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Biomedical subjects

M Horie

Publications and source records attributed to M Horie.

At least 55 records · Page 3Linked to original sources
PubMed1999

Cloning, expression, and chromosomal mapping of the human 14-3-3gamma gene (YWHAG) to 7q11.23.

The 14-3-3 family of proteins exerts diverse influences on the signal transduction pathways of cells. We have newly identified a human cDNA encoding the gamma subtype of the 14-3-3 family of genes. The deduced amino acid sequence of human 14-3-3gamma was identical to that of rat 14-3-3gamma. The human 14-3-3gamma gene (HGMW-approved symbol YWHAG) is highly expressed in brain, skeletal muscle, and heart. By fluorescence in situ hybridization analysis, the human 14-3-3gamma gene was mapped to chromosome 7q11.23. Radiation hybrid mapping has shown that this gene is localized 2.33 cR telomeric to D7S1870, a polymorphic marker located at the most telomeric end of the common deletion region of Williams-Beuren syndrome (WBS). This suggests that haploinsufficiency of 14-3-3gamma may not contribute to the WBS phenotype. However, information regarding the precise chromosomal location of a member of the 14-3-3 family of genes will aid in examining the relationship between this family of proteins and human disorders.

14-3-3 Proteins↗
PubMed1999

Pax-6 is required for thalamocortical pathway formation in fetal rats.

Pax-6, a transcription regulatory factor, has been demonstrated to play important roles in eye, nose, and brain development by analyzing mice, rats, and humans with a Pax-6 gene mutation. We examined the role of Pax-6 with special attention to the formation of efferent and afferent pathways of the cerebral cortex by using the rat Small eye (rSey2), which has a mutation in the Pax-6 gene. In rSey2/rSey2 fetuses, cortical efferent axons develop with normal trajectory, at least within the cortical anlage, when examined with immunohistochemistry of the neuronal cell adhesion molecule TAG-1 and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling from the cortical surface. A remarkable disorder was found in the trajectory of dorsal thalamic axons by immunostaining of the neurofilament and the neural cell adhesion molecule L1 and DiI labeling from the dorsal thalamus. In normal rat fetuses, dorsal thalamic axons curved laterally in the ventral thalamus without invading a Pax-6-immunoreactive cell cluster in the ventral part of the ventral thalamus. These axons then coursed up to the cortical anlage, passing just dorsal to another Pax-6-immunoreactive cell cluster in the amygdaloid region. In contrast, in rSey2/rSey2 fetuses, dorsal thalamic axons extended downward to converge in the ventrolateral corner of the ventral thalamus and fanned out in the amygdaloid region without reaching the cortical anlage. These results suggest that Pax-6-expressing cell clusters along the thalamocortical pathway (ventral part of the ventral thalamus and amygdala) are responsible for the determination of the axonal pathfinding of the thalamocortical pathway.

Afferent Pathways↗
PubMed1999

On the mechanism of genistein-induced activation of protein kinase A-dependent Cl- conductance in cardiac myocytes.

Genistein, an inhibitor of protein tyrosine kinase (PTK), enhanced the activation of the cardiac isoform of the protein kinase A (PKA)-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance in guinea-pig ventricular cells. We examined the mechanism(s) underlying this excitatory action of genistein by using patch-clamp techniques. The CFTR Cl- conductance, activated by isoproterenol (ISO, 10 nM; [Cl-] 153 mM extracellular, 21 mM intracellular; 36 degrees C), was enhanced by 20 microM genistein. Daidzein, a structural analogue of genistein with little inhibitory action on PTK, also enhanced CFTR Cl- currents. After maximal activation of the Cl- conductance by a cocktail of adenosine 3', 5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine and okadaic acid or vanadate plus forskolin in the pipette, genistein was no longer stimulatory but was rather slightly inhibitory at 100 microM. Direct exposure of myocytes to higher concentrations of genistein (50-100 microM) elicited outwardly rectifying currents with a reversal potential of -47 mV in the absence of ISO. In the presence of 50 microM H-89, a PKA inhibitor, genistein had no effect. Vanadate in the pipette at a concentration (100 microM) inhibiting phosphotyrosine phosphatases alone did not prevent the action of genistein. In contrast, no conductance was activated by tyrphostins B42 or 51 or lavendustin A, other PTK inhibitors. Genistein's stimulation of cardiac CFTR Cl- conductance appears to be independent of the PTK pathway and to be due to its direct interaction with CFTR Cl- channels.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Detection of bikunin mRNA in limited portions of rat brain.

Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.

Animals↗
PubMed1999

Expression of kininogen mRNAs and plasma kallikrein mRNA by cultured neurons, astrocytes and meningeal cells in the rat brain.

Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astrocytes, and the expression in meningeal cells was enhanced by culture with prostaglandin E2 (PGE2) or dibutyryl cAMP (Bt2cAMP). Low-molecular-weight kininogen mRNA was not detected in these cultures of cells, even after treatment with PGE2. Although expression of high-molecular-weight kininogen mRNA was very low in these cultures of cells, PGE2 or Bt2cAMP markedly stimulated its expression in cultures of meningeal cells and slightly in neurons, but not in astrocytes. We also found that expression of plasma kallikrein mRNA was strong in cultures of meningeal cells and slight in astrocytes, but absent in neurons. These results suggest that cells in the leptomeninges/choroid plexus are major sources of kininogens in rat brain which may function as precursor proteins for kinins and/or potent cysteine proteinase inhibitors during cerebral inflammation.

Animals↗
PubMed1999

Characterization of a novel missense mutation in the pore of HERG in a patient with long QT syndrome.

INTRODUCTION: A new strategy to elucidate the molecular mechanisms underlying the long QT syndrome (LQTS) is now available with genetic mutational analyses and characterization of ion channel mutations. METHODS AND RESULTS: In a 26-year-old woman with LQTS, we identified a novel missense mutation in the pore of HERG by using polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) and sequencing of her genomic DNA. The mutation resulted in an amino acid substitution of a positively charged lysine for a highly conserved uncharged asparagine at codon 629 (N629K). Whole cell, patch clamp studies were conducted in COS7 cells by transfecting with wild-type (WT) and/or the mutant N629K HERG. The WT HERG produced an I(Kr)-like, E-4031-sensitive conductance with an inward rectification. In contrast, the cells transfected with the N629K HERG did not display any time-dependent current. Cotransfection of WT and N629K HERG (at a ratio of 1:1) produced a significantly smaller conductance when compared with WT HERG (WT 59.9 +/- 7.3 pA/pF [n = 22] vs WT+N629K 5.5 +/- 2.3 pA/pF [n = 11]; P < 0.01), but did not alter K+ ion selectivity and tail current-voltage dependence. Because aprindine hydrochloride was effective in preventing ventricular tachycardias, we also tested the effect of the drug on WT HERG (I(Kr)) and KvLQT1/KCNE1 (I(Ks)) currents expressed in COS7. CONCLUSION: Functional analyses of a novel missense mutation in the pore of HERG suggest that the mutation causes marked reduction of I(Kr) via a dominant negative effect.

Adult↗
PubMed1999

Genomic organization and chromosome location of the rat CD3zeta locus (Cd3z).

The structure and pattern of expression of the CD3zeta chain have apparently been conserved throughout mammalian evolution. The organization of the rat CD3zeta locus was determined by genomic cloning and nucleotide sequencing. Most of the rat CD3zeta coding region was similar to mouse and human CD3zeta sequences. Whereas the 3' region involving alternative splicing was relatively well conserved at the nucleotide level, the deduced amino acid sequences were different in rats, mice and humans due to the presence of deletion and insertion mutations. Alternative splicing products of the CD3zeta locus, such as CD3eta, CD3&theta; and CD3tau, which have been reported for mice, were not expressed by rat T cells. By using fluorescence in situ hybridization, we have localized rat CD3zeta to chromosome 13q22-->q23.

Alternative Splicing↗
PubMed1999

Inhibitory effects of hydrolyzable tannins from Melastoma dodecandrum Lour. on nitric oxide production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide and interferon-gamma.

An extract of Melastoma dodecandrum LOUR. with 80% aqueous acetone (MDL) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide (LPS) and recombinant mouse interferon-gamma (IFN-gamma). On further fractionation of the extract, the majority of the inhibitory activity was recovered in the 50% methanol extracts, which contained hydrolyzable tannins. Among the latter, casuarinin, casuarictin, pedunclagin and nobotannin B exhibited strong inhibitory activities toward NO production, with ID50 values between 2.0 and 5.1 microM. Both MDL and the purified tannins significantly reduced the induction of the inducible nitric oxide synthase (iNOS) protein in the course of macrophage activation with LPS and IFN-gamma. In addition, the NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by these tannins, with IC50 values around 30-130 microM, but not by MDL. These results suggest that MDL has the pharmacological ability to suppress NO production by activated macrophages and that the hydrolyzable tannins have major inhibitory activities.

