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Biomedical subjects

Y Ebina

Publications and source records attributed to Y Ebina.

At least 73 records · Page 4Linked to original sources
PubMed1995

Role of the steady-state Na+ channel current in pacemaker depolarizations in young embryonic chick ventricular myocytes.

To assess the age-related changes in kinetic properties of the cardiac Na+ channel, whole-cell voltage-clamp (v-c) experiments were conducted using 3-, 10- and 17-day-old embryonic chick ventricular heart cells. In line with the first-order kinetic model, kinetic parameters for the activation and inactivation of the channel were determined from the v-c results. Simulation studies using kinetic parameters so determined have reproduced the current-voltage relations and the steady-state inactivation characteristics observed in cells in the three age groups. The rate of depolarization of the simulated action potentials was also comparable to that experimentally recorded. In conclusion, the steady-state Na+ conductance can play a significant role in the automatic depolarizations observed in young embryonic ventricular cells.

Action Potentials↗
PubMed1994

Epidermal growth factor triggers the translocation of insulin-responsive glucose transporter (GLUT4).

In a novel cell line we developed for direct, sensitive detection of insulin-responsive glucose transporter (GLUT4) on the cell surface, we considered that insulin-activated phosphatidylinositol 3-kinase (PI 3-kinase) may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report here evidence that epidermal growth factor (EGF), which stimulates PI 3-kinase activity, also triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the EGF receptor. The EGF-dependent GLUT4 translocation is possibly mediated by two independent pathways: one by PI 3-kinase and the other by protein kinase C (PKC); the PI 3-kinase-mediated pathway predominates. Triggering of the GLUT4 translocation is not specific for insulin, rather it may be a common property of growth factors which activate PI 3-kinase.

Androstadienes↗
PubMed1994

Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase.

cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaffinity-purified to near homogeneity. Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase. Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7.

Amino Acid Sequence↗
PubMed1993

Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase.

We examined the role of 185-kDa insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) in the signaling pathway of insulin-stimulated GLUT4 translocation. We had already developed a novel cell line to detect GLUT4 on the cell surface, directly and sensitively (Kanai, F., Nishioka, Y., Hayashi, H., Kamohara, S., Todaka, M., and Ebina, Y. (1993) J. Biol. Chem. 268, 14523-14526). We stably expressed a mutant insulin receptor in which Tyr972 was replaced with phenylalanine. Insulin-stimulated tyrosyl phosphorylation of IRS-1 and GLUT4 translocation were decreased in cells expressing the mutant receptor, as compared to findings in cells expressing the normal receptor. Wortmannin, an inhibitor of PI3-kinase, inhibits the insulin-stimulated PI3-kinase activity and GLUT4 translocation at 50 nM, but not the NaF-stimulated GLUT4 translocation. These results suggest that the tyrosine phosphorylation of IRS-1 and activation of PI3-kinase may be involved in the signaling pathway of the insulin-stimulated GLUT4 translocation.

Androstadienes↗
PubMed1993

Direct demonstration of insulin-induced GLUT4 translocation to the surface of intact cells by insertion of a c-myc epitope into an exofacial GLUT4 domain.

Stimulation of glucose transport is the main physiological effect of insulin in target tissues. This effect is linked to translocation of the GLUT4 glucose transporter from an intracellular pool to the cell surface. To elucidate the molecular mechanisms involved in this effect, we developed a simple direct sensitive method to detect GLUT4 immunologically on the cell surface. cDNA containing GLUT4 inserted by a c-myc epitope in the first ectodomain (GLUT4myc) was constructed without disrupting the functions of GLUT4 and was expressed in 3T3-L1 and Chinese hamster ovary fibroblast cells. In response to insulin, the GLUT4myc expressed in 3T3-L1 adipocytes was translocated to the cell surface from the intracellular pool, as shown by assays of exofacial antibody binding against the myc epitope and of the uptake of 2-deoxyglucose. Insulin, guanosine 5'-O-(3-thiotriphosphate), guanylyl imidodiphosphate, NaF, and phorbol 12-myristate 13-acetate also induced the translocation of GLUT4myc in Chinese hamster ovary cells coexpressing the human insulin receptor.

