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Biomedical subjects

N Inagaki

Publications and source records attributed to N Inagaki.

At least 73 records · Page 4Linked to original sources
PubMed1998

Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

Amino Acid Sequence↗
PubMed1998

Involvement of Bruton's tyrosine kinase in FcepsilonRI-dependent mast cell degranulation and cytokine production.

We investigated the role of Bruton's tyrosine kinase (Btk) in FcepsilonRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcepsilonRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcepsilonRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcepsilonRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcepsilonRI signal transduction in mast cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Modulation of reconstituted ATP-sensitive K(+)-channels by GTP-binding proteins in a mammalian cell line.

1. The action of GTP-binding proteins on ATP-sensitive potassium (KATP) channels was investigated. KATP channels were expressed in a mammalian cell line (COS-1 cells) by cotransfecting vectors carrying the sulphonylurea receptor (SUR1) and BIR (Kir6.2), a member of the inward rectifier K+ channel family. G proteins were also tested on KATP channels composed of an isoform of SUR1, SUR2A, in combination with Kir6.2. 2. The alpha and beta gamma subunits of the GTP binding protein G1 were tested separately in inside-out patches under continuous recording. G alpha-11 increases the activity of SUR1-Kir6.2 and SUR2A-Kir6.2 channels by 200 and by 30%, respectively. 3. G alpha-12 does not increase the activity of SUR1-Kir6.2 channels, but increase the activity of SUR2A-Kir6.2 channels by 30%. 4. Control experiments showed that GTP gamma S, a specific activator of G proteins, and heat-inactivated G alpha-11 do not increase the single channel activity. 5. No effects of the other subunits (beta gamma) from either G11 or G12 on the single channel activity were observed. 6. The protein kinase C inhibitors H7 and an inhibitory peptide (FARKGALRQKNV) had no effect on the modulatory action of G alpha-11 on SUR1-Kir6.2 channels. 7. We conclude that both types of reconstituted KATP channels are modulated by G proteins.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin.

Although accumulating data reveal patterns of proliferation, migration, and differentiation of neuronal lineage cells in the developing brain, gliogenesis in the brain has not been well elucidated. In the rat brain, vimentin is selectively expressed in radial glia and in their progeny, not in oligodendrocytes or neurons from embryonic day 15 (E15) until postnatal day 15 (P15). Here we examined mitotic radial glial lineage cells in the rat brain E17-P7, using the monoclonal antibody 4A4, which recognizes vimentin phosphorylated by a mitosis-specific kinase, cdc2 kinase. In the neocortex, mainly radial glia in the ventricular zone, but not their progeny, underwent cell division. In contrast, not only radial glia but also various types of radial glial progeny including Bergmann glia continued to proliferate in the cerebellum. Radial glia in the neocortex divided horizontally, obliquely, and vertically against the ventricular surface. The percentage of the vertical division increased with progress in the stage of development, concurrently with the decrease of the population of horizontal divisions. Thus, the monoclonal antibody 4A4 provides an useful tool to label mitotic glia in the developing brain and revealed different patterns of gliogenesis in the neocortex and cerebellum. A possibility is discussed that the dynamics of mitotic orientation observed here may be related to the change of the pattern of gliogenesis during development.

Animals↗
PubMed1998

Characterization of antihistamines using biphasic cutaneous reaction in BALB/c mice.

Effects of 11 histamine H1 receptor antagonists on IgE-mediated biphasic cutaneous reaction in mice were examined. The immediate phase reaction (IPR) assessed at 1 hour after antigen application was significantly inhibited by all antihistamines examined. The inhibition of IPR by cetirizine and mequitazine were potent, but those by cyproheptadine and diphenhydramine were weak. The later phase reaction (LPR) assessed at 24 hours after antigen application was inhibited by chlorpheniramine, oxatomide, ketotifen, mequitazine, emedastine, terfenadine and azelastine. The inhibition of LPR by emedastine was potent, but those by ketotifen and terfenadine were only partial. Emedastine inhibited both IPR and LPR comparably. Present results indicate that H1 receptor activation is involved in the IPR of the biphasic cutaneous reaction, and that the blockade of H1 receptors at IPR does not contribute to the attenuation of following LPR. Histamine H1 receptor antagonists inhibiting the LPR have a property distinct from H1 receptor antagonism, which may have an additional benefit for the treatment of allergic diseases.

Animals↗
PubMed1998

Modulation of Th1- and Th2-like cytokine production from mitogen-stimulated human peripheral blood mononuclear cells by phosphodiesterase inhibitors.

