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C Bon

Publications and source records attributed to C Bon.

At least 109 records · Page 6Linked to original sources
PubMed1993

Altered synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats.

Nitric oxide (NO) is a messenger molecule that is produced in the brain from the metabolism of L-arginine to L-citrulline. Growing evidence suggests a physiological role for NO in long-term potentiation (LTP). Since LTP is a form of synaptic plasticity thought to be involved in learning and memory, we have tested whether inhibition of endogenous NO production affects memory capacities of rats. We found that the NO synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39] inhibitor N omega-nitro-L-arginine, at doses blocking LTP in hippocampal slices, impairs spatial learning in a radial arm maze and olfactory memory in a social recognition test. In contrast, N omega-nitro-L-arginine left shock-avoidance learning unaffected. These results indicate that NO is involved in some but not all forms of memory and further support the existence of a causal link between LTP and spatial learning.

Amino Acid Oxidoreductases↗
PubMed1993

Secretory phospholipase A2 is not required for arachidonic acid liberation during platelet activation.

The subcellular localization of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in resting and activated platelets, and their involvement in arachidonic acid liberation during platelet activation, were studied. The amounts of sPLA2 and cPLA2 recovered were not modified during platelet activation. sPLA2 was mainly associated with the organelles of resting platelets (71% of total activity) and was released into the extracellular medium during cell activation (60% of total activity), whereas the majority of cPLA2 was localized in the cytosol of resting and activated platelets. The secretion of sPLA2 correlated with the release of ATP. sPLA2-depleted platelets aggregated as much as control platelets and produced similar amounts of thromboxane B2 upon thrombin activation. These results indicate that sPLA2 is not involved in the liberation of arachidonic acid during platelet activation.

Adenosine Triphosphate↗
PubMed1993

Cell-associated and soluble phospholipases A2 increase during carrageenan and zymosan-induced pleurisy in rat.

Extra-cellular and cell-associated Ca(2+)-dependent phospholipases A2 and released thromboxane B2 were correlated to exudation and cell migration during rat pleurisy induced by carrageenan or zymosan. Extra-cellular phospholipase A2 was delayed with respect to acute inflammation, while cell-associated phospholipase A2 closely correlated with cell migration and thromboxane B2 levels. This confirms that the subcellular localization of phospholipases A2 is linked to their physiological action and, in particular, suggests that the cell-associated, rather than the extracellular enzyme, accounts for the production of eicosanoids.

Animals↗
PubMed1993

Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action.

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase-A2 subunit (CB) and a non-enzymic, non-toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its non-specific adsorption. Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase-A2 activity. In this study, we demonstrate that the more-toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less-toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more-toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less-toxic isoforms are less stable (Kd = 25 nM) and dissociate rapidly (half-life about 1 min), whereas the more-toxic isoforms are more stable (Kd = 4.5 nM) and dissociate more slowly (half-life 10-20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles. The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency.

Animals↗
PubMed1993

Snake-venom phospholipase A2 neurotoxins. Potentiation of a single-chain neurotoxin by the chaperon subunit of a two-component neurotoxin.

The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (beta-neurotoxins): single-chain beta-neurotoxins, such as agkistrodotoxin and ammodytoxin-A, and dimeric beta-neurotoxins, which, in the case of the best studied ones, crotoxin-like toxins, consist of the non-covalent association of a phospholipase A2 (CB) and a non-enzymatic chaperon (CA). Possible evolutionary relationships of these beta-neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex. CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti-CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone. A 10-fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA.agkistrodotoxin. Ammodytoxin A did not compete with the anti-CA mAb. These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).

Animals↗
PubMed1993

Competitive inhibition of phospholipase A2 activity by the platelet-activating factor antagonist Ro 19-3704 and evidence for a novel suppressive effect on platelet activation.

The compound Ro 19-3704 [3-4(R)-2-(methoxycarbonyl) oxy-3-(octadecylcarbamoyl)oxy-propoxy butylthiazolium iodide], initially described as an antagonist of platelet-activating factor, is reported here to directly inhibit rabbit platelet phospholipase (PL) A2 activity, with an IC50 value of 4 to 7 microM. Classical Michaelis-Menten analysis showed that inhibition was reversible and competitive, inasmuch as apparent Km values increased in the presence of Ro 19-3704 (from 0.2-0.4 to 2 microM), whereas Vmax values remained constant (200 +/- 20 nmol/min/10(9) cells). Ro 19-3704 inhibited platelet aggregation, PLA2 release and thromboxane B2 formation induced by thrombin (0.25 U/ml), with IC50 values of 8, 15 and below 5 microM, respectively. Aggregation and PLA2 release by arachidonic acid (100 microM) were also inhibited, but thromboxane B2 formation was unaffected, indicating that Ro 19-3704 does not inhibit cyclooxygenase. Platelet activation by collagen (5 micrograms/ml), the thromboxane mimetic U46619 ([15(S)-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid] 1 microM) and low concentrations of thrombin (0.05-0.1 U/ml) was also inhibited by Ro 19-3704. Inhibition of platelet activation was reversible, suggesting that its suppressive effect was not due to cytotoxicity. Finally, Ro 19-3704 did not stimulate cyclic AMP formation or inhibit phosphodiesterase activity. Ro 19-3704 is a competitive inhibitor of PLA2 activity, and is also endowed with a potent suppressive effect on platelet activation induced by different agonists.

