RESUMO
Serine peptidases and metallopeptidases are the primary toxins found in Bothrops snakes venoms, which act on proteins in the tissues of victims or prey, and release of peptides formed through proteolytic activity. Various studies have indicated that these peptides, released by the proteolytic activity of heterologous enzymes, generate molecules with unidentified functions, referred to as cryptids. To address this, we purified serine peptidases from Bothrops jararaca venom using molecular exclusion chromatography and then incubated them with the endogenous substrate myoglobin. As a control, we also incubated the substrate with trypsin. The resulting proteolytic fragments were analyzed, separated, and collected via HPLC. These fractions were then tested on cell cultures, the active fractions were sequenced (ALELFR and TGHPETLEK) and synthesized. After confirming their activity, the peptides underwent sequencing and synthesis for additional cell tests, including the increase of cell viability, cycle phases, proliferation, signaling, growth kinetics, angiogenesis, and migration. The results revealed that the synthesized peptides exhibited cellular repair properties, suggesting a potential role in tissue repair in the range of 0.05-5 µ M. Additionally, the effects of fragments resulting from myoglobin degradation isolated (ALELFR and TGHPETLEK) revealed a regenerative action on tissue.
Assuntos
Bothrops , Venenos de Crotalídeos , Mioglobina , Serina Proteases , Animais , Venenos de Crotalídeos/química , Serina Proteases/metabolismo , Serina Proteases/química , Mioglobina/metabolismo , Peptídeos/farmacologia , Peptídeos/química , Humanos , Sobrevivência Celular/efeitos dos fármacos , Bothrops jararacaRESUMO
Ivermectin (IVM) is a macrocyclic lactone used for the treatment of parasitic infections and widely used in veterinary medicine as endectocide. In mammals, evidence indicates that IVM interacts with γ-aminobutyric acid (GABA)-mediated chloride channels. GABAergic system is involved in the manifestation of sexual behavior. We previously found that IVM at therapeutic doses did not alter sexual behavior in male rats, but at a higher dose, the appetitive phase of sexual behavior was impaired. Thus, we investigated whether the reduction of sexual behavior that was previously observed was a consequence of motor or motivational deficits that are induced by IVM. Data showed significant decrease in striatal dopaminergic system activity and lower testosterone levels but no effects on sexual motivation or penile erection. These findings suggest IVM may activate the GABAergic system and reduce testosterone levels, resulting in a reduction of motor coordination as consequence of the inhibition of striatal dopamine release.
Assuntos
Antiparasitários/toxicidade , Ivermectina/toxicidade , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Ereção Peniana/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Ratos Wistar , Serotonina/metabolismo , Comportamento Sexual Animal/efeitos dos fármacos , Testosterona/sangue , Ácido gama-Aminobutírico/metabolismoRESUMO
Incubation of plasma from the snake Crotalus durissus terrificus (CDTP) with trypsin generated two hypotensive peptides. The primary structure of the peptides was established for two sequences as: (Ser-Ile-Pro-Gln-Ala-Pro-Thr-Ser-Asn-Leu-Ile-Glu-Ala-Thr-Lys) and (Lys-Pro-Asp-Ala-Asn-Gln-Val-Leu-Ile-Gln-Val-Ile-Gly-Val). These peptides display homology with fragments of albumin from Trimeresurus flavoviridis. Bolus intra-arterial injection of the purified or the synthetic peptide produced a strong and sustained vasopressor response in the anaesthetized snake (CDT) and rats (Wistar); this hypotensive effect was also potentiated by captopril-an angiotensin-converting (0.1 mg/kg) enzyme inhibitor.
Assuntos
Anti-Hipertensivos/farmacologia , Crotalus , Peptídeos/farmacologia , Vasoconstritores/farmacologia , Sequência de Aminoácidos , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Captopril/farmacologia , Sinergismo Farmacológico , Feminino , Injeções Intra-Arteriais , Masculino , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Ratos , Ratos Wistar , Vasoconstritores/isolamento & purificaçãoRESUMO
Bacterial pathogenicity is a result of a combination of factors, including resistance to environmental threats and to the host's defenses, growth capability, localization in the host, tissue specificity, resource obtaining mechanisms and the bacterium's own defenses to aggression. A variety of bacterial components, often specific to each strain, are involved in the microorganism's survival, adhesion and growth in the host. Many of them are harmful and, therefore, are called virulence factors. The effects caused by the virulence factors determine the degree of aggressivity of the strain. In many cases the virulence factors are secreted proteins or enzymes, sometimes performing very specific functions. The enzymatic activity is directed to specific proteins from cell membranes, synaptic vesicle fusion proteins, among other important targets. One of the most toxic bacterial proteins is secreted by Clostridium botulinum, targeted to synaptic vesicle fusion proteins, cleaving them with a zinc-metalloprotease activity, which results in severe neurotoxic effects with a lethal dose as low as eight nanograms per kilogram of body weight. The tetanus neurotoxin acts in a similar way but is less active and Bacillus anthracis also presents a potent metalloprotease activity. In this work we describe a selection of these specially interesting and important bacterial proteins and proteases, stressing their relevance in the pathological process and in medical studies.
