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Dive into the research topics where Yoshito Ihara is active.

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Featured researches published by Yoshito Ihara.


Journal of the American Chemical Society | 2008

Effects of macromolecular crowding on glycoprotein processing enzymes.

Kiichiro Totani; Yoshito Ihara; Ichiro Matsuo; Yukishige Ito

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.


Free Radical Biology and Medicine | 2009

Glutathione S-transferase pi localizes in mitochondria and protects against oxidative stress.

Shinji Goto; Miho Kawakatsu; Shinichi Izumi; Yoshishige Urata; Kan Kageyama; Yoshito Ihara; Takehiko Koji; Takahito Kondo

Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the protection of cellular components against anti-cancer drugs or peroxidative stress. Previously we found that GST pi, an isoform of the GSTs, is transported into the nucleus. In the present study, we found that GST pi is present in mitochondria as well as in the cytosol and nucleus in mammalian cell lines. A construct comprising the 84 amino acid residues in the amino-terminal region of GST pi and green fluorescent protein was detected in the mitochondria. The mutation of arginine to alanine at positions 12, 14, 19, 71, and 75 in full-length GST pi completely abrogated the ability to distribute in the mitochondria, suggesting that arginine, a positively charged residue, is required for the mitochondrial transport of GST pi. Chemicals generating reactive oxygen species, such as rotenone and antimycin A, decreased cell viability and reduced mitochondrial membrane potential. The overexpression of GST pi diminished these changes. GST pi-targeting siRNA abolished the protective effect of GST pi on the mitochondria under oxidative stress. The findings indicate that the peptide signal is conducive to the mitochondrial localization of GST pi under steady-state conditions without alternative splicing or posttranslational modifications such as proteolysis, suggesting that GST pi protects mitochondria against oxidative stress.


Biochimica et Biophysica Acta | 2012

In vivo role of aldehyde reductase

Motoko Takahashi; Satoshi Miyata; Junichi Fujii; Yoko Inai; Shigemitsu Ueyama; Motoko Araki; Tomoyoshi Soga; Reiko Fujinawa; Chiaki Nishitani; Shigeru Ariki; Takeyuki Shimizu; Tomomi Abe; Yoshito Ihara; Morimitsu Nishikimi; Yasunori Kozutsumi; Naoyuki Taniguchi; Yoshio Kuroki

BACKGROUND Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. METHODS Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. RESULTS The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. CONCLUSIONS AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. GENERAL SIGNIFICANCE AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.


American Journal of Pathology | 2013

Epithelial Calreticulin Up-Regulation Promotes Profibrotic Responses and Tubulointerstitial Fibrosis Development

Niki Prakoura; Panagiotis K. Politis; Yoshito Ihara; Marek Michalak; Aristidis Charonis

Renal fibrosis is the common anatomical feature underlying the progression of chronic kidney disease, a leading cause of morbidity and mortality worldwide. In a previous study, we demonstrated that during development of renal fibrosis in a rat model of unilateral ureteric obstruction, calreticulin (CRT) is up-regulated in tubular epithelial cells (TECs). In the present study, we used in vitro and in vivo approaches to examine the role of CRT in TECs and its contribution to the progression of fibrosis. In cultured renal TECs, CRT overexpression induced acquisition of an altered, profibrotic cellular phenotype. Consistently, the opposite effects were observed for CRT knockdown. Subsequently, we confirmed that critical changes observed in vitro were also apparent in tubular cells in vivo in the animal model of unilateral ureteric obstruction. In agreement with these results, we demonstrate that substantial (50%) reduction in the expression of CRT reduced the development of tubulointerstitial fibrosis at a comparable level through regulation of inflammation, transcriptional activation, transforming growth factor β1-associated effects, and apoptosis. In summary, our findings establish that CRT is critically involved in the molecular mechanisms that drive renal fibrosis progression and indicate that inhibition of CRT expression might be a therapeutic target for reduction of fibrosis and chronic kidney disease development.


