RESUMO
This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its V(max) (7.56nmoles/min) and K(M) (2.76mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75mM and 0.4muM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotalus/fisiologia , Fosfolipases A2/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Venenos de Crotalídeos/genética , Crotalus/genética , Dados de Sequência Molecular , Fosfolipases A2/química , Fosfolipases A2/farmacologia , Filogenia , Xanthomonas axonopodis/efeitos dos fármacosRESUMO
A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.
Assuntos
Acacia/química , Lectinas de Plantas/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Quitina/química , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Fabaceae , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Lectinas de Plantas/análise , Lectinas de Plantas/química , Alinhamento de Sequência , Análise de Sequência de Proteína , Espectrometria de Massas em TandemRESUMO
Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3',4'-OH flavonoids are known inhibitors of phospholipases A2.
Assuntos
Antivenenos/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Edema/tratamento farmacológico , Hemorragia/etiologia , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Polygalaceae/química , Animais , Antivenenos/isolamento & purificação , Bothrops , Venenos de Crotalídeos/toxicidade , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edema/etiologia , Hemorragia/tratamento farmacológico , Masculino , RatosRESUMO
Our purpose was to determine the blood amino acid concentration during insulin induced hypoglycemia (IIH) and examine if the administration of alanine or glutamine could help glycemia recovery in fasted rats. IIH was obtained by an intraperitoneal injection of regular insulin (1.0 U/kg). The blood levels of the majority of amino acids, including alanine and glutamine were decreased (P < 0.05) during IIH and this change correlates well with the duration than the intensity of hypoglycemia. On the other hand, the oral and intraperitoneal administration of alanine (100 mg/kg) or glutamine (100 mg/kg) accelerates glucose recovery. This effect was partly at least consequence of the increased capacity of the livers from IIH group to produce glucose from alanine and glutamine. It was concluded that the blood amino acids availability during IIH, particularly alanine and glutamine, play a pivotal role in recovery from hypoglycemia.
Assuntos
Alanina/sangue , Glicemia/biossíntese , Gluconeogênese/efeitos dos fármacos , Glutamina/sangue , Hipoglicemia/sangue , Insulina/farmacologia , Fígado/efeitos dos fármacos , Aminoácidos/sangue , Animais , Glicemia/análise , Combinação de Medicamentos , Hipoglicemia/induzido quimicamente , Injeções Intraperitoneais , Fígado/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 A, and diffracted to 1.75 A resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 A, and diffracted to 2.6 A resolution. The crotapotin crystal diffracted to 2.3 A resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.
Assuntos
Crotoxina/química , Fosfolipases A/química , Cristalização , Cristalografia por Raios X , Dimerização , Fosfolipases A2 , Conformação ProteicaRESUMO
In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotoxina/química , Fator de Ativação de Plaquetas/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Animais , Plaquetas/efeitos dos fármacos , Crotalus/metabolismo , Crotoxina/isolamento & purificação , Fibrinogênio/efeitos dos fármacos , Dados de Sequência Molecular , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Fator de Ativação de Plaquetas/isolamento & purificação , Fator de Ativação de Plaquetas/farmacologia , Agregação Plaquetária , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Trombina/isolamento & purificação , Trombina/farmacologia , Tosilina Clorometil Cetona/farmacologiaRESUMO
Crotamine, a neurotoxin present in the venom of the South American rattlesnake Crotalus durrisus terrificus exists as several polymorphic variants, as demonstrated by recombinant DNA technology (Smith and Schmidt, Toxicon 28 (1990) 575-585). We have isolated native crotamine by chromatography on Sephadex G75, and have purified two crotamine isoforms (F2 and F3) by a single step of RP-HPLC. Native crotamine and RP-HPLC fractions F2 and F3 produced skeletal muscle spasms and spastic paralysis in mice. At low glucose concentrations (2.8-5.6 mmol/l), none of the crotamines altered the insulin secretion by rat isolated islets. In the presence of 16.7 mmol glucose/l, F2 (5 microg/ml), but not F3, increased insulin secretion two-fold, whereas native crotamine (1.5, 5 and 16.5 microg/ml) potentiated the secretion dose-dependently. The increase in insulin secretion induced by F2 fraction (5 microg/ml) was similar to that obtained with 16.5 microg of native crotamine/ml. These results indicate that the mode of action of the F2 and F3 isoforms in beta-cells is different from that in muscle cells. This difference may be related to the binding affinity of each isoform for the Na(+) channels located in the beta-cell membrane. Crotamine isoforms may be valuable tools for studying the involvement of Na(+) channels in the mechanism of insulin secretion.