Animals↗
PubMed1992

Differential effects of presynaptic phospholipase A2 neurotoxins on Torpedo synaptosomes.

The effects of several phospholipase A2 neurotoxins from snake venoms were examined on purely cholinergic synaptosomes from Torpedo electric organ. The noncatalytic component A of crotoxin had no effect, whereas its phospholipase component B, used alone or complexed to component A, elicited a rapid and dose-dependent acetylcholine (ACh) release and a depolarization of the preparation. Subsequent ACh release evoked by high K+ levels or calcium ionophore was identical to the control after the action of component A but reduced after the action of crotoxin or of component B. These effects were not observed when the phospholipase A2 activity of the toxin was blocked either by replacing Ca2+ by Ba2+ (respectively, activator and inhibitor of phospholipase A2) or by alkylation of component B with p-bromophenacyl bromide. beta-Bungarotoxin, another very potent phospholipase A2 neurotoxin, induced release of little ACh, did not affect ionophore-evoked ACh release, but significantly reduced depolarization-induced ACh release. The single-chain phospholipase A2 neurotoxin agkistrodotoxin behaved like crotoxin component B. A nonneurotoxic phospholipase A2 from mammalian pancrease induced release of an amount of ACh similar to that released by crotoxin but did not affect the evoked responses. The obvious differences in effect of the various neurotoxins suggest that they exert their specific actions on the excitation-secretion coupling process at different sites or by different mechanisms.

Acetylcholine↗
PubMed1991

Multiplicity of acidic subunit isoforms of crotoxin, the phospholipase A2 neurotoxin from Crotalus durissus terrificus venom, results from posttranslational modifications.

Crotoxin, the major toxin of the venom of the South American rattlesnake, Crotalus durissus terrificus, is made of two subunits: component B, a basic and weakly toxic phospholipase A2, and component A, an acidic and nontoxic protein that enhances the lethal potency of component B. Crotoxin is a mixture of isoforms that results from the association of several isoforms of its two subunits. In the present investigation, we have purified four component A isoforms that, when associated with the same purified component B isoform, produced different crotoxin isoforms, all having the same specific enzymatic activity and the same lethal potency. We further determined by Edman degradation the polypeptide sequences of these four component A isoforms. They are made of three disulfide-linked polypeptide chains (alpha, beta, and gamma) that correspond to three different regions of a phospholipase A2 precursor. We observed that the polypeptide sequences of the various component A isoforms all agree with the sequence of an unique precursor. The differences between the isoforms result first by differences in the length of the various chains alpha and beta, indicating that component A isoforms are generated from the proteolytic cleavage of the component A precursor at very close sites, possibly by the combined actions of endopeptidases and exopeptidases, and second by the possible cyclization of the alpha-NH2 of the N-terminal glutamine residue of chains beta and gamma. These observations indicate that the component A isoforms are the consequence of different posttranslational events occurring on an unique precursor, rather than the expression of different genes.

Amino Acid Sequence↗
PubMed1991

Possible involvement of nitric oxide in long-term potentiation.

Long-term potentiation (LTP) in the hippocampus is known to involve NMDA (N-methyl-D-aspartate) receptors. Since activation of NMDA receptors in the cerebellum results in the formation of nitric oxide (NO), we studied the possible involvement of this messenger in hippocampal synaptic plasticity. We report here that the NO-synthase inhibitor, L-N omega-nitro-arginine, blocks LTP and that sodium nitroprusside, which releases NO, produces a long-lasting enhancement in synaptic efficacy which is not additive with tetanus-induced LTP.

Animals↗
PubMed1991

Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A2-like precursor.

We report the sequences of three cDNAs encoding the two subunits (CA and CB) of crotoxin, a neurotoxic phospholipase A2 from the venom of the South-American rattlesnake Crotalus durissus terrificus. CB is a basic and toxic phospholipase A2 and CA is an acidic, non toxic and non enzymatic three chain containing protein which enhances the lethal potency of CB. Two cDNAs encoding precursors of CB isoforms have been isolated from a cDNA library prepared from one venom gland. Both precursors are made of the same 16 residues signal peptide followed by a polypeptide of 122 amino acid residues. The two mature sequences differ from each other at eight positions and are in good agreement with the previous polypeptide sequence reported for CB. In the case of CA, the cDNA encodes a signal peptide identical to those found in CB precursors, followed by a polypeptide of 122 amino acids clearly homologous to phospholipases A2 and including three regions which correspond to the three chains of mature CA. This demonstrates that CA is generated from a phospholipase A2-like precursor, called pro-CA, by the removal of three peptides, leaving unchanged the molecule core cross-linked by disulfide bridges. The 5'-untranslated tracts of cDNAs encoding CA and CB are nearly identical and the 3'-untranslated tracts are very similar, suggesting that the mRNAs encoding the two crotoxin subunits may result from the alternative splicing of a single gene or from the existence of a recent gene conversion. Data have been analysed in light of recent results on other phospholipases A2 from different origins.

