RESUMO
The association between the clinical use of nitroglycerin (NTG) and headache has led to the examination of NTG as a model trigger for migraine and related headache disorders, both in humans and laboratory animals. In this study in mice, we hypothesized that NTG could trigger behavioural and physiological responses that resemble a common manifestation of migraine in humans. We report that animals exhibit a dose-dependent and prolonged NTG-induced thermal and mechanical allodynia, starting 30-60 min after intraperitoneal injection of NTG at 5-10 mg/kg. NTG administration also induced Fos expression, an anatomical marker of neuronal activity in neurons of the trigeminal nucleus caudalis and cervical spinal cord dorsal horn, suggesting that enhanced nociceptive processing within the spinal cord contributes to the increased nociceptive behaviour. Moreover, sumatriptan, a drug with relative specificity for migraine, alleviated the NTG-induced allodynia. We also tested whether NTG reduces the threshold for cortical spreading depression (CSD), an event considered to be the physiological substrate of the migraine aura. We found that the threshold of CSD was unaffected by NTG, suggesting that NTG stimulates migraine mechanisms that are independent of the regulation of cortical excitability.
Assuntos
Hiperalgesia/tratamento farmacológico , Nitroglicerina/toxicidade , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Sumatriptana/farmacologia , Vasodilatadores/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Hiperalgesia/induzido quimicamente , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Física , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
PURPOSE: Elastase, produced by Aspergillus fumigatus and A. flavus, is an important pathogenic factor in pulmonary aspergillosis. We investigated the possibility of using A. fumigatus-derived A. fumigatus elastase inhibitor (AFUEI) as a therapeutic agent. As native-AFUEI (N-AFUEI) has an extremely low yield, we generated a synthetic-AFUEI (S-AFUEI) and investigated whether S-AFUEI has a biological activity against A. fumigatus elastase (AFUE) and inhibits cytotoxicity. METHODOLOGY: A. fumigatus was cultured in Yeast Carbon Base (YCB) -elastin culture medium for 3-7 days, and AFUE was purified by chromatography using DE52 cellulose and Sephadex G-75 column. Elastolytic activity was examined using Glt-Ala-Ala-Pro-Leu-pNA (GAAPLNA) as the substrate. The hydrolytic activity of AFUE was determined using the characteristic substrates, fibrinogen and collagen (Type IV), and human cell cytotoxicity was measured colorimetrically. Furthermore, the inhibitory effect of S-AFUEI on these activities was examined. RESULTS: We confirmed that S-AFUEI demonstrated elastase inhibitory activity and heat stability equivalent to that demonstrated by N-AFUEI, and inhibited human collagen hydrolytic activity and human fibrinogen hydrolytic activity. Further, S-AFUEI inhibited cytotoxicity in AFUE human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), and human pulmonary alveolar epithelial cells (HPAEpiC). CONCLUSION: As S-AFUEI strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.
Assuntos
Antifúngicos/síntese química , Aspergillus fumigatus/química , Aspergillus fumigatus/enzimologia , Elastase Pancreática/antagonistas & inibidores , Células Epiteliais Alveolares/efeitos dos fármacos , Antifúngicos/farmacologia , Colágeno/metabolismo , Meios de Cultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Fibrinogênio/metabolismo , Temperatura Alta , Humanos , Hidrólise , Artéria Pulmonar/citologia , Aspergilose Pulmonar/tratamento farmacológico , Aspergilose Pulmonar/microbiologiaRESUMO
A kallikrein-like enzyme was isolated from Crotalus viridis viridis (Prairie rattlesnake) venom by Sephadex G-50, DEAE-Sephacel and heparin-Sepharose CL-6B column chromatography. The purified enzyme has a molecular mass of 32 kDa and an isoelectric point of 5.4. The enzyme catalyzed the hydrolysis of arginine esters, kallikrein substrates Pro-Phe-Arg-MCA and Z-Phe-Arg-MCA. The specificity of the enzyme's substrate requirement is demonstrated by the fact that no proteolytic activity was detected against either dimethyl casein or fibrinogen. The enzyme also cleaves kininogen analogs to release bradykinin. Although the enzyme induced contraction of the isolated rat uterus directly at high concentrations, more forceful contractions resulted when the reaction mixture of the enzyme and bovine plasma was applied to the uterus. The reaction mixture of 5.10(-11) M of the enzyme and plasma caused contractions equal to that of 10(-9) M of bradykinin. Additionally the enzyme demonstrated capillary permeability-increasing activity and hypotensive activity on the anesthetized rat, suggesting that the enzyme releases the dilator of the wall of capillaries from plasma. Uterine contraction, capillary permeability-increasing activity and arginine esterolytic activity were inhibited by diisopropyl fluorophosphate, indicating that the serine hydroxyl group is essential for enzymatic and biological activities. It was demonstrated that the NH2-terminal region of the enzyme has significant similarities in sequence with kallikrein-like enzymes from other snake venoms and porcine pancreatic kallikrein.
