RESUMO
We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development.
Assuntos
Proteínas de Transporte/genética , Dictyostelium/genética , Proteínas de Drosophila , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Protozoários/genética , Sequências Repetitivas de Ácido Nucleico , Transativadores , Sequência de Aminoácidos , Animais , Proteínas do Domínio Armadillo , Sequência de Bases , Proteínas de Transporte/metabolismo , Bovinos , Clonagem Molecular , DNA de Protozoário , Dictyostelium/fisiologia , Humanos , Proteínas de Insetos/química , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologia , Homologia de Sequência de AminoácidosRESUMO
An immunoassay for the detection of Clostridium difficile toxin A in stool samples (Clearview C. DIFF A; Unipath, UK) was evaluated against the cell cytotoxicity assay using 407 stool samples from patients suspected to have, or considered at risk of, antibiotic-associated diarrhoea. Of the samples tested, 98 were positive and 280 were negative by both tests (sensitivity 83.1%, specificity 96.9%). Following resolution of the 29 discrepant results, the sensitivity and specificity of the immunoassay were 91% and 98%, respectively, and the sensitivity for the cell cytotoxicity assay was calculated as 91.5%, with a specificity of 99%. The Clearview C. DIFF A test proved to be a rapid simple assay for the detection of Clostridium difficile toxin A in stool samples. The test was equally suited to single or batch testing, required minimal sample handling, and provided results within 30 min of applying the sample to the test unit.
