RESUMO
Androgenic-anabolic steroids (AAS) have been associated with an increased incidence of tendon rupture. The aim of this study was to compare the biomechanical properties of the rat calcaneal tendon (CT), superficial flexor tendon (SFT), and deep flexor tendon (DFT), and to determine the effect of jump training in association with AAS. Animals were separated into four groups: sedentary, trained, AAS-treated sedentary rats (AAS), and AAS-treated and trained animals. Mechanical testing showed that the CT differed from the DFT and SFT, which showed similar mechanical properties. Jump caused the CT to exhibit an extended toe region, an increased resistance to tensional load, and a decreased elastic modulus, characteristics of an elastic tendon capable of storing energy. AAS caused the tendons to be less compliant, and the effects were reinforced by simultaneous training. The DFT was the most affected by training, AAS, and the interaction of both, likely because of its involvement in the toe-off step of jumping, which we suggest is related to the rapid transmission of force as opposed to energy storage. In conclusion, tendons are differently adapted to exercise, but responded equally to AAS, showing reduced flexibility, which is suggested to increase the risk of tendon rupture in AAS consumers.
Assuntos
Anabolizantes/farmacologia , Nandrolona/análogos & derivados , Tendões/efeitos dos fármacos , Suporte de Carga/fisiologia , Anabolizantes/administração & dosagem , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Masculino , Nandrolona/administração & dosagem , Nandrolona/farmacologia , Decanoato de Nandrolona , Condicionamento Físico Animal , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Bothrops/fisiologia , Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Reações Cruzadas , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/toxicidade , Edema/induzido quimicamente , Edema/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Immunoblotting , Imunoglobulinas , Injeções Intraperitoneais , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Testes de NeutralizaçãoRESUMO
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structurefunction relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.
Assuntos
Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Antivenenos/imunologia , Bothrops/classificação , Metaloproteases/classificação , Metaloproteases/toxicidade , Venenos de Serpentes/imunologia , Anticorpos Neutralizantes , Colubridae , Elapidae , ViperidaeRESUMO
Bioactive compounds of great interest are found in the saliva of hematophagous organisms. While exploring a cDNA library derived from the salivary glands of the tick Amblyomma cajennense, a transcript that codes for a protein with unique structure (containing an N-terminal Kunitz-type domain and a C-terminus with no homology to any annotated sequences) was found. The recombinant mature form of this protein ( approximately 13.5kDa) was produced in Escherichia coli BL21 (DE3), and it was able to inhibit Factor Xa (FXa) and extend global blood clotting times in vitro and ex vivo. Static and dynamic predictions of its tertiary structure indicate regions that may be related to its FXa inhibitor function.
Assuntos
Inibidores do Fator Xa , Fator Xa/química , Ixodidae/química , Inibidores de Serina Proteinase/química , Animais , Clonagem Molecular , DNA Complementar/genética , Fator Xa/metabolismo , Humanos , Ixodidae/genética , Ixodidae/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Relação Estrutura-AtividadeRESUMO
Bioactive compounds of great interest are found in the saliva of hematophagous organisms. While exploring a cDNA library derived from the salivary glands of the tick Amblyomma cajennense, a transcript that codes for a protein with unique structure (containing an N-terminal Kunitz-type domain and a C-terminus with no homology to any annotated sequences) was found. The recombinant mature form of this protein (¡13.5 kDa) was produced in Escherichia coli BL21 (DE3), and it was able to inhibit Factor Xa (FXa) and extend global blood clotting times in vitro and ex vivo. Static and dynamic predictions of its tertiary structure indicate regions that may be related to its FXa inhibitor function.
Assuntos
Animais , Anticoagulantes , Fator Xa , Saliva/fisiologia , SalivaRESUMO
Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.
Assuntos
Colágeno/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Metaloendopeptidases/toxicidade , Sequência de Aminoácidos , Animais , Sítios de Ligação , Colágeno/química , Colágeno/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/metabolismo , Veneno de Bothrops jararacaRESUMO
The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35 degrees C. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35% in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-beta gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single approximately 60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.
Assuntos
Metaloproteinases da Matriz/metabolismo , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Natação/fisiologia , Remodelação Ventricular/fisiologia , Animais , Western Blotting , Peso Corporal , Regulação da Expressão Gênica , Ácido Láctico/sangue , Masculino , Metaloproteinases da Matriz/genética , Miocárdio/enzimologia , Cadeias Pesadas de Miosina/genética , Tamanho do Órgão , Condicionamento Físico Animal , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35°C. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35 percent in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-ß gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single ~60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.
Assuntos
Animais , Masculino , Ratos , Metaloproteinases da Matriz/metabolismo , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Natação/fisiologia , Remodelação Ventricular/fisiologia , Western Blotting , Peso Corporal , Regulação da Expressão Gênica , Ácido Láctico/sangue , Metaloproteinases da Matriz/genética , Miocárdio/enzimologia , Cadeias Pesadas de Miosina/genética , Tamanho do Órgão , Condicionamento Físico Animal , Reação em Cadeia da Polimerase , Ratos Wistar , RNA Mensageiro/análise , Fatores de TempoRESUMO
Snake venoms are relevant sources of toxins that have evolved towards the engineering of highly active compounds. In the last years, research efforts have produced great advance in their understanding and uses. Metalloproteases with disintegrin domains are among the most abundant toxins in many Viperidae snake venoms. This review will focus on the structure, function and possible applications of the metalloprotease and disintegrin domains.
Assuntos
Desintegrinas/química , Metaloproteases/química , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Desintegrinas/metabolismo , Humanos , Metaloproteases/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , SerpentesRESUMO
Snake venom metalloproteases (SVMPs) are a set of interesting enzymes that are one of the major components of venom affecting hemostasis. A great challenge since their discovery has been to find molecular features responsible for their hemorrhagic potency and many attempts have been made without any consistent result. Here we describe a series of comparisons between the catalytic domains of hemorrhagic and non-hemorrhagic SVMPs made with the help of bioinformatics. These involved sequence and structure-based multiple alignments, phylogenetic reconstruction, predicted physical and chemical properties, motif scanning and structural analyses. Although hemorrhagic activity seems to be complex, involving multiple factors, we found some molecular characteristics that may influence the toxic effects. Among these findings, it was possible to use a molecular surface feature to subdivide the P-I class in hemorrhagic and non-hemorrhagic SVMPs. It was also possible to suggest a role for the conserved Asp148 and Ser176 residues in the stabilization of the active site.