eIF5A has a function in the elongation step of translation in yeast

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

eIF5A interacts functionally with eEF2

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

Molecular characterization of Blastocrithidia culicis L17 ribosomal protein

Blastocrithidia culicis is a protozoan of the family Trypanosomatidae. It is a parasite of insects, but the presence of bacteriumlike endosymbionts in its cytoplasm led some investigators to study this protozoan. This trypanosomatid does not infect humans and although it is phylogenetically distant from Trypanosoma cruzi, it presents many morphological characteristics, which are similar. In previous studies our group showed the presence of a L27 ribosomal protein in T cruzi (named TcrL27) using a RT-PCR, which also resulted in the cloning, sequencing and expression of an unexpected ribosomal protein, L17, in Blastocrithidia culicis (BcL17). In this paper, Western blot analysis demonstrated that the anti-BcL17 antibody recognizes the presence of the same ribosomal protein either in Blastochritidia culicis and T. cruzi nuclear extracts. Besides, two similar bands (40 and 47 kDa) appeared also in T. cruzi isolated ribosomal proteins and B. culicis nuclear extract corroborating with the findings showed in the phylogenetic reconstruction. With respect to their localization within the ribosome, both the L17 and L27 ribosomal proteins appear to belong to the peptidyl-transferase site, and are therefore part of the key step in protein synthesis. Both ribosomal proteins bind spiramycin derivatives, being therefore compounds of the macrolides connection sites in the ribosome. These findings would open a possibility to better evaluate this issue.

Is there a role for eIF5A in translation?

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archaebacteria to mammals. This factor is the only cellular protein that undergoes an essential posttranslational modification dependent on the polyamine spermidine, called hypusination. This review focuses on the functional characterization of eIF5A. Although this protein was originally identified as a translation initiation factor, subsequent studies did not support a role for eIF5A in general translation initiation. eIF5A has also been implicated in nuclear export of HIV-1 Rev and mRNA decay, but these findings are controversial in the literature and may reflect secondary effects of eIF-5A function. Next, the involvement of eIF5A and hypusination in the control of the cell cycle and proliferation in various organisms is reviewed. Finally, recent evidence in favor of reconsidering the role of eIF5A as a translation factor is discussed. Future studies may reveal the specific mechanism by which eIF5A affects protein synthesis.

eIF5A binds to translational machinery components and affects translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archebacteria to mammals. Although this protein was originally identified as a translation initiation factor, subsequent experiments did not support a role for eIF5A in general translation. In this work, we demonstrate that eIF-5A interacts with structural components of the 80S ribosome, as well as with the translation elongation factor 2 (eEF2). Moreover, eIF5A is further shown to cofractionate with monosomes in a translation-dependent manner. Finally, eIF5A mutants show altered polysome profiles and are sensitive to translation inhibitors. Our results re-establish a function for eIF5A in translation and suggest a role for this factor in translation elongation instead of translation initiation. (c) 2006 Elsevier B.V. All rights reserved.

Metabolismo mitocondrial, radicais livres e envelhecimento

O envelhecimento pode estar associado ao maior acúmulo de lesões celulares decorrentes das espécies reativas do oxigênio e do nitrogênio derivadas do metabolismo mitocondrial. Com a progressão da idade, há acúmulo de proteínas, lipídeos, carboidratos e DNA oxidados em relação a organismos jovens, de acordo com a teoria dos radicais livres. Entretanto, nem sempre os idosos ou animais envelhecidos apresentam maior estresse oxidativo que os jovens. Este artigo discute o paradoxo da teoria dos radicais livres de acordo com a teoria da biogênese da manutenção adequada do metabolismo mitocondrial. Diversos fatores podem contribuir para a redução do estresse oxidativo, como a hormese induzida pela prática regular de exercícios físicos, a restrição calórica, a ingestão de antioxidantes nutricionais e o aumento da produção de antioxidantes celulares que. em conjunto, estes promovem a expressão das sirtuínas e das proteínas do choque térmico, protegendo a integridade e funcionalidade mitocondriais, reduzindo o estresse oxidativo e nitrosativo, o que está associado à redução do envelhecimento e aumento da longevidade.

Antibacterial effects of Brazilian and Bulgarian propolis and synergistic effects with antibiotics acting on the bacterial DNA and folic acid

Propolis is a honeybee product that has been used since ancient times because of its therapeutic effects. It can be used in the development of alternative therapies for the treatment of many diseases, and because propolis shows antibacterial action, this work was carried out in order to investigate a possible synergism between propolis and antibiotics acting on DNA (ciprofloxacin and norfloxacin) and on the metabolism (cotrimoxazole) against Salmonella Typhi. Propolis samples collected in Brazil and Bulgaria were compared in these assays, and the synergism was investigated by using 1/2 and 1/4 of the minimal inhibitory concentration for propolis and antibiotics, evaluating the number of viable cells according to the incubation time. Brazilian and Bulgarian propolis showed antibacterial activity, but no synergistic effects with the three tested antibiotics were seen. Previous works by our laboratory have revealed that propolis has synergistic effects with antibiotics, acting on the bacterial wall and ribosome, but it does not seem to interact with antibiotics acting on DNA or folic acid, and only a bacteriostatic action was seen in these assay conditions.

