Mechanisms of a delayed calcium uptake in mouse ischemic model

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We have reported that signal transduction pathway involving calcineurin and cydophilin D is a novel target for developing anti-ischemic drugs. For further analysis to clarify the role of both enzymes, we measured the intracellular calcium in pyramidal neuronal cells in hippocampal shoes after stimulation of hypoxia and low glucose with or without immunosuppressants cydosporin A or FK506. The results suggested that calcineurin activity might regulate the uptake of calcium through unknown calcium channel existed on plasma membranes.In addition, we found the new relation between calcineurin and hypoxia responsive factor (HIF), which was reported to be critical in inducing factors in angiogenesis, glycolytic metabolism, apoptosis, and erythrocytosis. This hypoxic factors deeply involved in mechanisms by which hypoxia played an important role in ischemic brain damage and degenerative diseases such as Parkinson disease and Alzheimer disease. In our experiments, calcineurin can stabilize the … More expression and activate the transcription of HIFs. We performed yeast two hybrid to discover novel factors regulating HIF and found novel positive clones which suppressed HIF2 transcription activity as well as expression. One of fished done identified is Int6/eIF3e, which involed in breast cancer caused by mouse mammalian tumor virus (MMTV). MMTV was integrated into genome at several integration sites and damaged the target genes. Int6 was one of the target gene. Further analysis using recombinant Int6 mutants showed that Int6 was a tumor suppressor by direct binding to HIF2 and the degradation. We demonstrated the potent angiogenesis in mouse skin after injection of si RNA which specifically suppressed HIF2. Under these results, we started analysis using mice transgened with a Int6 dominant negative mutant or siRNA expression vector.According to our specific aims in this research plan, we tried to establish a mouse ischemic model (forebrain mouse ischemic model). Two factors were critical One was the maintenance of body temperature. We equipped acryl bords surrounding microscope to keep 37℃. Another factor was selection of ischemic mouse after ligation of carotid arteries. Around 20 % of operated mice was observed to have enough blood supply through co-lateral blood flow by Doppler flow meter. It was regret that we could not evaluate PLC D1 KO mice nor NCX2 KO mice, but we plan to perform evaluation of Int6 siRNA or dominant negative Tg mice even after finishing this project. Less