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Takeshi Endo

Publications and source records attributed to Takeshi Endo.

At least 19 recordsLinked to original sources
PubMed2007

Role of Src-family kinases in formation and trafficking of macropinosomes.

Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.

Animals↗
PubMed2006

Designed surface construction by photo-induced vapor-phase assisted surface polymerization of vinyl monomers using immobilized free radical initiators.

To build up finely designed patterns on solid surfaces, consecutive vapor-phase assisted surface photo-polymerization (VASP) of methyl methacrylate and styrene was carried out under UV-irradiation through a stripe-patterned photo-mask on Si-wafer and Au-plate surfaces, resulting in the reproduction of designed and multi-layered patterns made of block copolymer chains grafted from the surfaces.

Journal Article↗
PubMed2006

Scavenging DPPH radicals catalyzed by binary noble metal-dendrimer nanocomposites.

Catalytic activity of gold-platinum, gold-palladium, and platinum-palladium dendrimer nanocomposites for scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was investigated. The gold-platinum and gold-palladium dendrimer nanocomposites were prepared via simultaneous reduction by sodium borohydride in the presence of poly(amidoamine) (PAMAM) dendrimers with amine or carboxyl terminal groups. The particles were not mixtures of monometallic particles but alloyed bimetallic particles. Bimetallic particles exhibited higher catalytic activity than monometallic ones.

Alloys↗
PubMed2006

Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells.

Neuronal differentiation in PC12 cells induced by nerve growth factor (NGF) requires sustained activation of ERK/MAP kinase pathway (Raf-MEK-ERK cascade). Although classical Ras (H-Ras, K-Ras, and N-Ras) activated by NGF signaling induces activation of ERK pathway, the activation is transient and not sufficient for PC12 cell differentiation. Instead, it has been widely accepted that NGF signaling-mediated Rap1 activation causes sustained activation of ERK pathway. There has been no direct evidence, however, that Rap1 participates in neuronal differentiation. Here we show that NGF signaling induces sustained activation of M-Ras and subsequent sustained activation of ERK pathway and the transcription factor CREB leading to PC12 cell differentiation. Exogenously expressed constitutively active mutant of M-Ras caused neurite outgrowth in PC12 cells and activating phosphorylation of ERK, whereas activated Rap1 did not. Knockdown of endogenous M-Ras by small interfering RNAs as well as the expression of a dominant-negative mutant of M-Ras interfered with NGF-induced neuritogenesis. Since MEK inhibitors prevented M-Ras-induced neurite outgrowth, ERK pathway participates in this differentiation pathway. Furthermore, M-Ras brought about ERK pathway-mediated activating phosphorylation of CREB and the CREB-mediated transcription. In addition, a dominant-negative mutant of CREB inhibited M-Ras-induced neuritogenesis. Taken together, NGF-induced PC12 cell differentiation requires M-Ras-ERK pathway-mediated activation of CREB. M-Ras was predominantly expressed in the hippocampus and cerebellum of mouse brain and in the gray matter of the spinal cord. All these properties of M-Ras were apparently indistinguishable from those of H-Ras. However, NGF stimulation caused transient activation of classical Ras proteins but sustained activation of M-Ras as well as sustained activating phosphorylation of ERK and CREB. Therefore, M-Ras is essential for neuronal differentiation in PC12 cells by inducing sustained activation of ERK pathway.

Animals↗
PubMed2006

Design of new phosphonic acid monomers for dental adhesives--synthesis of (meth) acryloxyalkyl 3-phosphonopropionates and evaluation of their adhesion-promoting functions.

