Translocation of PKC, Protein Phosphatase Inhibition and Preconditioning of Rabbit Cardiomyocytes

This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions. Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells, the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta. Preconditioning with adenosine (100 μM) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of the PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a post-incubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug, The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro

The Ombudsman: Verification of Citations: Fawlty Towers of Knowledge?

The prevalence of faulty citations impedes the growth of scientific knowledge. Faulty citations include omissions of relevant papers, incorrect references, and quotation errors that misreport findings. We discuss key studies in these areas. We then examine citations to “Estimating nonresponse bias in mail surveys,” one of the most frequently cited papers from the Journal of Marketing Research, to illustrate these issues. This paper is especially useful in testing for quotation errors because it provides specific operational recommendations on adjusting for nonresponse bias; therefore, it allows us to determine whether the citing papers properly used the findings. By any number of measures, those doing survey research fail to cite this paper and, presumably, make inadequate adjustments for nonresponse bias. Furthermore, even when the paper was cited, 49 of the 50 studies that we examined reported its findings improperly. The inappropriate use of statistical-significance testing led researchers to conclude that nonresponse bias was not present in 76 percent of the studies in our sample. Only one of the studies in the sample made any adjustment for it. Judging from the original paper, we estimate that the study researchers should have predicted nonresponse bias and adjusted for 148 variables. In this case, the faulty citations seem to have arisen either because the authors did not read the original paper or because they did not fully understand its implications. To address the problem of omissions, we recommend that journals include a section on their websites to list all relevant papers that have been overlooked and show how the omitted paper relates to the published paper. In general, authors should routinely verify the accuracy of their sources by reading the cited papers. For substantive findings, they should attempt to contact the authors for confirmation or clarification of the results and methods. This would also provide them with the opportunity to enquire about other relevant references. Journal editors should require that authors sign statements that they have read the cited papers and, when appropriate, have attempted to verify the citations