The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11-59K protein

U11 and U12 interact cooperatively with the 5' splice site and branch site of pre-mRNA as a stable preformed di-snRNP complex, thereby bridging the 5' and 3' ends of the intron within the U12-dependent prespliceosome. To identify proteins contributing to di-snRNP formation and intron bridging, we investigated protein-protein and protein-RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12-65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C-terminal RRM and the U11-associated 59K protein via its N-terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3' half of U12 forms an extended stem-loop with a highly conserved seven-nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12-dependent, but not U2-dependent, pre-mRNA splicing. Taken together, these data suggest that U11/U12-65K and U11-59K contribute to di-snRNP formation and intron bridging in the minor prespliceosome.     

Association of ebola virus matrix protein VP40 with microtubules

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.     

Munich information center for protein sequences plant genome resources: a framework for integrative and comparative analyses 1(W)

With several plant genomes sequenced, the power of comparative genome analysis can now be applied. However, genome-scale cross-species analyses are limited by the effort for data integration. To develop an integrated cross-species plant genome resource, we maintain comprehensive databases for model plant genomes, including Arabidopsis (Arabidopsis thaliana), maize (Zea mays), Medicago truncatula, and rice (Oryza sativa). Integration of data and resources is emphasized, both in house as well as with external partners and databases. Manual curation and state-of-the-art bioinformatic analysis are combined to achieve quality data. Easy access to the data is provided through Web interfaces and visualization tools, bulk downloads, and Web services for application-level access. This allows a consistent view of the model plant genomes for comparative and evolutionary studies, the transfer of knowledge between species, and the integration with functional genomics data.     

A -1 frameshift in the HIV-1 env gene is enhanced by arginine deficiency via a hungry codon mechanism

Ribosomal frameshifting is used by various organisms to maximize protein coding potential of genomic sequences. It is commonly exploited by RNA viruses to overcome the constraint of their limited genome size. Frameshifting requires specific RNA structural features, such as a suitable heptanucleotide “slippery” sequence and an RNA pseudoknot. Previous genomic analysis of HIV-1 indicated the potential for several hidden genes encoded through frameshifting; one of these, overlapping the envelope gene, has an RNA pseudoknot just downstream from a slippery sequence, AAAAAGA that features an adenine quadruplet prior to a potential hungry arginine codon (AGA). This env-frameshift (env-fs) gene has been shown to encode a truncated glutathione peroxidase homologue, with both antioxidant and anti-apoptotic activities in transfected cells. Using a dual reporter cell-based frameshift assay, we demonstrate that the env-fs frameshift sequence is active in vitro. Furthermore, in arginine deficient media, env-fs frameshifting increased over 100% (p < 0.005), consistent with the hypothesized hungry codon mechanism. As a response to arginine deficiency, increased expression of the antioxidant viral GPx gene (env-fs) by upregulation of frameshifting could be protective to HIV-infected cells, as a countermeasure to the increased oxidative stress induced by arginine deficiency (because NO is a known scavenger of hydroxyl radical).     

Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver

Application of recently developed gene expression techniques using microarrays in toxicological studies (toxicogenomics) facilitate the interpretation of a toxic compound's mode of action and may also allow the prediction of selected toxic effects based on gene expression changes. In order to test this hypothesis, we investigated whether carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate characteristic sets of genes in a short term in vivo study and whether these deregulated genes represent defined biological pathways. Male Wistar rats were dosed with the four nongenotoxic hepatocarcinogens methapyrilene (MPy, 60 mg/kg/day), diethylstilbestrol (DES, 10 mg/kg/day), Wy-14643 (Wy, 60 mg/kg/day), and piperonylbutoxide (PBO, 1200 mg/kg/day). After 1, 3, 7, and 14 days, the livers were taken for histopathological evaluation and for analysis of the gene expression profiles on Affymetrix RG_U34A arrays. The expression profile of the four nongenotoxic carcinogens were compared to the profiles of the four genotoxic carcinogens 2-nitrofluorene (2-NF), dimethylnitrosamine (DMN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and aflatoxin B1 (AB1) from a similar study reported previously. By using statistical and clustering tools characteristically deregulated genes were extracted and functionally classified. Distinct cellular pathways were affected by the nongenotoxic carcinogens compared to the genotoxic carcinogens which at least partly correlated with the two-stage model of carcinogenesis. Characteristic to genotoxic carcinogens were a DNA damage response and the activation of proliferative and survival signaling. Nongenotoxic carcinogens showed responses to oxidative DNA or protein damage, as well as cell cycle progression and signs of regeneration. Many of the gene alterations found with the nongenotoxic carcinogens imply compound-specific mechanisms. Although neither a single gene nor a single pathway will be sufficient to discriminate the two classes of carcinogens, it became evident that combinations of pathway-associated gene expression profiles may be used to predict a genotoxic or nongenotoxic carcinogenic potential of a compound in short-term studies.     

Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi)

Carotenoid biosynthesis was examined in a phylloplane yeast identified by ITS, 18S and 28S rDNA analysis as a Dioszegia sp. close to D. takashimae. In well-aerated flask or fermentor cultures, this strain produced essentially a single pigment confirmed as the xanthophyll plectaniaxanthin by NMR analysis, at concentrations of 103-175 microgg(-1) biomass dry weight. Detailed studies showed increases in plectaniaxanthin concentrations in the presence of 5 mM hydrogen peroxide (1.8-fold), 50 and 100 microM duroquinone (3.1- and 3.7-fold, respectively), and 2% ethanol (4.9-fold). Whereas oxidative stress is known to enhance the biosynthesis of torularhodin or astaxanthin in other red yeasts where they are associated with an antioxidant function, this is the first report implicating plectaniaxanthin in a similar role. At reduced aeration, biosynthesis of plectaniaxanthin was suppressed and its putative precursor gamma-carotene accumulated. The carotenoid cyclase inhibitor nicotine (5-20 mM) inhibited plectaniaxanthin formation, with lycopene accumulating stoichiometrically. Hydroxy groups at C-1' and C-2' therefore seem to be introduced late in plectaniaxanthin biosynthesis, following cyclization of the beta-ionone ring.     

Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.     

Disruption of Sertoli-germ cell adhesion function in the seminiferous epithelium of the rat testis can be limited to adherens junctions without affecting the blood-testis barrier integrity: an in vivo study using an androgen suppression model

During spermatogenesis, both adherens junctions (AJ) (such as ectoplasmic specialization (ES), a testis-specific AJ type at the Sertoli cell-spermatid interface (apical ES) or Sertoli-Sertoli cell interface (basal ES) in the apical compartment and BTB, respectively) and tight junctions (TJ) undergo extensive restructuring to permit germ cells to move across the blood-testis barrier (BTB) as well as the seminiferous epithelium from the basal compartment to the luminal edge to permit fully developed spermatids (spermatozoa) to be sloughed at spermiation. However, the integrity of the BTB cannot be compromised throughout spermatogenesis so that postmeiotic germ cell-specific antigens can be sequestered from the systemic circulation at all times. We thus hypothesize that AJ disruption in the seminiferous epithelium unlike other epithelia, can occur without compromising the BTB-barrier, even though these junctions, namely TJ and basal ES, co-exist side-by-side in the BTB. Using an intratesticular androgen suppression-induced germ cell loss model, we have shown that the disruption of AJs indeed was limited to the Sertoli-germ cell interface without perturbing the BTB. The testis apparently is using a unique physiological mechanism to induce the production of both TJ- and AJ-integral membrane proteins and their associated adaptors to maintain BTB integrity yet permitting a transient loss of cell adhesion function by dissociating N-cadherin from beta-catenin at the apical and basal ES. The enhanced production of TJ proteins, such as occludin and ZO-1, at the BTB site can supersede the transient loss of cadherin-catenin function at the basal ES. This thus allows germ cell depletion from the epithelium without compromising BTB integrity. It is plausible that the testis is using this novel mechanism to facilitate the movement of preleptotene and leptotene spermatocytes across the BTB at late stage VIII through early stage IX of the epithelial cycle in the rat while maintaining the BTB immunological barrier function.