Relationship between the pharmacological action of crotoxin and its phospholipase activity.
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Animals↗
Biomedical subjects
C Bon
Publications and source records attributed to C Bon.
Synergism of the two subunits of crotoxin.
Crotoxin, a potent neurotoxin from the venom of Crotalus durissus terrificus, is composed of an acidic subunit which is non-toxic and enzymatically inactive and a basic subunit which possesses a phospholipase activity and low toxicity. It is shown that crotoxin very efficiently blocks the cholinergic post-synaptic response of the isolated electroplaque from Electrophorus electricus and of cellular microsacs fromTorpedo marmorata. This post-synpatic effects was investigated by studying the binding of crotoxin and its isolated subunits to acetylcholine receptor-rich membranes from Torpedo and by analysing the relationship between its catalytic activity and its pharmacological effects on Electrophorus electroplaque. The mechanism of action of crotoxin could be divided into two distinct steps: a quasi irreversible binding step, which has no blocking action by itself, and a catalytic step, which irreversibly inhibits the postsynaptic response. These results suggest that the non-enzymatic subunit of crotoxin enhances the pharmacological efficiency of the phospholipase by preventing its adsorption to non-saturable binding sites, restricting its binding to specific critical target sites. These sites are distinct from the cholinergic receptor sites, but probably closely related to them, as suggested by their approximately equal number and by the fact that after crotoxin action the receptor appears to be blocked in a molecular form very similar to the desensitized state. The mechanism proposed explains in simple terms the synergistic action of the two subunits of crotoxin at the level of the cholinergic receptor-ionophore assembly.
Animals↗
Isolation of "ceruleotoxin" from Bungarus fasciatus venoms.
Ceruleotoxin is a potent neurotoxin which irreversibly blocks the neuromuscular transmission at a postsynaptic level, without preventing the binding of acetylcholine to its receptor. We have originally purified this toxin from a batch of venom obtained from the Pasteur Institute, which was thought to be Bungarus caeruleus venom. Recently Noble and co-workers have observed that the protein composition of Bungarus caeruleus provided by Miami Serpentarium significantly differed from that of the Pasteur Institute batch, which they concluded therefore to be of a different origin. In order to clarify this point, the venom composition of various Bungarus species from several origins have been analysed by electrophoresis and by electrofocusing on polyacrylamide gels. Although individual variations exist between samples of the same snake species, the venom from Bungarus caeruleus, Bungarus fasciatus and Bungarus multicinctus possess distinct and characteristic protein compositions. The results of this study allowed us to identify unambiguously the batch used to purify the ceruleotoxin, as a Bungarus fasciatus venom. We identified a neurotoxin similar to ceruleotoxin in each of the five samples of Bungarus fasciatus venoms that we tested. On the contrary we did not detect such a toxin either in Bungarus caeruleus or in Bungarus multicinctus venoms. All purified ceruleotoxins are acidic proteins with a high toxicity (their LD50 by intravenous injection in mice are from 0.04 to 0.06 mg per kg), which irreversibly block the postsynaptic response of Electrophorus electricus electroplaque to cholinergic agonists. They also possess a phospholipase A2 activity (200 nmoles of egg lecithins hydrolysed per min per mg of protein). In this respect, ceruleotoxin is analogous to crotoxin and beta-bungarotoxin.
Animals↗
Partial purification of the Glycera convoluta venom components responsible for its presynaptic effects.
The crude extract of glands appended to the jaws of the polychaete annelid Glycera convoluta induces an important increase in the spontaneous quantal transmitter release on frog and crayfish neuromuscular junctions and on Torpedo nerve-electroplaque junctions. The venom similarly triggers acetylcholine (ACh) release from synaptosomes purified from Torpedo electric organ. At the frog neuromuscular junction, the reproducibility, the reversibility and the dose-dependence of the venom action permit a quantitative evaluation of the effect. The crude venom extract has been fractionated by gel-filtration. The effect on transmitter release has been found in a high molecular weight fraction distinct from those which contain the protease and phospholipase activities.
Acetylcholine↗
Postsynaptic effects of crotoxin and of its isolated subunits.
Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of alpha-toxin from Naja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non-saturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.
Animals↗
Crotoxin: a possible mechanism of action.
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Acetylcholine↗
Chemical and pharmacological characterization of toxic polypeptides from the venom of Bungarus caeruleus.
Five toxic fractions have been purified to homogeneity from the venom of Bungarus ceruleus by chromatography on CM-Sephadex, QAE-Sephadex and Bio-Rex 70 followed by filtration on Sephadex G-50 and Biogel P-30. One of them blocks synaptic transmission at the pre-synaptic level and is identified as a beta-bungarotoxin similar to that described by Lee from the venom of B. multicinctus. Three other ones are short-chain (type I) alpha-toxins which block the response of the isolated electroplaque from Electrophours electricus to bath-applied carbamylcholine. These basic polypeptides of 61-62 amino acids contain four disulfide bridges. A postsynaptically acting toxin, different from the alpha-toxins, has also been purified from the same venom. This ceruleotoxin is acidic and made up of several subunits.
