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Y Ohya

Publications and source records attributed to Y Ohya.

At least 145 records · Page 8Linked to original sources
PubMed1991

Two yeast genes encoding calmodulin-dependent protein kinases. Isolation, sequencing and bacterial expressions of CMK1 and CMK2.

We have isolated two genes from Saccharomyces cerevisiae that both encode a calmodulin-dependent protein kinase (CaM kinase). The CMK1 gene has been cloned by hybridization using an oligonucleotide probe synthesized on the basis of the peptide sequence of purified yeast CaM kinase (Londesborough, J. (1989) J. Gen. Microbiol. 135, 3373-3383). The other gene, CMK2, which is homologous to CMK1, has been isolated by screening at low stringency with a CMK1 fragment as a probe. The CMK2 product expressed in bacteria shows Ca(2+)- and CaM-dependent protein kinase activity, indicating that CMK2 also encodes a CaM kinase. The CMK1 and CMK2 products expressed in bacteria were found to have different biochemical properties in terms of autoregulatory activity and preference for yeast CaM or bovine CaM for maximal activity. Antibody raised against a peptide fragment of the CMK1 protein cross-reacts with the CMK2 product. Immunoblotting with this antibody indicated that the CMK1 and CMK2 products have apparent molecular masses of 56 and 50 kDa, respectively, in yeast cells. The predicted amino acid sequences of the two CMK products exhibit highest similarity with mammalian calmodulin-dependent multifunctional protein kinase II (CaM kinase II): the similarity within the N-terminal catalytic domain is about 40%, whereas that within the rest of the sequence is 25%. These data indicate that yeast has two kinds of genes encoding CaM kinase isozymes whose structural and functional properties are closely related to those of mammalian CaM kinase II. Another gene may be substituted for function of the CMK1 and CMK2 kinase in vivo, since elimination of both kinase genes is not lethal.

Amino Acid Sequence↗
PubMed1991

Half-calmodulin is sufficient for cell proliferation. Expressions of N- and C-terminal halves of calmodulin in the yeast Saccharomyces cerevisiae.

Calmodulin (CaM) has been shown to be an essential component for progression of nuclear division in the yeast Saccharomyces cerevisiae (Ohya, Y., and Anraku, Y. (1989) Curr. Genet. 15, 113-120). To define the functional domain of the molecule required for cell proliferation, we constructed plasmids expressing a series of N- and C-terminal halves of the CaM under the control of the galactose-inducible GAL1 promoter. These plasmids were introduced into a cmd1-disrupted yeast haploid strain, and the growth properties of the cells depending on the half-CaMs were examined. Plasmids expressing the N-terminal half (Ser1-Leu76) and the C-terminal half (Leu85-Cys147), which each maintain two complete EF-hand structures, complemented the growth defect of the cmd1 null mutation, whereas those expressing shorter regions of C- and N-terminal CaM did not. The half-CaMs that complemented the cmd1 null mutation were found to be approximately 6-fold overexpressed relative to expression of native CaM by the wild-type CMD1 gene. The levels of expression of the half CaMs with the true CMD1 promoter were not sufficient for complementation. These results demonstrate that half-CaMs (either the N- or the C-terminal) are capable of supporting growth of yeast cells when they are suitably overproduced. Cells depending solely on half-CaMs all showed a temperature-sensitive growth phenotype, suggesting that half-CaMs cannot carry out all the cellular functions of the complete CaM molecule.

Base Sequence↗
PubMed1991

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in prenatal stage on the dopamine system in the postnatal mouse brain.

A dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP), administered to a pregnant female was found to affect postnatally the catecholamine metabolism of the pups. MPTP (5 mg/kg body weight/day) was administered to pregnant C57 Black BYA mice daily for 7 days between the 12th and 18th day of gestation. Dopamine levels and tyrosine hydroxylase (TH) activity were measured in the whole brain from the pups sacrificed after birth. In MPTP-treated pups at 7 days of age, TH total activity (TH activity/brain) did not change (92% of the control value), while TH specific activity (TH activity/mg protein) was increased to 163% of that in control mice. Thus, TH homospecific activity (TH activity/mg TH protein) doubled compared to the control mice. At 28 days of age, both the total activity and the specific activity of TH in the brains of postnatal mice were reduced to 50% and 78% of the control, respectively. Dopamine concentration in the striatum was also reduced significantly. Reduction in the TH activity and dopamine concentration were also observed at the age of 12 weeks. These data suggest that the prenatal exposure to MPTP induced a prolonged reduction of TH activity in the brains of mice with a transient increase of TH homospecific activity during the postnatal period.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1991

Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells.

The possible involvement of a GTP-binding protein in the regulation of Ca2+ channels by angiotensin II (Ang II) in vascular muscle cells was investigated by the whole-cell voltage-clamp method. Single cells were freshly isolated from guinea pig portal vein. The pipette solution contained high Cs+ to inhibit K+ currents and thereby isolate the Ca2+ channel current. Ba2+ (2 mM) was in the bath solution as a charge carrier for the Ca2+ channel. Application of Ang II (0.1-100 nM) produced an increase in peak amplitude of the Ba2+ current, with a shift of the current-voltage curve in the negative direction. These effects were inhibited by pretreatment with an antagonist of the Ang II receptor, [Sar1,Ile8]-Ang II. Presence of 0.1 mM GTP in the pipette solution stabilized the Ang II action, but 0.3-1.0 mM GDP-beta-S and 1.0 mM GTP-gamma-S inhibited it. GTP-gamma-S alone produced a slowly progressing increase in the basal (unstimulated) current amplitude. Preincubation of muscle tissues with pertussis toxin (1 micrograms/ml, for up to 6 hours at 36 degrees C) or intracellular application of preactivated pertussis toxin (1 micrograms/ml) plus NAD (1 mM) did not inhibit the Ang II action. Cholera toxin (10 micrograms/ml) also had no effect on the Ang II action. These results suggest that the Ang II stimulation of Ca2+ channels in smooth muscle of guinea pig portal vein may be mediated by a G protein that is insensitive to both pertussis toxin and cholera toxin.

Angiotensin II↗
PubMed1991

DNA diagnosis of falciparum malaria.

We have developed DNA diagnosis using Universal probe system for the rapid detection of Plasmodium falciparum parasite in blood. We chose the DHFR-TS (dihydrofolate reductase-thymidylate synthase) gene as target for detection which are the junction part (410 bp) of and the DHFR part (790 bp). In the parasite, there is only one copy of target sequence, therefore, the target sequences were amplified by PCR (polymerase chain reaction) to increase the sensitivity. Our hybridization method consists of two probes; a primary probe prepared from a chimeric phage-plasmid vector (pUCf1) containing sequence complementary to the target, and a biotin-labeled secondary probe complementary to a portion of the primary probe, which is detected by the BCIP/NBT method. We showed that the 410 bp was more sensitive than the 790 bp as a target of P. falciparum, and the limit of detection was 10(3) parasites in 1 ml human blood using 410 bp junction part. We also constructed double PCR systems using junction part of DHFR-TS gene. By amplification of the 410 bp of the junction part and reamplification of 228 bp of inside sequence of the 410 bp, as little as 10 parasites in 10 microliters human blood was sufficient for specific detection.

Animals↗
PubMed1990

A multinuclear magnetic resonance study of a cls11 mutant showing the Pet- phenotype of Saccharomyces cerevisiae.

