41 results on '"Harada, Y."'
Search Results
2. Single-Molecule Analysis of the Actomyosin Motor Using Nano-Manipulation
- Author
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Ishijima, A., primary, Harada, Y., additional, Kojima, H., additional, Funatsu, T., additional, Higuchi, H., additional, and Yanagida, T., additional
- Published
- 1994
- Full Text
- View/download PDF
3. Force-Generating Domain of Myosin Motor
- Author
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Itakura, S., primary, Yamakawa, H., additional, Toyoshima, Y.Y., additional, Ishijima, A., additional, Kojima, T., additional, Harada, Y., additional, Yanagida, T., additional, Wakabayashi, T., additional, and Sutoh, K., additional
- Published
- 1993
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4. Identification and biological activity of dihydro-leukotriene B4: A prominent metabolite of leukotriene B4 in the human lung
- Author
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Kumlin, M., primary, Falck, J.R., additional, Raud, J., additional, Harada, Y., additional, Dahlén, S.-E., additional, and Granström, E., additional
- Published
- 1990
- Full Text
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5. Identification and biological activity of dihydro-leukotriene B4: A prominent metabolite of leukotriene B4in the human lung
- Author
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Kumlin, M., Falck, J.R., Raud, J., Harada, Y., Dahlén, S.-E., and Granström, E.
- Abstract
Exogenous [3H]leukotriene B4(LTB4) was converted into several polar and non-polar metabolites in the chopped human lung. One of the major metabolites was identified as 5(S),12-dihydroxy-6,8,14-eicosatrienoic acid (10,11-dihydro-LTB4) by means of co-chromatography with authentic standards, ultraviolet spectrometry and gas chromatography-mass spectrometry. Analysis on chiral straight phase HPLC revealed the presence of both the 12(S) and 12(R) epimers of dihydro-LTB4. Dihydro-LTB4was also formed from endogenously generated LTB4in ionophore A23187 stimulated incubations. The dihydro metabolites were approximately 100 times less potent than LTB4in causing guinea pig lung strip contraction and leukocyte-dependent inflammation in the hamster cheek pouch in vivo.
- Published
- 1990
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6. Epidermal loss of Bcl6 exacerbates MC903-induced atopic dermatitis-like skin inflammation.
- Author
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Kanemaru K, Nagasawa K, Kunugi Y, Tanaka A, Ikeoku S, Tai Y, Harada Y, and Nakamura Y
- Subjects
- Mice, Animals, Epidermis metabolism, Skin metabolism, Keratinocytes metabolism, Cytokines metabolism, Inflammation pathology, Proto-Oncogene Proteins c-bcl-6 metabolism, Dermatitis, Atopic, Calcitriol analogs & derivatives
- Abstract
Atopic dermatitis (AD) is a chronic inflammatory skin disease where Th2-type immune responses are dominant. In the lesional skin of AD, keratinocytes show differentiation defects and secrete proinflammatory cytokines and chemokines, amplifying Th2-type responses in AD. We previously reported that inducible loss of B-cell lymphoma 6 (Bcl6), a transcription repressor and a master transcriptional regulator of follicular helper T cells and germinal center B cells, in the whole body results in upregulation of Th2-related cytokines in mouse skin. However, the role of Bcl6 in keratinocytes remains to be clarified. Here, we observed that BCL6 positively regulates the expression of keratinocyte differentiation markers and plasma membrane localization of adherence junctional proteins in keratinocyte cell culture. Although keratinocyte-specific loss of Bcl6 alone did not induce AD-like skin inflammation, it aggravates MC903-induced AD-like skin inflammation in mice. In addition, Bcl6 expression is decreased in the epidermis of lesional skin from MC903-induced AD-like skin inflammation in mice. These results strongly suggest that Bcl6 downregulation in keratinocytes contributes to the development and aggravation of AD-like skin inflammation in mice., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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7. High expression of PKCλ and ALDH1A3 indicates a poor prognosis, and PKCλ is required for the asymmetric cell division of ALDH1A3-positive cancer stem cells in PDAC.
- Author
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Kasai T, Tamori S, Takasaki Y, Matsuoka I, Ozaki A, Matsuda C, Harada Y, Sasaki K, Ohno S, and Akimoto K
- Subjects
- Humans, Asymmetric Cell Division, Cell Line, Tumor, Aldehyde Dehydrogenase 1 Family metabolism, Neoplastic Stem Cells pathology, Pancreatic Neoplasms, Pancreatic Neoplasms pathology, Carcinoma, Pancreatic Ductal pathology
- Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the cancer with the poorest prognosis. One of the major properties reflecting its poor prognosis is high-grade heterogeneity, which leads to insensitivity to anticancer treatments. Cancer stem cells (CSCs) acquire phenotypic heterogeneity, generating abnormally differentiated cells by asymmetric cell division. However, the detailed mechanism leading to phenotypic heterogeneity is largely unknown. Here, we showed that PDAC patients with co-upregulation of PKCλ and ALDH1A3 had the poorest clinical outcome. PKCλ knockdown by DsiRNA in the ALDH1
high population of PDAC MIA-PaCa-2 cells attenuated the asymmetric distribution of the ALDH1A3 protein. To monitor asymmetric cell division of ALDH1A3-positive PDAC CSCs, we established stable Panc-1 PDAC clones expressing ALDH1A3-turboGFP (Panc-1-ALDH1A3-turboGFP cells). In addition to MIA-PaCa-2-ALDH1high cells, turboGFPhigh cells sorted from Panc-1-ALDH1A3-turboGFP cells showed asymmetric cell propagation of ALDH1A3 protein. PKCλ DsiRNA in Panc-1-ALDH1A3-turboGFP cells also attenuated the asymmetric distribution of ALDH1A3 protein. These results suggest that PKCλ regulates the asymmetric cell division of ALDH1A3-positive PDAC CSCs. Furthermore, Panc-1-ALDH1A3-turboGFP cells can be useful for the visualization and monitoring of CSC properties such as asymmetric cell division of ALDH1A3-positive PDAC CSCs in time-lapse imaging., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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8. Lipid droplet accumulation and adipophilin expression in follicular thyroid carcinoma.
