PubMed · 13099400
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J DANKMEIJER, R SPRUIT. 1953-09-05. [Radioautography].. https://pubmed.ncbi.nlm.nih.gov/13099400/
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We examined ultraviolet (UV) irradiation and cisplatin treatment damage formation and repair efficiency in the p53 tumor suppressor gene of various cultured cell lines and lymphocytes using a nonradioactive multiplex long quantitative polymerase chain reaction (QPCR) assay, which amplified a 7-kb fragment of the target gene and a 500-bp fragment of the template control to successfully increase the sensitivity and reliability of the assay. The multiplex long QPCR detected a lesion frequency of 0.63 lesions/10kb/10J/m(2) in the p53 gene of fibroblast cells. In addition, the multiplex long QPCR assay detected pronounced differences in the repair of UV damage in the p53 gene among repair-proficient CRL-1475 cells and repair-deficient XP-A and XP-C cells. The multiplex long QPCR assay was also evaluated as a sensitive assay for the detection of DNA damage induced by cisplatin. The data indicated that the lesion frequency in the p53 gene was 1.27-1.75 times higher in the H23 cisplatin-sensitive cell than in the H1435 cisplatin-resistant cell at the IC(70) dose. After 8-h and 24-h repair periods, only 13 and 75% of cisplatin-induced damage had been removed in the H23 cells, whereas these values were 92 and 100% in the H1435 cells. In addition, our data indicate that multiplex long QPCR is a sensitive method for validly estimating repair in freshly isolated lymphocytes. The results suggest that the current protocol of the multiplex long QPCR method can be used to assess the damage formation and repair efficiency of various agents at biologically relevant doses and to allow a more precise determination of gene-specific repair in disease susceptibility and drug resistance in epidemiological studies.
We present the first images of beta autoradiography obtained with the high-resolution hybrid pixel detector consisting of the Medipix2 single photon counting read-out chip bump-bonded to a 300 microm thick silicon pixel detector. This room temperature system has 256 x 256 square pixels of 55 microm pitch (total sensitive area of 14 x 14 mm2), with a double threshold discriminator and a 13-bit counter in each pixel. It is read out via a dedicated electronic interface and control software, also developed in the framework of the European Medipix2 Collaboration. Digital beta autoradiograms of 14C microscale standard strips (containing separate bands of increasing specific activity in the range 0.0038-32.9 kBq g(-1)) indicate system linearity down to a total background noise of 1.8 x 10(-3) counts mm(-2) s(-1). The minimum detectable activity is estimated to be 0.012 Bq for 36,000 s exposure and 0.023 Bq for 10,800 s exposure. The measured minimum detection threshold is less than 1600 electrons (equivalent to about 6 keV Si). This real-time system for beta autoradiography offers lower pixel pitch and higher sensitive area than the previous Medipix1-based system. It has a 14C sensitivity better than that of micro channel plate based systems, which, however, shows higher spatial resolution and sensitive area.
The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community.