Induced sputum genes associated with spirometric and radiological disease severity in COPD ex-smokers.

BACKGROUND: Induced sputum is used to sample inflammatory cells, predominantly neutrophils and macrophages, from the airways of COPD patients. The author's aim was to identify candidate genes associated with the degree of airflow obstruction and the extent of emphysema by expression profiling, and then to confirm these findings for selected candidates using PCR and protein analysis. METHODS: Two sputum studies were performed in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 2-4 COPD ex-smokers from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) cohort. First, gene array profiling at baseline in samples from 148 patients. The findings were replicated in a separate population of 176 patients using real-time PCR. The findings for one selected gene IL-18R were further analysed using immunohistochemistry in lung tissue and induced sputum from patients outside the ECLIPSE cohort. RESULTS: Gene expression profiling revealed changes in 277 genes associated with GOLD stage 2 versus 3 and 4, and 198 genes with changes associated with the degree of emphysema (p < 0.01 for each gene). Twelve of these candidate genes were analysed by PCR in the replication cohort, with significant changes (p < 0.05) observed for 11 genes. IL-18R protein expression was higher on alveolar macrophages in lung tissue of COPD patients (mean 23.2%) compared to controls (mean ex-smokers 2% and non-smokers 2.5%). CONCLUSION: Gene expression profiling in sputum cells identified candidate genes that may play roles in molecular mechanisms associated with COPD. The replication by PCR and protein in different studies confirms these findings, and highlights a potential role for IL-18R upregulation in severe COPD.

Identification of kinase inhibitors by an ATP depletion method.

ATP is one of the substrates of luciferase. ATP concentrations can be measured by quantitating the light output from a luciferase reaction. As kinases also use ATP, it is possible to assay kinase activity through the loss of luminescence in a coupled luciferase reaction. We have applied this luminescence-based ATP depletion approach to a model serine/threonine kinase. We find that the method may be run as an endpoint assay, in which ATP detection reagents (containing luciferase and luciferin) are added at the end of the reaction, or in a kinetic mode, where the ATP detection reagents are present throughout the reaction. The ATP depletion approach is capable of detecting kinase inhibitors. Six inhibitors of the model kinase, previously identified using other screening methods, are also active in the luminescence-based approach and display a similar rank order of potency. An advantage of the method is that kinase inhibitors, because they increase luminescence (by reversing the enzyme-dependent loss of signal), are immediately distinguishable from compounds such as luciferase inhibitors and luminescence quenchers, which further reduce the luminescence. The compound collections that we screened were rich in compounds that reduced luminescence. Compounds that have dual kinase and luciferase inhibitory activity, or kinase inhibitory activity combined with luminescence quenching, might be missed by being classified as false negatives. We show that the kinetic form of the assay can be used to minimize this possibility.

Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe.

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling. Several different types of mutant were identified. Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2). Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway. Although previously shown to regulate other MAP kinase pathways in Sz. pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.