RESUMO
Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca(2+) and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca(2+)], derived from intracellular stores, followed, only in myotubes, by a large Ca(2+) influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca(2+) permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca(2+) mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death.
Assuntos
Bothrops , Cálcio/metabolismo , Venenos de Crotalídeos/análise , Venenos de Crotalídeos/farmacologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/toxicidade , Murinae , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismoRESUMO
The effect of four myotoxins isolated from Bothrops snake venoms on the release of peroxidase trapped in large multilamellar liposomes was studied and correlated to their phospholipase A2, myotoxic and anticoagulant activities. The four myotoxins affected negatively-charged liposomes in a dose-dependent way, having no effect on positively-charged liposomes. Conditions that inhibited phospholipase A2 activity, i.e., substitution of calcium by EDTA, reduced liposome-disrupting activity of Bothrops asper myotoxin I and Bothrops atrox myotoxin, both of which have high phospholipase A2 activity, but did not affect the action of B. asper myotoxin II and Bothrops moojeni myotoxin II, which have extremely low phospholipase A2 activity. However, all myotoxins disrupted to some extent negatively-charged liposomes under conditions where phospholipase A2 activity was abolished. Since these toxins behave as amphiphilic proteins in charge-shift electrophoresis, it is suggested that membrane-disorganization is at least partially due to a non-enzymatic penetration and alteration of bilayers. There was no strict correlation between liposome-disrupting activity and myotoxicity in vivo. Thus, although both effects probably depend on the toxins' ability to disturb membranes, it is likely that variation in complexity between skeletal muscle plasma membrane and liposome bilayers are the basis for this difference. The anticoagulant effect seems to depend on the ability of the toxins to enzymatically degrade phospholipids, since only B. asper myotoxin I and B. atrox myotoxin prolonged the plasma recalcification time.
Assuntos
Venenos de Crotalídeos/farmacologia , Peroxidase do Rábano Silvestre/metabolismo , Lipossomos , Músculos/efeitos dos fármacos , Fosfolipases A/metabolismo , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Cálcio/farmacologia , Creatina Quinase/metabolismo , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/toxicidade , Ácido Egtázico/farmacologia , Eletroforese em Gel de Ágar , Camundongos , Camundongos Endogâmicos , Músculos/enzimologia , Músculos/patologia , Fosfatidilcolinas , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , OvinosRESUMO
Myotoxin II is a group II Lys49 phospholipase A(2) (PLA(2)) isolated from the venom of the snake Bothrops asper. Previous studies on a synthetic peptide derived from its heparin-binding, cationic/hydrophobic sequence 115-129 demonstrated a direct functional role of this particular region in the in vitro cytolytic and bactericidal actions of the protein. Nevertheless, no significant myonecrosis has been observed after local intramuscular injection of peptide 115-129 (p115-129) in mice. Since the membrane-damaging action of p115-129 was proposed to depend on its amphiphilic character, the present study examined the effects of substituting its cluster of three tyrosine residues by tryptophan residues, on its toxic/pharmacological activities in vitro and in vivo. This substitution resulted in a drastic enhancement of the membrane-damaging activities of the peptide (p115-W3), together with the clear expression of myotoxic activity in vivo. Both the heparin-binding and antigenic characteristics of p115-129 were essentially conserved in p115-W3, suggesting that the modification did not lead to radical structural alterations. In addition to myotoxicity, cytotoxicity, and bactericidal action, p115-W3 exerted edema-forming activity in the mouse footpad assay. Thus, the synthetic 13-mer p115-W3 reproduced all the known toxic effects of myotoxin II. In spite of its potent membrane-damaging actions, p115-W3 did not acquire direct hemolytic activity upon mouse erythrocytes, an effect which is not present in myotoxin II, but that has been ascribed to the presence of tryptophan in other cationic, membrane-damaging peptides such as mellitin from bee venom. The myotoxic activity of p115-W3 herein described constitutes the first example of a short, PLA(2)-based linear synthetic peptide with the ability to reproduce this effect of a parent protein in vivo. This finding is in clear support of the proposed relevance of the C-terminal region 115-129 in all the membrane-damaging mechanisms exerted by myotoxin II, including the myotoxic mechanism.
