RESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disorder; however, there is no treatment able to prevent the loss of dopaminergic neurons or its consequences. Trophic factors such as NGF and BDNF has positive effects on different disorders of the brain, including neurodegeneration. Additionally, studies have suggested the use of venom peptides as a therapeutic strategy for neurological disorders. Therefore, in the present study, we investigated the neuroprotective activity of a peptide isolated from Bothrops atrox venom and its trophic ability by using a cellular model of dopaminergic neurotoxicity induced by 1-methyl-4-phenylpyridinium (MPP(+)) in PC12 cells. We showed that it decreased the activities of the apoptotic proteases caspase-9 (mitochondrial) and caspase-3 (executor) and increased cell viability and proliferation in this model. Additionally, it increased neuritogenesis in non-treated PC12 cells (neuronal model) as well as in PC12 cells treated with the dopaminergic neurotoxin. The amino acid sequence of the peptide was identified as Glutamic acid-Valine-Tryptophan (Glu-Val-Trp). These findings suggest that this tripeptide has the potential to protect against the dopaminergic neurons loss and that trophic stimulation of neuroplasticity might be involved in its mechanism of neuroprotection.
Assuntos
Bothrops/metabolismo , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Peptídeos/farmacologia , Peçonhas/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Ácido Glutâmico/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Células PC12 , Doença de Parkinson/metabolismo , Ratos , Triptofano/farmacologia , Valina/farmacologiaRESUMO
The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164 I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 µg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and ß-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and ß-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.
Assuntos
Venenos de Crotalídeos/enzimologia , Fibrinólise , Metaloproteases/metabolismo , Sequência de Aminoácidos , Animais , Bothrops , Ponto Isoelétrico , Espectrometria de Massas , Metaloproteases/química , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The neurodegenerative diseases are important causes of morbidity and mortality in Western countries. Common mechanisms of toxicity involving mitochondrial damage have been suggested; however, a definitive treatment has not yet been found. Therefore, there has been great interest in the development of mitochondria-targeted protective compounds for the treatment of neuropathies. Animal toxins represent a promising source of new molecules with neuroprotective activity and potential to originate new drugs. We present here the effects of a low-molecular-mass peptides fraction (Ba-V) from Bothrops atrox snake venom, on rat brain mitochondrial function. Ba-V did not induce the mitochondrial swelling and moreover, was as effective as cyclosporin A (CsA) to inhibit the calcium/phosphate-induced swelling, which indicates its potential to prevent the mitochondrial permeability transition (MPT). The membrane electrochemical potential, the oxygen consumption during states-3 and -4 respirations as well as the respiratory control ratio (RCR) were not affected by Ba-V. Additionally, Ba-V did not induce reactive oxygen species (ROS) generation. Interestingly, Ba-V did not protect against the generation of ROS induced by t-BOH, which suggests a protection mechanism other than ROS scavenging. Given the important role of the mitochondrial damage and, more specifically, of MPT, in the development of neuropathies, Ba-V might be useful in the future strategies for the treatment of these diseases.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Dilatação Mitocondrial/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Proteínas de Répteis/uso terapêutico , Animais , Encéfalo , Brasil , Avaliação Pré-Clínica de Medicamentos , Peróxido de Hidrogênio/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Peso Molecular , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/efeitos adversos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Fosforilação Oxidativa/efeitos dos fármacos , Peptídeos/efeitos adversos , Peptídeos/química , Peptídeos/isolamento & purificação , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Répteis/efeitos adversos , Proteínas de Répteis/química , Proteínas de Répteis/isolamento & purificaçãoRESUMO
Hemostatically active snake venom metalloproteinases (SVMPs) perturb the blood coagulation cascade at specific points and due to their potential application as thrombolytic agents, the fibrin(ogen)olytic non-hemorrhagic SVMPs have been employed as biochemical tools in coagulation research and diagnosis. Structural studies complemented by the design of metalloproteinase inhibitors have been instrumental in understanding their stereo specificity and action mechanism. We present here, details of the crystal structure of BmooMPalpha-I, a 22.6 kDa non-hemorrhagic P-I class SVMP isolated from Bothrops moojeni venom, determined at 1.76 A resolution. In this structure, the catalytic zinc ion displays an unusual octahedral coordination formed by the three canonical histidines (His(142), His(146) and His(152)) and additionally, by three solvent molecules. Comparative sequence and structural studies indicate that the motif comprising amino acid segments 153-164 and 167-176 adjacent to the methionine-turn is a salient feature that differentiates both non and hemorrhagic P-I class SVMPs and could directly be involved in the development of the hemorrhagic activity.
Assuntos
Bothrops/fisiologia , Metaloproteases/química , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Cristalização , Eletroforese em Gel de Poliacrilamida , Hemorragia/induzido quimicamente , Metaloproteases/antagonistas & inibidores , Metaloproteases/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Especificidade por Substrato , Venenos de Víboras/farmacologia , Difração de Raios X , Zinco/químicaRESUMO
Ethanolic extract of leaves of Galactia glauscescens (GGE) at concentration of 100 and 500 microg/ml prevented the neuromuscular paralysis induced by Crotalus durissus terrificus venom on mouse phrenic nerve-diaphragm preparation.