Amino Acids↗
Ceruleotoxin: a possible marker of the cholinergic ionophore.
Ceruleotoxin, a toxin component of Bungarus caeuleus venom, blocks in vivo the depolarisation caused by carbamylcholine on the isolated electroplaque from Electrophorus electricus and in vitro in increase of 22Na+ and 42K+ efflux caused by cholinergie agonists on excitable receptor-rich microsacs prepared from Torpedo marmorata electric organ. However, at variance with the typical alpha-toxins, ceruleotoxin does not interfere with the binding of [3H]acetylcholine and of alpha-[3H]toxin to the cholinergic receptor site. Ceruleotoxin also slightly modifies 35SO 2 4-and [14C]glucose efflux. Some of its effects on the postsynaptic membrane may be related to the presence of a phospholipase activity present in the preparation and are discussed in those terms. It is proposed that ceruleotoxin selectively blocks the electrogenic action of acetylcholine on the postsynaptic membrane without binding to the cholinergic receptor site, and might serve as an useful tool to characterise the ion translocating device, the ionophore, associated with the cholinergic receptor site.
Acetylcholine↗
[Cytogenetic study of 350 mental deficient patients at medico-pedagogic institutions].
This report brings to light the results of cytogenetic study carried out on 350 mentally retarded persons (medium: Group I, deep Group II). In 8,2% and 12,56% of the cases a chromosomic abnormality is observed (mainly trisomy 21). These percentages are in correlation with the results of other studies. Ante and peri natal pathology plays a very important part among exogenous factors. In numerous cases a multifactors etiology can be found as the cause of this mental retardation, the effect of an initial organic mental retardation being aggravated by precocious affective perturbations. For those cases without apparent etiology, a teratologic origin may be thought as their cause.
Adolescent↗
Effects of alpha-toxins from Bungarus multicinctus and Bungarus caeruleus on cholinergic responses in Aplysia neurons.
The effects of alpha-bungarotoxin from Bungarus multicinctus and Bungarus caeruleus were studied on three types of cholinergic response in Aplysia central neurones. These responses are the result of three distinct changes in ionic permeability: selective increases in permeabiliyt to Na, Cl and K, respectively. It was shown that 10(-5) M alpha-bungarotoxin from B. multicinctus completely blocks the response resulting from an increase in Cl permeability, while having no effect on either of the other two responses types (even when the toxin concentration was increased to 5 X 10(-5) M). The block of the Cl-dependent response by alpha-bungarotoxin is reversible. None of the three response types were affected by similar concentrations of alpha-toxin from B. caeruleus. Higher concentrations were not systematically tested. These results contradict reports of other authors on the action of alpha-bungarotoxin on molluscan acetylcholine (ACh) responses. Possible reasons for the discrepancies between our findings and those published by other authors are discussed.
Animals↗
Ceruleotoxin: an acidic neurotoxin from the venom of Bungarus caeruleus which blocks the response to a cholinergic agonist without binding to the cholinergic receptor site.
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Action Potentials↗
Envenoming by viper bites in France: clinical gradation and biological quantification by ELISA.
Viper bites are frequent in France but the evaluation of the severity of envenomings and consequently patient treatment has not yet been properly evaluated. The purpose of this study was to measure venom antigens in blood and/or urine of bitten patients and to establish a quantitative relationship with clinical observations. A prospective enquiry was conducted in 1990 in France to collect epidemiological, clinical and biological data from hospitals. Urine and blood samples were tested for their content of Vipera aspis venom antigens by a sandwich enzyme linked immunosorbent assay (ELISA). One hundred and two charts were analysed, from patients presenting documented viper bites. Oedema was the prominent local feature (81 cases). Systemic signs consisted of vomiting and/or diarrhoea (22 cases), slight or severe hypotension (15 cases), shock (2 cases) and bleeding (1 case). A relationship was observed between these systemic signs and the extent of the oedema, which permitted the establishment of a grading scale. Grade 0 (no envenoming) was identified by fang marks and absence of oedema and local reaction; grade 1 (mild envenoming) by local oedema and absence of systemic symptoms; grade 2 (moderate envenoming) by regional oedema and moderate systemic symptoms; and grade 3 (severe envenoming) by extensive oedema and severe systemic symptoms. Quantification of venom antigens in blood or urine of patients by ELISA revealed a significant correlation between clinical signs of envenoming and the level of venom antigens in blood or urine. This indicated that the ELISA test is a useful and predictive tool for clinically grading viper envenomings.
