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Biomedical subjects

E Miyamoto

Publications and source records attributed to E Miyamoto.

At least 109 records · Page 6Linked to original sources
PubMed1996

Cyclosporin A stimulation of glucose-induced insulin secretion in MIN6 cells.

Effects of the immunosuppressant cyclosporin A (CsA), a specific inhibitor of Ca2+/calmodulin-dependent protein phosphatase (PP2B), were examined with regard to the induction of insulin secretion from MIN6 cells, a glucose-responsive cell line derived from mouse insulinoma. CsA had no effect on basal insulin secretion from MIN6 cells, but did increase glucose-, tolbutamide-, and KCl-induced insulin secretion. Treatment of the cells with CsA resulted in a dose-dependent increase in insulin secretion, which was maximal at 3 microM. CsA inhibited PP2B activity in a dose-dependent manner, and the increase in insulin secretion correlated with the decrease in PP2B activity. In 32P-labeled cells, treatment with CsA for 30 min increased phosphorylation of synapsin I-like protein by 50 +/- 5.7%. As revealed by one-dimensional phosphopeptide mapping of 32P-labeled synapsin I-like protein, treatment with CsA for 30 min increased phosphorylation of site II of synapsin I-like protein by 59 +/- 8%, which is phosphorylated by calmodulin kinase II. Messenger RNAs, which hybridize with complementary DNAs of calcineurin A and B subunits from rat brain, were detected in MIN6 cells. Western blot analysis showed a 61-kDa band, which interacts with rat brain calcineurin A antibody. Similar increases in secretagogue-induced insulin secretion with CsA were observed for HIT-T15 cells. These results suggest that CsA stimulates glucose-induced insulin secretion by inhibiting the activity of PP2B, an event that may be involved in mechanisms governing glucose-induced insulin secretion via dephosphorylation of synapsin I-like protein in MIN6 cells.

Animals↗
PubMed1996

[Evaluation of the training of the anesthesiologist].

In order to evaluate both the knowledge and the degree of competence acquired by anesthesiology trainees, we constructed a checklist and began using it in January 1994. Twenty-one trainees participated in a 3-month program ending in February 1995. By means of the checklist, we attempted to evaluate the extent of their individual attainment. The total number of anesthesia administered was 50.9 +/- 7.8 cases. The total anesthesia time was 212.0 +/- 32.8 hours, and 76.2 % of the anesthesia methods applied involved either nitrous oxide-sevoflurane or nitrous oxide-isoflurane. Using a 5-level rating scale, the self-assessment by the supervising doctor tended to be one point higher than the assessment by the trainees. We plan to improve this checklist to improve the results of training.

Anesthesia, Inhalation↗
PubMed1995

In situ dephosphorylation of tau by protein phosphatase 2A and 2B in fetal rat primary cultured neurons.

Using antibodies recognizing the phosphorylation state of specific sites, phosphorylation states of tau were monitored in fetal rat primary cultured neurons. When cultured neurons were treated with okadaic acid (OA) or calyculin A (CalA) at concentrations sufficient to inhibit protein phosphatase 2A (PP2A), phosphorylation of Ser-199/Ser-202 (numbered according to the human tau 441) and Ser-235 increased. On the other hand, treatment with Ca2+ ionophore, A23187, induced dephosphorylation of Ser-199/Ser-202, Thr-205, Ser-396 and Ser-404, and this dephosphorylation was repressed by inhibitors of protein phosphatase 2B (PP2B), cyclosporin A and FK506. These results indicate that PP2A and PP2B are differentially involved in dephosphorylation of tau in neurons.

Animals↗
PubMed1995

Calcium/calmodulin-dependent protein kinase II and protein phosphatase 2B (calcineurin) immunoreactivity in the rat hippocampus long after ischemia.

