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Y Nonomura

Publications and source records attributed to Y Nonomura.

At least 19 recordsLinked to original sources
PubMed1995

Biochemical and pharmacological studies with KT7692 and LY294002 on the role of phosphatidylinositol 3-kinase in Fc epsilon RI-mediated signal transduction.

Wortmannin inhibited phosphatidylinositol 3-kinase (P13-kinase) and Fc epsilon RI-mediated histamine secretion in RBL-2H3 cells to a similar degree, with IC50 values of 3 and 2 nM, respectively. Although P13-kinase is an acknowledged regulator of intracellular trafficking and secretion, wortmannin has proved to be a difficult drug to use in assessing the role of P13-kinase because it inhibits another important enzyme, myosin light-chain kinase (MLCK; IC50 = 200 nM). In the present study we synthesized a unique derivative of wortmannin, O-acetyl-delta 16-wortmannin-17-ol (KT7692), that has an inhibitory potency against PI3-kinase one-hundredth that of wortmannin, but retains a similar potency to wortmannin against MLCK. Histamine secretion was influenced 100-fold more by wortmannin than by KT7692.2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a structurally different PI3-kinase inhibitor from wortmannin, inhibited PI3-kinase with an IC50 of 2 microM but had little effect on MLCK activity in this concentration range. LY294002 also inhibited histamine secretion in RBL-2H3 cells with an IC50 of 5 microM. These results provide further evidence that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion after stimulation of Fc epsilon RI. Furthermore KT7692 in combination with wortmannin and LY294002 would be a powerful tool for clarifying the involvement of PI3-kinase as distinct from that of MLCK in signal transduction systems of various cellular responses.

Androstadienes↗
PubMed1995

The influence of dystrophin on lateral diffusion of proteins in sarcolemma of L-185 and C2 myoblasts and mature striated muscle cells of rats and mice, as measured by FRAP technique.

The expression of dystrophin can be suppressed in cultured skeletal muscle cells (coded L-185 from rat, and C2 from mouse) after a proper genetic manipulation. The influence of presence or absence of dystrophin on the lateral diffusion constant of Con-A-receptors was studied in the cell membrane of such cells (and also of mature skeletal muscle fibres of rat and mouse) by means of the fluorescence recovery after photobleaching (FRAP) technique, applying a novel fluorescent label called Con-A-BODIPY-FI conjugate. It has been established that the normal maturation, of myoblasts into skeletal muscle fibres involves a significant decrease of the mobility of Con-A receptors in the sarcolemma. In the absence of dystrophin, this maturation process cannot take place; the membrane proteins display an increasing mobility during the culture time, which is of lethal effect for these cells.

Animals↗
PubMed1995

An inhibitory role for phosphatidylinositol 3-kinase in insulin secretion from pancreatic B cell line MIN6.

Phosphatidylinositol 3-kinase (PI3-kinase) has been implicated in the regulation of vesicular transport. We examined the roles of PI3-kinase in the glucose-induced insulin secretion from the pancreatic beta cell line MIN6 by using wortmannin, a potent inhibitor of PI3-kinase. Low concentrations of wortmannin markedly potentiated the glucose-induced insulin secretion. This effect was probably mediated by PI3-kinase inhibition. Furthermore, wortmannin completely canceled the suppressive effect of insulin-like growth factor-I on insulin secretion from MIN6 cells. On the basis of these results, we discuss a possible role of PI3-kinase in the negative feed-back regulation of insulin secretion.

Androstadienes↗
PubMed1995

Pharmacological study of stem-cell-factor-induced mast cell histamine release with kinase inhibitors.

Stem cell factor (SCF) is a ligand for c-kit receptor and has a critical role in the development of mast cells. In this study, we investigated the effect of a panel of kinase inhibitors on SCF-induced histamine release from rat peritoneal mast cells. Genistein, an inhibitor of tyrosine kinases, inhibited SCF-induced histamine release with IC50 of 1.6 x 10(-5) M. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), inhibited histamine release stimulated with SCF dose-dependently with IC50 of 4 x 10(-9) M. KT5926, an inhibitor of myosin light chain (MLC) kinase, reduced histamine release with IC50 of 1.8 x 10(-7) M. Staurosporine, an inhibitor of protein kinases, also inhibited SCF-induced histamine release with IC50 of 6.5 x 10(-8) M. These results show the early involvement of tyrosine kinase and PI3 kinase and the possible role of MLC kinase in the late secretory phase in the signaling pathway used by SCF.

Alkaloids↗
PubMed1995

Myosin-actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells.

