ROCK inhibitor, Y-27632, reduces FBS-induced structural alteration in organ-cultured mesenteric artery

Background Chronic treatment with fetal bovine serum (FBS) causes gradual vasoconstriction, vascular wall thickening, and contractility reduction in organ-cultured vascular tissues. We have previously demonstrated that Rho-associated kinase (ROCK) inhibitors prevent the functional alterations of small arteries in response to the FBS treatment. Here, we tested a further hypothesis that the chronic inhibition of ROCK has a protective effect on FBS-induced structural alterations. Methods To verify the new hypothesis, the rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor and then western blot, immunohistochemistry, apoptosis assay, and electron microscopy were performed using organ-cultured arterial rings. Results Chronic treatment with Y-27632 maintained the arterial diameter by preventing FBS-induced gradual arterial constriction during organ culture. Y-27632 also reduced the apoptosis and the loss of contractile myosin and actin filaments of smooth muscle cells. In addition, Y-27632 protected the morphological integrity between the endothelial cell layer and smooth muscle cell layer by preventing endothelial cell detachment and platelet endothelial cell adhesion molecule (PECAM) expression decrement. Conclusions Chronic ROCK inhibition provides protective effects against FBS-stimulated structural in addition to functional alterations of vascular smooth muscle cells and endothelial cells. These results strongly suggest that the RhoA/ROCK signaling is crucial for maintaining the structural and functional phenotypes of vasculature, and hence, chronic ROCK inhibition may provide protective effects on excessive growth factor-related vascular diseases including hypertension and atherosclerosis

e-Learningにおけるポートフォリオ評価法の動向とその応用

本研究の目的は、ポートフォリオ評価法の理論的枠組みと、ポートフォリオ評価法をe-Learningに導入した研究の知見を概観し、ポートフォリオ評価法を応用したe-Learning環境のデザインについて考察することにある。ポートフォリオ評価法の研究は勃興期に位置づけられる。多くの研究が学習者中心型アプローチの立場から、ポートフォリオ評価法をe-Learning環境に導入するフレームワークを検討し、一部の研究はその効果を検証している段階にある。ポートフォリオを作成することや、そのための支援ツールの実装に注目が集まっている。ポートフォリオ評価法の理念に立ち返れば、学習者と熟達者間、あるいは学習者相互によるポートフォリオの検討やその評価、学習活動の改善に向けた内省や計画立案といった学習活動を行うためのコミュニケーション環境を充実させることが重要である。また、様々な学習方法に対応して、その学習プロセスに対する内省を促進するようなポートフォリオ作成を支援することが、e-Leamingにおける学習環境デザインに必要である。The aim of this paper is to examine the design of learning environments involving portfolio assessment applied in e-learning with recent studies and frameworks. Research in this field is largely in its early stages, and the study of schemes for utilizing portfolio assessment for e-learning takes the learner-centered approach. Some studies have made progress by taking account of new frameworks for portfolio utilization. The authors examine the appropriate learning environments for e-learning using portfolio assessment from the viewpoint of assessment theory and philosophy. In conclusion, an adequate communication environment between learners and experts is required for the promotion of learning. Such communication includes portfolio conferences, evaluation of learner portfolios and reflective planning for learning improvement. The design of portfolios appropriate to the instructional design is also needed in order to promote learner awareness of the learning process

Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction.

A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions. Also, the knowledge is expected to facilitate devising innovative strategies that more precisely monitor and normalize organ functions by targeting individual smooth muscles. Evidence has established a current paradigm that the myosin light chain phosphatase (MLCP) is a master regulator of smooth muscle responsiveness to stimuli. Cellular MLCP activity is negatively and positively regulated in response to G-protein activation and cAMP/cGMP production, respectively, through the MYPT1 regulatory subunit and an endogenous inhibitor protein named CPI-17. In this article we review the outcomes from two decade of research on the CPI-17 signaling and discuss emerging paradoxes in the view of signaling pathways regulating smooth muscle functions through MLCP

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogen-targeting subunit (G(M)) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1·PP1C·P-CPI-17, leading to an increase in smooth muscle contraction