106 results on '"Yamashita T"'
Search Results
2. Inhibition of Intercellular Communication via Gap Junction in Cultured Aortic Endothelial Cells by Elevated Glucose and Phorbol Ester
- Author
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Inoguchi, T., primary, Ueda, F., additional, Umeda, F., additional, Yamashita, T., additional, and Nawata, H., additional
- Published
- 1995
- Full Text
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3. The Gene Responsible for LEC Hepatitis, Located on Rat Chromosome 16, Is the Homolog to the Human Wilson Disease Gene
- Author
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Sasaki, N., primary, Hayashizaki, Y., additional, Muramatsu, M., additional, Matsuda, Y., additional, Ando, Y., additional, Kuramoto, T., additional, Serikawa, T., additional, Azuma, T., additional, Naito, A., additional, Agui, T., additional, Yamashita, T., additional, Miyoshi, I., additional, Takeichi, N., additional, and Kasai, N., additional
- Published
- 1994
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4. Evidence That α-Fetoprotein Suppresses the Immunological Function in Transgenic Mice
- Author
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Yamashita, T., primary, Nakane, A., additional, Watanabe, T., additional, Miyoshi, I., additional, and Kasai, N., additional
- Published
- 1994
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5. High Level Expression of Human α-Fetoprotein in Transgenic Mice
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Yamashita, T., primary, Kasai, N., additional, Miyoshi, I., additional, Sasaki, N., additional, Maki, K., additional, Sakai, M., additional, Nishi, S., additional, and Namioka, S., additional
- Published
- 1993
- Full Text
- View/download PDF
6. Role of tubulin C-terminal tail on mechanical properties of microtubule.
- Author
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Nowroz S, Nasrin SR, Kabir AMR, Yamashita T, Kusumoto T, Taira J, Tani M, Ichikawa M, Sada K, and Kakugo A
- Subjects
- Polymerization, Tubulin metabolism, Microtubules metabolism
- Abstract
Tubulin C-terminal tail (CTT) is a disordered segment extended from each tubulin monomer of αβ tubulin heterodimers, the building blocks of microtubules. The tubulin CTT contributes to the cellular function of microtubules such as intracellular transportation by regulating their interaction with other proteins and cell shape regulation by controlling microtubule polymerization dynamics. Although the mechanical integrity of microtubules is crucial for their functions, the role of tubulin CTT on microtubule mechanical properties has remained elusive. In this work, we investigate the role of tubulin CTTs in regulating the mechanical properties of microtubules by estimating the persistence lengths and investigating the buckling behavior of microtubules with and without CTT. We find that microtubules with intact CTTs exhibit twice the rigidity of microtubules lacking tubulin CTTs. Our study will widen the scope of altering microtubule mechanical properties for its application in nano bio-devices and lead to novel therapeutic approaches for neurodegenerative diseases with altered microtubule properties., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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7. Aldo-keto reductase family 1 member B10 is regulated by nucleos(t)ide analogues for chronic hepatitis B.
- Author
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Orita N, Kawaguchi K, Honda M, Shimode T, Hayakawa N, Terashima T, Komura T, Nishikawa M, Horii R, Nio K, Shimakami T, Takatori H, Arai K, Sakai Y, Yamashita T, Mizukoshi E, Kaneko S, Kagaya T, and Yamashita T
- Subjects
- Humans, Lamivudine therapeutic use, Tenofovir, Antiviral Agents pharmacology, Antiviral Agents therapeutic use, Aldo-Keto Reductases, Hepatitis B, Chronic complications, Hepatitis B, Chronic drug therapy, Liver Neoplasms pathology, Aldo-Keto Reductase Family 1 member B10, Carcinoma, Hepatocellular pathology
- Abstract
The number of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients persists even under nucleos(t)ide analogues (NAs) treatment. Aldo-keto reductase family 1 member B10 (AKR1B10) expression has been reported in advanced chronic liver diseases as well as cancer tissues. We observed an association between related to HCC incidence and serum AKR1B10 by analyzing patients under treatment with NAs. Serum AKR1B10 levels measured by ELISA were higher in HCC cases under NA treatment compared with non-HCC cases and were associated with lamivudine- and adefovir pivoxil-, but not entecavir- or tenofovir alafenamide-treated cases. The latter drugs did not increase AKR1B10 values even in HCC cases, suggesting that they influence the reduction of AKR1B10 in any cases. This analysis was supported by in-vitro examination, which showed reduced AKR1B10 expression by entecavir and tenofovir via immunofluorescence staining. In conclusion there was a relationship between HBV-related HCC incidence and AKR1B10 under nucleos(t)ide analogues, especially in the use of lamivudine and adefovir pivoxil, but entecavir and tenofovir had suppressive effects of AKR1B10., 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 © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
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8. Glucosylceramide in T cells regulates the pathology of inflammatory bowel disease.
- Author
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Komuro M, Nagane M, Endo R, Nakamura T, Miyamoto T, Niwa C, Fukuyama T, Harashima H, Aihara N, Kamiie J, Suzuki R, and Yamashita T
- Subjects
- Animals, Colitis chemically induced, Colitis drug therapy, Colitis pathology, Dextran Sulfate toxicity, Disease Models, Animal, Glucosylceramides administration & dosage, Glucosylceramides genetics, Glucosyltransferases genetics, Humans, Inflammatory Bowel Diseases metabolism, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Nanoparticles administration & dosage, Nanoparticles chemistry, T-Lymphocytes pathology, Mice, Glucosylceramides metabolism, Glucosyltransferases metabolism, Inflammatory Bowel Diseases pathology, T-Lymphocytes metabolism
- Abstract
Inflammatory bowel disease (IBD) is a chronic inflammatory disease in the colon characterized by excessive activation of T cells. Glycosphingolipids (GSLs) are composed of lipid rafts in cellular membranes, and their content is linked to immune cell function. In the present study, we investigated the involvement of GSLs in IBD. Microarray data showed that in IBD patients, the expression of only UDP-glucose ceramide glucosyltransferase (UGCG) decreased among the GSLs synthases. Ad libitum access to dextran sulfate sodium (DSS) resulted in decreased UGCG and glucosylceramide (GlcCer) content in mesenteric lymph nodes and T cells from the spleen. Furthermore, the knockdown of Ugcg in T cells exacerbated the pathogenesis of colitis, which was accompanied by a decrease in Treg levels. Treatment with GlcCer nanoparticles prevented DSS-induced colitis. These results suggested that GlcCer in T cells is involved in the pathogenesis of IBD. Furthermore, GlcCer nanoparticles are a potential efficacious therapeutic target for IBD patients., Competing Interests: Declaration of competing interest Azabu University and Hokkaido University hold patents on the usage of GlcCer nanoparticles. MK, MN, ER, TN, and TY are the inventors in these patents. Remaining authors do not declare any competing interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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9. Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.
- Author
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Takahashi D, Matsunaga E, Yamashita T, Caaveiro JMM, Abe Y, and Ueda T
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- Amyloid genetics, Amyloid metabolism, Antioxidants metabolism, Antioxidants pharmacology, Catechin analogs & derivatives, Catechin metabolism, Catechin pharmacology, Dose-Response Relationship, Drug, Flavonoids chemistry, Humans, Immunoglobulin Light-chain Amyloidosis genetics, Immunoglobulin lambda-Chains chemistry, Immunoglobulin lambda-Chains genetics, Kinetics, Magnetic Resonance Spectroscopy, Molecular Structure, Mutation, Protein Binding, Protein Stability drug effects, Quercetin chemistry, Quercetin pharmacology, Time Factors, Amyloid antagonists & inhibitors, Flavonoids pharmacology, Immunoglobulin Light-chain Amyloidosis metabolism, Immunoglobulin lambda-Chains metabolism, Quercetin analogs & derivatives
- Abstract
AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with the contents of this article., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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10. Endothelial ganglioside GM3 regulates angiogenesis in solid tumors.