Energetic and intermediary metabolism was studied in a Pet- mutant of Saccharomyces cerevisiae with a calcium-sensitive phenotype that shows an inability to grow when cultured in a medium containing non-fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild-type cells incubated with [1,3-13C]glycerol or [2-13]acetate, and analyzed by 31P, 13C and 1H NMR. 31P- and 1H-NMR spectra showed significant differences between cls11 and wild-type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild-type cells). 13C-NMR spectra revealed major differences in the steady-state labelling of glutamate carbons. For incubations with [1,3-13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl-CoA C2 is 43% C13 labelled in wild-type and 10% 13C labelled in mutant cells, respectively. For incubations with [2-13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild-type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl-CoA was observed in the mutant cells (89%) compared to the wild-type cells (97%). These results are discussed in terms of pleiotropic defects in non-fermentable carbon metabolism in mutant cells.

Acetates↗
PubMed1990

Effects of azelastine on contraction of guinea pig tracheal smooth muscle.

Azelastine [4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepine-4-yl)-1(2H)-phthalazinone hydrochloride] is a new anti-asthmatic drug. We examined the mechanism of its inhibitory action on guinea pig tracheal smooth muscle contraction by measuring membrane potential and isometric force using intracellular microelectrodes and a micro-force transducer. The mean resting membrane potential of guinea pig tracheal muscle cells was -54 mV. Perfusion with 20 mM tetraethylammonium (TEA) caused membrane depolarization and elicited spontaneous action potentials. Azelastine (1-100 microM) suppressed both the amplitude and maximal rate of rise of the action potentials in a concentration-dependent manner. Complete abolition occurred at 100 microM. Similarly, azelastine (0.1-100 microM) inhibited and abolished 50 mM KCl-induced contractions. These results suggest that azelastine may inhibit voltage-dependent Ca2+ channels. Next, pretreatment of tracheal muscle (for 15 min) with azelastine (0.01-100 microM) inhibited subsequent acetylcholine (ACh) (0.01-100 microM)-induced contractions. Azelastine, 100 microM, completely abolished the ACh-induced contractions. In contrast, high concentrations of Ca2+ channel antagonists diltiazem (10-100 microM) or nifedipine (20 microM), and Ca2(+)-free solution, only partially depressed the ACh contractions suggesting that azelastine has an additional effect on intracellular Ca2+ release. In Ca2(+)-free solution (containing 0.5 mM EGTA), azelastine (1-100 microM) depressed and abolished the transient contractions induced by 10 microM ACh. We conclude that azelastine inhibits airway constriction by inhibiting both voltage-sensitive Ca2+ slow channels on the cell membrane and Ca2+ release from a intracellular storage site.

Action Potentials↗
PubMed1990

Transplacentally-transported 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) affects the catecholamine and indoleamine levels in the fetal mouse brain.

The effects of a dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the amounts of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were examined in the whole brains of fetal mice and maternal mice after its administration to pregnant mice. DA and DOPAC concentrations were decreased significantly in both the fetal and maternal brains. At 3 hr after injection, reduction of the DOPAC concentration was more marked than that of DA in both the fetal and maternal brains. Increase of 5-HT concentration was observed until 12 hr after injection in the fetal brains and 6 hr in the maternal brains. These results indicate that 1-methyl-4-phenyl-pyridinium ion (MPP+) and MPTP affect the levels of catechol- and indoleamines in the brain of premature stage as well as in the mature brain.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1990

Transcriptional analysis of the flagellar regulon of Salmonella typhimurium.

In Salmonella typhimurium, nearly 50 genes are involved in flagellar formation and function and constitute at least 13 different operons. In this study, we examined the transcriptional interaction among the flagellar operons by combined use of Mu d1(Apr Lac) cts62 and Tn10 insertion mutants in the flagellar genes. The results showed that the flagellar operons can be divided into three classes: class I contains only the flhD operon, which is controlled by the cAMP-CAP complex and is required for expression of all of the other flagellar operons; class II contains seven operons, flgA, flgB, flhB, fliA, fliE, fliF, and fliL, which are under control of class I and are required for the expression of class III; class III contains five operons, flgK, fliD fliC, motA, and tar. This ordered cascade of transcription closely parallels the assembly of the flagellar structure. In addition, we found that the fliD defect enhanced expression of the class III operons. This suggests that the fliD gene product may be responsible for repression of the class III operons in the mutants in the class II genes. These results are compared with the cascade model of the flagellar regulon of Escherichia coli proposed previously (Y. Komeda, J. Bacteriol. 170:1575-1581, 1982).