Assuntos
Venenos de Crotalídeos/análise , Calicreínas/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Relação Dose-Resposta a Droga , Ponto Isoelétrico , Cininas/metabolismo , Masculino , Dados de Sequência Molecular , Peso Molecular , Ratos , Ratos Endogâmicos , Especificidade por SubstratoRESUMO
A kinin-releasing enzyme was isolated from Bitis arietans (puff adder) venom by Sephadex G-100 and DEAE-cellulose column chromatographies. The kinin-releasing enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. Its molecular mass is approximately 45 kDa with an isoelectric point of 6.5. Kinin-releasing enzyme possesses proteolytic activity which hydrolyzes the Leu6-Cys7, His10-Leu11 and Ala14-Leu15 bonds of the B chain of oxidized insulin and the A alpha and B beta chain of fibrinogen. Kinin-releasing and benzoyl-L-arginine ethyl ester hydrolytic activities of this enzyme were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.
Assuntos
Peptídeo Hidrolases/isolamento & purificação , Venenos de Víboras/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Estabilidade Enzimática , Cinética , Peso Molecular , Peptídeo Hidrolases/metabolismo , Venenos de Víboras/metabolismoRESUMO
Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.
Assuntos
Venenos de Crotalídeos/análise , Venenos de Crotalídeos/isolamento & purificação , Animais , Cromatografia , Venenos de Crotalídeos/metabolismo , Dipeptídeos/metabolismo , Eletroforese , Fibrinogênio/metabolismo , Hemorragia/induzido quimicamente , Camundongos , Peso Molecular , Necrose/induzido quimicamente , Especificidade por SubstratoRESUMO
We investigated the pathophysiological role of nitric oxide synthesized by inducible nitric oxide synthase in the brain, by injecting lipopolysaccharide directly into the rat cerebral cortex/hippocampus. The levels of nitric oxide metabolites, nitrite and nitrate, began to increase in a dose-dependent manner with a 3-h lag, and reached approximately seven-fold the basal levels 8 h after the direct injection of lipopolysaccharide (5 microg). The lipopolysaccharide-induced increase in nitrite and nitrate levels was inhibited by treatment with the specific inducible nitric oxide synthase inhibitor aminoguanidine. The protein synthesis inhibitor cycloheximide delayed the onset of the increase in nitric oxide metabolite levels, and reduced the peak levels. Lipopolysaccharide increased Ca2+-independent, but not Ca2+-dependent, nitric oxide synthase activity in the brain. Intense nicotinamide adenine dinucleotide phosphate-diaphorase activity was observed in round cells in the vicinity of the site of injection of lipopolysaccharide 8 h after the injection. Neuronal death was observed seven days after the injection of lipopolysaccharide. Spatial memory, as assessed by performance in a water maze task and spontaneous alternation behavior in a Y-maze, was significantly impaired in rats which had had previous bilateral injections of lipopolysaccharide into the hippocampus. The lipopolysaccharide-induced neuronal death and spatial memory impairments were prevented by aminoguanidine. These results suggest that direct injection of lipopolysaccharide into the brain causes an induction of inducible nitric oxide synthase in vivo. Furthermore, it is suggested that nitric oxide produced by inducible nitric oxide synthase is responsible for the lipopolysaccharide-induced brain dysfunction.