Calcium/calmodulin-dependent protein kinase II (kinase II) and protein phosphatase 2B (calcineurin) immunoreactivity in the rat hippocampus was studied 100 days after ischemic damage to hippocampal CA1 pyramidal neurons. One-hundred days after ischemia, only a few CA1 pyramidal neurons survived and they exhibited enhanced kinase II and calcineurin immunoreactivity in their basal and apical dendrites. The stratum lucidum of the CA3 (mossy fiber terminal area) had enhanced kinase II and calcineurin immunoreactivity. These results suggest activity-dependent regulation and redistribution of kinase II and calcineurin after intervention in neuronal circuitry.

Animals↗
PubMed1995

Preparation of tau from the peripheral nerve: presence of insoluble low molecular weight tau with high phosphorylation.

Purely axonal tau protein of the peripheral nervous system (PNS) obtained from adult rat ventral roots was composed of both high (HMW) and low molecular weight (LMW) isoforms. While the PNS-specific HMW isoform (110 kDa) was soluble, 60-70% of the LMW isoforms with apparent molecular weights of 67 kDa, 62 kDa and 58 kDa was insoluble. When analyzed by two-dimensional electrophoresis, these axonal LMW isoforms corresponded to the most acidic species among the large number of isoforms found in brain microtubule-associated tau. Immunoreactivities towards phosphorylation-dependent antibody tau-1 and the two anti-phosphopeptide antibodies (PP1 and PP2) indicate that PNS axonal tau is highly phosphorylated at Ser190, Ser193, and Ser387, which are the sites shown to be phosphorylated in fetal brain tau and tau comprising the paired helical filaments of Alzheimer's disease.

Animals↗
PubMed1995

Increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in the induction of long-term potentiation.

Induction of long-term potentiation in the CA1 region of hippocampal slices is associated with increased activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D. (1993) J. Biol. Chem. 268, 7863-7867). Here we report that application of high but not low frequency stimulation to two groups of afferents in the CA1 region of 32P-labeled slices resulted in the phosphorylation of two major substrates of this enzyme, synapsin I and microtubule-associated protein 2, as well as in the autophosphorylation of CaM kinase II. Furthermore, immunoblotting analysis revealed that long term potentiation induction was associated with an increase in the amount of CaM kinase II in the same region. All these changes were prevented when high frequency stimulation was applied in the presence of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These results indicate that activation of CaM kinase II is involved in the induction of synaptic potentiation in both the postsynaptic and presynaptic regions.

Animals↗
PubMed1995

Augmented expression of atrial myosin light chain 1 in ventricular aneurysms of human: enzyme immunoassay for atrial myosin light chain 1.

We established an enzyme immunoassay (EIA) for atrial myosin light chain 1 (ALC1) using monoclonal antibodies KA1 and KB1, which were specific for ALC1 and for both ALC1 and ventricular myosin light chain 1, respectively. The serum ALC1 levels of healthy subjects were 0.28 +/- 0.14 ng/ml (mean +/- SD). The tissue ALC1 levels of normal adult human atria were much higher than those of ventricles (p < 0.01, 2,120 +/- 1,200 in right atria, 2,180 +/- 1,450 in left atria vs. 36.0 +/- 20.2 in right ventricles, 37.7 +/- 15.3 in left ventricles, ng/mg of proteins). The tissue ALC1 levels of ventricular aneurysms were significantly higher than those of normal ventricles (p < 0.01, 206.7 +/- 101.8). These results indicate that ALC1 is augmented in aneurysms and that the EIA provides a useful tool to investigate the roles of ALC1.

Adult↗
PubMed1995

Stimulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase with brain gangliosides.

The holoenzyme of cAMP-dependent protein kinase (cAMP-kinase) partially purified from the particulate fraction of rat brain was stimulated by gangliosides. Among various gangliosides tested, GM1 was most potent, giving Ka value of 19.5 microM. The maximal activation of the kinase was obtained with 100 microM GM1 using kemptide as substrate. Gangliosides inhibited the kinase activity of the catalytic subunit of cAMP-kinase. Of various substrates tested, the ganglioside-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I and myelin basic protein, but not histone H1 and casein. The molecular mechanisms of the stimulatory effect of gangliosides were investigated. The kinase activated with GM1 was inhibited by the addition of PKItide, a specific inhibitor for cAMP-kinase. However, GM1 did not dissociate the holoenzyme into the catalytic and regulatory subunits and did not interfere with the binding ability of cAMP to the holoenzyme. These results suggest that the gangliosides can directly activate cAMP-kinase in a different manner from cAMP.

