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Coagulant and anticoagulant activity of eight standard W.H.O. snake venoms
370
7th European Symposium
which had been abolished by prior treatment with heat (56°C, 30 min), had similar effects on phagocytosis in vivo and in vitro as had SLO in the active form used in our experiments described above. coli and on LT production A. H09TACKA, V. MAJTAN and J. KAROOEK (Research Institute of Preventive Medicine, Bratislava, C.S .S .R .) .
IXfluence of culture media composition on the growth of E.
THE AUTHORS compared the growth of E. coli and production of thermolabile enterotoxin (LT) in different culture media (complete, semisynthetic, synthetic) without antibiotics, as well as in the presence of lincomycin (45 and 90 lAg/ml) . They observed that optimal conditions for growth were not in correspondence with optimal conditions for production of LT . It was shown that the minimal synthetic medium with lincomycin was also suitable for production of LT .
Differences in effectiveness of streptococcal pyrogenic exotoxins on immune system cells. V.
Hi IBALOVA,' D. GERLACH 2 J.-H. OZEGOWSKI,= M. POSPf§IL' and P. PETRA§' ('Institute of Hygiene and Epidemiology and 'Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia, and 'Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of GDR, Jena, G.D .R . COMPARISON of the effectiveness of streptococcal pyrogenic exotoxin types A (SPE A) and C (SPE C) in reactions of cell-mediated immunity in vitro disclosed characteristic differences, depending on the system studied. SPE C was several orders more effective as a stimulant of the production of endogenous pyrogen and migration inhibition factor and as a mitogen for rabbit blood lymphocytes. For human blood lymphocytes, on the contrary, SPE C was less effective, both in newborns and, especially, in adults, in whom sensitization to SPE A (but not to SPE C) due to previous streptococcal infections was a contributory factor . Mousethymocytes were quite refractory to the mitogenic effect of SPE C in most mouse strains . In the latter system an analogy between the effect of SPE C and the staphylococcal toxic-shock-syndrome toxin-1 (TSST-1) was observed . Pathophysiological consequences of the differentiated action of the individual SPE types are discussed.
The use of immunoblotting with monoclonal antibodies in studies of snake venom. D. IDDON,' R. D. G. THEAKSTON' and M. HOMMEL = ( W. H. O. Collaborative Centre for the Control of Antivenoms and 'Wolfson Tropical Immunology Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K . By VIRTUE of their high specificity, monoclonal antibodies are powerful tools for the study of antigen/antibody interactions . Use of monoclonal antibodies in venom research should result in elucidation of many of the mechanisms of snake venom poisoning, how the body's immune system responds to venom antigens and, ultimately, should assist in the treatment of human envenomation . Immunoblotting (TOWBIN et al., 1979) is a very useful technique for analysing the antigen binding characteristics of monoclonal antibodies . Separation of proteins by the SDS Page system of LAEMMLi and FAvRE (1973), prior to immunoblotting, allows high resolution and gives information regarding their molecular weights. The treatment of samples with a reducing agent (in our case dithiothreitol) before electrophoresis brought about destruction of antigenic determinants in Echis carinatus (carpet viper) venom, causing loss of immunoreactivity of the target antigen with appropriate monoclonal antibodies . The currently available methods for visualising antigenic proteins on immunoblots, employing radioactively labelled or enzyme-conjugated probes, were compared using non-reduced venom in order to determine the optimum procedure for venom monoclonal antibody characterisation studies . Examples are presented using a panel of monoclonal antibodies directed towards the venom of Echis carinatus. REFERENCES LAEMMLI, U. K. anti FAvRE, M. F. (1973) J. molec. Biol. 80, 575. TowBIN, H., STACHLIN, T. and GORDON, J. (1979) Proc. natn . Acad.
Sci. U.S.A.
Coagulant and anticoagulant activity of eight standard W.H.O. snake venoms. F. (Institute of Pathophysiology, Charles University, Prague, C.S .S .R.) .
76, 4350 .
KORNAUK and E. TABORSKA
CLOT rING activity was assayed with two methods: W.H .O . recommended and a modification of Quick's teat . By both methods five venoms were procoagulant and three were inactive . The values of Minimal Clotting Dose (MCD), as well as the sequence of the toxins with respect to the activity, differed with the two methods. The discrepancy in MCD is explained by the different substrate-enzyme relation and the required clotting time for one MCD (60 min and 20 min, respectively). Quick's method seems to be more suitable for the antivenom control. Anticoagulant activity in vivo was assayed by determination of the Minimal Defibrinating Dose (MDD) on mice (W .H .O . method) and on rats . With the W.H .O . technique the MDD could be assayed on only three
7th European Symposium
37 1
venoms, in the other four the MDD being higher than the LDx. In rats the MDD ranged from 0.15 pg/ml to 20 pg/ml in the five venoms . A different susceptibility to the clotting enzymes in mice and rats, as well as the different amounts of venom required per body weight for anticoagulabilty, explains the discrepancy. MDD determination on rats seems to be more suitable for the antivenom control.
