Phosphorylation State of hsp27 and p38 MAPK During Preconditioning and Protein Phosphatase Inhibitor Protection of Rabbit Cardiomyocytes

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit. Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylaled. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation. SB203580 accelerated ischemic hsp27 dephosphorylation and injury. IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (\u3e 60-90 min) ischemia

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