Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level

Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both beta-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEEI-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEEI-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:147 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7. (c) 2005 Elsevier SAS. All rights reserved.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

TURBULENCE AND THE FORMATION OF FILAMENTS, LOOPS, AND SHOCK FRONTS IN NGC 1275

NGC 1275, the central galaxy in the Perseus cluster, is the host of gigantic hot bipolar bubbles inflated by active galactic nucleus (AGN) jets observed in the radio as Perseus A. It presents a spectacular H alpha-emitting nebulosity surrounding NGC 1275, with loops and filaments of gas extending to over 50 kpc. The origin of the filaments is still unknown, but probably correlates with the mechanism responsible for the giant buoyant bubbles. We present 2.5 and three-dimensional magnetohydrodynamical (MHD) simulations of the central region of the cluster in which turbulent energy, possibly triggered by star formation and supernovae (SNe) explosions, is introduced. The simulations reveal that the turbulence injected by massive stars could be responsible for the nearly isotropic distribution of filaments and loops that drag magnetic fields upward as indicated by recent observations. Weak shell-like shock fronts propagating into the intracluster medium (ICM) with velocities of 100-500 km s(-1) are found, also resembling the observations. The isotropic outflow momentum of the turbulence slows the infall of the ICM, thus limiting further starburst activity in NGC 1275. As the turbulence is subsonic over most of the simulated volume, the turbulent kinetic energy is not efficiently converted into heat and additional heating is required to suppress the cooling flow at the core of the cluster. Simulations combining the MHD turbulence with the AGN outflow can reproduce the temperature radial profile observed around NGC 1275. While the AGN mechanism is the main heating source, the SNe are crucial to isotropize the energy distribution.

Reprint of ""Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury""

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields alpha-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields a-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at I week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at I week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

G??nero, ra??a e espa??os de poder: o caso de Maria Ant??nia e Geraldo

O caso relata uma situa????o de promo????o em ??rg??o p??blico na qual dois funcion??rios ??? uma mulher negra e um homem branco - se mostram como candidatos ao cargo. S??o apresentados os perfis e habilidades profissionais dos dois servidores e, ao final, a decis??o tomada pelo Secret??rio, chefe de ambos. O estudo suscita discuss??es sobre pr??ticas discriminat??rias nos espa??os de poder das institui????es p??blicas e o papel do dirigente para combater ou difundir tais pr??ticas. Pode ser aplicado em cursos sobre lideran??a, gest??o de pessoas e ??tica e servi??o p??blico

Projeto cria????o e revitaliza????o dos espa??os de conviv??ncia na UFPA

O Projeto Cria????o e Revitaliza????o dos Espa??os de Conviv??ncia na UFPA agrega professores, alunos e t??cnicos administrativos comprometidos com a Universidade e com os bens p??blicos. A equipe multidisciplinar mobiliza os usu??rios da Institui????o bem como os respons??veis pela gest??o do campus universit??rio e pela manuten????o da sua infraestrutura a produzirem a????es concretas de grande efeito multiplicador capazes de promover mudan??as em situa????es que estavam prejudicando a rotina acad??mica da Institui????o

Exclusi??n social y pol??ticas de inclusi??n. Algunos elementos sobre la realidad europea y espa??ola

La globalizaci??n econ??mica ha impactado fuertemente en el mercado laboral europeo, abriendo puertas para el trabajo precario. Mientras el capital se torna global, el trabajo permanece local y m??s sumiso a los flujos econ??micos mundiales. Adem??s, los patrones de la sociedad de consumo se han consolidado, promoviendo nuevas desigualdades sociales y aumentando los riesgos de exclusi??n y vulnerabilidad social. La ca??da progresiva del industrialismo tambi??n afecta las principales bases de la estructura social como los roles de g??nero o las relaciones familiares. Diferentes redes aparecen como clave para la inclusi??n, especialmente en el actual contexto de crisis del Estado de Bienestar Social en que se encuentra Europa. Es decir, el an??lisis de la exclusi??n social debe tener en cuenta la evoluci??n de las pol??ticas sociales en toda Europa y la tendencia actual de descentralizaci??n y focalizaci??n de los ser vicios sociales. El objetivo de este art??culo es, por lo tanto, no s??lo describir el fen??meno de la exclusi??n social, sino tambi??n lanzar ideas para construir nuevos criterios de redefinici??n de las pol??ticas sociales.