Animals↗
PubMed1995

Dephosphorylation of abnormal sites of tau factor by protein phosphatases and its implication for Alzheimer's disease.

The abnormally phosphorylated forms of tau factor are major constituents of neurofibrillary tangles in Alzheimer's disease brain. In order to investigate protein phosphatases which are related to dephosphorylation of abnormal phosphorylation sites, we examined the dephosphorylation of tau factor phosphorylated by three proline-directed type protein kinases. Tau factor phosphorylated by cdc2 kinase and tau protein kinase II was dephosphorylated by the holoenzyme of protein phosphatase 2A and calcineurin, while either the catalytic subunit of protein phosphatase 2A or protein phosphatase 2C could not catalyze the dephosphorylation. From the kinetic analysis, we concluded that tau factors phosphorylated by the protein kinases serve as good substrates for protein phosphatase 2A and calcineurin. On the other hand, tau factor phosphorylated by glycogen synthase kinase 3 alpha was dephosphorylated by the catalytic subunit of protein phosphatases 2A as well as the holoenzyme of protein phosphatase 2A and calcineurin. It has been reported that serines 199, 202 and 396 according to the numbering of the longest human tau isoform are among the major abnormal phosphorylation sites of tau factor. We synthesized two phosphopeptides which contained phosphoserines 199 and 202 or phosphoserine 396 and prepared the polyclonal antibodies specific for the phosphopeptides. Using these antibodies, we confirmed that the holoenzyme of protein phosphatase 2A and calcineurin could dephosphorylate phosphoserines 199, 202 and 396 in tau factor. The catalytic subunit of protein phosphatase 2A could dephosphorylate phosphoserine 396 but not phosphoserines 199 and 202. Neurofibrillary tangles in Alzheimer's disease brain were immunostained with both antibodies but the normal neurons in the normal aged brains were not. The results suggest that protein phosphatase 2A and calcineurin can be involved in the dephosphorylation of abnormal phosphorylation sites in tau factor and that the dephosphorylation of phosphoserine 396 is differently regulated from phosphoserines 199 and 202.

Aged↗
PubMed1995

Glutamate-induced loss of Ca2+/calmodulin-dependent protein kinase II activity in cultured rat hippocampal neurons.

The exposure of cultured rat hippocampal neurons to 500 microM glutamate for 20 min induced a 55% decrease in the total Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca(2+)-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N-methyl-D-aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca2+ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.

Animals↗
PubMed1995

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by proline-directed protein kinases and its dephosphorylation.

We identified two major substrates for the proline-directed protein kinases--cdc2 kinase and tau protein kinase II (TPKII)--in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80-kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine-rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80-kDa protein and purified MARCKS, we concluded that the 80-kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two-dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline-directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.

Animals↗
PubMed1995

Activation of mitogen-activated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors.

Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca2+ ionophore, remained intact. Lavendustin A, a tryrosine kinase inhibitor, was without effect. In experiments with 32P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c-raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca(2+)-dependent pathways and that the activation of Raf-1 is not involved.

Animals↗
PubMed1995

Dephosphorylation of fetal-tau and paired helical filaments-tau by protein phosphatases 1 and 2A and calcineurin.

