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Comparative study of the edema-forming activity of Costa Rican snake venoms and its neutralization by a polyvalent antivenom
Second American Symposium
143
artificial lipid membranes produced an increase in membrane conductance through the formation of ionpermeable channels, modulated by the direction and size of the datrical potential differences across the membrane . Similarly to Latrodertus t., Steatodap. venom wan able to stimulate the release of traaamitters from rat brain synaptosomes and neuroaecretory cells . Polydonal antibodies to alpha-latrotoxin did not cross-react in the immunadiffusion teat with Steatoda p. venom . Likewise, an immunofluorexence assay for alpha-latrotoxin with Steatoda p. cephalothor~ax sections was negative, suggesting the absence of alpha-latrotoxin in its venom glands . A molecule of Steatodap. venom highly toxic to insects has been partially characterized as an acidic high molecular weight protein .
Myonecrasis induced by Bothrops saper venom: pathogenesis and trratmart. JosEMnRh Gtm~rzttt z /Instituto Clodomiro Picado, Facultad de Microbiologie, Universidad de Costa Rica, San Jasl, Costa Rirn). STUDIFS performed on the pathogenesis and treatment of myonecrosis induced by Bothrops riper venom are reviewed . A myotoxin has beta isolated from this venom ; it is a monomeric, basic phoapholipase A, which, besides myotoxicity, is anticoagulant and induces edema. The toxin affects akdetai muscle fibers by acting initially on the plasma membrane, disrupting its integrity and causing an efflux of creative and creative kinase and an influx of calcium . This calcium influx is probably responsible for many alterations that eventually result is irreversible cell injury . After plasma membrane damage, the neat of the organelles are affected . Initially myofilaments form dense, clumped manses, which are then redistributed in the cellular space . Mitochondria appear swollen and contain flooculent densities. At later time periods mitochrondria are disrupted . There are many small veaides in the cytoplasm, probably as a consequence of :arcoplasmic reticulum damage . Eventually, necrotic fibers are invaded by phagocytic cells, which may be responsible for further myoförillar degradation . Experiments performed in vitro suggest that the toxin affats muscle cells even in conditions where phoapholipase A activity is inhibited . Besides myotoxidty induced by myotoxias that affect directly akdetal muscle cells, the venom also causes myotoxicity indiraxly, as a consequence of the iachemia resulting from disruption of capillary vessels by hemorrhagic components . Regarding treatment, the polyvalent antivenom used in Costa Rica contains antibodies against B. riper myotoxin . Howtver, if antivenom is administered after venom injection, neutralization of myoaecrosis is only partial, suggesting that the rapid development of mantic damage makes difficult the neutralization by aativenoms .
Isolation mrd partial biochemical and pharmacological chaiacterisatton of a myotaxin jrom the venom of the snake Bothropa aummifer . JosE Mtaxte GunE44Q~ BAUno Loatorv~re and Luts Canins (Inatituto Clodovtiro Picado, Facuhad de Microbiologiâ, Universidad de Coats Rica, San JosE, Costa Rica) .
A titvoroxnv from the venom of the snake Bothrops nummjfer was purified to homogeneity by ion-atchange chromatography on CM~ephadex . The toxin is a basic dieter, with a subunit molauhtr weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis . The toxin lacks phosphoGpase A, aàivity when tested oa egg Yolk lecithin and skeletal muscle homogenates. It induces akdetal muscle damage both In vitro and In vivo. When injected intramuscularly it promotes a drastic increase in serum creative kinase levels ; the iaozyme CKMM is responsible for thin increment . A rapid release of creative and creative kinase was observed when mouse gaatrocnemius muscle wan incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' is the plasma membrane of muscle cells . Moreover, analysis of the done-response data indicates that the mytoxin affala muscle aarcolemma by a 'one hit' mechanism . Skiletal mastic cells are affected by the toxin when calcium is eliminated from the medium . The myotoxin ha: as imravenous t.n of 66.3 pg/16- l8 g min and induces edema when injated in the footpad of min . On the other hand, it is not dually hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes . The myotoxin shows a partial immunologic identity with a myotoxic phoapholipase A isolated from Bothrop~s asps venom . The polyvalent antivenom produced in Costa Rica forms a precipitin arc when confronted with this myotoxin. Supported by Vioerrectoria de Inveatigaci8n, Universidad de Costa Rica .
