockers in the node.
Pathogenesis o f tetanus, clinically relevant new concepts. K. TAKANO and F. KIRCHNER (Department of Physiology and Pathophysiology, University of GOttingen, G6ttingen, F.R.G.). THIS STUDY gives a review of the pathogenesis of the hyperactivity of the motor system during tetanus intoxication primarily of our own experiments on animals. Tetanus toxin at doses of 1 - 100,000 mouse MLD/kg was injected into the muscle or vein of the cat, rabbit or rat for experimental local or general tetanus. Tetanus toxin blocked not only the inhibitory synapses of the spinal motoneurones as had been believed, but also the excitatory synapses at lower doses of tetanus toxin. At the minimal dose which caused local tetanus the monosynaptic reflex was always depressed or blocked. Presynaptic inhibition could be observed in local tetanus during the period when the monosynaptic reflex was present. Many types of spinal inhibition were left intact in general tetanus. The rhythmic activity of alpha as well as gamma motoneurones in the spinal cord in general tetanus could not be observed distally to the level of spinal transection. We conclude that clinically relevant tetanus is most likely of central rather than of spinal origin.
Chemical modification o f phospholipase A2 from the venom o f king brown snake, Pseudechis australis. C. TAKASAKI, A. YANAGITA, A. SUGAMAand N. TAMIYA (Department of Chemistry, Tohoku University, Sendal, Japan). Pseudechis australis phospholipase A2, Pa-I l, (phospholipase activity, 3030 units/mg protein; LDs00.23 gg/g of mouse) is chemically modified and its enzymic and lethal activities are studied. (1) Carbamoylation of lysyl residues. The enzyme protein (with 14 lysyl residues) is carbamoylated with K'~CNO and a mixture of monocarbamoyl derivatives is separated by chromatography on a CM-52 column. Mono-carbamoyl derivatives on 58-, 63-, 81- and 85- lysine residues are isolated and other derivatives obtained in mixtures. 63-carbamoyl lysine Pa-11 shows one tenth of the enzymic and lethal activities as compared to the original protein. 58-carbamoyl lysine Pa-11 has 30°70 higher enzymic activity and slightly lower lethal activity. 81- and 85-carbamoyl derivatives have the same activity as the original protein. (2) Carhoxymethylation of methionine. Pa-ll is carboxymethylated at its only methionine-8 with ICH2COOH in 6 M guanidine at pH 3.0. The derivative shows 7070 and less than 4070 of the enzymic and lethal activities, respectively. (3) Nitrophenylsulphenylation of tryptophan. Pa-I ! is treated with nitrophenylsulphenyl chloride. The modification resulted in complete loss of both enzymic and lethal activities. These results suggest that the enzymic activity is indispensable for the lethal activity, although there is no proportional relationship between the two activities.
Anticholinergic action o f a pedicellarial glycoprotein from the sea urchin Toxopneustes pileolus. M. TAKEI,~ K. ENDOH,l H. NAKAGAWA2 and A. KIMURA2 (~Department of Pharmacology, School of Pharmacy, Tokushima University of Arts and Science, and 2Department of Health Science, Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770, Japan). A PEDICELLARIALglycoprotein (con A fraction) from the sea urchin Toxopneustespileolus has been fractionated by a combination of gel chromatography, ion-exchange chromatography and lectin affinity chromatography. The purpose of this study was to characterize the pharmacological properties of con A fraction in the isolated skeletal muscle preparation. We have found that the pedicellarial extract and gel chromatographic fraction antagonize acetylcholine-inducedcontraction of frog rectus ahdominis. Con A fraction also had the same effect as these fractions. The inhibitory effect of con A fraction was approximately 10 times higher than that of gel chromatographic fraction. In the high K ÷-induced contraction, however, no appreciable effect of con A fraction was observed. Likwise, in the presence of AChE inhibitor, the effect of con A fraction was completely blocked. The present data suggest that the inhibitory action of con A fraction are mediated through the cholinergic receptor sites.