Search PubMed⌕ Search

Biomedical subjects

H Nonaka

Publications and source records attributed to H Nonaka.

At least 55 records · Page 3Linked to original sources
PubMed1995

A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.

The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.

Amino Acid Sequence↗
PubMed1995

Synthesis of 2-imidazolidinylidene propanedinitrile derivatives as stimulators of gastrointestinal motility--III.

Recently, we reported that a ranitidine derivative 2 (fumarate: KW-5092), which had a 2-imidazolidinylidene propanedinitrile moiety (A), showed potent gastrointestinal motility enhancing activity. We have also found that introduction of substituents such as benzyl or 4-fluorobenzyl (i.e., giving 3 or 4) at the N-3 position of the moiety (A) significantly increased this activity. In this study, novel 2-imidazolidinylidene propanedinitrile derivatives possessing a thioether 5-15 were prepared and evaluated for in vitro assays; acetylcholinesterase (AChE) inhibitory activity and potentiating action on electrically induced contractions of guinea pig ileum. Compound 5, in which a nitrogen atom of compound 2 was replaced by a sulfur atom, was more potent than 2 in these tests. Also, in a series of thioether derivatives, introduction of substituents at the N-3 position of the 2-imidazolidinylidene propanedinitrile moiety markedly influenced both activities. In particular, compounds 12 and 13, which showed an excellent potency during in vitro study (AChE IC50 = 3.6 and 2.7 nM; ES. EC30 = 2.1 and 2.5 nM, respectively), were found to be more active in the enhancement of gastrointestinal motility in anesthetized rabbits than their corresponding parent compounds 3 and 4, respectively. In addition, compounds 12 and 13 showed lower affinity for the histamine H2-receptor than ranitidine. Therefore, these compounds may be potent and selective stimulators of gastrointestinal motility.

Acetylcholinesterase↗
PubMed1994

Molecular cloning and characterization of yeast rho GDP dissociation inhibitor.

We have previously isolated rho GDP dissociation inhibitor (rho DGI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids. rho GDI inhibits the GDP/GTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.

Amino Acid Sequence↗
PubMed1994

KF17837 ((E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine), a potent and selective adenosine A2 receptor antagonist.

8-(3,4-Dimethoxystryryl)-1,3-dipropyl-7-methylxanthine exhibited high affinity and selectivity for adenosine A2A receptors in binding assay using rat striatal A2A receptors labeled with [3H]2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosin e (CGS21680). The affinity was stereo selective: the E isomer, KF17837, showed a Ki value of 1.0 +/- 0.057 nM for the A2A receptors, whereas the Z isomer showed much lower affinity. KF17837 had 62-fold selectivity for the A2A receptors versus rat forebrain A1 receptors labeled with [3H]N6-cyclohexyladenosine (CHA). KF17837 was rapidly photoisomerized to form a stable equilibrium mixture (18% E - 82% Z), KF17837S, which showed Ki values of 7.9 +/- 0.055 nM and 390 +/- 68 nM for the A2A and A1 receptors, respectively. The inhibition type was competitive for [3H]CGS21680 binding. In rat pheochromocytoma PC12 cells KF17837S antagonized cAMP accumulation induced by 1 microM CGS21680 via the A2A receptors, with an IC50 value of 53 +/- 10 nM. cAMP accumulation induced by 10 microM 5'-N-ethylcarboxamidoadenosine via the A2B receptors in Jurkat cells (human T-cell line) was inhibited by KF17837S with an IC50 value of 1500 +/- 290 nM. These results indicate that KF17837S (and hence KF17837) is a highly potent and selective adenosine A2A receptor antagonist.

Adenosine↗
PubMed1994

Synthesis of 2-imidazolidinylidene propanedinitrile derivatives as stimulators of gastrointestinal motility--II.

In a previous paper, we reported that a novel ranitidine derivative 2 (fumarate: KW-5092), which had a 2-imidazolidinylidene propanedinitrile moiety (A), showed potent gastrointestinal motility enhancing activity. In order to obtain more potent gastrointestinal motility enhancing agents than compound 2 and to examine the effects of various substituents both at a nitrogen atom (B) in the 2-imidazolidinylidene propanedinitrile moiety and a basic nitrogen atom (C), compounds 5-29 were synthesized and evaluated for acetylcholinesterase (AChE) inhibitory activity and potentiating action on electrically stimulated contractions of guinea pig ileum. Introduction of alkyl, benzyl, aryl or acyl groups to the nitrogen (B) or (C), remarkably influenced both activities. Among these, compounds 14 and 15 showed more potent AChE inhibitory activity (IC50 = 6.7, 6.8 nM, respectively) than compound 2 and were active in potentiating action on the ileal contraction (EC30 = 9.5, 11 nM, respectively) together with a negligible histamine H2-receptor blocking property. Furthermore, these compounds were found to be more effective in the enhancement of gastrointestinal motility in anesthetized rabbits than compound 2.

