Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrinmediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP-and Src kinasedependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.

Nitric oxide synthase oxygenase domains (NOS ox ) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS ox reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal β-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra-to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS ox molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. Here we demonstrate that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. Our in vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3' UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting.

Background: Microglial activation plays an important role in the pathogenesis of neurodegenerative disorders. Results: Taylor-Couette-Poiseuille flow-modified saline (RNS60) inhibits microglial inflammation via type 1A phosphatidylinositol 3-kinase-Akt-CREB-mediated up-regulation of IB␣ and inhibition of NF-B activation. Conclusion: These results delineate a novel biological function of a physically modified saline. Significance: RNS60 may be of therapeutic benefit in neurodegenerative disorders.

Intracellular parasitic protozoans of the genus Leishmania depend for their survival on the elaboration of enzymic and other mechanisms for evading toxic free-radical damage inflicted by their phagocytic macrophage host. One such mechanism may involve superoxide dismutase (SOD), which detoxifies reactive superoxide radicals produced by activated macrophages, but the role of this enzyme in parasite survival has not yet been demonstrated. We have cloned a SOD gene from L. tropica and generated SOD-deficient parasites by expressing the corresponding antisense RNA from an episomal vector. Such parasites have enhanced sensitivity to menadione and hydrogen peroxide in axenic culture, and a markedly reduced survival in mouse macrophages. These results indicate that SOD is a major determinant of intracellular survival of Leishmania.