Comparative study qj the edemo-jorming activity of Costa Rk~an snake vr~nts and its neutralization by a polyvalent antivrnom. José Mwatw Gtrnéate~z, Gusrwo Rais, BalJNO 1..OMOK18, José A . Gerté and Lins Cannas (Inatituto Clodomiro Plcado, Facultad de Microbiologta, Universidad de Costa Rica, San JosE, Conta Rica) .
THe eot:Mw-tnoR~ttra activities of eight Costa Rican crotaline venoma and its neutralization by a polyvalent antiveaom was studied using the mouse footpad teat . All of the veaoms iaduad edema, the highest activity
144
Second American Symposium
being presrnt in the vrnoms of Bothrops picadoi and Bothrop~s lateralis . Whrn experiments were performed with preincubation of venom and antivrnom, neutralization of edema was poor . Moreover, it was observed that, with some venons, edema increased whrn large doses of antivrnom were used . This effect was also observed whrn some vrnoms were incubated with coral snake antivrnom, suggesting that vrnoms may release some pharmacologically active components from antivrnom, since the latter contains tress of alpha-2 and beta globulins. Based on these findings, an ahanative approadt to study neutralization of edema was used ; in this new method antivrnom was injected intravrnoualy before venom administration, thereby avoiding preincubadon . With this technique a much better neutralization of edema was observed, although with some vrnoms it was still poor . Vrnoms contain low molecular weight factors that induce edema, suggesting that lack of immuaogenicity of some componrnts may cause a poor neutralization . However, such rnmponrnts are responsible for only a minor portion of the edema induced by crude vrnoms . It is suggested that experimrnts in which vrnom and antivrnom are preincubated in leafing the neutralization of edema should be avoided and that a more adequate approach may be an independent inoculation of vrnom and antivrnom. Supported by Viarrectorla de Iavestigaddn, Universidad de Costa Rica .
Bruin synaptosornal rreurotransmitta uptakelrrlease 4lfateaf by Mojave toxin and its subrenits Jossrx H~ttA~s, JoxH M~t.ts and At.NV L . Bttmert (Departmrnt of Chemistry, Arizona State University, Tempe, AZ 85287, U .S .A .). Ca"-dependent and phorpholipase A,-active protein snake toxins, e .g. ß-bungarotoxin and crotoxin, have received marked sttrntion for their preayneptic inhibition of neurotransmission in neuromuscular and mammalian central all membranes . Results of a neurotransmitter release study of a purified toxin isolated from the Mojave rattlesnake (Crotalus srutulahv srutulatus), which has lethality comparable to ß-bungarotoxin and crotoxin, reveal a large and immodiau linear release of radio-labdled norepinephrine sad raotonin but a leaner and delayed (2 min) release of aoetylcholine in isolated rat brain synaptosomes. The delayed effort induced by intact Mojave toxin is in contrast to the immediate release reported for P-bungatotoudn (Gtrxn$asma and Jermrrv, 1981) . Further, the late release induced by Mojave toxin does not agree with the inhibition of acaylcholine release reported by Gor" ~ ~r4,axwutorm tt al. (1980) . The Mojave toxin basic aubunit, posrasittg phospholipase A, activity, also inducts release of aatyldtoline equivalent to the intact toxin . Results of the uptake study show intact Mojave tourin to have diffenntial inhibitory effects as all of the dassical neurotrensmittera . Uptake is biphaak. In the preaena of toxin, uptake is rapid in the first phase, reselling about S09s of control for all neurotransmitters except ACh (289x) and Epi ß09s) . The low level inhibition of ACh uptake (289s at S min) by Mojave toxin is in marked eoatrart to ß-bungarotoxin, which induaa almost complete (%9r) inhibition (Gaerrr end $TUMPL, 1973) . Quite different maxima for inhibition wen observed after the second phare of uptake (up to 20 min). The results were : highest, NE (709x); intermediate, GABA (6090, DA (S 19~) and S-HT (619x); lowest, ACh (439x) and Epi (469x) . The non-lethal acid aubunit derived from native Mojave toxin does not affecx neurotransitta uptake, whereas We lethal but leas tactic phospholipase A,-active basic rubunit has one half of the inhibitory effort of the intact toxin . The rerttlta obtained in this study reveal distinctive differrnaa between Mojave toxin end other prerynaptic neurotoxins . GoP~L~trntstflvNCOrre, P ., Hwwooon, B. J., Hot.eaoottt:, S . E ., M~nstt, N . A ., oe S~rrr~xw, S . S . and Tu, A. T. (1980) Br. J. Pharmar. 69, 421 . Ga~xt, L. D. aad STUA~L, W. E. (1973) ln: Aeatomkal Neuroendrocrinology p . 443 (Sntw~t., W . E . and Ga~rrr, L . C ., Edr) . Basel: S. Kerga. Outvueasex, C . B . and Jetvoex, D . J . (1981) J. 1Veurocherrt . 3f, 938 .
