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T Miyakawa

Publications and source records attributed to T Miyakawa.

At least 19 recordsLinked to original sources
PubMed1995

Adaptation to high-salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase.

Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin, PP2B) of Saccharomyces cerevisiae is implicated in adaptation to high-salt conditions. Calcineurin mediates high salt-induced expression of the ENA1/PMR2 gene encoding the P-type ATPase, which is suggested to be involved in Na+ efflux. We identified the PDE1 gene encoding the low-affinity cAMP phosphodiesterase as a multicopy suppressor of the Li(+)- and Na(+)-sensitive calcineurin null mutant, suggesting that cAMP is a negative regulator of adaptation to high-salt stress. Genetic analysis indicated that calcineurin and cAMP act antagonistically in a common pathway for adaptation. The bcy1 disruption, which leads to constitutive cAMP-dependent protein kinase (PKA) activity inhibited high NaCl-induced expression of the ENA1/PMR2 gene, caused an elevation of the intracellular Na+ level and a growth defect in high-NaCl medium, all of which were analogous to the defects of a calcineurin mutant. A reduced cAMP level resulting from multiple copies of the PDE1 gene caused increased expression of the ENA1/PMR2 gene in response to high NaCl. We propose a model for the regulation of cation homeostasis, in which calcineurin antagonizes PKA to activate transcription of the ENA1/PMR2 gene in response to high-salt conditions.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Cloning and characterization of the Saccharomyces cerevisiae SVS1 gene which encodes a serine- and threonine-rich protein required for vanadate resistance.

A novel Saccharomyces cerevisiae (Sc) SVS1 gene was cloned as a multicopy suppressor of vanadate (Vn) sensitivity (VnS) due to a calcineurin (CaN) null mutation. SVS1 encoded a 260-amino-acid protein abundant in Ser and Thr residues, with a putative signal sequence at the N terminus. Deletion of SVS1 resulted in increased sensitivity to Vn, but not to other metallic ions or drugs. Northern analysis of the SVS1 mRNA indicated that the induction of the gene occurred specifically in the response to Vn. These results suggested that Sc has a mechanism to enhance the tolerance to Vn by increasing the expression of SVS1. The results of genetic experiments suggested that CaN and the Svs1 proteins act in separate pathways to enhance the tolerance to Vn.

Amino Acid Sequence↗
PubMed1995

Protein phosphatase 2B of Saccharomyces cerevisiae is required for tolerance to manganese, in blocking the entry of ions into the cells.

The role of protein phosphatase 2B (PP2B/calcineurin) of Saccharomyces cerevisiae in the tolerance to divalent cations was investigated. PP2B-deficient mutants were found to be sensitive to MnCl2, but not to ZnCl2, CuCl2, NiCl2 and CoCl2. By measuring both manganese uptake and its efflux, it was found that the sensitivity of the mutant cells was due to an increase in manganese uptake and that the wild-type cells were able to prevent manganese entry into the cells, rather than export it in a more efficient manner. In the presence of the immunosuppressant FK506, the behavior of wild-type cells became similar to that of PP2B mutants. Out of various divalent cations tested, externally added magnesium ions were able to block manganese uptake in both wild-type and PP2B mutant strains.

Biological Transport↗
PubMed1995

Are advanced glycation end-products associated with amyloidosis in Alzheimer's disease?

Recent studies have suggested that advanced glycation end-products (AGE) of the Maillard reaction are associated with amyloidosis in Alzheimer's disease. To evaluate this possibility, the present immunohistochemical study was undertaken to locate AGE in cerebral cortices of Alzheimer's disease, using a monoclonal antibody specific for AGE-proteins. Deposits of beta-amyloid protein within cores of classic senile plaques and vascular walls in amyloid angiopathy showed no AGE-positive reaction, while primitive plaques, coronas of classic plaques and some glial cells were positive for AGE. These findings are inconsistent with the suggestion that AGE may be involved primarily in amyloidosis in Alzheimer's disease.

Aged↗
PubMed1995

Enhanced susceptibility of audiogenic seizures in Fyn-kinase deficient mice.