Glycobiology | 2010

C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells

Yoshito Ihara; Shino Manabe; Midori Ikezaki; Yoko Inai; In-Sook Matsui; Yuriko Ohta; Eiji Muroi; Yukishige Ito

The thrombospondin type 1 repeat (TSR) is a functional module of proteins called TSR superfamily proteins (e.g., thrombospondin, F-spondin, mindin, etc.) and includes a conserved Trp-x-x-Trp (W-x-x-W) motif, in which the first Trp residue is preferably modified by C-mannosylation. We previously reported that synthesized C-mannosylated TSR-derived peptides (e.g., C-Man-WSPW) specifically enhanced lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells. In this study, we searched for the proteins that bind to C-mannosylated TSR-derived peptides in RAW264.7 cells and identified heat shock cognate protein 70 (Hsc70). The binding affinity of Hsc70 for C-mannosylated peptides in solution was higher than that for the peptides without C-mannose. The binding was influenced by a nucleotide-induced conformational change of Hsc70, and C-mannosylated peptides preferred the substrate-binding domain of Hsc70. Furthermore, in RAW264.7 cells, addition of Hsc70 stimulated cellular signaling to produce tumor necrosis factor-alpha, via transforming growth factor-beta-activated kinase 1, and the Hsc70-induced signaling was enhanced more in the presence of the peptides with C-mannose than that without C-mannose, suggesting functional interaction between Hsc70 and the C-mannosylated peptides in the cells. Together, these results demonstrate a novel function of the C-mannosylation of TSR-derived peptides in terms of interaction with Hsc70 to regulate cellular signaling.


Biochemical and Biophysical Research Communications | 2016

Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

Midori Ikezaki; Natsuki Higashimoto; Ko Matsumura; Yoshito Ihara

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells.


Bioscience, Biotechnology, and Biochemistry | 2011

Calreticulin inhibits prion protein PrP-(23-98) aggregation in vitro.

Noriyuki Shiraishi; Yoko Inai; Yoshiaki Hirano; Yoshito Ihara

Because prion protein PrP-(23–98) was recently found to polymerize into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions, we tested to determine whether calreticulin (CRT) inhibits PrP-(23–98) aggregation in vitro. The results indicated that CRT suppressed PrP-(23–98) aggregation, and that CRT-mediated solubilization occurred in the aggregates.


Endocrinology | 2017

Calreticulin Is Involved in Invasion of Human Extravillous Trophoblasts Through Functional Regulation of Integrin β1

Madoka Yamamoto; Midori Ikezaki; Saori Toujima; Naoyuki Iwahashi; Mika Mizoguchi; Sakiko Nanjo; Sawako Minami; Yoshito Ihara; Kazuhiko Ino

Calreticulin (CRT), a molecular chaperone in the endoplasmic reticulum (ER), plays a variety of roles in cell growth, differentiation, apoptosis, immunity, and cancer biology. It has been reported that CRT is expressed in the human placenta, although its function in placental development is poorly understood. Appropriate invasion of extravillous trophoblasts (EVTs) into the maternal decidua is necessary for successful pregnancy. The objective of the present study was to investigate the expression and functional role of CRT in EVTs using the human EVT cell line HTR8/SVneo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in the human placenta in the early stage of pregnancy and localized to the EVTs. CRT knockdown markedly suppressed the invasion ability of HTR8/SVneo cells. Furthermore, the adhesion to fibronectin was suppressed in the CRT-knockdown cells via the dysfunction of integrin α5β1. In the CRT-knockdown cells, terminal sialylation and fucosylation were decreased, and the core galactose-containing structure was increased in the N-glycans of integrin β1. In addition, the expression levels of several critical glycosyltransferases were changed in the CRT-knockdown cells, consistent with the changes in the N-glycans. These results showed that CRT regulates the function of integrin β1 by affecting the synthesis of N-glycans in HTR8/SVneo cells. Collectively, the results of the present study demonstrate that the ER chaperone CRT plays a regulatory role in the invasion of EVTs, suggesting the importance of CRT expression in placental development during early pregnancy.


Protein and Peptide Letters | 2009

Proteinase K-resistant aggregates of recombinant prion protein PrP-(23-98) are toxic to cultured cells.

Noriyuki Shiraishi; Yoko Inai; Yoshito Ihara

Here, we show for the first time that non-fibrillar and spherical aggregates produced from PrP-(23-98) in the presence of NADPH plus copper ions are toxic to cultured cells and induce apoptotic signals. It is also confirmed that endogenous cellular PrP isoform is not required for toxicity to occur.


Biochemistry | 2009

The recognition motif of the glycoprotein-folding sensor enzyme UDP-Glc:glycoprotein glucosyltransferase.

Kiichiro Totani; Yoshito Ihara; Takashi Tsujimoto; Ichiro Matsuo; Yukishige Ito

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Midori Ikezaki

Wakayama Medical University

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Yoko Inai

Wakayama Medical University

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Ichiro Matsuo

National Institute of Advanced Industrial Science and Technology

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Kazuhiko Ino

Wakayama Medical University

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Madoka Yamamoto

Wakayama Medical University

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