Assuntos
Neurotoxinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfolipases A/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Creatina Quinase/sangue , Venenos de Crotalídeos/química , Endotélio/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fosfolipases A2 do Grupo II , Técnicas Imunoenzimáticas , Injeções Intramusculares , Lisina/química , Membranas/efeitos dos fármacos , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Proteínas de Répteis , Triptofano/química , Tirosina/químicaRESUMO
Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Creatina Quinase/sangue , Edema/sangue , Edema/induzido quimicamente , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2 do Grupo II , Injeções Intramusculares , L-Lactato Desidrogenase/metabolismo , Camundongos , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Músculos/patologia , Necrose , Fosfolipases A/isolamento & purificação , Fosfolipases A/toxicidade , Fosfolipases A2 , Proteínas de Répteis , Alinhamento de SequênciaRESUMO
Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.
Assuntos
Venenos de Crotalídeos/toxicidade , Músculo Esquelético/efeitos dos fármacos , Fosfolipases A/toxicidade , Animais , Bothrops , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia por Troca Iônica , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Edema/induzido quimicamente , Dose Letal Mediana , Lipossomos , Masculino , Camundongos , Músculo Esquelético/patologia , Necrose , Fosfolipases A/isolamento & purificaçãoRESUMO
The reduction of the tetrazolium compound MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was used as the basis for the development of a simple method for the quantitative estimation of metabolically active skeletal muscle tissue remaining after in vivo venom-induced myonecrosis. Using the venom of the snake Micrurus nigrocinctus as a potent myotoxic agent, this MTT-based technique was evaluated in comparison with available methods based on the measurement of creatine kinase (CK) activity, and a quantitative histological technique considered as a reference. Homogenates of the gastrocnemius muscle prepared in the presence of 1% Triton X-100 reduced MTT and this activity correlated closely with the number of viable cells in the tissue, as determined by histological evaluation. After venom injection, residual MTT-reducing activity of muscle homogenates showed higher correlation to the myonecrosis index obtained by histological analysis, than residual muscle CK activity. Using the new MTT-based assay, the ability of an anti-M. nigrocinctus equine antivenom to neutralize venom myotoxins was studied. The myotoxic activity of the venom was completely neutralized using 4 ml antivenom/mg venom, with a 50% effective dose (ED50) value of about 2.5 ml/mg. The MTT-based method described should be useful in the estimation and standardization of anti-myotoxic potency of antivenoms, and in the screening of other neutralizing agents, as a convenient and reliable alternative to the time-consuming quantitative histological methods.
Assuntos
Antivenenos/imunologia , Venenos Elapídicos/toxicidade , Músculos/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Animais , Creatina Quinase/metabolismo , Venenos Elapídicos/imunologia , Camundongos , Testes de NeutralizaçãoRESUMO
Basic phospholipases A2 from the venom of Bothrops asper exhibit skeletal muscle damaging activity in vivo, and cytolytic activity to a variety of cell types in culture. Glycosaminoglycans of the heparin/heparan sulfate family were found to be potent blockers of the cytolytic action in vitro, and, as well, to be able to neutralize the muscle damaging activity of purified myotoxins and crude venom in vivo. However, the neutralizing effect of heparins was more potent in vitro than in vivo. The cytolytic activity of myotoxin II (a lysine-49 phospholipase A2 isoform) and its inhibition by heparin was characterized. The neutralizing effect of heparin did not depend on its anticoagulant activity, since both standard heparin and heparin with low affinity for antithrombin (LA-heparin) had a similar efficiency. Heparan sulfate and low molecular mass heparin (5 kDa) also neutralized myotoxin II. In contrast, different heparin-derived disaccharides were unable to block cytolysis, implying a requirement for a longer carbohydrate chain structure for the interaction with the protein. By affinity chromatography and gel diffusion, it was demonstrated that heparins form a complex with all isoforms of basic venom myotoxins, held at least in part by electrostatic interactions. The phospholipase A2 activity of myotoxin III, a related aspartate-49 isoform from the same venom, was unaffected by heparins, despite the fact that its myotoxic activity was inhibited, indicating a dissociation of the two actions.