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Suzuki M, Nagane M, Kato K, Yamauchi A, Shimizu T, Yamashita H, Aihara N, Kamiie J, Kawashima N, Naito S, and Yamashita T
- Subjects
- Animals, Cattle, Cell Line, Tumor, Cell Survival genetics, Cells, Cultured, Kaplan-Meier Estimate, Mammary Neoplasms, Experimental blood supply, Mammary Neoplasms, Experimental genetics, Mammary Neoplasms, Experimental metabolism, Mice, Inbred C57BL, Mice, Knockout, N-Acetylgalactosaminyltransferases genetics, N-Acetylgalactosaminyltransferases metabolism, Neovascularization, Pathologic genetics, Sialyltransferases genetics, Sialyltransferases metabolism, Tumor Burden genetics, Polypeptide N-acetylgalactosaminyltransferase, Mice, Endothelial Cells metabolism, G(M3) Ganglioside metabolism, Neovascularization, Pathologic metabolism
- Abstract
Cancer cells require oxygen and nutrients for growth, making angiogenesis one of the essential components of tumor growth. Gangliosides, constituting membrane lipid rafts, regulate intracellular signal transduction and are involved in the malignancy of cancer cells. While endothelial cells, as well as cancer cells, express vast amounts of gangliosides, the precise function of endothelial gangliosides in angiogenesis remains unclear. In this study, we focused on gangliosides of vascular endothelial cells and analyzed their functions on tumor angiogenesis. In human breast cancer, GM3 synthase was highly expressed in vascular endothelial cells as well as immune cells. Angiogenesis increased in GM3S-KO mice. In BAEC, RNA interference of GM3S showed increased cellular invasion and oxidative stress tolerance through activation of ERK. In the breast cancer model, GM3-KO mice showed an increase in tumor growth and angiogenesis. These results suggest that the endothelial ganglioside GM3 regulates tumor angiogenesis by suppressing cellular invasion and oxidative stress tolerance in endothelial cells., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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11. Comparison of culture media for human intestinal organoids from the viewpoint of pharmacokinetic studies.
- Author
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Yokota J, Yamashita T, Inui T, Nomoto R, Kishimoto W, Nakase H, and Mizuguchi H
- Subjects
- Cell Culture Techniques methods, Cells, Cultured, Duodenum metabolism, Humans, Organoids cytology, Pharmacokinetics, Culture Media metabolism, Duodenum cytology, Organoids metabolism
- Abstract
Human intestinal organoids are expected to be applied in pharmaceutical research. Various culture media for human intestinal organoids have been developed, but it remains unclear which media are preferable for pharmacokinetic studies. Here, we cultured human intestinal organoids with three major culture media that are already used widely around the world: the medium of Sato et al. (S-medium; reported in 2011), Fujii et al. (F-medium; 2018), and Miyoshi et al. (M-medium; 2013). The growth of human intestinal organoids cultured in S-medium was faster than that in F- or M-medium. The gene expression levels of most pharmacokinetic-related enzymes or transporters in human intestinal organoids cultured in M-medium were higher than those in S- or F-medium, and comparable to those in the adult human small intestine. The level of cytochrome P450 (CYP) 3A4 activity was also highest in human intestinal organoids cultured in M-medium. Collectively, the results underscored the importance of selection and optimization of culture medium for various applications using human intestinal organoids, including pharmacokinetic studies., 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 © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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12. Novel phospho-specific monoclonal antibodies reveal differential regulation of tyrosine phosphorylation within the immunoreceptor tyrosine-based activation motif of the Fc receptor γ subunit leading to fine tuning of Syk activation.
- Author
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Yamashita Y and Yamashita T
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antibodies, Phospho-Specific immunology, Cells, Cultured, Mast Cells metabolism, Mice, Inbred C57BL, Phosphorylation, Receptors, IgG chemistry, Signal Transduction, Tyrosine chemistry, Mice, Antibodies, Monoclonal metabolism, Antibodies, Phospho-Specific metabolism, Immunoreceptor Tyrosine-Based Activation Motif, Mast Cells immunology, Receptors, IgG metabolism, Syk Kinase metabolism, Tyrosine metabolism
- Abstract
The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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13. Presence of ES1 homolog in the mitochondrial intermembrane space of porcine retinal cells.
- Author
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Utsumi S, Sakamoto K, Yamashita T, Tomita H, Sugano E, Ishida K, Ishiyama E, and Ozaki T
- Subjects
- Amino Acid Sequence, Animals, Eye Proteins chemistry, Mitochondrial Proteins chemistry, Mitochondrial Proteins metabolism, Photoreceptor Cells, Vertebrate cytology, Photoreceptor Cells, Vertebrate metabolism, Photoreceptor Cells, Vertebrate ultrastructure, Retina ultrastructure, Solubility, Eye Proteins metabolism, Mitochondrial Membranes metabolism, Retina cytology, Sequence Homology, Amino Acid, Swine metabolism
- Abstract
ES1 homologs are conserved among prokaryotes and eukaryotes, and the gene expression of ES1 homologs has been confirmed in diverse mammalian tissues. However, the localization and function of mammalian ES1 proteins remain poorly understood. ES1 protein was found specifically expressed in the cone cells of zebrafish and was proposed to contribute to the formation of mega mitochondria. We also observed mega mitochondria in the cone cells of porcine retinas, which raised the question regarding the localization of the porcine ES1. Therefore, in the present study, we aimed to determine the localization of ES1 in porcine retinas. We prepared a rabbit polyclonal antibody against the ES1 C-terminal and performed western blotting analysis and immunoelectron microscopy. The ES1 was found to be localized mainly in the mitochondrial intermembrane space of the porcine retinal cells. Immunopositive signals for ES1 were observed in the mitochondria of almost all retinal cells, and not specifically in cone cells. Our results and the ES1 sequences indicated that the glyoxalase III activity of ES1 might contribute to the stable functionality of the active mitochondria in a protective manner., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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14. PrP (122-139) is a covert mitochondrial targeting signal of prion protein and it specifically triggers the perinuclear clustering of mitochondria in neuronal culture cells.
- Author
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Shimizu T, Kozuka Y, Kusano M, Nagane M, Yamashita T, and Hachiya N
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- Animals, COS Cells, Cell Line, Chlorocebus aethiops, HeLa Cells, Humans, Mice, Mitochondria metabolism, Neurons cytology, Neurons metabolism, Prion Diseases metabolism, Prion Diseases pathology, Prion Proteins metabolism, Mitochondria pathology, Neurons pathology, Prion Proteins analysis
- Abstract
In many neurodegenerative diseases, mitochondria are actively involved in the onset and/or progression of diseases because the energy depletion of the neuronal cells directly leads to the dysfunction and degeneration of cells. In the case of prion diseases, mitochondrial involvement has been reported recently and evidence that prion protein (PrP) is localized in mitochondria is increasing. Despite these findings, the precise molecular mechanism by which PrP targets mitochondria remains unclear. PrP is a secretory protein and does not have a pre-sequence that targets the mitochondria, therefore, we thought that there was a covert signal in the amino acid sequence of PrP. To find the sequence, we constructed various GFP-fused PrP-truncations and colocalization with mitochondria was verified by live-cell imaging. Consequently, we found that 18 amino acids, PrP (122-139), are indispensable for the mitochondrial targeting of PrP. In addition, fluorescent microscopy observation revealed that PrP-localized mitochondria were accumulated at the perinuclear region in neuronal cells such as mouse neuroblastoma Neuro2a (N2a) and prion persistent infection N2a strain (ScN2a), anterograde movement of the mitochondria toward the cell membrane was completely inhibited because of the stacking of PrP on the outer membrane. The cristae formation of perinuclear accumulated mitochondria was disappeared indicating the reduced mitochondrial activity. Surprisingly, PrP-dependent mitochondrial perinuclear accumulation was specifically occurred on neuronal cells, whereas in epithelial HeLa cells and fibroblast COS-7 cells, no perinuclear accumulation observed even after the mitochondrial targeting of PrP., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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15. Induction of Trop-2 expression through the binding of galectin-3 to MUC1.
- Author
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Yamashita T, Mori Y, Alzaaqi SM, Yashiro M, Sawada T, Hirakawa K, and Nakada H
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- Blood Proteins, Galectin 3 genetics, Galectins, Gene Expression Regulation, Neoplastic, HCT116 Cells, Humans, MCF-7 Cells, Mucin-1 genetics, Neoplasms genetics, Neoplasms metabolism, Protein Binding, Sp1 Transcription Factor metabolism, Antigens, Neoplasm genetics, Cell Adhesion Molecules genetics, Galectin 3 metabolism, Mucin-1 metabolism, Up-Regulation
- Abstract
Both mucin 1 (MUC1) and trophoblast cell surface antigen 2 (Trop-2) are overexpressed in various epithelial tumor cells, and their high expression is correlated with a poor prognosis. Both proteins were expressed in a human breast cancer cell line, MCF-7 cells, but neither was in a human colon cancer cell line, HCT116 cells. When MUC1 cDNA was introduced into HCT116 cells (HCT116/MUC1), expression of Trop-2 was induced. Reciprocally, treatment of MCF-7 cells with MUC1 siRNA reduced the level of Trop-2. Mithramycin A, an inhibitor of specificity protein 1 (Sp1) transcription factor, effectively inhibited the expression of Trop-2. Consistently, treatment with Sp1 siRNA reduced the expression of Trop-2. To reveal the relationship between MUC1 and Sp1, coimmunoprecipitation assays were performed. Sp1 was coimmunoprecipitated with MUC1 and the level of coimmunoprecipitated Sp1 increased in relation to the level of induced Trop-2. It is known that galectin-3 is an endogenous ligand of MUC1. Binding of galectin-3 to MUC1 elevated the recruitment of Sp1 to MUC1, and knockdown of galectin-3 reduced the level of Trop-2. These results suggest that the binding of galectin-3 to MUC1 enhances the recruitment of Sp1, leading to promotion of the transcription of Trop-2., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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16. Presence of calpain-5 in mitochondria.