A study on the cause of death due to sea nettle venom . C . Y. LEE,' S. Y. LEE,= W. W. LIN,' Y. M. CHEN' and J.
W. BURNETI3 (Departments of 'Pharmacology and 'Clinical Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O .C ., and 'Division of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland 21201, U.S .A .) . THE vENom of sea nettle (Chrysaora quinquecirrha) contains several components which exhibit cardiotoxic and neurotoxic activities (BURNETT and GOLDNER, 1969 ; WARmcx et al., 1981). The effects of the venom were further studied on the isolated atrial, aortic ring and langendorff heart preparations of rats, in addition to its cardiovascular effects in anesthetized mice . The venom (0 .5 -1 .0 mg/kg i.v .) produced a prompt increase in arterial blood pressure with ischemic ECG changes and conduction abnormalities, followed by circulatory failure . Artificial respiration failed to maintain theblood pressure . In the Langendorff preparation, 1-10 pg of the venom injected into the perfusion system produced a marked coronary vasospasm and a decrease in contractility and ventricular rate . In the presence of nifedipine (10-' M) or verapamil (10-' M) these effects were largely attenuated . In the atrial preparation, however, no appreciable effects were observed up to 3 pg/ml. At 10 Mg/ml the venom caused a gradual increase in the diastolic tension and a decrease in both contractility and contraction rate . In the aortic ring preparation the venom (1-10 pg/ml) caused a gradual and sustained increase of tension. No recovery was observed after wash-out of the venom. This effect was largely attenuated by nifedipine or verapamil, but not by phenoxybenzamine, atropine, indomethacin, tetrodotoxin or low Na' medium . In Ca"-free medium no increase in tension was produced by the venom. It is concluded that the primary cause of death produced by sea nettle venom is coronary vasospasm, rather than its direct cardiotoxic effects . The vasoconstriction appears to be due to an increase in Ca" influx through voltage-dependent Ca" channels . REFERENCES BuRNETT, J. W. and GOLDNER, R. (1969) Proc. Soc. exp. Biol. Med. 132, 535. WARNICK, J. F., WEINREICH, D. and BURNETT, J . W. (1981) Toxicon 19, 361 .
Scorpion toxins: toxin VII of Tityus serrulatus as a proof that there exists a continuous series of variants having pharmacological selectivities. M. E. DE LImA," M. F. MARTIN,' C. R. DIN& and H. ROCHAT' (Laboratoire de
Biochimie, Facult6 de M6decine Nord, Boulevard Pierre Drammard, 13326 Marseille Cedex 15, France, and 'Departmento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, M.G ., Brazil) . STUDIES of amino acid, immunological (DuFroN and ROCHAT, 1984) and relative conformational data (DuFroN et al., 1986) show that scorpion toxins constitute a series of related conformational variants, whoseresemblances can be related to their pharmacological specificities . In the present work we have verified that Tityus serrulatus toxin VII (Ts VII), the most potent P-toxin to mice purified from the venom of this South American scorpion, is also toxic for blowfly larvae of Muss domestica and completely displaces the radiolabelled insect toxin of Androctonus australis Hector (["'I)AaH IT) from its binding site on a synaptosomal fraction of fly head . We found that the a-toxins (i .e . AaH II), which have been shown to not compete with (125-I)-AaH IT (CORDON et al., 1981), does not displace ["I]Ts VII and has no apparent toxicity to blowfly larvae . Binding studies on the synaptosomal fraction of fly head and tests of toxicity on larvae with two ß-toxins (Css II and Css VI) from Centruroides skffusus syjfusus venom show that Css II is not toxic for larvae and does not displace ['bI]Ts VII from its site, while Css VI, slightly toxic in the same assay, displaces ["I]Ts VII. On the other hand, ['nI]TS VII is displaced by Css VI and Css II on a rat synaptosomal fraction with different affinities . The results support the existence of a correlated series of scorpion toxins and suggest that Ts VII, showing both mammalian and insect toxicities, may be a link which eliminates the gap present between Centruroides P-toxins and AaH IT . REFERENCES DUFFON, M. J. and ROCHAT, H . (1984) J. Molec. Evol. 20, 120. DUFrON, M. J., DRAKE, A. F. and ROCHAT, H. (1986) Biochim. biophys. Acta 569, 16 . CORDON, D., JOUER, E., COURAUD, F. and ZLOTKIN, E. (1984) Blochim. biophys. Acta 775, 349.