Assuntos
Venenos de Crotalídeos/química , Glicosaminoglicanos/farmacologia , Fosfolipases A/química , Animais , Bothrops , Citotoxinas/química , Fosfolipases A2 do Grupo II , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Camundongos , Neurotoxinas/química , Testes de Neutralização , Fosfolipases A2 , Proteínas de RépteisRESUMO
An enzyme immunoassay for the detection of serum IgG antibodies to H. pylori whole bacterial sonicate was used in a study of dyspeptic adult patients (n = 165), apparently healthy blood donors (n = 154), and children (n = 66) from Costa Rica, where a very high incidence of gastric carcinoma is observed. The mean antibody levels and frequency distributions were similar in the dyspeptic patients and the blood donors. Meanwhile lower antibody levels to H. pylori were more frequently observed in children than in adults, suggesting an age-dependent increase in seroprevalence, as described in studies from other populations.
Assuntos
Anticorpos Antibacterianos/sangue , Dispepsia/microbiologia , Helicobacter pylori/imunologia , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Feminino , Humanos , Técnicas Imunoenzimáticas , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-IdadeRESUMO
The edema effect induced in mice by venoms of Crotalus durissus durissus and Lachesis muta stenophrys was studied. Minimum edema-forming doses were 11 and 5 micrograms, respectively. Edema developed very rapidly after injections of both venoms and reached a maximum at 6 hr. Neutralizing activity was tested by preincubation of the venoms with polyvalent antivenom. The edema induced by the venom of L. m. stenophrys was partially neutralized, whereas that induced by the venom of C. d. durissus was not neutralized.
Assuntos
Antivenenos/farmacologia , Venenos de Crotalídeos/toxicidade , Edema/induzido quimicamente , Animais , Edema/prevenção & controle , Camundongos , Fatores de TempoRESUMO
Seven murine monoclonal antibodies against Bothrops asper myotoxin were produced and partially characterized. They revealed the presence of at least four cross-reacting basic components in crude venom, with a common subunit mol. wt of 16,000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, but slight differences in charge. These myotoxin-related components might be isoforms of the toxin. By Western blotting and enzyme-immunoassay binding techniques, differences in the reactivities with basic venom fractions were observed among monoclonal antibodies, suggesting differences in epitope specificities. Three antibodies cross-reacted with B. nummifer crude venom. Two monoclonal antibodies were utilized to develop a two-site enzyme-immunoassay for myotoxin detection at the nanogram level. Three of the antibodies (one IgM and two IgG1) showed ability to neutralize myotoxicity of purified myotoxin in preincubation type experiments.
Assuntos
Anticorpos Monoclonais/biossíntese , Venenos de Crotalídeos/imunologia , Músculos/efeitos dos fármacos , Toxinas Biológicas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Reações Cruzadas , Venenos de Crotalídeos/análise , Imunodifusão , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Testes de NeutralizaçãoRESUMO
The ability of pre-existing antibodies to neutralize locally-acting toxins of Bothrops asper snake venom was investigated. Hemorrhage, myonecrosis, and edema were markedly reduced in actively immunized mice, although none of these effects was completely abolished. In mice passively immunized with equine antivenom, hemorrhage was prevented completely, while myonecrosis and edema were partially reduced. Pre-existing antibodies did not modify the early stage (< 3 hr) of venom-induced edema, but significantly accelerated the normalization of this effect within 24 hr. Passive administration of antivenom either 5 or 120 min before venom injection gave similar results, suggesting that the presence of antibodies in the intravascular compartment may fully neutralize locally acting toxins, in this experimental animal model. Overall, the homologous or heterologous origin of antibodies was not a significant factor influencing their in vivo neutralizing efficiency against local venom effects. Antibody titrations by enzyme-immunoassay using purified toxins and whole venom indicated that serum from actively-immunized mice had a higher proportion of anti-myotoxin antibodies than equine antivenom.
