RESUMO
Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.
Assuntos
Fosfoproteínas Fosfatases/química , Monoéster Fosfórico Hidrolases/química , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/enzimologia , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Citocinese , DNA/química , Regulação para Baixo , Citometria de Fluxo , Teste de Complementação Genética , Glutationa Transferase/metabolismo , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Mitose , Fenótipo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático , TemperaturaRESUMO
The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.
