Quasispecies of hepatitis C virus genotype 1 and treatment outcome with Peginterferon and Ribavirin

The majority of patients with chronic hepatitis C fail to respond to antiviral therapy. The genetic basis of this resistance is unknown. The quasispecies nature of HCV may have an important implication concerning viral persistence and response to therapy. The HCV nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy. To evaluate whether the NS5A quasispecies pre-treatment composition of HCV 1a/1b is related to responsiveness to combined pegylated interferon (PEG-IFN) and Ribavirin therapy, detailed analyses of the complete NS5A were performed. Fifteen full-length NS5A clones were sequenced from 11 pretreatment samples of patients infected with genotype 1 HCV (3 virological sustained responders, 4 non-responders, and 4 end-of-treatment responders). Our study could not show a significant correlation between the mean number of mutations in HCV NS5A before treatment and treatment outcome, and the phylogenetic construction of complete NS5A sequences obtained from all patients failed to show any clustering associated with a specific response pattern. No single amino acid position was associated with different responses to therapy in any of the NS5A regions analyzed, and mutations were clustered downstream the ISDR, primarily in the V3 region. We observed that the CRS and NLS regions of the NS5A protein were conflicting for some variables analyzed, although no significant differences were found. If these two regions can have antagonistic functions, it seems viable that they present different mutation profiles when compared with treatment response. The patient sample that presented the lowest genetic distance values also presented the smallest number of variants, and the most heterogeneous pattern was seen in the end-of-treatment patients. These results suggest that a detailed molecular analysis of the NS5A region on a larger sample size may be necessary for understanding its role in the therapy outcome of HCV 1a/1b infection. (C) 2008 Elsevier B.V. All rights reserved.

The evolutionary dynamics of the Helena retrotransposon revealed by sequenced Drosophila genomes

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

At the interface: Crystal structures of phospholipases A(2)

The protein content of many snake venoms often includes one or more phospholipases A(2) (PLA(2)). In recent years a growing number of venoms from snakes of Agkistrodon, Bothrops and Trimeresurus species have been shown to contain a catalytically inactive PLA(2)-homologue in which the highly conserved aspartic acid at position 49 (Asp49) is substituted by lysine (Lys49). Although demonstrating little or no catalytic activity, these Lys49-PLA(2)s disrupt membranes by a Ca2+-independent mechanism of action. In addition, this family of PLA(2)s demonstrates myotoxic and cytolytic pharmacological activities, however the structural bases underlying these functional properties are poorly understood. Through the application of X-ray crystallography in combination with biophysical and bioinformatics techniques, we are studying structure/function relationships of Lys49-PLA(2)s. We here present results of a systematic X-ray crystallographic and amino acid sequence analysis study of Lys49-PLA(2)s and propose a model to explain the Ca2+ independent membrane damaging activity. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Detecção e genotipagem de papilomavírus humano em lesões de queratoacantoma solitário de pacientes imunocompetentes

FUNDAMENTOS: O queratoacantoma é neoplasia cutânea benigna que incide preferencialmente em indivíduos de pele clara, faixa etária elevada, acometendo áreas fotoexpostas. Além da exposição à radiação ultravioleta, sua etiologia é relacionada a diversos carcinógenos, entre eles a infecção pelo papilomavírus humano (HPV). OBJETIVOS: Avaliar a prevalência do DNA do HPV, bem como seus genótipos, em lesões de queratoacantoma solitário de pacientes imunocompetentes. MÉTODOS: Foram estudados queratoacantomas de pacientes sem evidências de imunocomprometimento, excisados entre 1996 e 2000 em hospital universitário. Realizaram-se cortes histológicos, desparafinização e extração de DNA desses fragmentos. Os espécimes positivos para DNA de HPV foram submetidos ao seqüenciamento gênico, para determinação do genótipo. RESULTADOS: Foram estudados 58 pacientes com idade média de 64,5±13,8 anos. A proporção entre os sexos foi semelhante, e as localizações mais comuns foram os membros superiores (50%) e a face (27,6%). Detectou-se DNA de HPV em 48 (82,7%) fragmentos de queratoacantomas, sendo os genótipos 6, 11 e 16 os prevalentes. CONCLUSÕES: A alta prevalência do achado de DNA de HPV em lesões de queratoacantoma solitário pode sugerir a participação viral em sua oncogênese.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide-phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intertnedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (alpha/beta)(8) barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro-Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular characterization of Acidithiobacillus ferrooxidans and A-thiooxidans strains isolated from mine wastes in Brazil

