RESUMO
Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects.
Assuntos
Casearia/química , Contração Muscular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Brasil , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Diafragma/efeitos dos fármacos , Diafragma/inervação , Diafragma/fisiologia , Técnicas In Vitro , Masculino , Metanol/química , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Extratos Vegetais/químicaRESUMO
Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA(2), but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.
Assuntos
Anticorpos/imunologia , Anticorpos/metabolismo , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/toxicidade , Coração/efeitos dos fármacos , Fosfolipases A/toxicidade , Animais , Anticorpos/genética , Anticorpos/isolamento & purificação , Coagulação Sanguínea/imunologia , Venenos de Crotalídeos/imunologia , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Fosfolipases A2 do Grupo II , Humanos , Cinética , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fosfolipases A/imunologia , Fosfolipases A/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Répteis , SolubilidadeRESUMO
Toxin gamma is a basic, low-molecular-weight, neurotoxic protein, isolated from the venom of the Brazilian scorpion, Tityus serrulatus. Raman spectra (400-1800 cm-1 region) of this toxin in both the lyophilized state and in 0.1 M acetate buffer (pH 4.5) and the infrared spectrum (700-4000 cm-1 region) of a solid film were investigated. From the vibrational spectra, it can be concluded that the polypeptide backbone of toxin gamma consists of a mixture of the different secondary structures, with predominance of beta-sheet, followed by unordered structure and alpha-helix, with some evidence of beta-turn structures. The four disulfide bridges assume the gauche-gauche-gauche conformation of the CCSSCC fragments. The intensity ratio of the doublet at 853 and 828 cm-1 suggests that four out of the five tyrosine residues are exposed. The three tryptophan residues are exposed on the surface, and the single methionine residue assume the gauche-gauche conformation. Toxin gamma retains full activity in the pH 4.5-7.5 range, but is almost completely inactivated at pH 11.5.
Assuntos
Venenos de Escorpião , Espectrofotometria Infravermelho , Análise Espectral Raman , Animais , Dissulfetos , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Metionina , Camundongos , Fenilalanina , Conformação Proteica , Venenos de Escorpião/toxicidade , Triptofano , TirosinaRESUMO
A procedure for the preparation of highly purified sheep prothrombin is described. The purified zymogen, when subjected to disc gel electrophoresis in polyacrylamide, gave rise to one single band. Only alanine was found as N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. The isoelectric point, as determined by isoelectric focusing in polyacrylamide gel slab, was shown to be 4.9-5.0. Non-chromatographed, but not the purified zymogen, could be converted into active thrombin in half-saturated trisodium citrate seeded with thrombin. Pure sheep prothrombin showed 5.6% of neutral sugars and the following amino acid composition: Ala35, Arg44, Asx54-55, -Cys24, Glx72, Gly53-54, His8, Ile19, Leu45, Lys31, Met7, Phe23, Pro36, Ser34, Thr29-29, Trp16, Tyr19 and Val33, which accounts for a molecular weight of about 66 000 (amino acids only). The molecular weight as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after reduction by 2-mercaptoethanol, was shown to be 77 000 +/- 3000 (carbohydrates included).
Assuntos
Protrombina , Aminoácidos/análise , Animais , Ativação Enzimática , Focalização Isoelétrica , Peso Molecular , Protrombina/isolamento & purificação , Protrombina/metabolismo , OvinosRESUMO
TsTX-V, a new neurotoxin from Tityus serrulatus scorpion venom able to induce a prolongation of the inactivation of Na+ channels, has been purified to homogeneity. The venom was chromatographed on CM-cellulose-52 and 13 fractions were first collected. A subsequent stepwise elution chromatography of fraction XI afforded, among other toxins, highly purified TsTX-V, which showed a single band by PAGE, SDS-PAGE or isoelectric focusing, a distinctive amino acid composition, mol. wt. = 7230, pI = 8.0 and i.v. LD50 = 94 +/- 7 micrograms/kg in mice. TsTX-V induced a long lasting hypertension in anesthetized rats and prolonged the action potential of the B fibers of the rabbit vagus nerve at 0.03 microgram/ml. At 0.3 microgram/ml and higher concentrations it caused also a nerve depolarization. These effects on nerve membranes were irreversible and could be suppressed by tetrodotoxin (200-500 nM). Nerve fibers depolarized by high extracellular K+(15-30mM) concentrations still displayed long duration action potentials after TsTX-V treatment. It is suggested that TsTX-V blocks the Na+ channel inactivation system probably as an alpha-toxin.