Assuntos
Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Peptídeo Hidrolases/metabolismo , Fatores de Virulência/metabolismo , Animais , Bactérias/enzimologia , Bactérias/patogenicidade , Humanos , VirulênciaRESUMO
Incubation of plasma from the snake Bothrops jararaca (BJP) with trypsin generated two hypotensive peptides. The primary structure of the peptides was established for three sequences as: Asn-Pro-Phe-Val-Asp-Ala (fraction 13), Ser-Lys-Pro-Asn-Met-Ser-Asp-Glu-Ser-Leu-Ala-Val-Ala-Ile (fraction 14), Asn-Pro-Phe- Val-Asp-Ala (fraction 15). These peptides display homology with fragments of albumin from Trimeresurus flavoviridis. A bolus intra-arterial injection of the purified or the synthetic peptide produced a strong and sustained vasopressor response in the anaesthetized snake B. jararaca and Wistar rats; this hypotensive effect was also potentiated by captopril, an angiotensin-converting enzyme inhibitor (0.1 mg/kg). The natural concentrations of these peptides in plasma need to be determined and could play a physiological role in snake blood pressure regulation.
Assuntos
Anti-Hipertensivos/farmacologia , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Bothrops , Bradicinina/metabolismo , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Feminino , Hipotensão/induzido quimicamente , Masculino , Oligopeptídeos/sangue , Oligopeptídeos/isolamento & purificação , Peptídeos/sangue , Peptídeos/isolamento & purificação , Ratos , Ratos WistarRESUMO
TsTX is an á-type sodium channel toxin that stimulates the discharge of neurotransmitters from neurons. In the present study we investigated which neurotransmitters are released in the hippocampus after TsTX injection and if they are responsible for electrographic or histopathological effects. Microdialysis revealed that the toxin increased glutamate extracellular levels in the hippocampus; however, levels of gamma-aminobutyric acid (GABA), glycine, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were not significantly altered. Neurodegeneration in pyramidal cells of hippocampus and electroencephalographic alterations caused by the toxin were blocked by pretreatment with riluzole, a glutamate release inhibitor. The present results suggest a specific activity of TsTX in the hippocampus which affects only glutamate release.
Assuntos
Humanos , Animais , Ratos , Hipocampo , Neurotransmissores , Venenos de Escorpião/toxicidadeRESUMO
The present study evaluated four phospholipase A2 (PLA2) (Mlx-8, Mlx-9, Mlx-11 and Mlx-12) isolated from Micrurus lemniscatus snake venom (Elapidae). The effects of intrahippocampal administration of these toxins have been determined on behavior, electroencephalography, and neuronal degeneration in rats. These four PLA2 toxins induced motor and EEG alterations in a dose-dependent manner. Behavioral convulsions were characterized by clonic movements and were often accompanied by EEG alterations. Mlx toxins were convulsive but weakly epileptogenic, since low rates of seizure discharges were observed in EEG records. Neuronal injury seemed to depend on the dose of the toxin used. The highest doses of toxins caused severe intoxication and death of some animals. The injury of hippocampal cells was characterized by massive neuronal loss and hippocampal gliosis. A high occurrence of compulsive scratching was observed. Moreover, the onset of seizures induced by Mlx toxins was markedly delayed. The similarities between the effects of Mlx PLA2s and those isolated from other Elapidae snakes venoms suggest that their toxicity are probably due to their specific binding to neuronal membranes and to the catalysis of phospholipid hydrolysis, producing lysophospholipids and fatty acids. These compounds likely disturb the membrane conformation, causing a marked increase in the release of neurotransmitters and concurrent inhibition of vesicle fission and recycling. These toxins can be a useful tool to investigate properties of endogenous secretory PLA2s and therefore may be important both to study mechanisms involved in neurotransmitter release at nerve terminals and to explain the convulsive properties of PLA2s toxins.