Monoclonal antibodies against saxitoxin inhibit binding of 'H aaxitoxin to the sodium dtannel. JoxN F . HEWETaoN and Jo~rave E . Bawr+~ aQ (United Stator Army Medical Research Institute of Infectious Diseases, Fort Derrick, Frederick, MD 21701-3011, U .S .A .) . Moxoci.oNN. aatbodies Prepared against raxitoxin conjugated as keyhole limpet hemocyaain was initially screened by a solid-phase EL1SA and thrn evaluated for their ability to inhibit binding of raxitoutin to the sodium channel. Hybrids were originally screened by as ELISA, with :a:titoxin oogjugated via bovine raum albumin to a solid phase, and 33 loner selected on the basis of podtive aaßtoudn binding; only 18 of these dottea wen also poridve for inhibition of radiolabdled saxitoxln by a sodium dtannd binding array. These 18 hybrids were redoned, with 11 secondary domes picked on the heals of the ELISA . Binding of there 11 aaxitoxin antibodies on
143
artificial lipid membranes produced an increase in membrane conductance through the formation of ionpermeable channels, modulated by the direction and size of the datrical potential differences across the membrane . Similarly to Latrodertus t., Steatodap. venom wan able to stimulate the release of traaamitters from rat brain synaptosomes and neuroaecretory cells . Polydonal antibodies to alpha-latrotoxin did not cross-react in the immunadiffusion teat with Steatoda p. venom . Likewise, an immunofluorexence assay for alpha-latrotoxin with Steatoda p. cephalothor~ax sections was negative, suggesting the absence of alpha-latrotoxin in its venom glands . A molecule of Steatodap. venom highly toxic to insects has been partially characterized as an acidic high molecular weight protein .
Myonecrasis induced by Bothrops saper venom: pathogenesis and trratmart. JosEMnRh Gtm~rzttt z /Instituto Clodomiro Picado, Facultad de Microbiologie, Universidad de Costa Rica, San Jasl, Costa Rirn). STUDIFS performed on the pathogenesis and treatment of myonecrosis induced by Bothrops riper venom are reviewed . A myotoxin has beta isolated from this venom ; it is a monomeric, basic phoapholipase A, which, besides myotoxicity, is anticoagulant and induces edema. The toxin affects akdetai muscle fibers by acting initially on the plasma membrane, disrupting its integrity and causing an efflux of creative and creative kinase and an influx of calcium . This calcium influx is probably responsible for many alterations that eventually result is irreversible cell injury . After plasma membrane damage, the neat of the organelles are affected . Initially myofilaments form dense, clumped manses, which are then redistributed in the cellular space . Mitochondria appear swollen and contain flooculent densities. At later time periods mitochrondria are disrupted . There are many small veaides in the cytoplasm, probably as a consequence of :arcoplasmic reticulum damage . Eventually, necrotic fibers are invaded by phagocytic cells, which may be responsible for further myoförillar degradation . Experiments performed in vitro suggest that the toxin affats muscle cells even in conditions where phoapholipase A activity is inhibited . Besides myotoxidty induced by myotoxias that affect directly akdetal muscle cells, the venom also causes myotoxicity indiraxly, as a consequence of the iachemia resulting from disruption of capillary vessels by hemorrhagic components . Regarding treatment, the polyvalent antivenom used in Costa Rica contains antibodies against B. riper myotoxin . Howtver, if antivenom is administered after venom injection, neutralization of myoaecrosis is only partial, suggesting that the rapid development of mantic damage makes difficult the neutralization by aativenoms .