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

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

Molecular evidence of ribosomal DNA (rDNA) amplification of a minichromosome derived from Drosophila arizonae in D. mulleri-D. arizonae hybrid males

We present data supporting cytogenetic observations on nucleolar dominance in hybrids between Drosophila arizonae and D. mulleri. Our approach was to compare the rDNA restriction patterns between the parental species and their hybrids. Results demonstrated that the minichromosome attached to the nucleolus in hybrid males is derived from D. arizonae.

Fluorescence in situ hybridization with rDNA probes on chromosomes of two nucleolus organizer region phenotypes of a species of Eigenmannia (Pisces, Gymnotoidei, Sternopygidae)

Nucleolus organizer regions (NORs) were analysed in two related and geographically close populations of Eigenmannia sp.1 (Pisces, Gymnotoidei, Sternopygidae) using silver staining and fluorescence in situ hybridization (FISH). The two populations differed in their AS-NOR phenotypes, displaying fixed differences in the NOR-bearing chromosome pairs. FISH with rDNA probes showed that these differences were due to the location of rDNA cistrons. This finding, showing fixed NOR differences between two populations belonging to the same species in a connected river system, is highly significant in terms of evolutionary change, possibly indicating an initial step of genetic differentiation. This result also has important implications from the cytosystematic point of view, as NORs usually have a very constant karyotypic location in fish species and have been used as species-specific chromosome markers.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

The thrombocyte aggregation process in the turtle Phrynopys hilarii (Chelonia). An ultrastructural study

This study investigates the thrombocyte aggregation process in the South American fresh water turtle (Phrynopys hilarii) using electron microscopy. Blood was taken from surgically exposed lateral neck vessels often turtles Phrynopys hilarii during the spring and summer seasons, when the mean temperature is 37°C. Blood samples were fixed with Karnovsky solution for processing by transmission electron microscopy. The turtle thrombocytes were spindle-shaped with lobulated nuclei. Prominent vesicles and canaliculi were found throughout the cytoplasm. The cytoplasm organelles showed an agranular endoplasmatic reticulum, Golgi complex near the centrioles and scattered free ribosomes. These cells are similar to bird thrombocytes but distinct from fish and frog thrombocytes. Blood clotting time was 5 min ± 30 sec measured by the Lee and White method. Structural alterations resulting from the aggregation process occurred after activation. Thrombocytes developed numerous filopodial projections, an increased number of vacuoles and changed from spindle to spherical shape. P. hilarii thrombocytes have different morphologic characteristics compared to other non-mammalian vertebrate cells. These cells can participate in the aggregation process, as observed in birds.

Análise filogenética de Phytophthora capsici Leonian do estado de São Paulo baseada na seqüência de nucleotídeos da região ITS-5.8S rDNA

Phylogenetic analysis through morphological and cultural traits is considered inconsistent and hard to be scientifically accepted once there is no way to establish the direction of evolution on morphological traits. Molecular markers are suitable for phylogenetic analysis. The sequencing of ITS1 and ITS2 (internal transcribed spacers) regions and of 5.8S gene from ribosomal DNA were used to estimate the genetic variation and distance of 10 Phytophthora capsici isolates. The amount of genetic variation amongst isolated P. capsici from distinct regions of São Paulo State was 0.1 to 1.6 and 0.1 to 1.1% at ITS1 and ITS2, respectively, and a phylogenetic distance of about 78.5% between P. capsici and Phytophthora spp. was observed.

Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil

Ehrlichia canis has a worldwide distribution, but clinical manifestations may vary geographically. We selected 129 dogs to determine prevalence of ehrlichiosis in dogs with anemia, thrombocytopenia, or ticks presented to a Veterinary Teaching Hospital in South Brazil. Of the 129 dogs, 68 carried the brown dog tick (Rhipicephalus sanguineus), 61 had thrombocytopenia (platelet count <150,000/μl), and 19 had anemia (PCV < 22%). Twenty dogs fulfilled more than one inclusion criteria. Ehrlichiosis was diagnosed by positive amplification of ehrlichial DNA by PCR using primers ECC and ECB that amplify a sequence of the 16S rRNA gene. Presence of E. canis was confirmed by cleavage of the amplified DNA using endonucleases HaeIII and AvaI. Fourteen of 68 (21%) dogs with ticks had ehrlichiosis, whereas 12 of 61 (20%) dogs presented with thrombocytopenia and 4 of 19 (21%) anemic dogs had ehrlichiosis. Similar results were obtained in dogs with thrombocytopenia and anemia (one of eight positive) and in dogs with thrombocytopenia and ticks (two of seven positive). All four dogs with anemia and ticks, and the dog that fulfilled all inclusion criteria yield no amplification of ehrlichial DNA by PCR. Based on our results, one in each five dogs infested by the brown dog tick, with anemia or thrombocytopenia had ehrlichosis. Contrary to widespread believe, ehrlichiosis was not the main cause for thrombocytopenia in our region. © 2003 Elsevier B.V. All rights reserved.