Polyamine depletion with the ornithine decarboxylase inhibitor a-difluoromethyl ornithine (DFMO), prevents Rac1 activation causing the formation of a thick actin cortex at the cell periphery and inhibits migration of intestinal epithelial cells. In the present study, we demonstrate that MEK activation by EGF increased Rac1 activation, dissociation of intercellular contacts, and migration in both control and polyamine-depleted cells, while U0126, a specific inhibitor of MEK1, prevented disruption of junctions as well as EGF-induced Rac1 activation. Constitutively active MEK1 (CA-MEK) expression altered cell-cell contacts in control and polyamine depleted cells. The expression of constitutively active Rac1 (CA-Rac1) restored b-catenin to the cell periphery and prevented the formation of actin cortex and caused the appearance of F-actin stress fibers in polyamine-depleted cells. Inhibition of Rac activation by NSC23766, a specific inhibitor of Tiam1, an upstream guanidine nucleotide exchange factor for Rac1, reproduced the b-catenin localization and actin structure of polyamine-depleted cells. Tiam1 localized more extensively with b-catenin at the cell periphery in CA-Rac1 cells compared to vector cells. Polyamine depletion decreased the expression of E-cadherin to a greater extent compared to b-catenin. Subcellular fractionation further confirmed our immuno-localization and western blotting observations. These data suggest that EGF acting through MEK1/ERK to activate Rac1 regulates cell-cell contacts. Thus, decreased migration in polyamine depleted cells may be due to the inhibition of Tiam1 activation of Rac1 and the subsequent decreased expression of b-catenin and E-cadherin leading to reduced cell-cell contacts. Cell Motil.

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Vaidya, Rajiv J., Ramesh M. Ray, and Leonard R. Johnson. MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm.

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.

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 3AE4 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.