- Author
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Iwamoto T, Ishiyama E, Ishida K, Yamashita T, Tomita H, and Ozaki T
- Subjects
- Animals, Cytosol metabolism, Electrons, Gene Expression Profiling, Gene Expression Regulation, Inflammation, Microscopy, Immunoelectron, Photoreceptor Cells, Vertebrate metabolism, Subcellular Fractions, Swine, Calpain metabolism, Mitochondria metabolism, Photoreceptor Cells metabolism, Retina metabolism
- Abstract
Calpains are Ca
2+ -dependent cysteine proteases that are widely distributed in animal tissues and modulate a variety of cellular processes. There are 15 members of the calpain family in mammals. In animal cells, there are three types of calpains, viz., calpain-1, calpain-2, and calpain-10 in the mitochondria. The three types of calpains have been shown to play significant roles in pathophysiological conditions, including in apoptosis- and necrosis-like cell death. One of the severe retinal diseases, autosomal dominant neovascular inflammatory vitreoretinopathy, is known to be induced by mutations of the calpain-5 gene. However, the distribution of calpain-5 in the retina has not been elucidated. Therefore, in the present study, we determined the localization of calpain-5 in the porcine retina. We detected calpain-5 in the inner segment of photoreceptor cells using immunohistochemistry. With immunoelectron microscopy, calpain-5 was localized in the mitochondria of photoreceptor cells. Western blot analyses showed that calpain-5 was present in each mitochondrial subfraction. Furthermore, we showed that the molecular weight of mitochondrial calpain-5 was slightly smaller than cytosolic one. Our results demonstrated that a novel mitochondrial calpian, calpain-5, was localized in the mitochondria of retinal photoreceptor cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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17. Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.
- Author
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Yasuoka Y, Izumi Y, Nagai T, Fukuyama T, Nakayama Y, Inoue H, Horikawa K, Kimura M, Nanami M, Yanagita K, Oshima T, Yamazaki T, Uematsu T, Yamamura R, Kobayashi N, Shimada Y, Nagaba Y, Nakanishi T, Yamashita T, Mukoyama M, Sato Y, Kawahara K, and Nonoguchi H
- Subjects
- Animals, Cell Hypoxia, Erythropoietin genetics, Glycosylation, Kidney Tubules, Collecting cytology, Male, Nephrons cytology, RNA, Messenger genetics, Rats, Sprague-Dawley, Renin-Angiotensin System, Up-Regulation, Aldosterone metabolism, Erythropoietin metabolism, Fludrocortisone metabolism, Kidney Tubules, Collecting metabolism, Nephrons metabolism
- Abstract
Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
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18. Distinct notch signaling expression patterns between nucleoside and nucleotide analogues treatment for hepatitis B virus infection.
- Author
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Wang Z, Kawaguchi K, Honda M, Sakai Y, Yamashita T, Mizukoshi E, and Kaneko S
- Subjects
- Adult, Aged, Antiviral Agents chemistry, Antiviral Agents pharmacology, Female, Hep G2 Cells, Hepatitis B genetics, Hepatitis B metabolism, Hepatitis B virus physiology, Humans, Male, Middle Aged, Nucleosides chemistry, Nucleosides pharmacology, Nucleotides chemistry, Nucleotides pharmacology, Receptors, Notch metabolism, Signal Transduction drug effects, Antiviral Agents therapeutic use, Gene Expression Regulation drug effects, Hepatitis B drug therapy, Hepatitis B virus drug effects, Nucleosides therapeutic use, Nucleotides therapeutic use, Receptors, Notch genetics
- Abstract
Nucleos(t)ide analogues therapies are currently approved for the treatment of chronic hepatitis B virus (HBV) infection, which effectively suppress HBV replication and correlate with the anti-HBV-specific immune response. Notch signaling serves pleiotropic roles in the immune system that also contribute to virus-specific immunity. In this study, we assessed Notch signal-related gene expression after administrating nucleoside or nucleotide analogues to HBV-replicating cells and clinical liver tissues. We found distinct Notch signaling expression patterns under nucleos(t)ide analogues therapies, with high expression for nucleotide analogues (adefovir pivoxil or tenofovir disoproxil fumarate) and low expression for nucleoside analogues (lamivudine or entecavir) in the presence of HBV infection. Furthermore, activation of mammalian target of rapamycin (mTOR)-Akt (Ser473) phosphorylation was also observed after nucleotide analogue treatment. In conclusion, nucleoside and nucleotide analogues displayed different patterns of Notch signaling activity under HBV infection, and the induction of mTORC2-Akt (Ser473) phosphorylation may contribute to nucleotide analogues-mediated Notch signaling activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
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19. Myotube-derived factor promotes oligodendrocyte precursor cell proliferation.
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Nakasone A, Muramatsu R, Kato Y, Kawahara Y, and Yamashita T
- Subjects
- Animals, Cell Line, Cell Proliferation, Mice, Inbred C57BL, Muscle Fibers, Skeletal cytology, RNA metabolism, Muscle Fibers, Skeletal metabolism, Oligodendrocyte Precursor Cells cytology, Oligodendrocyte Precursor Cells metabolism
- Abstract
Muscle cells secrete numerous molecules that function as endocrine hormones and regulate the functions of distant organs. Myelination in the central nervous system (CNS) is regulated by peripheral hormones. However, the effects of muscle-derived molecules on myelination have not been sufficiently analyzed. In this study, we show that muscle-releasing factors promote proliferation of oligodendrocyte precursor cells (OPCs), which is an element of myelination process. Supernatants of mouse myotube cultures stimulated bromodeoxyuridine (BrdU) incorporation into mouse OPCs. Mouse myotube supernatants did not enhance mouse OPC transmigration and myelin basic protein (MBP) expression. RNA sequencing identified candidate genes with hormonal functions that were expressed in mouse myotubes. These data support the possibility that hormonal molecules secreted by myotubes contribute to OPC proliferation and myelination., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
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20. Aging impairs beige adipocyte differentiation of mesenchymal stem cells via the reduced expression of Sirtuin 1.
- Author
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Khanh VC, Zulkifli AF, Tokunaga C, Yamashita T, Hiramatsu Y, and Ohneda O
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- Aged, Aged, 80 and over, Cellular Senescence, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Humans, Infant, Infant, Newborn, Signal Transduction, Sirtuin 3 metabolism, Tissue Donors, Tumor Suppressor Protein p53 metabolism, Adipocytes, Beige cytology, Adipocytes, Beige metabolism, Aging metabolism, Cell Differentiation, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism, Sirtuin 1 metabolism
- Abstract
In the body, different types of adipose tissue perform different functions, with brown and beige adipose tissues playing unique roles in dissipating energy. Throughout life, adipocytes are regenerated from progenitors, and this process is impaired by aging. One of the progenitors of adipocytes are mesenchymal stem cells (MSCs), which have recently become a promising tool for stem cell therapy. However, whether or not aging impairs the brown/beige adipocyte differentiation of adipose tissue-derived MSCs (AT-MSCs) remains unclear. In the present study, we isolated AT-MSCs from two different age groups of donors (infants and elderly subjects) and examined the effects of aging on the AT-MSC brown/beige adipocyte differentiation ability. We found that none of the AT-MSCs expressed Myf5, which indicated the beige (not brown) differentiation ability of cells. Of note, an inverse correlation was noted between the beige adipocyte differentiation ability and age, with AT-MSCs derived from elderly donors showed the most severely reduced function due to induced cellular senescence. The impaired expression of Sirtuin 1 (Sirt1) and Sirt3 proved to be responsible for the induction of senescence in elderly AT-MSCs; however, only Sirt1 was directly involved in the regulation of beige adipocyte differentiation. The overexpression of Sirt1 impaired the p53/p21 pathway, thereby preventing elderly AT-MSCs from entering senescence and restoring the beige differentiation ability. Thus, our study represents the important role of Sirt1 and senescence in the regulation of beige adipocyte differentiation during aging., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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21. Corrigendum to "SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells" [Biochem. Biophys. Res. Commun. 493/2 (2017) 1010-1017].
