Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Crotoxin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1995

Specificity and binding affinity of an anti-crotoxin combinatorial antibody selected from a phage-displayed library.

A crotoxin-specific, monoclonal, high-affinity, single-chain antibody variable region (scFv) was generated by combinatorial methods using Pharmacia's Recombinant Phage Antibody System. A high-affinity clone, designated A10G, was selected, and its DNA sequence was determined. Protein A10G showed high reaction specificity, with only the closely related rattlesnake neurotoxins, concolor toxin and Mojave toxin, showing cross-reactivity out of eleven group II phospholipase A2s (PLA2s) screened. No group I PLA2s cross-reacted in enzyme-linked immunosorbent assays. The gene coding for A10G was subcloned into an expression vector, and the resulting expressed nonfusion protein, designated A10GPE, was renatured and purified to apparent homogeneity. Dissociation constants of A10G with intact crotoxin and crotoxin basic subunit were determined to be 7 x 10(-10) and 6.8 x 10(-9) M, respectively. When A10GPE was preincubated with either the basic subunit or intact crotoxin at molar ratios of up to 5:1, no inhibition of phospholipase activity was observed. Expressed protein, however, could partially neutralize the lethality of Mojave toxin, a crotoxin homolog, in mice.

Amino Acid Sequence↗
PubMed1989

Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab).

Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate.

Antibodies↗
PubMed1989

The effect of crotoxin on the longitudinal muscle-myenteric plexus preparation of the guinea pig ileum.

The effects of crotoxin, the neurotoxin of the venom of the South American rattlesnake (Crotalus durissus terrificus), was studied by using the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. Crotoxin (0.02-4.0 microM) caused depression of the twitch response of the electrically stimulated preparation. This transitory depression depended on the concentration of crotoxin; since crotoxin diminished the output of acetylcholine, this depression may be due to the inhibition of the release of acetylcholine from the plexus. Crotoxin also induced an early contraction, followed by relaxation; as the contraction was inhibited by aspirin and indomethacin, it may have resulted from the release of prostaglandin. In addition, a late persistent contracture was observed after the early contraction. The contracture was resistant to blockage by muscarinic, histamine and serotonin antagonists, to hexamethonium, a non-depolarizing ganglionic blocking substance and to tetrodotoxin, a sodium channel blocker. The contracture was blocked by an elevated concentration of calcium (10 mM) and by verapamil, a calcium channel blocker.

Acetylcholine↗
PubMed1987

Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus.

Crotalus durissus terrificus venoms collected either from individual snakes or from a large number of animals (more than 30) have been fractionated by high performance liquid chromatography on gel-filtration and ion exchange columns. The chromatographic patterns obtained with individual venom samples indicated that each Crotalus durissus terrificus snake synthesizes five to ten different crotoxin isoforms in widely variable relative proportions. Furthermore, the heterogeneity of venom samples collected from a large number of snakes did not appear significantly larger than that observed with venoms obtained from individual snakes. The comparison of the chromatographic patterns that we obtained with the various (individual and pooled) venoms allowed us to identify about 15 crotoxin isoforms, which may result from the expression of isogenes, since two amino acid variants have been reported to occur at several positions in the sequence of crotoxin component B. These observations confirm the existence of numerous molecular isoforms of crotoxin and suggest that an individual Crotalus durissus terrificus snake possesses several genes coding for the various crotoxin isoforms. The heterogeneity of venom samples collected from a large number of animals is explained, in a large measure, by the complexity of the venom obtained from the individual snakes.

Animals↗
PubMed1990

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions.

Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca2+ plus tubocurarine or 0.5 mM Ca2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Protection against the lethal effects of Crotalus durissus terrificus (South American rattlesnake) venom in animals immunized with crotoxin.

Mice and rabbits were immunized against crotoxin (the neurotoxic component isolated from Crotalus durissus terrificus venom) using small amounts of antigen in a water-in-oil emulsion. Following boosting (three times at 21-day intervals) a high titre of antibodies against crotoxin was obtained. Crotoxin immunoglobulin G antibody recognizes whole venom antigen at a level comparable with that of crotoxin antigen, using the ELISA method for antibody detection. The antibodies generated by crotoxin were capable of providing 100% protection against challenge with 11 and 50 i.p. LD50 doses of whole venom in mice. When 100 i.p. LD50 doses of whole venom were injected survival was 77.8%.

Animals↗
PubMedDate not supplied

Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies.

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the "toxic" and the "catalytic" sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.

Amino Acid Sequence↗
PubMed2004

Immunosuppresive role of principal toxin (crotoxin) of Crotalus durissus terrificus venom.