Mice with a mutation in fyn genes were examined for their susceptibility to acoustically primed audiogenic seizures. Homozygous mutant (fynz/fynz) mice were significantly more likely to have seizures and to show the stronger seizure syndrome (clonus). These results indicate that the susceptibility of acoustically primed audiogenic seizures is enhanced in the Fyn kinase deficient mice.

Animals↗
PubMed1995

Dephosphorylation of abnormal sites of tau factor by protein phosphatases and its implication for Alzheimer's disease.

The abnormally phosphorylated forms of tau factor are major constituents of neurofibrillary tangles in Alzheimer's disease brain. In order to investigate protein phosphatases which are related to dephosphorylation of abnormal phosphorylation sites, we examined the dephosphorylation of tau factor phosphorylated by three proline-directed type protein kinases. Tau factor phosphorylated by cdc2 kinase and tau protein kinase II was dephosphorylated by the holoenzyme of protein phosphatase 2A and calcineurin, while either the catalytic subunit of protein phosphatase 2A or protein phosphatase 2C could not catalyze the dephosphorylation. From the kinetic analysis, we concluded that tau factors phosphorylated by the protein kinases serve as good substrates for protein phosphatase 2A and calcineurin. On the other hand, tau factor phosphorylated by glycogen synthase kinase 3 alpha was dephosphorylated by the catalytic subunit of protein phosphatases 2A as well as the holoenzyme of protein phosphatase 2A and calcineurin. It has been reported that serines 199, 202 and 396 according to the numbering of the longest human tau isoform are among the major abnormal phosphorylation sites of tau factor. We synthesized two phosphopeptides which contained phosphoserines 199 and 202 or phosphoserine 396 and prepared the polyclonal antibodies specific for the phosphopeptides. Using these antibodies, we confirmed that the holoenzyme of protein phosphatase 2A and calcineurin could dephosphorylate phosphoserines 199, 202 and 396 in tau factor. The catalytic subunit of protein phosphatase 2A could dephosphorylate phosphoserine 396 but not phosphoserines 199 and 202. Neurofibrillary tangles in Alzheimer's disease brain were immunostained with both antibodies but the normal neurons in the normal aged brains were not. The results suggest that protein phosphatase 2A and calcineurin can be involved in the dephosphorylation of abnormal phosphorylation sites in tau factor and that the dephosphorylation of phosphoserine 396 is differently regulated from phosphoserines 199 and 202.

Aged↗
PubMed1995

Functional analysis of the promoter of the Saccharomyces cerevisiae multidrug resistance gene YDR1, which encodes a member of the ATP binding cassette (ABC) superfamily.

The Saccharomyces cerevisiae gene YDR1/PDR5/STS1, which encodes a member of the ATP binding cassette (ABC) superfamily, is important for cross-resistance to apparently unrelated drugs. The expression of YDR1 is induced by various drugs and heat shock [Hirata et al., Curr. Genet., 26, 285-294 (1994)]. By deletion analysis of the 5'-noncoding region of the YDR1 gene, two drug responsive regions (-604 to -485 and -335 to -275) were identified with fluphenazine and cycloheximide. Three conserved palindrome sequences were found in these regions.

ATP-Binding Cassette Transporters↗
PubMed1995

The physiological roles of membrane ergosterol as revealed by the phenotypes of syr1/erg3 null mutant of Saccharomyces cerevisiae.

Ergosterol is a major sterol component of fungal plasma membranes. The effects of disrupting the Saccharomyces cerevisiae SYR1/ERG3 gene, which encodes sterol C-5 desaturase, an enzyme of ergosterol biosynthesis pathway, were markedly different for different S. cerevisiae strains and growth temperatures. The null mutation of SYR1 (delta syr1) in strain RAY-3A had only a slight effect on the growth rate at 28 degrees C. However, at this temperature, the same mutation caused poor growth in strain KA-311A and no growth in strain W303-1A. The delta syr1 disruptant of these strains were able to grow at 37 degrees C, as well as their parental strains. Moreover, the growth of the delta syr1 disruptant of W303-1A and KA-311A strains were severely inhibited at 16 degrees C. These results indicated that ergosterol is essential for growth at low temperatures, and the effects of the gene disruption are variable by the genetic background. The growth defect at low temperatures appeared to be due to the defect of tryptophan uptake in the delta syr1 mutants. The delta syr1 mutants were sensitive to a wide variety of drugs, chemicals, and ions, suggesting that yeast ergosterol is important as permeability barrier against various chemical stresses.

