35 results on '"Hirano M."'
Search Results
2. Exon Skipping Caused by a Base Substitution at a Splice Site in the GTP Cyclohydrolase I Gene in a Japanese Family with Hereditary Progressive Dystonia/DOPA-Responsive Dystonia
- Author
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Hirano, M., primary, Tamaru, Y., additional, Nagai, Y., additional, Ito, H., additional, Imai, T., additional, and Ueno, S., additional
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- 1995
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3. A Protein Kinase C Isozyme, NPKC-ϵ, Is Involved in the Activation of NF-κ-B by 12-O-Tetradecanoylphorbol-13-Acetate (TPA) in Rat 3Y1 Fibroblasts
- Author
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Hirano, M., primary, Hirai, S., additional, Mizuno, K., additional, Osada, S., additional, Hosaka, M., additional, and Ohno, S., additional
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- 1995
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4. Temperature-Dependent Ca2+ Mobilization Induced by Hypoxia-Hypoglycemia in the Monkey Hippocampal Slices
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Yamashima, T., primary, Takita, M., additional, Akaike, S., additional, Hirano, M., additional, Miyakawa, A., additional, Miyazawa, A., additional, Kudo, Y., additional, and Yoshioka, T., additional
- Published
- 1994
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5. A New Variant Cu/Zn Superoxide Dismutase (Val7→Glu) Deduced from Lymphocyte mRNA Sequences from Japanese Patients with Familial Amyotrophic Lateral Sclerosis
- Author
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Hirano, M., primary, Fujii, J., additional, Nagai, Y., additional, Sonobe, M., additional, Okamoto, K., additional, Araki, H., additional, Taniguchi, N., additional, and Ueno, S., additional
- Published
- 1994
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6. Spontaneous differentiation of human induced pluripotent stem cells to odorant-responsive olfactory sensory neurons.
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Kikuta H, Tanaka H, Ozaki T, Ito J, Ma J, Moribe S, and Hirano M
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- Humans, Cells, Cultured, Receptors, Odorant genetics, Receptors, Odorant metabolism, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Cell Differentiation, Olfactory Receptor Neurons metabolism, Olfactory Receptor Neurons cytology, Odorants analysis
- Abstract
Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPSCs), can differentiate into almost all cell types and are anticipated to have significant applications in the field of regenerative medicine. However, there are no reports of successfully directing iPSCs to become functional olfactory sensory neurons (OSNs) capable of selectively receiving odorant compounds. In this study, we employed dual SMAD inhibition and fibroblast growth factor 8 (FGF-8, reported to dictate olfactory fates) along with N-2 and B-27 supplements in the culture medium to efficiently induce the differentiation of iPSCs into neuronal cells with olfactory function through olfactory placode. Temporal gene expression and expression of OSN-specific markers during differentiation indicated that the expression of olfactory marker proteins and various olfactory receptors (ORs), which are markers of mature OSNs, was observed after approximately one month of differentiation culture, irrespective of the differentiation cues, suggesting differentiation into OSNs. Cells that exhibited specific responses to odorant compounds were identified after administering odorant compounds to differentiated iPSC-derived OSNs. This suggests the spontaneous generation of functional OSNs expressing diverse ORs that respond to odorant compounds from iPSCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)
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- 2024
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7. Stratified analysis of lectin-like chaperones in the folding disease-related metabolic syndrome rat model.
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Hirano M, Imagawa A, and Totani K
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- Animals, Lectins metabolism, Male, Molecular Chaperones metabolism, Protein Folding, Rats, Rats, Zucker, Calnexin metabolism, Calreticulin metabolism, Metabolic Syndrome metabolism, Obesity metabolism, Proteostasis Deficiencies metabolism
- Abstract
The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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8. Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand.
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Hirano M, Adachi Y, Ito Y, and Totani K
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- Acetylglucosamine chemistry, Animals, Binding Sites, Chickens, Endoplasmic Reticulum metabolism, Glucosyltransferases chemistry, Glycoproteins chemistry, Glycosylation, Hot Temperature, Hydrophobic and Hydrophilic Interactions, Immunoglobulins chemistry, Ligands, Molecular Chaperones chemistry, Protein Binding, Protein Folding, Recombinant Proteins chemistry, Calreticulin metabolism
- Abstract
Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (-OH, -Gly-NH2, and -Gly-Glu-(t)Bu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order -Gly-Glu-(t)Bu > -Gly-NH2 > -OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by the folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system., (Copyright © 2015 Elsevier Inc. All rights reserved.)
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- 2015
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9. Analytical method for determining relative chaperone activity using an ovalbumin-conjugated column.
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Hirano M, Kato Y, Imagawa A, and Totani K
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- Animals, Calreticulin chemistry, Cattle, Chemistry Techniques, Analytical, Chromatography, Endoplasmic Reticulum Chaperone BiP, Heat-Shock Proteins analysis, Molecular Chaperones analysis, Protein Binding, Protein Denaturation, Protein Folding, Serum Albumin chemistry, Endoplasmic Reticulum chemistry, Heat-Shock Proteins chemistry, Molecular Chaperones chemistry, Ovalbumin chemistry
- Abstract
Investigating the relative efficiencies of molecular chaperones is important for understanding protein biosynthesis inside a cell. We developed an analytical method for estimating relative chaperone activity under physiological, multi-chaperone conditions using a protein-conjugated column. A chaperone mixture was subjected to chromatography on a column conjugated with denatured ovalbumin, and the elution positions of target chaperones were compared using western blotting to determine the relative affinity of each chaperone for the denatured protein. Because molecular chaperones should be eluted according to their strength of association with the denatured ovalbumin in the column, the elution position must accord with the chaperone activity and can be used as an indicator of relative chaperone activity. We found that the column procedure was effective in an assay of a mixture of calreticulin and BiP, the molecular chaperones in the endoplasmic reticulum; the assay showed that calreticulin associated with denatured ovalbumin more strongly than BiP., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2015
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10. A single amino acid gates the KcsA channel.