3T3 Cells↗
PubMed1993

Relationship between resting cytosolic Ca2+ and responses induced by N-methyl-D-aspartate in hippocampal neurons.

Cytosolic calcium concentrations ([Ca2+]i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca2+]i showed greater [Ca2+]i responses to N-methyl-D-aspartate (NMDA) and K+ depolarization. There was a strong relationship between resting [Ca2+]i and the maximal changes in [Ca2+]i (delta[Ca2+]i), which fit the our proposed equation to describe this relationship.

Animals↗
PubMed1993

[Gating properties of cardiac Na channels: from the macroscopic Na current viewpoint].

The sodium channel plays essential roles in the initiation and propagation of action potentials (APs) in excitable tissues including the heart, nerves, and muscles. Na channels in these tissues undergo so-called activation and then inactivation upon step-depolarizations of the cell membrane. Hodgkin and Huxley, early in the 1950s, proposed a mathematical model to describe such events, which was based on voltage-clamp (V-C) data on axonal membranes. However, for the next 30 years or so since the pioneering work of the above workers, electrophysiological studies of the Na channel kinetics in the heart had relied exclusively on AP data (Vmax) as an indirect measure of the Na current instead of V-C data due to difficulty in determining V-C from the complex geometry of cardiac tissues. However, recent development of an isolation procedure for preparing single heart cells and the use of single patch-pipettes for high resolution V-C experiments on these cells have made direct recording of Na channel currents also possible in the heart. Voltage-clamp studies carried out for the last decade have provided several lines of evidence supporting the view that the Na channel properties in the heart of any animal species are somehow more complex than in the axonal membrane and hence showing that Hodgkin-Huxley model can not be directly applied to describe the Na channel behavior in the former type of tissues. Here, we review recent results from V-C studies on Na channel properties with special reference to the macroscopic Na current in cardiac tissues.

Animals↗
PubMed1992

Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.

Animals↗
PubMed1992

Report of a patient with POEMS/Takatsuki/Crow-Fukase syndrome associated with focal spinal pachymeningeal amyloidosis.

The authors present the results of an autopsy of a 67-year-old Japanese man with POEMS/Takatsuki/Crow-Fukase syndrome (P/T/CFS) diagnosed in 1972. Each component of the syndrome was gradually recognized after the resection of lumbar vertebral solitary plasmacytoma in 1967. The patient died in 1989 of generalized infection and renal failure. Autopsy revealed in the vertebral canal between the fifth and seventh thoracic vertebrae dorsal pachymeningeal fibrosis, with prominent amyloid deposition that oppressed the spinal cord. This condition was consistent with the final neurologic manifestation of the patient, bilateral motor and sensory disturbance below the sixth thoracic level. Myelopathy remained clinically unnoticed because neurologic disturbance had begun as peripheral polyneuropathy. Normocellular marrow with heterogeneously scattered lambda light chain-positive plasma cells and degeneration of the myelinated fibers of sciatic nerve also were observed. This is the first report of focal spinal amyloidosis associated with P/T/CFS.

Adult↗
PubMed1992

Identification of two enhancer elements in the gene encoding the type 1 glucose transporter from the mouse which are responsive to serum, growth factor, and oncogenes.

The type 1 glucose transporter (GLUT1) gene encodes an integral membrane glycoprotein responsible for facilitating transfer of glucose across plasma membrane and is rapidly activated by serum, growth factors, and by oncogenic transformation. To elucidate the molecular mechanisms of regulation of GLUT1 gene expression, we isolated and characterized the mouse GLUT1 gene. DNA elements regulating transcription of the gene were analyzed in transient expression assays after transfection of NIH/3T3 cells with a low background chloramphenicol acetyltransferase (CAT) vector system pSVOOCAT. We identified two enhancer elements; the first one is located 2.7 kilobases upstream of the cap site of the gene which contains the homologous sequences with two 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs), a serum response element, a cyclic AMP-responsive element (CRE) and three GC boxes, and the second one is located in the second intron of the gene which contains the homologous sequences with two TREs and one CRE. With the promoter alone the transcription of the gene is activated by src, only slightly activated by ras and is not activated by serum and platelet-derived growth factor. When the gene is accompanied by one of these enhancers, the transcription is activated by all these stimuli.