Animals↗
PubMed1998

Isolation and characterization of a new member of the human Ly6 gene family (LY6H).

The Ly6 family of genes encodes glycosylphosphatidylinositol-anchored cell surface glycoproteins expressed on various types of cells. Intriguing patterns of expression of Ly6 genes on specific subpopulations of lymphoid and myeloid cells suggest that Ly6 molecules may be involved in the development and homeostasis of hematopoietic cells. We have isolated a new member of the human Ly6 gene family, LY6H, from a human fetal brain cDNA library. Fluorescence in situ hybridization and radiation hybrid analyses assigned LY6H to chromosome 8, where other members of the Ly6 gene family are also located. Northern analysis revealed that LY6H is highly expressed in particular subdivisions of human brain and also in MOLT-3 and -4 acute lymphoblastic leukemia cells. These data suggest that LY6H may play a role(s) in both the central nervous system and the immune system.

Amino Acid Sequence↗
PubMed1998

The antiarrhythmic agent cibenzoline inhibits KATP channels by binding to Kir6.2.

We reported previously that cibenzoline, an antiarrhythmic agent, inhibits the ATP-sensitive K+ (KATP) channels of pancreatic beta-cells through a binding site distinct from that for glibenclamide. In the present study, we have determined the locus of the action of cibenzoline on KATP channels reconstituted with mutant Kir6.2 and SUR1. We expressed a C-terminal truncated Kir6.2 (Kir6. 2DeltaC26) with and without SUR1 in COS7 cells. Both Kir6.2DeltaC26 and Kir6.2DeltaC26 + SUR1 formed functional KATP channels. Glibenclamide inhibited Kir6.2DeltaC26 + SUR1 channels but failed to inhibit Kir6.2DeltaC26. In contrast, cibenzoline inhibited equally Kir6.2DeltaC26 and Kir6.2DeltaC26 + SUR1 channels, in a dose-dependent manner, the half-maximal concentrations of channel inhibition being 22.2 +/- 6.1 and 30.9 +/- 9.4 microM, respectively. Furthermore, we determined also that [3H]cibenzoline bound to Kir6. 2DeltaC26. These findings confirm that cibenzoline inhibits KATP channels by a novel inhibitory mechanism in which cibenzoline directly affects the pore-forming Kir6.2 subunit rather than the SUR1 subunit.

ATP-Binding Cassette Transporters↗
PubMed1998

Cytoplasmic terminus domains of Kir6.x confer different nucleotide-dependent gating on the ATP-sensitive K+ channel.

1. In order to investigate the structural basis for the nucleotide-dependent gating of ATP-sensitive K+ channels (KATP), Kir6.1 (uKATP-1), Kir6.2 (BIR1) and chimeric channels were co-expressed with a common subtype of sulphonylurea receptor, SUR1, in COS7 cells. Representing the amino terminal domain-transmembrane domain-carboxyl-terminal domain of Kir6.1 as 1-1-1 and of Kir6.2 as 2-2-2, chimeric Kir6.x channels were constructed by swapping the amino and/or carboxyl terminal domains between Kir6.1 and Kir6.2 to give the chimeric x-1-x channels 1-1-2, 2-1-1 and 2-1-2, and the chimeric x-2-x channels 2-2-1, 1-2-2 and 1-2-1. 2. Inside-out patch clamp experiments revealed that both wild-type Kir6.1 and Kir6.2 formed inwardly rectifying K+ channels. Single-channel conductances were 36.3 and 66.1 pS, respectively. Chimeric x-1-x channels, whose transmembrane domain was that of Kir6.1, showed similar ion-pore properties to wild-type Kir6.1. Likewise, chimeric x-2-x channels had similar ion-pore properties to wild-type Kir6.2. 3. Wild-type Kir6.1 and Kir6.2 possessed distinct gating properties towards intracellular nucleotides. The activity of Kir6.1 was entirely dependent on Mg2+ and nucleotide diphosphates (NDPs) such as UDP. In contrast, Kir6.2 was activated upon excision of patch membrane. When Kir6.2 underwent rundown, UDP reactivated the channel. 4. In order to eliminate UDP dependence from Kir6.1, it was necessary to replace both N- and C-termini; chimera 2-1-2 opened in UDP-free conditions. With Kir6.2, substitution of the N-terminus with that of Kir6.1 conferred UDP dependence on chimeras 1-2-2 and 1-2-1. Chimera 2-2-1 opened in UDP-free conditions, but UDP potentiated the channel activity by > 20-fold. 5. The kinetics of UDP-dependent activation were significantly different between Kir6.1 and Kir6.2. Kir6.1 maximally activated by UDP was sensitive to intracellular ATP, although its ATP sensitivity was significantly lower than that of Kir6.2 measured in identical conditions. The kinetics of UDP-dependent activation and ATP sensitivity could be transferred between Kir6.1 and Kir6.2 only when both N- and C-termini were replaced. We therefore concluded that nucleotide-dependent gating was regulated by the N- and C-terminal domains irrespective of the transmembrane domains.

