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C Bon

Publications and source records attributed to C Bon.

At least 91 records · Page 5Linked to original sources
PubMed1996

Re-examination of crotoxin-membrane interactions.

The interaction of crotoxin with synaptic membranes from Torpedo marmorata has been re-examined, using radioiodinated toxin. In competition experiments, the 'saturable binding' is usually calculated by subtracting the non-saturable binding, determined in the presence of an excess of unlabelled crotoxin, from total binding. Paradoxically, we observed a notable increase of the 'saturable binding' of 125I-crotoxin, defined in this manner in the presence of a high concentration of certain competitors (e.g. crotoxin subunit B, agkistrodotoxin, ammodytoxin and ammodytin I2). This potentiation effect was analysed by competition and cross-linking experiments. The dissociation of the basic crotoxin subunit CB, which differs markedly from crotoxin in its membrane-binding characteristics, was found to be the main reason for the observed phenomenon. 125I-CB could be released from 125I-crotoxin by exchange with a molecule having sufficient affinity towards CA (e.g. CB or agkistrodotoxin) or it could be dissociated from the specific crotoxin membrane-binding site(s) by a competitor molecule. Our results, therefore, suggest a reinterpretation of previous 125I-crotoxin binding studies.

Animals↗
PubMed1996

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.

Acetylcholinesterase↗
PubMed1996

Human metabotropic glutamate receptor 1: mRNA distribution, chromosome localization and functional expression of two splice variants.

We have isolated clones from a human brain cDNA library coding for two splice variants of mGluR1. The combined sequences account for 6864 base pairs (bp) of the approximately 7 kilobase (kb) transcript seen on northern blots of human brain mRNA. The distribution of mGluR1 mRNA in human brain is similar to that found in rat brain and the gene for mGluR1 is localized on human chromosome 6. The mRNA for mGluR1 alpha contains an open reading frame that codes for a 1194 amino acid protein, which is slightly smaller than rat mGluR1 alpha. The major structural difference noted between the human and rat mGluR1 is a deletion of 21 nucleotides which would result in the loss of seven amino acids in the middle of a proline- and glutamine-rich region of the C-terminal tail of mGluR1 alpha. The 85 bp exon that results in the HmGluR1 beta splice variant was found to code for a protein with two amino acid differences compared to the rat receptor. Functional and pharmacological characterization of heterologously expressed human mGluR1 was performed using electrophysiological recordings from Xenopus oocytes and calcium imaging in HEK-293 cells. No major differences were found in the response of human mGluR1 to typical agonists and antagonists compared to the rat, or in the behavior of the two splice variants.

Alternative Splicing↗
PubMed1996

Electrophysiological actions of GABAB agonists and antagonists in rat dorso-lateral septal neurones in vitro.

1. The actions of GABAB-receptor agonists and antagonists on rat dorso-lateral septal neurones in vitro were recorded with intracellular microelectrodes. 2. In the presence of 1 microM tetrodotoxin to prevent indirect neuronal effects caused by action potential-dependent neurotransmitter release, bath application of baclofen (0.1-30 microM) or SK&F 97541 (0.01-3 microM) evoked concentration-dependent hyperpolarizations which reversed close to the potassium equilibrium potential; the EC50S were 0.55 and 0.05 microM, respectively. No significant desensitization was observed during prolonged agonist exposure (< or = 10 min). 3. Hyperpolarizations induced by baclofen were antagonized in a competitive manner by the following GABAB-receptors antagonists (calculated pA2 values in parentheses): CGP 36742 (4.0), 2-OH saclofen (4.2), CGP 35348 (4.5), CGP 52432 (6.7) and CGP 55845A (8.3). Responses to SK&F 97541 were also antagonized by CGP 55845A (pA2 = 8.4). 4. The amplitude of the late, GABAB receptor-mediated inhibitory postsynaptic potential (i.p.s.p.) was reduced by the GABAB antagonists as follows (means +/- s.e.mean): CGP 55845A (1 microM) 91 +/- 5%, CGP 52432 (1 microM) 64 +/- 5%, CGP 35348 (100 microM) 82 +/- 5%, CGP 36742 (100 microM) 76 +/- 8%, and 2-OH saclofen (100 microM) 68 +/- 3%. 5. It is concluded that neurones in the rat dorso-lateral septal nucleus express conventional GABAB receptors, which are involved in the generation of slow inhibitory postsynaptic potentials. CGP 55845A is the most potent GABAB receptor antagonist described in this brain area.

Analysis of Variance↗
PubMed1996

Prothrombin time sensitivity and specificity to mild clotting factor deficiencies of the extrinsic pathway: evaluation of eight commercial thromboplastins.