Enzyme-Linked Immunosorbent Assay↗
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↗
Clinical indicators of envenoming and serum levels of venom antigens in patients bitten by Bothrops lanceolatus in Martinique. Research Group on Snake Bites in Martinique.
An enzyme-linked immunosorbent assay was developed to measure venom antigen levels in the serum of 40 patients bitten by Bothrops lanceolatus. The grading system used for the severity of envenomation (grades 1 to 4, minor to major) was predominantly based on the presence of local signs. Serum venom levels increased with the grade of severity (P < 0.001, by Spearman's rank correlation test); they were 6 +/- 6 ng/mL (mean +/- SD) in clinically non-envenomed patients (grade 1, n = 3), 7.6 +/- 11.7 (n = 17), 44.3 +/- 41.8 (n = 17), and 80.3 +/- 34.1 ng/mL (n = 3) in patients diagnosed as grade 2, 3 and 4 respectively. However, venom antigens could not be detected in the serum of 54% of patients who showed clinical signs of envenomation. Most patients diagnosed as grade 2, 3 or 4 were given 20, 40 and 60 mL of a monospecific F(ab')2 antivenom, respectively. Venom concentrations > or = 15 ng/mL were observed in all patients with progressive aggravation of swelling despite the use of early antivenom therapy. No venom was detectable in blood samples taken after completion of serotherapy. All patients recovered. These results confirm the efficacy of both the clinical severity scoring system used and the therapeutic regimen.
Adolescent↗
Coagulant and anticoagulant activities of Bothrops lanceolatus (Fer de lance) venom.
Bothrops lanceolatus venom contains caseinolytic, phospholipase, esterase and haemorrhagic activities. We have investigated the coagulant and anticoagulant actions of B. lanceolatus venom on human citrated plasma and on purified plasma components. Although B. lanceolatus venom up to 50 microg/ml was unable to clot citrated plasma, at concentrations > or = 5 microg/ml the venom dose-dependently clotted purified human fibrinogen, indicating the presence of a thrombin-like enzyme. Human plasma (final concentration > or = 12.5%) dose-dependently inhibited the venom-induced fibrinogen clotting. This finding suggested that endogenous plasma protease inhibitors can affect the venom's action on fibrinogen. To investigate this possibility, B. lanceolatus venom was incubated with different plasma protease inhibitors and the activity on fibrinogen tested. alpha(2)-Macroglobulin and alpha(1)-antitrypsin did not interfere with the coagulant activity of the venom whereas the antithrombin-III/heparin complex partially inhibited this activity. A non-toxic, acidic phospholipase A(2) purified from B. lanceolatus venom prolonged the activated partial thromboplastin time in human plasma from 39.7+/-0.5 s (control with saline) to 60.2+/-0.9 s with 50 microg of PLA(2) (p<0.001), suggesting an anticoagulant activity associated with this enzyme. This anticoagulant activity may account for some of the effects of the venom on blood coagulation.
Animals↗
Snake venom proteins acting on hemostasis.
The venoms of Viperidae and Crotalidae snakes are a rich source of proteins with activity against various factors involved in coagulation and fibrinolysis. These proteins are very specific for their molecular targets, resistant to physiological inhibitors and stable in vitro and in vivo. They have therefore proved to be useful for diagnostic tests. Based on sequence similarities, these snake venom proteins have been classified into various families, such as serine proteinases, metalloproteinases, C-type lectins, disintegrins and phospholipases A(2). The various members of a given family, although structurally similar, act selectively on different blood coagulation factors. This opens up the possibility of characterizing the structural elements involved in target molecule recognition. Thus, snake venom proteins provide excellent models for studies of structure-function relationships.
Amino Acid Sequence↗
Quantitation of venom antigens from European vipers in human serum or urine by ELISA.
We describe an enzyme-linked immunosorbent assay (ELISA) to quantitate venom antigens in human serum and urine, and thus to help evaluate the severity of envenomation due to viper bites. This assay, which is performed with commercially available polyclonal Fab'2s in a double-sandwich method, is rapid, simple, and specific for antigens of European vipers (Vipera aspis, Vipera berus, and Vipera ammodytes). No cross-reactivity was observed with other snake venoms or human serum proteins. It showed a good linear response over a wide range of concentrations of venom antigens (from 1 to 100 ng/mL). It was very sensitive, with detection limits of 7 and 2 ng/mL for Vipera aspis venom in serum and urine, respectively. This ELISA is also easily reproducible; the coefficients of variation determined at different concentrations of venom (50, 25, and 5 ng/mL) did not exceed 10% in serum and 14% in urine samples collected from different donors. This test was applied to determine the concentrations of venom in the serum of patients bitten by a viper in France and to follow its elimination as a function of time. The method is adaptable to other venoms by using other specific immunoglobulins.
Antigens↗