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Y Ohya

Publications and source records attributed to Y Ohya.

At least 19 recordsLinked to original sources
PubMed1995

STT3, a novel essential gene related to the PKC1/STT1 protein kinase pathway, is involved in protein glycosylation in yeast.

Mutations of genes involved in the STT1/PKC1 pathway in yeast show staurosporine and temperature sensitivities (stt) which are suppressed by the addition of 1 M sorbitol [Yoshida et al., Mol. Gen. Genet. 242 (1994) 631-640]. Among the stt mutants, stt3-2 shares this phenotype. The STT3 gene encodes a novel 718-amino-acid protein with significant homology to potential transmembrane proteins of Caenorhabditis elegans and mouse mandibular condyle (about 80% homologous and 60% identical). Unlike the STT1/PKC1 gene, STT3 is essential for cell growth irrespective of osmotic support. Pulse-chase experiments show that the sst3 mutants are defective in protein glycosylation. The stt3 mutants are sensitive to hygromycin B and resistant to sodium orthovanadate, whose phenotypes are common to those defective in protein glycosylation.

Alkaloids↗
PubMed1995

STT10, a novel class-D VPS yeast gene required for osmotic integrity related to the PKC1/STT1 protein kinase pathway.

We report the genetic and biochemical properties of a staurosporine (ST)- and temperature-sensitive mutant, stt10, of Saccharomyces cerevisiae. The stt10 mutant shows an osmoremedial phenotype in a medium with 1 M sorbitol. ST sensitivity of the stt10 mutant was suppressed by overexpression of PKC1/STT1, showing the genetic interactions of STT10 with the PKC1/STT1 pathway. The nucleotide sequence of STT10 predicts a hydrophilic protein composed of 577 amino acids that possesses 20-25% sequence similarity with yeast Slp1/Vam5p, Sec1p and Sly1p, and nematode Unc-18. The stt10 deletion mutant is viable and shows a typical class-D vacuolar protein sorting defective (vps) phenotype. Vacuoles from stt10 cells have a normal vacuolar H(+)-ATPase activity, but are defective in vacuolar acidification. Genetic studies of yeast mutants carrying delta stt10, delta bck1, stt1/pkc1 or stt4 have revealed that their functions are phenotypically related to maintenance of cellular osmotic integrity.

Alkaloids↗
PubMed1995

Cooperation of calcineurin and vacuolar H(+)-ATPase in intracellular Ca2+ homeostasis of yeast cells.

Saccharomyces cerevisiae VMA genes, encoding essential components for the expression of vacuolar membrane H(+)-ATPase activity, are involved in intracellular ionic homeostasis and vacuolar biogenesis. We report here that the immunosuppressants FK506 and cyclosporin A cause general growth inhibition of the vma3 mutant. Upon addition of the drugs, the mutant grew neither in the presence of more than 5 mM Ca2+ nor above pH 6.0. The action of the immunosuppressants is dependent on their binding proteins and ascribable to inhibition of calcineurin activity; a mutation of a calcineurin subunit (cnb1) shows synthetic lethal interaction with the vma mutation. The addition of FK506 decreases the cytosolic free concentration of Ca2+ in the vma3 mutant cells. Consequently, FK506 induces an 8.9-fold elevation of a nonexchangeable Ca2+ pool. These results suggest that calcineurin controls calcium homeostasis by repression of Ca2+ flux into a cellular compartment(s) and that the vacuolar H(+)-ATPase is essential for cell growth cooperating with calcineurin to regulate the cytosolic free concentration of Ca2+.

Base Sequence↗
PubMed1995

The CLS2 gene encodes a protein with multiple membrane-spanning domains that is important Ca2+ tolerance in yeast.

Genetic screening of Saccharomyces cerevisiae mutants defective in Ca2+ homeostasis identified cls2, which exhibits a specific Ca(2+)-sensitive growth phenotype. We describe here the CLS2 gene and a multicopy suppressor (named BCL21, for bypass of CLS2) of the cls2 mutation. The CLS2 gene encodes a polypeptide of 410 amino acid residues, and its hydropathy profile indicates that the predicted Cls2 protein (Cls2p) contains ten putative membrane spanning regions. Immunofluorescent staining of the yeast cells expressing epitope-tagged Cls2p suggests that Cls2p is localized to endoplasmatic reticulum (ER) membrane. A cls2 disruption strain is viable, but shows a Ca(2+)-sensitive phenotype like the original cls2 mutants. BCL21 suppresses the cls2 disruption mutation, indicating that the multicopy suppression does not require the Cls2p. Suppression of cls2 was observed even after introduction of a single-copy plasmid harboring BCL21. The BCL21 gene encodes a protein of 382 amino acid residues and is identical to the SUR1 gene. sur1 was originally isolated as a suppressor of rvs161, which has reduced viability in nutrient starvation conditions. Possible mechanisms of the multicopy suppression are discussed.