Adenosine Triphosphate↗
PubMed1998

Simultaneous determination of five macrolide antibiotics in meat by high-performance liquid chromatography.

A simple and rapid method using high-performance liquid chromatography (HPLC) for the simultaneous determination of five macrolides (josamycin, kitasamycin, mirosamicin, spiramycin and tylosin) in meat has been developed. The drugs were extracted with 0.3% metaphosphoric acid-methanol (7:3, v/v), and the extracts were cleaned up on a Bond Elut SCX (500 mg) cartridge. The HPLC separation was performed on a Puresil 5C18 column (150 x 4.6 mm I.D.) with a gradient system of 0.025 M phosphate buffer (pH 2.5)-acetonitrile as the mobile phase at a flow-rate of 1.0 ml/min. The drugs were detected at 232 mn for josamycin, kitasamycin, mirosamicin and spiramycin, and 287 mn for tylosin. The calibration graphs were rectilinear from 2.5 to 100 ng for each drug. The recoveries at the level of 1.0 microgram/g were 70.8-90.4%, and detection limits were 0.05 microgram/g for each drug.

Animals↗
PubMed1998

Cystic fibrosis transmembrane conductance regulator mediates sulphonylurea block of the inwardly rectifying K+ channel Kir6.1.

1. Recombinant ATP-sensitive K+ channels (KATP channels) were heterologously expressed in the NIH3T3 mouse cell line, and the electrophysiological properties were studied using patch-clamp techniques. 2. The NIH3T3 cell lines transfected with the inwardly rectifying K+ channel Kir6.1 alone or with both Kir6.1 and cystic fibrosis transmembrane conductance regulator (CFTR) exhibited time-independent K+ currents with weak inward rectification. In contrast, no measurable K+ conductance was observed in mock-transfected cells or in cells transfected with CFTR alone. Regardless of co-transfection with Kir6.1, the transfection with CFTR produced a Cl- conductance that was activated by cell dialysis with cAMP (1 mM). The conductance was reversibly suppressed by glibenclamide (30 microM). 3. Whole-cell currents at +60 mV were blocked in a concentration-dependent manner by Ba2+ ions with similar IC50 values: 89.3 +/- 23.3 microM (Kir6.1 alone) and 67.3 +/- 24.9 microM (Kir6.1-CFTR). 4. The currents recorded from Kir6. 1-transfected cells were not affected by glibenclamide, whereas glibenclamide did inhibit the conductance expressed in cells co-transfected with CFTR (IC50 = 35.9 +/- 6.6 microM). 5. In the cell-attached mode with a 150 mM K+ pipette solution, both Kir6.1- and Kir6.1-CFTR-transfected cells displayed a class of K+ channels showing weak inward rectification and a slope conductance of 50.7 +/- 1.0 and 52.4 +/- 4.9 pS, respectively. 6. In the inside-out mode, the single-channel currents recorded from both types of cells were not inhibited by intracellular ATP (1 mM). However, glibenclamide was found to block the single-channel activities in the co-transfected cells.

3T3 Cells↗
PubMed1998

Genistein inhibits slow component delayed-rectifier K currents via a tyrosine kinase-independent pathway.