- Author
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Kato T, Khanh VC, Sato K, Takeuchi K, Carolina E, Yamashita T, Sugaya H, Yoshioka T, Mishima H, and Ohneda O
- Published
- 2018
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22. Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells.
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Yamashita T, Takayama K, Sakurai F, and Mizuguchi H
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- Batch Cell Culture Techniques methods, Cells, Cultured, Equipment Design, Equipment Failure Analysis, Humans, Tissue Engineering instrumentation, Tissue Engineering methods, Batch Cell Culture Techniques instrumentation, Bioreactors, Cell Differentiation physiology, Hepatocytes cytology, Hepatocytes physiology, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells physiology
- Abstract
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10
9 cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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23. SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells.
- Author
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Kato T, Khanh VC, Sato K, Takeuchi K, Carolina E, Yamashita T, Sugaya H, Yoshioka T, Mishima H, and Ohneda O
- Subjects
- Adipose Tissue drug effects, Animals, Cells, Cultured, Chemokine CXCL12 analysis, Humans, Mesenchymal Stem Cells cytology, Mice, Inbred C57BL, Adipose Tissue cytology, Chemokine CXCL12 genetics, Down-Regulation drug effects, Glucocorticoids pharmacology, Mesenchymal Stem Cell Transplantation, Mesenchymal Stem Cells drug effects, Wound Healing drug effects
- Abstract
Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs. In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors. These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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24. Cardiomyocyte-released factors stimulate oligodendrocyte precursor cells proliferation.
- Author
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Kuroda M, Muramatsu R, and Yamashita T
- Subjects
- Animals, Brain metabolism, Cell Differentiation, Cells, Cultured, Central Nervous System metabolism, Coculture Techniques, Culture Media, Conditioned, Extracellular Signal-Regulated MAP Kinases metabolism, Mice, Mice, Inbred C57BL, Multiple Sclerosis metabolism, Myelin Sheath metabolism, Phosphatidylinositol 3-Kinases metabolism, Phosphorylation, Cell Proliferation, Myocytes, Cardiac cytology, Oligodendroglia cytology
- Abstract
The heart produces multiple diffusible factors that are involved in a number of physiological processes, but the action of these factors on the central nervous system is not well understood. In this study, we found that one or more factors released by cardiomyocytes promote oligodendrocyte precursor cell (OPC) proliferation in vitro. Mouse OPCs co-cultured with mouse cardiomyocytes showed higher proliferative ability than OPCs cultured alone. In addition, cardiomyocyte-conditioned media was sufficient to promote OPC proliferation. The phosphorylation of phosphatidylinositol (PI) 3-kinase and extracellular signal-regulated kinase (ERK) in OPCs is necessary for the enhancement of OPC proliferation by cardiomyocyte-conditioned media. These data indicate that heart-derived factors have the ability to directly regulate the function of central nervous system (CNS) cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
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25. The protection of rat retinal ganglion cells from ischemia/reperfusion injury by the inhibitory peptide of mitochondrial μ-calpain.
- Author
-
Ozaki T, Yamashita T, Tomita H, Sugano E, and Ishiguro S
- Subjects
- Amino Acid Sequence, Animals, Apoptosis drug effects, Apoptosis Inducing Factor antagonists & inhibitors, Apoptosis Inducing Factor metabolism, Blotting, Western, Calpain metabolism, Glaucoma metabolism, Glaucoma physiopathology, Microscopy, Confocal, Mitochondrial Proteins metabolism, Ophthalmic Solutions, Peptides administration & dosage, Peptides chemical synthesis, Protective Agents administration & dosage, Protective Agents chemical synthesis, Protective Agents pharmacology, Rats, Sprague-Dawley, Reperfusion Injury physiopathology, Retina drug effects, Retina metabolism, Retina physiopathology, Retinal Ganglion Cells metabolism, Calpain antagonists & inhibitors, Mitochondrial Proteins antagonists & inhibitors, Peptides pharmacology, Reperfusion Injury metabolism, Retinal Ganglion Cells drug effects
- Abstract
Intracellular Ca(2+)-dependent cysteine proteases such as calpains have been suggested as critical factors in retinal ganglion cell (RGC) death. However, it is unknown whether mitochondrial calpains are involved in RGC death. The purpose of the present study was to determine whether the inhibition of mitochondrial μ-calpain activity protects against RGC death during ischemia/reperfusion (I/R) injury. This study used a well-established rat model of experimental acute glaucoma involving I/R injury. A specific peptide inhibitor of mitochondrial μ-calpain, Tat-μCL, was topically applied to rats via eye drops three times a day for 5 days after I/R. RGC death was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The truncation of apoptosis-inducing factor (AIF) was determined by western blot analyses. Retinal morphology was determined after staining with hematoxyline and eosin. In addition, the number of Fluoro Gold-labeled RGCs in flat-mounted retinas was used to determine the percentage of surviving RGCs after I/R injury. After 1 day of I/R, RGC death was observed in the ganglion cell layer. Treatment with Tat-μCL eye drops significantly prevented the death of RGCs and the truncation of AIF. After 5 days of I/R, RGC death decreased by approximately 40%. However, Tat-μCL significantly inhibited the decrease in the retinal sections and flat-mounted retinas. The results suggested that mitochondrial μ-calpain is associated with RGC death during I/R injury via truncation of AIF. In addition, the inhibition of mitochondrial μ-calpain activity by Tat-μCL had a neuroprotective effect against I/R-induced RGC death., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
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26. Improved transduction efficiencies of adeno-associated virus vectors by synthetic cell-permeable peptides.
- Author
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Tabata K, Sugano E, Murakami F, Yamashita T, Ozaki T, and Tomita H
- Subjects
- Amino Acid Sequence, Animals, Blotting, Western, Cell Line, Cell-Penetrating Peptides chemical synthesis, Dose-Response Relationship, Drug, Embryo, Mammalian cytology, Fibroblasts cytology, Fibroblasts metabolism, Genetic Vectors genetics, Macaca fascicularis, Microscopy, Fluorescence, Phosphorylation drug effects, Rats, Reproducibility of Results, Skin cytology, Skin metabolism, p38 Mitogen-Activated Protein Kinases metabolism, Cell-Penetrating Peptides pharmacology, Dependovirus genetics, Fibroblasts drug effects, Skin drug effects, Transduction, Genetic methods
- Abstract
Various serotypes of adeno-associated virus (AAV) vectors have been used for gene therapy and as research tools. Among these serotypes, the AAV type 2 vector has been used successfully in human gene therapies. However, the transduction efficiency of AAV2 depends on the cell type, and this poses a problem in the efficacy of gene therapy. To improve the transduction efficiency of AAV2, we designed a small peptide consisting of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor peptide and the HIV-Tat sequence Tat-Y1068. Pre- or co-treatment of CYNOM-K1 cells from cynomolgus monkey embryo skin with Tat-Y1068 increased the transduction efficiencies in a dose-dependent manner and caused p38 phosphorylation. The transduction efficiency of AAV2 into the rat fibroblast cell line RAT-1 highly expressing EGFR was less than the transduction efficiency of AAV2 into CYNOM-K1 cells. Tat-Y1068 increased the transduction efficiency in RAT-1 cells in the same manner as in CYNOM-K1 cells. In conclusion, cell-permeable peptides possessing the EGFR tyrosine kinase inhibitor function might serve as a useful ingredient of AAV2 vector solution for increasing the transduction efficiency of gene therapies., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
27. Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs.
- Author
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Tu TC, Yamashita T, Kato T, Nagano M, Trinh NT, Hamada H, Sato F, Ohneda K, Matsuo-Takasaki M, and Ohneda O
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Endothelial Progenitor Cells cytology, Mesenchymal Stem Cells cytology, Wound Healing
- Abstract
Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
28. Microvesicles enhance the mobility of human diabetic adipose tissue-derived mesenchymal stem cells in vitro and improve wound healing in vivo.