1. Effects of phosphodiesterase (PDE) inhibitors on the production of IFN-gamma, IL-2, IL-4 and IL-5 by phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) were investigated. In addition, we investigated the effects of dibutyrylcyclic AMP (dbcAMP) and a beta-adrenoceptor agonist on production of these cytokines. 2. Type IV, type III and nonselective PDE inhibitors were effective at inhibiting the production of IFN-gamma and IL-2 production in a dose-dependent manner. In contrast, IL-4 and IL-5 production was inhibited by only the highest concentration of type IV inhibitor, and other agents had no effect on the production. 3. Similarly, dbcAMP inhibited the production of IFN-gamma and IL-2 more potently than IL-4 and IL-5. 4. The addition of a beta-adrenoceptor agonist increased the inhibitory effect of PDE inhibitors tested on the production of IFN-gamma and IL-2. 5. These results indicate that PDE inhibitors or cAMP-elevating agents modulate Th1 cytokine more effectively than Th2 cytokine production.

Adrenergic beta-Agonists↗
PubMed1998

The effects of anti-asthma drugs on mediator release from cultured human mast cells.

BACKGROUND: A method for generating human mast cells in vitro was recently established. Little is known about the pharmacological profiles of allergic mediator release from cultured mast cells. OBJECTIVE: The main objective was to investigate the nature of cultured mast cells from a pharmacological point of view. We examined the effect of anti-asthma drugs on the release of histamine, sulfidoleukotrienes (LTs) and prostaglandin D2 (PGD2) from the cultured mast cells. METHODS: Using the method established by Saito et al. we cultured cord blood mononuclear cells in the presence of 80 ng/mL stem cell factor (SCF), 50 ng/mL interleukin-6 (IL-6) and 300 nmol/L prostaglandin E2 (PGE2), and obtained almost pure (> 99%) mast cells. We sensitized cultured mast cells with immunoglobulin E (IgE)-rich serum, and then treated them with some anti-asthma drugs before challenge with anti-human IgE. Released histamine, LTs and PGD2 were measured by high-performance liquid chromatography, commercial enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA) systems, respectively. RESULTS: The cultured mast cells released histamine, LTs and PGD2 following immunological stimulation through IgE. The mast cell stabilizing agents disodium cromoglycate (DSCG, 1 mmol/L) and azelastine (100 micromol/L) significantly inhibited the release of these three mediators. The beta-adrenoceptor agonists isoproterenol, salbutamol, and clenbuterol also inhibited all three mediators' release in a concentration-dependent manner. The non-selective and selective phosphodiesterase (PDE) inhibitors theophylline, rolipram, and cilostazol had no significant effect on mediator release at clinically useful concentrations. BAY x 1005 (a 5-lipoxygenase-activating protein inhibitor) inhibited the LTs release, whereas indomethacin (a cyclo-oxygenase I and II inhibitor) and NS-398 (a cyclo-oxygenase II inhibitor) inhibited PGD2 release. CONCLUSIONS: The present results indicate that cultured mast cells release histamine, LTs and PGD2 following IgE crosslinking. Anti-asthma drugs showed a characteristic suppression of the release of each mediator. The suppressive actions of these drugs are similar to their pharmacological actions on human lung mast cells. These results suggest that cultured mast cells are useful for the analysis of function and pharmacological profiles of lung mast cells.

Anti-Asthmatic Agents↗
PubMed1998

Possible role of nitric oxide in IgE-mediated allergic cutaneous reaction in mice.

The role of nitric oxide (NO) in the development of allergic cutaneous reactions in mice was investigated. A biphasic cutaneous reaction composed of an immediate-phase edema and a late-phase edema was evoked by epicutaneous application of 2,4-dinitrofluorobenzene on the ear of mice passively sensitized with mouse anti-dinitrophenol monoclonal IgE. Two NO synthase inhibitors, L-NG-nitroarginine methyl ester (L-NAME) and NG-monomethyl-L-arginine, significantly inhibited both phases of the IgE-mediated biphasic cutaneous reaction. Simultaneous treatment with L-arginine attenuated the inhibitory effect of L-NAME. An NO donor, 3-morpholinosydononimine-N-ethylcarbamide, caused a potent edematous reaction in the mouse ear. Furthermore, L-NAME inhibited the cutaneous reactions caused by both interleukin-1beta and tumor necrosis factor-alpha, putative mediators of the late-phase edema in the biphasic cutaneous reaction. These results indicate that a gaseous mediator, NO, participates, at least in part, in the development of ear edema in the IgE-mediated biphasic cutaneous reaction in mice.

Animals↗
PubMed1998

Pharmacological modulation of LPS-induced MIP-1 alpha production by peripheral blood mononuclear cells.

