Biomedical subjects

M Kasuga

Publications and source records attributed to M Kasuga.

At least 109 records · Page 6Linked to original sources

PubMed1998

Local expression of immunoregulatory IL-12p40 gene prolonged syngeneic islet graft survival in diabetic NOD mice.

Local production of immunosuppressive cytokines will be one of the most suitable therapeutic strategies against organ-specific autoimmune diabetes. To establish such a new therapy, we constructed recombinant adenoviral vectors with inserted mIL-12p40 (Ad.IL-12p40) and mIL-10 (Ad.IL-10). Sufficient amounts of IL-12p40 and IL-10 were secreted by relevant adenovirus-transfected nonobese diabetic (NOD) islets. Shortly after transfection, 400 NOD islets transfected with Ad.IL-12p40 or Ad.IL-10 were transplanted under the renal capsule of a newly diabetic NOD mouse. NOD mice with IL-12p40-producing islet grafts kept normoglycemia in all of 14 grafted mice for over 4 wk after transplantation. In contrast, NOD mice with IL-10-producing islet grafts became diabetic in all of six grafted mice within 2 wk af-ter transplantation. Reverse transcription-PCR analysis revealed that local production of IL-12p40 led to the decrease of interferon-gamma and the augmentation of transforming growth factor-beta at the graft site. These results suggest that IL-12 plays an important role in the destruction of islet cells at the inflamed site of autoimmunity. Such a local blockade of IL-12 would be a useful gene therapy for human autoimmune diabetes.

Adenoviridae↗

PubMed1998

Posttranscriptional control of adipocyte differentiation through activation of phosphoinositide 3-kinase.

Differentiation of adipocytes is an important aspect of energy homeostasis. Although the transcriptional regulation of adipocyte differentiation is relatively well characterized, the subsequent molecular events remain unclear. The activity of phosphoinositide (PI) 3-kinase precipitated with antibodies to phosphotyrosine has now been shown to increase transiently during adipocyte differentiation of 3T3-F442A and of 3T3-L1 cells. PI 3-kinase activity precipitated with antibodies to insulin receptor substrate 1 (IRS1) and association of subunits of PI 3-kinase with IRS1 were also increased at this stage of differentiation, suggesting that IRS1 contributes to PI 3-kinase activation. Inhibition of the activation of PI 3-kinase by expression of dominant negative mutant subunits of the enzyme prevented adipogenesis, as assessed by lipid accumulation and expression of key adipocyte proteins such as GLUT4, adipsin, and aP2, suggesting that PI 3-kinase activation is essential for adipocyte differentiation. However, these mutant proteins did not affect either the expression of the transcription factor PPARgamma at the mRNA or protein level or the increase in the abundance of mRNAs encoding the adipocyte marker proteins. These results demonstrate that adipocyte differentiation is regulated at the posttranscriptional level and that activation of PI 3-kinase is required for this regulation.

3T3 Cells↗

PubMed1998

An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression.

In higher plants, a cis-acting element, DRE/CRT, is involved in gene expression responsive to drought and low-temperature stress. To understand signal transduction pathways from the cold stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB1A and CBF1 in Arabidopsis thaliana. DREB1A and CBF1 were shown to be involved in low-temperature-responsive gene expression. We screened an Arabidopsis genomic DNA library with the cDNA fragment of DREB1A as a probe and isolated DREB1A and 2 related genes, DREB1B (= CBF1) and DREB1C. These were arrayed in the order B, A, C in an 8.7 kb region of Arabidopsis chromosome 4. Northern blot analysis using gene-specific probes showed that the 3 DREB1 genes are induced mainly by cold stress but not by osmotic stress in leaves, roots, and stems. Several conserved sequences were found in the promoter regions of all 3 genes. The beta-glucuronidase (GUS) reporter gene driven by the DREB1 promoters was induced at transcriptional level by low temperature in transgenic Arabidopsis plants.

Amino Acid Sequence↗

PubMed1998

Requirement of the serine-threonine kinase Akt for heat treatment-induced activation of p70 S6 kinase.

p70 S6 kinase plays an important role in growth factor-induced translational control and in cell cycle progression. Although the mechanism of p70 S6 kinase regulation is not fully understood, phosphorylation of serine and threonine residues of the enzyme is essential for its activation. The possible role of the serine-threonine kinase Akt in the activation of p70 S6 kinase induced by exposure of cells to heat has now been investigated. Overexpression of a mutant Akt1 (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine was shown to exert a dominant negative effect on Akt activation induced by platelet-derived growth factor (PDGF) or by heat treatment in CHO cells. Akt-AA also inhibited p70 S6 kinase activation induced by these stimuli. However, Akt-AA had no effect on the activation of p70 S6 kinase induced by 12-O-tetradecanoylphorbol 13-acetate, which did not stimulate Akt activity in these cells. These data suggest that Akt is required for heat treatment-induced activation of p70 S6 kinase.