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Hirano M, Okuno D, Onishi Y, and Ide T
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- Hydrogen-Ion Concentration, Microscopy, Fluorescence, Mutagenesis, Site-Directed, Potassium Channels genetics, Ion Channel Gating, Potassium Channels physiology
- Abstract
The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel., (Copyright © 2014 Elsevier Inc. All rights reserved.)
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- 2014
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11. Regulation of SIRT1 determines initial step of endometrial receptivity by controlling E-cadherin expression.
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Shirane A, Wada-Hiraike O, Tanikawa M, Seiki T, Hiraike H, Miyamoto Y, Sone K, Hirano M, Oishi H, Oda K, Kawana K, Nakagawa S, Osuga Y, Fujii T, Yano T, Kozuma S, and Taketani Y
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- Actins metabolism, Cell Line, Tumor, Endometrium metabolism, Female, Glycodelin, Glycoproteins metabolism, Humans, Inhibitor of Apoptosis Proteins metabolism, Pregnancy Proteins metabolism, RNA, Small Interfering genetics, Sirtuin 1 genetics, Survivin, Cadherins biosynthesis, Embryo Implantation, Endometrium physiology, Sirtuin 1 metabolism
- Abstract
Sirtuin 1 (SIRT1), originally found as a class III histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. We examined the role of SIRT1 in the regulation of uterine receptivity using Ishikawa and RL95-2 endometrial carcinoma cell lines. Exogenous expression of SIRT1 significantly enhanced E-cadherin expression, while small interfering RNA-mediated depletion of endogenous SIRT1 resulted in a significant reduction of E-cadherin expression. A SIRT1 activator resveratrol elevated E-cadherin expression in a dose dependent manner, while SIRT1 repressors nicotinamide and sirtinol exhibited a dose dependent reduction of E-cadherin expression. We also showed that both forced expression of SIRT1 and activation of SIRT1 promote E-cadherin-driven reporter gene constructs, and SIRT1 is localized at E-cadherin promoter containing E-box elements in Ishikawa cells. Using an in vitro model of embryo implantation, we demonstrate that exogenous expression of SIRT1 and stimulation of SIRT1 activity resulted in the Ishikawa cell line becoming receptive to JAR cell spheroid attachment. Furthermore, resveratrol enhanced E-cadherin and Glycodelin protein expression at sites of intercellular contact, suggesting an additive role of resveratrol in promoting implantation. The initial step of human reproduction depends on the capacity of an embryo to attach and implant into the endometrial wall, and these results revealed the novel mechanism that activation and increased expression of SIRT1 play an important role in uterine receptivity., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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12. Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity.
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Villarroya J, Lara MC, Dorado B, Garrido M, García-Arumí E, Meseguer A, Hirano M, and Vilà MR
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- Cell Line, Tumor, Cell Respiration, DNA Replication, Electron Transport Complex IV metabolism, Gene Silencing, Gene Targeting, Humans, Mitochondria genetics, Mitochondrial Diseases genetics, Nucleotides metabolism, Thymidine Kinase metabolism, Transcription, Genetic, DNA, Mitochondrial genetics, Gene Expression, Mitochondria enzymology, Thymidine Kinase genetics
- Abstract
The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1(-) cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase γ, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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13. A poly(dimethylsiloxane)-based device enabling time-lapse imaging with high spatial resolution.
- Author
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Hirano M, Hoshida T, Sakaue-Sawano A, and Miyawaki A
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- Cell Cycle, HeLa Cells, Humans, Dimethylpolysiloxanes chemistry, Microscopy, Fluorescence instrumentation, Microscopy, Fluorescence methods
- Abstract
We have developed a regulator-free device that enables long-term incubation of mammalian cells for epi-fluorescence imaging, based on a concept that the size of sample to be gassed and heated is reduced to observation scale. A poly(dimethylsiloxane) block stamped on a coverslip works as a long-lasting supplier of CO(2)-rich gas to adjust bicarbonate-containing medium in a tiny chamber at physiological pH, and an oil-immersion objective warms cells across the coverslip. A time-lapse imaging experiment using HeLa cells stably expressing fluorescent cell-cycle indicators showed that the cells in the chamber proliferated with normal cell-cycle period over 2 days.
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- 2010
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14. Naturally- and experimentally-designed restorations of the Parkin gene deficit in autosomal recessive juvenile parkinsonism.