- Author
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Hayakawa M, Taylor JN, Nakao R, Mochizuki K, Sawai Y, Hashimoto K, Tabata K, Kumamoto Y, Fujita K, Konishi E, Hirano S, Tanaka H, Komatsuzaki T, and Harada Y
- Subjects
- Humans, Lipid Droplets metabolism, Perilipin-2, Phosphatidylinositol 3-Kinase metabolism, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism, TOR Serine-Threonine Kinases metabolism, Adenocarcinoma, Follicular metabolism, Adenocarcinoma, Follicular pathology, Thyroid Neoplasms pathology
- Abstract
Follicular neoplasms of the thyroid include follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA). However, the differences in cytological findings between FTC and FTA remain undetermined. Here, we aimed to evaluate the accumulation of lipid droplets (LDs) and the expression of adipophilin (perilipin 2/ADRP/ADFP), a known LD marker, in cultured FTC cells. We also immunohistochemically compared adipophilin expression in the FTC and FTA of resected human thyroid tissues. Cultured FTC (FTC-133 and RO82W-1) possessed increased populations of LDs compared to thyroid follicular epithelial (Nthy-ori 3-1) cells. In vitro treatment with phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling inhibitors (LY294002, MK2206, and rapamycin) in FTC-133 cells downregulated the PI3K/Akt/mTOR/sterol regulatory element-binding protein 1 (SREBP1) signaling pathway, resulting in a significant reduction in LD accumulation. SREBP1 is a master transcription factor that controls lipid metabolism. Fluorescence immunocytochemistry revealed adipophilin expression in the LDs of FTC-133 cells. Immunohistochemical analysis of surgically resected human thyroid tissues revealed significantly increased expression of adipophilin in FTC compared with FTA and adjacent non-tumorous thyroid epithelia. Taken together, LDs and adipophilin were abundant in cultured FTC; the evaluation of adipophilin expression can help distinguish FTC from FTA in surgical specimens., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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9. N-glycan branching enzymes involved in cancer, Alzheimer's disease and COPD and future perspectives.
- Author
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Taniguchi N, Ohkawa Y, Maeda K, Kanto N, Johnson EL, and Harada Y
- Subjects
- Humans, N-Acetylglucosaminyltransferases genetics, Polysaccharides, Alzheimer Disease, 1,4-alpha-Glucan Branching Enzyme, Neoplasms, Pulmonary Disease, Chronic Obstructive
- Abstract
Over the past 3 decades, our group has been involved in studies related to the biosynthesis of N-glycan branching and related glycosyltransferases and have purified most of these Golgi-derived enzymes to homogeneity using classical purification methods and cloned the cDNA of GnT-III, IV, V, VI and Fut8 except GnT-IX(Vb) which was obtained by homology cloning. Based primarily on our data, we briefly summarize the significance of three major enzymes and discuss perspectives for future studies on the occasion of Ernesto's 90th birthday celebration., Competing Interests: Declaration of competing interest The authors declare that we have no financial interests/personal relationships which may be considered as potential competing interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
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10. Oligomerization of Ca 2+ /calmodulin-dependent protein kinase kinase.
- Author
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Fukumoto Y, Harada Y, Ohtsuka S, Kanayama N, Magari M, Hatano N, Sakagami H, and Tokumitsu H
- Subjects
- Animals, Binding Sites, COS Cells, Calcium-Calmodulin-Dependent Protein Kinase Kinase genetics, Calcium-Calmodulin-Dependent Protein Kinase Kinase metabolism, Calcium-Calmodulin-Dependent Protein Kinase Type 1 genetics, Calcium-Calmodulin-Dependent Protein Kinase Type 1 metabolism, Chlorocebus aethiops, Cloning, Molecular, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Gene Expression Regulation, Genetic Vectors chemistry, Genetic Vectors metabolism, Glutathione Transferase genetics, Glutathione Transferase metabolism, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Phosphorylation, Protein Binding, Protein Multimerization, Rats, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Signal Transduction, Calcium-Calmodulin-Dependent Protein Kinase Kinase chemistry, Calcium-Calmodulin-Dependent Protein Kinase Type 1 chemistry, Glutathione Transferase chemistry, Recombinant Fusion Proteins chemistry
- Abstract
Ca
2+ /calmodulin-dependent protein kinase kinases (CaMKKα and β) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His-CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG-CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST-CaMKKα/His-CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca2+ /CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG-CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2022
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11. An improved macromolecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under stressed conditions.
- Author
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Miyagi T, Yamanaka Y, Harada Y, Narumi S, Hayamizu Y, Kuroda M, and Kanekura K
- Abstract
Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes in their conformation and biological functions. Disturbed LLPS homeostasis in MLOs is thought to associate with fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes in the degree of crowding in MLOs. However, it has not been investigated well due to the lack of an appropriate method. To address this, we developed a genetically encoded macromolecular crowding sensor CRONOS (crowding sensor with mNeonGreen and mScarlet-I) that senses the degree of macromolecular crowding in MLOs using a fluorescence resonance energy transfer (FRET) system. CRONOS is a bright biosensor with a wide dynamic range and successfully detects changes in the macromolecular volume fraction in solution. By fusing to the scaffold protein of each MLO, we delivered CRONOS to MLO of interest and detected previously undescribed differences in the degree of crowding in each MLO. CRONOS also detected changes in the degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for the determination of molecular crowding and detection of pathological changes in MLOs in live cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
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12. Spontaneous antibody production caused by regulatory T cell deficiency occurs through a germinal center-independent pathway.
