cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.

The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

Catalytically critical nucleotide in domain 5 of a group II intron.

Domain 5 (D5) is a small hairpin structure within group II introns. A bimolecular assay system depends on binding by D5 to an intron substrate for self-splicing activity. In this study, mutations in D5 identify two among six nearly invariant nucleotides as being critical for 5' splice junction hydrolysis but unimportant for binding. A mutation at another site in D5 blocks binding. Thus, mutations can distinguish two D5 functions: substrate binding and catalysis. The secondary structure of D5 may resemble helix I formed by the U2 and U6 small nuclear RNAs in the eukaryotic spliceosome. Our results support a revision of the previously proposed correspondence between D5 and helix I on the basis of the critical trinucleotide 5'-AGC-3' present in both. We suggest that this trinucleotide plays a similar role in promoting the chemical reactions for both splicing systems.

Two helices plus a linker: a small model substrate for eukaryotic RNase P.

Using precursor tRNA molecules to study RNA-protein interactions, we have identified an RNA motif recognized by eukaryotic RNase P (EC 3.1.26.5). Analysis of circularly permuted precursors indicates that interruptions in the sugar-phosphate backbone are not tolerated in the acceptor stem, in the T stem-loop, or between residues A-9 and G-10. Prokaryotic RNase P will function with a minihelix consisting of the acceptor stem connected directly to the T stem-loop. Eukaryotic RNase P cannot use such a minimal substrate unless a linker sequence is added in the gap where the D stem and anticodon stem-loop were deleted.

Estudos sobre a organização genômica, evolução e expressão de microRNAs

Os microRNAs (miRNAs) são pequenos RNAs não codificadores de proteínas presentes na maioria dos eucariotos. Esses RNAs regulam a expressão gênica em nível pós-transcricional através do silenciamento de mRNAs-alvo que possuem sítios complementares às suas sequências, atuando em praticamente todos os processos celulares. Embora a estrutura e função dos miRNAs estejam bem caracterizadas, aspectos relacionados à sua organização genômica, evolução e atuação em doenças são tópicos que apresentam enormes lacunas. Nesta tese, utilizamos abordagens computacionais para investigar estes temas em três trabalhos. No primeiro, processamos e integramos um vasto volume de dados publicamente disponíveis referentes aos miRNAs e genes codificadores de proteínas para cinco espécies de vertebrados. Com isso, construimos uma ferramenta web que permite a fácil inspeção da organização genômica dos miRNAs em regiões inter e intragênicas, o acesso a dados de expressão de miRNAs e de genes codificadores de proteínas (classificados em genes hospedeiros e não hospedeiros de miRNAs), além de outras informações pertinentes. Verificamos que a ferramenta tem sido amplamente utilizada pela comunidade científica e acreditamos que ela possa facilitar a geração de hipóteses associadas à regulação dos miRNAs, principalmente quando estão inseridos em genes hospedeiros. No segundo estudo, buscamos compreender como o contexto genômico e a origem evolutiva dos genes hospedeiros influenciam a expressão e evolução dos miRNAs humanos. Nossos achados mostraram que os miRNAs intragênicos surgem preferencialmente em genes antigos (origem anterior à divergência de vertebrados). Observamos que os miRNAs inseridos em genes antigos têm maior abrangência de expressão do que os inseridos em genes novos. Surpreendentemente, miRNAs jovens localizados em genes antigos são expressos em um maior número de tecidos do que os intergênicos de mesma idade, sugerindo uma vantagem adaptativa inicial que pode estar relacionada com o controle da expressão dos genes hospedeiros, e como consequência, expondo-os a contextos celulares e conjuntos de alvos diversos. Na evolução a longo prazo, vimos que genes antigos conferem maior restrição nos padrões de expressão (menor divergência de expressão) para miRNAs intragênicos, quando comparados aos intergênicos. Também mostramos possíveis associações funcionais relacionadas ao contexto genômico, tais como o enriquecimento da expressão de miRNAs intergênicos em testículo e dos intragênicos em tecidos neurais. Propomos que o contexto genômico e a idade dos genes hospedeiros são fatores-chave para a evolução e expressão dos miRNAs. Por fim, buscamos estabelecer associações entre a expressão diferencial de miRNAs e a quimioresistência em câncer colorretal utilizando linhagens celulares sensíveis e resistentes às drogas 5-Fluoruracil e Oxaliplatina. Dentre os miRNAs identificados, o miR-342 apresentou níveis elevados de expressão nas linhagens sensíveis à Oxaliplatina. Com base na análise dos alvos preditos, detectamos uma significativa associação de miR-342 com a apoptose. A superexpressão de miR-342 na linhagem resistente SW620 evidenciou alterações na expressão de genes da via apoptótica, notavelmente a diminuição da expressão do fator de crescimento PDGFB, um alvo predito possivelmente sujeito à regulação direta pelo miR-342.

Optimized methods for extracting circulating small RNAs from long-term stored equine samples.

Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.

List of size fractionated eukaryotic plankton community samples and associated metadata (Database W1)

The present data set provides an Excel file in a zip archive. The file lists 334 samples of size fractionated eukaryotic plankton community with a suite of associated metadata (Database W1). Note that if most samples represented the piconano- (0.8-5 µm, 73 samples), nano- (5-20 µm, 74 samples), micro- (20-180 µm, 70 samples), and meso- (180-2000 µm, 76 samples) planktonic size fractions, some represented different organismal size-fractions: 0.2-3 µm (1 sample), 0.8-20 µm (6 samples), 0.8 µm - infinity (33 samples), and 3-20 µm (1 sample). The table contains the following fields: a unique sample sequence identifier; the sampling station identifier; the Tara Oceans sample identifier (TARA_xxxxxxxxxx); an INDSC accession number allowing to retrieve raw sequence data for the major nucleotide databases (short read archives at EBI, NCBI or DDBJ); the depth of sampling (Subsurface - SUR or Deep Chlorophyll Maximum - DCM); the targeted size range; the sequences template (either DNA or WGA/DNA if DNA extracted from the filters was Whole Genome Amplified); the latitude of the sampling event (decimal degrees); the longitude of the sampling event (decimal degrees); the time and date of the sampling event; the device used to collect the sample; the logsheet event corresponding to the sampling event ; the volume of water sampled (liters). Then follows information on the cleaning bioinformatics pipeline shown on Figure W2 of the supplementary litterature publication: the number of merged pairs present in the raw sequence file; the number of those sequences matching both primers; the number of sequences after quality-check filtering; the number of sequences after chimera removal; and finally the number of sequences after selecting only barcodes present in at least three copies in total and in at least two samples. Finally, are given for each sequence sample: the number of distinct sequences (metabarcodes); the number of OTUs; the average number of barcode per OTU; the Shannon diversity index based on barcodes for each sample (URL of W4 dataset in PANGAEA); and the Shannon diversity index based on each OTU (URL of W5 dataset in PANGAEA).

The sidereal messenger.

Vol. 1, no. 7 and no. 8 are paginated so that v. 1, no. 8 comes before v. 1, no. 7.

The Messenger of mathematics

Mode of access: Internet.

The Messenger of peace.

Publication suspended, 1895-1899.

The Messenger.

Mode of access: Internet.

The Boston weekly messenger, a journal of politics, agriculture, literature and miscellaneous intelligence.

Mode of access: Internet.

Poe and the Southern literary messenger,

Mode of access: Internet.

The Western literary messenger.

Published: Buffalo [N.Y.] : Thomas & Lathrop, Sept. 1854-