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Y Ohya

Publications and source records attributed to Y Ohya.

At least 109 records · Page 6Linked to original sources
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

Diverse essential functions revealed by complementing yeast calmodulin mutants.

Calmodulin, a cytoplasmic calcium-binding protein, is indispensable for eukaryotic cell growth. Examination of 14 temperature-sensitive yeast mutants bearing one or more phenylalanine to alanine substitutions in the single essential calmodulin gene of yeast (CMD1) revealed diverse essential functions. Mutations could be classified into four intragenic complementation groups. Each group showed different characteristic functional defects in actin organization, calmodulin localization, nuclear division, or bud emergence. Phenylalanine residues implicated in calmodulin localization and nuclear division are located in the amino-terminal half of the protein, whereas those implicated in actin organization and bud emergence are located in the carboxyl-terminal half.

Actins↗
PubMed1994

A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae.

A staurosporine-sensitive mutation (stt1) in yeast has been found in the PKC1 gene that encodes a protein kinase C homologue (Yoshida, S., Ikeda, E., Uno, I., and Mitsuzawa, H. (1992) Mol. Gen. Genet. 231, 337-344). We report here another staurosporine-sensitive mutant, stt4, which shows very similar phenotypes to that of the stt1 mutant. The stt4 temperature-sensitive mutant arrests mostly in G2/M phase at 37 degrees C, and the stt4 deletion mutant shows an osmoremedial phenotype. Staurosporine sensitivity of the stt4 mutant was suppressed by overexpression of PKC1/STT1, indicating genetic interaction between stt4 and pkc1/stt1. The nucleotide sequence of STT4 predicts a hydrophilic protein composed of 1,900 amino acid residues, with 26% sequence identity to the yeast VPS34 gene product and 27% to the catalytic subunit of mammalian phosphatidylinositol (PI) 3-kinase, respectively. Cell homogenates of the stt4 deletion mutant show normal PI3-kinase activity but lack most of the PI4-kinase activity that is detected in the wild-type. We conclude that STT4 encodes a yeast PI4-kinase that functions in the PKC1 protein kinase pathway.

1-Phosphatidylinositol 4-Kinase↗
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↗
PubMed1993

VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae.

Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. However, the Vma13 polypeptide is not required for targeting of the other V-ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar membranes isolated from delta vma13 cells. The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme. This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.

Amino Acid Sequence↗
PubMed1993

Pro-major basic protein has three types of sugar chains at the pro-portion.

The amino-acid sequence of purified recombinant pro-major basic protein from Chinese hamster kidney cells was determined to verify the primary structure and glycosylation sites. Reduced and S-carboxamidemethylated protein was first digested with Achromobacter proteinase I. Each peptide was characterized by amino-acid analysis and amino-acid sequence analysis. We could identify all the peptides which were expected from the pro-major basic protein cDNA sequence. Sequence analysis and deglycosylation study revealed that Ser-8, Thr-9, Ser-46 and Asn-70 were glycosylated. The results indicated that proMBP has three types of sugar chains, O-glycoside, N-glycoside and glycosaminoglycan, in the pro-portion.

Amino Acid Sequence↗
PubMed1993

VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.

vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants. The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains. A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants. Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts. None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal. The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions. Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa. However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.

Amino Acid Sequence↗
PubMed1993

Factors related to QT interval prolongation during probucol treatment.

To clarify factors related to the QT interval prolongation produced by probucol, multivariate analysis was applied to clinical and laboratory data retrospectively obtained from 89 patients with hypercholesterolaemia, who had taken probucol for more than 3 months. The corrected QT interval (QTc) increased from 0.410s before treatment to 0.431 s after the administration of probucol; the total cholesterol level decreased from 267 mg.dl-1 to 212 mg.dl-1. None of the patients demonstrated new arrhythmia. The QTc after probucol was independently correlated with sex, serum albumin level and baseline QTc. Changes in QTc after probucol were independently correlated with the presence of ischaemic heart disease, baseline QTc, and a change in the total cholesterol level. The results suggest that a prolonged QTc is likely to appear in female patients, and in patients with a long baseline QTc or with a low serum albumin. It is also suggested that marked lengthening of the QTc is likely to occur in patients with ischaemic heart disease or with a short baseline QTc. Probucol can be used safely in patients with hypercholesterolaemia, but ECG monitoring may be necessary, especially in female patients, as well as in those with hypoalbuminaemia or with ischaemic heart disease.