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Asai H, Hirano M, Kiriyama T, Ikeda M, and Ueno S
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- Aged, Buthionine Sulfoximine pharmacology, Cell Survival, Cells, Cultured, Cyclin E metabolism, DNA Damage genetics, Female, Fibroblasts drug effects, Gene Deletion, Humans, Male, Oligonucleotides, Antisense genetics, Open Reading Frames genetics, Oxidative Stress genetics, SKP Cullin F-Box Protein Ligases, Transfection, Exons genetics, Fibroblasts metabolism, Parkinsonian Disorders genetics, Ubiquitin-Protein Ligases genetics
- Abstract
Intranuclear events due to mutations in the Parkin gene remain elusive in autosomal recessive juvenile parkinsonism (ARJP). We identified a mutant PARKIN protein in fibroblast cultures from a pair of siblings with ARJP who were homozygous for the exon 4-deleted Parkin gene. Disease was mild in one patient and debilitating in the other. The detected mutant, encoded by a transcript lacking exon 3 as well as exon 4, is an in-frame deletion that removes 121 aa, resulting in a 344-aa protein (PaDel3,4). Cell culture and transfection studies revealed negative correlations between expression levels of PaDel3,4 and those of cell cycle proteins, including cyclin E, CDK2, ppRb, and E2F-1, and demonstrated that GFP-PaDel3,4 entered nucleus and ubiquitinated cyclin E as a part of SCF(hSel-10) ligase complex in the patient cells. In addition, nuclear localization signal-tagged PaDel3,4 expressed in the transfected patient cells most effectively ubiquitinated cyclin E and reduced DNA damage, protecting cells from oxidative stress. Antisense-oligonucleotide treatment promoted skipping of exon 3 and thus generated PaDel3,4, increasing cell survival. Collectively, we propose that naturally- and experimentally-induced exon skipping at least partly restores the mutant Parkin gene deficit, providing a molecular basis for the development of therapeutic exon skipping., (Copyright 2009 Elsevier Inc. All rights reserved.)
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- 2010
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15. Restoration of nuclear-import failure caused by triple A syndrome and oxidative stress.
- Author
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Kiriyama T, Hirano M, Asai H, Ikeda M, Furiya Y, and Ueno S
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- Active Transport, Cell Nucleus genetics, Adrenal Insufficiency therapy, Amino Acid Sequence, Cells, Cultured, DNA Ligase ATP, DNA Ligases genetics, DNA Ligases metabolism, DNA-Binding Proteins genetics, Dry Eye Syndromes therapy, Esophageal Achalasia therapy, Fibroblasts metabolism, Humans, Molecular Sequence Data, Nuclear Proteins genetics, Nuclear Proteins metabolism, Oxidative Stress, Syndrome, X-ray Repair Cross Complementing Protein 1, Adrenal Insufficiency metabolism, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Dry Eye Syndromes metabolism, Esophageal Achalasia metabolism, Nuclear Localization Signals genetics
- Abstract
Triple A syndrome is an autosomal recessive neurological disease, mimicking motor neuron disease, and is caused by mutant ALADIN, a nuclear-pore complex component. We recently discovered that the pathogenesis involved impaired nuclear import of DNA repair proteins, including DNA ligase I and the cerebellar ataxia causative protein aprataxin. Such impairment was overcome by fusing classical nuclear localization signal (NLS) and 137-aa downstream sequence of XRCC1, designated stretched NLS (stNLS). We report here that the minimum essential sequence of stNLS (mstNLS) is residues 239-276, downsized by more than 100 aa. mstNLS enabled efficient nuclear import of DNA repair proteins in patient fibroblasts, functioned under oxidative stress, and reduced oxidative-stress-induced cell death, more effectively than stNLS. The stress-tolerability of mstNLS was also exerted in control fibroblasts and neuroblastoma cells. These findings may help develop treatments for currently intractable triple A syndrome and other oxidative-stress-related neurological diseases, and contribute to nuclear compartmentalization study.
- Published
- 2008
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16. Functional characterization of human COQ4, a gene required for Coenzyme Q10 biosynthesis.
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Casarin A, Jimenez-Ortega JC, Trevisson E, Pertegato V, Doimo M, Ferrero-Gomez ML, Abbadi S, Artuch R, Quinzii C, Hirano M, Basso G, Ocaña CS, Navas P, and Salviati L
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- Amino Acid Sequence, Blotting, Northern, Chromosomes, Human, Pair 9 genetics, Cloning, Molecular, Genetic Complementation Test, Green Fluorescent Proteins analysis, Green Fluorescent Proteins genetics, HeLa Cells, Humans, Isoenzymes analysis, Isoenzymes biosynthesis, Isoenzymes genetics, Mitochondrial Proteins analysis, Mitochondrial Proteins genetics, Molecular Sequence Data, Mutation, Saccharomyces cerevisiae Proteins genetics, Transcription Initiation Site, Transcription, Genetic, Ubiquinone analysis, Ubiquinone biosynthesis, Ubiquinone genetics, Mitochondria enzymology, Mitochondrial Proteins metabolism, Ubiquinone analogs & derivatives
- Abstract
Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a severe multisystem disorders presenting as progressive encephalomyopathy and nephropathy. The COQ4 gene encodes an essential factor for biosynthesis in Saccharomyces cerevisiae. We have identified and cloned its human ortholog, COQ4, which is located on chromosome 9q34.13, and is transcribed into a 795 base-pair open reading frame, encoding a 265 amino acid (aa) protein (Isoform 1) with a predicted N-terminal mitochondrial targeting sequence. It shares 39% identity and 55% similarity with the yeast protein. Coq4 protein has no known enzymatic function, but may be a core component of multisubunit complex required for CoQ biosynthesis. The human transcript is detected in Northern blots as a approximately 1.4 kb single band and is expressed ubiquitously, but at high levels in liver, lung, and pancreas. Transcription initiates at multiple sites, located 333-23 nucleotides upstream of the ATG. A second group of transcripts originating inside intron 1 of the gene encodes a 241 aa protein, which lacks the mitochondrial targeting sequence (isoform 2). Expression of GFP-fusion proteins in HeLa cells confirmed that only isoform 1 is targeted to mitochondria. The functional significance of the second isoform is unknown. Human COQ4 isoform 1, expressed from a multicopy plasmid, efficiently restores both growth in glycerol, and CoQ content in COQ4(null) yeast strains. Human COQ4 is an interesting candidate gene for patients with isolated CoQ(10) deficiency.