7th European Symposium
which had been abolished by prior treatment with heat (56°C, 30 min), had similar effects on phagocytosis in vivo and in vitro as had SLO in the active form used in our experiments described above. coli and on LT production A. H09TACKA, V. MAJTAN and J. KAROOEK (Research Institute of Preventive Medicine, Bratislava, C.S .S .R .) .
IXfluence of culture media composition on the growth of E.
THE AUTHORS compared the growth of E. coli and production of thermolabile enterotoxin (LT) in different culture media (complete, semisynthetic, synthetic) without antibiotics, as well as in the presence of lincomycin (45 and 90 lAg/ml) . They observed that optimal conditions for growth were not in correspondence with optimal conditions for production of LT . It was shown that the minimal synthetic medium with lincomycin was also suitable for production of LT .
Differences in effectiveness of streptococcal pyrogenic exotoxins on immune system cells. V.
Hi IBALOVA,' D. GERLACH 2 J.-H. OZEGOWSKI,= M. POSPf§IL' and P. PETRA§' ('Institute of Hygiene and Epidemiology and 'Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia, and 'Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of GDR, Jena, G.D .R . COMPARISON of the effectiveness of streptococcal pyrogenic exotoxin types A (SPE A) and C (SPE C) in reactions of cell-mediated immunity in vitro disclosed characteristic differences, depending on the system studied. SPE C was several orders more effective as a stimulant of the production of endogenous pyrogen and migration inhibition factor and as a mitogen for rabbit blood lymphocytes. For human blood lymphocytes, on the contrary, SPE C was less effective, both in newborns and, especially, in adults, in whom sensitization to SPE A (but not to SPE C) due to previous streptococcal infections was a contributory factor . Mousethymocytes were quite refractory to the mitogenic effect of SPE C in most mouse strains . In the latter system an analogy between the effect of SPE C and the staphylococcal toxic-shock-syndrome toxin-1 (TSST-1) was observed . Pathophysiological consequences of the differentiated action of the individual SPE types are discussed.
The use of immunoblotting with monoclonal antibodies in studies of snake venom. D. IDDON,' R. D. G. THEAKSTON' and M. HOMMEL = ( W. H. O. Collaborative Centre for the Control of Antivenoms and 'Wolfson Tropical Immunology Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K . By VIRTUE of their high specificity, monoclonal antibodies are powerful tools for the study of antigen/antibody interactions . Use of monoclonal antibodies in venom research should result in elucidation of many of the mechanisms of snake venom poisoning, how the body's immune system responds to venom antigens and, ultimately, should assist in the treatment of human envenomation . Immunoblotting (TOWBIN et al., 1979) is a very useful technique for analysing the antigen binding characteristics of monoclonal antibodies . Separation of proteins by the SDS Page system of LAEMMLi and FAvRE (1973), prior to immunoblotting, allows high resolution and gives information regarding their molecular weights. The treatment of samples with a reducing agent (in our case dithiothreitol) before electrophoresis brought about destruction of antigenic determinants in Echis carinatus (carpet viper) venom, causing loss of immunoreactivity of the target antigen with appropriate monoclonal antibodies . The currently available methods for visualising antigenic proteins on immunoblots, employing radioactively labelled or enzyme-conjugated probes, were compared using non-reduced venom in order to determine the optimum procedure for venom monoclonal antibody characterisation studies . Examples are presented using a panel of monoclonal antibodies directed towards the venom of Echis carinatus. REFERENCES LAEMMLI, U. K. anti FAvRE, M. F. (1973) J. molec. Biol. 80, 575. TowBIN, H., STACHLIN, T. and GORDON, J. (1979) Proc. natn . Acad.
Sci. U.S.A.
Coagulant and anticoagulant activity of eight standard W.H.O. snake venoms. F. (Institute of Pathophysiology, Charles University, Prague, C.S .S .R.) .
76, 4350 .
KORNAUK and E. TABORSKA
CLOT rING activity was assayed with two methods: W.H .O . recommended and a modification of Quick's teat . By both methods five venoms were procoagulant and three were inactive . The values of Minimal Clotting Dose (MCD), as well as the sequence of the toxins with respect to the activity, differed with the two methods. The discrepancy in MCD is explained by the different substrate-enzyme relation and the required clotting time for one MCD (60 min and 20 min, respectively). Quick's method seems to be more suitable for the antivenom control. Anticoagulant activity in vivo was assayed by determination of the Minimal Defibrinating Dose (MDD) on mice (W .H .O . method) and on rats . With the W.H .O . technique the MDD could be assayed on only three
7th European Symposium
37 1
venoms, in the other four the MDD being higher than the LDx. In rats the MDD ranged from 0.15 pg/ml to 20 pg/ml in the five venoms . A different susceptibility to the clotting enzymes in mice and rats, as well as the different amounts of venom required per body weight for anticoagulabilty, explains the discrepancy. MDD determination on rats seems to be more suitable for the antivenom control.
