The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.

A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S5methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.

No abstract

Use of the nonpathogenic yeastSaccharomyces boulardii is a thermophilic, nonpathogenic yeast administered in Western Europe for the prevention and treatment of a variety of diarrheal diseases (17, 29). However, the mechanisms by which S. boulardii controls diarrhea remain elusive. The efficacy of this yeast has been attributed to several of its properties, such as its effect on the mucosa leading to an increase in dissaccharidase activity (8) or stimulation of the immune response (7). In animals, administration of S. boulardii provides protection against intestinal lesions caused by several diarrheal pathogens (10,33). In vitro studies have demonstrated that S. boulardii exerts antagonistic activity against various bacterial pathogens (6). Recent studies have reported the adhesion of the Salmonella enterica serovars Typhimurium and Enteritis and of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli to S. boulardii (24,25).EPEC is a major cause of diarrhea in the developing world (31, 34). The pathogenesis of EPEC infections involves a three-stage process. (i) EPEC adheres initially to intestinal epithelial cells in a pattern described as localized adherence (36). This pattern of adherence, characterized by microcolonies of bacteria associated with the epithelial cells, is dependent on the expression of the bacterial type IV bundle-forming pilus (BFP) (3). (ii) Next, the bacteria induce signal transduction pathways in host cells, leading to an elevation in the intracellular levels of Ca 2ϩ and inositol triphosphate (16, 23) and the phosphorylation of cellular proteins (4, 35, 41). (iii) These signaling events culminate in the formation of attachingand-effacing lesions which are characterized by localized degeneration of the microvilli, intimate contact between the bacteria and the infected cell, and the assembly of highly organized cytoskeletal structures in the epithelial cells just beneath the attached bacteria, forming cuplike pedestals (22,27,30). EPEC is also able to induce its internalization by nonphagocytic epithelial cells (2, 15).The aim of our study was to investigate in vitro the effect of S. boulardii against EPEC infection using the T84 cell line derived from a colon carcinoma. This cell line has been extensively used to elucidate the mechanism of EPEC-induced diarrhea. EPEC infection results in a modification of the T84 barrier function, characterized by a drop in transepithelial resistance, an increase in permeability, and modification of the distribution of the tight junction-associated protein 37). Our study reveals that S. boulardii maintains the barrier function and the viability of EPEC-infected T84 cells. Although the yeast does not modify the number of cell-associated bacteria, it reduces the number of intracellular bacteria. The phosphorylation of several proteins induced by EPEC in T84 cells is diminished in the presence of S. boulardii. Finally, the yeast interferes with the ERK1/2 mitogen-activated protein (MAP) kinase pathway that, as demonstrated in this study, is ...

No abstract

Insulin stimulates the phosphorylation of its own receptor. In the work reported here, the kinase activity responsible for the insulin-stimulated phosphorylation of the insulin receptor was localized. In a first approach, partially purified insulin receptors derived from normal rat hepatocytes were immunoprecipitated with antibodies specific for the insulin receptor; thereafter, the immunoprecipitates were incubated with [gamma-(32)P]-ATP in the absence or presence of insulin (1 muM). NaDodSO(4)/polyacrylamide gel electrophoretic analysis of the immunoprecipitates under reducing conditions revealed autophosphorylation of the beta subunit (M(r) 95,000) of the insulin receptor; the alpha subunit (M(r) 130,000) was not phosphorylated. Further, insulin specifically increased 3- to 4-fold the labeling of its own receptor beta subunit, indicating that anti-receptor antibodies precipitate a functional and insulin-stimulable protein kinase that appears to be independent of cyclic AMP and calcium. To localize more precisely the insulin receptor-related kinase activity, we searched for an ATP-binding site on solubilized insulin receptors. By using covalent labeling with oxidized [alpha-(32)P]ATP, a labeled polypeptide with precisely the same electrophoretic mobility as that of the beta subunit of the insulin receptor (M(r) 95,000) was specifically immunoprecipitated with anti-receptor antibodies. Further, its appearance was prevented when the immunoprecipitation was preceded by incubation with unlabeled insulin. In conclusion, we have shown that an insulin-stimulated phosphorylation site and an ATP-binding site coexist on the beta subunit of the insulin receptor. The simultaneous presence of these two sites on the same receptor subunit indicates that the insulin receptor acts as its own protein kinase.

Fractalkine displays features that distinguishes it from the other chemokines. In particular, besides its chemoattractant action it promotes, under physiologic flow, the rapid capture and the firm adhesion of a subset of leukocytes or intervenes in the neuron/microglia interaction. This study verified that indeed the human

Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.