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- 2008
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17. Novel reciprocal regulation of cAMP signaling and apoptosis by orphan G-protein-coupled receptor GPRC5A gene expression.
- Author
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Hirano M, Zang L, Oka T, Ito Y, Shimada Y, Nishimura Y, and Tanaka T
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- Apoptosis physiology, Cells, Cultured, Gene Expression Regulation physiology, Humans, Cyclic AMP metabolism, Myocytes, Smooth Muscle cytology, Myocytes, Smooth Muscle metabolism, Receptors, G-Protein-Coupled metabolism, Signal Transduction physiology
- Abstract
GPRC5A is a member of G-protein-coupled receptors, which was originally identified as an all-trans-retinoic acid-induced gene. Although recent studies reported that this gene was highly expressed in the cancer cell lines and that GPRC5A might positively regulate cell proliferation, its mechanism remains unknown. We investigated the upstream and downstream signaling of GPRC5A and its biological function, and found that cAMP signaling is the novel GPRC5A induction pathway. When GPRC5A gene was overexpressed, intracellular cAMP concentration was decreased, and Gsalpha gene expression was downregulated. On the other hand, RNA interference of GPRC5A increased mRNA levels of Gsalpha and intracellular cAMP, reduced cell number, and induced apoptosis. Conversely, cell number was increased by GPRC5A overexpression. We first report the novel negative feedback model of cAMP signaling through GPRC5A gene expression. This evidence explains one of the mechanisms of the GPRC5A-regulated cell growth in some cancer cell lines.
- Published
- 2006
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18. Involvement of Gi/o in the PAR-4-induced NO production in endothelial cells.
- Author
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Momota F, Hirano K, Hirano M, Nishimura J, and Kanaide H
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- Animals, Calcium Signaling drug effects, Calcium Signaling physiology, Cattle, Cells, Cultured, Egtazic Acid analogs & derivatives, Egtazic Acid pharmacology, Endothelium, Vascular drug effects, Indicators and Reagents pharmacology, Ionomycin pharmacology, Ionophores pharmacology, Oligopeptides physiology, Pertussis Toxin pharmacology, Thrombin physiology, Endothelium, Vascular cytology, Endothelium, Vascular metabolism, GTP-Binding Protein alpha Subunits, Gi-Go physiology, Nitric Oxide biosynthesis, Receptors, Thrombin physiology
- Abstract
We investigated the involvement of G(i/o) protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH(2) (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 microM, 0.1U/ml, and 1 microM, respectively. Ionomycin elevated [Ca(2+)](i) in a concentration-dependent manner. However, AYPGKF-NH(2) and thrombin induced no [Ca(2+)](i) elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca(2+)](i) elevation induced by 1 microM ionomycin, while it had no significant effect on the AYPGKF-NH(2)-induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH(2)-induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca(2+) signal and also that G(i/o) is involved in such NO production in vascular endothelial cells.
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- 2006
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19. Akt plays a central role in the anti-apoptotic effect of estrogen in endothelial cells.
- Author
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Koga M, Hirano K, Hirano M, Nishimura J, Nakano H, and Kanaide H
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- Animals, Cattle, Cells, Cultured, Endothelial Cells cytology, Endothelial Cells drug effects, Endothelium, Vascular cytology, Gene Products, tat genetics, Gene Products, tat metabolism, Humans, Mutation, Peptides genetics, Peptides metabolism, Protein Serine-Threonine Kinases genetics, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-akt, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Tumor Necrosis Factor-alpha pharmacology, Apoptosis physiology, Endothelial Cells metabolism, Estrogens metabolism, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism
- Abstract
Estrogen has been reported to inhibit apoptosis in vascular endothelial cells. However, its precise mechanism still remains to be elucidated. Here we determined the role of Akt in the anti-apoptotic effect of estrogen. 17Beta-estradiol prevented the apoptosis induced by TNF-alpha in bovine aortic endothelial cells, as evaluated by double staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. Introducing a dominant negative mutant of Akt by using a cell-penetrating peptide of Tat protein inhibited the anti-apoptotic effect of estrogen in a concentration-dependent manner, and resulted in the complete inhibition of the anti-apoptotic effect of 17beta-estradiol at 1nM and higher concentrations. The dominant negative mutant without the cell-penetrating peptide and Tat peptide-conjugated protein A had no effect. The intracellular protein transduction was confirmed by immunoblot analysis. Our observations thus provide first direct evidence that Akt plays a central role in the anti-apoptotic effect of estrogen in vascular endothelial cells.
- Published
- 2004
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20. Loss of function mechanism in aprataxin-related early-onset ataxia.