Antifungal Agents↗
PubMed1994

Stress-induced transcriptional activation mediated by YAP1 and YAP2 genes that encode the Jun family of transcriptional activators in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae YAP2 gene encoding an AP-1-like transcriptional activator protein was cloned by selection for genes that confer pleiotropic drug resistance when present in high copy number. The novel YAP2 gene encodes a protein of 45827 daltons and is homologous in part to a known transcriptional activator protein encoded by YAP1/PDR4/SNQ3/PAR1. Homology was found only in both terminal regions. The N-terminal portion contains a region rich in basic amino acids, followed by a "leucine zipper" motif. Overexpression of YAP2 led to the induction of expression of an AP-1 recognition element (ARE)-dependent promoter. The yap1 disruptant has been shown to be sensitive to H2O2. In this study, we demonstrated that the yap1 disruptant is also unable to grow in medium containing 150 microM cadmium, whereas the yap2 disruptant exhibited no significant phenotypes. However, YAP2 in high copy number did suppress cadmium sensitivity, but not H2O2 sensitivity of the yap1 disruptant. YAP1 was able to mediate both cadmium- and H2O2-induced transcriptional activation of an ARE-dependent promoter. A high-copy-number plasmid bearing YAP2 mediated cadmium-induced transcriptional activation of this promoter. The inductions were prevented by the antioxidant N-acetyl-L-cysteine.

Acetylcysteine↗
PubMed1994

Saccharomyces cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is required for multidrug resistance.

A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.

ATP-Binding Cassette Transporters↗
PubMed1994

Re-evaluation of classic senile plaques by three-dimensional analysis.

We evaluated the proportion of classic plaques among all of the senile plaques in four Alzheimer brains (frontal, temporal, occipital and hippocampal areas) by the usual method of two-dimensional analysis using a single methenamine silver-stained section and three-dimensional analysis using a set of serial sections. Three-dimensional analysis showed the number and percentage of classic plaques to be 2-5 times greater than those revealed by two-dimensional analysis. In the hippocampal area of one case, no classic plaques were found by two-dimensional analysis but three-dimensional analysis showed that some classic plaques were present. From these findings, it is suggested that three-dimensional analysis using serial sections is indispensable for subclassifying senile plaques.

Aged↗
PubMed1994

Increased fearfulness of Fyn tyrosine kinase deficient mice.

Fyn-deficient mice were produced by inserting the beta-galactosidase gene (lacZ) into the fyn gene locus. The homozygously Fyn kinase-deficient (fynz/fynz) mice exhibited stronger light aversion in the light-dark choice test and higher fear-response scores in the novelty preference and passive avoidance tests than did the heterozygously Fyn-deficient (+/fynz) mice. These results indicate that fynz/fynz mice are hyperresponsive to fear-inducing stimuli.

Animals↗
PubMed1994

Identification and analysis of the Saccharomyces cerevisiae SYR1 gene reveals that ergosterol is involved in the action of syringomycin.

A 2.5 kb DNA fragment of the Saccharomyces cerevisiae SYR1 gene was cloned by complementation of the syr1 mutations that simultaneously lead to resistance to the phytotoxin syringomycin and sensitivity of growth to high Ca2+ concentrations. Sequencing of this fragment revealed a single open reading frame encoding a polypeptide of 365 amino acids. Four hydrophobic regions each separated by hydrophilic regions were present in the protein. SYR1 was identical to ERG3, which is suggested to encode C-5 sterol desaturase required for ergosterol biosynthesis. The protein product of SYR1 was identified by Western blot analysis as a protein of 40 kDa in the particulate fraction. Gene disruption experiments demonstrated that elimination of SYR1/ERG3 is not lethal, but results in membrane C-5 desaturated sterol deficiencies, resistance to syringomycin and sensitivity to high Ca2+. The syr1 mutant cells had significantly decreased ability for syringomycin binding. The results indicated that C-5 desaturated sterols are involved in the binding of syringomycin to the cell, and the lack of the sterols in the mutant membrane results in sensitivity to high Ca2+ and an increased rate of cellular Ca2+ influx.