Prothrombin-time (PT) sensitivity and specificity to mild clotting factor II, V, VII and X deficiencies have rarely been studied. We therefore carried out a prospective study, in 350 patients, of eight commercial thromboplastins (CTs) in their ability to detect mild clotting factor deficiencies, notably in factor VII. In each patient the factor II, V, VII and X clotting activities and PT performed with each CT were determined. For each CT, PT sensitivity and specificity in detecting factor deficiencies below 0.5 U/ml or below 0.4 U/ml were determined at various PTs, and then Receiver Operator Characteristic curves constructed. At optimum PT threshold level (sensitivity = specificity), exactitude varied from 0.64 to 0.74 (p < 0.01) and from 0.67 to 0.81 (p < 0.0001) in detecting deficiencies below 0.5 and 0.4 U/ml respectively. In conclusion, this study shows the limits of the PT test as performed with 8 CTs in patients with mild clotting factor deficiencies. The impact of such differences in sensitivity and specificity on monitoring certain patients subjects to decrease in coagulation factor, and, in particular, of those under low dose oral anticoagulant, remains to be determined.

Blood Coagulation Disorders↗
PubMed1995

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes.

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.

Amino Acid Sequence↗
PubMed1995

A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning.

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Amides↗
PubMed1995

Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum.

An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23-25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.

Amino Acid Sequence↗
PubMed1995

An activator of blood coagulation factor X from the venom of Bungarus fasciatus.

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Animals↗
PubMed1994

Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2.

The ability of platelet secretory phospholipase A2 (sPLA2) to induce platelet activation was investigated. sPLA2 (group II) contained in an activated platelet supernatant, as well as high concentrations of purified recombinant platelet sPLA2, failed to induce platelet activation. Furthermore, sPLA2 did not modify platelet activation induced by various agonists. The possible relationship between the failure of this enzyme to induce platelet activation and its origin (mammalian) or its structural group (group II) was then investigated, using pancreatic PLA2s (group I) and venom PLA2s from groups I, II and III. All venom PLA2s induced platelet activation that was accompanied by the liberation of arachidonic acid and was abolished by aspirin. In contrast, as observed for platelet sPLA2, enzymes from hog or bovine pancreas were unable to induce platelet activation even when used at high concentrations. Interestingly, PLA2 able to induce platelet activation efficiently hydrolyse phosphatidylcholine, while those inactive on platelets did not. Taken together, these results suggest that the catalytic activity of added PLA2 is necessary but not sufficient to induce platelet activation. Moreover, the ability of PLA2 to induce platelet activation is not related to its structural group (I, II, III) but rather to its origin (venom vs. mammalian) and capacity to hydrolyse phosphatidylcholine, the major phospholipid of the outer leaflet of the plasma membrane.

Animals↗
PubMed1994

The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus.

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a beta-neurotoxin which consists of the non-covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non-enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro-CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus. The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post-translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.

Amino Acid Sequence↗
PubMed1994

Purification of an acidic phospholipase A2 from Bothrops lanceolatus (fer de lance) venom: molecular and enzymatic properties.

The acidic phospholipase A2 from Bothrops lanceolatus venom has been purified by gel filtration on Sephadex G-50 and ion exchange chromatography on CM-cellulose. Analysis by FPLC on Mono-Q column of the purified phospholipase A2 indicated that it is a mixture of several isoenzymes. The two major isoforms consist of a single polypeptide chain with mol. wts of 14,500 and 15,000, which slightly differ in their isoelectric point (4.9 and 5.3) and amino acid composition. However, enzymatic and pharmacological properties of the various isoenzymes are identical. The phospholipase from B. lanceolatus venom is characterized by a progressive increase in the rate of hydrolysis when enzymatic activity is determined with crude egg yolk as substrate in the absence of detergent. This phenomenon, which is not observed with mixed micelles of lecithin-detergent, is not due to the presence of a phospholipase A2 inhibitor in the venom, as previously suggested by several investigators in the case of other Bothrops and Cobra venoms. It is rather a catalytic characteristics of B. lanceolatus venom phospholipase, the enzymatic activity of which depends on the physical state of phospholipids. Bothrops lanceolatus acidic phospholipase A2 is non-toxic.

Amino Acids↗
PubMed1994

Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA2 subunit of crotoxin and neutralize its pharmacological activity.

Crotoxin and ammodytoxin A are snake venom neurotoxic phospholipases A2. Polyclonal antibodies against three synthetic peptides selected from the C-terminal part of the primary structure of ammodytoxin A were tested by ELISA for their interaction with crotoxin and its subunits, CA and CB. All three antipeptide antibodies reacted specifically with corresponding parts of ammodytoxin A and CB, either native or reduced. Conversely, polyclonal antibodies produced against ammodytoxin A and CB reacted strongly with all three peptides, suggesting that they constitute at least a part of natural epitopes in both proteins. All antipeptide antibodies reacted also with the corresponding peptides derived from CB by cyanogen bromide cleavage. The biological activity of the immune complexes was tested. No significant change in the enzymatic activity of CB, ammodytoxin A or crotoxin was observed with any of the three antipeptide antibodies. These antibodies were, however, able to protect mice against the lethal potency of CB and to prolong survival time of mice injected with crotoxin. These antipeptide antibodies were assayed in vitro for their protective effect against the action of CB or crotoxin on synaptosomes from Torpedo marmorata electric organ. They partly inhibited the acetylcholine release induced by both proteins. These results indicate that the C-terminal part of CB is likely to be involved in the pharmacological action of crotoxin.

