RESUMO
BACKGROUND: Oesophageal adenocarcinoma or Barrett's adenocarcinoma (EAC) is increasing in incidence and stratification of prognosis might improve disease management. Multi-colour fluorescence in situ hybridisation (FISH) investigating ERBB2, MYC, CDKN2A and ZNF217 has recently shown promising results for the diagnosis of dysplasia and cancer using cytological samples. METHODS: To identify markers of prognosis we targeted four selected gene loci using multi-colour FISH applied to a tissue microarray containing 130 EAC samples. Prognostic predictors (P1, P2, P3) based on genomic copy numbers of the four loci were statistically assessed to stratify patients according to overall survival in combination with clinical data. RESULTS: The best stratification into favourable and unfavourable prognoses was shown by P1, percentage of cells with less than two ZNF217 signals; P2, percentage of cells with fewer ERBB2- than ZNF217 signals; and P3, overall ratio of ERBB2-/ZNF217 signals. Median survival times for P1 were 32 vs 73 months, 28 vs 73 months for P2; and 27 vs 65 months for P3. Regarding each tumour grade P2 subdivided patients into distinct prognostic groups independently within each grade, with different median survival times of at least 35 months. CONCLUSIONS: Cell signal number of the ERBB2 and ZNF217 loci showed independence from tumour stage and differentiation grade. The prognostic value of multi-colour FISH-assays is applicable to EAC and is superior to single markers.
Assuntos
Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/patologia , Hibridização in Situ Fluorescente/métodos , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/mortalidade , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA de Neoplasias/genética , Neoplasias Esofágicas/mortalidade , Feminino , Dosagem de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-myc/genética , Receptor ErbB-2/genética , Transativadores/genéticaRESUMO
Although drug-resistance markers have been used frequently for gene-disruption mutagenesis in Streptococcus pneumoniae, none has yet been shown to be free of dependence on local transcription for its expression. Indeed, the erythromycin-resistance marker (erm), originating in pAM beta 1, has been used as an indicator of local transcription on several occasions. A procedure is demonstrated for evaluation of the autonomous expression of such a marker by placing it in a consistent background, at the pneumococcal ami (aminopterin resistance) locus, in combination with active or inactive alleles of the ami promotor (pA). Using this test platform, a chloramphenicol-resistance marker (cat) and a spectinomycin-resistance marker used in streptococcal gene disruption studies and derived from pJS3 and pDL269, respectively, were shown to depend on local transcriptional signals for expression when placed in the pneumococcal chromosome as single-copy genes. To overcome this limitation, new drug-resistance cassettes were designed and constructed, using pA as a model for synthetic promoters for the erm and cat genes. Both new cassettes were shown, by the same procedure, to be expressed after insertion in the pneumococcal chromosome, independent of local transcription. A new insertion-duplication vector, pEVP3, incorporating the new cat cassette and a lacZ reporter derived from pTV32, was also constructed. The ami test platform was used to demonstrate both the autonomous expression of cat and the reporter function of lacZ in chromosomal copies of pEVP3.
Assuntos
Resistência Microbiana a Medicamentos/genética , Marcadores Genéticos , Mutagênese Insercional/métodos , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Aminopterina/farmacologia , Sequência de Bases , Resistência ao Cloranfenicol/genética , Eritromicina/farmacologia , Estudos de Avaliação como Assunto , Genes Reporter , Vetores Genéticos , Óperon Lac , Dados de Sequência Molecular , Espectinomicina/farmacologiaRESUMO
Although more than a dozen new proteins are produced when Streptococcus pneumoniae cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random lacZ transcriptional fusion library was constructed in S. pneumoniae by using the insertional lacZ reporter vector pEVP3. Screening the library for clones with competence-specific beta-galactosidase (beta-Gal) production yielded three insertion mutants with induced beta-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the lacZ reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts of Bacillus subtilis com operons. One locus contained open reading frames (ORFs) homologous to the comEA and comEC genes in B. subtilis but lacked a comEB homolog. A second locus contained four ORFs with homology to the B. subtilis comG gene ORFs 1 to 4, but comG gene ORFs 5 to 7 were replaced in S. pneumoniae with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an erm cassette for gene disruptions.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos/genética , Streptococcus pneumoniae/genética , Transformação Bacteriana/genética , beta-Galactosidase/metabolismo , Sequência de Aminoácidos , Genes Reporter/genética , Óperon Lac/genética , Dados de Sequência Molecular , Mutagênese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
The regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, but the only molecular elements of the system whose specific role have been identified are an extracellular peptide signal and an ABC-transporter required for its export. Here we show that transcription of comC, the gene encoding a predicted 41-residue precursor peptide that is thought to be processed and secreted as the 17-residue mature competence activator, increased approximately 40-fold above its basal level of expression in response to exogenous synthetic activator, consistent with earlier experiments indicating that the activator acts autocatalytically. We also describe two new genes, comD and comE, that encode members of histidine protein kinase and response-regulator families and are linked to comC. Disruption of comE abolished both response to synthetic activator peptide and endogenous competence induction.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Complexos Multienzimáticos , Streptococcus pneumoniae/genética , Transformação Bacteriana/genética , Sequência de Aminoácidos , Genes Bacterianos/genética , Histidina Quinase , Dados de Sequência Molecular , Família Multigênica/genética , Fases de Leitura Aberta/genética , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The association between fluoroquinolone susceptibility and DNA mutations coding for amino acid substitutions in the quinolone resistance-determining region was assessed with 44 clinical isolates of Streptococcus pneumoniae. Twenty-three strains bore at least one amino acid substitution. Only seven strains with mutations were suggested by diminished susceptibility to ciprofloxacin (MIC, > or =2 microg/ml).