Electrocardiography↗
PubMed1993

Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.

Geranylgeranyl transferase I (GGTase I), which modifies proteins containing the sequence Cys-Ali-Ali-Leu (Ali: aliphatic) at their C-termini, is indispensable for growth in the budding yeast Saccharomyces cerevisiae. We report here that GGTase I is no longer essential when Rho1p and Cdc42p are simultaneously overproduced. The lethality of a GGTase I deletion is most efficiently suppressed by provision of both Rho1p and Cdc42p with altered C-terminal sequences (Cys-Ali-Ali-Met) corresponding to the C-termini of substrates of farnesyl transferase (FTase). Under these circumstances, the FTase, normally not essential for growth of yeast, becomes essential.

Alkyl and Aryl Transferases↗
PubMed1993

Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.

Alkyl and Aryl Transferases↗
PubMed1993

Age-related changes in endothelium-dependent hyperpolarization in the rat mesenteric artery.

This study was designed to determine the age-related changes in the endothelium-dependent hyperpolarization to acetylcholine (ACh) and its contribution to relaxation in the isolated mesenteric artery from normotensive and hypertensive rats. Membrane potentials and contractions were recorded in arteries from male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) that were 5-6 wk old (young), 6-8 mo old (adult), and 20-26 mo old (aged). Endothelium-dependent hyperpolarizations produced by ACh, applied both at the resting state of the membrane and under conditions of depolarization with norepinephrine (10(-5) M), were markedly impaired in aged WKY rats, adult SHR, and aged SHR. Endothelium-dependent relaxations to ACh in arterial rings precontracted with 10(-5) M norepinephrine were also impaired in aged WKY rats, adult SHR, and aged SHR even in the presence of indomethacin. Furthermore, in these rats, N omega-nitro-L-arginine, an inhibitor of nitric oxide formation, showed potent inhibitory effects on the relaxations, whereas the 20 mM high K+ solution that reduces hyperpolarization had less pronounced effects. Hyperpolarizations and relaxations to cromakalim (10(-5) M), a K(+)-channel opener, were on the whole preserved in aged rats. It would thus appear that the endothelium-dependent hyperpolarization to ACh is reduced with aging as well as by hypertension, and this would, in part, account for the impaired relaxation to ACh in arteries of both aged rats and hypertensive rats.

Acetylcholine↗
PubMed1993

Voltage-dependent Ca2+ channels in resistance arteries from spontaneously hypertensive rats.

Alterations in voltage-dependent Ca2+ channels in the arterial smooth muscle cells of spontaneously hypertensive rats (SHR) were investigated using the whole-cell voltage clamp and compared with Wistar-Kyoto (WKY) rats. Single cells were freshly isolated from resistance mesenteric arteries from 4- to 5-week-old (young) and 16- to 18-week-old (adult) SHR. Elevated blood pressure was only evident in adult SHR, not in young SHR. In young rats, the Ca2+ channel current density (current amplitude normalized by cell capacitance) was significantly higher (P < .01) in SHR than in WKY rats at the command potential of -10 mV or higher (with 50 mmol/L Ba2+): The current density at 20 mV was -16.8 +/- 1.1 pA/pF in SHR (n = 38 cells) and -11.0 +/- 0.8 pA/pF in WKY rats (n = 30 cells). In adult rats, the difference in current densities disappeared: -15.9 +/- 1.3 pA/pF in SHR (n = 25 cells) and -15.6 +/- 1.5 pA/pF in WKY rats (n = 29 cells). The ratio of maximal amplitude of T-type current to that of L-type current was low in young SHR (0.10 +/- 0.01) compared with the other three groups (0.16 to 0.20). Neither the activation curve nor the steady-state inactivation curve of SHR was different from that of age-matched WKY rats. However, the activation curves in adult rats were shifted to a hyperpolarized direction compared with those of young rats in both strains. These results suggest that the increased activity of voltage-dependent L-type Ca2+ channels of resistance arteries in young SHR may be related to the development of hypertension. The changes observed in adult rats may be due to a secondary modification of the channel during maturation and the presence of hypertension.

Animals↗