Towards an assessment of character interdependence in avian RNA phylogenetics: A general secondary structure model for the avian mitochondrial 16S rRNA

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

Silencing of mitochondrial alternative oxidase gene of Aspergillus fumigatus enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.

Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase

Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The Delta aifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 mu M FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 mu M FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Phylogeny, species limits, and biogeography of the Brazilian lizards of the genus Eurolophosaurus (Squamata : Tropiduridae) as inferred from mitochondrial DNA sequences

Phylogenetic relationships and divergence times for 10 populations of the three recognized ""species"" of Brazilian lizards of genus Eurolophosaurus were estimated from 1229 bp of cyt b, COI, 12S, and 16S rRNA mitochondrial gene segments. Eurolophosaurus is monophyletic and the basal split within the genus separates E divaricatus from a clade comprising E amathites and E nanuzae. Three populations of E divaricatus, which occurs along the western bank of Rio S (a) over tildeo Francisco, were consistently grouped together. Oil the east bank of the river, E amathites and E nanuzae from state of Bahia were recovered as the sister group of E nanuzae populations from state of Minas Gerais. The paraphyly of E nanuzae and the high divergence levels among populations of E divaricatus strongly suggest that species limits in Eurolophosaurus should be revised. Even considering an extreme evolutionary rate of 2.8% sequence divergence per million years for the four gene segments analyzed together, E. divaricatus would have separated from the two other species by at least 5.5 my ago, and E. amathites from E nanuzae populations from Bahia and Minas Gerais, respectively, by 1.5 and 3.5 my. The paleolacustrine hypothesis and changes in the course of the river potentially explain faunal divergence in the area, but divergences are much older than previously admitted. (C) 2007 Elsevier Inc. All rights reserved.

Role of the Mitochondrial Mutations, m. 827A > G and the Novel m. 7462C > T, in the Origin of Hearing Loss

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m. 1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m. 827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m. 7472insC mutation and three probands presented a novel variant, m. 7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

Genetic structure, phylogeny, and biogeography of Brazilian eyelid-less lizards of genera Calyptommatus and Nothobachia (Squamata, Gymnophthalmidae) as inferred from mitochondrial DNA sequences

Calyptommatus and Nothobachia genera of gymnophthalmid lizards are restricted to sandy open habitats on Sao Francisco River margins, northeastern Brazil. Phylogenetic relationships and geographic distribution of the four recognized species of Calyptommatus were analyzed from partial mitochondrial cyt b, 12S, and 16S rRNA genes sequencing, taking allopatric populations of the monotypic Nothobachia ablephara as the outgroup. In Calyptommatus a basal split separated C. sinebrachiatus, a species restricted to the eastern bank of the river, from the three other species. In this clade, C. confusionibus, found on western margin, was recovered as the sister group of the two other species, C. leiolepis and C. nicterus, from opposite margins. According to approximate date estimations, C. sinebrachiatus would have separated from the other congeneric species by 4.4-6.5 my, and C. nicterus, also from eastern bank, would be diverging by 1.8-2.6 my from C. leiolepis, the sister species on the opposite margin. C. confusionibus and C. leiolepis, both from western sandy areas, would be differentiating by 2.8-5.0 my. Divergence times of about 3.0-4.0 my were estimated for allopatric populations of Nothobachia restricted to western margin. Significant differences in 16S rRNA secondary structure relatively to other vertebrates are reported. Distinct evolutionary patterns are proposed for different taxa in those sandy areas, probably related to historical changes in the course of Sao Francisco River. (C) 2010 Elsevier Inc. All rights reserved.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

The mitochondrial genome of the stingless bee Melipona bicolor (Hymenoptera, Apidae, Meliponini): sequence, gene organization and a unique tRNA translocation event conserved across the tribe Meliponini

At present a complete mtDNA sequence has been reported for only two hymenopterans, the Old World honey bee, Apis mellifera and the sawfly Perga condei. Among the bee group, the tribe Meliponini (stingless bees) has some distinction due to its Pantropical distribution, great number of species and large importance as main pollinators in several ecosystems, including the Brazilian rain forest. However few molecular studies have been conducted on this group of bees and few sequence data from mitochondrial genomes have been described. In this project, we PCR amplified and sequenced 78% of the mitochondrial genome of the stingless bee Melipona bicolor (Apidae, Meliponini). The sequenced region contains all of the 13 mitochondrial protein-coding genes, 18 of 22 tRNA genes, and both rRNA genes (one of them was partially sequenced). We also report the genome organization (gene content and order), gene translation, genetic code, and other molecular features, such as base frequencies, codon usage, gene initiation and termination. We compare these characteristics of M. bicolor to those of the mitochondrial genome of A. mellifera and other insects. A highly biased A+T content is a typical characteristic of the A. mellifera mitochondrial genome and it was even more extreme in that of M. bicolor. Length and compositional differences between M. bicolor and A. mellifera genes were detected and the gene order was compared. Eleven tRNA gene translocations were observed between these two species. This latter finding was surprising, considering the taxonomic proximity of these two bee tribes. The tRNA Lys gene translocation was investigated within Meliponini and showed high conservation across the Pantropical range of the tribe.

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

The genus Brycon, the largest subunit of the Bryconinae, has 42 valid species distributed from southern Mexico to the La Plata River in Argentina. Henochilus is a monotypic genus, comprising a single species (H. wheatlandii) found in the upper Rio Doce basin. In the present study, partial sequences of the mitochondrial gene 16S were obtained for fifteen species of Brycon and for Henochilus wheatlandii. The results showed that the genus Brycon is paraphyletic, since Henochilus is the sister-group of B. ferox and B. insignis. The most basal species analyzed were the trans-Andean species B. henni, B. petrosus, and B. chagrensis.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Extraction and purification of total RNA from banana tissues (small scale)

Introduction. This protocol aims at preparing total RNA for gene expression analysis by Northern blots, RT-PCR and real-time quantitative PCR; cDNA isolation by RTPCR; and cDNA library construction. The principle, key advantages, starting plant material, time required for obtaining total RNA and expected results are presented. Materials and methods. This part describes the required materials and the 27 steps necessary for preparing RNA from peel and pulp fruit tissue: preparation of plant tissue powder, preparation of the complete RNA extraction buffer and isolation of RNA from ground banana fruit tissue. Results. Extraction of total RNA by the method described makes it possible to achieve electrophoresis under denatured conditions and in vitro reverse transcription. An example for Northern blot analysis is illustrated.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.