Amino Acid Sequence↗
PubMed1995

Calmodulin-dependent protein kinase II and calmodulin are required for induced thermotolerance in Saccharomyces cerevisiae.

We show here that yeast mutants lacking calmodulin-dependent protein kinase II fail to fully acquire induced thermotolerance. A similar result was also obtained with mutants depending solely on either the N-terminal half or the C-terminal half of calmodulin. These findings indicate that both calmodulin-dependent protein kinase II and calmodulin are required for induced thermotolerance.

Calcium↗
PubMed1995

Cu/Zn superoxide dismutase-like immunoreactivity is present in Lewy bodies from Parkinson disease: a light and electron microscopic immunocytochemical study.

Copper-zinc superoxide dismutase (SOD1)-like immunoreactivity has been demonstrated in Lewy body-like inclusions (LIs) in brain tissues from patients with familial and sporadic amyotrophic lateral sclerosis. Using immunocytochemistry, we studied Lewy bodies (LBs), the original inclusions from which the term LI was derived, in five patients with Parkinson disease (PD). Surprisingly, many LBs were immunostained by an antibody against SOD1. There were two types of staining pattern: a diffuse pattern, and a peripheral pattern with an unstained core. An immunoelectron microscopic study demonstrated that the immunoreactive products were restricted to the fibrillary profiles, sparing the unstructured core. Our results showed that SOD1-like immunoreactivity occurred frequently in LBs and LIs, suggesting that a common cytopathological process is responsible for the formation of LB-type neuronal intracytoplasmic inclusions. Our results also suggest that SOD1 plays a role in the neurodegeneration associated with PD.

Aged↗
PubMed1995

Causative allergens of allergic rhinitis in Japan with special reference to silkworm moth allergen.

To determine the importance of silkworm moth allergens, we tested 267 patients with allergic rhinitis with CAP-RAST, a new assay system for detecting specific IgE in sera. We used the following allergen sources: house-dust mite, Candida, Alternaria, cat dander, orchard grass, ragweed, mugwort, Japanese hops, Japanese cedar, Japanese cypress, cotton, silk, larval chironomid midge, adult chironomid midge, and silkworm moth. As expected, the highest positive rate of reaction, as determined by CAP-RAST, was 73.8% for Japanese cedar, followed by 53.2% for mite and 50.6% for Japanese cypress. Although the positive rate for silkworm moth was not as high as for cedar pollen, one-third of patients had specific IgE against silkworm moth. The positive rate of reaction to silkworm moth was much higher than that to chironomid. It was interesting to note that the patients reported had not been documented as having frequent contact with silkworm moth allergen. Correlations between silkworm moth and silk, and silkworm moth and chironomid midge were found to be significant (P < 0.001). Silkworm moth allergen showed the third highest reaction rate in patients with severe symptoms. These results suggest that silkworm moth allergen should be considered to be important in patients with allergic rhinitis in Japan.

Adolescent↗
PubMed1995

Inhibitory action of betaxolol, a beta 1-selective adrenoceptor antagonist, on voltage-dependent calcium channels in guinea-pig artery and vein.