Animals↗
PubMed1994

Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.

The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.

Actins↗
PubMed1994

Tacrine increases stimulation-evoked acetylcholine release from rat hippocampal slices.

We examined the effects of tacrine (9-amino-1,2,3,4-tetrahydroacridine) on endogenous acetylcholine (ACh) release from rat hippocampal slices. Tacrine (more than 1 microM) increased the measurable amount of basal ACh release. On the other hand, in the presence of physostigmine (50 microM; under this condition, cholinesterase activity was inhibited), tacrine did not enhance the basal ACh release. Tacrine at more than 100 microM increased the submaximal electrical stimulation-evoked release of ACh in both the absence and presence of physostigmine (50 microM). This effect of tacrine was abolished by a combination of atropine (100 mM) and physostigmine. These results indicate that a high-dose of tacrine increases cholinergic neurotransmission not only by inhibition of cholinesterase but also by increasing ACh release through an atropine-like effect, perhaps by blockade of part of the process of muscarinic autoinhibition.

Acetylcholine↗
PubMed1994

[Immunocytochemistry in the diagnosis of acute myeloid leukemia (M0)].

We examined leukemic blasts from 5 cases of AML-M0 diagnosed according to The French-American-British (FAB) classification for expression of immunological markers as well as myeloperoxidase (MPO) using flow cytometry (FCM) and immunocytochemistry (ICC). In one patient, the myeloid antigens, CD13 and CD33, were negative on FCM, but apparently positive in the cytoplasm by ICC, leading to a diagnosis of AML-M0. We examined MPO with anti-MPO monoclonal antibody in four patients by ICC, and could detect 3% or more MPO positive rates in all cases. These findings indicate that immunological studies for MPO and myeloid markers using ICC are very useful for the diagnosis of AML-M0. Two of 5 patients achieved CR, but they relapsed soon or after one year, respectively. The treatment outcomes suggest that the AML-M0 is an AML subtype with poor prognosis.

Adolescent↗
PubMed1994

Binding of [3H]KF17837S, a selective adenosine A2 receptor antagonist, to rat brain membranes.

The potential of 8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-[3H] methylxanthine ([3H]Kf17837S) as a highly selective antagonist radioligand for the adenosine A2A receptor was examined and compared with the properties of the adenosine A2A receptor agonist radioligand 2-[p-(2-[3H]carboxyethyl)phenethylamino]-5'-N-ethyl- carboxamidoadenosine ([3H]CGS21680). [3H]KF17837S specific binding to rat striatal membranes was saturable and reversible. Saturation studies showed that the binding of [3H]KF17837S occurred at a single site, with high affinity (Kd, 7.1 +/- 0.91 nM) and limited capacity (Bmax, 1.3 +/- 0.23 pmol/mg of protein). Adenosine receptor antagonist ligands competed with the binding of 1 nM [3H]KF17837S with the following order of activity: CGS15943 > KF17837S > N-[2-(dimethylamino)ethyl]-N-methyl- 4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)benzenesul fonamide > or = xanthine amine congener > 8-cyclopentyl-1,3-dipropylxanthine > 8-(noradamantan-3-yl)-1,3-dipropylxanthine > caffeine. Adenosine receptor agonists inhibited [3H] KF17837S binding in the following order: 5'-N-ethylcarboxamidoadenosine > or = CGS21680 > 2-phenylaminoadenosine > or = (R)- N6-phenylisopropyladenosine > N6-cyclopentyladenosine > (S)- N6-phenylisopropyladenosine. The Ki values of the antagonists for [3H]KF17837S binding and the rank order of potency were similar to those for [3H]CGS21680 binding. The affinities of the agonists were lower with [3H]KF17837S binding than with [3H] CGS21680 binding. However, a strong positive correlation (r = 0.98) was observed between the pharmacological profiles for these two radioligand assays. The inhibition curve for CGS21680 was best fitted to a two-component binding model and addition of GTP shifted the inhibition curve to the right, suggesting that [3H]KF17837S labeled two agonist coupling states. Other pharmacological agents had negligible affinities for the [3H]KF17837S binding site. Autoradiographic study of [3H]KF17837S binding using rat brain sections revealed that the binding site was highly enriched in the striatal region. These data indicate that [3H] KF17837S labels the adenosine A2A receptor in rat brain.

Adenosine↗
PubMed1994

RES-701-1, a novel, potent, endothelin type B receptor-selective antagonist of microbial origin.