- Author
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Tai Y, Takano A, Haga K, Koshida K, and Harada Y
- Subjects
- Animals, Forkhead Transcription Factors genetics, Forkhead Transcription Factors immunology, Germinal Center immunology, Immunoglobulin E immunology, Immunoglobulin G immunology, Mice, Inbred C57BL, Mice, Knockout, Antibody Formation, T-Lymphocytes, Regulatory immunology
- Abstract
Foxp3
+ regulatory T cells (Tregs) are essential for the prevention of autoantibody and allergen-specific IgE production. Treg deficiency causes an elevation of the serum levels of these pathogenic antibodies, accompanied by spontaneous germinal center (GC) formation. However, it remains to be determined whether excessive and pathogenic antibody production induced by Treg deficiency requires a GC response. Here, we demonstrate that spontaneous antibody production observed in Foxp3 conditional-knockout mice did not need GC formation. Foxp3 and Bcl6 conditional-double knockout mice exhibited spontaneous elevations of IgG1, IgG2c, and IgE levels even though they showed impaired production of IgG1 and IgE specific for the immunized antigen. Furthermore, the IgG1 and IgE antibodies specific for auto- and food-antigens were produced independently of GCs. These data suggested that a GC response was unnecessary for pathogenic antibody production caused by Treg deficiency., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
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13. Dysregulation of humoral immunity in Foxp3 conditional-knockout mice.
- Author
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Tai Y, Sakamoto K, Takano A, Haga K, and Harada Y
- Subjects
- Animals, Antibody Formation immunology, B-Lymphocytes immunology, Forkhead Transcription Factors metabolism, Gene Deletion, Germinal Center immunology, Lymphocyte Activation immunology, Mice, Knockout, T-Lymphocytes, Helper-Inducer immunology, Forkhead Transcription Factors deficiency, Immunity, Humoral
- Abstract
Foxp3
+ regulatory T cells (Tregs) are crucial for maintaining tolerance to self-antigens and preventing autoimmune diseases. Loss of Foxp3 expression leads to autoimmunity and disrupts humoral immune responses, including hyperproduction of immunoglobulin E (IgE). Elucidation of how Tregs control antibody production can lead to the development of new therapies for autoimmune and allergic diseases. However, premature death of Foxp3-deficient mice makes it difficult to analyze the roles of Tregs in humoral immunity of adult mice. In this study, we developed Foxp3 conditional-knockout mice (Foxp3flox R26CreERT2 ) in which the Foxp3 gene was inducibly deleted by tamoxifen administration. After oral administration of tamoxifen, titers of immunoglobulins, particularly IgG2c and IgE, were increased in Foxp3flox R26CreERT2 mice compared with that in controls. Under these conditions, CD4+ T cells from Foxp3flox R26CreERT2 mice had increased expression of several activation markers, including inducible costimulator and CD40 ligand, as well as the cytokines interleukin 4 and interferon gamma. In addition, the proportions of T follicular helper (Tfh) cells and germinal center (GC) B cells were increased in Foxp3flox R26CreERT2 mice compared with those in controls. These results indicated that Tregs controlled excessive or pathogenic antibody production by suppressing Tfh cell differentiation and GC formation. Furthermore, these data suggested that Foxp3flox R26CreERT2 mice could be a useful tool for screening therapeutic agents., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2019
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14. Circular IRE-type RNAs of the NR5A1 gene are formed in adrenocortical cells.
- Author
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Ohe K, Tanaka T, Horita Y, Harada Y, Yamasaki T, Abe I, Tanabe M, Nomiyama T, Kobayashi K, Enjoji M, and Yanase T
- Subjects
- Adrenal Cortex cytology, Adrenal Cortex Neoplasms genetics, Adrenal Cortex Neoplasms metabolism, Adrenal Cortex Neoplasms pathology, Base Sequence, Binding Sites genetics, Cell Line, Tumor, Endoplasmic Reticulum Stress, Endoribonucleases antagonists & inhibitors, Endoribonucleases metabolism, Exons, Gene Expression, Humans, Models, Biological, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, RNA Splicing, RNA, Messenger genetics, RNA, Messenger metabolism, Sulfonamides pharmacology, Thiophenes pharmacology, Adrenal Cortex metabolism, Endoribonucleases genetics, Protein Serine-Threonine Kinases genetics, RNA, Circular biosynthesis, RNA, Circular genetics, Steroidogenic Factor 1 genetics
- Abstract
The recently discovered circular RNAs (circRNAs) are mostly formed by back-splicing where the downstream 5' splice site splices to the upstream 3' splice site by conventional pre-mRNA splicing. These circRNAs regulate gene expression by acting as sponges for micro-RNAs or RNA-binding proteins. Here we show that the NR5A1 (previously called Ad4BP or SF-1) gene which is exclusively expressed in the adrenal cortex and steroidogenic tissue can form atypical circRNAs by unconventional splicing. Two stem loops with inositol-requiring protein-1α (IRE1α) cleavage sites are connected by an IRE1α cleavage site to form a circRNA (circIRE RNA). From total RNA of normal human adrenal cortex, we detected a circIRE RNA with connected ends by IRE1α cleavage sites in exon 6 and exon 1 (circIRE NR5A1 ex6-1 RNA). circIRE NR5A1 ex6-1 RNA was not detected in the adrenocortical cancer cell line, H295R. When IRE1α was expressed in H295R cells a different circIRE NR5A1 RNA connecting IRE1-cleavage sites in exon 7 and exon 1 was detected (circIRE NR5A1 ex7-1 RNA). The expression of this circIRE RNA was inhibited by the IRE1 inhibitor 1, STF-083010, implicating that it was formed via the ER stress pathway, where IRE1α is a major factor. This is the first report of this type of circular RNA connected by IRE1-cleavage sites found to be expressed in mammalian cells in a tissue-specific manner. To our surprise, the concomitant expression of NR5A1 was increased by IRE1α implicating that NR5A1 was not subjected to IRE1-dependent decay of mRNA (RIDD) but rather activating a transcriptional regulatory network to cope with ER stress in steroidogenic tissue reminiscent to XBP1 in other tissue. We believe this is the first report of such tissue-specific transcriptional cascade responding to ER stress as well as the novel finding of circular RNAs connected by IRE1α cleavage sites expressed in mammalian tissue., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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15. Three populations of adult Leydig cells in mouse testes revealed by a novel mouse HSD3B1-specific rat monoclonal antibody.