Assuntos
Encéfalo/fisiopatologia , Córtex Cerebral/fisiologia , Hipocampo/fisiologia , Lipopolissacarídeos/toxicidade , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Óxido Nítrico Sintase/biossíntese , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Colina O-Acetiltransferase/metabolismo , Cicloeximida/farmacologia , Indução Enzimática , Guanidinas/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Cinética , Lipopolissacarídeos/administração & dosagem , Masculino , Microdiálise , Microinjeções , Atividade Motora/efeitos dos fármacos , Nitratos/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo III , Nitritos/metabolismo , Especificidade de Órgãos , Ratos , Ratos Wistar , Fatores de Tempo , ômega-N-Metilarginina/farmacologiaRESUMO
The activation of N-methyl-D-aspartate receptors induces the synthesis of nitric oxide, which activates soluble guanylate cyclase and leads to the formation of cyclic GMP in the brain. The inhibition of nitric oxide production, as well as the blockade of N-methyl-D-aspartate receptors, has been reported to prevent the induction of hippocampal long-term potentiation and learning and memory formation in vivo, although the effects of inhibitors of nitric oxide synthase are still controversial. We investigated the putative role of nitric oxide and cyclic GMP in dizocilpine-induced memory impairment in mice. The nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and 7-nitro indazole, as well as dizocilpine, a non-competitive N-methyl-D-aspartate receptor antagonist, dose-dependently impaired spatial working memory in mice, assessed by their spontaneous alternation behavior in a Y-maze. The inhibitory effects of both NG-nitro-L-arginine methyl ester and dizocilpine on their behavior were completely reversed by 8-bromo-cyclic GMP. Cyclic GMP levels in the cerebellum were reduced by treatment with dizocilpine. NG-Nitro-L-arginine methyl ester and 7-nitro indazole reduced cyclic GMP levels in the cerebral cortex/hippocampus and cerebellum, and the suppressive effect of NG-nitro-L-arginine methyl ester on cyclic GMP levels in the cerebral cortex/hippocampus was reversed by co-treatment with L-arginine. Cyclic AMP levels in the brain were not affected by treatment with either dizocilpine, NG-nitro-L-arginine methyl ester, or 7-nitro indazole. Neither NG-nitro-L-arginine methyl ester nor L-arginine had any effect on monoamine and acetylcholine metabolism in the brain. These results suggest that the reduction in nitric oxide/cyclic GMP production in the brain may be responsible for dizocilpine-induced impairment of spontaneous alternation behavior in a Y-maze.
Assuntos
Comportamento Animal/efeitos dos fármacos , GMP Cíclico/metabolismo , Maleato de Dizocilpina/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Óxido Nítrico/farmacologia , Animais , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos , NG-Nitroarginina Metil Éster/farmacologiaRESUMO
Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.
Assuntos
Venenos de Crotalídeos/química , Metaloendopeptidases/química , Serpentes , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Brometo de Cianogênio , Dissulfetos/química , Hidrólise , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Conformação ProteicaRESUMO
A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.
Assuntos
Metaloendopeptidases/química , Metaloendopeptidases/fisiologia , Peptídeos/isolamento & purificação , Inibidores da Agregação Plaquetária/isolamento & purificação , Venenos de Serpentes/enzimologia , Especificidade por Substrato , Peçonhas/isolamento & purificação , Sequência de Aminoácidos , Animais , Desintegrinas , Metaloendopeptidases/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/fisiologiaRESUMO
Hemorrhagic toxin g (HT-g) was isolated from Crotalus atrox (western diamondback rattlesnake) venom using a five-step purification procedure to obtain approximately equal to 5.9 mg of purified HT-g from 2.0 g of crude venom. The purified toxin was homogeneous by disc electrophoresis on polyacrylamide gel at pH 8.3 and 4.3, and by isoelectric focusing. HT-g possessed lethal, hemorrhagic and proteolytic activities. These activities of toxin were inhibited by ethylenediamine-tetraacetic acid (EDTA), 1,10-phenanthroline or ethyleneglycol (beta-amino-ethyl) N,N,N',N'-tetracetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). Its molecular weight was approximately 60,000 and the isoelectric point was 6.8. The toxin contains 516 amino acid residues. HT-g did not coagulate fibrinogen to fibrin; however, the toxin hydrolysed the A alpha-chain or B beta-chain of fibrinogen without cleaving the gamma-chain. HT-g produced only local hemorrhage in internal organs such as the intestine, heart and liver.