Assuntos
Anticorpos Heterófilos/imunologia , Venenos de Crotalídeos/toxicidade , Edema/terapia , Animais , Anticorpos Heterófilos/uso terapêutico , Formação de Anticorpos , Antivenenos/uso terapêutico , Bothrops , Venenos de Crotalídeos/administração & dosagem , Modelos Animais de Doenças , Hemorragia/prevenção & controle , Cavalos , Imunização Passiva , Técnicas Imunoenzimáticas , Camundongos , VacinaçãoRESUMO
The region comprising amino acid residues 115-129 of myotoxin II, a Lys49 phospholipase A2 from the venom of Bothrops asper, was previously shown to constitute a heparin binding site, and to be associated with its toxic activities. The corresponding synthetic peptide, KKYRYYLKPLCKK, was coupled to diphtheria toxoid as a carrier, and utilized as an immunogen in mice, to explore the possible protection from the myotoxic activity induced by myotoxin II in vivo. Mice receiving peptide-carrier injections produced antibodies to peptide 115-129, which cross-reacted to myotoxin II, as determined by enzyme-immunoassay. In contrast, no antibodies against peptide 115-129 were detected in mice immunized with myotoxin II, despite the strong antibody response to the whole antigen. Thus, region 115-129 of myotoxin II is not an immunodominant B-cell epitope in the mouse. After immunization with conjugated peptide or myotoxin II, mice were challenged with myotoxin II, and the extent of myonecrosis was estimated by determining their plasma creatine kinase activity, in comparison to non-immunized mice. After the challenge, both the group immunized with myotoxin II, and the group immunized with peptide 115-129, had a significant reduction of myonecrosis. These results demonstrate that region 115-129 of myotoxin II constitutes a neutralizing epitope, and provide further evidence for the relevance of this region in its myotoxic effect in vivo.
Assuntos
Bothrops/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fragmentos de Peptídeos/imunologia , Fosfolipases A/toxicidade , Venenos de Serpentes/toxicidade , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Creatina Quinase/sangue , Reações Cruzadas , Toxoide Diftérico/química , Epitopos de Linfócito B , Imunização/métodos , Epitopos Imunodominantes , Técnicas Imunoenzimáticas , Camundongos , Dados de Sequência Molecular , Necrose , Fosfolipases A2 , Venenos de Serpentes/imunologiaRESUMO
A new muscle damaging toxin, myotoxin II, was purified from the venom of Bothrops asper by ion-exchange chromatography on CM-Sephadex C-25. The toxin is a dimeric, basic protein with a monomer mol.wt of 13,341, according to the amino acid composition, and 16,000 on the basis of SDS-polyacrylamide gel electrophoretic mobility. It has a high number of aspartate and lysine residues, as well as of hydrophobic amino acids. Upon i.m. injection into mice, the toxin induces myonecrosis and increase in serum creatine kinase levels. In addition, myotoxin II induces edema in the mouse foot pad. Immunochemical tests, mol.wt, and amino acid composition indicate a high degree of homology between myotoxin II and a previously characterized myotoxin from this venom, myotoxin I. However, in contrast to myotoxin I, myotoxin II lacks phospholipase A2 and anticoagulant activities in vitro.
Assuntos
Venenos de Crotalídeos/análise , Músculos/efeitos dos fármacos , Neurotoxinas/análise , Aminoácidos/análise , Animais , Anticoagulantes , Creatina Quinase/sangue , Venenos de Crotalídeos/toxicidade , Edema/induzido quimicamente , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2 do Grupo II , Imunoquímica , Camundongos , Músculos/patologia , Neurotoxinas/toxicidade , Fosfolipases A/análise , Fosfolipases A2 , Proteínas de Répteis , OvinosRESUMO
Several myotoxins have been isolated from Bothrops snake venoms during the last 10 years. All of them are group II basic phospholipases A2, although some lack enzymatic activity (i.e. Lys-49 variants). These myotoxins appear as an antigenically related family of proteins occurring in many, but not all, Bothrops venoms, bearing a close structural and antigenic relationship to toxins found in other crotalid venoms of the genera Agkistrodon and Trimeresurus. Myotoxins are quantitatively important venom components in some Bothrops species. Intramuscular injection of Bothrops myotoxins leads to a rapid series of drastic degenerative events, probably initiated at the plasma membrane level, which culminate in a selective skeletal muscle necrosis. This in vivo specificity contrasts with the ability of myotoxins to lyse many types of cells in culture. Muscle damage, as well as cytolysis and liposome disruption, occur in conditions where phospholipase A2 activity is inhibited, although enzymatic activity might enhance myotoxin actions. A membrane receptor for Bothrops myotoxins has not been identified yet. A working hypothesis on the mechanism of action is proposed. Current evidence suggests that these toxins interact with biological membranes via a molecular region distinct from their known catalytic site. The active region is likely to be formed by a combination of basic and hydrophobic amino acid residues near the C-terminus of the protein, which allow electrostatic interaction and bilayer penetration. These events may lead to membrane destabilization and loss of selective permeability to ions such as calcium, both of which appear to be important mediators in the process of muscle necrosis.