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.

Yeast biodiversity from oleic ecosystems: Study of their biotechnological properties

The aim of this study was to know the yeast biodiversity from fresh olive (Olea europaea L.) fruits, olive paste (crush olives) and olive pomace (solid waste) from Arbequina and Cornicabra varieties. Yeasts were isolated from fruits randomly harvested at various olive groves in the region of Castilla La Mancha (Spain). Olive paste and pomace, a byproduct of the processing of this raw material, were also collected in sterile flasks from different oil mills. Molecular identification methodology used included comparison of polymerase chain reaction (PCR) amplicons of their 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2 followed by restriction pattern analysis (RFLP). For some species, sequence analysis of the 5.8S rDNA gene was necessary. The results were compared to sequences held in public databases (BLAST). These techniques allowed to identify fourteen different species of yeasts, belonging to seven different genera (Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces, Saccharomyces, Candida, Torulaspora) from the 108 yeast isolates. Species diversity was thus considerable: Pichia caribbica, Zygosaccharomyces fermentati (Lachancea fermentati) and Pichia holstii (Nakazawaea holstii) were the most commonly isolated species, followed by Pichia mississippiensis, Lachancea sp., Kluyveromyces thermotolerans and Saccharomyces rosinii. The biotechnological properties of these isolates, was also studied. For this purpose, the activity of various enzymes (beta-glucosidase, beta-glucanase, carboxymethylcellulase, polygalacturonase, peroxidase and lipase) was evaluated. It was important that none of species showed lipase activity, a few had cellulase and polygalacturonase activities and the majority of them presented beta-glucanase, beta-glucosidase and peroxidase activities. (C) 2010 Elsevier Ltd. All rights reserved.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Tuberculosis diagnosis after bleach processing for early stage tuberculosis laboratory capacity building

The diagnosis of tuberculosis is seriously hampered in the absence of standard biosafety laboratory facilities for specimen concentration and Mycobacterium tuberculosis culture. Within a laboratory twinning arrangement, heat-fixed direct smear and sediment from 74 bleach-processed and 20 non-processed specimens from Cumura Hospital, Guinea-Bissau, were sent to Lisbon for molecular evaluation of rifampicin resistance. Sequence analysis of a 369 base-pair ppoB locus detected 3.2% (3/94) resistant specimens. To our knowledge, this represents the first report on the molecular analysis of M. tuberculosis from bleach-processed sputum, an alternative to current diagnostic practice in low-resource settings.

Genetically Diverse Coronaviruses in Captive Bird Populations in a Brazilian Zoological Park

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

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups

The genetic relatedness among 96 invasive Escherichia coli belonging to several serogroups and 13 non-invasive of several serotypes that share the same O antigen was investigated by multilocus enzyme electrophoresis analysis. The invasive strains were isolated in different parts of the world and most of them recovered from dysentery. Twenty-nine electrophoretic types were distinguished and the most invasive strains were found to belong to two major lineages. These results suggested that the invasive ability in these strains has evolved in divergent chromosomal backgrounds, presumably through the horizontal spread of plasmid-borne invasion genes. The maintenance of invasive phenotypes in separate lineages suggests that this ability confers a selective advantage to invasive strains. Copyright (C) 1999 Federation of European Microbiological Societies.

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.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5′ sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.