We have reported that many sites of tau in fetal brain (fetal-tau) as well as in paired helical filaments (PHF-tau) are phosphorylated. In the present study, we used site-specific antibodies and peptide mapping to examine protein phosphatases involved in dephosphorylation of fetal-tau and PHF-tau. Immunoblot analysis and electrophoretic mobility showed that protein phosphatases 1 and 2A and calcineurin could dephosphorylate fetal-tau and PHF-tau. Phosphoserines 199, 202, 396, and 413 and phosphothreonine 231, numbered according to the longest human tau isoform, were dephosphorylated, as shown by the immunoblot analysis. Phosphoserine 422 was dephosphorylated by protein phosphatase 2A and calcineurin, but not by protein phosphatase 1. Peptide mapping with Achromobacter lyticus protease 1 showed that phosphoserines 199, 202, 235, and 396 and phosphothreonine 231 were dephosphorylated by protein phosphatases. Fetal-tau was more rapidly dephosphorylated by protein phosphatase 2A and calcineurin than PHF-tau. Interestingly, PHF-tau which had not been solubilized with guanidine HCl was little dephosphorylated by protein phosphatases. Thus, PHF-tau in neurofibrillary tangles of Alzheimer's disease brain is likely to be resistant to dephosphorylation by protein phosphatases.

Amino Acid Sequence↗
PubMed1995

Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.

Animals↗
PubMed1995

Modifications of neurofilament proteins by possible metabolites of allyl chloride in vitro.

Chronic exposure to allyl chloride (ALL) is known to produce a central-peripheral distal axonopathy. In relation to the mechanism(s), the present study was conducted to examine the abilities of ALL and its putative metabolites, i.e., epichlorohydrin, glycerol alpha-monochlorohydrin, allyl alcohol and acrolein to cross-link proteins in vitro. Neurofilament-riched cytoskeletal proteins (1mg/ml) and ovalbumin (10mg/ml) were incubated with 160 mM tested chemicals except for acrolein at 0.5 mM and 1 mM. Time-dependent studies by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that not only ALL, but also acrolein and epichlorohydrin exerted chemical modifications on axonal cytoskeletal proteins; while only acrolein-treated ovalbumin could manifest evidence of polymerization of the protein. Immunoblotting of PAGE-separated proteins confirmed that the high molecular weight proteins on the top of SDS-PAGEs were NF antigen-contained covalent cross-linked materials.

1-Propanol↗
PubMed1995

Increased angiotensin converting enzyme activity in left ventricular aneurysm of patients after myocardial infarction.

OBJECTIVE: Angiotensin converting enzyme (ACE) inhibitors have been shown to improve left ventricular dysfunction and survival in patients with chronic myocardial infarction. The aim of this study was to examine the ACE activity in infarcted tissues in such patients in comparison with non-diseased tissues from control subjects obtained at necropsy. METHODS: ACE activity was measured in the left ventricles and right atrial auricles of patients (n = 9) with chronic myocardial infarction obtained at left ventricular aneurysmectomy, and in the hearts of control subjects at necropsy (n = 10). RESULTS: In non-diseased hearts, the ACE activity was highest in right atria and auricles [2.4(SEM 0.2), 2.2(0.3) nmol.mg-1 protein.min-1, NS, respectively], followed by left atria [1.7(0.2)], left auricles [1.5(0.1)], right ventricles [1.0(0.2)], and left ventricles [0.5(0.1)]. The ACE activity was significantly increased in aneurysmal tissues of patients with chronic myocardial infarction relative to left ventricles of control subjects [4.2(0.4) v 0.5(0.1) nmol.mg-1 protein.min-1, P < 0.01]. There was, however, no difference in the ACE activity of right atrial auricles between patients with chronic myocardial infarction and control subjects [2.8(0.5) v 2.2(0.3), NS]. In patients with chronic myocardial infarction, the ACE activity was higher in left ventricles than in right auricles (P < 0.01). The ACE activities in the infarcted and control ventricles were negatively correlated with the membrane protein content (r = -0.77, P < 0.01). CONCLUSIONS: In non-diseased human hearts, the ACE activity is higher in atria than in ventricles and higher in the right than in the left ventricle. Furthermore, the ACE activity in aneurysmal left ventricular tissue after myocardial infarction is higher than in non-diseased left ventricular myocardium. These results suggest that the local ACE in the human heart may play an important role in the pathophysiological state after myocardial infarction.

Adult↗