CYP24A1 mutants were generated , and the presence of the desired mutations was verified by DNA sequencing. Transfection Human wild-type and mutant CYP24A1 constructs were or stably transfected into V79-4 Chinese hamster lung fibroblast cellular material transiently. For steady transfections, a targeted integration method mediated by Flippase recombination enzyme was utilized ; for transient transfections, CYP24A1 constructs containing pcDNA5/FRT were transfected into native cellular material directly, and enzyme activity was assayed. Experimental information are provided in the Supplementary Appendix. Cell Analysis and Lifestyle of CYP24A1 Activity Details of cell-tradition experiments and analyses of CYP24A1 activity are provided in the Supplementary Appendix.Gregg Fonarow, a professor of cardiology at the University of California, Los Angeles, cautioned that this study only found a link, and did not prove that high degrees of stress or depression caused heart attacks or deaths. ‘Depression and stress have previously been found to be associated with the development of cardiovascular disease and also fatal and non-fatal heart attacks and strokes in women and men already suffering from heart disease,’ he stated. This scholarly study, however, was not able to find a cause-and-effect relationship between heart disease, stress and depression, and an increased risk for heart death or attack, he said..