Assuntos
Venenos de Crotalídeos/química , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Espectrometria de Massas , Estrutura Molecular , Peso Molecular , Isoformas de Proteínas/química , Ratos , Homologia de Sequência , América do SulRESUMO
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
The crystal structure of neurotoxin Ts1, a major component of the venom of the Brazilian scorpion Tityus serrulatus, has been determined at 1.7 A resolution. It is the first X-ray structure of a highly toxic anti-mammalian beta-toxin. The folding of the polypeptide chain of Ts1 is similar to that of other scorpion toxins. A cysteine-stabilised alpha-helix/beta-sheet motif forms the core of the flattened molecule. All residues identified as functionally important by chemical modification and site-directed mutagenesis are located on one side of the molecule, which is therefore considered as the Na+channel recognition site. The distribution of charged and non-polar residues over this surface determines the specificity of the toxin-channel interaction. Comparison to other scorpion toxins shows that positively charged groups at positions 1 and 12 as well as a negative charge at position 2 are likely determinants of the specificity of beta-toxins. In contrast, the contribution of the conserved aromatic cluster to the interaction might be relatively small. Comparison of Ts1 to weak beta-toxins from Centruroides sculpturatus Ewing reveals that a number of basic amino acid residues located on the face of the molecule opposite to the binding surface may account for the high toxicity of Ts1.
Assuntos
Neurotoxinas/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Insetos , Modelos Moleculares , Dados de Sequência Molecular , Neurotoxinas/toxicidade , Conformação Proteica , Venenos de Escorpião/toxicidade , Homologia de Sequência de AminoácidosRESUMO
In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A2 (PLA2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA2s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA2s such as from the Bothrops snake venom. Also, this PLA2 induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and p-BPB showed a similar reduction. The p-BPB did not reduce significantly the myotoxic activity induced by the PLA2, but the anhydrous acetic acid treatment and the pre-incubation of PLA2 with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiforae (Gram-negative), which was drastically reduced by incubation of this PLA2 with p-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or beta-wing region may be involved in the binding of this PLA2 to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Fosfolipases A/farmacologia , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Crotalus , Masculino , Dados de Sequência Molecular , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
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 effects of Bothrops pirajai snake venom on the mouse extensor digitorum longus (EDL) preparation were examined using myographic, histopathological and biochemical approaches. B. pirajai venom (10, 25 or 50 microg/ml) dose dependently and irreversibly blocked the contractile response of indirectly stimulated EDL muscle. Histopathological analysis of EDL muscle incubated with venom showed dose-dependent damage with a loss of the normal tissue structure and the appearance of highly dark, edematous fibers together with myofibrils in various stages of condensation. At high doses of venom (50 microg/ml), loss of muscle cells was observed. In non-stimulated EDL, B. pirajai venom (10 and 50 microg/ml) caused a time-dependent release of CK which was maximal after 120 min. These results suggest that a component(s) present in the B. pirajai venom has a direct myolytic action on the skeletal muscle.
