Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3′-phosphoinositides on membrane trafficking in yeast and mammalian cells.     

Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal

The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF⁶⁵ to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF⁶⁵ to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF⁶⁵ can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.     

Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver

The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.     

Proteinase inhibitors reduce basement membrane degradation by human breast cancer cell lines.

The relative importance of different proteinases, and their inhibition, in the breakdown of human endothelial basement membrane (BM) by MDA-MB-231 and MCF7ADR human breast cancer cell lines has been studied using 35S-labelled BM-coated 96-well culture plates. Basement membrane degradation (BMD) was independent of cell proliferation above the seeding density. Inhibitors of aspartic (pepstatin and PD 134678-0073) and cysteine proteinases (E64) had little effect on BMD under normal culture conditions, suggesting that cathepsins D, B and L have only a minor role. In contrast, inhibitors of urokinase-type plasminogen activator (uPA) and/or plasminogen activation to plasmin (aprotinin, amiloride, EACA, tranexamic acid, anti-uPA antibody) all reduced BMD by MDA-MB-231 cells by approximately 30-40%, but only in the presence of serum or plasminogen. BB94, an inhibitor of matrix metalloproteinases (MMPs), also reduced BMD by about 30% under these conditions but was similarly effective in serum-free medium. Combinations of BB94 with any of the uPA/plasminogen activation inhibitors in serum-containing medium had additive effects, while BB94 with pepstatin and E64 under serum-free conditions reduced BMD to 16% of control. Serum-containing conditioned medium exhibited appreciable BMD, largely due to aprotinin-inhibitable activity. Although small reductions in cell proliferation were seen with some inhibitors, the combination of BB94 with E64 or E64d reduced the cell population by about 60% under serum-containing conditions. These in vitro observations suggest that combinations of proteinase inhibitors, particularly of uPA/plasminogen activation and MMPs, may merit clinical evaluation as potential antimetastatic therapy for breast cancer.     

Dissociation in the recovery from neglect dyslexia and visuospatial unilateral neglect: a case report

The recovery of the ability to read of a patient affected by persistent visuospatial neglect suggests the functional independence of the two phenomena. Neglect dyslexia seems to be an example of a dissociation between an implicit and explicit knowledge of the characteristics of the stimulus.

---

Sommario Il recupero della capacità di lettura in un paziente con persistente neglect visuo-spaziale suggerisce l'indipendenza funzionale dei due fenomeni. La neglect dyslexia sembra costituire un esempio di dissociazione tra conoscenza implicita ed esplicita delle caratteristiche dello stimolo.