Expression Of Functional Type Research Articles

IFN-ε Mediates TNF-α-Induced STAT1 Phosphorylation and Induction of Retinoic Acid-Inducible Gene-I in Human Cervical Cancer Cells

Retinoic acid inducible gene-I (RIG-I) plays important roles during innate immune responses to viral infections and as a transducer of cytokine signaling. The mechanisms of RIG-I up-regulation after cytokine stimulation are incompletely characterized. It was previously reported that IFN-gamma induces the expression of RIG-I in endothelial cells. In this study, we characterized the mechanism of type I IFN-mediated up-regulation of RIG-I in HeLa cells and found that, in addition to type I IFN, TNF-alpha, a cytokine that regulates innate immune responses, induced expression of RIG-I. To investigate whether TNF-alpha- and type I IFN-mediated up-regulations of RIG-I were causally related, we studied the kinetics of these responses. Our results were consistent with a model in which TNF-alpha functioned upstream of type I IFNs. The ability of TNF-alpha to up-regulate RIG-I required protein synthesis, expression of functional type I IFNRs, and STAT1 signaling. We also found that IFN-epsilon was the only IFN isoform expressed constitutively in HeLa cells and that its expression was up-regulated in response to stimulation with TNF-alpha. The mechanism of up-regulation involved stabilization of IFN-epsilon mRNA in the absence of transcriptional activation. Silencing the expression of IFN-epsilon attenuated STAT1 expression and phosphorylation and inhibited RIG-I expression, providing additional support for the participation of IFN-epsilon upstream of STAT1. Our findings support a sequential mechanism whereby TNF-alpha leads to stabilization of IFN-epsilon mRNA, increased IFN-epsilon synthesis, engagement of type I IFNRs, increased STAT1 expression and phosphorylation, and up-regulation of RIG-I expression. These findings have implications for our understanding of the immune responses that follow cytokine stimulation.

Expression of functional type 1 protease-activated thrombin receptors by mouse primary palatal mesenchymal cells in vitro

Development of the primary palate involves a series of processes including cell growth, differentiation, and morphogenesis. To study the molecular and cellular processes during mouse primary palatogenesis, mesenchymal cells were isolated from the primary palate of BALB/cBy embryos (day-11, hour 20). Most of the primary palatal mesenchymal (PPM) cells were morphologically similar to fibroblasts. The population doubling time was about 36 h. At concentrations of 5 and 10 unit/ml, α-thrombin significantly stimulated the proliferation of these palatal cells by 2- to 2.4-fold compared to untreated controls over a 72 hour incubation period. Reverse transcriptase-polymerase chain reaction using primers based on the mouse type 1 protease-activated thrombin receptor (PAR1) detected PAR1 mRNA in the PPM cells, the authenticity of which was confirmed by partial DNA sequencing. Blocking of the α-thrombin proteolytic site with the highly specific inhibitor D-phenylalanyl-prolyl-arginyl chloromethyl ketone significantly suppressed the mitogenic effect of thrombin on the PPM cells by 71%. These results suggest that PAR1 is present on PPM cells in the mouse embryo and that serine protease activity is important for the receptor activation.

Expression of type 1 metabotropic glutamate receptors in Purkinje neurons of mice

Expression of functional type 1 metabotropic glutamate receptors (mGluR1) was studied in Purkinje neurons in cerebellar slices of mice. Extracellular application of 100 μM of a specific activator of G protein-coupled mGluR, (1S, 3R)-ACPD, triggered a transient elevation of [Ca2+]i in Purkinje neurons. The amplitudes of [Ca2+]i transients in the dendrites of Purkinje neurons were considerably higher than those in the somata, whereas the amplitudes of glutamate-triggered [Ca2+]i transients were similar in these cellular compartments. This suggests the preferential localization of mGluR and/or InsP3-sensitive Ca2+ stores in the dendrites of Purkinje neurons. By applying a single-cell reverse-transcriptase polymerase chain reaction (RT-PCR) technique, we also demonstrated that mRNA, responsible for the expression of mGluR1 type of glutamate receptors, is transcribed in Purkinje neurons; these receptors are likely to be responsible for (1S,3R)-ACPD-evoked calcium signalling.

Hyperadhesive mutant of type 1-fimbriated Escherichia coli associated with formation of FimH organelles (fimbriosomes).

The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae. Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa) subunit protein of type 1 fimbriae. Phenotypically, the mutant demonstrated an eightfold-higher mannose-specific hemagglutination titer than the parent strain. On electron microscopy, the mutant strain expressed the same number of fimbriae as the parent strain. However, numerous 10-nm-diameter rounded structures (fimbriosomes) were observed both closely associated with fimbriae and in the culture medium. Fimbriosomes isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner. Dissociation of the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electrophoresis. Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of dissociated type 1 fimbriae and also blocked the adherence of other strains of type 1 fimbriated E. coli to eucaryotic cells. These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant of the D-mannose-sensitive adhesion of type 1 fimbriae.

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.

The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit

The role of type 1 fimbriae in the mannose-sensitive attachment of Escherichia coli to eucaryotic cells was investigated by deletion mutation analysis of a recombinant plasmid, pSH2, carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. A mutant, pUT2002, containing a deletion remote from the structural gene encoding the 17-kilodalton subunit protein of type 1 fimbriae failed to agglutinate guinea pig erythrocytes even though the bacteria expressed fimbriae morphologically and antigenically indistinguishable from those produced by the intact recombinant plasmid. Fimbriae isolated from pUT2002 failed to agglutinate guinea pig erythrocytes, but reacted with a monoclonal antibody specific for quaternary structural determinants of type 1 fimbriae. Moreover, the dissociated fimbrial subunits from this mutant were indistinguishable from normal fimbriae by their migration during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, by their reactivity with a monoclonal antibody directed against a subunit-specific epitope, and in enzyme-linked immunosorbent assays with monospecific antisera. These results indicate that the adhesive functions in type 1 fimbriae are dependent on a factor(s) encoded by a gene other than those required for synthesis, assembly, and expression of the structural 17-kilodalton subunit.