Adenoviridae↗

PubMed1998

Inhibition of insulin-induced GLUT4 translocation by Munc18c through interaction with syntaxin4 in 3T3-L1 adipocytes.

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.

3T3 Cells↗

PubMed1998

Endomorphins have orexigenic and anxiolytic activities in mice.

This study was designed to investigate the effects of endomorphin 1 and 2, recently identified mu-opioid receptor selective peptides, on food intake and anxiety in non-food-deprived mice. The intracerebroventricular (i.c.v.) injection of either endomorphin 1 or 2 (0.03-30 nmol) increased food intake in a dose-related manner. A significant increase was observed 20 min after i.c.v. injection of endomorphin 1 or 2 and continued for 4 h. In the elevated plus maze test, the i.c.v. injection of endomorphin 1 (30 nmol) significantly decreased the normal preference for the closed arms. These results suggest that endomorphin produces orexigenic and anxiolytic effects, and that the mu-opioid receptor contributes to the regulation of feeding and anxiety in mice.

Animals↗

PubMed1998

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase.

SHPS-1 is an approximately 120 kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.

ADP Ribose Transferases↗

PubMed1998

Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases.

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.

Animals↗

PubMed1998

Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation.

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.

Amino Acid Sequence↗

PubMed1998

Effect of the Kobe earthquake on stress and glycemic control in patients with diabetes mellitus.

OBJECTIVE: To examine the effects of the Kobe, Japan, earthquake, a life-threatening event, on stress and glycemic control in diabetic patients. PATIENTS AND METHODS: Hemoglobin A1c levels before and after the earthquake were evaluated in diabetic patients in Kobe (N = 157; magnitude, 7.2) and in Osaka, Japan, as a control (N = 277; magnitude, 4.2), where little damage to houses and traffic facilities occurred. Glycosylated hemoglobin levels were also compared with those of 2 years before and 1 year after the earthquake. The General Health Questionnaire (GHQ) and a self-administered questionnaire regarding damage to houses and relatives killed or injured were used to assess psychological and mental stresses on earthquake survivors. RESULTS: Glycemic control was aggravated in diabetic patients after the earthquake in Kobe but not in Osaka. THe GHQ scores were significantly higher in the patients in Kobe than those in Osaka. Increased hemoglobin A1c concentrations and high scores on the GHQ were especially evident in diabetic patients with severe damage to houses and/or with relatives killed or injured. CONCLUSION: These results suggest an association between chronic, life-threatening stress and the worsening of metabolic control in patients with diabetes mellitus.

Adult↗

PubMed1998

Administration of IL-4 prevents autoimmune diabetes but enhances pancreatic insulitis in NOD mice.

The present study demonstrated that the administration of recombinant interleukin-4 (rIL-4) prevented overt diabetes in nonobese diabetic (NOD) mice whose T cells produced relatively low amounts of IL-4. However, massive insulitis was observed in rIL-4-treated NOD mice. The flow cytometric analysis of islet-infiltrating T cells revealed that the number of CD45RBlowCD4+ T cells was significantly increased by in vivo administration of rIL-4. By measuring the cytokine production of splenic T cells after stimulation, it was shown that CD45RBlowCD4+ T cells predominantly produced IL-4 and IL-10 but produced less IL-2 and interferon-gamma (IFN-gamma). A semiquantitative reverse-transcriptase polymerase chain reaction assay revealed a higher expression of IL-4 and IL-10 mRNA and an apparent decrease in IFN-gamma mRNA in the islets of NOD mice which were administered rIL-4. These results suggested that autoreactive CD45RBlowCD4+ T helper 2 (Th2)-like cells which developed following rIL-4 administration were predominant in the infiltrate of the islets, and overt diabetes was prevented. On the other hand, when splenocytes from rIL-4-treated NOD mice were transferred to irradiated NOD recipients, along with splenocytes from diabetic NOD mice, all of the recipient mice became diabetic within 8 weeks after transfer. Considered together, a supplement of rIL-4 administered to NOD mice may protect against autoimmune diabetes by facilitating the development of Th2-like autoreactive T cells in the islets.

Animals↗

PubMed1998

Differential expression of SHP2, a protein-tyrosine phosphatase with SRC homology-2 domains, in various types of renal tumour.