Assuntos
Comportamento Animal/efeitos dos fármacos , Venenos Elapídicos/química , Eletroencefalografia/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Fosfolipases A2/análise , Fosfolipases A2/farmacologia , Animais , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Venenos Elapídicos/classificação , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Espectrometria de Massas/métodos , Camundongos , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Síndromes Neurotóxicas/patologia , RatosRESUMO
Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/química , Metaloendopeptidases/farmacologia , Metaloproteases/farmacologia , Sequência de Aminoácidos , Animais , Benchmarking , Fatores de Coagulação Sanguínea , Células Cultivadas , Venenos de Crotalídeos/farmacologia , Células Endoteliais/efeitos dos fármacos , Hemorragia/induzido quimicamente , Humanos , Metaloendopeptidases/química , Metaloproteases/química , Camundongos , Dados de Sequência Molecular , Veneno de Bothrops jararacaRESUMO
Total RNA from lipopolysaccharide (LPS)-stimulated rat macrophages used to treat protoplasts from an Aspergillus nidulans strain originated the RT2 regenerated strain, whose culture supernatant showed anti-inflammatory activity in Wistar rats. The protein fraction presenting such anti-inflammatory activity was purified and biochemically identified. The screening of the fraction responsible for such anti-inflammatory property was performed by evaluating the inhibition of carrageenan-induced paw edema in male Swiss mice. Biochemical analyses of the anti-inflammatory protein used chromatography, carbohydrates quantification of the protein sample, amino acids content analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Total sugar quantification revealed 32 percent glycosylation of the protein fraction. Amino acid analysis of such fraction showed a peculiar pattern presenting 29 percent valine. SDS-PAGE revealed that the protein sample is pure and its molecular weight is about 40kDa. Intravenous injection of the isolated substance into mice significantly inhibited carrageenan-induced paw edema. The isolated glycoprotein decreased carrageenan-induced paw edema in a prostaglandin-dependent phase, suggesting an inhibitory effect of the isolated glycoprotein on prostaglandin synthesis.
Assuntos
Camundongos , Animais , Anti-Inflamatórios , Aspergillus nidulans , Aspergilose , GlicoproteínasRESUMO
The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.
Assuntos
Bothrops , Venenos de Crotalídeos/genética , Variação Genética , Metaloendopeptidases/genética , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Cromatografia Líquida , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Cisteína/química , Desintegrinas/química , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Metaloendopeptidases/química , Metaloendopeptidases/efeitos dos fármacos , Metaloendopeptidases/isolamento & purificação , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Veneno de Bothrops jararacaRESUMO
PURPOSE: TsTx is a scorpion alpha-type toxin that binds to site 3 of the Na+ channels in a voltage-dependent mode, slowing or blocking the inactivation mechanism of these channels (Possani et al., Eur J Biochem 1999). This binding increases depolarization time of the channel and consequently induces excessive neurotransmitter release. Previously we reported that hippocampal injection of TsTx induces clonic convulsions, electrographic seizures, and hippocampal damage. This investigation was designed to characterize the long-term behavioral, electroencephalographic (EEG), and histopathologic features after a single TsTx injection into the rat hippocampus. METHODS: Cannulas and electrodes were stereotaxically implanted in the CA1 dorsal hippocampus of rats. Three days after surgery, the animals were injected into the hippocampus with 1 microl of TsTx, 2 microg (n = 9) or 0.1 M phosphate buffer (n = 5). After injection, EEG records and behavioral observations were made during 10 h. For a period of 4 months, the animals were observed through direct visual observation for 8 h/day, 5 days/week for occurrence of convulsive seizures. At the end of the experiment, the animals were processed for histologic analyses. Sections 40- and 20-microm thick were stained according to neo-Timm or Nissl methods, respectively. Cell counts in the cresyl violet-stained sections were performed within the hippocampal pyramidal cell layers (CA1, CA3, and CA4) and granule cell of the dentate gyrus. RESULTS: Fifteen minutes after TsTx injection, facial automatisms, rearing, masticatory jaw movements, sniffing, and wet-dog shakes were observed. In approximately 1 h, limbic convulsions characterized by forelimb clonus, rearing, and falling after generalized clonic convulsion with jumping, wild running, and falling were observed. EEG showed isolated spikes and clusters of spikes that started in the hippocampus and evolved to the cortex, isolated seizures, and epileptic discharges delayed 1-2 min. These recurred repeatedly characterizing the status epilepticus (SE). SE was characterized in 77.5% of animals. Seizures were no longer observed 24 h after the injection. Spontaneous recurrent seizures (SRSs) occurred 31-49 days after TsTx injection. All animals that showed SE in the acute period developed SRSs. Facial myoclonus, generalized clonus, forelimb clonus, rearing, and falling characterized the seizures. The seizure frequency was 1-2 per animal per week. All rats injected with TsTx had significantly fewer cells in CA1, CA3, and CA4 subfield of the hippocampal formation compared with the animals of the control group (p < 0.05, analysis of variance, followed by Tukey test). Neo-Timm-positive granules, normally absent in the supragranular layer, were present in dentate gyrus of rats with TsTx-induced SRSs. CONCLUSIONS: Our results suggest that SRSs observed in this study may be a consequence of the TsTx-induced SE. All animals injected with the toxin showed massive neuronal loss in the hippocampal subfields CA1, CA3, and CA4, but only those that had SRSs showed mossy fiber sprouting in the supragranular layer of the dentate gyrus. This shows synaptic reorganization that also is observed in human epileptogenic tissue. These results indicate that TsTx toxin may be a useful tool for studies on neuronal lesions and/or experimental epilepsy. Studies on the mechanisms involved are under investigation.