- Author
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Yokoyama C, Chigi Y, Baba T, Ohshitanai A, Harada Y, Takahashi F, and Morohashi KI
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Cell Lineage, Fluorescent Antibody Technique, Male, Mice, Protein Isoforms analysis, Rats, Testis embryology, Leydig Cells cytology, Multienzyme Complexes analysis, Progesterone Reductase analysis, Steroid Isomerases analysis, Testis cytology
- Abstract
Leydig cells play a pivotal function in the synthesis of a male sex steroid, testosterone. The ability of the steroid production is dependent on the expression of the steroidogenic genes, such as HSD3B (3β-hydroxysteroid dehydrogenase/Δ5- Δ4 isomerase). It has been established that two different types of Leydig cells, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs), are developed in mammalian testes. FLCs and ALCs are characterized by different sets of marker gene expression. In the case of mouse Leydig cells, Hsd3b1 (Hsd3b type 1) is expressed both in FLCs and ALCs whereas Hsd3b6 (Hsd3b type 6) is expressed in ALCs but not in FLCs. However, because the antibodies established so far for HSD3B were unable to distinguish between the HSD3B1 and HSD3B6 isoforms, it remained unclear whether both of them are expressed in every ALC. Therefore, in the present study, we generated a rat monoclonal antibody specific for mouse HSD3B1. Intriguingly, this monoclonal antibody together with an antibody specific for HSD3B6 identified three populations of ALCs based on the expression levels of these HSD3Bs., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
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16. Natural antibody against neuroblastoma of TH-MYCN transgenic mice does not correlate with spontaneous regression.
- Author
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Kawakubo N, Harada Y, Ishii M, Souzaki R, Kinoshita Y, Tajiri T, Taguchi T, and Yonemitsu Y
- Subjects
- Animals, Antibodies chemistry, Biological Products chemistry, Humans, Mice, Mice, Transgenic, N-Myc Proto-Oncogene Protein genetics, Neuroblastoma pathology, Tumor Cells, Cultured, Antibodies immunology, Biological Products immunology, N-Myc Proto-Oncogene Protein immunology, Neuroblastoma immunology
- Abstract
The mechanism underlying the spontaneous regression of neuroblastoma is unclear. Although it was hypothesized that this regression occurs via an immunological mechanism, there is no clinical evidence, and no animal models have been developed to investigate the involvement of immune systems, especially natural antibodies, against neuroblastoma. We performed an immunological analysis of homo- and heterozygous TH-MYCN transgenic mice as a model of aggressive neuroblastoma. Mice with no or small (<5 mm) tumors showed higher antibody titers in plasma than mice with large (>5 mm) tumors. A significant negative correlation was observed between the tumor diameter and the titer of antitumor antibody. This antibody had complement-dependent cytotoxicity but not antibody-dependent cellular cytotoxicity against neuroblastoma cells. Moreover, B-cell depletion had no effect on the tumor incidence in vivo. We revealed that TH-MYCN transgenic mice have a natural antibody against neuroblastoma that correlate with tumor size. However, this antibody does not correlate with the spontaneous regression of neuroblastoma. Thus, the function of the natural antibody is limited., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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17. A novel mouse model for tracking the fate of CXCR5-expressing T cells.
- Author
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Takebe T, Sakamoto K, Higami Y, and Harada Y
- Subjects
- Adoptive Transfer, Animals, B-Lymphocytes immunology, Cell Differentiation, Cell Movement immunology, Germinal Center cytology, Germinal Center immunology, Immunity, Humoral, Immunologic Memory, Mice, Mice, Inbred C57BL, Mice, Transgenic, Models, Animal, Models, Immunological, Ovalbumin immunology, Receptors, CXCR5 genetics, T-Lymphocytes, Helper-Inducer cytology, T-Lymphocytes, Helper-Inducer physiology, Receptors, CXCR5 metabolism, T-Lymphocytes, Helper-Inducer immunology
- Abstract
The germinal center (GC) reaction, a critical process in the humoral immune response, requires follicular helper T (Tfh) cells. Tfh cells express the master transcription factor Bcl6 and chemokine receptor CXCR5, which enable them to migrate from the T cell zone to B cell follicles and interact with GC B cells. However, CXCR5 is downregulated when Tfh cells become memory cells. Therefore, it is difficult to track Tfh cells continuously in vivo. In this study, we generated a mouse strain, Cxcr5
CreERT2 R26Tomato , in which the fluorescent protein tdTomato is inducibly expressed in CXCR5+ cells by tamoxifen administration. After the oral administration of tamoxifen, most Tfh cells in Peyer's patches (PP) from Cxcr5CreERT2 R26Tomato mice were tdTomato+ . To track antigen-specific Tfh cells in vivo, OVA-specific OT-II T cells derived from Cxcr5CreERT2 R26Tomato mice were transferred to wild-type mice, and the recipient mice were immunized with OVA followed by tamoxifen administration. CXCR5+ T cells became tdTomato+ and were mainly located in B cell follicles and GC areas 8 days after immunization. Four weeks after immunization, tdTomato+ OT-II T cells migrated from B cell follicles to the T-B border area and T cell zone after CXCR5 downregulation and CCR7 upregulation. These results indicate that Cxcr5CreERT2 R26Tomato mice are a useful tool for studying the cell fate of differentiated Tfh cells in vivo and therefore have implications for the development of therapeutic strategies for infectious and autoimmune diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2018
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18. Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides.
- Author
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Suzuki T and Harada Y
- Subjects
- Animals, Biological Transport, Active, Cytosol metabolism, Endoplasmic Reticulum metabolism, Glycosylation, Humans, Models, Biological, Oligosaccharides chemistry, Polysaccharides chemistry, Protein Folding, Pyrophosphatases metabolism, Dolichols metabolism, Oligosaccharides metabolism, Polysaccharides metabolism
- Abstract
There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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19. Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers.
- Author
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Harada Y, Garenáux E, Nagatsuka T, Uzawa H, Nishida Y, Sato C, and Kitajima K
- Subjects
- Animals, Binding Sites, Carbohydrate Sequence, Dermatan Sulfate metabolism, Glycolipids chemistry, Glycosaminoglycans chemistry, HSP70 Heat-Shock Proteins chemistry, Heparin metabolism, Heparitin Sulfate metabolism, Mice, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Binding, Protein Structure, Tertiary, Glycolipids metabolism, Glycosaminoglycans metabolism, HSP70 Heat-Shock Proteins metabolism
- Abstract
Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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20. Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament.