We examined the potential role of myosin and actin in the release of human immunodeficiency virus type 1 (HIV-1) from infected cells. Wortmannin (100 nM to 5 microM), an effective inhibitor of myosin light chain kinase, blocked the release of HIV-1 from infected T-lymphoblastoid and monocytoid cells in a concentration-dependent manner. Cytochalasin D, a reagent that disrupts the equilibrium between monomeric and polymeric actin, also partially inhibited the release of HIV-1 from the infected cells. At the budding stage, myosin and HIV-1 protein were detected in the same areas on the plasma membrane by using dual-label immunofluorescence microscopy and immunoelectron microscopy. In the presence of 5 microM wortmannin, viral components were observed on the plasma membrane by using immunofluorescence microscopy and electron microscopy, implying that wortmannin did not disturb the transport of viral proteins to the plasma membrane but rather inhibited budding.

Actins↗
PubMed1994

A substrate of ecto-protein kinase is microtubule-associated protein 1B in cortical cell cultures undergoing synaptogenesis.

Synapse formation between cultured rat cortical neurons is inhibited by the continuous application of K-252b, an ecto-protein kinase inhibitor, which cannot permeate the cell membrane. In order to identify the phosphorylated membrane proteins which are necessary for synapse formation, endogenous substrates for ecto-protein kinase activity were investigated. To detect phosphorylation of proteins containing extracellular domains, [gamma-33P]ATP was applied to the medium for brief periods. Proteins were then separated by SDS polyacrylamide gel electrophoresis and detected by autoradiography. Some bands showed immediate phosphorylation and this phosphorylation was suppressed by the addition of K-252b to the medium. We examined partial amino acid sequences of these substrates. The band with the highest molecular weight, whose phosphorylation was strongly inhibited by K-252b, was identified as microtubule-associated protein (MAP) 1B. These results suggest the possibility that the phosphorylation of extracellular domains of MAP1B is involved in synaptogenesis between cortical neurons.

Adenosine Triphosphate↗
PubMed1994

Analysis of the mechanism for acetylcholine release at the synapse formed between rat sympathetic neurons in culture.

Superior cervical ganglion neurons (SCGNs) were isolated from 7-day-old rat SCG and cultured in MEM containing horse serum, fetal calf serum, and nerve growth factor. In this culture condition, it is well known that the SCGNs form cholinergic synapse. In 3-4 weeks cultured neurons, immunofluorescent staining for synaptophysin, a small synaptic vesicle associated protein, showed the presence of synaptophysin as small dots on the surface of the soma. Postsynaptic potentials could be recorded in 50-80% of the neurons responding to evoked action potentials elicited in neighboring neurons. Because of its relatively large cell size and the short distance to the terminal, this synapse is a useful model for studying the mechanisms of acetylcholine (ACh) release by introducing substances such as antibodies or selective inhibitors into the presynaptic neuron by means of the whole-cell clamp technique. In this model synapse we tested the possible role of myosin in ACh release. The distribution of myosin was studied by the immunofluorescent staining technique. Myosin was recognized by the anti-myosin II IgG at the same synaptic terminals that showed the presence of synaptophysin with its antibody. The functional blockade of myosin by the antibody itself, and that of myosin light chain kinase (MLCK) by a pseudosubstrate inhibitor of MLCK, SM-1, or by a selective inhibitor of MLCK, wortmannin, induced depression of synaptic transmission in a dose-dependent manner. These indicate that phosphorylation of myosin by MLCK may be necessary for ACh release mechanisms.

Acetylcholine↗
PubMed1993

Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells.

The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and leukotriene release following activation of the tyrosine kinase Lyn together with phosphatidylinositol 3-kinase (PI3-kinase). Wortmannin inhibited the activity of partially purified PI3-kinase from calf thymus, as well as the PI3-kinase activity in anti-PI3-kinase p85 immunoprecipitates from RBL-2H3 cells, at a concentration as low as 1.0 nM and with IC50 values of 3.0 nM, but did not inhibit PI4-kinase activity. The inhibition of PI3-kinase by wortmannin was irreversible. Wortmannin inhibited both Fc epsilon RI-mediated histamine secretion and leukotriene release up to 80% with IC50 values of 2.0 and 3.0 nM, respectively. Wortmannin inhibited PI3-kinase activity in intact cells up to 80% with an IC50 value of 2.0 nM, which is almost equal to those for PI3-kinase in vitro and for histamine secretion and leukotriene release. With anti-wortmannin antibody, we have shown that wortmannin binds to the 110-kDa protein, but not to PI3-kinase 85-kDa regulatory subunit both in vitro and in whole cells. Furthermore, there was a positive correlation between the potencies of wortmannin derivatives as inhibitors of PI3-kinase and as inhibitors of histamine secretion. Wortmannin had no effect on the activation of the tyrosine kinase Lyn. These results suggest that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion following stimulation of Fc epsilon RI and that wortmannin blocks these responses through direct interaction with the catalytic subunit of this enzyme.