Assuntos
Venenos de Crotalídeos/química , Músculo Esquelético/patologia , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Fosfolipases A/imunologia , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Toxinas Biológicas/isolamento & purificaçãoRESUMO
Antibodies against Bothrops asper myotoxin were purified from a polyvalent antivenom by affinity chromatography. These antibodies neutralized most of the myotoxic activity of crude B. asper venom, as judged by histologic evaluation of skeletal muscle and determination of plasma creatine kinase levels in mice. When tested by immunoelectrophoresis, purified antibodies formed two superimposed bands of precipitation against an homogeneous (by SDS-PAGE analysis) preparation of B. asper myotoxin, as well as against crude B. asper venom. Ouchterlony immunodiffusion analysis of purified antibodies showed two precipitation bands with a pattern of complete immunologic identity between samples of crude B. asper venoms from specimens collected in the Atlantic and Pacific regions of Costa Rica. In addition, these antibodies precipitated when reacted against venom of newborn B. asper specimens from the Pacific region, but not against venom of newborn specimens from the Atlantic region. Purified antibodies were tested by immunodiffusion against eleven different snake venoms from Costa Rica. Only the venom of B. schlegelii cross-reacted, indicating the presence in this venom of components immunologically related to B. asper myotoxin. Analysis of purified antibodies to B. asper myotoxin by agarose electrophoresis and by SDS-PAGE suggests the presence of both IgG and IgM on the basis of electrophoretic position and molecular weight of the bands. Results obtained in neutralization experiments suggest that this myotoxin is a major factor in the development of local myonecrosis induced by crude B. asper venom.
Assuntos
Anticorpos/isolamento & purificação , Antivenenos/isolamento & purificação , Venenos de Crotalídeos/imunologia , Músculos/efeitos dos fármacos , Animais , Venenos de Crotalídeos/toxicidade , Imunodifusão , Imunoeletroforese , Camundongos , Músculos/patologia , Testes de NeutralizaçãoRESUMO
The presence of components antigenically related to Bothrops asper myotoxin was investigated by Western blotting and immunoelectrophoretic techniques. B. asper myotoxin is a non-glycosylated monomeric phospholipase A with a molecular weight by SDS-PAGE of 16,000 and isoelectric point of pH 9.8-10.0. Results showed that proteins in the venoms of B. nummifer, B. godmani, B. schlegelii, B. picadoi, and Agkistrodon bilineatus were recognized by monospecific antibodies to B. asper myotoxin raised in rabbit and sheep. Western blotting indicated that cross-reacting proteins have a molecular weight of 16,000, with the exception of that of B. picadoi, which is of 24,000 mol. wt. However, immunoelectrophoresis indicated that these components are highly heterogeneous in charge, ranging from basic to acidic proteins. The cross-reacting component(s) present in newborn B. asper venom has a different charge from that of the 'adult-type'. Venoms from newborn specimens showed an additional cross-reacting band of 18,000 mol. wt. Myotoxin is an abundant component in adult B. asper venom. Myotoxin-antimyotoxin complexes had different electrophoretic mobilities in rocket immunoelectrophoresis depending upon the species in which monospecific immune sera were produced.