Assuntos
Substituição de Aminoácidos , Anti-Infecciosos/farmacologia , DNA Topoisomerases Tipo II/genética , Streptococcus pneumoniae/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Fluoroquinolonas , Marcadores Genéticos , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
The in vitro activity of the novel 8-methoxyquinolone, moxifloxacin, against Streptococcus pneumoniae was evaluated, and the intracellular targets of this agent were studied. Analysis of mutant strains selected with moxifloxacin demonstrated that first-step mutants bore amino acid substitutions at position 81 in the GyrA subunit of DNA gyrase. This suggests that, unlike older fluoroquinolone agents such as ciprofloxacin and levofloxacin, but similar to other C-8 substituted quinolones like sparfloxacin and gatifloxacin, moxifloxacin targets the GyrA subunit of DNA gyrase as an initial lethal event. Such a mechanism results in high activity against increasingly common S. pneumoniae strains bearing substitutions in DNA topoisomerase IV. Moxifloxacin was active with an MIC of = 0.25 mg/L against S. pneumoniae clinical isolates, and against mutants, selected in the laboratory with ciprofloxacin or levofloxacin, that bore a Ser-79-->Phe/Tyr substitution in ParC. The moxifloxacin MIC for strains with mutations in the structural genes for both DNA gyrase subunit GyrA and DNA topoisomerase IV subunit ParC did not exceed 2 mg/L, a level within clinically achievable serum concentrations for this agent. We also found that moxifloxacin is a poor substrate for active efflux in S. pneumoniae. Therefore, the high activity of moxifloxacin against S. pneumoniae appears to be a result of both enhanced activity against DNA gyrase and topoisomerase IV, and reduced efflux from the bacterial cell.
Assuntos
Anti-Infecciosos/farmacologia , Compostos Aza , Fluoroquinolonas , Infecções Pneumocócicas/microbiologia , Quinolinas , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Anti-Infecciosos/metabolismo , DNA Topoisomerases Tipo I/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Resistência Microbiana a Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Moxifloxacina , Mutação , Reação em Cadeia da Polimerase , Reserpina/farmacologia , Análise de Sequência de DNA , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79-->Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 microg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV.
Assuntos
Anti-Infecciosos/farmacologia , DNA Topoisomerases Tipo II/genética , Fluoroquinolonas , Mutação , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Antituberculosos/farmacologia , Ciprofloxacina/farmacologia , DNA Girase , DNA Topoisomerase IV , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos , Levofloxacino , Testes de Sensibilidade Microbiana , Naftiridinas/farmacologia , Ofloxacino/farmacologia , Fenótipo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificação , Transformação BacterianaRESUMO
We developed a simplified assay for estimating efflux by measuring the effect of reserpine on the growth of Streptococcus pneumoniae and Staphylococcus aureus over 7 h. Reserpine enhanced ciprofloxacin and levofloxacin 17 to 68%. The hydrophobic drug trovafloxacin and the drug moxifloxacin, with a bulky C-7 substituent but hydrophilicity similar to that of levofloxacin, showed little (0 to 11%) reserpine-enhancing effect. The ease of resistant mutant strain selection correlated with efflux susceptibility.