The unique cyclic peptide designated RES-701-1 blocked the binding of 125I-labeled endothelin (ET)-1 to bovine cerebellar membranes. ETB receptors are predominant in bovine cerebellum. However, in bovine lung membranes, where both ETA and ETB receptors are expressed, RES-701-1 inhibited 125I-ET-1 binding by up to 70%; RES-701-1, in the presence of the ETA-selective antagonist BQ-123 at 1 microM, displaced 125I-ET-1 binding completely. With membranes from transfected Chinese hamster ovary cells expressing the human ETA or ETB receptors, RES-701-1 inhibited 125I-ET-1 binding to the ETB receptor with an IC50 value of 10 nM but had no effect on 125I-ET-1 binding to the ETA receptor. Thus, RES-701-1 is highly specific for the ETB receptor; it has no effect on a number of other receptors. RES-701-1 selectively inhibited the ET-1-induced increase in intracellular Ca2+ concentration in COS-7 cells expressing the ETB receptor but did not inhibit the Ca2+ transient in ETA-expressing cells. When injected intravenously (250 nmol/kg) into anesthetized rats, RES-701-1 abolished the initial depressor response to ET-1 but enhanced the subsequent pressor response. These results suggest that RES-701-1 is a potent and specific antagonist for the ETB receptor and that RES-701-1 will be a powerful tool for understanding the physiological roles of this receptor.

Animals↗
PubMed1993

5-HT3 receptor antagonists. 3. Quinoline derivatives which may be effective in the therapy of irritable bowel syndrome.

A series of quinolinecarboxylic acid derivatives has been previously described as a new class of 5-HT3 receptor antagonists due to deviation of a carbonyl moiety from the place of an aromatic ring in their minimum-energy conformations. These derivatives were evaluated in a wrap-restraint stress-induced defecation model in rats. Reference compounds, ondansetron (1), granisetron (2), and YM060 (4), potently inhibited a stress-induced increase in stools excreted from fed rats (ID50 = 0.27, 0.12, and 0.0052 mg/kg, po, respectively). However, quinoline derivatives exhibited different activities depending on structural class. 4-Hydroxyquinoline-3-carboxylic acid derivatives 5 and 6a possess high affinity for the 5-HT3 receptor (Ki = 6.1 and 1.5 nM, respectively) and exhibit potent activity in the Bezold-Jarisch (B-J) reflex test (ED50 = 0.0017 and 0.000 10 mg/kg, i.v., respectively), but they did not effectively inhibit the increase in fecal pellet output at the dose of 1 mg/kg, po. On the other hand, most of 1-substituted 2-oxoquinoline-4-carboxylates 10 showed less potent activity in the B-J reflex test than 1 or 2 but inhibited restraint stress-induced defecation more potently than 1 or 2. The ID50 value of endo-8-methyl-8- azabicyclo[3.2.1]oct-3-yl 1-isobutyl-2-oxo-1,2-dihydro-4- quinolinecarboxylate 10e was 0.013 mg/kg, po. With respect to the selected compounds 6a and 10e, effects of 5-HT- and thyrotropin-releasing hormone (TRH)-induced defecation, castor oil-induced diarrhea and wrap-restraint stress-induced colonic propulsion in rats were examined. These 5-HT3 receptor antagonists did not effectively inhibit castor oil-induced diarrhea, which has been reported not to be mediated via the 5-HT3 receptor. Although 10e showed 800-fold decreased potency compared with 4 in the B-J reflex test, 10e exhibited activity as potent as 4 in 5-HT- and TRH-induced defecation assays; 10e exhibited 7-fold increased potency compared with 4 in wrap-restraint stress-induced colonic propulsions. From these results, 10e appears to interact selectively with 5-HT3 receptors in the gastrointestinal system and might be effective in the therapy of irritable bowel syndrome (IBS).

Animals↗
PubMed1993

Adenosine A1 antagonists. 3. Structure-activity relationships on amelioration against scopolamine- or N6-((R)-phenylisopropyl)adenosine-induced cognitive disturbance.