- Author
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Hirano M, Furiya Y, Kariya S, Nishiwaki T, and Ueno S
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- Acid Anhydride Hydrolases genetics, Age of Onset, Ataxia epidemiology, DNA-Binding Proteins metabolism, Evolution, Molecular, Humans, Immunoblotting, Immunohistochemistry, Mutation, Neoplasm Proteins genetics, Nuclear Proteins metabolism, RNA, Messenger metabolism, Sequence Homology, Ataxia genetics, Ataxia metabolism, DNA-Binding Proteins genetics, Nuclear Proteins genetics
- Abstract
Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is an autosomal recessive form of cerebellar ataxia that occurs most commonly in Japan but is also frequently seen in Europe. This disease is caused by mutations in the aprataxin gene, but the functions of the gene product and the pathogenic mechanism remain unclear. The present study provides experimental evidence that the histidine triad (HIT) domain in aprataxin has enzymatic activity that is negatively regulated by the intramolecular interaction of the N-terminal domain. Furthermore, the reduction in HIT activity seen in all the disease-causing mutants tested, and the correlation between the reduced activity and the severe phenotype, support that aprataxin's physiological function is associated with its catalytic activity. Our findings suggest that the clinical phenotypes are caused by a loss of aprataxin function, attributable largely to diminished HIT activity but partially to a reduction in the levels of gene products.
- Published
- 2004
- Full Text
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21. Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency.
- Author
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Martí R, Nishigaki Y, and Hirano M
- Subjects
- Base Sequence, Biochemistry methods, Dose-Response Relationship, Drug, Humans, Kinetics, Molecular Sequence Data, Mutation, Nucleosides chemistry, Point Mutation, Reference Values, Substrate Specificity, Thymidine metabolism, Time Factors, Deoxyuridine blood, Thymidine Phosphorylase deficiency
- Abstract
Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.
- Published
- 2003
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22. Pathogenesis of the deafness-associated A1555G mitochondrial DNA mutation.
- Author
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Giordano C, Pallotti F, Walker WF, Checcarelli N, Musumeci O, Santorelli F, d'Amati G, Schon EA, DiMauro S, Hirano M, and Davidson MM
- Subjects
- Adult, Cell Division, Cells, Cultured, Clone Cells, Female, Fibroblasts cytology, Fibroblasts physiology, Humans, Kinetics, Middle Aged, Point Mutation, Polymorphism, Restriction Fragment Length, DNA, Mitochondrial genetics, Deafness genetics, RNA, Ribosomal genetics
- Abstract
The pathogenic mechanisms of the A1555G mitochondrial DNA mutation in the 12S rRNA gene, associated with maternally inherited sensorineural deafness, are largely unknown. Previous studies have suggested an involvement of nuclear factor(s). To address this issue cybrids were generated by fusing osteosarcoma cells devoid of mtDNA with enucleated fibroblasts from two genetically unrelated patients. Furthermore, to determine the contribution, if any, of the mitochondrial and nuclear genomes, separately or in combination, in the expression of the disease phenotype, transmitochondrial fibroblasts were constructed using control and patient's fibroblasts as nuclear donors and homoplasmic mutant or wild-type cybrids as mitochondrial donors. Detailed analysis of mutant and wild-type cybrids from both patients and transmitochondrial fibroblast clones did not reveal any respiratory chain dysfunction suggesting that, if nuclear factors do indeed act as modifier agents, they may be tissue-specific. However, in the presence of high concentrations of neomycin or paromomycin, but not of streptomycin, mutant cells exhibit a decrease in the growth rate, when compared to wild-type cells. The decrease did not correlate with the rate of synthesis or stability of mitochondrial DNA-encoded subunits or respiratory chain activity. Further studies are required to determine the underlying biochemical defect.
- Published
- 2002
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23. Minimal requirements for the nuclear localization of p27(Kip1), a cyclin-dependent kinase inhibitor.
- Author
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Zeng Y, Hirano K, Hirano M, Nishimura J, and Kanaide H
- Subjects
- Amino Acid Sequence, Amino Acids metabolism, Animals, Aorta metabolism, Cyclin-Dependent Kinase Inhibitor p27, Cytosol metabolism, Endothelium, Vascular metabolism, Green Fluorescent Proteins, HeLa Cells, Humans, Luminescent Proteins metabolism, Microscopy, Confocal, Microtubule-Associated Proteins chemistry, Microtubule-Associated Proteins genetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Plasmids, Recombinant Fusion Proteins metabolism, Swine, Transfection, Cell Cycle Proteins, Cell Nucleus metabolism, Microtubule-Associated Proteins biosynthesis, Tumor Suppressor Proteins
- Abstract
p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1)., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
24. Synthetic phytanyl-chained glycolipid vesicle membrane as a novel matrix for functional reconstitution of cyanobacterial photosystem II complex.
- Author
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Baba T, Minamikawa H, Hato M, Motoki A, Hirano M, Zhou D, and Kawasaki K
- Subjects
- Freeze Fracturing, Light, Membrane Lipids chemical synthesis, Membrane Lipids chemistry, Membranes, Artificial, Microscopy, Electron, Oxygen metabolism, Photosynthetic Reaction Center Complex Proteins radiation effects, Photosynthetic Reaction Center Complex Proteins ultrastructure, Cyanobacteria metabolism, Glycolipids chemical synthesis, Glycolipids chemistry, Photosynthetic Reaction Center Complex Proteins metabolism
- Abstract
The vesicles composed of synthetic phytanyl-chained glycolipid and natural sulfoquinovosyldiacylglycerol at 9:1 molar ratio were successfully applied to functional reconstitution of photosystem II complex (PS II) from a thermophilic cyanobacterium. The synthetic glycolipid employed was one of our model archaeal diether lipids, 1, 3-di-O-phytanyl-2-O-(beta-D-maltotriosyl)glycerol. The light-induced oxygen-evolving activity of PS II reconstituted in the glycolipid vesicles was approximately 6-fold higher than that reconstituted in several phosphatidylcholine vesicles. The present results reveal the first evidence that a well-designed synthetic glycolipid is effective for the functional reconstitution of complicated and labile membrane protein complexes, such as PS II., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
25. Expression, subcellular localization, and cloning of the 130-kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells.
