Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca++ stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2α. Since TCTP has been characterized as an antiapoptotic and Ca++-binding protein, we asked whether it is involved in protecting cells from Ca++-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2α phosphorylation.

Análise da expressão heteróloga de protéinas com domínios de ligação a RNA em Leishmania infantum

Os tripanossomatídeos possuem uma combinação não usual de mecanismos moleculares, e seus processos de regulação de expressão gênica ocorreram a nível pós-transcricional. Acredita-se que essa regulação envolva tanto o controle da estabilidade dos mRNAs, como sua tradução em proteínas, eventos em que atua a proteína de ligação à cauda poli-A (PABP - Poly-A Binding Protein), uma das principais proteínas de ligação a RNAs em eucariotos. Um grande número destas proteínas está presente nos tripanosomatídeos, se caracterizando por possuírem domínios típicos de ligação a RNA, como o domínio RRM (RNA Recognition Motif). Dentre estas se destacam as proteínas de ligação a sequências ricas em uridina (UBPs), que se mostraram capazes de interagir com homólogos de PABP. Outras proteínas hipotéticas contendo domínios de ligação a RNA foram identificadas em ensaios que buscavam parceiros diferenciais para os três homólogos de PABP de Leishmania. Este trabalho se propôs a contribuir na caracterização funcional das proteínas UBPs e das proteínas hipotéticas, através da otimização de sua expressão de forma heteróloga em L. infantum, fusionadas ao epítopo HA. Para isto, os genes codificantes dos três homólogos de UBPs, e de cinco outras proteínas de ligação a RNA que parecem interagir com PABPs, foram amplificados e clonados em vetor de expressão de Leishmania. As construções geradas foram transfectadas em L. infantum e a expressão de seis destas proteínas avaliada. Os resultados obtidos mostram uma variação no reconhecimento das proteínas geradas com anticorpos comerciais anti-HA, que parecem depender da sequência de aminoácidos da sua extremidade C-terminal. Diferenças significativas nos seus níveis de expressão também foram observadas. Entre os três homólogos de UBP, dois destes se mostraram mais abundantes enquanto que os três são representados por mais de uma banda, indicando possíveis modificações pós-traducionais

Cost733cat: a database of weather and circulation type classifications

A new database of weather and circulation type catalogs is presented comprising 17 automated classification methods and five subjective classifications. It was compiled within COST Action 733 "Harmonisation and Applications of Weather Type Classifications for European regions" in order to evaluate different methods for weather and circulation type classification. This paper gives a technical description of the included methods using a new conceptual categorization for classification methods reflecting the strategy for the definition of types. Methods using predefined types include manual and threshold based classifications while methods producing types derived from the input data include those based on eigenvector techniques, leader algorithms and optimization algorithms. In order to allow direct comparisons between the methods, the circulation input data and the methods' configuration were harmonized for producing a subset of standard catalogs of the automated methods. The harmonization includes the data source, the climatic parameters used, the classification period as well as the spatial domain and the number of types. Frequency based characteristics of the resulting catalogs are presented, including variation of class sizes, persistence, seasonal and inter-annual variability as well as trends of the annual frequency time series. The methodological concept of the classifications is partly reflected by these properties of the resulting catalogs. It is shown that the types of subjective classifications compared to automated methods show higher persistence, inter-annual variation and long-term trends. Among the automated classifications optimization methods show a tendency for longer persistence and higher seasonal variation. However, it is also concluded that the distance metric used and the data preprocessing play at least an equally important role for the properties of the resulting classification compared to the algorithm used for type definition and assignment.

Long run relationships between City office rents and the economy in the UK: creating a database for research

This paper sets out progress during the first eighteen months of doctoral research into the City of London office market. The overall aim of the research is to explore relationships between office rents and the economy in the UK over the last 150 years. To do this, a database of lettings has been created from which a long run index of City office rents can be constructed. With this index, it should then be possible to analyse trends in rents and relationships with their long run determinants. The focus of this paper is on the creation of the rent database. First, it considers the existing secondary sources of long run rental data for the UK. This highlights a lack of information for years prior to 1970 and the need for primary data collection if earlier periods are to be studied. The paper then discusses the selection of the City of London and of the time period chosen for research. After this, it describes how a dataset covering the period 1860-1960 has been assembled using the records of property companies active in the City office market. It is hoped that, if successful, this research will contribute to existing knowledge on the long run characteristics of commercial real estate. In particular, it should add a price dimension (rents) to the existing long run information on stock/supply and investment. Hence, it should enable a more complete picture of the development and performance of commercial real estate through time to be gained.