- Author
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Harada Y, Furukawa K, Asari T, Chin S, Ono A, Tanaka T, Mizukami H, Murakami M, Yagihashi S, Motomura S, and Ishibashi Y
- Subjects
- Adipogenesis genetics, Aged, Cell Differentiation genetics, Cell Separation, Chondrogenesis genetics, Clone Cells, Female, Flow Cytometry, Humans, Male, Mesenchymal Stem Cells metabolism, Middle Aged, Ossification, Heterotopic metabolism, Cell Lineage genetics, Longitudinal Ligaments metabolism, Longitudinal Ligaments pathology, Mesenchymal Stem Cells pathology, Ossification, Heterotopic pathology, Osteogenesis genetics
- Abstract
Ectopic bone formation is thought to be responsible for ossification of the posterior longitudinal ligament of the spine (OPLL). Mesenchymal stem cells (MSCs) were isolated from spinal ligaments and shown to play a key role in the process of ectopic ossification. The purpose of this study was to explore the capacity of these MSCs to undergo lineage commitment and to assess the gene expression changes between these committed and uncommitted MSCs between OPLL and non-OPLL patients. Spinal ligament-derived cells were obtained from OPLL patients or patients with cervical spondylotic myelopathy (non-ossified) for comparison (n=8 in each group). MSCs from the two patient cohorts were evaluated for changes in colony forming ability; osteogenic, adipogenic and chondrogenic differentiation potential; and changes in gene expression following induction with lineage-specific conditions. We show that the osteogenic differentiation potential was significantly higher in MSCs from OPLL patients than in those from non-OPLL patients. In addition, alkaline phosphatase activity and several osteogenic-related genes expressions (bone morphogenetic protein 2, runt-related transcription factor 2 and alkaline phosphatase) were significantly higher in the OPLL group than in the non-OPLL group. However, single cell cloning efficiency, adipogenic and chondrogenic differentiation, and the expression of adipogenic and chondrogenic-related genes were equivalent between MSCs harvested from OPLL and non-OPLL patient samples. These findings suggest an increase in the osteogenic differentiation potential of MSCs from OPLL patients and that this propensity toward the osteogenic lineage may be a causal factor in the ossification in these ligaments., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2014
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21. Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells.
- Author
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Emi Y, Harada Y, and Sakaguchi M
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Cytoplasm metabolism, Dogs, Hep G2 Cells, Humans, Isoleucine metabolism, Madin Darby Canine Kidney Cells, Molecular Sequence Data, Multidrug Resistance-Associated Protein 2, Mutant Proteins chemistry, Mutant Proteins metabolism, Protein Sorting Signals, Protein Structure, Tertiary, Protein Transport, Structure-Activity Relationship, Subcellular Fractions metabolism, Cell Membrane metabolism, Cell Polarity, Epithelial Cells cytology, Epithelial Cells metabolism, Leucine metabolism, Multidrug Resistance-Associated Proteins chemistry, Multidrug Resistance-Associated Proteins metabolism
- Abstract
Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E(295)EVEALI(301), which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L(300) and I(301) residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E(295), E(296), and E(298) residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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22. Immunohistochemical localization of mesenchymal stem cells in ossified human spinal ligaments.
- Author
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Chin S, Furukawa K, Ono A, Asari T, Harada Y, Wada K, Tanaka T, Inaba W, Mizukami H, Motomura S, Yagihashi S, and Ishibashi Y
- Subjects
- Collagen metabolism, Humans, Immunohistochemistry, Ligaments metabolism, Mesenchymal Stem Cells cytology, Ossification, Heterotopic, Spine metabolism
- Abstract
Mesenchymal stem cells (MSCs) have been isolated from various tissues and used for elucidating the pathogenesis of numerous diseases. In our previous in vitro study, we showed the existence of MSCs in human spinal ligaments and hypothesized that these MSCs contributed to the pathogenesis of ossification of spinal ligaments. The purpose of this study was to use immunohistochemical techniques to analyze the localization of MSCs in ossified human spinal ligaments in situ. Ossified (OLF) or non-ossified ligamentum flavum (non-OLF) samples from the thoracic vertebra were obtained from patients who had undergone posterior spinal surgery. Serial sections were prepared from paraffin-embedded samples, and double immunofluorescence staining was performed using antibodies against markers for MSCs (CD73, CD90 and CD105), endothelial cells (CD31), pericytes (α-smooth muscle actin), and chondrocytes (S100). Immunolocalization of MSCs was observed in the perivascular area and collagenous matrix in spinal ligaments. Markers for MSCs and pericytes were co-expressed in the perivascular area. Compared with non-OLF, OLF had a large amount of neovascularization in the fragmented ligament matrix, and a high accumulation of MSCs around blood vessels. The prevalence of MSCs in OLF within collagenous matrix was significantly higher than that in non-OLF. Chondrocytes near the ossification front in OLF also presented expression of MSC markers. MSCs may contribute to the ectopic ossification process of OLF through endochondral ossification., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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23. PLCε cooperates with the NF-κB pathway to augment TNFα-stimulated CCL2/MCP1 expression in human keratinocyte.
- Author
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Harada Y, Edamatsu H, and Kataoka T
- Subjects
- Animals, Cell Line, Gene Knockdown Techniques, Humans, Keratinocytes drug effects, Mice, Phosphoinositide Phospholipase C genetics, Tumor Necrosis Factor-alpha pharmacology, Chemokine CCL2 biosynthesis, Keratinocytes metabolism, NF-kappa B metabolism, Phosphoinositide Phospholipase C metabolism
- Abstract
Phospholipase Cε (PLCε) is a unique class of PLC regulated by both Ras family small GTPases and heterotrimeric G proteins. We previously showed by using mice bearing its null or transgenic allele that PLCε plays a crucial role in various forms of skin inflammation through upregulation of proinflammatory cytokine production from keratinocytes. However, molecular mechanisms how PLCε augments cytokine production were largely unknown. We show here using cultured human keratinocyte PHK16-0b cells that induction of the expression of chemokine (C-C motif) ligand 2 (CCL2) following stimulation with tumor necrosis factor (TNF)α, which primarily depends on the activation of the NF-κB pathway, is abrogated by small interfering RNA-mediated knockdown of PLCε. Enforced expression of PLCε causes substantial CCL2 expression and cooperates with low level TNFα stimulation to induce marked overexpression of CCL2, both of which are only partially blocked by pharmacological inhibition of the NF-κB signaling. However, PLCε knockdown exhibits no effect on both the NF-κB-cis-element-mediated transcription per se and the post-translational modifications of NF-κB implicated in transcriptional regulation, suggesting that PLCε constitutes a yet unknown signaling pathway distinct from the NF-κB pathway. This pathway can cooperate with the NF-κB pathway to achieve a synergistic TNFα-stimulated CCL2 induction in keratinocytes., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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24. Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma.