Assuntos
Bothrops/fisiologia , Creatina Quinase/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Venenos de Serpentes/toxicidade , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Técnicas In Vitro , Camundongos , Músculo Esquelético/química , Músculo Esquelético/patologia , MiografiaRESUMO
Crotoxin from Crotalus durissus cascavella venom was purified by a combination of molecular exclusion chromatography (Superdex 75 column) and HPLC molecular exclusion (Protein Pack 300SW column). Neurotoxic and myotoxic effects from C. durissus cascavella whole venom and its main fraction, the crotoxin-like, were studied in the chick biventer cervicis (CBC) nerve-muscle preparation. Both venom and its crotoxin showed significant (p < 0.05) blockade of neuromuscular transmission at concentrations as low as 0.2-1, 5 and 25 microg/ml, but no significant effect has been shown with a concentration of 0.04 microg/ml (n = 5 each). The time required to produce 50% neuromuscular blockade with the venom and its crotoxin was 53.6+/-8.2 and 65.9+/-4.9 min (0.2 microg/ml), 29.7+/-1.9 and 34.3+/-1.9 min (1 microg/ml), 24.8+/-1.6 and 21.1+/-1.5 min (5 microg/ml), 20.9+/-3.7 and 20.1+/-1.4 min (25 microg/ml), respectively. The addition to the incubation bath of acetylcholine (55 and 110 microM) or KCl (20.1 mM), either before or after the venom or the crotoxin induced contracture in the presence of a total blockade, in all the concentrations used. Morphological analysis showed that the damage caused by C. durissus cascavella venom is stronger than that caused by crotoxin. The myonecrotic picture was more marked at higher venom and crotoxin doses (1, 5 or 25 microg/ml). Only at 25 microg/ml concentrations of the venom and crotoxin, marked muscle fiber changes were detected. We concluded that the crotoxin-like and the whole venom from C. durissus cascavella possess a preponderant and quite potent neurotoxic action in this preparation, and a myotoxic action which is observed only at higher doses.
Assuntos
Venenos de Crotalídeos/toxicidade , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Neurotoxinas/toxicidade , Animais , Galinhas , Relação Dose-Resposta a Droga , Masculino , Junção Neuromuscular/efeitos dos fármacosRESUMO
The fractionation of Didelphis albiventris serum by DEAE-Sephadex A50 yields a fraction (DA2) which protects the opossum against Bothrops venom. One polypeptide (DA2-II) responsible for this protection was isolated from fraction DA2 by ion exchange chromatography and biochemically characterized. DA2-II is a 43,000 mol. wt glycoprotein with the following N-terminal sequence: LKAMDTTPPLKIKKEPVK. Pairwise comparison of the amino acid sequence with four anti-hemorrhagic factors isolated from other opossum species indicated that DA2-II possesses high similarity (60-80%) with these proteins.
Assuntos
Antivenenos/farmacologia , Proteínas Sanguíneas/isolamento & purificação , Venenos de Crotalídeos/toxicidade , Gambás/sangue , Sequência de Aminoácidos , Animais , Antivenenos/química , Antivenenos/isolamento & purificação , Proteínas Sanguíneas/química , Proteínas Sanguíneas/farmacologia , Bothrops , Fracionamento Químico , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso MolecularRESUMO
Bothropstoxin-I (BthTX-I) and bothropstoxin-II (BthTX-II) are Lys-49 and Asp-49 phospholipases A(2) (PLA(2)s), respectively, isolated from Bothrops jararacussu venom. Piratoxin-I (PrTX-I) is a Lys-49 PLA(2) isolated from Bothrops pirajai venom. In this study, the ability of BthTX-I, BthTX-II and PrTX-I to recruit leucocytes into the rat pleural cavity and potential mechanisms underlying this effect were investigated. Intrapleural injection of either BthTX-I or PrTX-I (10-100 microg/cavity each) caused a significant leucocyte infiltration at 12 h after injection. The maximal cell migration was observed with the dose of 30 microg/cavity (14.9+/-15.5 and 17.6+/-1. 