SHP2, a widely distributed protein-tyrosine phosphatase with src homology-2 (SH2) domains, is highly expressed in the brain and may play a role in synaptic communications or cellular proliferation. In this study, we examined SHP2 protein expression in 110 renal cell tumours of various histological subtypes, including clear, granular, papillary, chromophobe, collecting duct, and sarcomatoid-type renal cell carcinoma (RCC), and oncocytoma. SHP2 was expressed predominantly in normal distal tubules and collecting ducts, and positivity in various types of renal tumours was as follows: clear cell RCC, 0% (0/77 cases); granular, 7.7% (1/13); papillary, 50% (3/6); sarcomatoid, 0% (0/1); chromophobe, 85.7% (6/7); collecting duct carcinoma, 0% (0/2); oncocytoma, 100% (4/4). Clear and granular-type RCCs showed a very low but positive expression of SHP2. Chromophobe RCC and oncocytoma showed the highest rates and strongest intensities of SHP2 protein on immunostaining. SHP2 may serve as a powerful marker in detecting rare tumours. Estimates of its expression may be useful in histological diagnosis.

Adenoma, Oxyphilic↗

PubMed1998

Triglyceride metabolism in heterozygote of Watanabe heritable hyperlipidemic rabbit.

The present study was conducted in order to examine the role of low-density lipoprotein (LDL)-receptor activity in very-low-density lipoprotein (VLDL) triglyceride metabolism in vivo. Fructose-feeding (10% in drinking water) for 2 weeks resulted in elevated plasma triglyceride in heterozygote of Watanabe heritable hyperlipidemic (WHHL) rabbit (WHHLH) associated with suppressed fractional catabolic rate (FCR) of plasma triglyceride, whereas Japanese white (JW) rabbit with normal LDL receptor activity showed no remarkable change in plasma triglyceride turnover after fructose-feeding, suggesting an involvement of LDL receptor activity on triglyceride metabolism. Thereafter, in order to stimulate cellular LDL receptor activity, fluvastatin, a new 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase inhibitor, was administered orally (1.52 +/- 0.26 mg/kg) to fructose-fed WHHLH. Significant suppression of triglyceride secretion rate (TGSR) was observed after treatment. However, since plasma triglyceride level was markedly suppressed, FCR of plasma triglyceride was significantly elevated by fluvastatin. Thus, it is speculated from the present data that LDL receptor activity is significantly involved in VLDL triglyceride metabolism in rabbits.

Animals↗

PubMed1998

Neuropeptide Y produces anxiety via Y2-type receptors.

In the present study, the effects of intracerebroventricular (ICV) NPY, [Leu31, Pro34]NPY and NPY13-36 have been evaluated with respect to anxiety in mice in the elevated plus maze. NPY had opposing effects on behavior, depending on the doses used. NPY decreased the normal preference for the closed arms of the maze at 0.7 nmol, indicating an anxiolytic effect; however, at 7 pmol NPY further increased the preference for the closed arm, indicating an anxiogenic effect. [Leu31, Pro34]NPY, a Y1-type receptor agonist, significantly reduced the preference for the closed arms at 70 pmol. NPY13-36, a Y2-type receptor agonist, significantly intensified the preference at 20 pmol. It has been demonstrated that NPY produces not only an anxiolytic effect via Y1-type receptors, but also an anxiogenic effect via Y2-type receptors. The time course of these NPY actions are quite different and the anxiogenic effect was observed only shortly after ICV NPY injection.

Animals↗

PubMed1998

Motilin increases food intake in mice.

The effect of motilin on food intake was investigated in nonfood-deprived mice. A significant increase in food intake was observed 1 h after ICV administration of motilin (3 nmol/mouse) and continued for 2 h. This effect was attenuated markedly by the motilin receptor antagonist GM-109 (0.3-3 nmol/mouse) in a dose-related manner. GM-109 alone had no effect on food intake. These results indicate that motilin receptors are present in the brain and may have a role in the regulation of food intake.

Animals↗

PubMed1998

Anxiolytic effect of motilin and reversal with GM-109, a motilin antagonist, in mice.

There have been few reports on the effects of the brain-gut peptide motilin on the central nervous system (CNS). We administered motilin intracerebroventricularly to mice and investigated the effect of motilin on anxiety using an elevated plus-maze. Motilin produced a significant decrease in anxiety with an inverted U-shaped dose response. To determine whether the anxiolytic effect of motilin was mediated via motilin receptors in the brain, the effect of GM-109, a novel motilin receptor antagonist, was investigated. GM-109 showed a significant and dose-dependent antagonism on the motilin-induced anxiolytic effect. GM-109 administered alone had no effect on anxiety. These results suggest that motilin receptors are present in the brain and may have a role in anxiety and emotion.

Animals↗

PubMed1998

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase.

BACKGROUND: Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. RESULTS: One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. CONCLUSION: Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

Amino Acid Sequence↗

PubMed1998

Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.

Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.

Adaptation, Physiological↗