- Author
-
Trinh NT, Yamashita T, Tu TC, Kato T, Ohneda K, Sato F, and Ohneda O
- Subjects
- Animals, Cell Movement, Cell-Derived Microparticles pathology, Cell-Derived Microparticles physiology, Female, Mesenchymal Stem Cells pathology, Mice, Mice, Inbred C57BL, Treatment Outcome, Adipocytes cytology, Cell-Derived Microparticles transplantation, Diabetes Mellitus, Type 2 pathology, Mesenchymal Stem Cell Transplantation methods, Mesenchymal Stem Cells physiology, Wound Healing physiology
- Abstract
Microvesicles (MVs) derived from mesenchymal stem cells showed the ability to alter the cell phenotype and function. We previously demonstrated that type 2 diabetic adipose tissue-derived mesenchymal stem cells (dAT-MSCs) increase in cell aggregation and adhesion in vitro and impair wound healing in vivo. However, the characterization and function of MVs derived from human non-diabetic AT-MSCs (nAT-MSCs) remain unknown. In this study, we characterized nAT-MSC-derived MVs and their function after the transfection of dAT-MSCs with MVs using the scratch assay and a flap mouse model. We found that human nAT-MSC-derived MVs expressed MSC-surface markers and improved dAT-MSC functions by altering the expression of genes associated with cell migration, survival, inflammation, and angiogenesis as well as miR29c and miR150. Remarkably, the transfection of dAT-MSCs with nAT-MSC-derived MVs improved their migration ability in vitro and wound healing ability in a flap mouse model. These results demonstrate a promising opportunity to modify the function of dAT-MSCs for therapeutic stem cell application in diabetic patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
29. NME2 associates with PTPσ to transduce signals from chondroitin sulfate proteoglycans.
- Author
-
Hamasaki H, Fujitani M, and Yamashita T
- Subjects
- Animals, Cell Nucleus enzymology, Cerebral Cortex cytology, Chondroitin Sulfate Proteoglycans pharmacology, Cytoplasm enzymology, Gene Knockdown Techniques, HEK293 Cells, Humans, Mass Spectrometry, Mice, NM23 Nucleoside Diphosphate Kinases genetics, Neurites drug effects, Neurites metabolism, Neurons enzymology, Neurons ultrastructure, Receptor-Like Protein Tyrosine Phosphatases, Class 2 genetics, Chondroitin Sulfate Proteoglycans metabolism, NM23 Nucleoside Diphosphate Kinases metabolism, Neurites physiology, Receptor-Like Protein Tyrosine Phosphatases, Class 2 metabolism
- Abstract
Chondroitin sulfate proteoglycans (CSPGs) are a major component of glial scars, inhibiting axonal growth in the central nervous system. Protein tyrosine phosphatase, receptor type S (PTPσ) has been identified as a receptor for CSPGs, whereas its downstream signaling pathway remains to be fully understood. Here, we report that nucleoside diphosphate kinase 2 (NME2) interacts with PTPσ. We screened proteins associated with PTPσ by mass spectrometry, and obtained NME2. Immunoprecipitation analysis revealed that NME2 associated with the PTPσ intracellular domain in HEK-293T cells. NME2 was expressed in the cytoplasm and nucleus of cortical neurons, and knockdown of NME2 in the cortical neurons completely rescued neurite outgrowth inhibition induced by CSPGs. These results demonstrate that NME2 associates with PTPσ to elicit neurite outgrowth inhibition in response to CSPGs., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
30. Repulsive guidance molecule A suppresses angiogenesis.
- Author
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Harada K, Fujita Y, and Yamashita T
- Subjects
- Angiogenic Proteins metabolism, Cells, Cultured, GPI-Linked Proteins metabolism, Humans, Cell Movement physiology, Endothelial Cells cytology, Endothelial Cells physiology, Neovascularization, Physiologic physiology, Nerve Tissue Proteins metabolism, Vascular Endothelial Growth Factor A metabolism
- Abstract
The repulsive guidance molecule-a (RGMa) is a membrane-associated glycoprotein that has diverse functions in the developing and adult central nervous system. Here, we show that RGMa suppresses new blood vessel formation. Treatment of human umbilical artery endothelial cells (HUAEC) on Matrigel with recombinant RGMa inhibits vascular endothelial growth factor (VEGF)-induced and VEGF-independent tubular formation and migration. RGMa enhances adhesion presumably through dephosphorylation of focal adhesion kinase (FAK) at tyrosine-397. Neogenin, an RGMa receptor, in HUAEC is required for the effect of RGMa. In vivo Matrigel plug assay reveals that treatment with recombinant RGMa suppresses angiogenesis. Thus, we conclude that RGMa inhibits angiogenesis in vitro and in vivo suggesting that its manipulation would be an efficient therapeutic strategy for pro-angiogenic conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
31. Wnt16 regulates osteoclast differentiation in conjunction with Wnt5a.
- Author
-
Kobayashi Y, Thirukonda GJ, Nakamura Y, Koide M, Yamashita T, Uehara S, Kato H, Udagawa N, and Takahashi N
- Subjects
- Animals, Calcitriol pharmacology, Coculture Techniques, Mice, Mice, Knockout, Osteoclasts drug effects, Osteoprotegerin genetics, Wnt-5a Protein, Cell Differentiation physiology, Osteoclasts cytology, Wnt Proteins physiology
- Abstract
The canonical Wnt/β-catenin signaling pathway in osteoblast-lineage cells inhibits osteoclastogenesis through the expression of osteoprotegerin (Opg), a decoy receptor of receptor activator of Nf-κb (Rank) ligands. Wnt5a, a typical non-canonical Wnt ligand, enhances the expression of Rank in osteoclast precursors, which, in turn, promotes the Rank ligand (Rankl)-induced formation of osteoclasts. In contrast, Wnt16 and Wnt4 have been shown to inhibit the Rankl-induced formation of osteoclasts through non-canonical Wnt signals. However, the relationships among these Wnt ligands in osteoclastogenesis remained to be elucidated. We herein showed that Wnt16, but not Wnt4, inhibited the Rankl-induced osteoclastogenesis in bone marrow-derived macrophage (BMM) cultures. Wnt3a and Wnt4 inhibited the 1α,25-dihydroxy vitamin D3 (1,25D3)-induced osteoclastogenesis in co-cultures prepared from wild-type mice, but not in those from Opg(-/-) nice. Wnt16 inhibited the 1,25D3-induced formation of osteoclasts in both wild-type and Opg(-/-) co-cultures. Wnt16, Wnt4, and Wnt3a failed to inhibit the pit-forming activity of osteoclasts. Wnt16 failed to inhibit the Wnt5a-induced expression of Rank in osteoclast precursors. In contrast, Wnt5a abrogated the inhibitory effects of Wnt16 on Rankl-induced osteoclastogenesis. These results suggested that Wnt16 inhibited osteoclastogenesis, but not the function of osteoclasts and that Wnt16, an inhibitory Wnt ligand for osteoclastogenesis, regulates bone resorption in conjunction with Wnt5a., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
32. Eph receptor A10 has a potential as a target for a prostate cancer therapy.
- Author
-
Nagano K, Yamashita T, Inoue M, Higashisaka K, Yoshioka Y, Abe Y, Mukai Y, Kamada H, Tsutsumi Y, and Tsunoda S
- Subjects
- Antibodies, Monoclonal immunology, Antineoplastic Agents immunology, Antineoplastic Agents therapeutic use, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Cell Line, Tumor, Cell Survival, Colonic Neoplasms drug therapy, Colonic Neoplasms pathology, Drug Delivery Systems methods, Female, Humans, Male, Prostatic Neoplasms pathology, Receptors, Eph Family immunology, Treatment Outcome, Antibodies, Monoclonal therapeutic use, Biomarkers, Tumor metabolism, Breast Neoplasms metabolism, Colonic Neoplasms metabolism, Prostatic Neoplasms drug therapy, Prostatic Neoplasms metabolism, Receptors, Eph Family metabolism
- Abstract
We recently identified Eph receptor A10 (EphA10) as a novel breast cancer-specific protein. Moreover, we also showed that an in-house developed anti-EphA10 monoclonal antibody (mAb) significantly inhibited proliferation of breast cancer cells, suggesting EphA10 as a promising target for breast cancer therapy. However, the only other known report for EphA10 was its expression in the testis at the mRNA level. Therefore, the potency of EphA10 as a drug target against cancers other than the breast is not known. The expression of EphA10 in a wide variety of cancer cells was studied and the potential of EphA10 as a drug target was evaluated. Screening of EphA10 mRNA expression showed that EphA10 was overexpressed in breast cancer cell lines as well as in prostate and colon cancer cell lines. Thus, we focused on prostate cancers in which EphA10 expression was equivalent to that in breast cancers. As a result, EphA10 expression was clearly shown in clinical prostate tumor tissues as well as in cell lines at the mRNA and protein levels. In order to evaluate the potential of EphA10 as a drug target, we analyzed complement-dependent cytotoxicity effects of anti-EphA10 mAb and found that significant cytotoxicity was mediated by the expression of EphA10. Therefore, the idea was conceived that the overexpression of EphA10 in prostate cancers might have a potential as a target for prostate cancer therapy, and formed the basis for the studies reported here., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