6x10(6) cells/cavity, respectively). Leucocyte counts consisted mainly of mononuclear cells, but significant amounts of neutrophils and eosinophils were also observed. Intrapleural injection of BthTX-II (10-100 microg/cavity) caused a marked leucocyte infiltration at 6 and 12 h after injection. The maximal response was observed with the dose of 100 microg/cavity (57.3+/-3.4x10(6) cells/cavity, 6 h). The leucocyte counts were mainly composed of neutrophils and mononuclear cells. The treatment of either BthTX-I (30 microg/cavity, 12 h) or BthTX-II (30 microg/cavity, 6 h) with the PLA(2) inhibitor p-bromophenacyl bromide (p-BPB) had no effect on the total and differential leucocyte counts induced by these proteins. The same treatment partially reduced the PrTX-I-induced pleural leucocyte infiltration. In rats depleted of the histamine and 5-hydroxytryptamine (5-HT) stores by chronic treatment with compound 48/80, the total leucocyte counts in response to BthTX-I, BthTX-II and PrTX-I was not significantly affected compared to control animals. In addition, BthTX-I, BthTX-II and PrTX-I (100 microg/ml each) significantly degranulated pleural mast cells in vitro leading to the release of [(14)C]5-hydroxytryptamine ([(14)C]5-HT). p-BPB and heparin (50 IU/ml) significantly reduced the [(14)C]5-HT release induced by these PLA(2)s. Our results demonstrate that BthTX-I, BthTX-II and PrTX-I recruit leucocyte into the pleural cavity of the rat by mechanisms unrelated to enzymatic activity and pleural mast cell degranulation.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinófilos/fisiologia , Neutrófilos/fisiologia , Fosfolipases A/farmacologia , Pleura/fisiologia , Acetofenonas/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Fosfolipases A2 do Grupo II , Histamina/metabolismo , Contagem de Leucócitos , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas de Répteis , Serotonina/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Bothtrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate Mr of 14000 and 27000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 14 half-cystine residues. Its isoelectric point and extinction coefficient (E(1.0 mg/ml) (1.0 cm) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A2 (PLA2) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK... revealed high homology with other Lys 49 PLA2-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI, which diffracted to a maximal resolution of 1.6 A, were obtained and indicated the presence of a dimer in the asymmetrical unit.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/química , Fosfolipases A/análise , Fosfolipases A/isolamento & purificação , Sequência de Aminoácidos , Animais , Edema/induzido quimicamente , Eletroforese em Gel de Poliacrilamida , Membro Posterior , Dados de Sequência Molecular , Peso Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Miofibrilas/efeitos dos fármacos , Necrose , Fosfolipases A/química , Fosfolipases A/toxicidade , Fosfolipases A2 , Ratos , Difração de Raios XRESUMO
Acute renal failure is one the most common systemic complications after snakebite, however, its pathogenesis remains obscure. In this study we evaluated the renal effects of Bothrops moojeni venom and its myotoxins (Bmtx-I and BmtxII) in rat isolated perfused kidneys. The myotoxins were purified by ion-exchange chromatography and reverse phase HPLC. The whole venom (10 microg/ml) and myotoxins (5 microg/ml) were added to the perfusion system 30 min after the beginning of each perfusion. The renal effects were compared to a control group perfused with modified Krebs-Henseleit solution alone. B. moojeni venom decreased the perfusion pressure (PP), renal vascular resistance (RVR), and the percent sodium, potassium and chloride tubular transport (%TNa(+), %TK(+), %TCl(-)). In contrast, the venom increased the urinary flow (UF), glomerular filtration rate (GFR), and the sodium, potassium and chloride excretion (ENa(+), EK(+), ECl(-)). The renal effects of myotoxin I was very similar to those of the whole venom, but there was an increase rather than a decrease in the PP and RVR. Myotoxin II had no effect on renal physiology, except for a transient decrease in %TK(+). In conclusion, B. moojeni venom caused intense alterations in renal physiology, including a drop in vascular resistance associated with diuresis, natriuresis and kaliuresis. Bmtx-I had an opposite effect when compared to whole venom, showed in the parameters of PP and RVR. Bmtx-II had a mild effect in %TK(+). The apparent inability of Bmtx-II to induce the renal effect similarly to Bmtx-I should be explained by the absence in the Bmtx-II of the C-terminal lysine rich region.