- Author
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Yamaguchi K, Feril LB Jr, Tachibana K, Takahashi A, Matsuo M, Endo H, Harada Y, and Nakayama J
- Subjects
- Animals, Cell Line, Tumor, Female, Humans, Interferon-beta metabolism, Mice, Mice, Inbred BALB C, Mice, Nude, Ultrasonics, Genetic Therapy methods, Interferon-beta genetics, Melanoma therapy, Skin Neoplasms genetics, Transfection methods
- Abstract
We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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25. Innate immune system still works at diapause, a physiological state of dormancy in insects.
- Author
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Nakamura A, Miyado K, Takezawa Y, Ohnami N, Sato M, Ono C, Harada Y, Yoshida K, Kawano N, Kanai S, Miyado M, and Umezawa A
- Subjects
- Animals, Microscopy, Electron, Scanning, Moths immunology, Moths ultrastructure, Phagocytosis, Pupa immunology, Pupa physiology, Pupa ultrastructure, Adaptation, Physiological immunology, Body Temperature Regulation immunology, Immunity, Innate, Moths physiology
- Abstract
Diapause is most often observed in insects and is a physiologically dormant state different from other types of dormancy, such as hibernation. It allows insects to survive in harsh environments or extend longevity. In general, larval, pupal, or adult non-diapausing insects possess an innate immune system preventing the invasion of microorganisms into their bodies; however, it is unclear whether this system works under the dormant condition of diapause. We here report the occurrence of innate cellular reactions during diapause using pupae of a giant silkmoth, Samia cynthia pryeri. Scanning electron microscopic analysis demonstrated the presence of two major types of cells in the body fluid isolated from the thoracic region of a pupa. Phagocytosis and encapsulation, characteristics of innate cellular reactions, by these cells were observed when latex beads as foreign targets were microinjected into the internal portion of a pupa. Such behavior by these cells was still observed even when pupae were continuously chilled at 4°C. Our results indicate that innate cellular reactions can work in diapausing insects in a dormant state., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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26. Vitellogenin C-terminal fragments participate in fertilization as egg-coat binding partners of sperm trypsin-like proteases in the ascidian Halocynthia roretzi.
- Author
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Akasaka M, Harada Y, and Sawada H
- Subjects
- Acrosin genetics, Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA, Complementary genetics, Enzyme Precursors genetics, Female, Male, Molecular Sequence Data, Ovum physiology, Protein Structure, Tertiary, Serine Endopeptidases genetics, Urochordata metabolism, Vitellogenins genetics, Acrosin metabolism, Enzyme Precursors metabolism, Fertilization, Serine Endopeptidases metabolism, Spermatozoa enzymology, Urochordata physiology, Vitellogenins metabolism
- Abstract
Sperm trypsin-like proteases are known to play important roles in fertilization, but their detailed functions are still unknown. We previously explored the binding partners of sperm trypsin-like proteases, HrProacrosin and HrSpermosin, in the ascidian Halocynthia roretzi, and we isolated several candidate proteins on the vitelline coat. We found that some of these proteins are identical to the C-terminal coding region (CT) and von Willebrand factor type D (vWF-D) domain of vitellogenin. We also found that CT on the vitelline coat disappears after fertilization. Vitellogenin is a large lipid transfer protein that is enzymatically processed during vitellogenesis. Although the processed domains including phosvitin and lipovitellin are known to function as yolk nutrient proteins, the roles of the CT and vWF-D domain remain elusive. Our results showed that the CT and vWF-D domain of vitellogenin are processed and attached to the vitelline coat, which in turn participate in fertilization as the binding partners of sperm proteases., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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27. Identification of yeast Art5 as a multicopy suppressor for the mitochondrial translocator maintenance protein Tam41.
- Author
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Harada Y, Tamura Y, and Endo T
- Subjects
- Carrier Proteins genetics, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Mitochondria metabolism, Mitochondrial Precursor Protein Import Complex Proteins, Mitochondrial Proteins genetics, Protein Stability, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Suppression, Genetic, Carrier Proteins metabolism, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Normal mitochondrial protein import requires multiple translocator complexes in the outer and inner mitochondrial membrane. Tam41 is a peripheral inner membrane protein that is involved in the structural maintenance of the inner membrane translocator the TIM23 complex. Here we identified an arrestin-related protein Art5 as a multicopy suppressor for the Tam41-deficient yeast mutant, which exhibited the deteriorated TIM23 complex and temperature-sensitive growth defects. Overexpression of Art5 suppressed growth defects of tam41Delta cells and partially restored the destabilized TIM23 complex structure in tam41Delta mitochondria, so that the defects in mitochondrial protein import via the TIM23 complex were partially recovered. Deletion of the ART5 gene in turn exhibited synthetic growth defects with the TAM41 deletion. Art5 as a functional partner for Tam41 will provide a starting point to reveal the precise function of Tam41 in the maintenance of the TIM23 complex., (2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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28. Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy.
- Author
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Ogawa M, Harada Y, Yamaoka Y, Fujita K, Yaku H, and Takamatsu T
- Subjects
- Animals, Cytochromes b analysis, Cytochromes b metabolism, Cytochromes c analysis, Cytochromes c metabolism, Cytoplasm enzymology, Cytoplasm ultrastructure, Female, Fibrosis, Image Interpretation, Computer-Assisted, Myocardial Infarction enzymology, Myocardial Infarction pathology, Rats, Rats, Wistar, Coronary Vessels enzymology, Coronary Vessels ultrastructure, Microscopy, Confocal methods, Myocytes, Cardiac enzymology, Myocytes, Cardiac ultrastructure, Spectrum Analysis, Raman methods
- Abstract
Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM combined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy-hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results suggest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart.