33. Vascular endothelial cells promote cortical neurite outgrowth via an integrin β3-dependent mechanism.
- Author
-
Sumimoto S, Muramatsu R, Fujii S, and Yamashita T
- Subjects
- Animals, Cell Enlargement, Cells, Cultured, Rats, Rats, Wistar, Cell Communication physiology, Cerebral Cortex physiology, Endothelial Cells physiology, Integrin beta3 metabolism, Neurites physiology, Oligopeptides metabolism
- Abstract
The interaction of neurons with their non-neuronal milieu plays a crucial role in the formation of neural networks, and wide variety of cell-contact-dependent signals that promote neurite elongation have been identified. In this study, we found that vascular endothelial cells promote neurite elongation in an integrin β3-dependent manner. Vascular endothelial cells from the cerebral cortex promoted neurite elongation of cortical neurons in a cell contact-dependent manner. This effect was mediated by arginine-glycine-aspartic acid (RGD), a major recognition sequence for integrins. Pharmacological blockade of integrin β3 abolished the neurite elongation effect induced by the endothelial cells. Immunocytochemical analysis revealed that integrin β3 was expressed on cultured cortical neurons. These results demonstrate that the neurite elongation promoted by vascular endothelial cells requires integrin β3. Vascular endothelial cells may therefore play a role in the development and repair of neural networks in the central nervous system., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
34. Cathelicidin antimicrobial peptide inhibits fibroblast migration via P2X7 receptor signaling.
- Author
-
Kumagai S, Matsui K, Kawaguchi H, Yamashita T, Mohri T, Fujio Y, and Nakayama H
- Subjects
- Animals, Anti-Inflammatory Agents pharmacology, Cell Movement, Cells, Cultured, Dose-Response Relationship, Drug, Fibrosis, MAP Kinase Signaling System, Male, Mice, Mice, Inbred BALB C, Phosphorylation, Rats, Cathelicidins, Antimicrobial Cationic Peptides pharmacology, Fibroblasts drug effects, Receptors, Purinergic P2X7 metabolism, Signal Transduction
- Abstract
Fibrosis is one of the most common pathological alterations in heart failure, and fibroblast migration is an essential process in the development of cardiac fibrosis. Experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease characterized by inflammatory cell infiltration followed by healing without residual fibrosis. However, the precise mechanisms mediating termination of inflammation and nonfibrotic healing remain to be elucidated. Microarray analysis of hearts from model mice at multiple time points after EAM induction identified several secreted proteins upregulated during nonfibrotic healing, including the anti-inflammatory cathelicidin antimicrobial peptide (CAMP). Treatment with LL-37, a human homolog of CAMP, activated MAP kinases in fibroblasts but not in cardiomyocytes, indicating that fibroblasts were the target of CAMP activity. In addition, LL-37 decreased fibroblast migration in the in vitro scratch assay. P2X7 receptor (P2X7R), a well-known receptor for LL-37, was involved in LL-37 mediated biological effect on cardiac fibroblasts. Stimulation of BzATP, a P2X7R agonist, activated MAPK in fibroblasts, whereas the P2X7R antagonist, BBG, as well as P2X7R deletion abolished both LL-37-mediated MAPK activation and LL-37-induced reduction in fibroblast migration. These results strongly suggest that CAMP upregulation during myocarditis prevents myocardial fibrosis by restricting fibroblast migration via activation of the P2X7R-MAPK signaling pathway., (Copyright © 2013. Published by Elsevier Inc.)
- Published
- 2013
- Full Text
- View/download PDF
35. Improvement in protocol to generate homogeneous glutamatergic neurons from mouse embryonic stem cells reduced apoptosis.
- Author
-
Fujiki R, Sato A, Hata K, Tashiro F, Yasuhara N, Miyazaki J, Yoneda Y, Fujitani M, and Yamashita T
- Subjects
- Animals, Apoptosis, Brain-Derived Neurotrophic Factor pharmacology, Caspase 3 metabolism, Cell Survival, Embryonic Stem Cells drug effects, Fibroblast Growth Factors pharmacology, Mice, Tubulin metabolism, Cell Culture Techniques, Embryonic Stem Cells cytology, Glutamic Acid metabolism, Neurogenesis, Neurons cytology
- Abstract
Obtaining a homogenous population of central nervous system neurons has been a significant challenge in neuroscience research; however, a recent study established a retinoic acid-treated embryoid bodies-based differentiation protocol that permits the effective generation of highly homogeneous glutamatergic cortical pyramidal neurons from embryonic stem cells. We were able to reproduce this protocol regarding the purity of glutamatergic neurons, but these neurons were not sufficiently healthy for long-term observation under the same conditions that were originally described. Here, we achieved a substantial improvement in cell survival by applying a simple technique: We changed the medium for glutamatergic neurons from the original complete medium to commercially available SBM (the Nerve-Cell Culture Medium manufactured by Sumitomo Bakelite Co. Ltd.) and finally succeeded in maintaining healthy neurons for at least 3 weeks without decreasing their purity. Because SBM contains glial conditioned medium, we postulated that brain-derived neurotrophic factor or basic fibroblast growth factor is the key components responsible for pro-survival effect of SBM on neurons, and examined their effects by adding them to CM. As a result, neither of them had pro-survival effect on pure glutamatergic neuronal population., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
36. Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect.
- Author
-
Ueyama H, Muraki-Oda S, Yamade S, Tanabe S, Yamashita T, Shichida Y, and Ogita H
- Subjects
- Amino Acid Sequence, Asian People genetics, HEK293 Cells, Haplotypes, Humans, Japan, Male, Molecular Sequence Data, RNA, Messenger genetics, Alternative Splicing, Color Vision Defects genetics, Cone Opsins genetics, Exons genetics, Mutation, Missense, Rod Opsins genetics
- Abstract
We have analyzed L/M visual pigment gene arrays in 119 Japanese men with protanopia color vision defect and found that five had a normal gene order of L-M. Among the five men, two (identified as A376 and A642) had apparently normal L genes. To clarify their L gene defect, the whole L or M gene from A376 and control subjects was cloned in an expression vector. Total RNA extracted from the transfected HEK293 cells was analyzed by Northern blot and reverse transcription-polymerase chain reaction. The product from the cloned L gene of A376 was smaller than the normal control due to the absence of exon 3. To investigate such exon-skipping at splicing, minigenes of exon 3 accompanying introns 2 and 3 were prepared from A376, A642, and control subjects. The minigenes of A376 (L) and A642 (L) showed the product lacking exon 3 only, while the minigene of normal control N44 (L) showed the product retaining exon 3 only. Exchanging of introns 2 and 3 between the A376 (L) and N44 (L) minigenes showed that the skipping of exon 3 was caused by the exon itself. Seven differences in exon 3 between A376 (L) and N44 (L) were all within already-known polymorphisms as follows: G(151-3), C(153-1), G(155-3), A(171-1), T(171-3), G(178-1) and G(180-1) in A376 (L) and A642 (L), and A(151-3), A(153-1), C(155-3), G(171-1), G(171-3), A(178-1) and T(180-1) in N44 (L). An in vitro mutagenesis experiment with these nucleotides in the minigenes showed that exon 3 was completely skipped at splicing only in the haplotype observed in A376 (L) and A642 (L). These results suggest that complete skipping of exon 3 at splicing, due to the unique haplotype of the exon, causes loss of expression of L-opsin in these men., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
37. Annexin A4 is a possible biomarker for cisplatin susceptibility of malignant mesothelioma cells.
- Author
-
Yamashita T, Nagano K, Kanasaki S, Maeda Y, Furuya T, Inoue M, Nabeshi H, Yoshikawa T, Yoshioka Y, Itoh N, Abe Y, Kamada H, Tsutsumi Y, and Tsunoda S
- Subjects
- Annexin A4 genetics, Cell Line, Tumor, Gene Expression Regulation, Neoplastic, Gene Knockdown Techniques, Humans, Neoplasms, Mesothelial genetics, Annexin A4 metabolism, Antineoplastic Agents pharmacology, Biomarkers, Pharmacological metabolism, Cisplatin pharmacology, Drug Resistance, Neoplasm, Neoplasms, Mesothelial metabolism
- Abstract
Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
38. Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system.