Androstadienes↗
PubMed1992

Enhancing effect of wortmannin on muscarinic stimulation of phospholipase D in rat pheochromocytoma PC12 cells.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK), enhanced carbachol-induced formation of [3H]phosphatidylethanol ([3H]PEt), a marker of phospholipase D (PLD) activity, in [3H]palmitic acid-labeled PC12 cells. The apparent EC50 value was 1.5 microM, and the effect was maximal at 3 microM and slightly attenuated at higher concentration. Wortmannin alone had no significant effect on [3H]PEt formation. The enhancing effect of wortmannin was observed at the initial increasing phase of [3H]PEt formation but not at the subsequent plateau phase. Wortmannin enhanced also phorbol ester-induced PLD activation. Although the precise mechanism remains to be clarified, these results suggest that MLCK may be involved in PLD regulation in PC12 cells.

Androstadienes↗
PubMed1992

Molecular cloning of the antibacterial protein of the giant African snail, Achatina fulica Férussac.

An expression cDNA library was constructed with poly(A)-rich RNA extracted from the collar of the giant African snail, Achatina fulica Férussac. A 1.9-kbp cDNA clone encoding a precursor of antibacterial glycoprotein of the snail, achacin, was isolated from the cDNA expression library. The cDNA sequence contains an open reading frame with 1593-nucleotide residues. The deduced amino acid sequence of this achacin precursor starts with a 29-residue leader peptide followed by a 502-residue mature peptide (56 kDa) with four possible N-glycosylation sites, Asn-Xaa-Ser or Asn-Xaa-Thr. The Northern-blot analysis proved that the achacin precursor was specifically expressed in the tissue of snail collar and processed to mature achacin. cDNA inserts encoding achacin precursor were subcloned into expression plasmids. Three kinds of expressed polypeptides were cross-reacted with rabbit antiserum raised against achacin. The largest polypeptide (M(r) 63,000) should be the achacin precursor.

Amino Acid Sequence↗
PubMed1992

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.

Androstadienes↗
PubMed1992

Inhibition of Ca(2+)-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells.

To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K(+)-evoked catecholamine (CA) release. The IC50 for high K(+)-evoked CA release was 1 microM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca(2+)-dependent exocytosis was inhibited in a dose-dependent manner (IC50, 1 microM). These findings suggest the implication of MLCK in the Ca(2+)-triggered process in the machinery of exocytosis.

Acetylcholine↗
PubMed1992

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.

Animals↗
PubMed1992

Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.

Alkaloids↗
PubMed1992

Molecular shape of dystrophin.

The molecular shape of dystrophin has been reported to be a 175 nm flexible rod [Pons, F. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7851-7855] or a 120 nm dumbbell [Murayama, T. et al. (1990) Proc. Jpn. Acad. 66B, 96-99]. The present work revealed that 100 nm flexible rods with or without spheres were predominant in highly purified dystrophin preparations. When the sample was subjected to gel filtration, dystrophin oligomers were isolated just after the void volume and the fraction largely consisted of dumbbell-shaped molecules. From various rotary-shadowed images, it was suggested that dystrophin is a rod with spheres at both ends, approximately 110 nm long and 2 nm wide. It appeared that this monomer binds to another monomer in a staggered way, forming a dimer, and the dimers associate with each other side-by-side, forming a dumbbell-shaped tetramer, 130 nm long and 5 nm wide. The tetramers form an end-to-end aggregate. It seemed that the dumbbell structure was not affected by alkaline (pH 11) treatment to dissociate dystrophin associated glycoproteins, but was deteriorated by detergent, NP-40, Triton X-100, or CHAPS, used for solubilization of membrane-bound dystrophin.

Alkalies↗
PubMed1991

Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.

Actins↗
PubMed1991

The Ca2(+)-dependent actin filament-severing activity of 74-kDa protein (adseverin) resides in its NH2-terminal half.

Calcium sensitive actin severing protein, adseverin, with Mr 74,000, was cleaved into two fragments of Mr 42,000 and Mr 39,000 by V8 protease and trypsin, and both fragments were purified by high performance (pressure) liquid chromatography ion-exchange column chromatography. To understand how adseverin can sever actin filaments, we identified the actin-binding domains. The NH2 termini of native adseverin and the Mr 42,000 fragment were confirmed to be blocked by amino acid sequencing. Twelve amino acids of the Mr 39,000 fragment were sequenced from the NH2 terminus; the sequence of this part had a homology to the hinge region between segments 3 and 4 of gelsolin and villin. Thus, the Mr 42,000 fragment is the NH2-terminal half (N42), and the Mr 39,000 fragment is the COOH-terminal half (C39). Each fragment was examined for actin-severing, -nucleating, -capping, and phospholipid binding activities with and without calcium. N42 contained a calcium-dependent actin-severing activity regulated by phospholipid. C39 bound to G-actin in a calcium-dependent manner, but had no severing activity. The sequence homology and similar functional domain structure suggest a common structural basis for the calcium- and phospholipid-regulated actin-severing properties shared by adseverin, gelsolin, and villin.

Actin Cytoskeleton↗