In the present study, we investigated the effects of some anti-asthmatic drugs on the production of the CC chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), in response to lipopolysaccharide (LPS) by peripheral blood mononuclear cells (PBMC). MIP-1 alpha production was induced by LPS in a concentration-dependent fashion and reached the maximum at 10 micrograms/ml LPS (27.5 +/- 2.3 ng MIP-1 alpha/10(6) PBMC). At a submaximal concentration of LPS (1 microgram/ml), the release of MIP-1 alpha increased with time and reached the maximum 24 h after LPS stimulation. Actinomycin D and cycloheximide inhibited MIP-1 alpha production completely, but glucocorticoids did not completely inhibit MIP-1 alpha production, with a maximum inhibition of 70%. We examined the effect of beta-stimulants and phosphodiesterase inhibitors, which upregulate intracellular cyclic AMP levels, on MIP-1 alpha production. When PBMC were treated with beta-stimulants alone, beta-stimulants showed a slightly inhibitory effect on MIP-1 alpha production. However, the coadministration of roliplam significantly potentiated the inhibitory effect of beta-stimulants on MIP-1 alpha production. Moreover, db-cAMP suppressed MIP-1 alpha production dose-dependently. The above data indicate that the production of MIP-1 alpha is regulated by cyclic AMP and that cyclic AMP could provide a useful target for therapeutic treatment in asthmatic diseases and other diseases where MIP-1 alpha is involved in their etiology.

Adrenergic beta-Agonists↗
PubMed1998

Antiallergic action of betotastine besilate (TAU-284) in animal models: A comparison with ketotifen.

The effects of betotastine besilate (betotastine: TAU-284), a novel antiallergic drug, on homologous passive cutaneous anaphylaxis (PCA), mediator-induced cutaneous reaction, antigen-induced asthmatic responses and platelet-activating factor (PAF)-induced airway eosinophilia in several animal models, were compared to ketotifen. Betotastine (0.1 mg/kg, p.o.) and ketotifen (1 mg/kg, p.o.) inhibited both rat PCA and histamine-induced cutaneous reaction, whereas they showed little effect on serotonin-induced cutaneous reaction. Betotastine (0.3 mg/kg, p.o.) and ketotifen (1 mg/kg, p.o. ) significantly inhibited antigen-induced bronchoconstriction in guinea pigs which had been passively sensitized with guinea pig IgE antibody. In actively sensitized guinea pigs, the immediate and late phase increase in airway resistance (Rrs) were observed within 5 min and between 4 and 7 h after the aeroantigen challenge. Betotastine (1 mg/kg, p.o.) inhibited both responses. Ketotifen (1 mg/kg, p.o.) inhibited the immediate phase response, but did not affect the late phase response. Exposure of guinea pigs to aerosolized PAF increased the number of eosinophils in bronchoalveolar lavage fluid 24 h after the stimulation. Betotastine (3-10 mg/kg, p.o.) dose-dependently inhibited PAF-induced accumulation of eosinophils in the bronchoalveolar cavity. In contrast, cetirizine (10 mg/kg, p.o.) showed a tendency to inhibit eosinophil accumulation, and ketotifen (10 mg/kg, p.o.) and terfenadine (10 mg/kg, p.o.) did not have any affect. These results indicate that betotastine could be useful in the treatment of allergic disease such as bronchial asthma.

Animals↗
PubMed1998

Effects of vitamin B6 deficiency on cytokine levels and lymphocytes in mice.

The effects of vitamin B6 (B6) deficiency on cytokine levels and proportions of lymphocyte subsets in BALB/c mice were investigated. The proportion of lymphocytes from the thymus and spleen of mice given no B6, that were CD4+ CD8- T cells, was larger than in mice given B6, and the ratio of CD8+ to CD4+ T cells in the thymus of mice given no B6 was lower. The concentrations of interleukin-5 and -10 in spleen cells stimulated in vitro with concanavalin A were significantly higher in the mice with B6 deficiency, as was their plasma corticosterone concentrations. These results suggested that B6 is necessary to maintain cytokine levels and lymphoid function in the thymus and spleen of mice.

Animals↗
PubMed1998

ATP-sensitive potassium channels: structures, functions, and pathophysiology.

ATP-sensitive potassium channels (KATP channels) play important roles in various tissues by coupling cell metabolic status to electrical activity. Recently, molecular biological and electrophysiological techniques have revealed the molecular basis of the KATP channels to be a complex of the Kir6.0 subunit, a member of the inwardly rectifying K+ channel subfamily Kir6.0, and the sulfonylurea receptor (SUR) subunit, a member of ATP-binding cassette (ABC) superfamily; the functional diversity of the various KATP channels is being determined by a combination of the Kir6.0 subunit (Kir6.1 or Kir6.2) and the SUR subunit (SUR1 or SUR2) comprising it. Recent studies of the KATP channels have suggested mechanisms of KATP channel regulation and pathophysiology and also a new model in which ABC proteins regulate the functional expression of ion channels.

