RESUMO
The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3beta have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3beta (Ser9) phosphorylation. Pretreatment with 20 microM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3beta inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3beta and Rac1.
Assuntos
Movimento Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Mucosa Intestinal/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Poliaminas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Citoesqueleto/química , Células Epiteliais/citologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Intestino Delgado/citologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , RatosRESUMO
AIMS: To develop a novel, rapid and effective screening method for chitinase producing bacteria. METHODS AND RESULTS: A simple and rapid technique for screening of potential chitinolytic bacteria has been developed using the chitin binding dye calcofluor white M2R in chitin agar. Microorganisms possessing high chitinolytic potential gave a clear zone under ultraviolet light after 24-48 h of incubation. This method was successfully applied for isolating the hyperchitinase mutant of Alcaligenes xylosoxydans. The mutant Alc. xylosoxydans EMS 33 was found to produce 3.4 times more chitinase than the wild type. CONCLUSIONS: In this study, the screening method for chitinase producing bacteria has been developed and it was applied to screen chitinase-overproducing mutant of Alc. xylosoxydans. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel screening method for chitinase producer is more sensitive, rapid, user-friendly and reliable, which can also be used for screening of recombinants having chitinase gene.
Assuntos
Alcaligenes/enzimologia , Bactérias/enzimologia , Técnicas Bacteriológicas , Quitina/metabolismo , Quitinases/metabolismo , Alcaligenes/genética , Alcaligenes/isolamento & purificação , Alcaligenes/efeitos da radiação , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Benzenossulfonatos/química , Benzenossulfonatos/farmacologia , Quitina/química , Quitina/isolamento & purificação , Contagem de Colônia Microbiana , Mutação , Sensibilidade e EspecificidadeRESUMO
Alcaligenes xylosoxydans protected pigeonpea from Fusarium wilt in a pot experiment and field trials. When seeds of pigeonpea (C. cajan) were treated with A. xylosoxydans and sown in soil infested with Fusarium, the incidence of wilt was reduced by 43.5% and resulted in 58% higher grain yield. The antifungal activity of A. xylosoxydans was based on chitinase production and was comparable in efficacy to commercial antifungal agents such as benlate, monitor WP, thiram and bavistin.