Assuntos
Neurotoxinas/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , Canais de Sódio/efeitos dos fármacos , Potenciais de Ação , Aminoácidos/análise , Animais , Pressão Sanguínea/efeitos dos fármacos , Dose Letal Mediana , Camundongos , Peso Molecular , Neurotoxinas/química , Neurotoxinas/farmacologia , Coelhos , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Nervo Vago/efeitos dos fármacosRESUMO
Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of 86Rb+ release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 micrograms/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 microM veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Potássio/metabolismo , Venenos de Escorpião/química , Alquilação , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Ilhotas Pancreáticas/metabolismo , Dados de Sequência Molecular , Oxirredução , Canais de Potássio/efeitos dos fármacos , Ratos , Radioisótopos de Rubídio , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Homologia de Sequência , Veratridina/farmacologiaRESUMO
Crystals of a myotoxic phospholipase A(2) from Bothrops neuwiedi pauloensis have been obtained. They diffracted at 2.5 A resolution using a synchrotron radiation source and belong to space group P3(1)21. Preliminary analysis shows that there are two molecules in the asymmetric unit.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Neurotoxinas/química , Fosfolipases A/química , Animais , Brasil , Cristalização , Proteínas de Répteis , Difração de Raios XRESUMO
Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).
Assuntos
Anticoagulantes/farmacologia , Crotoxina/química , Crotoxina/metabolismo , Fosfolipases A/química , Fosfolipases A/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Creatina Quinase/sangue , Venenos de Crotalídeos/química , Crotalus , Crotoxina/farmacologia , Edema/induzido quimicamente , Escherichia coli/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Cinética , Dose Letal Mediana , Lipossomos/química , Camundongos , Dados de Sequência Molecular , Peroxidases/metabolismo , Fosfolipases A/farmacologia , Subunidades ProteicasRESUMO
A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NH units/mg of protein , a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroesis, E 1.0 mg/ml 1.0 cm, 280 nm = 1.45 at pH 7.0 and the following amino acid composition: Asx 51, Thr 38, Glx 62, Pro 23, Gly44, Ala 25, Half-Cys 30, Val 35, Met 3, Ile 30, Leu 32, Tyr 19, Phe 22, Lys 36, His 8, Arg 28, and Trp 13, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine was found as the only N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.
Assuntos
Protrombina , Aminoácidos/análise , Animais , Eletroforese Descontínua , Humanos , Peso Molecular , Protrombina/isolamento & purificação , Protrombina/metabolismo , Especificidade da Espécie , SuínosRESUMO
The complete amino acid sequence of the 121 amino acid residues of piratoxin II, a phospholipase A(2) like myotoxin from Bothrops pirajai venom, is reported. PrTX-II is a basic protein with a molecular mass of 13740 Da, a calculated pI of 9.03, but an experimental pI of 8.4 +/- 0.2, showing sequence similarity with other bothropic (90-99%) or non-bothropic ( approximately 80%) Lys49 PLA(2)-like myotoxins. This similarity falls to approximately 70% when this sequence is aligned with that of Asp49 PLA(2). Due to the substitution of Asp49 by Lys49 and alterations in the calcium binding loop structure, as the replacement of Gly32 by Leu32, piratoxin-II shows no PLA(2) activity when assayed on egg yolk. Piratoxin-II showed the same primary structure as piratoxin-I, except that it has Lys116 for Leu116. Despite this slightly higher basicity at the C-terminal region, piratoxin-II was shown to be less myotoxic than piratoxin-I. The change Leu --> Lys induced an alteration of the molecule surface shape and probably of the environment charge high enough to slightly decrease the myotoxic activity. When aligned with B. jararacussu bothropstoxin-I and with B. asper Basp-II, piratoxin-II revealed a single (position 132) and a quintuple (positions 17, 90, 111, 120 and 132) amino acid substitution, respectively, suggesting a common evolutionary origin for these three myotoxins.