- Author
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Hirano M, Niiro N, Hirano K, Nishimura J, Hartshorne DJ, and Kanaide H
- Subjects
- Amino Acid Sequence, Animals, Aorta, Thoracic, Base Sequence, Cell Division, Cells, Cultured, Cloning, Molecular, DNA biosynthesis, DNA, Complementary, Endothelium, Vascular cytology, Gene Library, Immunoblotting, Macromolecular Substances, Molecular Sequence Data, Molecular Weight, Myosin-Light-Chain Phosphatase, Phosphoprotein Phosphatases analysis, Phosphoprotein Phosphatases genetics, Recombinant Proteins analysis, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Swine, Endothelium, Vascular enzymology, Phosphoprotein Phosphatases metabolism
- Abstract
In endothelial cells in situ and in primary culture, immunoblot analysis revealed an expression of the 130-kDa subunit of myosin phosphatase, similar to the myosin phosphatase targeting subunit (MYPT) of smooth muscle. Screening of an endothelial cell cDNA library yielded a clone encoding an NH2-terminal fragment of 89.6 kDa, closely related to smooth muscle MYPT1. Two isoforms differing by a central insert of 56 residues were detected. In growing cells, MYPT1 was localized on stress fiber, but at confluence the localization pattern changed and MYPT1 was distributed close to the cell membrane and at cell-cell contacts. The membrane localization of MYPT1 suggested a target other than myosin and raised the possibility that MYPT1 may be involved in dephosphorylation of alternative substrate(s). These distinct mechanisms would also be dependent on the growth state of the endothelial cells, i.e., regulation of actin-myosin interactions in growing cells and an unknown function in cells at confluence., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
26. Restricted photorelease of biologically active molecules near the plasma membrane.
- Author
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Suga T, Hirano M, Takayanagi M, Koshimoto H, and Watanabe A
- Subjects
- Aniline Compounds metabolism, Animals, Calcium metabolism, Fluorescent Dyes metabolism, Glioma, Glutamic Acid metabolism, Inositol 1,4,5-Trisphosphate metabolism, Mice, Neuroblastoma, Rats, Second Messenger Systems, Tumor Cells, Cultured, Xanthenes metabolism, Cell Membrane metabolism, Photolysis
- Abstract
An evanescent wave of ultraviolet light was successfully used to release biologically active molecules from caged compounds in living cells. The evanescent wave was generated by the total internal reflection in a limited region near the plasma membrane attached to the illuminated interface. At first, the photolysis efficiency of the evanescent wave of ultraviolet laser light was studied using caged glutamic acid in vitro. Then, caged Ca2+ introduced in the living cultured cell was similarly photolyzed by the evanescent wave and the resulting elevations of the concentration of intracellular Ca2+ in the proximity of the plasma membrane and in the cytosol were observed with a simultaneously introduced fluorescent calcium indicator. Inositol trisphosphate can also be photoreleased near the plasma membrane, which enables study of the temporal and spatial pathways of signal transduction. The method developed here provides a useful tool for studying signal transduction near the plasma membrane in a living cell., (Copyright 1998 Academic Press.)
- Published
- 1998
- Full Text
- View/download PDF
27. Differential splicing of the GTP cyclohydrolase I RNA in dopa-responsive dystonia.
- Author
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Hirano M, Imaiso Y, and Ueno S
- Subjects
- Adult, Base Sequence, DNA Primers genetics, DNA, Complementary genetics, Dystonia drug therapy, Exons, Female, Humans, Levodopa therapeutic use, Molecular Sequence Data, Nucleic Acid Conformation, RNA Precursors chemistry, RNA Precursors genetics, RNA Precursors metabolism, Tissue Distribution, Alternative Splicing, Dystonia genetics, Dystonia metabolism, GTP Cyclohydrolase genetics, RNA genetics, RNA metabolism
- Abstract
We characterized the GTP cyclohydrolase I (GTP-CH-I) gene in a patient with hereditary progressive dystonia with marked diurnal fluctuation/dopa-responsive dystonia (HPD/DRD). The sequence analysis revealed a C to A transversion, which predicts a novel missense mutation (Thr186Lys). Unexpectedly, this base change, occurring in the middle of exon 5, resulted in a production of the novel transcript lacking exon 5 and a part of exon 6. Three different transcripts of the GTP-CH-I gene, previously reported in the human liver, were also present in the peripheral lymphocytes from the patient and controls. Quantitative comparison of the truncated-subunit mRNA and the wild-type one implied that differential splicing regulates the GTP-CH-I enzyme activity, leading to the clinical variations in HPD/DRD. The patient showed a unique clinical symptom, suggesting that the nigrostriatal dopaminergic system is more affected than previously thought in HPD/DRD.
- Published
- 1997
- Full Text
- View/download PDF
28. Molecular cloning and expression of a novel peptide (LN1) gene: reduced expression in the renal cortex of lupus nephritis in MRL/lpr mouse.
