RESUMO
OBJECTIVE: Our poor understanding of how inflammatory mediators can affect osteoblast behavior led us to investigate the tumor necrosis factor (TNF)α-induced focal adhesion kinase (FAK) and Src phosphorylation. MATERIAL AND METHODS: MC3T3-E1 pre-osteoblast cells were harvested at specific time points after either TNFα treatment or RAW267 stimulated conditioned medium, and thereafter cell extracts were prepared for Immunoblotting assay. ELISA detected TNFα content at conditioned medium. Tumor necrosis factor-α-neutralizing antibodies also were used. RESULTS: It was possible to show that TNFα provokes attenuation at Y-phosphorylation of both FAK (at Y397 ) and Src (at Y416 ) proteins (P < 0.05), suggesting a decrease in their activities. The very similar profile was observed when osteoblasts were incubated with conditioned medium from lipopolysaccharide (LPS)-stimulated macrophages, it being significantly different than control (FAK and Src, P < 0.05). Nevertheless, in order to validate these findings, we decided to pre-incubate osteoblasts with anti-TNFα neutralizing antibody (2 µg ml(-1) ) prior exposing to conditioned medium. Importantly, our results revealed that there was a diminution on those conditioned medium effects when the same biological parameters were evaluated (P < 0.05). Moreover, we also showed that TNFα impairs osteoblast adhesion, suggesting an interesting role on osteoblast performance. CONCLUSIONS: Altogether, these results suggest that LPS-stimulated macrophage mediators attenuate both FAK and Src activations in osteoblast, suggesting a novel role for TNFα on osteoblast performance.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Osteoblastos/enzimologia , Fator de Necrose Tumoral alfa/fisiologia , Quinases da Família src/metabolismo , Células 3T3 , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Camundongos , FosforilaçãoRESUMO
Silica gel surface was chemically modified with beta-diketoamine groups by reacting the silanol from the silica surface with 3-aminopropyl-triethoxysilane and 3-bromopentanedione. With this material, copper ions were adsorbed from aqueous solutions. The chemical analysis of the silica-gel-immobilized acetylacetone provided a quantity of 0.67 mmol g-1 of organic groups attached to the support and 0.63 mmol g-1 of copper. This material was used as a stationary phase in IMAC (immobilized metal affinity chromatography), to separate alpha-lactoalbumin from bovine milk whey. The results showed an efficient separation in the chromatographic column. The possibility of reutilization of the stationary phase was also investigated.