Amino Acid Sequence↗
PubMed1991

Immunochemical cross-reactivity of two phospholipase A2 neurotoxins, agkistrodotoxin and crotoxin.

Polyclonal rabbit antisera were raised against the phospholipase A2 neurotoxin agkistrodotoxin (AGTX) from Agkistrodon blomhoffii brevicaudus venom and against the phospholipase A2 subunit (component-B, CB) of crotoxin from Crotalus durissus terrificus venom. Anti-AGTX antibodies cross-reacted strongly with crotoxin and crotoxin-like molecules and more weakly with other phospholipases A2 from the venoms of Viperidae and Crotalidae. On the other hand, anti-CB antibodies cross-reacted with AGTX, and also recognized ammodytoxin A and the phospholipase A2 from Vipera berus venom, but not other phospholipases A2 from Crotalidae and Viperidae. Anti-AGTX and anti-CB antibodies were able to inhibit the phospholipase A2 activity and to neutralize the lethal potency of the homologous and heterologous toxins (AGTX or crotoxin). Immunoaffinity chromatography columns were used to isolate anti-AGTX antibodies which recognized CB (91% of the total anti-AGTX antibodies), and anti-CB antibodies which recognized AGTX (52% of the total anti-CB antibodies). Immunochemical investigations performed with each type of antibody indicated that the majority of AGTX antigenic determinants are present on crotoxin component-B and on phospholipases A2 from Viperidae venoms, and that some of these determinants are involved in the neutralization of lethal potency and in the inhibition of enzymatic activity of AGTX and crotoxin.

Animals↗
PubMed1991

Crotoxin, a phospholipase A2 neurotoxin from snake venom, interacts with epithelial mammary cells, is internalized and induces secretion.

Prolactin (PRL) induces liberation of arachidonic acid (AA) from phospholipids of lactating mammary epithelial cells and stimulates casein secretion. In order to investigate the possible involvement of phospholipase A2 (PLA2) activity in the hormonal control of casein secretion by PRL, we examined the effects of crotoxin, a PLA2 neurotoxin from snake venom, on mammary epithelial cells. Crotoxin is made of two subunits: a basic PLA2 with low toxicity (component B, CB) and an acidic, non-toxic and enzymatically inactive component A (CA) which enhances the pharmacological action of CB. While CA is inactive, the PLA2 subunit (CB) induces an accumulation of secretory products in the lumen of mammary acini, an extensive development of the Golgi apparatus. The secretion of newly synthesized casein is increased in the presence of CB and this effect is inhibited by nordihydroguaiaretic acid (NDGA) and caffeic acid, two inhibitors of the lipoxygenase pathway which also prevent stimulation of secretion by PRL. Further, CB transiently induces the release of radiolabelled AA from mammary tissues previously labelled with [14C]AA, the highest release being observed between 15 s and 5 min of contact with CB and CA. Immunofluorescence labelling by anti-CB antibodies of epithelial mammary tissues previously incubated with CA, CB or a combination of CA and CB indicates that CB binds to epithelial cells and is internalized, at least in part, and that CA enhances both CB binding and its internalization. These observations emphasize the involvement of PLA2 in the control of casein secretion and suggest that PLA2 acts intracellularly.

Animals↗
PubMed1989

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids.

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.

Cations, Divalent↗
PubMed1989

A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin.

Phospholipase A2 activity can be determined fluorometrically in the presence of serum albumin using phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid. In the water reaction medium 10-pyrene phospholipids form vesicles and the monomer fluorescence of the pyrene is negligible due to pyrene-pyrene interaction. Upon phospholipid hydrolysis 10-pyrenyldecanoic acids are produced and tightly bind to albumin so that a monomer pyrene fluorescence is observed. We obtained an excellent parallelism between hydrolysis determined by a classical extraction method and that followed by direct and continuous spectrofluorometric recording of the monomer emission of pyrene. This assay can measure picomole amounts of phospholipids hydrolyzed per minute so that picogram quantities of phospholipases A2 from pancreas or from venoms can be measured. Phospholipase activity remains proportional to enzyme concentration over three orders of magnitude. The method can be used to quantify the phospholipase A2 activity of crude extracts of low specific activity.

Hydrolysis↗
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

Binding of crotoxin, a presynaptic phospholipase A2 neurotoxin, to negatively charged phospholipid vesicles.

Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of less than 1 microM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to less than 1 microM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.

Chemical Phenomena↗
PubMed1989

Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin.

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.

Crotoxin↗