1. The effects of betaxolol, (+/-)-1-[4-[2-(cyclopropylmethoxy) ethyl] phenoxy]-3-(isopropylamino)-2-propanol hydrochloride, a beta 1-selective adrenoceptor antagonist, on voltage-dependent Ca2+ channels were investigated in single smooth muscle cells from guinea-pig mesenteric artery and portal vein using a whole-cell variant of the patch-clamp technique. Ca2+ channel currents were recorded with bath solutions contained 10 mM Ba2+ for arterial cells and 2 mM Ca2+ for venous cells. 2. Betaxolol inhibited Ca2+ channel currents dose-dependently in both mesenteric artery cells and portal vein cells. The two isomers, (+)-betaxolol and (-)-betaxolol (relative beta-antagonistic efficacies of 0.1 and 1, respectively), had similar potencies for inhibiting Ca2+ channel currents in portal vein cells. Propranolol did not inhibit the currents. Thus the inhibitory action of betaxolol on Ca2+ channel currents was independent of the beta-adrenoceptor. 3. The inhibitory action of betaxolol on Ca2+ channel currents was compared with that of diltiazem and of nifedipine in mesenteric artery cells. The current inhibition depended on the stimulation frequency with all drugs (use-dependent block). All drugs also accelerated the current decay and shifted the voltage-dependent inactivation curve in a negative direction. 4. In conclusion, betaxolol inhibited Ca2+ channel currents in vascular smooth muscle cells. The mode of inhibitory action was similar to that of diltiazem and nifedipine. Our results suggest that betaxolol is a unique beta-adrenoceptor antagonist that has a direct inhibitory action on voltage-dependent Ca2+ channels in vascular smooth muscle cells.

Animals↗
PubMed1995

Cloning and nucleotide sequence of the calmodulin-encoding gene (cmdA) from Aspergillus oryzae.

A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A. nidulans as a hybridization probe. The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A. oryzae. The nucleotide sequence analysis showed that the gene consists of five introns and six exons. Although the nucleotide sequence homology with that of A. nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A. nidulans and chicken, respectively. The cDNA encoding A. oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene.

Amino Acid Sequence↗
PubMed1995

Ambulatory blood pressure monitoring in elderly hypertensives treated with the new calcium antagonist, pranidipine (OPC-13340).

Pranidipine (OPC-13340), a new dihydropyridine calcium antagonist, was given to 9 elderly hypertensive inpatients aged 64-79 years. Once-daily administration of pranidipine (1-2 mg) for 1-2 weeks decreased the 24-h average BP significantly from 167/92 mmHg to 150/83 mmHg without any change in pulse rate (PR) or the variabilities of BP and PR. The reduction of BP was observed exclusively during daytime (171/95 mmHg to 153/86 mmHg, p < 0.01 for SBP, p < 0.05 for DBP), while BP reduction during nighttime was significant only for DBP (157/84 mmHg to 146/79 mmHg, p > 0.05 for SBP, p < 0.05 for DBP). The analysis of the circadian rhythm by the cosinor method revealed that the acrophases of BP and PR were not changed significantly by the treatment with pranidipine. No adverse effects such as flushing and headache developed during the treatment. These results suggest that once-daily treatment with pranidipine is safe and exerts a sufficient antihypertensive effect during daytime with mild reduction of nighttime BP in elderly hypertensives. Furthermore, it does not alter the circadian patterns or variabilities of BP and PR. Thus, pranidipine may be useful as a monotherapy for elderly hypertensives.

Aged↗
PubMed1994

Conditional lethality of a yeast strain expressing human RHOA in place of RHO1.

The yeast RHO1 GTPase, which has 72% amino acid sequence identity with its human counterpart, RHOA, is essential for growth, although the reason has not been investigated. We report here that yeast strains that rely solely on expression of human RHOA in place of RHO1 are able to grow at 23 degrees C but grow neither at 37 degrees C nor in the presence of 300 mM CaCl2 even at 23 degrees C. Measurements of steady-state protein levels indicate that inability to grow at the restrictive temperature is not due to instability of the protein. Homolog scanning with the two GTPases identified a small, 27-residue region of RHO1 which, when substituted into RHOA, confers full function in yeast. This region corresponds to the alpha 3-helix loop 7 region of RAS; the same region was reported to determine specificity of function between GTPases of the RAB family, Sec4p and Ypt1p. By examining the phenotype of RHOA substitution strains at nonpermissive temperature, we found evidence suggesting that the normal function of RHO1 is to maintain osmotic integrity.

Adenosine Diphosphate Ribose↗
PubMed1994

Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae.

Loss of yeast protein kinase C function results in three distinct phenotypes: staurosporine sensitivity, cell lysis and blockage of cell cycle progression at the G2/M boundary. Genetic analysis of the PKC1/STT1 protein kinase C gene and its interactions with STT4, encoding an upstream phosphatidylinositol 4-kinase, and BCK1, encoding a downstream protein kinase, reveal that they form part of a single pathway. However, the BCK1-20 mutation (a gain-of-function mutation of BCK1) or overexpression of PKC1 cannot suppress all of the phenotypes caused by the loss of STT4 function, strongly suggesting the existence of a branch point between STT4 and PKC1. We also describe the MSS4 gene, a multicopy suppressor of the temperature-sensitive stt4-1 mutation. MSS4 is predicted to encode a hydrophilic protein of 779 amino acid residues and is essential for cell growth. Based on genetic and biochemical data, we suggest that MSS4 acts downstream of STT4, but in a pathway that does not involve PKC1.

