Measurement of DNA damage in rat urinary bladder transitional cells: Improved selective harvest of transitional cells and detailed Comet assay protocols

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin–EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA–protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4 h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.     

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.     

The cost of stress: Dry matter partitioning changes with seasonal supply of water and nitrogen to dryland wheat

Spring wheat cv. ‘Gutha’ was grown in continuous wheat (W/W) and narrow-leafed lupin (L. angustifolius L. cv. Yandee)-wheat (L/W) rotation on a yellow earth over mottled clay (Arenic Fragiudult) in a mediterranean climate for two years. The first year had a higher than average rainfall with adequate soil water until anthesis. The second year was very dry (only 232 mm total rainfall) and soil water contents were low throughout the growing season. Nitrogen fertilizer (+N) treatments were included in both years. In the first year an adjacent experiment compared the effects of loosening a pronounced traffic pan which existed on the site (LS)versus unloosened (US). In the first year roots contained more dry matter than tops in the early vegetative stage in all crops and then declined exponentially to a ratio of 0.1 in the US and LS treatments. In the second year however, the decline was both linear and much less, so that root:shoot ratios at harvest were still between 0.4 and 0.8. There was a consistent trend in root:shoot ratios from the most favourable (LS) to least favourable (W/W-N) treatments over the combined two years’ data, and this was also found in grain yield, with a higher yield in year one from the LS than US, and the lowest yield in year two from the W/W-N treatment. The proportion of total biomass recovered from below ground was substantially higher than is commonly reported from studies carried out in temperate, high fertility soils, but probably still under-estimates of the true amount of dry matter in roots because of inadequacies of sampling, washing and storage techniques. Root length densities were much greater in the drier year, especially in the surface 0.1-m, and based on theoretical considerations, much greater than required for extraction of available water. The effect of environmental conditions on the relative size of cereal crop carbon sinks are discussed in relation to these results.