- Published
- 2009
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29. Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon.
- Author
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Fukunaga R, Harada Y, Hirao I, and Yokoyama S
- Subjects
- Base Pairing, Models, Chemical, Mutation, Nucleic Acid Conformation, Phosphoserine chemistry, Protein Conformation, Pyrimidines chemistry, RNA, Transfer, Amino Acyl genetics, RNA, Transfer, Amino Acyl metabolism, Serine-tRNA Ligase chemistry, Serine-tRNA Ligase genetics, Serine-tRNA Ligase metabolism, Thiophenes chemistry, Aminoacylation, Anticodon chemistry, Phosphoserine metabolism, Protein Biosynthesis, Pyrroles chemistry, RNA, Transfer, Amino Acyl chemistry
- Abstract
An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K(m)=47.1muM and a k(cat)=0.151s(-1), which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA(Cys) with the GCA anticodon (26.9muM and 0.111s(-1)). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.
- Published
- 2008
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30. Influenza A virus non-structural protein 1 (NS1) interacts with cellular multifunctional protein nucleolin during infection.
- Author
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Murayama R, Harada Y, Shibata T, Kuroda K, Hayakawa S, Shimizu K, and Tanaka T
- Subjects
- Cell Line, Tumor, Humans, Nucleolin, Influenza A virus physiology, Influenza, Human metabolism, Influenza, Human virology, Lung Neoplasms metabolism, Lung Neoplasms virology, Phosphoproteins metabolism, RNA-Binding Proteins metabolism, Viral Nonstructural Proteins metabolism
- Abstract
Influenza A virus non-structural protein 1 (NS1) is the most important viral regulatory factor that controls cellular processes to facilitate viral replication. To gain further insight into the role of NS1, we tried to find novel cellular factors that interact with NS1. The complexes of NS1 and target proteins were pulled down from an infected cell lysate using anti-NS1 (A/Udorn/72) single-chain Fv and identified by peptide mass fingerprinting analysis. We identified nucleolin, a multifunctional major nucleolar protein, as a novel NS1-binding protein. The RNA-binding domain of NS1 was responsible for this binding, as judged by a GST (glutathione S-transferase) pull-down assay with the GST-fused functional domains of NS1. By laser confocal microscopy, we observed the co-localization of NS1 with nucleolin most clearly in the nucleoli, indicating that NS1 is interacting with nucleolin during infection. Our results suggest a novel function of NS1, namely, affecting cellular events via interaction with nucleolin.
- Published
- 2007
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31. Retinoic acid-inducible G protein-coupled receptors bind to frizzled receptors and may activate non-canonical Wnt signaling.
- Author
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Harada Y, Yokota C, Habas R, Slusarski DC, and He X
- Subjects
- Animals, Embryonic Development drug effects, Embryonic Development physiology, Protein Binding, Signal Transduction drug effects, Signal Transduction physiology, Frizzled Receptors metabolism, Receptors, G-Protein-Coupled metabolism, Tretinoin pharmacology, Wnt Proteins metabolism, Xenopus laevis embryology, Xenopus laevis metabolism, Zebrafish embryology, Zebrafish metabolism
- Abstract
Frizzled (Fz) seven-pass transmembrane receptors are Wnt receptors and function in a variety of developmental pathways. Here we identify retinoic acid-inducible gene-1, 2, 3, and 4 (RAIG1, 2, 3, and 4) as potential Fz binding proteins. RAIG proteins are seven-pass transmembrane receptors, and Xenopus RAIG2, 3, and 4 are expressed in early gastrula. XRAIG2 can activate small GTPases, such as RhoA, Rac, and Cdc42, and c-jun N-terminal kinase, thus exhibit activities that overlap with non-canonical Wnt/Fz signaling. Injection of XRAIG2 mRNA into Xenopus embryo causes a severe shortened and bent body axis due to defective gastrulation movements, reminiscent of abnormal non-canonical Wnt signaling. XRAIG2 affects convergent extension in activin-treated animal caps, which can be partially rescued by co-injection of a dominant-negative form of Cdc42. In zebrafish embryo, XRAIG2 also causes Ca(2+) flux, one of the consequences of non-canonical Wnt signaling. These results suggest a possible crosstalk/integration between Wnt/Frizzled and RAIG signal transduction pathways.
- Published
- 2007
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32. Complex formation of 70-kDa heat shock protein with acidic glycolipids and phospholipids.
- Author
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Harada Y, Sato C, and Kitajima K
- Subjects
- Animals, Mice, Protein Structure, Tertiary, Recombinant Proteins chemistry, Sulfoglycosphingolipids chemistry, Glycolipids chemistry, HSP70 Heat-Shock Proteins chemistry, Phospholipids chemistry
- Abstract
A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces.
- Published
- 2007
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33. Lanthionine introduction into nukacin ISK-1 prepeptide by co-expression with modification enzyme NukM in Escherichia coli.
- Author
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Nagao J, Harada Y, Shioya K, Aso Y, Zendo T, Nakayama J, and Sonomoto K
- Subjects
- Alanine chemistry, Alanine metabolism, Amino Acid Sequence, Escherichia coli genetics, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Molecular Weight, Peptides, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Alanine analogs & derivatives, Bacteriocins chemistry, Bacteriocins metabolism, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Hydro-Lyases chemistry, Hydro-Lyases metabolism, Protein Engineering methods, Sulfides chemistry, Sulfides metabolism
- Abstract
We demonstrated lanthionine introduction into hexa-histidine-tagged (His-tagged) nukacin ISK-1 prepeptide NukA by modification enzyme NukM in Escherichia coli. Co-expression of nukA and nukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis showed that the prepeptide was converted into a postulated peptide with decrease in mass of 72Da which resulted from dehydration of four amino acids. Characterization of the resultant prepeptide indicated the presence of unusual amino acids, such as dehydrated amino acid, lanthionine or 3-methyllanthionine, in its C-terminal propeptide moiety. The modified prepeptide encompassing the leader peptide attached to the post-translationally modified propeptide moiety was readily obtained by one-step purification. Our findings will thus be a powerful tool for introducing unusual amino acids aimed at peptide engineering and also helpful to provide new insight for further understanding of lanthionine-forming enzymes for lantibiotics.