The aims of this study were to synthesize new phosphonic acid monomers, and to examine their bonding performance. Four kinds of newly designed phosphonic acid monomers were synthesized, and seven experimental composite-type adhesive resins comprising the synthesized monomers (3 wt%), with BPO/DEPT/BPBA initiator were prepared. Tensile bond strengths of the adhesive resins to enamel and metal were measured with a universal testing machine at a crosshead speed of 1.0 mm/ min. The synthesized adhesive monomers were light yellow viscous liquids with 32.5%-49.3% yields, and identified by 1H NMR, IR and elemental analysis to be (meth)acryloxyalkyl 3-phosphonopropionates [R-P(=O) (OH)2] (5-MPPP, 6-MHPP, 6-AHPP, 10-MDPP). It was found that the newly developed phosphonic acid monomers with BPO/DEPT/BPBA initiator attained strong adhesion to both unetched, ground enamel and sandblasted Ni-Cr alloy with good durability. They exhibited significantly higher bond strengths than conventional phosphorous-containing monomers such as MEPP and VBPA (p <0.01). These findings indicate that the experimental phosphonic acid monomer-containing adhesive resins have potential prosthodontic and orthodontic applications, especially as self-etching, non-rinsing orthodontic adhesive resins.

Adhesives↗
PubMed2005

Synthesis and catalytic activity of gold-silver binary nanoparticles stabilized by PAMAM dendrimer.

Gold-silver binary nanoparticles, which feed atomic ratios of gold to silver were 3:1, 1:1, and 1:3, were prepared. These particles were stabilized by amine-terminated (generation (G) 3.0 and 5.0) and carboxyl-terminated (G 3.5 and 5.5) poly(amidoamine) (PAMAM) dendrimers in water. UV-vis spectra indicate that the particles are not mere physical mixtures of monometallic particles or core/shell type but alloy. According to transmission electron microscope (TEM) observation, the mean diameters of the particles were 7-10 nm for silver particles and 3-4 nm for both gold and alloy particles, respectively. Catalytic activities for reduction of p-nitrophenol were investigated by monitoring the absorbance at 400 nm during the reaction. They were proportional to the feed ratio of gold in the particles and showed a maximum at the ratio of Au:Ag=3:1.

Journal Article↗
PubMed2005

alpha-Helical polypeptide microcapsules formed by emulsion-templated self-assembly.

alpha-Helical peptide microcapsules were prepared by the emulsion-templated self-assembly of amphiphilic poly(gamma-benzyl L-glutamate)s (PBLG) 1. By mixing solutions of 1 in dichloromethane (in the form of a sodium salt) with water, oil-in-water emulsions were obtained. Spontaneous stripping of the dichloromethane phase caused a decrease in the diameter of the microdroplets and finally stable microcapsules formed. The microcapsules contain an inner aqueous phase as observed by confocal laser scanning microscopy (CLSM). Binding of hydrophobic pyrene molecules to the polypeptide shell was also demonstrated. The present polypeptide microcapsules are stable even after drying in air and they would serve as supramolecular vehicles for both hydrophobic and water-soluble molecules.

Capsules↗
PubMed2005

ATP as building blocks for the self-assembly of excitonic nanowires.

Supramolecular nanowires are specifically self-assembled from adenosine 5'-triphosphate (ATP) and dichloro-substituted thiacarbocyanine dyes. Spectral blue-shift and induced circular dichroism with exciton coupling are observed upon mixing the dye and ATP molecules in water. These observations indicate the formation of chiral 1/ATP supramolecular assemblies with excitation energy delocalized in the parallel-oriented (H-aggregated) chromophores. Interestingly, formation of H-aggregates is facilitated most in the presence of ATP and not by other nucleotides (ADP or AMP). In electron microscopy, aqueous 1/ATP mixture gives developed nanowires with a minimum width of ca. 10 nm and lengths of several micrometers. The ATP-directed nanowires exhibit reversible thermal self-assembly accompanied by supramolecular thermochromism. This is the first example of ATP-based supramolecular nanoassemblies, and the use of small biomolecules as building blocks for functional supermolecules provides a new perspective in the design of bio-nanomaterials.

Adenosine Triphosphate↗
PubMed2005

Assays for functional properties of Rab34 in macropinosome formation.

