RESUMO
The Asr gene family is present in Spermatophyta. Its members are generally activated under water stress. We present evidence that tomato ASR1, one of the proteins of the family, accumulates in seed during late stages of embryogenesis, a physiological process characterized by water loss. In vitro, electrophoretic assays show a homo-dimeric structure for ASR1 and highlight strong non-covalent interactions between monomers prone to self-assemble. Direct visualization of single molecules by atomic force microscopy (AFM) confirms that ASR1 forms homodimers and that uncovers both monomers and dimers bind double stranded DNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Água/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Dimerização , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Microscopia de Força Atômica , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Plasmídeos/metabolismo , Plasmídeos/ultraestrutura , Ligação Proteica , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
AIMS: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. METHODS AND RESULTS: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. CONCLUSIONS: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.
Assuntos
Enterobacteriaceae/fisiologia , Escherichia coli/fisiologia , Administração Oral , Animais , Antibacterianos/análise , Bacteriocinas/análise , Bacteriófagos/metabolismo , Colicinas/análise , Enterobacter/fisiologia , Escherichia/fisiologia , Fezes/microbiologia , Feminino , Ácidos Hidroxâmicos/análise , Klebsiella/fisiologia , Masculino , Camundongos , Microscopia Eletrônica/métodos , Morganella/fisiologia , Myoviridae , Plasmídeos/ultraestrutura , Salmonella/fisiologia , Shigella/fisiologia , Shigella flexneri/fisiologia , Sideróforos/análise , Yersinia/fisiologiaRESUMO
The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMVbeta-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMVbeta-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA-protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against beta-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to beta-galactosidase induced by DNA-protein complexes was also detected.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Plasmídeos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Administração Intranasal , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Células COS , Linhagem Celular , Esquemas de Imunização , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/administração & dosagem , Plasmídeos/metabolismo , Plasmídeos/ultraestrutura , TransfecçãoRESUMO
Analysis of bacterial plasmid profiles has been shown to be very important in epidemiological studies, especially those involving outbreaks of nosocomial infections. The molecular weight size of unknown plasmids is determined by comparing their band pattern obtained in agarose gel electrophoresis with those obtained with plasmids that have been used as molecular weight or size standards. In this study, we determined the size of plasmids present in clinical samples of Enterobacter cloacae comparing their electrophoresis mobility with seven plasmids of known size, using three different mathematical methods. For plasmids with molecular weight ranging from 2 kb to 100 kb. The most accurate determinations were obtained by power-function. Analyses using the exponential variables obtained with these plasmids were accurate for two types of plasmids, those with size ranging from 50 kb to 100 kb and those with size ranging from 2 kb to 30 kb. We also observed discrepancies among the methodologies described, including one used by a computer software designed for calculating the size of plasmids DNA.