Assuntos
Fibras Musgosas Hipocampais/efeitos dos fármacos , Fibras Musgosas Hipocampais/fisiopatologia , Plasticidade Neuronal , Neurotoxinas/farmacologia , Venenos de Escorpião/farmacologia , Convulsões/induzido quimicamente , Animais , Eletroencefalografia , Fibras Musgosas Hipocampais/patologia , Ratos , Convulsões/patologia , Convulsões/fisiopatologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologiaRESUMO
Scorpion venom neurotoxins are responsible for toxicity and pharmacological effects. They are active in sodium and potassium channels leading to an increase in the release of neurotransmitters, such as glutamate. Glutamate is found in large quantities in the hippocampus (HPC) and is involved in the long-term potentiation (LTP) induction. The HPC is known to be related to certain kinds of memory. The aim of this study is to evaluate the effects of Tityus serrulatus TS-8F toxin on rat behavior with emphasis on learning and memory. We analyzed the effects of different doses of TS-8F on rat behavior in home cages, open-field (habituation), inhibitory avoidance, T-maze, and hippocampus morphology. In the first two experiments, 0.05µg/animal dose of TS-8F did not cause convulsion but led to a decrease in locomotion (LO) frequency in the open-field first session. During the second session, rats receiving 0.03µg/animal TS-8F showed a decrease in LO and rearing frequency (RE); controls only showed decreased LO; and those receiving 0.05µg/animal showed no significant changes. In inhibitory avoidance, T-maze, and HPC morphology experiments no significant differences were observed. It is concluded that TS-8F may exert some influence in rat learning and memory and seems to be useful as a pharmacological tool. Further research is required to elucidate all possible uses of this toxin.
Assuntos
Ratos , Animais , Masculino , Toxinas Biológicas , Aprendizagem , Memória , Neurotoxinas , Venenos de Escorpião/efeitos adversos , Venenos de Escorpião/toxicidade , Ratos Wistar , Convulsões/induzido quimicamenteRESUMO
The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA
Assuntos
Proteus mirabilis , Metaloendopeptidases , Proteínas de Bactérias , Proteus mirabilis , Espectrometria de Massas , Especificidade por Substrato , Virulência , Metaloendopeptidases , Hidrólise , Proteínas de Bactérias/análiseRESUMO
The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized beta-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the beta-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.
Assuntos
Proteínas de Bactérias/metabolismo , Metaloendopeptidases/metabolismo , Proteus mirabilis/enzimologia , Proteínas de Bactérias/análise , Hidrólise , Espectrometria de Massas , Metaloendopeptidases/análise , Proteus mirabilis/patogenicidade , Especificidade por Substrato , VirulênciaRESUMO
The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 microM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 microM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.
Assuntos
Proteínas de Bactérias/metabolismo , Metaloendopeptidases/metabolismo , Proteus mirabilis/enzimologia , Proteínas de Bactérias/análise , Caseínas/química , Hidrólise , Espectrometria de Massas , Infecções por Proteus/microbiologia , Proteus mirabilis/genética , Proteus mirabilis/patogenicidade , Análise de Sequência de Proteína , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.