Assuntos
Venenos de Crotalídeos/toxicidade , Crotalus/fisiologia , Fabaceae/química , Extratos Vegetais/farmacologia , Animais , Diafragma/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Camundongos , Bloqueio Neuromuscular , Junção Neuromuscular/efeitos dos fármacos , Nervo Frênico/efeitos dos fármacos , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
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
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
For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA2s.
Assuntos
Asparagina , Bothrops , Venenos de Crotalídeos/toxicidade , Fosfolipases A/toxicidade , Animais , Catálise , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Cristalografia por Raios X , Cinética , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Fosfolipases A2 , Conformação ProteicaRESUMO
The electrophile Ca(2+) is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca(2+) free and bound states at 0.97 and 1.60 A resolutions, respectively. In the Ca(2+) bound state, the Ca(2+) ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca(2+), a water molecule occupies the position of the Ca(2+) ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation.
Assuntos
Cálcio/metabolismo , Fosfolipases A/química , Fosfolipases A/metabolismo , Animais , Sítios de Ligação , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Cristalização , Cristalografia por Raios X/métodos , Fosfolipases A2 do Grupo IV , Ligação de Hidrogênio , Modelos Moleculares , Fosfolipases A2 , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Numerous plants are used as snakebite antidotes in Brazilian folk medicine, including Casearia sylvestris Swartz, popularly known as guaçatonga. In this study, we examined the action of a hydroalcoholic extract from C. sylvestris on the neuromuscular blockade caused by bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu venom, in mouse isolated phrenic nerve-diaphragm (PND) preparations. Aqueous (8 and 12 mg/ml, n=4 and 5, respectively) and hydroalcoholic (12 mg/ml, n=12) extracts of the leaves of C. sylvestris caused facilitation in PND preparations followed by partial neuromuscular blockade. BthTX-I (20 mg/ml, n=4) caused 50% paralysis after 65±15 min (mean ± S.E.M). Preincubation (30 min at 37°C) of BthTX-I (20 mg/ml, n=4) with a concentration of the hydroalcoholic extract (4 mg/ml) that had no neuromuscular activity, such as the control (n=5), prevented the neuromuscular blockade caused by the toxin. This protection may be mediated by compounds such as flavonoids and phenols identified by thin-layer chromatography and colorimetric assays
Assuntos
Animais , Masculino , Camundongos , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Mordeduras de Serpentes , Venenos de Serpentes , Bloqueio NeuromuscularRESUMO
Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.
Assuntos
Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Fitoterapia , Extratos Vegetais/farmacologia , Tabernaemontana , Animais , Antivenenos/administração & dosagem , Antivenenos/uso terapêutico , Creatina Quinase/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , Raízes de Plantas , Ratos , Ratos WistarRESUMO
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
Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which binds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4A resolution to a crystallographic residual of 18.6% (R(free)=26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and structural differences are observed in both the domains in the putative Ca(2+)and carbohydrate binding regions.
Assuntos
Venenos de Crotalídeos/química , Crotalus , Lectinas Tipo C/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Venenos de Crotalídeos/metabolismo , Cristalografia por Raios X , Dissulfetos/química , Lectinas Tipo C/metabolismo , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas , Alinhamento de SequênciaRESUMO
Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 A, and diffraction data were collected to 2.7 A. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.
Assuntos
Venenos de Crotalídeos/química , Lectinas Tipo C/química , Animais , Crotalus , Cristalização , Cristalografia por Raios X , Dissulfetos/química , Modelos Moleculares , Estrutura Quaternária de Proteína , Subunidades ProteicasRESUMO
Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 homolog with no apparent catalytic activity, was first isolated from Bothropsjararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29. 44. 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys5O-Cysl31, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA2s.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Neurotoxinas/química , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Venenos de Crotalídeos/genética , Dissulfetos , Fosfolipases A2 do Grupo II , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipases A2 , Conformação Proteica , Proteínas de RépteisRESUMO
Bothrops jararacussu venom and its major toxin, bothropstoxin-I (BthTX-I), possess myotoxic and neurotoxic activities. The ability of commercial equine antivenom to neutralize these activities was studied in mouse isolated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations by indirect stimulation (0.1 HZ, 0.2 ms). The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 70 +/- 11.5 min and 58 +/- 8 min for B. jararacussu venom (50 microg/mL), and 31 +/- 6 min and 30 +/- 3 min for BthTX-I (20 microg/mL). After a 120-min incubation, the creatine kinase (CK) concentrations in the EDL preparations were 3464 +/- 346 U/L and 3422 +/- 135 U/L following exposure to venom (50 microg/mL) and BthTX-I (20 microg/mL), respectively. Antivenom neutralized the neuromuscular blockade induced by the venom and toxin in PND preparations in a dose-dependent fashion, but only partially neutralized this effect in EDL. Antivenom also effectively prevented the venom- and toxin-induced release of CK from EDL. In contrast, histological analysis showed that the morphological damage caused by B. jararacussu venom and BthTX-I in the EDL was only partially prevented by the anti- venom. These results indicate that commercial equine antiserum fully protects against the neurotoxic action of B. jararacussu and BthTX-I in PND preparations, but only partially protects against the neurotoxic and myotoxic actions of the venom and its toxin in EDL preparations. Care must therefore be exercized in extrapolating results from different preparations even when similar pharmacological or physiological responses are involved.