Adenosine Triphosphate↗
PubMed1998

Protective effect of oral administration of a traditional medicine, Juzen-Taiho-To, and its components on lethal Candida albicans infection in immunosuppressed mice.

Protective effects of a kampo medicine, Juzen-taiho-to (TJ-48) and its herbal components against experimental candidiasis in cyclophosphamide-induced immunosuppressive mice were investigated. ICR mice were immunosuppressed by intraperitoneal treatment with cyclophosphamide (day-4) and were orally given TJ-48 or one of its 10 herbal components for 4 consecutive days (day-4--1). They were then challenged intravenously with a lethal dose of Candida albicans (day 0). An oral dose of 1 g/kg/day of TJ-48 prolonged their life span. A similar protective effect was obtained by treatment with its component drugs Ginseng radix, Glycyrrhizae radix, Atractylodis lancea rhizoma or Cnidii rhizoma. These herbal components were suggested to have a main role in the protective effect of Juzen-taiho-to against Candida infection.

Adjuvants, Immunologic↗
PubMed1997

Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria.

We have investigated the subcellular localization of the inwardly rectifying K+ channel subunit Kir6.1 (uKATP-1). Immunoblot analysis of the mitochondrial fractions prepared from rat skeletal muscle and liver detected a single band of Kir6.1 at 51 kDa, the intensity of which was stronger than that found in the total homogenate of each tissue. By immunofluorescence staining, the labelling for Kir6.1 was observed as a dispersed array of fine dots throughout all the tissues examined in the rat, including skeletal muscle, cardiac muscle, liver, and pancreas. Electron-microscopic examination revealed that the punctate staining distribution was due to a specific labelling of Kir6.1 in the mitochondria. Immuno-positive colloidal gold particles were scattered over the mitochondria, suggesting that Kir6.1 was located on the inner membrane. Although gold particles were not observed at plasma membrane, a 47 kDa protein was detected in the isolated plasma membrane vesicles by immunoblot analysis against Kir6.1. These results suggest that Kir6.1 might be a subunit of the ATP-sensitive K+ channel in the mitochondrion, as well as in the plasma membrane.

Adenosine Triphosphate↗
PubMed1997

Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells.

We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel.

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Adenosine Triphosphate↗
PubMed1997

Inhibitory mechanisms of beta-adrenoceptor agonists for immunoglobulin E-mediated experimental allergic reactions in rats.

Inhibitory mechanisms of isoproterenol and clenbuterol for immunoglobulin E (IgE)-mediated experimental allergic reactions in rats were studied. IgE-mediated passive cutaneous anaphylaxis, histamine-induced cutaneous reaction and serotonin-induced cutaneous reaction were evoked at the same time in the same rats. Isoproterenol administered intravenously immediately before challenge inhibited all these reactions significantly. Clenbuterol administered intravenously 0-3 h before challenge also significantly inhibited the three cutaneous reactions. The inhibition was maximum when the drug was given 1 h before challenge. Passive cutaneous anaphylaxis was always inhibited more potently than histamine-induced cutaneous reaction and serotonin-induced cutaneous reaction by these beta-adrenoceptor agonists. Passive peritoneal anaphylaxis was caused by injecting an antigen intravenously. Isoproterenol administered intravenously immediately before challenge inhibited the reaction significantly. Clenbuterol administered intravenously 0-3 h before challenge also significantly inhibited passive peritoneal anaphylaxis, maximally so when given 1 h before challenge. In vitro IgE-dependent histamine release from sensitized peritoneal mast cells or mesenteric mast cells was not affected by isoproterenol and clenbuterol. Mouse monoclonal IgE, a foreign protein, administered intravenously decreased rapidly in the circulation. About 50% of the mouse IgE given disappeared in 20 min. The decrease of mouse IgE was partly but significantly inhibited by the beta-adrenoceptor agonists, and the inhibition was abolished by simultaneous treatment with propranolol. These results indicate that direct inhibition of mast cell activation does not contribute to the potent inhibition of in vivo allergic reactions in rats by beta-adrenoceptor agonists, and that inhibition of the allergic cutaneous reaction is partially explained by the inhibition of vascular permeability increases caused by mast cell mediators. Penetration of intravenously administered antigen from blood vessels to peripheral tissues to cause mast cell activation might be inhibited by beta-adrenoceptor agonists, and this could play some role in inhibiting intravenous antigen-induced allergic reactions in rats. Clenbuterol exhibited its maximum action with some latency in vivo, suggesting that some time-requiring process may be involved in the manifestation of its action.

Adrenergic beta-Agonists↗