Assuntos
Venenos de Crotalídeos/análise , Venenos de Crotalídeos/isolamento & purificação , Aminoácidos/análise , Animais , Venenos de Crotalídeos/farmacologia , Venenos de Crotalídeos/toxicidade , Fibrinogênio/metabolismo , Hemorragia/etiologia , Temperatura Alta , Ponto Isoelétrico , Camundongos , Peso MolecularRESUMO
The role of glial cells in nitric oxide production in the cerebellum of conscious rats was investigated with a glial selective metabolic inhibitor, fluorocitrate. The levels of nitric oxide metabolites (nitrite plus nitrate) in the dialysate following in vivo microdialysis progressively increased to more than 2-fold the basal levels during a 2-h infusion of fluorocitrate (1 mM), and the increase persisted for more than 2 h after the treatment. Pretreatment with N(G)-nitro-L-arginine methyl ester attenuated the fluorocitrate-induced increase in nitric oxide metabolite levels. None of the glutamate receptor antagonists, including D(-)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione, and (+/-)-alpha-methyl-4-carboxyphenylglycine, inhibited the fluorocitrate-induced increase. The L-arginine-induced increase was significantly reduced by fluorocitrate treatment, while N-methyl-D-aspartate, (+)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and trans-(+/-)-1-amino-(1S,3R)-cyclopentane-dicarboxylic acid increased nitric oxide metabolites levels in the fluorocitrate-treated rats, as much as in control animals. These results suggest that glial cells play an important role in modulating nitric oxide production in the cerebellum by regulating L-arginine availability.
Assuntos
Citratos/farmacologia , Neuroglia/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Aconitato Hidratase/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Benzoatos/farmacologia , Cerebelo/química , Cerebelo/enzimologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Masculino , N-Metilaspartato/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Neuroglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Nitroarginina/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Especificidade por Substrato , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Pseudomonas aeruginosa elastase, a Bacillus subtilis thermolysin-like zinc-proteinase was examined for hemorrhagic activity and its effect on muscle and endothelial cells. Subcutaneous and intramuscular injections of elastase into mice caused severe hemorrhage with an acute increase of creatine phosphokinase activity in serum. The elastase also possessed fibrinogenolytic and fibrinolytic activities. The Aalpha and Bbeta chains of fibrinogen were completely hydrolyzed as demonstrated by their electrophoretic disappearance on SDS polyacrylamide gels. The pathological study indicates that elastase induces changes in the structure of the vascular wall and causes leakage of the plasma component and red and white blood cells into the extravascular tissue. This is further supported by results showing injury to cultured endothelial cells and macrophages. These data indicate that P. aeruginosa elastase directly affects endothelial cells and destroys the basement membrane of blood vessels to cause hemorrhage. Since fibrinogenolytic activity is an additional component of this elastase and this activity induces the hemorrhagic tendency, the damage in tissues could become increasingly severe.
Assuntos
Hemorragia/induzido quimicamente , Doenças Musculares/induzido quimicamente , Elastase Pancreática/toxicidade , Pseudomonas aeruginosa/química , Animais , Bovinos , Células Cultivadas , Creatina Quinase/metabolismo , Endopeptidases/química , Endotélio Vascular/patologia , Fibrinogênio/química , Fibrinogênio/efeitos dos fármacos , Fibrinolíticos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Camundongos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Doenças Musculares/enzimologia , Doenças Musculares/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
Mucrotoxin A was purified from the lyophilized venom of Trimeresurus mucrosquamatus using gel filtration on a Sephadex G-100 column, followed by chromatography on CM-Sephadex C-50 and DEAE-Sephadex A-50. By these procedures, 14 mg of purified preparation could be obtained from 1 g of crude venom. The purified preparation was homogeneous by disc electrophoresis on polyacrylamide gel at pH 8.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. Mucrotoxin A possessed both lethal and hemorrhagic activities, but it did not show caseinolytic activity. Its molecular weight was approximately 94,000 and the isoelectric point was 4.3. Mucrotoxin A contains approximately 3 moles of Ca and 2 moles of Zn per mole of toxin. The amino acid composition of Mucrotoxin A was determined. No carbohydrate was present.