Assuntos
Antivenenos/farmacologia , Venenos de Crotalídeos/toxicidade , Músculo Esquelético/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Nervo Frênico/efeitos dos fármacos , Animais , Bothrops , Diafragma/efeitos dos fármacos , Diafragma/patologia , Diafragma/fisiologia , Estimulação Elétrica , Técnicas In Vitro , Masculino , Camundongos , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/fisiologia , Nervo Frênico/fisiologiaRESUMO
In Brazilian folk medicine, victims of bites by poisonous animals are usually treated with plant extracts derived from the diverse national flora. The chemical and pharmacological properties of most extracts were yet not investigated. In the rural community of Assis-SP, the root bark of Tabernaemontana catharinensis ("leiteiro", "cow milk") is applied to the site of the snake bite and believed to neutralize the effect of the venom. We report here the ability of the lyophilized aqueous extract (AE) and of a pure compound obtained from the ethanolic extract of T. catharinensis to inhibit the lethal and myotoxic activities of C. d. terrificus (South American rattlesnake) venom. Doses of 10 mg AE/100 g, injected (i.m., rat) 20 s after injecting (i.m.) the venom and that of 2.5 mg AE/100 g, incubated for 1 h at 25 degrees C with the venom before injection (i.m.) were able to neutralize the lethal activity of 2LD50. These data indicate that T. catharinensis could be used as a source of a model molecule able to neutralize the lethality and myotoxicity induced by C. d. terrificus venom. Its ethanolic extract was then fractionated on a silica gel 60 chromatography column affording fractions A to F. Fraction A consisted basically of non-polar compounds, terpenes and sterols. Fraction D showed a pronounced antiophidian activity which was later correlated with the presence of the quaternary alkaloid 12-methoxy-4-methylvoachalotine in this fraction. This alkaloid was isolated and inhibited 100% lethality when injected 20 s after 2 LD50 at 1.7 mg/100 g.
Assuntos
Alcaloides/farmacologia , Antídotos/farmacologia , Carbazóis/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Plantas Medicinais/química , Árvores/química , Alcaloides/química , Alcaloides/isolamento & purificação , Animais , Antídotos/química , Antídotos/isolamento & purificação , Carbazóis/química , Carbazóis/isolamento & purificação , Cromatografia em Camada Fina , Creatina Quinase/sangue , Venenos de Crotalídeos/intoxicação , Dose Letal Mediana , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Intoxicação/tratamento farmacológico , Intoxicação/mortalidade , Ratos , Ratos WistarRESUMO
The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 120 amino acid residues (Mr = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA2's and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA2 activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA2's have been considered to be devoid of this enzymatic activity.
Assuntos
Ácido Aspártico/química , Venenos de Crotalídeos/química , Fosfolipases A/química , Fosfolipases A/metabolismo , Sequência de Aminoácidos , Animais , Bothrops , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2 do Grupo II , Hidrólise , Dados de Sequência Molecular , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Proteínas de Répteis , Serina Endopeptidases/metabolismoRESUMO
Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 microg/paw) and bothropstoxin-II (12.5-50 microg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 microg/site) and bothropstoxin-II (0.125-5 microg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 microg/ml) and bothropstoxin-II (3 and 10 microg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.
Assuntos
Degranulação Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Mastócitos/efeitos dos fármacos , Fosfolipases A/farmacologia , Animais , Venenos de Crotalídeos/metabolismo , Edema/induzido quimicamente , Fosfolipases A2 do Grupo II , Masculino , Mastócitos/citologia , Fosfolipases A/química , Fosfolipases A/metabolismo , Fosfolipases A2 , Ratos , Ratos Wistar , Proteínas de Répteis , Pele/patologiaRESUMO
Bothropstoxin I (BthTX-I) from the venom of Bothrops jararacussu is a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49-->Lys substitution, disrupts the integrity of lipid membranes by a Ca2+-independent mechanism. The crystal structures of two dimeric forms of BthTX-I which diffract X-rays to resolutions of 3.1 and 2.1 angstroms have been determined. The monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal alpha-helical regions and the tips of the beta-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in "open" and "closed" dimer conformations. Spectroscopic investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface. The possible relevance of this structural transition in the Ca2+-independent membrane damaging activity is discussed.