Efeito do espaçamento na produtividade de biomassa de Taxi-branco (Sclerolobium paniculatum vogel)

Visando determinar o efeito de diferentes espaçamentos sobre o comportamento silvicultural do taxi-branco (Sclerolobium paniculatum Vogel) foi instalado experimento no Campo Experimental do Cerrado pertencente ao Centro de Pesquisa Agroflorestal do Amapá - Embrapa Amapá, no Estado do Amapá, Brasil. Foram empregadas quatro repetições em experimento delineado em blocos casualisados e sete tratamentos correspondentes aos espaçamentos: 2,0mx1,0m; 1,5mx1,5m; 3,0mx1,0m; 2,0mx2,0m; 2,5mx2,0m; 3,0mx2,0m; 3,0mx2,5m. As análises foram feitas à idade de sete anos. Os resultados evidenciaram diferenças não significativas para os parâmetros altura, DAP, sobrevivência, e número de fustes. Quanto ao parâmetro biomassa foi detectado somente dois grupos de médias estatisticamente diferentes. A menor produção foi obtida no espaçamento 3,0mx2,0m enquanto que a maior produção foi verificada no espaçamento 2,0mx1,0m com 1.355kg semelhante estatisticamente das produções obtidas nos espaçamentos 1,5mx1,5m com 1.351 kg e 3,0mx1,0m com 1.091 kg havendo portanto concordância plena entre produção de biomassa e densidade de plantio. De um modo geral, nota-se que o espaçamento silvicultural mais indicado para o taxi-branco nas condições de cerrado amapaense é o 3,0mx1,0m, visto que além de utilizar menor número de plantas e portanto deve apresentar menor custo de produção, atingiu produtividade de biomassa semelhante estatisticamente aos menores espaçamentos.

Variação espaço-temporal na atividade forrageira da Saúva (Atta laevigata)

Na Amazônia, a regeneração natural da floresta em pastos e cultivos abandonados pode ser afetada pelas saúvas, pois estas são importantes predadoras das plântulas e das sementes das espécies arbóreas que eventualmente ali se estabelecem. Entretanto, pouco se sabe sobre como as chances de uma dada planta ser atacada ou de uma semente ser removida pelas saúvas varia no tempo e no espaço. Com este intuito, estabeleci uma parcela de 80 χ 120 m, subdividida em quadrantes de 10 χ 10m, em um campo abandonado próximo a Manaus. Na periferia desta parcela haviam 3 ninhos da saúva Atta laevigata. Ao longo de 1 ano, determinei quais plantas (das espécies arbóreas somente) foram atacadas pelas formigas. Determinei também como variou a atividade de coleta de sementes por A. laevigata. Para isto utilizei grãos de feijão (Phaseolus vulgaris). Iscas, com cerca de 20 feijões cada, foram distribuídas pela parcela ao longo do ano. Finalmente, determinei se plantas (mudas de Bellucia imperialis) estabelecidas em solo desnudo tinham uma maior chance de serem atacadas do que aquelas estabelecidas em solo com cobertura de gramíneas. Os resultados mostram que a atividade de coleta de sementes foi espacialmente agregada. A maior atividade foi observada nas regiões sudeste e noroeste da parcela, em geral, próximo aos ninhos. Forte variação temporal na coleta de sementes também foi observada. O pico máximo de atividade ocorreu em meados de julho e o de menor atividade em meados de janeiro. A atividade de corte seguiu um padrão similar. Houve uma clara diminuição no número de plantas atacadas entre novembro e janeiro. A proporção de plantas atacadas por parcela variou entre 6 e 89%. A incidência de ataques por saúvas sobre mudas de B. imperialis não variou em função da cobertura vegetal (com ou sem gramíneas). Isto possivelmente em função da forte heterogeneidade espacial na atividade de A. laevigata, que encobriu qualquer efeito devido a cobertura vegetal.

RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination.

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.

Correlation of male genital filaments and female spermathecal ducts in New World sand flies of the Lutzomyia intermedia species complex (Diptera: Psychodidae, Phlebotominae)

The lengths of the male genital filaments and female spermathecal ducts were measured in phlebotomine sand flies of the Lutzomyia intermedia species complex and the ratios between these characters calculated. Ratios for L. intermedia s. s. from Northeast vs Southeast Brazil (Espírito Santo and Minas Gerais), Espírito Santo/Minas Gerais vs Rio de Janeiro/São Paulo and L. intermedia vs L. neivai were significantly different at P < 0.1, 0.05 and 0.01 respectively when compared using ANOVA. The spermathecal ducts and genital filaments of L. intermedia were significantly longer than those of L. neivai (P < 0.01) and could be used to differentiate these species. The taxonomic and biological significance of these differences is discussed.

RecA-DNA helical filaments in genetic recombination.

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.