Gene expression profiling on global cDNA arrays gives hints concerning potential signal transduction pathways involved in cardiac fibrosis of renal failure

Cardiac remodelling with interstitial fibrosis in renal failure, which so far is only poorly understood on the molecular level, was investigated in the rat model by a global gene expression profiling analysis. Sprague-Dawley rats were subjected to subtotal nephrectomy (SNX) or sham operation (sham) and followed for 2 and 12 weeks, respectively. Heart-specific gene expression profiling, with RZPD Rat Unigene-1 cDNA arrays containing about 27 000 gene and EST sequences revealed substantial changes in gene expression in SNX compared to sham animals. Motor protein genes, growth and differentiation markers, and extracellular matrix genes were upregulated in SNX rats. Obviously, not only genes involved in cardiomyocyte hypertrophy, but also genes involved in the expansion of non-vascular interstitial tissue are activated very early in animals with renal failure. Together with earlier findings in the SNX model, the present data suggest the hypothesis that the local renin-angiotensin system (RAS) may be activated by at least two pathways: (a) via second messengers and Gproteins (short-term signalling); and (b) via motor proteins, actins and integrins (longterm signalling). The study documents that complex hybridization analysis yields reproducible and promising results of patterns of gene activation pointing to signalling pathways involved in cardiac remodelling in renal failure. The complete array data are available via http://www.rzpd.de/cgi-bin/services/exp/viewExpressionData.pl.cgi.     

An aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica is attenuated and immunogenic in a mouse model of infection

We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.     

Lack of a role of the interferon-stimulated response element-like region in interferon alpha -induced suppression of Hepatitis B virus in vitro

The antiviral effect of interferon-alpha (IFNalpha) on hepatitis B virus (HBV) is well documented in vitro and in vivo, but the mechanisms involved are elusive. Recently, an interferon-stimulated response like element (ISRE) competent for binding of interferon-stimulated gene factor-3gamma (p48) has been identified in the HBV enhancer I region. Mutation of this element was shown to abrogate IFNalpha-mediated reduction of HBV X-gene promoter-driven reporter gene expression. This suggested a role of the ISRE and of p48 in IFNalpha-induced antiviral activity against productive HBV infection. Here, we analyzed the antiviral effect of both IFNalpha and enhanced p48 expression on complete HBV genomes containing the wild-type or mutated ISRE. In human hepatoma cells transfected with both genomes, viral RNA and replicative intermediates were reduced by IFNalpha treatment to a similar degree. Enhanced p48 expression increased IFNalpha-induced suppression of HBV RNA significantly from 75 +/- 22.5% to 46 +/- 9.8%, but this was independent of the integrity of the ISRE-like region. These data imply that p48 neither mediates the antiviral activity of IFNalpha against HBV nor down-regulates enhancer I activity by binding directly to the HBV ISRE-like region, but rather argue for an indirect role of p48.     

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.