Bacterial Proteins↗
PubMed1994

Morphological investigation of neurofibrillary tangles in Alzheimer's disease.

There are two types of neurofibrillary tangles, namely, a twisted tubule and a straight or uniform tubule in the same cell in the brain with Alzheimer's disease. However, in some parts of the same fibril, both the twisted and straight tubules clearly could be seen as a continuation of each other. This finding suggests that both of the tubules have a deep relation to each other, especially at the molecular level. This might also suggest the possibility of both tubules which mutually transform into each other.

Aged↗
PubMed1994

Deletion polymorphism of the angiotensin I-converting enzyme gene is associated with serum ACE concentration and increased risk for CAD in the Japanese.

BACKGROUND: The angiotensin I-converting enzyme (ACE) is a key component of the renin-angiotensin system thought to be important in the pathogenesis of hypertension and cardiovascular disease. Deletion polymorphism in the ACE gene may be a risk factor for myocardial infarction in the Caucasian population. However, this finding has not yet been investigated in the Japanese population. METHODS AND RESULTS: A 287-bp insertion/deletion polymorphism in intron 16 of the ACE gene was examined by polymerase chain reaction in a cross-sectional study of 100 healthy subjects and 178 patients with coronary artery disease (CAD) (70 angina pectoris, 108 myocardial infarction), whose serum ACE levels were concomitantly measured. Polymorphism of the ACE gene was characterized by three genotypes: two deletion alleles (genotype DD), two insertion alleles (genotype II), and heterozygous alleles (genotype ID). No differences could be detected among the three genotypes for total cholesterol, HDL cholesterol, and body mass index. Serum ACE levels were 11.4 +/- 2.7, 14.5 +/- 3.5, and 16.6 +/- 4.6 IU/mL for genotypes II, ID, and DD, respectively. In the study population, the genotype DD was more closely associated with CAD than the other two genotypes (ID and II). The frequency of deletion alleles was higher (0.58) in the CAD group than in healthy control subjects (0.42) (P < .05). Furthermore, multivessel disease was more strongly associated with deletion alleles than with insertion alleles (P < .05). CONCLUSIONS: A deletion polymorphism of the ACE gene is associated with serum ACE activity and increased risk for CAD in the Japanese.

Adult↗
PubMed1994

[Three patients from two families with familial Creutzfeldt-Jakob disease having a point mutation in the prion protein gene at codon 200 (Glu-->Lys)].

We present three patients with Creutzfeldt-Jakob disease (CJD). They lived in Fuji city and its neighboring towns in the eastern part of Shizuoka prefecture. Patient 1 and patient 2 were cousins. Patient 1 developed the illness at the age of 50 in 1987 and died 13 months later. Patient 2 became ill at the age of 73 in 1989 and died seven months later. Patient 3 was related to a familial CJD cases in Yamanashi prefecture, known as Akai's "H" family (Akai et al in 1979, Yamamoto et al in 1986). She became ill at the age of 78 in 1990 and died four months later. Their clinical features were common; rapidly progressive dementia, generalized myoclonus, and periodic synchronous discharges on electroencephalographies. They were autopsied and neuropathologically diagnosed as typical CJD. Molecular genetic analysis of the prion protein (PrP) gene was performed on patient 2 and patient 3 using their frozen brain sections. The results showed a point mutation in the PrP gene at codon 200; GAG to AAG (Glu-->Lys). The eastern part of Shizuoka prefecture is adjacent to Yamanashi prefecture where a large number of patients with CJD including familial cases has been found during the recent 15 years. This study suggests that the patients with CJD in both Yamanashi and Shizuoka prefecture should be re-evaluated by analysis of the PrP gene.

Aged↗