Assuntos
Venenos de Crotalídeos/enzimologia , Lisina/química , Fosfolipases A/química , Sequência de Aminoácidos , Fosfolipases A2 do Grupo II , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Propriedades de SuperfícieRESUMO
The aqueous extract from the leaves of Casearia mariquitensis (C. m.), a plant found in Brazilian open pastures, was assayed for its ability to inhibit some hematological and hemostatic effects induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi pauloensis. The aqueous extract from C. m. was able to neutralize the hematological alterations induced by the crude venom (C.V.) upon erythrocytes when the venom was incubated at a ratio of 1:10 (w/w, venom/extract), but it did not neutralize the platelet decreasing ability of C.V. The plasma fibrinogen concentration decreased approximately 36% and 83% when 0.6 LD(50) of the C.V. or neuwiedase, respectively, were injected by i.p. route in mice, and the aqueous extract from C. m. was able to inhibit this effect. The Bbeta fibrinogen chain was protected against degradation caused by crude venom and neuwiedase when the venom or toxin were incubated with C. m. extract. We also observed that this extract exerted a very slight effect on the clotting time, prolonging it only to a little extent. The pulmonary hemorrhage induced by neuwiedase when injected intravenously with 0.6 LD(50) was completely inhibited when this toxin was incubated with the extract at a ratio of 1:10 (w/w, toxin/extract). It is concluded that C. m. displays components able to inhibit some hematological and systemic alterations induced by C.V.
Assuntos
Casearia/química , Inibidores Enzimáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Venenos de Víboras/antagonistas & inibidores , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/enzimologia , Eritrócitos/efeitos dos fármacos , Fibrinogênio/análise , Fibrinogênio/metabolismo , Fibrinólise/efeitos dos fármacos , Hematócrito , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Camundongos , Extratos Vegetais/farmacologia , Venenos de Víboras/enzimologiaRESUMO
The pathological alterations induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi, were studied in mice. Neuwiedase was devoid of hemorrhagic activity when tested in the skin up to a dose of 200 microgram, and also after intramuscular injection in the gastrocnemius. However, it induced bleeding when applied onto the mouse cremaster muscle in intravital microscopy experiments, and caused pulmonary hemorrhage when injected intravenously at doses higher than 5 microgram/g. Median lethal dose (LD(50)) by the intravenous route was 5 microgram/g, whereas LD(50) of crude venom was 0.47 microgram/g. After intramuscular injection, neuwiedase induced a mild myotoxic effect, evidenced histologically and by the increment in plasma creatine kinase activity, but it was devoid of hemorrhagic and thrombotic effects. In contrast, crude B. neuwiedi venom induced prominent hemorrhage and myonecrosis in gastrocnemius muscle. Both venom and neuwiedase induced an inflammatory reaction in muscle tissue characterized by abundant polymorphonuclear leukocytes. Moreover, a conspicuous edema developed in the foot pad after subcutaneous injection of neuwiedase. Anti-neuwiedase antibodies produced in rabbits were effective in the neutralization of hemorrhagic activity of crude venom, evidencing immunological cross-reactivity between neuwiedase and other hemorrhagic metalloproteinases present in the venom, and suggesting that metalloproteinases devoid of, or having low, hemorrhagic activity could be good immunogens to generate antibodies effective against high molecular mass metalloproteinasas having potent hemorrhagic activity. It is concluded that neuwiedase, despite its lack of hemorrhagic effect when injected in the gastrocnemius muscle, contributes to local tissue damage by inducing edema, inflammatory infiltrate and mild myotoxicity, and by degrading extracellular matrix components. In addition, large doses of neuwiedase may contribute to pulmonary bleeding