Assuntos
Peptídeos/análise , Proteus mirabilis/enzimologia , Proteínas de Bactérias , Metaloendopeptidases , Endopeptidases/isolamento & purificação , Infecções por Proteus/microbiologia , Proteus mirabilis/genética , Proteus mirabilis/patogenicidade , Espectrometria de Fluorescência , Espectrometria de Massas , Especificidade por Substrato , Proteínas de Bactérias/análise , Metaloendopeptidases/análise , Cinética , Caseínas/análise , HidróliseRESUMO
Scorpion venoms contain low molecular weight basic polypeptides, neurotoxins, that are the principal toxic agents. These toxins act on ion channels, promoting a derangement that may result in an abnormal release of neurotransmitters. In the present study we investigated some of the effects of the F, H and J fractions isolated from Tityus serrulatus scorpion venom on the central nervous system of rodents. The venom was partially purified by gel filtration chromatography. The neurotoxic effect of these fractions was studied on convulsive activity after intravenous injection, and on electrographic activity and neuronal integrity of rat hippocampus when injected directly into this brain area. The results showed that intravenous injection of the F and H fractions induced convulsions, and intrahippocampal injection caused electrographic seizures in rats and neuronal damage in specific hippocampal areas. Fraction J injected intravenously reduced the general activity of mice in the open field but induced no changes when injected into the brain. These results suggest that scorpion toxins are able to act directly on the central nervous system promoting behavioural, electrographic and histological modifications.
Assuntos
Neurotoxinas/toxicidade , Venenos de Escorpião/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Fracionamento Químico , Cromatografia em Gel , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Injeções Intravenosas , Masculino , Camundongos , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificação , Convulsões/induzido quimicamenteRESUMO
It has been previously shown that the crude venom of Tityus serrulatus can cause convulsions. This study was designed to investigate the neurotoxic effects of B, C, G, and K fractions isolated from this venom. Intravenous injection of these fractions in mice (0.6 - 6.0 mg/kg body weight) showed that the C fraction is a potent convulsant and G fraction decreased the threshold for tonic hand limb extension elicited by transauricular electroshock. Unilateral injection of B, C, and K fractions, but not G fraction, into the spikes and epileptic discharges that began in the hippocampus and evolved to the cortex. The following motor signs were observed: movements of facial muscles, wet dog shake, immobility, myoclonus, wild-running with clonus, and in some cases, loss of postural control. Intrahippocampal injection of B, C, and K fraction, but not G fraction, caused neuronal loss at the injection site as well as in other hippocampal areas. The effect of these fractions on epileptiform activity and on neuronal loss was dose-dependent. The severity of the epileptiform activity in the ipsilateral hippocampus correlated with the severity of the neuronal loss. The electrographic, behavioral, and histological changes induced by b, C, and K fractions were similar to those obtained with other drugs that are commonly used to induce convulsion. The convulsant effects of the crude venom may be caused by the fractions studied in this work.
Assuntos
Masculino , Ratos , Camundongos , Comportamento Animal/efeitos dos fármacos , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Convulsões/induzido quimicamente , Venenos de Escorpião/toxicidade , Convulsões/induzido quimicamente , Estudos de Casos e Controles , Ratos Wistar , Análise de Variância , Relação Dose-Resposta a Droga , Venenos de Escorpião/isolamento & purificaçãoRESUMO
The effects of intrahippocampal administration of a neuropeptide (TS-8F toxin) isolated from Tityus serrulatus scorpion venom have been determined on behavior, limbic seizures, and neuronal degeneration in rats. Behavioral observation showed orofacial automatism, wet dog shakes, and myoclonus. Concomitantly, the electroencephalographic record showed high-frequency and high-voltage spikes that evolved to seizure activity in the hippocampus and cortex. Seven days after TS-8F toxin microinjection, neuronal damage was observed in CA1 and CA2 pyramidal cells and in granular cells of the dentate gyrus. The results suggest that TS-8F toxin may be responsible, at least in part, by the epileptic effects observed with the crude venom. Thus, this toxin may be a useful tool in the study of some neurobiological process.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neuropeptídeos/toxicidade , Neurotoxinas/toxicidade , Venenos de Escorpião/toxicidade , Sequência de Aminoácidos , Animais , Comportamento Animal/fisiologia , Encéfalo/fisiopatologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Dados de Sequência Molecular , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Neuropeptídeos/química , Neuropeptídeos/isolamento & purificação , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Escorpião/química , Convulsões/induzido quimicamenteRESUMO
Toxin-gamma (Tgamma) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tgamma was approximately 200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tgamma (1.2 vs. 12 microg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+ concentration ([Ca2+]o), when 30 microM veratridine plus 0.45 microM Tgamma were used. Tgamma (0.45 microM) doubled the basal uptake of 45Ca2+, whereas veratridine (100 microM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tgamma and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+ channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.