Isolation mrd partial biochemical and pharmacological chaiacterisatton of a myotaxin jrom the venom of the snake Bothropa aummifer . JosE Mtaxte GunE44Q~ BAUno Loatorv~re and Luts Canins (Inatituto Clodovtiro Picado, Facuhad de Microbiologiâ, Universidad de Coats Rica, San JosE, Costa Rica) .
A titvoroxnv from the venom of the snake Bothrops nummjfer was purified to homogeneity by ion-atchange chromatography on CM~ephadex . The toxin is a basic dieter, with a subunit molauhtr weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis . The toxin lacks phosphoGpase A, aàivity when tested oa egg Yolk lecithin and skeletal muscle homogenates. It induces akdetal muscle damage both In vitro and In vivo. When injected intramuscularly it promotes a drastic increase in serum creative kinase levels ; the iaozyme CKMM is responsible for thin increment . A rapid release of creative and creative kinase was observed when mouse gaatrocnemius muscle wan incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' is the plasma membrane of muscle cells . Moreover, analysis of the done-response data indicates that the mytoxin affala muscle aarcolemma by a 'one hit' mechanism . Skiletal mastic cells are affected by the toxin when calcium is eliminated from the medium . The myotoxin ha: as imravenous t.n of 66.3 pg/16- l8 g min and induces edema when injated in the footpad of min . On the other hand, it is not dually hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes . The myotoxin shows a partial immunologic identity with a myotoxic phoapholipase A isolated from Bothrop~s asps venom . The polyvalent antivenom produced in Costa Rica forms a precipitin arc when confronted with this myotoxin. Supported by Vioerrectoria de Inveatigaci8n, Universidad de Costa Rica .
Comparative study qj the edemo-jorming activity of Costa Rk~an snake vr~nts and its neutralization by a polyvalent antivrnom. José Mwatw Gtrnéate~z, Gusrwo Rais, BalJNO 1..OMOK18, José A . Gerté and Lins Cannas (Inatituto Clodomiro Plcado, Facultad de Microbiologta, Universidad de Costa Rica, San JosE, Conta Rica) .
THe eot:Mw-tnoR~ttra activities of eight Costa Rican crotaline venoma and its neutralization by a polyvalent antiveaom was studied using the mouse footpad teat . All of the veaoms iaduad edema, the highest activity
144
Second American Symposium
being presrnt in the vrnoms of Bothrops picadoi and Bothrop~s lateralis . Whrn experiments were performed with preincubation of venom and antivrnom, neutralization of edema was poor . Moreover, it was observed that, with some venons, edema increased whrn large doses of antivrnom were used . This effect was also observed whrn some vrnoms were incubated with coral snake antivrnom, suggesting that vrnoms may release some pharmacologically active components from antivrnom, since the latter contains tress of alpha-2 and beta globulins. Based on these findings, an ahanative approadt to study neutralization of edema was used ; in this new method antivrnom was injected intravrnoualy before venom administration, thereby avoiding preincubadon . With this technique a much better neutralization of edema was observed, although with some vrnoms it was still poor . Vrnoms contain low molecular weight factors that induce edema, suggesting that lack of immuaogenicity of some componrnts may cause a poor neutralization . However, such rnmponrnts are responsible for only a minor portion of the edema induced by crude vrnoms . It is suggested that experimrnts in which vrnom and antivrnom are preincubated in leafing the neutralization of edema should be avoided and that a more adequate approach may be an independent inoculation of vrnom and antivrnom. Supported by Viarrectorla de Iavestigaddn, Universidad de Costa Rica .
