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Koichi Takeuchi

Publications and source records attributed to Koichi Takeuchi.

29 records · Page 2Linked to original sources
PubMed2003

Morphologic characterization of green fluorescent protein in embryonic, neonatal, and adult transgenic rats.

Transgenic (Tg) animals with reporter genes are useful models in which to study cell lineage and the process of differentiation into tissues. We developed the green fluorescent protein (GFP)-Tg rat, which is more suitable for transplantation and stem cell research because it is larger than mice. We found that marker gene expression was dependent on each organ and developmental stage. In this study we describe GFP expression in various tissues from embryonic, neonatal, and adult animals. GFP expression in brain, lung, liver, and islet tissues was restricted to early developmental stages, but it was continuously strong in the exocrine pancreas, kidney, and cardiac and skeletal muscles. The CAG promoter that was presumed to induce ubiquitous protein expression might be responsible for the differences in expression.

Animals↗
PubMed2003

Efficient gene transfer of a simian immuno-deficiency viral vector into cardiomyocytes derived from primate embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cells continually proliferate and can generate large numbers of differentiated cells. Genetic manipulation of transplantable cells derived from primate ES cells offers considerable potential for development research and regenerative cell therapy. However, protocols for efficient gene transfer into primate ES-cell-derived cells have not yet been established. METHODS: Spontaneously contracting areas were derived from cynomolgus monkey ES cells. Features of cardiomyocytes in the area were analyzed according to gene expression (RT-PCR), morphology (immunostaining and electron microscopy), and function (intracellular calcium transience). Beating cells were transduced using a simian immunodeficiency virus (SIV) vector expressing enhanced green fluorescence protein (EGFP), then transplanted into ischemic rat myocardium. RESULTS: Beating cells derived from monkey ES cells displayed gene expression, ultrastructural and functional properties of early-stage cardiomyocytes. Highly efficient (97% cardiac phenotype) and stable transduction of these ES-cell-derived cardiomyocytes was achieved using SIV vector without altering contractile function. In addition, transduced cardiomyocytes survived in the myocardium of a rat myocardial infarction model. CONCLUSIONS: A lentiviral vector system based on SIV represents a useful vehicle for genetic modification of cardiomyocytes derived from primate ES cells, and can extend the application of primate ES cells to gene therapy.

Animals↗
PubMed2003

Effect of denuded endothelial cells on arginine amidase activity released from rabbit arteries.

1. We examined the secretion of arginine amidase activity from rabbit aorta and ear arteries. 2. The amount of arginine amidase activity spontaneously secretion from the aorta was significantly less than that secreted from the ear artery. Dermatan sulphate significantly facilitated the secretion of arginine amidase activity from both the aorta and ear artery. 3. The dermatan sulphate-enhanced secretion of arginine amidase activity from the aorta and ear artery was reduced by denudation of the endothelium. 4. These findings may indicate that spontaneous release of arginine amidase activity from vascular smooth muscle and the enhancement of secretion of arginine amidase activity produced by dermatan sulphate depends on the endothelium.

Animals↗
PubMed2002

Synergism between stem cell factor and granulocyte-macrophage colony-stimulating factor on cell proliferation by induction of cyclins.

Synergism between stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be essential for hematopoietic cell proliferation. Since HML-2 cells proliferate exponentially in the presence of SCF and GM-CSF together, we analyzed the molecular mechanism of the interaction between these two factors in the cells. An immediate-early gene product, c-myc, was additively upregulated in HML-2 cells by addition of a combination of SCF and GM-CSF. c-myc antisense oligonucleotides effectively suppressed cell proliferation and downregulated the induction of D3, E, A, and B cyclins in HML-2 cells stimulated with the two-factor combination. HML-2 cells arrested at the G0/G1 phase with SCF alone and expressed modest amounts of c-myc and cyclin D3, but not cyclin E. With GM-CSF treatment alone, the cells could not progress to the G2/M phase and expressed c-myc, cyclin D3 and cyclin E but not cyclins A or B. The addition of the counterpart cytokine resulted in cell cycle completion by induction of the deficient cyclins. Taken together, it appears that the induction of c-myc is an indispensable event in the proliferation of HML-2 cells and that the cytokines SCF and GM-CSF interact reciprocally for expression of all cyclins required for cell cycle progression.