Assuntos
Bothrops/fisiologia , Venenos de Crotalídeos/toxicidade , Nefropatias/induzido quimicamente , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Venenos de Crotalídeos/análise , Fosfolipases A2 do Grupo II , Técnicas In Vitro , Nefropatias/fisiopatologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/fisiopatologia , Masculino , Dados de Sequência Molecular , Perfusão , Fosfolipases A/análise , Pressão , Ratos , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Circulação Renal/fisiologia , Proteínas de Répteis , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Micção/efeitos dos fármacos , Urodinâmica , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologiaRESUMO
The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 micro g/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 micro g/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 micro M acetylcholine alone and cumulative concentrations of 1 micro M to 10 mM were unaffected. At venom concentrations higher than 50 micro g/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24 degrees C, the venom (50 g/ml) produced only partial neuromuscular blockade (30.7 +/- 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.
Assuntos
Bothrops , Venenos de Crotalídeos/intoxicação , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Junção Neuromuscular/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Galinhas , Relação Dose-Resposta a Droga , Cloreto de Potássio/farmacologia , Fatores de TempoRESUMO
BACKGROUND: Nitric oxide is a key signalling molecule in the pathogenesis of inflammation, but its role in acute pancreatitis and related abdominal pain induced by secretory phospholipase A2 (sPLA2 ) from Crotalus durissus terrificus (Cdt) venom has not been investigated. METHODS: Male Wistar rats were i.v. injected with L-NAME (20 mg/kg), aminoguanidine (AG, 50 mg/kg), 7-nitroindazole (7-NI, 10 mg/kg) or vehicle 10 min before or 60 min after the injection of sPLA2 (300 µg/kg) into the common bile duct. After 4 h of sPLA2 injection, abdominal hyperalgesia and inflammation were assessed in addition to serum amylase, nitrite/nitrate (NOx), pancreas lipoperoxidation and 3-nitrotyrosine (3-NT) contents. RESULTS: sPLA2 -induced acute pancreatitis, related abdominal hyperalgesia, hyperamylasemia and increased concentration of NOx were not correlated with lipoperoxidation or increased 3-NT in the pancreas. Pretreatment with all the nitric oxide synthase (NOS) inhibitors significantly reduced abdominal mechanical hyperalgesia, but only iNOS blockade by AG suppressed pancreas oedema and serum NOx increase. The therapeutic approach with all the NOS inhibitors produced a similar reduction pattern of the abdominal hyperalgesia, but AG treatment also inhibited serum hyperamylasemia and NOx concentrations and pancreatic myeloperoxidase. The nNOS blockade by 7-NI treatment also inhibited myeloperoxidase activity in both pancreas and lung. CONCLUSIONS: Therapeutic blockade of iNOS or nNOS provides benefits in terms of inhibition of the acute pancreatitis-related abdominal hyperalgesia, while iNOS inhibition also ameliorates the inflammatory cell influx to the pancreas and reduces the resultant hyperamylasemia and NOx levels, thus representing alternative pharmacological strategies for treatment of clinical pancreatitis associated with increased PLA2 .
Assuntos
Inibidores Enzimáticos/uso terapêutico , NG-Nitroarginina Metil Éster/uso terapêutico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Dor/tratamento farmacológico , Dor/etiologia , Pancreatite/complicações , Pancreatite/tratamento farmacológico , Fosfolipases A2 Secretórias , Animais , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Pâncreas/enzimologia , Pâncreas/patologia , Pancreatite/enzimologia , Peroxidase/metabolismo , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3′,4′-OH flavonoids are known inhibitors of phospholipases A2.