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Amy Wang John L. Robertson Steven D. Holladay Alan H. Tennant Andrea J. Lengi S. Ansar Ahmed William R. Huckle Andrew D. Kligerman 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2007,634(1-2):51-59
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. 相似文献
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Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium. 总被引:2,自引:0,他引:2
A Gupta B A Whitton A P Morby J W Huckle N J Robinson 《Proceedings. Biological sciences / The Royal Society》1992,248(1323):273-281
Metal-tolerant cyanobacteria have been isolated from metal-polluted aquatic environments and also selected in culture, but no genes which confer metal tolerance have been described. To investigate the possibility that amplification of a prokaryotic metallothionein gene (smtA), or rearrangement of the smt locus, could be involved in the development of Cd tolerance in Synechococcus PCC 6301, Cd-tolerant lines were selected by stepwise adaptation of a Synechococcus culture. An increase in smtA gene copy number and the appearance of unique additional smtA restriction fragments (both larger and smaller) were detected in these tolerant lines (tolerant to 0.8 microM Cd, 1.3 microM Cd and 1.7 microM Cd). Stepwise adaptation was repeated by using a culture of Synechococcus PCC 6301 inoculated from a single plated colony to obtain four new lines (tolerant to 1.4 microM Cd, 1.8 microM Cd, 2.6 microM Cd and 3.2 microM Cd). Amplification of the smtA gene and development of unique smtA restriction fragments (larger and smaller) were once again detected in these tolerant lines. Amplification and rearrangement of the smt locus were only detected in the seven Cd-tolerant lines, with no evidence of amplification or rearrangement in the non-tolerant lines from which they were derived. As a control, another gene, psaE, was also monitored in these cell lines. There was no evidence of amplification or rearrangement of psaE in the non-tolerant or any of the Cd-tolerant lines. 相似文献
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