3T3 Cells↗
PubMed1992

N-methyl-D-aspartate increases cytosolic Ca2+ via G proteins in cultured hippocampal neurons.

The changes of the cytosolic Ca2+ concentrations ([Ca2+]i) induced by N-methyl-D-aspartate (NMDA) in fura-2-loaded cultured hippocampal neurons from rat embryos were investigated by the fast application method, using a fine pipe under extracellular Mg(2+)-free conditions. In the presence of Ca2+, NMDA, at concentrations in excess of 3 microM, induced a biphasic increase of [Ca2+]i, which consisted of an initial increase with a second rise that occurred after cessation of drug application. Under Ca(2+)-free conditions, NMDA (greater than 100 microM) in the absence of glycine or NMDA (greater than 50 microM) in the presence of glycine (greater than 10 microM) induced intracellular Ca2+ mobilization, which was blocked by 30 microM 2-amino-5-phosphonovaleric acid (APV) and reduced by islet-activating protein. When the neurons were superfused with Ca(2+)-free solution, the application of 3-10 microM NMDA, which had been dissolved in Ca(2+)-containing solution, induced the second phase [Ca2+]i increase, whereas application of kainate, quisqualate, or stimulation by 50 mM K+ did not. Islet-activating protein, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and D-sphingosine reduced the second phase [Ca2+]i increase. These results suggest that NMDA-induced intracellular Ca2+ mobilization is potentiated by the initial entry of Ca2+ into the cells and is regulated in an islet-activating protein-sensitive manner.

Animals↗
PubMed1991

Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase.

Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo.

Animals↗
PubMed1991

A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene.

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.

Animals↗
PubMed1991

Impairment of bone formation with aluminum and ferric nitrilotriacetate complexes.

The deleterious effects of aluminum(AL) and iron(Fe) on bone formation were studied in the presence of nitrilotriacetate (NTA) as a chelator. Both Al-NTA (1.0-1.5 mg Al/kg/day, n = 12)- and ferric nitrilotriacetate (Fe-NTA) (2.0 mg/kg/day, n = 4)-treated Wistar rats showed renal insufficiency blood urea nitrogen [BUN] levels of 25 +/- 8.8-20 +/- 0.7 compared to 12 +/- 0.7-11 +/- 0.4 mg/dl), osteomalacia with a relative osteoid volume of 31.5 +/- 5.6-13.2 +/- 2.4 compared to 4.6 +/- 1.8-0.83 +/- 0.12%, and bone growth retardation (3.1 +/- 0-3.0 +/- 0.2 compared to 3.4 +/- 0-3.3 +/- 0.1 cm) in 24 control rats. Dietary vitamin E(VE) supplementation prevented the Fe-NTA-induced impairment, but not the Al-NTA toxicity. Aluminum was deposited at the interface between osteoid and mineralized bone, while Fe was deposited in the osteoblasts and osteoclasts. There seems to be a positive correlation between hypophosphatemia and osteomalacia but carboxy-terminal parathyroid hormone (C-PTH) and calcium (Ca) levels in the serum were not related to the degree of osteomalacia. Administration of Al-NTA results in more bone Al deposition than that of aluminum chloride (AlCl3) (450 +/- 40 compared to 211 +/- 18 mg/kg fat-free dry weight). The Fe-NTA bone change is related to VE-preventable cellular injury, being consistent with the notion that Fe-NTA toxicity is caused by lipid peroxidation. Al-NTA can be used as an animal model of renal osteodystrophy. Osteodystrophy by Al in chronic renal failure may be mediated by the intrinsic chelator or chelating substance(s) retained in the body fluid due to renal insufficiency.

Aluminum↗
PubMed1990

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene.

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

Adolescent↗