Improving promiscuous mammalian cell entry by the baculovirus AcMNPV

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells we show that entry is favoured by low pH and increasing the available cell surface area through transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268-281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell to cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies suggesting additional factors also govern entry.

Trypanosoma cruzi: parasite shed vesicles increase heart parasitism and generate an intense inflammatory response

Trypanosoma cruzi trypomastigotes continuously shed into the medium plasma membrane fragments sealed as vesicles enriched in glycoproteins of the gp85 and trans-sialidase (TS) superfamily and alpha-galactosyl-containing glycoconjugates. Injection of a vesicle fraction into BALB/c mice prior to T. cruzi infection led to 40% of deaths on the 16th day post-infection and 100% on day 20th whereas 20% of untreated animals survived for more than 30 clays. The vesicle-treated animals developed severe heart pathology, with intense inflammatory reaction and higher number of amastigote nests. Analysis of the inflammatory infiltrates 15 days after infection showed predominance of TCD4(+) lymphocytes and macrophages, but not of TCD8(+) cells, as well as a decrease of areas labeled with anti-iNOS antibodies as compared to the control. Higher levels of IL-4 and IL-10 mRNAs were found in the hearts and higher IL-10 and lower NO levels in splenocytes of vesicles pretreated animals. Treatment of mice with neutralizing anti-IL-10 or anti-IL-4 antibodies precluded the effects of pre-inoculation of membrane vesicles on infection. These results indicate that T. cruzi shed membrane components increase tissue parasitism and inflammation by stimulation of IL-4 and IL-10 synthesis and thus may play a central role in the pathogenesis of Chagas` disease acute phase. (c) 2008 Elsevier Masson SAS. All rights reserved.

Avaliação do potencial mutagênico da água do açude de Lucrécia (RN- Brasil): um enfoque na relação saúde e ambiente

The water quality of many reservoirs in the world has been reduced due to percolation of contaminants to water, which can have natural or anthropogenic origin, increasing the level of genotoxic compounds in aquatic ecosystems. This fact has contributed to the reduction of environmental quality, and commitment the health of living beings that inhabit these ecosystems, including the human population. In this backdrop of reduced water quality, is the Lucrecia dam, which is a major surface water reservoirs by volume of semi-arid region of Rio Grande do Norte, and that has shown contamination by heavy metals, cyanobacteria toxic and the natural presence of Radon. The population that use this source has been showing high rates of cancer, popularly associated with the consumption of this water, with a prevalence about three times higher compared to the whole state of Rio Grande do Norte. Based on this, the present study aimed to evaluate the mutagenic potencial of surface water from the Lucrecia dam, using the Micronucleus Test in Tradescantia pallida (Trad-MN) and in human peripheral blood lymphocytes (CBMN) assay, as well as identify the concentrations of some heavy metals in this water. Water samples were collected on a dry season and a rainy season, in two distinct points. Moreover, in order to bring a completely view about the relationship of man-health-environment in this local, through the knowledge of knowing / acting environmental from residents of Lucrecia, and the use and perceptions they have about the dam of your city, a study of Environmental Perception was carried out with local residents. The results obtained for the both micronucleus test, showed significant results for the three points analyzed. The strongest mutagenic effect was observed in the dry season for both assays. Chemical analyses detected an increase of heavy metal levels in different points and season above the maximum allowed by legislation. Regarding the study of Environmental Perception with local residents, it was observed the knowledge of the environment that the residents have, as well as the strong ties and perceptions with the dam of the city. Thus, the combination of these two aspects (the genetic toxicity tests conducted in the dam together with analysis of environmental perception with the residents of Lucrecia) allowed to draw a more complete diagnosis on the local situation

Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the jaboticabal strain of Anaplasma marginale

Anaplasma is a tick-borne ehrlichial pathogen of cattle that causes the disease, anaplasmosis. In the present study, a total of 11 Anaplasma marginale seronegative calves were assigned into two groups: one immunized (G1, n = 6) and one nonimmunized-control (G2, n = 5). Six calves were immunized by using a DNA vaccine containing the gene of a major surface protein, MSP1b, encoded by the plasmid identified as pcDNA3.1/MSPIb. Calves received three intramuscular inoculations of 100 mug of pcDNA3.1/MSP1b at a 20-day interval. The control group received buffer phosphate at the same schedule as the experimental group. The immune response elicited by immunization with pcDNA3.1/MSP1b was evaluated in mice and calves. Twenty days following initial immunization, specific serum antibody from four BALB/c mice bound MSP1b in inummoblots. Sixty days after the last immunization, all calves were challenged with cryopreserved A. marginale at a dose of 10(4) parasites/mL/animal by intravenous injection. Results of packed cell volume (PCV) and detection of infected erythrocytes in all experimental groups revealed that the decrease of PCV and detection of infected erythrocytes occurred at 28 to 42 days after challenge. Mean temperature values did not increase over 39.85degreesC. Antibodies developed by immunized bovines from G2 were detected 14 days after challenge. MSP1b was characterized during the immunization period and MSP2 was the most predominant polypeptide at the challenge period. DNA of A. marginale was detected in all groups just after challenge by nested PCR assay. It can be concluded that all immunized bovines were partially protected against homologous challenge.

