RNAspace.org: An integrated environment for the prediction, annotation, and analysis of ncRNA

The annotation of noncoding RNA genes remains a major bottleneck in genome sequencing projects. Most genome sequences released today still come with sets of tRNAs and rRNAs as the only annotated RNA elements, ignoring hundreds of other RNA families. We have developed a web environment that is dedicated to noncoding RNA (ncRNA) prediction, annotation, and analysis and allows users to run a variety of tools in an integrated and flexible manner. This environment offers complementary ncRNA gene finders and a set of tools for the comparison, visualization, editing, and export of ncRNA candidates. Predictions can be filtered according to a large set of characteristics. Based on this environment, we created a public website located at http://RNAspace.org. It accepts genomic sequences up to 5 Mb, which permits for an online annotation of a complete bacterial genome or a small eukaryotic chromosome. The project is hosted as a Source Forge project (http://rnaspace.sourceforge.net/).     

Habitat Loss,Not Fragmentation,Drives Occurrence Patterns of Canada Lynx at the Southern Range Periphery

Peripheral populations often experience more extreme environmental conditions than those in the centre of a species'' range. Such extreme conditions include habitat loss, defined as a reduction in the amount of suitable habitat, as well as habitat fragmentation, which involves the breaking apart of habitat independent of habitat loss. The ‘threshold hypothesis’ predicts that organisms will be more affected by habitat fragmentation when the amount of habitat on the landscape is scarce (i.e., less than 30%) than when habitat is abundant, implying that habitat fragmentation may compound habitat loss through changes in patch size and configuration. Alternatively, the ‘flexibility hypothesis’ predicts that individuals may respond to increased habitat disturbance by altering their selection patterns and thereby reducing sensitivity to habitat loss and fragmentation. While the range of Canada lynx (Lynx canadensis) has contracted during recent decades, the relative importance of habitat loss and habitat fragmentation on this phenomenon is poorly understood. We used a habitat suitability model for lynx to identify suitable land cover in Ontario, and contrasted occupancy patterns across landscapes differing in cover, to test the ‘threshold hypothesis’ and ‘flexibility hypothesis’. When suitable land cover was widely available, lynx avoided areas with less than 30% habitat and were unaffected by habitat fragmentation. However, on landscapes with minimal suitable land cover, lynx occurrence was not related to either habitat loss or habitat fragmentation, indicating support for the ‘flexibility hypothesis’. We conclude that lynx are broadly affected by habitat loss, and not specifically by habitat fragmentation, although occurrence patterns are flexible and dependent on landscape condition. We suggest that lynx may alter their habitat selection patterns depending on local conditions, thereby reducing their sensitivity to anthropogenically-driven habitat alteration.     

Reconstruction of environment and history of plant use during the late Mesolithic (Ertebølle culture) at the inland settlement of Bökeberg III,southern Sweden

Pollen, plant macrofossils and charcoal were analysed from a lake-sediment sequence, including a refuse layer, from the Late Mesolithic settlement at Bökeberg III, southern Sweden. The chronology was established by means of AMS-dated plant macroremains. The results of the biostratigraphical studies indicate two settlement phases (A and B), at ca. 6650-6400 B.P. (5560-5320 cal. B.C.) and ca. 6150-5800 B.P. (5200-4680 cal. B.C.), respectively. The two settlement phases are associated with periods of low lake-level contemporaneous with the second major period of low lake levels during the Holocene in southern Sweden, and thus with a period of generally drier climate. The pollen analytical data suggest only minor human impact on the local vegetation during the two settlement phases. Three elm declines at ca. (1) 6200 B.P. (5200-5100 cal. B.C.), (2) 5450 B.P. (4340 cal. B.C.), and (3) 5150 B.P. (3980 cal. B.C.) are discussed. Elm decline 3 is synchronous with the classical north-west European elm decline. Elm declines 2 and 3 may be due to outbreaks of elm disease rather than to strong human impact or climate change. The charcoal analyses show that wood of a wide range of species was collected for fuel or other purposes. During phase A, plants used included acorns, hazelnuts and, possibly, Cornus sanguinea, and also Prunus spinosa, Sorbus aucuparia and Rubus idaeus. There is convincing evidence that Cladium mariscus was used for thatching. The second occupation phase, B, is characterised by the use of hazelnuts for food. The possible use of several other identified species is discussed.     

