Molecular Biology

For continuous culturing, MEF cultures were split: or. After h, the media was replaced with a second batch of viral supernatant and the cells were split hlater and harvested at the indicated time points.Pulsefield gel electrophoresis and ingel detection of telomeric DNA.Clamped homogenous electric field gel electrophoresis of mouse DNA was performed essentially as described previously.Cells were resuspended in PBS and mixed: to obtain cells per agarose plug, digested with mg ml proteinase K in TENS buffer and washed extensively in TE buffer. Following digestion, plugs were washed in TE and equilibrated in. TBE for hwith the following settings: initial pulse, min; final pulse, min; V cm at C.Ingel hybridization with a PATP endlabelled oligonucleotide and subsequent denaturation and hybridization steps were performed as described.Cells grown on coverslips were fixed in paraformaldehyde, permeabilized in. Jacks for p mice.The incidence of chromosome end fusions with telomeric signals was noted on the indicated number of metaphases.NATURE. COM NATURECELLB IOLOGY This technique was used to study peripheral blood cells from three volunteers after physical activity.The test subjects had to run on a treadmill and were checked for blood pressure and ECG, lactate concentration and creatine kinase activity.Blood was taken before and several times during and after the run.In a first multiple step test, the volunteers ran as long as possible with increasing speed.In a second test they had to run for min with a fixed individual speed which was defined to ensure an aerobic metabolism.In the first test, the white blood cells of all subjects showed increased ONA migration in the SCG assay.The effect was seen hafter the end of the exercise and reached its maximum hlater.After h, DNA migration decreased to about control level.The distribution of DNA migration among cells clearly demonstrated that the majority of white blood cells exhibited increased DNA migration and that the effect was not only due to a small fraction of damaged cells.From the same blood samples, blood cultures were set up to study a possible effect on the frequency of sister chromatid exchanges, another indicator for genotoxic effects.However, there was no significant increase in SCE in any of the cultures.In the second exercise, during aerobic metabolism, the effect on DNA migration was not seen.Thus, these preliminary results indicate that physical activity above the aerobicanaerobic threshold causes alterations in white blood cells which can be detected as DNA migration in the SCG assay.Although the biological significance of this effect is not yet clear, our results may have implications for the use of the SCG assay in biomonitoring and underline the necessity to carefully evalute the physiological state of the persons under study.This assay has already been used in both in vitro and in vivo studies to investigate DNA damage and repair induced by various agents in a variety of mammalian cells. In die course of biomonitoring studies in our laboratories, we repeatedly found unexpected effects in the SCG test with a control person.There were no indications of exposure to genotoxic agents or any health problem.The only obvious association was a sports activity on the day before the test was performed.

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