We have shown that Rab34/Rah participates in the promotion of macropinosome formation. Here we describe procedures for the analyses of intracellular localization and some functional properties of Rab34. Rab34 lacks a consensus sequence of the fourth motif for GTP/GDP binding and GTPase activities. Indeed, GTPase assay shows that wild-type Rab34 has extremely weak GTPase activity in vitro. However, Rab34 exhibits appreciable GTPase activity in vivo probably due to the presence of specific GTPase-activating protein (GAP) activity in cells. Specific intracellular localization of Rab34 is easily detected by the expression of epitope-tagged or enhanced green fluorescent protein (EGFP)-tagged protein. It is colocalized with actin filaments to membrane ruffles and membranes of nascent macropinosomes, which are formed from the ruffles. By contrast, Rab5 is not associated with the ruffles or nascent macropinosomes but present in endosomes at later stages. The function of Rab34 in macropinosome formation is analyzed by the transfection of wild-type, constitutively active, and dominant-negative mutants of Rab34 in fibroblasts followed by treatment with platelet-derived growth factor (PDGF) or phorbol ester. These analyses indicate that Rab34 is required for efficient macropinosome formation.

Amino Acid Sequence↗
PubMed2005

Preparation and catalytic activity of Au-Pd, Au-Pt, and Pt-Pd binary metal dendrimer nanocomposites.

Catalytic activity of Au-Pt, Au-Pd, and Pt-Pd dendrimer nanocomposites for reduction of p-nitrophenol was investigated in water. The bimetallic dendrimer nanocomposites were prepared by simultaneous reduction with sodium borohydride in the presence of poly(amidoamine) (PAMAM) dendrimers with amine and carboxyl terminal groups. Average diameters of the obtained particles were 2-4 nm by transmission electron microscopy. From UV-vis spectroscopy, it was found that the particles were not mixtures of monometallic particles but binary ones. X-ray photoelectron spectroscopy showed that formation of binary composite particles prevents palladium atoms from oxidation. The Au-Pd and Pt-Pd binary particles exhibited higher catalytic activity than monometallic ones. On the other hand, catalytic activity of Au-Pt binary particle was comparable to that of platinum nanoparticles.

Borohydrides↗
PubMed2004

Myocyte differentiation generates nuclear invaginations traversed by myofibrils associating with sarcomeric protein mRNAs.

Certain types of cell both in vivo and in vitro contain invaginated or convoluted nuclei. However, the mechanisms and functional significance of the deformation of the nuclear shape remain enigmatic. Recent studies have suggested that three types of cytoskeleton, microfilaments, microtubules and intermediate filaments, are involved in the formation of nuclear invaginations, depending upon cell type or conditions. Here, we show that undifferentiated mouse C2C12 skeletal muscle myoblasts had smoothsurfaced spherical or ellipsoidal nuclei, whereas prominent nuclear grooves and invaginations were formed in multinucleated myotubes during terminal differentiation. Conversion of mouse fibroblasts to myocytes by the transfection of MyoD also resulted in the formation of nuclear invaginations after differentiation. C2C12 cells prevented from differentiation did not have nuclear invaginations, but biochemically differentiated cells without cell fusion exhibited nuclear invaginations. Thus, biochemical differentiation is sufficient for the nuclear deformation. Although vimentin markedly decreased both in the biochemically and in the terminally differentiated cells, exogenous expression of vimentin in myotubes did not rescue nuclei from the deformation. On the other hand, non-striated premyofibrils consisting of sarcomeric actinmyosin filament bundles and cross-striated myofibrils traversed the grooves and invaginations. Time-lapse microscopy showed that the preformed myofibrillar structures cut horizontally into the nuclei. Prevention of myofibril formation retarded the generation of nuclear invaginations. These results indicate that the myofibrillar structures are, at least in part, responsible for the formation of nuclear grooves and invaginations in these myocytes. mRNA of sarcomeric proteins including myosin heavy chain and alpha-actin were frequently associated with the myofibrillar structures running along the nuclear grooves and invaginations. Consequently, the grooves and invaginations might function in efficient sarcomeric protein mRNA transport from the nucleus along the traversing myofibrillar structures for active myofibril formation.