Base Sequence↗
PubMed1990

Tocolytic agents act on calcium channel current in single smooth muscle cells of pregnant rat uterus.

Effects of Ca2+ channel blockers and clinically important tocolytic agents, Mg2+ and beta-agonists, were examined on the Ca2+ channel current recorded from freshly isolated single pregnant rat myometrial cells using the whole-cell voltage clamp method. Nifedipine inhibited the Ca2+ channel currents (Ba2+ current) dose-dependently. Inhibition by nifedipine was greater at higher (more positive) holding potentials and higher command potentials. According to the modulated receptor hypothesis, Kd values for resting state and inactivated state of channel were calculated to be 150-300 and 1.2-6.8 nM, respectively (at a command potential of -20 mV). Mg2+ applied in the bath dose-dependently inhibited the Ca2+ current recorded with 2 mM Ca2+ (Ki = 12 mM, at a command potential of -10 mV). Inhibition by Mg2+ was weaker at the higher command potentials. Voltage dependency observed for the Mg2+ block may be due to a shift of the activation curve (Mg2+ neutralizes the outer surface charge) and/or due to the location of the binding site for Mg2+ in the Ca2+ channel electric field. In contrast, high dose of isoproterenol (10 microM) did not produce significant change in the Ca2+ current. In summary, nifedipine and Mg2+ inhibited the Ca2+ channel, with opposite voltage dependencies, whereas isoproterenol had no effect on the Ca2+ current. Thus, beta-agonist may relax the uterine muscle by mechanisms other than inhibition of the Ca2+ channels.

Animals↗
PubMed1989

Uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the N-methyl-4-phenylpyridinium ion (MPP+) into fetal mouse brain through the placenta.

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to be taken up into fetal mice through the placenta from a maternal mouse. C57 black mice were mated and MPTP (30 mg/kg) was given intramuscularly at the 18th day of gestation. Definite amounts of MPTP were detected in fetal brains by assay using high-performance liquid chromatography (HPLC) at 1 h after MPTP injection, and much higher contents of MPTP were found in maternal brains, too. An oxidative product of MPTP, the N-methyl-4-phenylpyridinium ion (MPP+), was also detected in brains of both mother and fetus and its concentrations in their brains were followed at 1, 3, 6, 12, 24 h after MPTP injection. The time to reach the maximal MPP+ concentration in brains was different between mother and fetus; 1 h and 3 h respectively. In addition to brain, considerable amounts of MPP+ were found in fetal liver, maternal liver and kidney, and in the placenta.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1989

Functional expression of chicken calmodulin in yeast.

The coding region of a chicken calmodulin cDNA was fused to a galactose-inducible GAL1 promoter, and an expression system was constructed in the yeast Saccharomyces cerevisiae. Expression of calmodulin was demonstrated by purifying the heterologously expressed protein and analyzing its biochemical properties. When the expression plasmid was introduced into a calmodulin gene (cmd1)-disrupted strain of yeast, the cells grew in galactose medium, showing that chicken calmodulin could complement the lesion of yeast calmodulin functionally. Repression of chicken calmodulin in the (cmd1)-disrupted strain caused cell cycle arrest with a G2/M nucleus, as observed previously with a conditional-lethal mutant of yeast calmodulin. These results suggest that the essential function of calmodulin for cell proliferation is conserved in cells ranging from yeast to vertebrate cells.

Animals↗
PubMed1989

A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division.

The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12-15 h. The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12 h, no intracellular calmodulin protein was detectable. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. Thus, the defect was mainly in nuclear division. Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud. Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.

Amino Acid Sequence↗