A study on the cause of death due to sea nettle venom . C . Y. LEE,' S. Y. LEE,= W. W. LIN,' Y. M. CHEN' and J.
W. BURNETI3 (Departments of 'Pharmacology and 'Clinical Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O .C ., and 'Division of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland 21201, U.S .A .) . THE vENom of sea nettle (Chrysaora quinquecirrha) contains several components which exhibit cardiotoxic and neurotoxic activities (BURNETT and GOLDNER, 1969 ; WARmcx et al., 1981). The effects of the venom were further studied on the isolated atrial, aortic ring and langendorff heart preparations of rats, in addition to its cardiovascular effects in anesthetized mice . The venom (0 .5 -1 .0 mg/kg i.v .) produced a prompt increase in arterial blood pressure with ischemic ECG changes and conduction abnormalities, followed by circulatory failure . Artificial respiration failed to maintain theblood pressure . In the Langendorff preparation, 1-10 pg of the venom injected into the perfusion system produced a marked coronary vasospasm and a decrease in contractility and ventricular rate . In the presence of nifedipine (10-' M) or verapamil (10-' M) these effects were largely attenuated . In the atrial preparation, however, no appreciable effects were observed up to 3 pg/ml. At 10 Mg/ml the venom caused a gradual increase in the diastolic tension and a decrease in both contractility and contraction rate . In the aortic ring preparation the venom (1-10 pg/ml) caused a gradual and sustained increase of tension. No recovery was observed after wash-out of the venom. This effect was largely attenuated by nifedipine or verapamil, but not by phenoxybenzamine, atropine, indomethacin, tetrodotoxin or low Na' medium . In Ca"-free medium no increase in tension was produced by the venom. It is concluded that the primary cause of death produced by sea nettle venom is coronary vasospasm, rather than its direct cardiotoxic effects . The vasoconstriction appears to be due to an increase in Ca" influx through voltage-dependent Ca" channels . REFERENCES BuRNETT, J. W. and GOLDNER, R. (1969) Proc. Soc. exp. Biol. Med. 132, 535. WARNICK, J. F., WEINREICH, D. and BURNETT, J . W. (1981) Toxicon 19, 361 .
Scorpion toxins: toxin VII of Tityus serrulatus as a proof that there exists a continuous series of variants having pharmacological selectivities. M. E. DE LImA," M. F. MARTIN,' C. R. DIN& and H. ROCHAT' (Laboratoire de
Biochimie, Facult6 de M6decine Nord, Boulevard Pierre Drammard, 13326 Marseille Cedex 15, France, and 'Departmento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, M.G ., Brazil) . STUDIES of amino acid, immunological (DuFroN and ROCHAT, 1984) and relative conformational data (DuFroN et al., 1986) show that scorpion toxins constitute a series of related conformational variants, whoseresemblances can be related to their pharmacological specificities . In the present work we have verified that Tityus serrulatus toxin VII (Ts VII), the most potent P-toxin to mice purified from the venom of this South American scorpion, is also toxic for blowfly larvae of Muss domestica and completely displaces the radiolabelled insect toxin of Androctonus australis Hector (["'I)AaH IT) from its binding site on a synaptosomal fraction of fly head . We found that the a-toxins (i .e . AaH II), which have been shown to not compete with (125-I)-AaH IT (CORDON et al., 1981), does not displace ["I]Ts VII and has no apparent toxicity to blowfly larvae . Binding studies on the synaptosomal fraction of fly head and tests of toxicity on larvae with two ß-toxins (Css II and Css VI) from Centruroides skffusus syjfusus venom show that Css II is not toxic for larvae and does not displace ['bI]Ts VII from its site, while Css VI, slightly toxic in the same assay, displaces ["I]Ts VII. On the other hand, ['nI]TS VII is displaced by Css VI and Css II on a rat synaptosomal fraction with different affinities . The results support the existence of a correlated series of scorpion toxins and suggest that Ts VII, showing both mammalian and insect toxicities, may be a link which eliminates the gap present between Centruroides P-toxins and AaH IT . REFERENCES DUFFON, M. J. and ROCHAT, H . (1984) J. Molec. Evol. 20, 120. DUFrON, M. J., DRAKE, A. F. and ROCHAT, H. (1986) Biochim. biophys. Acta 569, 16 . CORDON, D., JOUER, E., COURAUD, F. and ZLOTKIN, E. (1984) Blochim. biophys. Acta 775, 349.