The effects of a variety of adenosine A1 and A2 antagonists on N6-((R)-phenylisopropyl)adenosine (R-PIA)- and scopolamine-induced amnesias were investigated in rodents in order to clarify the role of adenosine receptors in learning and memory. Some of the selective adenosine A1 antagonists exhibited antiamnesic activities at several doses where they did not induce an increase of spontaneous locomotion. These results suggest that the blockade of A1 receptors is more important than that of A2 receptors in learning and memory. Detailed studies of structure-activity relationships of adenosine A1 antagonists in two amnesia models demonstrated that there were three types of adenosine A1 antagonists: (A) Compounds 3-5 (8-substituted 1,3-dipropylxanthines) ameliorated the shortened latency in both models. (B) Compounds 7-11 (8-substituted 1,3-dialkylxanthines) and 19-21 (imidazo[2,1-i]purin-5(4H)-one derivatives) ameliorated the shortened latency in the (R)-PIA-induced amnesia model but not in the scopolamine-induced amnesia model. (C) Compounds 14-16 ameliorated the shortened latency in the scopolamine model but not in the (R)-PIA model. Aminophenethyl-substituted compounds C did not exhibit adenosine A1 antagonism in vivo presumably due to rapid metabolism. The dramatic change in the activities of A and B could not be explained by their simple pharmacokinetic differences because both types of compounds showed clear blockade of central adenosine A1 receptors in the (R)-PIA model. 8-(3-Dicyclopropylmethyl)-1,3-dipropylxanthine (5) (KF15372) was chosen for further studies and is currently under preclinical development as a cognition enhancer.

Adenosine↗
PubMed1993

Synthesis of 2-imidazolidinylidenepropanedinitrile derivatives as stimulators of gastrointestinal motility.

Ranitidine (1), the histamine H2-receptor antagonist, has been previously reported to increase gastric emptying and gastric motility by inhibition of acetylcholinesterase (AChE) and enhancement of acetylcholine (ACh) release. In order to obtain potent gastroprokinetic agents, a new series of ranitidine derivatives (5-32) possessing a nitrogen atom instead of a sulfur atom (B) was synthesized and their AChE inhibitory activity and potentiating action on electrically evoked contractions of guinea pig ileum were evaluated. Modification of substituents R1 and R2 markedly influenced the activities. In particular, compound 19, (1-[2-[[[5-(piperidinomethyl)-2-furanyl]methyl]amino]-ethyl]-2- imidazolidinylidene)propanedinitrile fumarate, showed 20 and 100 times more potent AChE inhibitory activity and potentiating action on the ileal contraction, respectively, than ranitidine. Furthermore, compound 19 (KW-5092) enhanced gastrointestinal motility in anesthetized rabbits along with a negligible histamine H2-receptor blocking activity.

Animals↗
PubMed1993

Effects of physostigmine and some nitric oxide-cyclic GMP-related compounds on muscarinic receptor-mediated autoinhibition of hippocampal acetylcholine release.

We have investigated the effects of (a) the cholinesterase inhibitor physostigmine and (b) drugs that are known to change intracellular cyclic GMP levels on the autoinhibition of acetylcholine release from rat hippocampal slices. Autoinhibition was triggered by submaximal electrical stimulation in both the absence and presence of physostigmine. The results obtained indicate that an unusual increase in the extracellular acetylcholine content, such as that induced by cholinesterase inhibition, is not essential for autoinhibition triggering. Dibutyryl cyclic GMP reduced significantly the stimulation-evoked acetylcholine release in the presence, but not in the absence, of atropine. Neither sodium nitroprusside nor glyceryl trinitrate exerted a dibutyryl cyclic GMP-like effect. NG-Nitro-L-arginine did not lessen the autoinhibition. These results indicate that an increase in the intracellular cyclic GMP level reduces acetylcholine release, and that the muscarinic receptor stimulation-nitric oxide synthesis-(soluble) guanylyl cyclase activation pathway is not involved in the cholinergic autoinhibition process.

Acetylcholine↗
PubMed1992

Characterization of thromboxane A2/prostaglandin H2 receptors and histamine H1 receptors in cultured guinea-pig tracheal smooth-muscle cells.

We characterized thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors and histamine H1 receptors in Guinea-pig cultured tracheal smooth-muscle cells (TSMC). [3H]SQ 29,548 (a TXA2 antagonist)-binding sites were saturable and a high affinity with a dissociation constant of 6.2 +/- 0.60 nM (mean +/- S.E.) and a receptor density of 46 +/- 4.6 fmol/10(6) cells. [3H]SQ 29548 binding was completely inhibited by TXA2 mimetics or antagonists. Intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SQ 29548 binding. [3H]Mepyramine (a H1 antagonist)-binding sites were also present in TSMC. [3H]Mepyramine had a single class of low-affinity-binding sites with a dissociation constant of 2.6 +/- 0.081 microM and a receptor density of 10.6 +/- 0.11 nmol/mg protein. [3H]Mepyramine binding in TSMC membrane was inhibited by H1 antagonists, but not by H2 antagonists. The inhibition constants of mepyramine in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H1 antagonists. All these observations provide evidence that TXA2/PGH2 receptors, mepyramine-binding sites and/or H1 receptors are expressed in cultured TSMC.

Animals↗