Actins↗
PubMed2004

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Actins↗
PubMed2004

Identification and characterization of epsilon-sarcoglycans in the central nervous system.

Alpha-, beta-, gamma-, and delta-sarcoglycans (SGs) are transmembrane glycoprotein components of the dystrophin-associated protein (DAP) complex, which is critical for the stability of the striated muscle cell membrane. Epsilon-SG was found as a homologue of alpha-SG, but unlike other SG members, it is ubiquitously expressed in various tissues as well as in striated muscle. Moreover, mutations in the epsilon-SG gene cause myoclonus-dystonia, indicating the importance of epsilon-SG for the function in the central nervous system. To gain insight into the role of epsilon-SG, its expression and subcellular distribution in mouse tissues and especially in the mouse brain were investigated. Analysis by reverse transcription-polymerase chain reaction showed four splice variants of epsilon-SG transcripts in the mouse brain, two of which are major transcript forms. One is a conventional form including exon 8 (epsilon-SG1), and the other is a novel form excluding exon 8 but including a previously unknown exon, 11b (epsilon-SG2). Immunoblot analysis using various mouse tissues indicated a broad expression pattern for epsilon-SG1, but epsilon-SG2 was expressed exclusively in the brain. Therefore, both epsilon-SG isoforms coexist in various regions of the brain. Furthermore, these isoforms were found in neuronal cells using immunohistochemical analysis. Subcellular fractionation of brain homogenates, however, indicated that epsilon-SG1 and epsilon-SG2 are relatively enriched in post- and pre-synaptic membrane fractions, respectively. These results suggest that the two epsilon-SG isoforms might play different roles in synaptic functions of the central nervous system.

Amino Acid Sequence↗
PubMed2004

Voltammetric study of sodium hypochlorite using dendrimer-stabilized gold nanoparticles.

Electrochemical activity of dendrimer-stabilized gold nanoparticles on a glassy carbon electrode (GCE) was studied by means of cyclic voltammetry of sodium hypochlorite. Dendrimer-stabilized gold nanoparticles were deposited onto a glassy carbon surface by electrophoresis and the deposition was observed by transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Both anodic and cathodic peak currents were found to increase after the deposition of dendrimer-stabilized gold nanoparticles. In the case of dendrimers deposited onto the GCE surface, the magnitude of the current was smaller than for dendrimer-stabilized-gold-nanoparticle-modified GCE. This indicates the enhancement in the rate of electrolysis due to gold nanoparticles.

Journal Article↗
PubMed2004

[Plasticity of skeletal muscle differentiation generating stem cell-like phenotype: possible application to cell therapy for muscular diseases].

Drug therapy, gene therapy, and cell therapy may be effective to degenerative muscular diseases caused by genetic mutations including muscular dystrophy. Stem cells including embryonic stem (ES) cells and adult stem cells (tissue stem cells) are generally believed to be applicable to cell therapy. However, both types of cells have several problems to be solved for the cell therapy. We have shown that the expression of SV40 large T antigen in terminally differentiated mouse C2 skeletal muscle myotubes induce mitosis and dedifferentiation. This finding has exploded the concept that terminally differentiated cells never proliferate. It remains to be determined, however, whether mature myofibers in vivo are capable of dedifferentiation and proliferation. Here we present the data showing that mouse myofibers dedifferentiate and proliferate to form many mononucleated cells. Eventually, these dedifferentiated cells redifferentiate and regenerate myofibers. The isolated dedifferentiated cells show stem cell-like phenotype. These cells are expected to be applied to the cell therapy for degenerative muscular diseases.

Animals↗