- Author
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Iwano M, Ueno S, Miyazaki M, Harada T, Nagai Y, Hirano M, Dohi Y, Akai Y, Kurioka H, and Dohi K
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Gene Expression, Humans, In Situ Hybridization, Lupus Nephritis etiology, Lupus Nephritis pathology, Male, Mice, Mice, Inbred BALB C, Mice, Inbred MRL lpr, Molecular Sequence Data, RNA, Messenger genetics, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Species Specificity, Time Factors, Tissue Distribution, Homeodomain Proteins genetics, Homeodomain Proteins isolation & purification, Kidney Cortex chemistry, Kidney Glomerulus chemistry, Lupus Nephritis genetics, Plant Proteins
- Abstract
A gene has been identified by mRNA differential display whose expression is reduced in the renal cortex of MRL/lpr mouse. The nucleotide sequence of the cDNA contains an open reading frame that encodes a protein of 338 amino acids (termed LN1). In situ hybridization showed that LN1 mRNA is present in glomeruli, and a 39 kDa protein was detected in the kidney by immunoblot. A human LN1 cDNA was also isolated, the deduced amino acid sequence of which is 78% identical to that of mouse LN1. Although the function of LN1 remains to be elucidated, its reduced expression may contribute to the pathogenesis of lupus nephritis.
- Published
- 1996
- Full Text
- View/download PDF
29. The carboxyl-terminal region is essential for Sec-A dimerization.
- Author
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Hirano M, Matsuyama S, and Tokuda H
- Subjects
- Adenosine Triphosphatases genetics, Adenosine Triphosphate metabolism, Bacterial Proteins genetics, Binding Sites, DNA Mutational Analysis, Dimerization, Recombinant Proteins metabolism, SEC Translocation Channels, SecA Proteins, Sequence Deletion, Structure-Activity Relationship, Adenosine Triphosphatases metabolism, Bacterial Proteins metabolism, Escherichia coli Proteins, Membrane Transport Proteins
- Abstract
SecA, comprising 901 amino acid residues, exists as a dimer. By means of size exclusion chromatography and chemical cross-linking analysis, five truncated SecA derivatives were examined to identify the region of SecA essential for dimer formation. Among them, only N95 (delta 832-901) retained SecA activity. N95 existed as a dimer, indicating that the carboxyl-terminal three cysteine residues are dispensable for physiological dimerization. Both N76 (delta 675-901) and N66 (delta 583-901) existed as monomers. Monomeric N76 was able to bind to ATP, indicating that the dimerization of SecA is not a prerequisite for ATP binding. However, the rate of ATP hydrolysis by N76 was 25% of that by SecA. C53 (delta 1-437) and C28 (delta 1-661) formed dimers irrespective of the presence or absence of 2-mercaptoethanol. C28, but not C53, also existed as an oligomer in the absence of 2-mercaptoethanol, suggesting that the 438-661 region present in C53 prevents intermolecular disulfide bond formation at the carboxyl-terminal cysteine residue. From these results, the region essential for the physiological dimer formation was concluded to be located in the 662-831 region of SecA.
- Published
- 1996
- Full Text
- View/download PDF
30. Exon skipping caused by a base substitution at a splice site in the GTP cyclohydrolase I gene in a Japanese family with hereditary progressive dystonia dopa responsive dystonia.
- Author
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Hirano M, Tamaru Y, Nagai Y, Ito H, Imai T, and Ueno S
- Subjects
- Adult, Base Sequence, Dystonia drug therapy, Frameshift Mutation, Humans, Japan, Male, Molecular Sequence Data, Polymerase Chain Reaction, RNA, Messenger chemistry, RNA, Messenger genetics, RNA-Directed DNA Polymerase, Dystonia genetics, Exons, GTP Cyclohydrolase genetics, Levodopa therapeutic use, Mutation, RNA Splicing
- Abstract
We report a novel mutation at a splice site in the GTP cyclohydrolase I gene in a Japanese family with hereditary progressive dystonia with marked diurnal fluctuation (HPD)/dopa responsive dystonia (DRD). Reverse transcriptase-initiated PCR (RT-PCR) of lymphocyte mRNA showed both normal and small size fragments in the HPD patient and his asymptomatic mother. Sequence analysis revealed that skip splicing of exon 1 to exon 3 occurred in the small fragment. The patient and his mother were heterozygous for G --> C substitution at conserved consensus sequence GT at 5' end of the intron 2. Quantitative RT-PCR showed that the expression of normal GTP cyclohydrolase I mRNA decreased in their lymphocytes, while the HPD patient had more expression of mutant GTP cyclohydrolase I mRNA than his asymptomatic mother.
- Published
- 1995
- Full Text
- View/download PDF
31. A protein kinase C isozyme, nPKC epsilon, is involved in the activation of NF-kappa B by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat 3Y1 fibroblasts.
- Author
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Hirano M, Hirai S, Mizuno K, Osada S, Hosaka M, and Ohno S
- Subjects
- Animals, Blotting, Western, Cell Line, Chloramphenicol O-Acetyltransferase biosynthesis, Fibroblasts, Luciferases biosynthesis, Plasmids, Point Mutation, Rats, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, Transfection, Isoenzymes metabolism, NF-kappa B biosynthesis, Protein Kinase C metabolism, Tetradecanoylphorbol Acetate pharmacology
- Abstract
In order to examine whether PKC is involved in the activation of NF-kappa B by TPA, we overexpressed a variety of PKC isozymes in rat 3Y1 fibroblasts and monitored the expression of the co-transfected reporter NF-kappa B gene. In contrast to TPA response element (TRE), where overexpression of a variety of PKC isozymes results in enhanced activation by TPA, activation of NF-kappa B by TPA is not enhanced by overexpression of PKC isozymes such as cPKC alpha, nPKC delta, or nPKC theta. However, the overexpression of nPKC epsilon does result in enhancement. A kinase-negative point mutant of nPKC epsilon, where Lys at the ATP binding site is altered to Arg, does not cause this enhancement of NF-kappa B activation. Further, the kinase-negative nPKC epsilon partially suppresses endogenous NF-kappa B activity. These results suggest that nPKC epsilon is specifically involved in the activation of NF-kappa B when cells are treated with TPA.