Electrical Properties of Textured Niobium-Doped Bismuth Titanate Ceramics

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

Expressão da proteína S1 recombinante do vírus da bronquite infecciosa em Saccharomyces cerevisiae para aplicação no imunodiagnóstico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará, Brazil

A babesiose, a anaplasmose e a tripanossomose são enfermidades relevantes, potencialmente causadoras de morbidade em bovinos, levando a perdas econômicas. A borreliose assume importância como zoonose potencial. O objetivo desse estudo foi determinar, por meio do ensaio de imunoadsorção enzimática (ELISA), a freqüência de anticorpos para Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax e Borrelia burgdorferi em bovinos da região nordeste do Estado do Pará, Brasil. Amostras de soro de 246 vacas dos municípios de Castanhal e São Miguel do Guamá foram usadas. ELISAs com antígeno bruto foram utilizados para detector anticorpos contra todos os agentes, exceto para A. marginale, para o qual um ELISA indireto com proteína principal de superfície 1a (MSP1a) foi usado. As freqüências de bovinos soropositivos foram: B. bigemina - 99,2%; B. bovis - 98,8%; A. marginale - 68,3%; T. vivax - 93,1% and B. burgdorferi -54,9% As freqüências de bovinos soropositivos para B. bovis e B. bigemina sugerem uma alta taxa de transmissão desses organismos por carrapatos, na região estudada, a qual pode ser classificada com sendo de estabilidade enzoótica para os hemoparasitos. A baixa freqüência de bovinos soropositivos para A. marginale pode ser atribuída a uma menor sensibilidade do ELISA com antígeno recombinante, ou uma menor taxa de inoculação da riquétsia pelos carrapatos, quando comparada àquelas observadas para Babesia sp. A alta freqüência de bovinos soropositivos para T. vivax indica que esse hemoprotozoário é prevalente em rebanhos do nordeste do Estado do Pará. O percentual de animais com anticorpos homólogos para B. burgdorferi indica a presenças deste espiroquetídeo transmitido por carrapatos na população de bovinos da região estudada.

Low genetic diversity associated with low prevalence of Anaplasma marginale in water buffaloes in Marajo Island, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Infection of water buffalo in Rio de Janeiro Brazil with Anaplasma marginale strains also reported in cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genetic diversity and molecular phylogeny of Anaplasma marginale studied longitudinally under natural transmission conditions in Rio de Janeiro, Brazil

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

Low genetic diversity of Anaplasma marginale in calves in an endemic area for bovine anaplasmosis in the state of São Paulo, Brazil

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

Diversidade genética do Anaplasma marginale em condições de transmissão natural

Pós-graduação em Medicina Veterinária - FCAV

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Elimination of chronic viral infection by blocking CD27 signaling

Neutralizing antibody (nAb) responses to lymphocytic choriomeningitis virus (LCMV) in mice and immunodeficiency virus and hepatitis C virus in humans are usually weak and slow to develop. This may be the result of structural properties of the surface glycoprotein, a low frequency of B cells with neutralizing specificity, and the necessity of prolonged affinity maturation of specific nAbs. In this study, we show that during LCMV infection, CD27 signaling on CD4+ T cells enhances the secretion of interferon-gamma and tumor necrosis factor-alpha. These inflammatory cytokines lead to the destruction of splenic architecture and immunodeficiency with reduced and delayed virus-specific nAb responses. Consequently, infection with the otherwise persistent LCMV strain Docile was eliminated after CD27 signaling was blocked. Our data provide a novel mechanism by which LCMV avoids nAb responses and suggest that blocking the CD27-CD70 interaction may be an attractive strategy to prevent chronic viral infection.

Characterization of sea urchin yolk glycoproteins

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, S. purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein was prepared. lmmunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa egg yolk glycoprotein during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified and peptide mapping revealed that they were cleavage products derived from the major yolk glycoprotein. The antibody identified a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: L. pictus, A. punctulata, and D. excentricus. However, eggs from other echinoderm classes and from chicken, frog, fruit fly, and nematode did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked, polymannose carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed, revealing striking similarities in pI and in amino acid and monosaccharide composition. Peptide mapping showed that the 160-kDa glycoprotein from the four echinoids are structurally homologous. The major yolk glycoprotein appeared to be proteolyzed by a thiol protease, which could be activated in yolk particles prepared from unfertilized eggs by low pH. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout embryonic development, and that externalization of the glycoproteins was not detectable. The yolk glycoprotein precursor began to be synthesized in premetamorphosis larvae, and continued in adult males and females. Both the yolk glycoproteins and the yolk platelets disappeared during larval development. This disappearance has special significance because there were no yolk proteins in the direct developing sea urchin, H. erthryogramma, which bypasses larval development and metamorphoses directly into an adult. ^