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3 '-S-phosphorothiolate linkages

3′-S-Phosphorothiolate (3′-SP) linkages have been incorporated into the DNA strand of both a DNA·RNA duplex and a DNA·DNA duplex. Thermal melting (Tm) studies established that this modification significantly stabilises the DNA·RNA duplex with an average increase in Tm of about 1.4 °C per modification. For two or three modifications, the increase in Tm was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on Tm. In contrast to the DNA·RNA duplex, the 3′-S-phosphorothiolate linkage destabilised the DNA·DNA duplex, irrespective of the arrangement of the 3′-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA·DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.

Synthesis of 3 '-S-phosphorothiolate oligonucleotides for their potential use in RNA interference

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3′-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5′ → 3′ synthesis is investigated, with initial solution-phase testing proving successful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced.

The Genomic Threading Database: a comprehensive resource for structural annotations of the genomes from key organisms

Currently, the Genomic Threading Database (GTD) contains structural assignments for the proteins encoded within the genomes of nine eukaryotes and 101 prokaryotes. Structural annotations are carried out using a modified version of GenTHREADER, a reliable fold recognition method. The Gen THREADER annotation jobs are distributed across multiple clusters of processors using grid technology and the predictions are deposited in a relational database accessible via a web interface at http://bioinf.cs.ucl.ac.uk/GTD. Using this system, up to 84% of proteins encoded within a genome can be confidently assigned to known folds with 72% of the residues aligned. On average in the GTD, 64% of proteins encoded within a genome are confidently assigned to known folds and 58% of the residues are aligned to structures.

The Genomic Threading Database

The Genomic Threading Database currently contains structural annotations for the genomes of over 100 recently sequenced organisms. Annotations are carried out by using our modified GenTHREADER software and through implementing grid technology.

The role of the international cocoa germplasm database and the international cocoa quarantine centre in information management and distribution of cocoa genetic resources

A range of physiological parameters (canopy light transmission, canopy shape, leaf size, flowering and flushing intensity) were measured from the International Clone Trial, typically over the course of two years. Data were collected from six locations, these being: Brazil, Ecuador, Trinidad, Venezuela, Côte d’Ivoire and Ghana. Canopy shape varied significantly between clones, although it showed little variation between locations. Genotypic variation in leaf size was differentially affected by the growth location; such differences appeared to underlie a genotype by environment interaction in relation to canopy light transmission. Flushing data were recorded at monthly intervals over the course of a year. Within each location, a significant interaction was observed between genotype and time of year, suggesting that some genotypes respond to a greater extent than others to environmental stimuli. A similar interaction was observed for flowering data, where significant correlations were found between flowering intensity and temperature in Brazil and flowering intensity and rainfall in Côte d’Ivoire. The results demonstrate the need for local evaluation of cocoa clones and also suggest that the management practices for particular planting material may need to be fine-tuned to the location in which they are cultivated.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana ‘Landsberg erecta’ plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant’s resilience to fluctuating relative humidity.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3; 1,4)-beta-D-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Breast, colorectal, and prostate cancer risk in the European Prospective Investigation into Cancer and Nutrition–Norfolk in relation to phytoestrogen intake derived from an improved database

Background: The characterization of phytoestrogen intake and cancer risk has been hindered by the absence of accurate dietary phytoestrogen values. Objective: We examined the risk of breast, colorectal, and prostate cancers relative to phytoestrogen intake on the basis of a comprehensive database. Design: Demographic and anthropometric characteristics, a medical history, and 7-d records of diet were collected prospectively from participants (aged 40–79 y) in the European Prospective Investigation into Cancer and Nutrition–Norfolk (EPIC-Norfolk). Five hundred nine food items were analyzed by liquid chromatography–mass spectrometry/mass spectrometry, and 13C3-labeled internal standards were analyzed for isoflavones (genistein, daidzein, glycitein, biochanin A, and formononetin), lignans (secoisolariciresinol and matairesinol), and enterolignans from gut microbial metabolism in animal food sources (equol and enterolactone). From the direct analysis, values for 10,708 foods were calculated. Odds ratios (ORs) for breast (244 cases, 941 controls), colorectal (221 cases, 886 controls), and prostate (204 cases, 812 controls) cancers were calculated relative to phytoestrogen intake. Results: Phytoestrogen intake was not associated with breast cancer among women or colorectal cancer among men. Among women, colorectal cancer risk was inversely associated with enterolactone (OR: 0.33; 95% CI: 0.14, 0.74) and total enterolignans (OR: 0.32; 95% CI: 0.13, 0.79), with a positive trend detected for secoisolariciresinol (OR: 1.60; 95% CI: 0.96, 2.69). A positive trend between enterolignan intake and prostate cancer risk (OR: 1.27; 95% CI: 0.97, 1.66) was attenuated after adjustment for dairy intake (OR: 1.19; 95% CI: 0.77, 1.82). Conclusion: Dietary phytoestrogens may contribute to the risk of colorectal cancer among women and prostate cancer among men.