The composition of the crotalic venom and the immunochemistry and/or pathophysiological characterization and main components were well studied. However, few studies have been carried out to investigate the effect of toxins of this venom on the development of the immune response. The objective of this work was to find out if venom or crotoxin of Crotalus durissus terrificus was able to modulate the immune response through its ability to change the mediators involved in the immune response by an unrelated antigen. We observed in the murine model, that venom as well as crotoxin have inhibitory effect on splenic cells proliferation induced by Con-A. Moreover, CB did not inhibit the proliferative response, suggesting that the integrity of crotoxin complex is necessary for the development of this phenomenon. Moreover, we showed that the effect on cellular proliferation was unrelated to cytotoxicity activity. We also observed that venom or crotoxin inhibited cytokine release induced in HSA immunised mice, mainly IL-2, IL-4 and IL-10, however, crotoxin did not inhibit the release of IFN-gamma. The involvement of T or B cells in the suppressive effect of venom was evaluated through the transference of purified splenic cells from venom-mice to normal mice that also produced low IgG1 anti-HSA levels, indicating the participation of these cells in this process. Mechanism of action of the crotalic venom on development of immune response to an unrelated antigen is much more complex, therefore it must not only involve the interaction of distinct cellular populations, but activation or inhibition of signalling proteins, need to be further investigated.

Animals↗
PubMed2002

Analysis of fatty acids released by crotoxin in rat brain synaptosomes.

Crotoxin, the main toxin of Crotalus durissus terrificus venom, exerts its lethal effect by blocking neurotransmission at the neuromuscular junction level through a triphasic mechanism. This effect seems to depend on its phospholipasic activity, suggesting that the mechanism of neurotransmission blockage may be related to fatty acids release in specific sites of the nervous terminal. In this work, we purified the fatty acids released by crotoxin's activity and this outline was compared with other phospholipases A(2), including CB, a subunit of crotoxin. Our results show a higher release of palmitate and arachidonate by crotoxin when compared to other phospholipases A(2). Since palmitate has a role in protein acylation processes and arachidonate participates in signal transduction events, these mechanisms may be related to the neurotoxic actions of crotoxin.

Animals↗
PubMed1998

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation.

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 microg/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum.

Animals↗
PubMed1991

Identification of a new binding protein for crotoxin and other neurotoxic phospholipase A2s on brain synaptic membranes.

Crotoxin and other neurotoxic phospholipase A2s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several others to synaptic membranes has been demonstrated previously. In this study we used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After 125I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of approximately 60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in a previous report. The membrane component in the conjugate was shown to be a single-chain protein of approximately 45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of around 1 x 10(-8) M. Mojave toxin and some other phospholipase A2s were also highly inhibitory to this conjugation, and notexin and others were less effective, while beta-bungarotoxin and pancreatic PLA2 were totally ineffective. We concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A2s.

Animals↗
PubMed2001

Effect of membrane composition and of co-encapsulation of immunostimulants in a liposome-entrapped crotoxin.

Crotoxin isolated from the venom of Crotalus durissus terrificus (South American rattlesnake) was incorporated into liposomes by the dehydration-rehydration vesicle method using different membrane compositions and the co-encapsulation of immunostimulants. Crotoxin was also encapsulated into liposomes formed from a non-phospholipid amphiphile, a mixture of polyoxyethylene 2-cetyl ether, dicetyl phosphate and cholesterol. The preparations were characterized in relation to stability, toxicity and the protection of mice against whole venom after immunization. All liposome preparations were quite stable, retaining more than 75% of the originally encapsulated crotoxin after 1 week of incubation at physiological temperature. Co-encapsulation with lipopolysaccharide increased the leakage of crotoxin. In contrast, co-encapsulation of the lipid moiety of lipopolysaccharide did not influence the stability of liposomes. Toxicity of liposomes was dependent on membrane composition. Liposomes made with phospholipids that were resistant to phospholipase A(2) activity were less toxic. Mice immunized with three doses of the 1 x LD50 of crotoxin encapsulated into liposomes, and with associated immunostimulants, were protected against challenge with 8 x subcutaneous LD50 of C. durissus terrificus venom. Using the same immunization schedule, liposomes made from a non-phospholipid mixture and without immunostimulants achieved 100% protection.

Adjuvants, Immunologic↗
PubMed2004

Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms.

Some myotoxic or neurotoxic PLA2s (phospholipases A2) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA2s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA2s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA2s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA2s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA2s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA2s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA2s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA2s. Structure-activity relationships at various regions of the PLA2 molecules were extensively discussed.

Amino Acid Sequence↗
PubMed1998

Irradiation of Crotalus durissus terrificus crotoxin with 60Co gamma-rays induces its uptake by macrophages through scavenger receptors.

