Background: The fractional concentration of nitric oxide (NO) in exhaled breath (Fe NO ) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no direct evidence to support the assumption and data from animal models suggest that non-inducible NOS systems have important roles in determining airway reactivity, regulating inflammation, and might contribute significantly to NO measured in exhaled breath. Methods: Bronchial epithelial cells were obtained from healthy, atopic, and asthmatic children by nonbronchoscopic brushing. Exhaled NO (Fe NO ) was measured directly using a fast response chemiluminescence NO analyser. RNA was extracted from the epithelial cells and real time polymerase chain reaction was used to determine the expression of NOS isoenzymes. NOS2 was examined in macrophages and epithelial cells by immunohistochemistry. Results: NOS1 mRNA was not detectable. NOS3 mRNA was detected in 36 of 43 samples at lower levels than NOS2 mRNA which was detectable in all samples. The median Fe NO was 15.5 ppb (95% CI 10 to 18.1). There was a significant correlation between Fe NO and NOS2 expression (R = 0.672, p,0.001). All epithelial cells exhibited NOS2 staining, whereas staining in the macrophages was variable and not related to phenotype. Conclusions: Only NOS2 expression was associated with Fe NO in respiratory epithelial cells obtained from children (R = 0.672; p,0.001). This suggests that Fe NO variability is largely determined by epithelial NOS2 expression with little contribution from other isoforms.

Non-bronchoscopic brushing in children is safe and easy to perform, and is not associated with any complications. Using this technique, adequate numbers of epithelial cells can be retrieved to allow cell culture, western blotting, real time PCR, and microarray analysis. The purpose of this study is to demonstrate the utility of non-bronchoscopic airway brushing to obtain and study epithelial cells and to encourage others so that we can accelerate our knowledge regarding the role of the epithelium in childhood respiratory disease.

Nuclear size and total RNA synthesis were compared in single lumbar motoneurons isolated from the grass frog . Transcription was found to correlate significantly, but not exclusively, with nuclear area or volume over a wide range of nuclear size, the largest nuclei having the highest mean transcriptional activity . Flow cytometric analysis of propidium iodide-stained nuclei excluded polyploidy or polyteny as an explanation for the increased transcription, but left open the possibility of a small increase in DNA with increasing nuclear size . Alternatively, motoneurons may increase transcription and nuclear size without increasing their DNA content, possibly by increasing the proportion of dispersed chromatin (euchromatin) . These two mechanisms for size-related changes in RNA synthesis in motoneurons present an interesting contrast to mechanisms used by many other large animal cells .Abbreviations used: SDS, sodium dodecyl sulfate; sucrose CG, sucrose in 1.7 mM sodium citrate buffer (pH 5.0) containing 15 mM glucose.

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