1-Phosphatidylinositol 4-Kinase↗
PubMed1994

Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene.

Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe-->Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl2 in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd 1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe-->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks.

Base Sequence↗
PubMed1994

Measurement of staphylokinase by enzyme-linked immunosorbent assay using monoclonal antibodies.

Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated. A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups. Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase. This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector". The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml. The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra- and inter-assay coefficients of variation, respectively. Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen. Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples.

Animals↗
PubMed1994

Modulation of circadian rhythm of blood pressure by cortisol in patients with hypopituitarism.

We conducted a study to determine the effect of exogenous cortisol on circadian blood pressure changes in patients with hypopituitarism. Under replacement with hydrocortisone of 15 to 25mg either once (8:00) or twice a day (8:00 and 20:00), and with prednisolone of 3.75 to 5mg once a day, the patients underwent non-invasive ambulatory blood pressure monitoring for 24 hours. The average 24-hour blood pressure before hydrocortisone replacement was 92.9 +/- 1.0 (systolic)/53.2 +/- 0.8mmHg (diastolic), while that after hydrocortisone replacement once or twice a day and prednisolone replacement significantly increased to 108.2 +/- 1.4/63.5 +/- 0.9mmHg, 109.1 +/- 1.6/62.3 +/- 1.0mmHg, and 105.4 +/- 1.2/62.3 +/- 0.9mmHg, respectively. Hydrocortisone replacement once a day showed a significant increase in day-night differences of blood pressure, while hydrocortisone replacement twice a day did not. There were no differences in nocturnal decrease in pulse rate between these two replacements. The daytime and nighttime urinary excretions of 17-hydroxycorticosteroids in hydrocortisone replacement once a day were 7.7mg/12hr (daytime) and 1.4mg/12hr (nighttime), respectively, while those in hydrocortisone replacement twice a day were 3.9mg/12hr (daytime) and 3.6mg/12hr (nighttime), respectively. Urinary 17-ketosteroids, epinephrine and norepinephrine did not show any differences between hydrocortisone replacement once and twice a day. These results suggest that hydrocortisone administration is one of the factors which modulate the circadian variation of blood pressure in patients with hypopituitarism, and may also suggest that the circadian change of cortisol secretion participates, at least in part, in the formation of an intrinsic circadian rhythm of blood pressure.

Blood Pressure↗
PubMed1994

Crossover comparison of the effects of enalapril and captopril on potassium homeostasis in patients with mild hypertension.

The effects of two types of angiotensin converting enzyme (ACE) inhibitors, enalapril (long-acting) and captopril (short-acting), on serum electrolytes and circadian rhythm of urinary electrolyte excretions were compared in relation to aldosterone status in patients with essential hypertension and normal renal function. Enalapril (5 mg once daily) and captopril (12.5 mg t.i.d.) were administered to 11 patients for 1 week each in a crossover fashion. Blood sampling in the early morning and 4-hour split urinary sampling for 24 hours were performed on the last day of control and each treatment periods. Enalapril and captopril significantly reduced blood pressure to similar levels. Enalapril but not captopril significantly inhibited plasma aldosterone concentration and urinary aldosterone excretion. Neither drug apparently altered serum or urinary Na levels. Both drugs significantly decreased urinary K excretion (p < 0.05, control: 44 +/- 4 mEq/day, captopril: 39 +/- 2 mEq/day, enalapril: 39 +/- 2 mEq/day; mean +/- SEM), but did not significantly alter serum K level (control: 4.1 +/- 0.1 mEq/l, captopril 4.2 +/- 0.2 mEq/l, enalapril 4.3 +/- 0.1 mEq/l). The circadian rhythm (acrophase) of urinary K excretion was not affected by either drug, while the amplitude was decreased by both, as assessed by the cosinor method. In summary, although enalapril caused more sustained inhibition of aldosterone secretion compared with captopril, both drugs showed similar effects on the K homeostasis in patients with mild essential hypertension.

Aldosterone↗