- Author
-
Ma L, Koyota S, Myoen Y, Yamashita T, Yatabe N, Koizumi Y, Aosasa M, Nishimichi N, Matsuda H, and Sugiyama T
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Chickens, Humans, Molecular Sequence Data, Peptide Library, Single-Chain Antibodies chemistry, Single-Chain Antibodies genetics, N-Acetylgalactosaminyltransferases immunology, Peptides immunology, Single-Chain Antibodies biosynthesis, Two-Hybrid System Techniques
- Abstract
Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
39. A subpopulation of endothelial progenitor cells with low aldehyde dehydrogenase activity attenuates acute ischemic brain injury in rats.
- Author
-
Nakamura K, Tsurushima H, Marushima A, Nagano M, Yamashita T, Suzuki K, Ohneda O, and Matsumura A
- Subjects
- Animals, Brain Infarction pathology, Brain Infarction surgery, Brain Ischemia pathology, Disease Models, Animal, Humans, Male, Rats, Rats, Sprague-Dawley, Reperfusion Injury pathology, Treatment Outcome, Aldehyde Dehydrogenase biosynthesis, Brain Ischemia surgery, Human Umbilical Vein Endothelial Cells enzymology, Human Umbilical Vein Endothelial Cells transplantation, Reperfusion Injury surgery, Stem Cell Transplantation methods, Stem Cells enzymology
- Abstract
Previous studies have examined the therapeutic effect of endothelial progenitor cells (EPCs) during the chronic phase of cerebral infarction in rats; however, few studies have investigated the effects of EPCs during the acute phase of infarction. In this study, we evaluated the therapeutic effect of EPCs with low aldehyde dehydrogenase activity (Alde-Low EPCs) in rats with acute cerebral infarction, and our results provide insight that may help to identify a therapeutic mechanism of EPCs for acute cerebral infarction. The administration of Alde-Low EPCs into rats with acute cerebral infarction results in the accumulation and migration of the Alde-Low EPCs into the infarct area and the subsequent decrease of infarct volume. Moreover, we found that the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) signaling pathway may regulate the accumulation of Alde-Low EPCs. The transplantation of Alde-Low EPCs may represent a potential treatment strategy for acute cerebral infarction., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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40. Impairment of neuropsychological behaviors in ganglioside GM3-knockout mice.
- Author
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Niimi K, Nishioka C, Miyamoto T, Takahashi E, Miyoshi I, Itakura C, and Yamashita T
- Subjects
- Animals, Attention Deficit Disorder with Hyperactivity genetics, Attention Deficit Disorder with Hyperactivity physiopathology, Emotions, Female, G(M3) Ganglioside genetics, Hyperkinesis genetics, Hyperkinesis physiopathology, Male, Maze Learning, Methylphenidate administration & dosage, Mice, Mice, Knockout, Behavior, Animal, G(M3) Ganglioside physiology, Motor Activity
- Abstract
The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
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41. Limited expression of reticulocalbin-1 in lymphatic endothelial cells in lung tumor but not in normal lung.
- Author
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Yoshida Y, Yamashita T, Nagano K, Imai S, Nabeshi H, Yoshikawa T, Yoshioka Y, Abe Y, Kamada H, Tsutsumi Y, and Tsunoda S
- Subjects
- Cell Line, Tumor, Endothelial Cells metabolism, Humans, Lymphatic Vessels pathology, Tissue Array Analysis, Calcium-Binding Proteins biosynthesis, Lung metabolism, Lung Neoplasms metabolism, Lymphatic Vessels metabolism
- Abstract
Lymphatic endothelial cells in tumors (T-LECs) are considered to have different characteristics from LECs in non-tumor tissues (N-LECs). However, differences between the two types have not been well analyzed at molecular level. In this report, we performed differential proteome analysis of T-LEC and N-LEC models prepared by cultivation of LECs in tumor conditioned medium. By expression profiling of identified proteins using tissue microarrays, reticulocalbin-1 was found to be expressed in clinical specimen-derived T-LECs and lung cancer cells but not N-LECs. It is suggested that reticulocalbin-1 may be an important molecule in understanding T-LEC function and control of lymphatic metastasis., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
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42. Axotomy induces axonogenesis in hippocampal neurons by a mechanism dependent on importin β.
- Author
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Ohara R, Hata K, Yasuhara N, Mehmood R, Yoneda Y, Nakagawa M, and Yamashita T
- Subjects
- Animals, Axotomy, Cells, Cultured, Dynactin Complex, Dyneins metabolism, Microtubule-Associated Proteins metabolism, Neurites physiology, Rats, Rats, Wistar, Axons physiology, Hippocampus injuries, Hippocampus physiology, Neurons physiology, beta Karyopherins metabolism
- Abstract
We characterize the previously unrecognized phenomenon of axotomy-induced axonogenesis in rat embryonic hippocampal neurons in vitro and elucidate the underlying mechanism. New neurites arose from cell bodies after axotomy and grew. These neurites were Tau-1-positive, and the injured axons showed negative immunoreactivity for Tau-1. Axonogenesis was delayed in these neurons by inhibiting the dynein-dynactin complex through the overexpression of p50. Importin β, which was locally translated after axotomy, was associated with the dynein-importin α complex and was required for axonogenesis. Taken together, these results suggest that retrograde transport of injury-induced signals in injured axons play key roles in the axotomy-induced axonogenesis of hippocampal neurons., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
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43. Enhanced glucose tolerance in the Brattleboro rat.
- Author
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Nakamura K, Yamashita T, Fujiki H, Aoyagi T, Yamauchi J, Mori T, and Tanoue A
- Subjects
- Animals, Glucose administration & dosage, Glucose Tolerance Test, Male, Mice, Rats, Rats, Brattleboro, Receptors, Vasopressin genetics, Arginine Vasopressin physiology, Blood Glucose physiology, Glucose Intolerance physiopathology, Receptors, Vasopressin physiology
- Abstract
[Arg(8)]-vasopressin (AVP) plays a crucial role in regulating body fluid retention, which is mediated through the vasopressin V(2) receptor in the kidney. In addition, AVP is involved in the regulation of glucose homeostasis via vasopressin V(1A) and vasopressin V(1B) receptors. Our previous studies demonstrated that vasopressin V(1A) receptor-deficient (V(1A)R-/-) and V(1B) receptor-deficient (V(1B)R-/-) mice exhibited hyperglycemia and hypoglycemia with hypoinsulinemia, respectively. These findings indicate that vasopressin V(1A) receptor deficiency results in decreased insulin sensitivity whereas vasopressin V(1B) receptor deficiency results in increased insulin sensitivity. In addition, vasopressin V(1A) and vasopressin V(1B) receptor double-deficient (V(1AB)R-/-) mice exhibited impaired glucose tolerance, suggesting that the effects of vasopressin V(1B) receptor deficiency do not influence the development of hyperglycemia promoted by vasopressin V(1A) receptor deficiency, and that the blockage of both receptors could lead to impaired glucose tolerance. However, the contributions of the entire AVP/vasopressin receptors system to the regulation of blood glucose have not yet been clarified. In this study, to further understand the role of AVP/vasopressin receptors signaling in blood glucose regulation, we assessed the glucose tolerance of AVP-deficient homozygous Brattleboro (di/di) rats using an oral glucose tolerance test (GTT). Plasma glucose and insulin levels were consistently lower in homozygous di/di rats than in heterozygous di/+ rats during the GTT, suggesting that the blockage of all AVP/vasopressin receptors resulting from the AVP deficiency could lead to enhanced glucose tolerance., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
44. Th1 cells promote neurite outgrowth from cortical neurons via a mechanism dependent on semaphorins.
- Author
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Ishii H, Kubo T, Kumanogoh A, and Yamashita T
- Subjects
- Animals, Antigens, CD genetics, Antigens, CD physiology, Coculture Techniques, Interferon-gamma pharmacology, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurites drug effects, Neurons drug effects, Semaphorins genetics, Cerebral Cortex cytology, Neurites physiology, Neurons physiology, Semaphorins physiology, Th1 Cells physiology
- Abstract
The roles of T lymphocytes in the central nervous system (CNS) are diverse; their roles in the injured CNS have been reported to be both detrimental and advantageous. Hence, an investigation of the effects of specific subsets of T cells on neurons may provide an insight into the interaction between the nervous system and the immune system. In the present study, we demonstrate that a specific subset of T lymphocytes enhanced neurite outgrowth in vitro. When cultured T helper type 1 (Th1) cells were co-cultured with cortical neurons, neurite outgrowth from neurons was enhanced; however, the same was not observed when Th2 or naïve T cells were used. We observed that the promotion of neurite outgrowth by Th1 cells was completely inhibited by anti-interferon γ (IFN-γ) neutralizing antibody, but that IFN-γ did not directly promote neurite growth. Furthermore, experiments using knockout mice revealed that semaphorin 4A (Sema4A) but not Sema7A was required for the effect produced by Th1 cells. These results demonstrate that Sema4A and IFN-γ expressed in Th1 cells play a critical role in enhancing neurite outgrowth from cortical neurons., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
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45. Differential gene expression profiling in blood from patients with digestive system cancers.