- Published
- 1995
- Full Text
- View/download PDF
32. A new variant Cu/Zn superoxide dismutase (Val7-->Glu) deduced from lymphocyte mRNA sequences from Japanese patients with familial amyotrophic lateral sclerosis.
- Author
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Hirano M, Fujii J, Nagai Y, Sonobe M, Okamoto K, Araki H, Taniguchi N, and Ueno S
- Subjects
- Adult, Aged, Amyotrophic Lateral Sclerosis enzymology, Base Sequence, DNA Primers, Female, Humans, Japan, Male, Middle Aged, Molecular Sequence Data, Superoxide Dismutase blood, Amyotrophic Lateral Sclerosis genetics, Glutamic Acid genetics, Lymphocytes enzymology, Mutation, RNA, Messenger genetics, Superoxide Dismutase genetics, Valine genetics
- Abstract
We have identified a new mutant Cu/Zn superoxide dismutase (SOD1) deduced from the nucleotide sequences of peripheral blood lymphocyte mRNA from Japanese patients with familial amyotrophic lateral sclerosis (FALS). Sequence analysis of reverse transcriptase-initiated PCR amplified mRNA revealed a heterozygosity indicative of one normal allele and one variant allele with a T-->A transversion. This base change led to replacement of valine by glutamic acid at position 7 of 153-residue SOD1 molecule, and produced a new restriction site for Alu I in the exon 1. Restriction fragment length polymorphism analysis confirmed the linkage of this mutation with this type of FALS. Both enzymatic activity and protein of the SOD1 were reduced in red blood cells from the patient.
- Published
- 1994
- Full Text
- View/download PDF
33. Quantification of intracellular free sodium ions by using a new fluorescent indicator, sodium-binding benzofuran isophthalate in guinea pig myocytes.
- Author
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Satoh H, Hayashi H, Noda N, Terada H, Kobayashi A, Yamashita Y, Kawai T, Hirano M, and Yamazaki N
- Subjects
- Animals, Guinea Pigs, In Vitro Techniques, Microscopy, Fluorescence, Muscles chemistry, Spectrometry, Fluorescence, Strophanthidin pharmacology, Benzofurans chemistry, Cytoplasm chemistry, Ethers, Cyclic chemistry, Fluorescent Dyes, Sodium analysis
- Abstract
Isolated guinea pig myocytes were loaded with the Na(+)-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI). The 340/380 nm fluorescence ratios were measured with fluorescence microscopy. The distribution of intracellular Na+ concentration ([Na+]i) was homogenous, and the mean resting [Na+]i was 8.4 +/- 0.5 mM. There was a significant relationship (r = 0.66, p less than 0.001) between elevation of [Na+]i and shortening of longitudinal length of the cells, during the perfusion of 100 microM strophanthidin. It is concluded that this method is suitable for measuring [Na+]i in isolated myocytes.
- Published
- 1991
- Full Text
- View/download PDF
34. Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes.
- Author
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Miyata H, Hayashi H, Suzuki S, Noda N, Kobayashi A, Fujiwake H, Hirano M, and Yamazaki N
- Subjects
- Animals, Fura-2, In Vitro Techniques, Indicators and Reagents, Microscopy, Fluorescence methods, Rats, Video Recording, Benzofurans administration & dosage, Calcium analysis, Fluoresceins administration & dosage, Myocardium cytology
- Abstract
Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.
- Published
- 1989
- Full Text
- View/download PDF
35. Evidence that the intervening sequence was excised as a linear molecule during D beta-J beta rearrangement in T-cell receptor beta chain gene loci.
- Author
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Ino T, Hirano M, and Kurosawa Y
- Subjects
- Base Sequence, Cell Line, Clone Cells, DNA Restriction Enzymes, Humans, Macromolecular Substances, Molecular Sequence Data, Nucleic Acid Hybridization, Genes, Introns, Receptors, Antigen, T-Cell genetics
- Abstract
In a previous paper (Proc. Natl. Acad. Sci. USA 84: 4264, 1987) we reported an unusual DNA rearrangement in T-cell receptor beta chain gene loci in cells from a patient with human T-cell leukemia. A D beta 1-J beta 2.3 junction was found on one chromosome, while the other chromosome kept the germline configuration. Although the DNA fragment located between the D beta 1 and J beta 2.3 loci should have disappeared from the cells, it was found on chromosome 6 as an inserted segment. We have now determined the nucleotide sequences bordering both sides of the inserted segment. The signal sequence for D beta-J beta rearrangement at the 5' side of J beta 1.2 gene seems to have been used for the insertion. The 3' end of the inserted segment corresponded to the edge of the signal heptamer at the 5' side of J beta 2.3 which was used for the initial D beta 1-J beta 2.3 joining. This indicates that, during D beta-J beta rearrangement, the intervening sequence was excised as a linear molecule.
- Published
- 1988
- Full Text
- View/download PDF
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