Assuntos
Venenos de Crotalídeos/análise , Epitopos/análise , Animais , Anticorpos Monoclonais , Reações Cruzadas , ImunoeletroforeseRESUMO
The binding of Bothrops asper myotoxin to mouse skeletal muscle was studied at both the light and electron microscope levels using the peroxidase anti-peroxidase technique. The toxin binds to muscle cell sarcolemma, and there are no intracellular binding sites. This study corroborates previous observations which suggested that the sarcolemma is the first site of action of B. asper myotoxin.
Assuntos
Venenos de Crotalídeos/metabolismo , Sarcolema/metabolismo , Animais , Técnicas Imunológicas , Masculino , Camundongos , Microscopia EletrônicaRESUMO
A myotoxin from the venom of the snake Bothrops nummifer was purified to homogeneity by ion-exchange chromatography on CM-Sephadex. The toxin is a basic dimer with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis. The toxin lacks phospholipase A2 activity when tested on egg yolk lecithin and skeletal muscle homogenates. It induces skeletal muscle damage both in vivo and in vitro. When injected i.m. it promotes a drastic increase in serum creatine kinase levels; the isozyme CK-MM is responsible for this increment. A rapid release of creatine kinase was observed when mouse gastrocnemius muscle was incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' in the plasma membrane of muscle cells. Moreover, analysis of the dose-response data indicated that the myotoxin affects muscle sarcolemma by a 'one hit' mechanism. Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium. The myotoxin has an i.v. LD50 of 3.9 mg/kg body weight in mice, and induces edema when injected in the foot pad. On the other hand, it is not directly hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes. The myotoxin shows partial immunologic identity with a myotoxic phospholipase A2 isolated from Bothrops asper venom. The polyvalent antivenom produced in Costa Rica forms a precipitation arc against B. nummifer myotoxin on immunoelectrophoresis.
Assuntos
Venenos de Crotalídeos/isolamento & purificação , Músculos/efeitos dos fármacos , Animais , Cromatografia por Troca Iônica , Creatina Quinase/sangue , Venenos de Crotalídeos/toxicidade , Eletroforese Descontínua , Imunoquímica , Imunoeletroforese , Técnicas In Vitro , Isoenzimas , Camundongos , Peso Molecular , Músculos/patologia , Fosfolipases A , Fosfolipases A2RESUMO
Group II phospholipase A(2) (PLA(2)) myotoxins found in the venoms of Crotalidae snakes can be divided into 'Asp49' and 'Lys49' isoforms, the latter being considered catalytically-inactive variants. Previous studies on one Lys49 isoform, myotoxin II from Bothrops asper, indicated that its myotoxic activity is due to the presence of a short cationic/hydrophobic sequence (115-129) near its C-terminus, which displays membrane-damaging properties. Since the C-terminal region of different group II PLA(2) myotoxins presents considerable sequence variability, synthetic peptides homologous to region 115-129 of myotoxin II, but corresponding to B. asper myotoxin III (Asp49), Agkistrodon piscivorus piscivorus Asp49 PLA(2) and Lys49 PLA(2), were studied to determine the possible functional relevance of such region for the toxic activities of these proteins. Results showed that both Lys49-derived peptides (p-BaK49 and p-AppK49) were able to lyse skeletal muscle C2C12 cells in culture, and to induce edema in the mouse footpad assay. Moreover, p-AppK49, which showed a markedly stronger cytotoxic potency than p-BaK49, additionally induced skeletal muscle necrosis when injected into mice. These observations unequivocally identify the sequence 115-129 (KKYKAYFKLKCKK) of the Lys49 PLA(2) of A. p. piscivorus as containing the key structural determinants needed for myotoxicity, and represent the first report of an unmodified, PLA(2)-derived short synthetic peptide with the ability to reproduce this effect of a parent toxin in vivo. On the other hand, the two Asp49-derived peptides did not show any toxic effects in vitro or in vivo, even at high concentrations. These findings suggests that Lys49 and Asp49 group II PLA(2)s might exert their myotoxic actions through different molecular mechanisms, by implying that the latter may not utilize their C-terminal regions as main membrane-destabilizing elements.