RESUMO
Enteroinvasive Escherichia coli (EIEC) and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.
Assuntos
Humanos , Citocinas , Escherichia coli , Regulação Bacteriana da Expressão Gênica/imunologia , Macrófagos , Shigella flexneri , Fatores de Virulência/biossíntese , Morte Celular , ELISPOT , Escherichia coli , Genes Bacterianos , Regulação Bacteriana da Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Shigella flexneri , Fatores de VirulênciaRESUMO
Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.
Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.
Assuntos
Azospirillum brasilense/isolamento & purificação , Hibridização Genética , Técnicas In Vitro , Fixação de Nitrogênio , Estruturas Vegetais , Técnica de Amplificação ao Acaso de DNA Polimórfico , Classificação , Genótipo , Métodos , MétodosRESUMO
The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.
Assuntos
Humanos , Técnicas de Tipagem Bacteriana , Escherichia coli/genética , Variação Genética , DNA Bacteriano/genética , Escherichia coli/classificação , Escherichia coli/patogenicidade , Fenótipo , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise de Sequência de DNA , Virulência/genéticaRESUMO
The genetic diversity of 41 typical and atypical enteropathogenic Ëscherichia coli" (EPEC) strains of the serogroup O55 was analysed by using the random amplified polymorphic DNA (RAPD) method. All typical EPEC O55 strains were grouped in two clusters (A and C) and belonged to serotype O55:H6, while cluster B included all atypical strains, which were of the serotype O55:H7. The three groups also included non-motile strains. RAPD may be a useful method for epidemiological studies on "E. coli" O55 infection
Assuntos
Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/patologia , Variação Genética/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico/normasRESUMO
Biotechnology owes a great deal to molecular biology. Even so, scientific discoveries are just a partial, although very important, component of modern biotechnology. To generate products and processes basic research needs integration with other fields, like process technologies, which are essential for large-scale production, or plant and animal breeding for dissemination of valuable genes identified through molecular markers. Moreover, several different agents must interact for the transformation of science into technological innovation: research centers, companies, government departments, financing organizations. The demand for biotechnologies in Brazilian agriculture serves here as a "case study". Technological trajectories and opportunities for inverstment are identifield in segments where biotechnology is relevant for competitiveness. The country's institutional framework for supporting research and development (R&D) activites related to commercial biotechnology is analyzed focusing the PADCT experience.
Assuntos
Humanos , Academias e Institutos/organização & administração , Biotecnologia/tendências , Biologia Molecular/tendências , Apoio à Pesquisa como Assunto/organização & administração , Agricultura , Brasil , Programas Governamentais , Projetos de PesquisaRESUMO
Recentemente demonstrou-se que a linhagem celular TM-4, derivada de células de Sertoli isoladas do testículo de camundongos imaturos BALB/c, secreta o antígeno H-Y. Essa evidência vem em apoio à hipótese de que o antígeno H-Y é o indutor do testículo em mamíferos. A disponibilidade de uma fonte de antígeno H-Y solúvel permitiu-nos desenvolver um imunoensaio sensível para a determinaçäo do antígeno H-Y em pacientes intersexuados. No entanto, a pesquisa de moléculas que controlam a determinaçäo do sexo em mamíferos (e em outros vertebrados) permanece um campo controvertido. O uso de sondas moleculares para o cromossomo Y, em conjunto com testes sorológicos, poderá permitir evidências mais conclusivas num futuro próximo