Amino Acid Sequence↗
PubMed1994

Viper bites in France: clinical and biological evaluation; kinetics of envenomations.

1. A second inquiry was conducted in France to collect more accurate epidemiological, clinical and biological data from patients hospitalized after a viper bite, as well as treatment that they received. Fifty-seven well documented cases were classified in four grades of increasing severity defined according to the clinical signs of envenomation. 2. Local and systemic signs of envenomation appeared during the first 3 h, but the severity of the envenomation was observed to increase during the 12-24 h following bites in 50% of moderate and severe cases. One fatal case was reported. Biological analysis revealed an hyperleukocytosis in patients with moderate and severe envenomations. 3. The average length of hospitalization was of 1.7 +/- 1.3 days for patients without signs of envenomation (grade 0) or presenting a minimal envenomation (grade 1), and statistically longer, 6.2 +/- 2.9 days, for patients presenting moderate (grade 2) or severe envenomation (grade 3). 4. Levels of venom antigens in serum samples regularly collected during hospitalization were determined by a sandwich ELISA test. The serum venom levels determined during the first 4 h following the bite correlated with the severity of the envenomation when the symptoms were determined at their worst, usually 12-24 h later. In fact, concentrations higher than 20 ng ml-1 predict a moderate or severe clinical evolution. 5. The pharmacokinetics of venom antigens was also investigated during human envenomations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Pharmacokinetics of Vipera aspis venom after experimental envenomation in rabbits.

Toxicokinetic studies of Vipera aspis venom were performed in rabbits after experimental envenomation. Venom proteins with a molecular weight greater than 6 kDa (high-molecular weight proteins) and which reacted in enzyme-linked immunosorbent assay with specific antiviper venom Fab'2, were also responsible for the lethal potency and the capillary permeability increasing activity of the venom. Conversely, low-molecular weight proteins were not detected by enzyme-linked immunosorbent assay and were pharmacologically inactive. The toxicokinetics of both classes of venom components were studied, using high-molecular weight and low-molecular weight radiolabeled proteins as well as enzyme-linked immunosorbent assay. After intravenous injection, Vipera aspis venom in plasma followed a biexponential decline with a distribution half-life of 0.7 hr and an elimination half-life of 12 hr. The distribution volume was 1.2 l.kg-1 and the systemic clearance was 84 ml.hr-1.kg-1. Venom levels in plasma after intramuscular injection of three doses (300, 500 and 700 micrograms/kg) of venom increased within the few hours after the venom administration to reach maximal values proportional to the injected doses. They subsequently followed a monoexponential decline, with an apparent terminal half-life of 32.5 hr. Absorption was a kinetically complex process, rapid during the first 24 hr and continued at a slower rate over the subsequent 72 hr. Bioavailability of venom was about 65%, regardless of the administered dose, and less than 5% of venom injected was excreted by the renal route.

Animals↗
PubMed1993

Model for the interaction of crotoxin, a phospholipase A2 neurotoxin, with presynaptic membranes.

Crotoxin is a phospholipase A2 neurotoxin that impairs the release of acetylcholine at neuromuscular junctions, primarily at the presynaptic level. It associates a phospholipase A2 subunit, CB, with a chaperon subunit, CA. We have shown elsewhere that the purely cholinergic synaptosomes from the Torpedo electric organ provided a convenient model to study the pharmacology of crotoxin and other related neurotoxins [Délot, E., & Bon, C. (1992) J. Neurochem. 58, 311-319]. In the present experiments, we labeled crotoxin with 125I and demonstrated saturable binding to Torpedo presynaptic membranes. In the range of concentrations that was effective on synaptosomes, crotoxin bound to a single class of sites without cooperativity. The binding was affected by divalent cations, and its kinetics was rather complex. We observed a competition between crotoxin and related neurotoxins, but not CB. Although CA could not bind, it could compete efficiently with crotoxin. We compare our results with those previously obtained by others on guinea pig brain membranes. On Torpedo membranes, as on all models tested, CB was the major species bound to the membrane, while CA remained in solution. However, the mechanism underlying this phenomenon had never been clarified. The major question is the time scale of the events, i.e., does CB bind before or after dissociating from CA? Our results indicate that the predominant pathway involves the formation of a ternary complex between crotoxin's subunits and the acceptor site preceding the release of CA.

Acetylcholine↗
PubMed1993

Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition.

A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.

Amino Acid Sequence↗