PURPOSE: To investigate the action of 2 kGy 60Co gamma-rays on crotoxin and its favoured uptake through scavenger receptor (ScvR) mouse peritoneal macrophages. MATERIALS AND METHODS: Native or irradiated crotoxin (iCTX) (50 microg/ml) dosed with 2 kGy 60Co gamma-rays (dose-rate 540 Gy/h) were offered to mouse peritoneal macrophages; their uptake was evaluated by immunohistochemistry and quantitative in situ ELISA. Receptors recognizing irradiated crotoxin were evaluated with specific ScvR blockers (Probucol and dextran sulphate) or with non-specific blocking using foetal calf serum (FCS). RESULTS: Immunohistochemical assays revealed more deeply staining intracytoplasmic vacuoles in macrophages incubated with iCTX. Using in situ ELISA with ScvR specific blockers, it was shown that the increased uptake of iCTX was blocked by Probucol or dextran sulphate, but not by FCS. On the other hand, the uptake of native crotoxin was decreased by FCS, but not affected by ScvR blockers. The morphology and viability of macrophages were preserved during the experiments. CONCLUSIONS: It is concluded that 60Co gamma-rays probably induced oxidative changes in crotoxin, driving this toxin towards ScvR mouse peritoneal macrophages. This suggests a different in vivo route of iCTX away from toxic neural sites by a preferential and rapid internalization and processing by macrophages, leading to the induction of a better immune response.

Animals↗
PubMed1992

The effect of crotoxin on the release of acetylcholine and lactate dehydrogenase from rat brain cortical slices.

1. We have studied the effects of crotoxin, the neurotoxin of the South American rattlesnake Crotalus durissus terrificus, on the release of acetylcholine and lactate dehydrogenase from rat brain cortical slices. 2. Crotoxin enhances the release of [3H]-acetylcholine from cortical slices (control values 92.8 +/- 5.9 and 150.3 +/- 11.7 DPM/mg and crotoxin values 199.1 +/- 7.0 and 336.0 +/- 26.0 DPM/mg, at 60 and 120 min incubation, respectively) in parallel with the release of lactate dehydrogenase (control values 50.4 +/- 16.8 and 80.3 +/- 19.5 U/mg and crotoxin values 162.5 +/- 39.1 and 355.7 +/- 38.2 U/mg, at 60 and 120 min incubation, respectively). Both effects are markedly reduced when substituting Sr2+ for Ca2+ in the incubation medium. 3. It is concluded that the phospholipase activity of crotoxin is responsible for the observed effects.

Acetylcholine↗
PubMed1979

The role of crotoxin subunits in tropical rattlesnake neurotoxic action.

The major toxin (crotoxin) of Crotalus durissus terrificus (neotropical rattlesnake) is known to be a reversible non-covalently associated complex consisting of an acidic and basic subunit. On separation biological activity is found only with the basic subunit, yet, although void of detectable biological activity, the acidic subunit is essential for the full neurotoxic activity of the complex. Recent evidence suggests that crotoxin A serves as a 'chaperone' to enhance the specificity of crotoxin B and, upon binding, crotoxin A is released to the medium. This study was designed to test this hypothesis. Dimethyl suberimidate, a bifunctional cross-linking agent, was used to irreversibly bind the two subunits. Disc electrophoresis, ion-exchange chromatography, molecular sieve chromatography, capillary isotachophoresis and isoelectric precipitation confirm the existence of an inter-subunit covalently cross-linked complex. The conversion of a dissociable complex to a non-dissociable complex abolished neurotoxicity. Although neurotoxicity was lost, phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), which is found associated with many presynaptic neurotoxins, was unaffected. The data in this paper add credence to the 'chaperone' concept of crotoxin A and the importance of the reversible nature of the complex for full expression of neurotoxicity.

Crotalid Venoms↗
PubMed1996

Influence of ionizing radiation on crotoxin: biochemical and immunological aspects.

Irradiation of crotoxin and its subunits with 2000 Gy of gamma-rays from 60Co source leads to aggregation and generation of lower mol. wt breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformational epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-react and neutralize crotoxin. Compared with native crotoxin, aggregates appear less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin.

Animals↗
PubMed1989

The importance of phospholipase A2 in the early induction by crotoxin of biphasic changes in endplate potentials at the frog neuromuscular junction.

In Ca2+-Ringer, crotoxin induced a rapid fall in amplitude of endplate potentials followed by a marked secondary rise but, in Ca2+-free, Sr2+-EGTA Ringer, crotoxin failed to elicit either response. In Ca2+-washout experiments, the onset of giant miniature endplate potentials indicated that binding of crotoxin occurred in Sr2+-EGTA Ringer. Facilitation of endplate potential amplitude due to a closely spaced twin impulse was increased to 162% of control near the peak of the crotoxin-induced secondary phase. These findings are consistent with the phospholipase A2 subunit acting specifically at the active zone.

Animals↗