RESUMO
PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.
Assuntos
Eletroforese/métodos , Fibroblastos/enzimologia , Metaloproteinase 2 da Matriz/análise , Sobrevivência Celular , Células Cultivadas , Gelatinases/fisiologia , Humanos , Lipoproteínas , Metaloproteinase 2 da Matriz/fisiologia , Ligamento Periodontal/citologia , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Fatores de Tempo , Receptores Toll-Like/análiseRESUMO
PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.
Assuntos
Humanos , Eletroforese/métodos , Fibroblastos/enzimologia , /análise , Sobrevivência Celular , Células Cultivadas , Gelatinases/fisiologia , Lipoproteínas , /fisiologia , Ligamento Periodontal/citologia , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Fatores de Tempo , Receptores Toll-Like/análiseRESUMO
Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.
Assuntos
Infecções Protozoárias do Sistema Nervoso Central/veterinária , Doenças do Cão/parasitologia , Leishmaniose Visceral/veterinária , Metaloproteinase 2 da Matriz/fisiologia , Animais , Infecções Protozoárias do Sistema Nervoso Central/enzimologia , Progressão da Doença , Doenças do Cão/enzimologia , Cães , Precursores Enzimáticos/sangue , Precursores Enzimáticos/líquido cefalorraquidiano , Precursores Enzimáticos/fisiologia , Feminino , Gelatinases/sangue , Gelatinases/líquido cefalorraquidiano , Gelatinases/fisiologia , Leishmaniose Visceral/enzimologia , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/líquido cefalorraquidiano , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/líquido cefalorraquidiano , Metaloproteinase 9 da Matriz/fisiologiaRESUMO
Proteases are well-recognized as virulence factors in different pathologies, resulting in tissue damage potential. Despite efforts over the past few years to identify mycobacterial protein antigens, there is little information regarding the role of mycobacterial proteinase activities. In this study, by zymography techniques, we have detected and partially studied some biochemical properties of Mycobacterium bovis proteases, such as pH dependency of activity and susceptibility to classical proteinase inhibitors. We observed optimal proteolytic activity at pH 8. Some proteinases were inhibited by classic inhibitors of serine proteases, such as PMSF, AEBSF, and 3-4 DCI. In some AEBSF pre-treated preparations we observed residual gelatinase activity in Rf 0.32. This gelatinase was stimulated by Zn2+ and inhibited by OPA (1 mM). This last effect was reversed by exposure to equimolar quantitative OPA/Zn+2 (1 mM/1 mM). These results suggest the existence of serine proteinase and metalloproteinase types in protein extracts of Mycobacterium bovis.