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/toxicidade , Venenos de Víboras/toxicidade , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Creatina Quinase/metabolismo , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/toxicidade , Edema/induzido quimicamente , Edema/tratamento farmacológico , Hemorragia/induzido quimicamente , Hemorragia/tratamento farmacológico , Técnicas In Vitro , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Dose Letal Mediana , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/imunologia , Camundongos , Músculos/efeitos dos fármacos , Músculos/patologia , Testes de Neutralização , Fatores de Tempo , Venenos de Víboras/antagonistas & inibidores , Venenos de Víboras/imunologiaRESUMO
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
Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Creatina Quinase/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Cristalografia por Raios X , Edema/induzido quimicamente , Técnicas In Vitro , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Fosfolipases A/isolamento & purificação , Fosfolipases A/farmacologia , Fosfolipases A/toxicidade , Fosfolipases A2 , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Conformação ProteicaRESUMO
1. The effect of Tityus serrulatus scorpion venom and its toxin components on the rabbit isolated corpus cavernosum was investigated by use of a bioassay cascade. 2. Tityus serrulatus venom (3-100 microg), acetylcholine (ACh; 0.3-30 nmol) and glyceryl trinitrate (GTN; 0.5-10 nmol) dose-dependently relaxed rabbit isolated corpus cavernosum preparations precontracted with noradrenaline (3 microM). The selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ; 30 microM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 microM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible. 3. The non-selective NO synthase (NOS) inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME; 10 microM) and NG-iminoethyl-L-ornithine (L-NIO; 30 microM) also increased the tone of the rabbit isolated corpus cavernosum and markedly reduced both ACh- and Tityus serrulatus venom-induced relaxations without affecting those evoked by GTN. The inhibitory effect was reversed by infusion of L-arginine (300 microM), but not D-arginine (300 microM). The neuronal NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM, 100 microM) did not affect either the tone of the rabbit isolated corpus cavernosum or the relaxations induced by ACh, bradykinin (Bk), Tityus serrulatus venom and GTN. TRIM was approximately 1,000 times less potent than L-NAME in inhibiting rabbit cerebellar NOS in vitro, as measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline. 4. The protease inhibitor aprotinin (Trasylol; 10 microg ml[-1]) and the bradykinin B2 receptor antagonist Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7, Oic8]-BK; 50 nM) did not affect the rabbit isolated corpus cavernosum relaxations induced by Tityus serrulatus venom. The ATP-dependent K+ channel antagonist glibenclamide (10 microm) and the Ca2+-activated K+ channel antagonists apamin (0.1 microM) and charybdotoxin (0.1 microM) also failed to affect the venom-induced relaxations. Similarly, the K+ channel blocker tetraethylammonium (TEA; 10 microM) had no effect on the venom-induced relaxations. 5. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose-dependent and non-tachyphylactic manner. Ruthenium red (30 microM), an inhibitor of capsaicin-induced responses, markedly reduced the relaxations caused by capsaicin, but failed to affect those induced by Tityus serrulatus venom. L-NAME (10 microM) had no effect on the capsaicin-induced relaxations of the rabbit isolated corpus cavernosum. 6. The sodium channel blocker tetrodotoxin (TTX; 1 microM) abolished the relaxations of the rabbit isolated corpus cavernosum induced by Tityus serrulatus venom without affecting those evoked by capsaicin, ACh and GTN. Tetrodotoxin (1 microM) also promptly reversed the response to the venom when infused during the relaxation phase. 7. The bioassay cascade of the toxin components purified from Tityus serrulatus venom revealed that only fractions X, XI and XII caused dose-dependent relaxations of the rabbit isolated corpus cavernosum and these were markedly reduced by either TTX (1 microM) or L-NAME (10 microM). 8. Our results indicate that Tityus serrulatus scorpion venom (and the active fractions X, XI and XII) relaxes rabbit corpus cavernosum via the release of NO. This release is specifically triggered by the activation of capsaicin-insensitive cavernosal non-adrenergic non-cholinergic (NANC) fibres, that may possibly be nitrergic neurones. Tityus serrulatus venom may therefore provide an important tool for understanding further the mechanism of NANC nitrergic nerve activation.