Assuntos
Venenos de Crotalídeos/análise , Hemorragia/induzido quimicamente , Proteínas/isolamento & purificação , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Venenos de Crotalídeos/isolamento & purificação , Camundongos , Proteínas/toxicidadeRESUMO
Ac5-Proteinase (15.2 mg) was isolated from Agkistrodon acutus venom (1 g) by column chromatography on Sephadex G-75, CM-Sephadex C-50 and CM-Cellulose. Ac5-Proteinase was homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3 and also by SDS-disc polyacrylamide gel electrophoresis. Ac1-, Ac2-, Ac3- and Ac5-proteinases possessed lethal and hemorrhagic activities, but Ac4-proteinase had no lethal activity. These activities were inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine. The molecular weights of Ac1-, Ac2-, Ac3-, Ac4- and Ac5-proteinases were approximately 24,500, 25,000, 57,000, 33,000 and 24,000, respectively. Ac1-, Ac2-, Ac4- and Ac5-proteinases did not contain any carbohydrates, but Ac3-proteinase contained 0.1% carbohydrate by weight.
Assuntos
Venenos de Crotalídeos/análise , Endopeptidases/isolamento & purificação , Hemorragia/induzido quimicamente , Peptídeo Hidrolases/isolamento & purificação , Aminoácidos/análise , Animais , Fenômenos Químicos , Química , Cromatografia em Gel , Venenos de Crotalídeos/toxicidade , Temperatura Alta , Imunodifusão , Focalização Isoelétrica , Dose Letal Mediana , Camundongos , Peso Molecular , CoelhosRESUMO
A lectin was isolated from the venom of Trimeresurus okinavensis (Himehabu) and the complete amino acid sequence was determined using clostripain, lysyl endopeptidase, and V8 protease. The hemagglutinating activity of this lectin are reported.
Assuntos
Venenos de Crotalídeos/química , Lectinas/isolamento & purificação , Lectinas/farmacologia , Trimeresurus , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/toxicidade , Eritrócitos/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Testes de Hemaglutinação , Humanos , Dados de Sequência Molecular , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
A lectin (APL) was purified from the venom of Agkistrodon piscivorus piscivorus (Eastern cottonmouth moccasin). APL is a disulfide-linked, homodimeric protein consisting of identical monomers of molecular weight 16,200. Native rabbit and human erythrocytes were agglutinated by APL and the activity was found to be calcium-dependent. Galactose, lactose, rhamnose and EGTA strongly inhibited the hemagglutination activity of APL. The complete amino acid sequence determined by Edman sequencing of the S-pyridylethylated derivative and its peptides derived from enzymatic digestion indicate the structure of APL to be highly homologous with lectins and the platelet glycoprotein Ib (GPIb)-binding proteins isolated from other snake venoms. These results suggest that APL belongs to the C-type beta-galactoside binding lectin family which possess structural similarities with the C-terminal carbohydrate-recognition domain (CRD) of animal membrane lectins.
Assuntos
Agkistrodon/fisiologia , Hemaglutinação/efeitos dos fármacos , Lectinas/toxicidade , Venenos de Serpentes/toxicidade , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Humanos , Lectinas/antagonistas & inibidores , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Coelhos , Alinhamento de Sequência , Venenos de Serpentes/químicaRESUMO
An arginine ester hydrolase (ME-5) was isolated from the venom of Trimeresurus mucrosquamatus by column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel and by isoelectric focusing, obtaining 1.4 mg of purified enzyme from 1 g of crude venom. The enzyme was homogeneous by SDS disc electrophoresis on polyacrylamide gel at pH 8.3. ME-5 is a glycoprotein which possesses both TAME hydrolase and capillary permeability-increasing activity, but it did not show clotting or bradykinin-releasing activities. Its molecular weight is approximately 33,000 and its isoelectric point is 6.48. The enzyme is stable to heat treatment and to pH changes between 5 and 9. Trimeresurus mucrosquamatus venom contains five arginine ester hydrolases, designated as ME-1, 2, 3, 4 and 5. Three of the five (ME-1 , 4 and 5) are inactivated by DFP, suggesting that the serine hydroxyl group is involved in enzymatic activity. All five arginine ester hydrolases showed capillary permeability-increasing activity, but none of the enzymes showed clotting activity. Their amino acid compositions were determined and all appear to be unique and distinct from those of other snake venoms.