- Author
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Honda M, Sakai Y, Yamashita T, Yamashita T, Sakai A, Mizukoshi E, Nakamoto Y, Tatsumi I, Miyazaki Y, Tanno H, and Kaneko S
- Subjects
- Aged, Cluster Analysis, Digestive System Neoplasms genetics, Female, Humans, Male, Middle Aged, Oligonucleotide Array Sequence Analysis, Digestive System Neoplasms blood, Digestive System Neoplasms diagnosis, Gene Expression Profiling methods
- Abstract
To develop a non-invasive and sensitive diagnostic test for cancer using peripheral blood, we evaluated gene expression profiling of blood obtained from patients with cancer of the digestive system and normal subjects. The expression profiles of blood-derived total RNA obtained from 39 cancer patients (11 colon cancer, 14 gastric cancer, and 14 pancreatic cancer) was clearly different from those obtained from 15 normal subjects. By comparing the gene expression profiles of cancer patients and normal subjects, 25 cancer-differentiating genes (p<5.0 x 10(-6) and fold differences >3) were identified and an "expression index" deduced from the expression values of these genes differentiated the validation cohort (11 colon cancer, 8 gastric cancer, 18 pancreatic cancer, and 15 normal subjects) into cancer patients and normal subjects with 100% (37/37) and 87% (13/15) accuracy, respectively. Although, the expression profiles were not clearly different between the cancer patients, some characteristic genes were identified according to the stage and species of the cancer. Interestingly, many immune-related genes such as antigen presenting, cell cycle accelerating, and apoptosis- and stress-inducing genes were up-regulated in cancer patients, reflecting the active turnover of immune regulatory cells in cancer patients. These results showed the potential relevance of peripheral blood gene expression profiling for the development of new diagnostic examination tools for cancer patients., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
46. Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects.
- Author
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Yamazaki Y, Imura A, Urakawa I, Shimada T, Murakami J, Aono Y, Hasegawa H, Yamashita T, Nakatani K, Saito Y, Okamoto N, Kurumatani N, Namba N, Kitaoka T, Ozono K, Sakai T, Hataya H, Ichikawa S, Imel EA, Econs MJ, and Nabeshima Y
- Subjects
- Adult, Age Factors, Aged, Aged, 80 and over, Animals, Antibodies, Monoclonal immunology, Calcium blood, Child, Female, Fibroblast Growth Factor-23, Glucuronidase genetics, Glucuronidase immunology, Humans, Klotho Proteins, Male, Mice, Middle Aged, Mutation, Aging blood, Enzyme-Linked Immunosorbent Assay, Glucuronidase blood
- Abstract
Background: Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation., Objectives: The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects., Results: We successively developed an ELISA to measure serum salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/mL) than those in adults (mean+/-SD; 562+/-146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls., Conclusion: We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
47. Novel protein engineering strategy for creating highly receptor-selective mutant TNFs.
- Author
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Nomura T, Abe Y, Kamada H, Inoue M, Kawara T, Arita S, Furuya T, Yoshioka Y, Shibata H, Kayamuro H, Yamashita T, Nagano K, Yoshikawa T, Mukai Y, Nakagawa S, Taniai M, Ohta T, Tsunoda S, and Tsutsumi Y
- Subjects
- Amino Acid Sequence, Cell Line, DNA Shuffling, Humans, Molecular Sequence Data, Mutation, Peptide Library, Receptors, Tumor Necrosis Factor, Type I metabolism, Receptors, Tumor Necrosis Factor, Type II metabolism, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha metabolism, Protein Engineering methods, Receptors, Tumor Necrosis Factor, Type I agonists, Receptors, Tumor Necrosis Factor, Type II agonists, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Tumor necrosis factor (TNF) plays important roles in host defense and in preventing tumor formation by acting via its receptors, TNFR1 and TNFR2, functions of which are less understood. To this end, we have been isolating TNF receptor-selective mutants using phage display technique. However, generation of a phage library with large repertoire (>10(8)) is impeded by the limited transformation efficiency of Escherichia coli. Therefore, it is currently difficult to create a mutant library containing amino acid substitutions in more than seven residues. To overcome this problem, here we have used two different TNF mutant libraries, each containing random substitutions at six selected amino acid residues, and utilized a gene shuffling method to construct a randomized mutant library containing substitutions at 12 different amino acid residues of TNF. Consequently, using this library, we identified TNF mutants with greater receptor-selectivity and enhanced receptor-specific bioactivity than the existing mutants.
- Published
- 2009
- Full Text
- View/download PDF
48. Endothelin promotes neurite elongation by a mechanism dependent on c-Jun N-terminal kinase.
- Author
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Uesugi N, Muramatsu R, and Yamashita T
- Subjects
- Animals, Cells, Cultured, Cerebral Cortex cytology, Cerebral Cortex physiology, Endothelins pharmacology, JNK Mitogen-Activated Protein Kinases antagonists & inhibitors, Neurons drug effects, Rats, Receptor, Endothelin A physiology, Endothelins physiology, JNK Mitogen-Activated Protein Kinases metabolism, Neurites physiology, Neurons physiology
- Abstract
Endothelin (ET), which is known as a vasoconstrictive peptide, is associated with a lot of biological functions. Although endothelin receptors are expressed in the central nervous system (CNS), little is known about the effects of endothelin on neuronal function. In this study, we reported that endothelins elongate cortical neurites via the endothelin A receptor. All the endothelin isoforms tested, endothelin-1, endothelin-2, and endothelin-3, promoted neurite elongation. ET-1-induced neurite elongation was specifically inhibited by treatment with BQ123, an antagonist for the endothelin A receptor. In addition, inhibition of ET-1-induced c-Jun N-terminal kinase (JNK) activation by treatment with SP600125, a JNK inhibitor, also prevented the ET-1-mediated promotion of neurite elongation. Thus, endothelin induces cortical neurite elongation through the endothelin A receptor by a mechanism dependent on JNK.
- Published
- 2009
- Full Text
- View/download PDF
49. Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury.
- Author
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Liu X, Hashimoto M, Horii H, Yamaguchi A, Naito K, and Yamashita T
- Subjects
- Animals, Cerebral Cortex cytology, Cerebral Cortex physiology, Membrane Proteins biosynthesis, Nerve Tissue Proteins antagonists & inhibitors, Nerve Tissue Proteins genetics, Neurites metabolism, Neurons metabolism, Neurons physiology, Rats, Rats, Wistar, Spinal Cord Injuries metabolism, Nerve Regeneration, Nerve Tissue Proteins physiology, Neurites physiology, Spinal Cord Injuries physiopathology
- Abstract
Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.
- Published
- 2009
- Full Text
- View/download PDF
50. Targeted deletion of the tybe IIb Na(+)-dependent Pi-co-transporter, NaPi-IIb, results in early embryonic lethality.
- Author
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Shibasaki Y, Etoh N, Hayasaka M, Takahashi MO, Kakitani M, Yamashita T, Tomizuka K, and Hanaoka K
- Subjects
- Animals, Embryonic Development genetics, Female, Gene Deletion, Gene Expression, Mice, Mice, Mutant Strains, Sodium-Phosphate Cotransporter Proteins, Type IIb genetics, Embryo Loss genetics, Sodium-Phosphate Cotransporter Proteins, Type IIb physiology
- Abstract
NaPi-IIb encodes a Na(+)-dependent Pi co-transporter, which is expressed in various adult tissues and mediates transport of extracellular Pi ions coupling with Na(+) ion. To define the role of NaPi-IIbin vivo, NaPi-IIb gene deficient mice were generated utilizing targeted mutagenesis, yielding viable, heterozygous NaPi-IIb mice. In contrast, homozygous NaPi-IIb mice died in utero soon after implantation, indicating that NaPi-IIb was essential for early embryonic development. In situ hybridization revealed NaPi-IIb mRNA expression in the parietal endoderm, followed by the visceral endoderm, at a time point prior to establishment of a functioning chorio-allantoic placenta. At the time point of functional placenta development, the main site of NaPi-IIb production resided in the labyrinthine zone, where embryonic and maternal circulations were in closest contact. Expression patterns of NaPi-IIb suggest that NaPi-IIb plays an important role in Pi absorption from maternal circulation.
- Published
- 2009
- Full Text
- View/download PDF
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