Bruin synaptosornal rreurotransmitta uptakelrrlease 4lfateaf by Mojave toxin and its subrenits Jossrx H~ttA~s, JoxH M~t.ts and At.NV L . Bttmert (Departmrnt of Chemistry, Arizona State University, Tempe, AZ 85287, U .S .A .). Ca"-dependent and phorpholipase A,-active protein snake toxins, e .g. ß-bungarotoxin and crotoxin, have received marked sttrntion for their preayneptic inhibition of neurotransmission in neuromuscular and mammalian central all membranes . Results of a neurotransmitter release study of a purified toxin isolated from the Mojave rattlesnake (Crotalus srutulahv srutulatus), which has lethality comparable to ß-bungarotoxin and crotoxin, reveal a large and immodiau linear release of radio-labdled norepinephrine sad raotonin but a leaner and delayed (2 min) release of aoetylcholine in isolated rat brain synaptosomes. The delayed effort induced by intact Mojave toxin is in contrast to the immediate release reported for P-bungatotoudn (Gtrxn$asma and Jermrrv, 1981) . Further, the late release induced by Mojave toxin does not agree with the inhibition of acaylcholine release reported by Gor" ~ ~r4,axwutorm tt al. (1980) . The Mojave toxin basic aubunit, posrasittg phospholipase A, activity, also inducts release of aatyldtoline equivalent to the intact toxin . Results of the uptake study show intact Mojave tourin to have diffenntial inhibitory effects as all of the dassical neurotrensmittera . Uptake is biphaak. In the preaena of toxin, uptake is rapid in the first phase, reselling about S09s of control for all neurotransmitters except ACh (289x) and Epi ß09s) . The low level inhibition of ACh uptake (289s at S min) by Mojave toxin is in marked eoatrart to ß-bungarotoxin, which induaa almost complete (%9r) inhibition (Gaerrr end $TUMPL, 1973) . Quite different maxima for inhibition wen observed after the second phare of uptake (up to 20 min). The results were : highest, NE (709x); intermediate, GABA (6090, DA (S 19~) and S-HT (619x); lowest, ACh (439x) and Epi (469x) . The non-lethal acid aubunit derived from native Mojave toxin does not affecx neurotransitta uptake, whereas We lethal but leas tactic phospholipase A,-active basic rubunit has one half of the inhibitory effort of the intact toxin . The rerttlta obtained in this study reveal distinctive differrnaa between Mojave toxin end other prerynaptic neurotoxins . GoP~L~trntstflvNCOrre, P ., Hwwooon, B. J., Hot.eaoottt:, S . E ., M~nstt, N . A ., oe S~rrr~xw, S . S . and Tu, A. T. (1980) Br. J. Pharmar. 69, 421 . Ga~xt, L. D. aad STUA~L, W. E. (1973) ln: Aeatomkal Neuroendrocrinology p . 443 (Sntw~t., W . E . and Ga~rrr, L . C ., Edr) . Basel: S. Kerga. Outvueasex, C . B . and Jetvoex, D . J . (1981) J. 1Veurocherrt . 3f, 938 .
Monoclonal antibodies against saxitoxin inhibit binding of 'H aaxitoxin to the sodium dtannel. JoxN F . HEWETaoN and Jo~rave E . Bawr+~ aQ (United Stator Army Medical Research Institute of Infectious Diseases, Fort Derrick, Frederick, MD 21701-3011, U .S .A .) . Moxoci.oNN. aatbodies Prepared against raxitoxin conjugated as keyhole limpet hemocyaain was initially screened by a solid-phase EL1SA and thrn evaluated for their ability to inhibit binding of raxitoutin to the sodium channel. Hybrids were originally screened by as ELISA, with :a:titoxin oogjugated via bovine raum albumin to a solid phase, and 33 loner selected on the basis of podtive aaßtoudn binding; only 18 of these dottea wen also poridve for inhibition of radiolabdled saxitoxln by a sodium dtannd binding array. These 18 hybrids were redoned, with 11 secondary domes picked on the heals of the ELISA . Binding of there 11 aaxitoxin antibodies on