Cell Cycle↗
PubMed2002

Size of side-chain at channel pore mouth affects Ca(2+) block of P2X(2) receptor.

Effects of amino acid replacement at the channel pore mouth of P2X(2) receptor/channel on multivalent cation channel block were investigated. When Asn(333) was replaced with various amino acid residues with neutral side chains (Gly, Ala, Val, Leu and Ile), the block by Ca(2+) was attenuated according to the sizes of the side chains. The block by La(3+) was also greatest with the Gly-substituted mutant, but this preference was not found for the block by other multivalent cations tested. The side chain at the channel pore mouth may interfere with the access of Ca(2+) block by steric hindrance.

Adenosine Triphosphate↗
PubMed2002

Soluble FLT-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer.

Vascular endothelial growth factor (VEGF), a bifunctional protein enhancing vascular permeability and stimulating endothelial growth, is thought to be responsible for fluid accumulation and angiogenesis in ascites tumors. To investigate the effects of stable expression of the soluble form of Flt-1 VEGF receptor (sFlt-1), a known endogenous inhibitor of VEGF, on the malignant ascites tumors, we cotransduced RMG-1 human ovarian cancer cells with adeno-associated virus vectors carrying the sFlt-1 cDNA and Neo gene or Neo gene alone and isolated both the sFlt-1-expressing clone and the Neo-expressing clone. In vitro growth characteristics were essentially the same. As expected, conditioned medium collected from the sFlt-1-expressing cells significantly inhibited the human umbilical vein endothelial cell proliferation in the presence of recombinant VEGF. Expression of sFlt-1 significantly suppressed RMG-1 cell-induced angiogenesis in vivo in the mouse dorsal air sac assay model. We then inoculated sFlt-1- or Neo alone-expressing cells i.p. into female BALB/c nude mice. The average volume of ascites fluid, number of leaked RBCs, and number of cancer cells were significantly lower in mice injected with sFlt-1-expressing cells than in the controls. Survival time was significantly prolonged in mice injected with sFlt-1-expressing cells. These results suggest that inhibition of VEGF activity by sFlt-1 expression may provide a means to control carcinomatous ascites and angiogenesis of malignant ascites tumors.

Animals↗
PubMed2002

Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism.

Lysophosphatidylcholine (lysoPC) is a component of oxidized low density lipoprotein (LDL) and is involved in the pathogenesis of atherosclerosis and inflammation. Previous studies demonstrated that lysoPC can induce various protein kinases including tyrosine kinases, protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in vascular endothelial cells. However, the role of lysoPC-activated kinases remains undefined. In this study, we examined the effect of lysoPC on apoptosis and investigated the role of lysoPC-activated protein kinases in human umbilical vein endothelial cells (HUVEC). The presence of apoptosis was evaluated by morphological criteria, MTT assay, and electrophoresis of DNA fragments showing the characteristic apoptotic ladder, TUNEL analysis, and quantified as the proportion of hypodiploid cells by flow cytometry. The lysoPC induced apoptosis in a time- and dose-dependent manner. It stimulated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and p38-MAPK in HUVEC. The use of specific pharmacologic inhibitors indicated that the p38-MAPK-signaling pathway (SB203580) is required for lysoPC-induced apoptotic signals. Furthermore, lysoPC-induced apoptosis was inhibited by DEVD-FMK (a caspas-3/CPP32 inhibitor), suggesting involvement of an important segment in the apoptosis. These results demonstrate that lysoPC induces apoptosis in human endothelial cells through a p38-MAPK-dependent pathway.

Analysis of Variance↗
PubMed2002

Fluvastatin enhances apoptosis in cytokine-stimulated vascular smooth muscle cells.

Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) have been shown to attenuate proliferation of vascular smooth muscle cells (VSMCs) by mechanisms independent of lipid reduction. In the current study, we investigated the effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis in unstimulated or cytokine-stimulated VSMCs. The presence of apoptosis in rat VSMCs was evaluated by electrophoresis of DNA fragments and 4'6'-diamidine-2'-phenylindole staining and quantified by flow cytometry. Fluvastatin but not pravastatin enhanced apoptosis in interleukin-1beta-stimulated VSMCs. The proapoptotic effect of fluvastatin was fully reversed by mevalonate and geranylgeranyl-pyrophosphate, and partially by farnesyl-pyrophosphate, but not by squalene. Inhibition of the extracellular signal-regulated protein kinase (ERK1/2) pathway significantly increased fluvastatin-enhanced apoptosis, whereas inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway significantly prevented this increase. However, fluvastatin showed no effect on the activity of ERK1/2 and p38-MAPK. Furthermore, fluvastatin-induced apoptosis was inhibited by YVAD-FMK (a caspase-1/interleukin-1beta-converting enzyme-like protease inhibitor) and DEVD-FMK (a caspase-3/CPP32 inhibitor), indicating involvement of an important segment in the apoptosis signaling pathway. These findings suggest that fluvastatin enhances apoptosis in cytokine-stimulated VSMCs and that protein prenylation, MAPK (ERK1/2 and p38-MAPK), and caspases are critically involved in the pathways of fluvastatin-enhanced apoptosis.

Animals↗
PubMed2002

Fluvastatin induces apoptosis in rat neonatal cardiac myocytes: a possible mechanism of statin-attenuated cardiac hypertrophy.

Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) have been shown to reduce atherosclerotic cardiovascular mortality and morbidity. Recent evidence indicates that statins may also exert direct effects on vascular wall cells (including endothelial cells and smooth muscle cells) independently of their hypocholesterolemic properties. However, little is known about whether statins have direct effects on myocardium. The effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis and protein synthesis in rat neonatal cardiac myocytes was investigated. The presence of apoptosis was evaluated by morphologic criteria, electrophoresis of DNA fragments, 4",6"-diamidine-2"-phenylindole (DAPI) staining, and TUNEL assay. Protein synthesis was measured by H-leucine incorporation into the cells. Fluvastatin, but not pravastatin, induced apoptosis in cardiac myocytes in a time- and dose-dependent manner. The pro-apoptotic effect of fluvastatin was reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate (GGPP), but not in the presence of squalene. The addition of protein prenylation inhibitor perillic acid and Rho-kinase inhibitor Y27632 significantly increased apoptosis. Fluvastatin decreased RhoA protein in the membrane fraction, whereas there were no significant changes of the RhoA protein in the cytosol fraction. Interleukin-1beta-stimulated H-leucine incorporation was completely inhibited by fluvastatin, but not by pravastatin. The findings suggest that fluvastatin induces apoptosis in cardiac myocytes via protein prenylation and the subsequent inhibition of Rho, and may play a role in the pathogenesis of cardiac hypertrophy and remodeling.

Animals↗
PubMed2001

Inclusions in novel perivascular macrophages (Mato's fluorescent granular perithelial cells) and neurons in the cerebral cortex of Hex A- and Hex B-deficient mice.

Beta-hexosaminidases are important enzymes for lipid and saccharide metabolism in the brain. In mice deficient in these enzymes, indigestible metabolic intermediates deposit in neurons. Inclusions such as membranous cytoplasmic bodies (MCB) and zebra bodies were seen in neurons of Tay-Sachs (TS) model mice, Sandhoff's disease (SD) model mice, and double knockout (DKO) mice. However, the cerebral perivascular macrophages discovered by Mato are active in the uptake of waste products and regarded as scavenger cells under steady-state conditions. We observed that indigestible components derived from neurons were taken up by the perivascular macrophages of TS mice by pinocytosis, but those of SD and DKO mice contained only pale inclusions and had marked vacuolations, and pinocytosis was rarely observed. Histochemically, the inclusions in the perivascular macrophages of TS mice were positive for the PAS stain, but those of SD and DKO mice were negative. In addition, the perivascular cells of TS mice expressed clear positive immunoreactivity against BM-8 and F4/80, but those of DKO mice had very weak BM-8 and F4/80 immunoreactivity. These differences between TS, SD, and DKO mice are based on their metabolism of oligosaccharides and glycosaminoglycans (GAG). Thus, hexosaminidase B is more important for keeping normal morphology and function of perivascular macrophages than hexosaminidase A. The foamy cells that appeared along the cerebral microvessels in lipidosis and saccharidosis were identified as perivascular macrophages (Mato's fluorescent granular perithelial cells: FGP cells).

Animals↗