Assuntos
Óxido Nítrico/fisiologia , Pênis/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Animais , Aprotinina/farmacologia , Atropina/farmacologia , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Pênis/inervação , Pênis/metabolismo , Bloqueadores dos Canais de Potássio , Coelhos , Receptores Adrenérgicos/metabolismo , Receptores Colinérgicos/metabolismo , Bloqueadores dos Canais de Sódio , Tetrodotoxina/farmacologiaRESUMO
The fractionation of Phoneutria nigriventer spider venom by gel filtration (Sephadex G-10-120) followed by ion-exchange chromatography (microgranular CM-cellulose-52) resulted in sixteen fractions (CI to CXVI) from which CVII+VIII, CIX and CX+XI caused dose-dependent and short-lived contractions of both arterial and venous rabbit vessels. Fraction CX+XI was further purified by a reverse phase HPLC, and a contractile polypeptide (PNV2) was isolated. The amino terminal sequence of PNV2 (LAKRADICQPGKTSQRACET) indicated that it represents a pure polypeptide consisting of a single chain. Furthermore, the amino acid analysis of PNV2 revealed the presence of four disulfide bridges, a high content in Lys (14%), Glx (11%), and the absence of His. The global amino acid composition showed that this polypeptide is composed of 102 residues (Trp not included) with a calculated molecular weight of 12,114. Whether this peptide is responsible for the vascular alterations observed in Phoneutria envenomation, such as lung edema and priapism, remains to be further investigated.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Neuropeptídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Sequência de Aminoácidos , Animais , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Coelhos , Venenos de Aranha/farmacologiaRESUMO
Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum (Didelphis albiventris) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point < 4. The two subunits were separated by ion-exchange HPLC. The N-terminal sequences of both subunits (LKAMDPTPXLWIETESP, where X is Arg-9 and Pro-9, respectively) showed a high degree of identity with other serum inhibitors isolated from different marsupials. Functional studies pointed out that ABC inhibits the hemorrhagic and proteolytic activities on fibrin, fibrinogen, and casein induced by the metalloproteases BaP1 and BaH4 isolated from B. asper venom. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A(2) isolated from the same venom. Moreover, it had inhibitory effects on the phospholipase A(2) activity of the crude venom as well as the isolated venom phospholipases A(2).
Assuntos
Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Neurotoxinas/antagonistas & inibidores , Gambás/sangue , Fosfolipases A/antagonistas & inibidores , Aminoácidos/química , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Bothrops , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Fosfolipases A2 do Grupo II , Hemorragia/prevenção & controle , Proteínas de Répteis , Análise de Sequência de ProteínaRESUMO
Piratoxin-I (PrTX-I) is a Lys-49 phospholipase (PLA(2)) homologue, isolated from Bothrops pirajai snake venom, that has no phospholipase activity. In this study, we investigated the in vivo oedematogenic activity of PrTX-I in both the rat and the rabbit as well as the ability of PrTX-I to activate rat mast cells in vitro. In the rat paw and skin, PrTX-I (3-100 microg/paw) induced a dose-dependent oedema that was associated with extensive mast cell degranulation. The involvement of mast cells in PrTX-I-mediated oedema formation in the rat was further confirmed by the findings that this protein significantly activated rat peritoneal mast cells in vitro, causing the release of [(14)C]5-hydroxytryptamine ([(14)C]5-HT; 51 +/- 1%). In the rabbit, PrTX-I (10-100 microg/site) also induced dose-dependent skin oedema formation that was not affected by either mepyramine (a histamine H(1) receptor antagonist) or cyproheptadine (1.0 microg/site), indicating that mast cells do not play a role in this animal species. The bradykinin B(2) receptor antagonist Hoe 140 (0.5 microg/site) and the platelet-activating factor (PAF) receptor antagonist WEB 2086 (200 microg/site) also failed to affect the PrTX-I-induced rabbit skin oedema, ruling out the involvement of kinins and PAF. The PLA(2) inhibitor p-bromophenacyl bromide greatly reduced the PrTX-I-induced oedema in both the rat and the rabbit, and also inhibited the rat in vitro mast cell activation induced by this PLA(2) homologue. The polyanions heparin and dermatan sulphate efficiently prevented oedema formation in both species, and heparin inhibited PrTX-I-induced rat mast cell degranulation. Our results are consistent with the suggestion that the cationic charge of PrTX-I plays a major role in the inflammatory responses induced by this PLA(2) homologue.
Assuntos
Venenos de Crotalídeos/farmacologia , Mediadores da Inflamação/farmacologia , Mastócitos/efeitos dos fármacos , Fosfolipases A/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Edema/induzido quimicamente , Fosfolipases A2 do Grupo II , Masculino , Coelhos , Ratos , Ratos Wistar , Proteínas de RépteisRESUMO
Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.
Assuntos
Venenos de Crotalídeos/isolamento & purificação , Animais , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/toxicidade , Dissulfetos , Substâncias Macromoleculares , Camundongos , Conformação ProteicaRESUMO
Viperidae snakes belonging to the genus Crotalus, subfamily Crotalinae, include the species durissus terrificus, durissus durissus, adamanteus, atrox, cerastes, horridus, molossus, scutulatus, viridis and others. All of them, except for the first 2, are found in North America. Crotalus atrox, or diamondback rattlesnakes, live in the southwest United States and in Mexico. This paper describes the fractionation of C. atrox venom in order to isolate and identify its hypotensive agents.