Investigação eletroquímica e aplicação de eletrodos de carbono vítreo modificados por filmes de poli-L-lisina na determinação de antioxidantes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biogênese mitocondrial e marcadores de estresse oxidativo em ratos treinados e suplementados com L-Arginina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

eIF5A has a function in the cotranslational translocation of proteins into the ER

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

eIF5A and EF-P: two unique translation factors are now traveling the same road

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeitos da suplementação da quercetina sobre o rendimento físico em esteira ergométrica e indicadores metabólicos do exercício exaustivo em atletas de futebol

Pós-graduação em Patologia - FMB

Análise da expressão gênica de pequenos RNAs derivados de tRNAs (tRFs) em plantas

Small non coding RNAs emerged as important characters in several biology aspects. Among then, the most studied are microRNAs (miRNAs) and short interfering RNAs (siRNAs), that regulate their target gene post-transcriptionally in plants, animals and RNAi pathway intermediates, respectively. Both of classes have similar biogenesis being processed by Dicer enzymes and subsequent association with Argonaute enzymes. In plants, miRNAs and siRNAs have important functions in development, genome integrity and biotic and abiotic stress responses. The advances in high-throughtput sequencing and in silico analisys provide the uncover of new small non coding RNAs classes, many of them with unknown functions and biogenesis. tRNA derived small RNAs (tRFs) are a small non coding RNA class, that have as precursor a tRNA molecule. These were uncovers in the last decade in many organisms and, recently, in plants. Recent works detected tRFs from different sizes, with different source portions of the mature tRNA molecule (5’ end; 3’ end, anti-codon loop) and some from the tRNA precursor (pre-tRNA), suggesting that may be a novel class of small RNA and not random degradation products. Works in humans showed that some tRFs are processed by the Dicer enzymes, have association with the Argonaute enzymes and cell differentiation, tumor appearance and gene silencing related functions. Works in Arabidopsis and pumpkin (Cucurbita maxima) showed, respectively, that the tRFs have nutritional stress response possible functions and long distance signaling function between source and drain tissues, and may affect the translation. The tRFs biogenesis in plants are, until now an unknown, absence information about it in the literature and its possible biological functions are few studied yet, making then interesting target for studies among the small non coding RNAs in plants

Production of active recombinant eIF5A: reconstitution in E. coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.

Estudo do papel da proteína eIF5A na elongação da tradução em S. cerevisiae: análise da relação funcional com o trna de alanina e sua participação na síntese proteica geral da célula

Pós-graduação em Biotecnologia - IQ

Transcriptome response signatures associated with the overexpression of a mitochondrial uncoupling protein (AtUCP1) in tobacco

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The putative Leishmania telomerase RNA (leishTER) undergoes trans-splicing and contains a conserved template sequence

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructure of the Interface Between Stages of Eimeria funduli/i> (Apicomplexa) and Hepatocytes of the Longnose Killifish, Fundulus similis

The interface between stages of Eimeria funduli and hepatocytes of the experimentally infected killifish Fundulus similis was studied ultrastructurally. Parasitophorous vacuoles (PV's) in which meronts, macrogamonts, and microgamonts developed were lined by an inner, smooth membrane and an outer, ribosome-studded membrane. The outer membrane bordered on the cytoplasm of the host cell, whereas the inner one limited the PV. The origins of these membranes have not been determined with certainty, but images were observed in which both membranes appeared to be continuous with the outer nuclear membrane of the host cell. Furthermore, the outer PV membrane was continuous with membranes of rough endoplasmic reticulum in the host cell. For stages which were rapidly growing or differentiating, the inner membrane blebbed into the PV. Blebbing ceased and ribosomes detached from the outer membrane after maturation of the meront or fertilization of the macrogamont. Blebbing appears to be a mechanism by which nutrients transfer from the host to the parasite. During sporogony, the inner PV membrane acquired a thin layer of electron dense material, but otherwise membranes lining the PV remained intact. The two PV membranes, probably together with dense material of parasitic origin lining the inner membrane, appear to serve as the oocyst wall enclosing the sporocysts until they are released in the intermediate host.

Calorie restriction increases cerebral mitochondrial respiratory capacity in a NO center dot-mediated mechanism: Impact on neuronal survival

Calorie restriction (CR) enhances animal life span and prevents age-related diseases, including neurological decline. Recent evidence suggests that a mechanism involved in CR-induced life-span extension is NO-stimulated mitochondrial biogenesis. We examine here the effects of CR on brain mitochondrial content. CR increased eNOS and nNOS and the content of mitochondria] proteins (cytochrome c oxidase, citrate synthase, and mitofusin) in the brain. Furthermore, we established an in vitro system to study the neurological effects of CR using serum extracted from animals on this diet. In cultured neurons, CR serum enhanced nNOS expression and increased levels of nitrite (a NO product). CR serum also enhanced the levels of cytochrome c oxidase and increased citrate synthase activity and respiratory rates in neurons. CR serum effects were inhibited by L-NAME and mimicked by the NO donor SNAP. Furthermore, both CR sera and SNAP were capable of improving neuronal survival. Overall, our results indicate that CR increases mitochondrial biogenesis in a NO-mediated manner, resulting in enhanced reserve respiratory capacity and improved survival in neurons. (C) 2012 Elsevier Inc. All rights reserved.

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc.

Serum from Calorie-Restricted Rats Activates Vascular Cell eNOS through Enhanced Insulin Signaling Mediated by Adiponectin

eNOS activation resulting in mitochondrial biogenesis is believed to play a central role in life span extension promoted by calorie restriction (CR). We investigated the mechanism of this activation by treating vascular cells with serum from CR rats and found increased Akt and eNOS phosphorylation, in addition to enhanced nitrite release. Inhibiting Akt phosphorylation or immunoprecipitating adiponectin (found in high quantities in CR serum) completely prevented the increment in nitrite release and eNOS activation. Overall, we demonstrate that adiponectin in the serum from CR animals increases NO center dot signaling by activating the insulin pathway. These results suggest this hormone may be a determinant regulator of the beneficial effects of CR.

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.