Assuntos
Hidrolases de Éster Carboxílico/isolamento & purificação , Venenos de Crotalídeos/análise , Aminoácidos/análise , Animais , Bradicinina/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia por Troca Iônica , Focalização Isoelétrica , Peso MolecularRESUMO
Mucrotoxin A from the venom of Trimeresurus mucrosquamatus was isolated in homogeneous form by a previously published method. Mucrotoxin A did not hydrolyze casein, however, when dimethylcasein was used as a substrate, the toxin cleaved the substrate. This toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The sites of cleavage in the oxidized B chain of insulin were identified as Ser(9)-His(10), His(10)-Leu(11), Ala(14)-Leu(15), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The toxin digested the A alpha chain of fibrinogen first, followed by hydrolysis of the B beta chain. The fact that no fibrin clot formed indicates that the sites of cleavage in the A alpha and B beta chains of fibrinogen by the toxin must be different from those cleaved by thrombin. Mucrotoxin A produced systemic hemorrhage in internal organs such as the heart and stomach.
Assuntos
Venenos de Crotalídeos/toxicidade , Animais , Creatina Quinase/sangue , Venenos de Crotalídeos/análise , Hemorragia/induzido quimicamente , Camundongos , Peptídeo Hidrolases/farmacologiaRESUMO
A thrombin-like proteinase, named bilineobin, was isolated from Agkistrodon bilineatus venom by Sephadex G-75, DEAE-Sephacel and Heparin-Sepharose CL-6B column chromatography. The purified enzyme has a mol. wt of 57,000 and catalysed the hydrolysis of arginine esters and thrombin substrates Boc-Val-Pro-Arg-MCA and Boc-Asp(OBz)-Pro-Arg-MCA. Although bilineobin converted fibrinogen into fibrin resulting in the production of fibrinopeptides, the activity was relatively low (0.65 NIH units/mg). Fibrinopeptides released upon hydrolysis by this proteinase were identified as fibrinopeptide A (FpA) and fibrinopeptide B (FpB) by measuring fast atom bombardment (FAB) mass spectra and amino acid sequence. This indicates that bilineobin hydrolyses the Arg(19)-Gly(20) bond in the A alpha chain and the Arg(21)-Gly(22) bond in the B beta chain of the bovine fibrinogen molecule. Kinetic study of FpA and FpB release reveals that bilineobin has a preference for cleaving the B beta chain. In addition, bilineobin is resistant to thrombin inhibitors such as hirudin. These suggest that the mechanism of action of bilineobin is similar but not identical to that of thrombin. It was demonstrated that the NH2-terminal region of bilineobin has significant similarities in sequence with thrombin-like proteinases from other snake venoms; however, only three residues were common with thrombin up to residue number 24.
Assuntos
Venenos de Crotalídeos/enzimologia , Endopeptidases/toxicidade , Serina Endopeptidases/metabolismo , Trombina/toxicidade , Sequência de Aminoácidos , Coagulação Sanguínea/efeitos dos fármacos , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia Líquida de Alta Pressão , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Fator XIII/metabolismo , Fibrinogênio/metabolismo , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Peso Molecular , Inibidores de Proteases/farmacologia , Espectrometria de Fluorescência , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Especificidade por Substrato , Trombina/antagonistas & inibidores , Trombina/isolamento & purificaçãoRESUMO
BACKGROUND AND OBJECTIVES: Interstitial cystitis (IC) is characterized clinically by lower abdominal pain, pain during urination, and increased frequency of urination. Treatment of the symptoms in IC remains challenging. We report effective treatment using lumbar sympathetic block for 2 patients with IC. CASE REPORT: A 63-year-old and 78-year-old woman were diagnosed with IC. Medical therapy with nonsteroidal anti-inflammatory drugs (NSAID), anticholinergics, and hydrodistention of the bladder failed to improve their symptoms. Subsequently, a continuous lumbar epidural block using 1% mepivacaine was used in these patients. A transient reduction of the symptoms in both patients was achieved. A lumbar sympathetic block with a neurolytic agent produced almost complete, and long-lasting relief of their symptoms. CONCLUSION: Lumbar sympathetic block using a neurolytic agent produced long-lasting pain relief in 2 patients with IC. Reg Anesth Pain Med 2001;26:271-273.