Persisting Neural and Endocrine Modifications Induced by a Single Fat Meal

1. High-fat diets, modify the neuroendocrine response and, when prolonged, result in positive energy balance and obesity. Little is known about the effects of fat on the mechanisms operating in the initial steps of the neural and endocrine disturbances. 2. The studies reported here were designed to access the impact of the consumption of a single exclusively animal fat meal (lard), 24 h following its ingestion a) on the response of the hypothalamic serotonergic system to a standard laboratory chow meal and b) on the circulating levels of glucose, insulin, and leptin. The release of serotonin in the extracellular medial hypothalamic space (including the paraventricular-PVN and ventromedian-VMH nuclei) was determined using electrochemical detection following HPLC in samples obtained in vivo by microdialysis, in nonanesthetized adult male Wistar rats. 3. A lard meal resulted in decreased hypothalamic serotonin release postprandially and attenuated (24 h later) the hypothalamic serotonin response that normally follows a balanced meal. 4. In permanently catheterized rats, postprandial glucose and insulin levels measured in samples obtained in vivo, were either not, or only slightly, modified after a lard meal, whereas plasma leptin levels were increased. Interestingly, 24 h after a meal, insulin and leptin levels were increased in those animals eating a fat meal compared with those eating chow. Next-day glucose levels remained identical after the absorption either of a chow, or a lard meal. 5. The changes induced by the fat meal on peripheral and central regulators of energy and glucose homeostasis represent either adaptive mechanisms or early alterations that could render the organism vulnerable to further insults.     

DNA damage measured by the single-cell gel electrophoresis (Comet) assay in mammals fed with mussels contaminated by the 'Erika' oil-spill

This research aimed to estimate potential genotoxicity for consumers resulting from the ingestion of seafood contaminated with polycyclic aromatic hydrocarbons (PAHs) released into the marine environment after the 'Erika' shipwreck along the coasts of south Brittany, in France. Mussels (Mytilus sp.) collected from sites on the Atlantic coast that were affected by the oil slick in various degrees, were used to feed rats daily for 2 and 4 weeks. DNA damage was measured by use of the Comet assay in the liver, bone marrow and blood of rats receiving food contaminated with 312 microg of 16PAHs/kg dry weight (d.w.) equivalent to 33.8 microg TEQs (toxic equivalent quantities to benzo(a)pyrene (BaP))/kg d.w. mussels, 569 microg/kg d.w. (83.6 microg TEQs/kg) and 870 microg/kg d.w. (180.7 microg TEQs/kg). A dose-effect-time relationship was observed between the amount of DNA damage in the liver and bone marrow of the rats and the PAH contamination level of the mussels. Genotoxicity increased during the period between 15 and 30 days in rats that received food at the highest two PAH levels. On the other hand, no significant change in liver and bone marrow of rats fed with mussels containing 33.8 microg TEQs/kg d.w. was recorded at 30 days compared with 15 days, indicating efficient DNA repair capacity at low levels of exposure. No signs of genotoxicity were found in peripheral blood. Globally, the observed effects were rather moderate. These results show that oil-contaminated food caused DNA damage in predators, and underline the bioavailability to consumers of pollutants in mussels contaminated with fuel oil. The usefulness of the Comet assay as a sensitive tool in biomonitoring studies analyzing responses of PAH transfer through food webs was also confirmed.     

hScrib interacts with ZO-2 at the cell-cell junctions of epithelial cells

In Drosophila, the tumor suppressor Scribble is localized at the septate junctions of epithelial cells. Its mammalian homologue, hScrib, is a basolateral protein likely associated to proteins of the cell-cell junctions. We report the direct interaction between hScrib and ZO-2, a junction-associated protein. This interaction relies on two PDZ domains of hScrib and on the C-terminal motif of ZO-2. Both proteins localise at cell-cell junctions of epithelial cells. A point mutation in the LRR of hScrib delocalises the protein from the plasma membrane and abrogates the interaction with ZO-2 but not with betaPIX. Tyrosine phosphorylation of hScrib does not impair the interaction with ZO-2. We show a direct link between two junctional proteins that are down-regulated during cancer progression.     

A novel mutation in the mitochondrial tRNA Asn gene associated with a lethal disease

We describe a lethal mitochondrial disease in a 10-month-old child who presented with encephalomyopathy. Histochemical and electron microscopy examinations of skeletal muscle biopsy revealed abnormal mitochondria associated with a combined deficiency of complexes I and IV. After excluding mitochondrial DNA deletions and depletion, direct sequencing was used to screen for mutation in all transfer RNA (tRNA) genes. A T-to-C substitution at position 5693 in the tRNA(Asn) gene was found in blood and muscle. Microdissection of muscle biopsy and its analysis revealed the highest level of this mutation in cytochrome c oxidase (COX)-negative fibres. We suggest that this novel mutation would affect the anticodon loop structure of the tRNA(Asn) and cause a fatal mitochondrial disease.     

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model. Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells. Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied. For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed. These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs.     

A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity

The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.