In single guinea pig ventricular cells, genistein, a potent inhibitor of protein tyrosine kinase (PTK), was found to suppress the delayed-rectifier K (IK) current. The present study was carried out to examine the underlying mechanism. Ventricular myocytes were voltage-clamped in the conventional whole-cell mode (36 degrees C). The amplitudes of tail and steady-state (2-s pulse) currents were measured as IK. Genistein (10-100 microM) reversibly inhibited both basal and intrapipette cAMP (1 mM)-enhanced IK currents in a concentration-dependent manner with a half-maximum inhibitory concentration (IC50) at approximately 30 microM. In contrast, lavendustin A (10 microM; n = 5) and tyrphostin 51 (100 microM; n = 5) had no effect on the currents. The inhibitory action of genistein was also seen after IK currents were activated by forskolin (500 nM) plus intrapipette orthovanadate (500 microM). The intrapipette cAMP-enhanced IK was also reduced to a lesser degree by daidzein, an inactive analogue of genistein. Envelope tail and short pulse protocols revealed that genistein inhibits the slow component of IK (IKs). Thus, the inhibitory action of genistein is not mediated via an inhibition of PTK but may be due to the block of IKs channels.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Accentuated antagonism by angiotensin II on guinea-pig cardiac L-type Ca-currents enhanced by beta-adrenergic stimulation.

To examine mechanism(s) underlying the accentuated antagonism by angiotensin II (A-II) on twitch tension, we recorded L-type Ca2+ currents (ICa,L) using conventional patch-clamp techniques in single, guinea-pig, ventricular myocytes. ICa,L was recorded by a step-pulse protocol after eliminating K+ conductances (internal Cs+ plus tetraethylammonium chloride and K+-free extracellular solution). A-II (100 nM) did not affect basal ICa,L, but inhibited ICa,L that had been enhanced (approximately 200% of control) by (ISO, isoproterenol 100 nM). The inhibitory action of A-II was concentration dependent (concentration eliciting 50% inhibition 88+/-9 pM, n=41) and the ISO-enhanced component of ICa,L was completely blocked by A-II at concentrations above 10 nM. CV-11974 (500 nM), an A-II type-1 receptor (AT1) antagonist, prevented the inhibitory action of A-II. Pre-incubation with pertussis toxin (PTX) abolished the inhibitory effect of A-II. A-II also inhibited the ICa, L enhanced by histamine (500 nM) and forskolin (1 microM), but failed to affect ICa,L enhanced by intracellular cyclic adenosine monophosphate (1 mM). The inhibitory action of A-II may therefore involve AT1 receptors/PTX-sensitive, guanine nucleotide-binding (G) proteins (Gi)/adenylate cyclase and partially explains the A-II-dependent accentuated antagonism of inotropy.

Adrenergic beta-Antagonists↗
PubMed1998

Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia.

We isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1-q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.

5-Aminolevulinate Synthetase↗
PubMed1998

Functional communication between cardiac ATP-sensitive K+ channel and Na/K ATPase.

INTRODUCTION: Functional interaction between K(ATP) channel and Na/K ATPase was studied in single guinea pig ventricular myocytes because both membrane molecules are known to be involved in ischemic episodes. METHODS AND RESULTS: K(ATP) channel currents were recorded at 36 degrees C by using whole cell, cell-attached, inside-out, and open cell-attached modes of patch clamp techniques on enzymatically isolated ventricular myocytes. In the whole cell mode, ouabain (1 microM) reversibly inhibited the K(ATP) currents induced by metabolic stress (ATP-free pipette solution and 1 mM NaCN), but not those activated by cromakalim (100 microM), a K(ATP) channel opener. In the cell-attached mode, ouabain concentration dependently inhibited K(ATP) channel opening induced by metabolic suppression (5.5 mM 2-deoxyglucose and 1 mM CN-). Half-inhibition concentration for ouabain was 21.0 +/- 5.5 nM and the Hill coefficient was 0.8 +/- 0.1 (n = 26). However, ouabain did not have an effect on the channel activity induced by cromakalim (100 microM). In the inside-out mode, ouabain applied to the internal side of membrane did not affect the channel. In the open cell-attached mode made by preincubation with streptolysin-O (0.08 U/mL), the K(ATP) channels were not activated by the metabolic inhibitors but were by reducing extracellular ATP concentrations, because subsarcolemmal ATP concentration could be controlled through tiny membrane holes. The channels thus activated were not suppressed by ouabain. CONCLUSION: The inhibition of Na/K ATPase by ouabain appeared to block the K(ATP) channels by accumulating subsarcolemmal ATP caused by a decrease of the transition from ATP to ADP. In the presence of ischemic episodes, the administration of digitalis compounds may affect the opening of K(ATP) channels, which is primarily protective against the development of irreversible myocardial damage.

Adenosine Diphosphate↗