- Published
- 2005
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34. Suicide gene therapy of human hepatoma and its peritonitis carcinomatosis by a vector of replicative-deficient herpes simplex virus.
- Author
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Iwai M, Harada Y, Ishii M, Kashima K, Mazda O, Tamura M, Wolfe D, Goin WF, and Glorioso JC
- Subjects
- Animals, Cell Division, Defective Viruses genetics, Female, Green Fluorescent Proteins, Humans, Indicators and Reagents metabolism, Kinetics, Liver Neoplasms, Experimental pathology, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mice, Mice, Nude, Mice, SCID, Microscopy, Fluorescence, Neoplasm Transplantation, Survival Rate, Thymidine Kinase genetics, Transfection, Tumor Cells, Cultured, Genetic Therapy methods, Genetic Vectors, Herpesvirus 1, Human genetics, Liver Neoplasms, Experimental therapy
- Abstract
Herpes simplex virus type 1 (HSV-1) deleted for the immediate-early gene was applied for treatment of hepatoma cells of SKHep 1 and Huh-7. Hepatoma cells were cultured in medium containing HSV1 expressing GFP gene (QOZ/HG) to determine its transfection rate, and both cell lines infected by MOI 1 of QOZ/HG were found to have high expression of GFP without cytotoxicity. Subcutaneous growth of SKHep 1 cell tumor in nude mice was significantly reduced by injection of replicative-deficient herpes virus (TOZ.1) containing Tk-gene with administration of GCV, in comparison with that of noninjected tumor. SCID mice of peritonitis carcinomatosis due to Huh-7 hepatoma cells infected with TOZ.1 could survive longer under administration of GCV than those without TOZ.1. Therefore replicative-deficient HSV1 is a useful vector for treatment of human hepatoma cells, and TOZ.1 with GCV may be applied to suicide gene therapy for hepatoma and peritonitis carcinomatosis of hepatoma cells.
- Published
- 2002
- Full Text
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35. Polyethylenimine-mediated suicide gene transfer induces a therapeutic effect for hepatocellular carcinoma in vivo by using an Epstein-Barr virus-based plasmid vector.
- Author
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Iwai M, Harada Y, Tanaka S, Muramatsu A, Mori T, Kashima K, Imanishi J, and Mazda O
- Subjects
- Animals, Ganciclovir administration & dosage, Gene Transfer, Horizontal, Genetic Vectors administration & dosage, Genetic Vectors chemistry, Genetic Vectors metabolism, Herpesvirus 1, Human enzymology, Herpesvirus 1, Human genetics, Humans, Liver Neoplasms, Experimental pathology, Mice, Mice, SCID, Neoplasm Transplantation, Polyethyleneimine administration & dosage, Polyethyleneimine chemistry, Survival Rate, Thymidine Kinase administration & dosage, Thymidine Kinase biosynthesis, Thymidine Kinase genetics, Transfection, Treatment Outcome, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols administration & dosage, Genetic Therapy methods, Herpesvirus 4, Human genetics, Liver Neoplasms, Experimental genetics, Liver Neoplasms, Experimental therapy
- Abstract
The present study aimed to establish a novel efficient nonviral strategy for suicide gene transfer in hepatocellular carcinoma (HCC) in vivo. We employed branched polyethylenimine (PEI) and combined it with Epstein-Barr virus (EBV)-based plasmid vectors. The HCC cells transfected with an EBV-based plasmid carrying the herpes simplex virus-1 thymidine kinase (HSV-1 Tk) gene (pSES.Tk) showed up to 30-fold higher susceptibilities to ganciclovir (GCV) than those transfected with a conventional plasmid vector carrying the HSV-1 Tk gene (pS.Tk). The therapeutic effect in vivo was tested by intratumoral injection of the plasmids into HuH-7 hepatomas transplanted into C.B-17 scid/scid mutant (SCID) mice and subsequent GCV administrations. Treatment with pSES.Tk, but not pS.Tk, markedly suppressed growth of hepatomas in vivo, resulting in a significantly prolonged survival period of the mice. These findings suggest that PEI-mediated gene transfer system can confer efficient expression of the suicide gene in HCC cells in vivo by using EBV-based plasmid vectors., (©2002 Elsevier Science (USA).)
- Published
- 2002
- Full Text
- View/download PDF
36. A hematopoietic-specific transmembrane protein, Art-1, is possibly regulated by AML1.
- Author
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Harada Y, Harada H, Downing JR, and Kimura A
- Subjects
- Amino Acid Sequence, Animals, Cell Differentiation, Cloning, Molecular, Core Binding Factor Alpha 2 Subunit, Enzyme Inhibitors pharmacology, Erythrocytes physiology, Hydroxamic Acids pharmacology, Leukemia, Erythroblastic, Acute, Membrane Proteins physiology, Mice, Molecular Sequence Data, Myeloid Cells metabolism, Oncogene Proteins, Fusion biosynthesis, Oncogene Proteins, Fusion genetics, RNA, Messenger biosynthesis, RUNX1 Translocation Partner 1 Protein, Tetracycline pharmacology, Tissue Distribution, Transcription Factors biosynthesis, Transcription Factors genetics, Tumor Cells, Cultured, DNA-Binding Proteins physiology, Erythrocytes metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Proto-Oncogene Proteins, Transcription Factors physiology
- Abstract
The functions of AML1 in hematopoietic differentiation are repressed by AML1-mutants including the AML1/ETO chimeric protein, which is seen in t(8;21) acute myeloid leukemia. Erythroid progenitors of the patients with t(8;21) AML expressed AML1/ETO. To investigate the effect of AML1/ETO in erythroid cells, we made a tetracycline-regulated AML1/ETO overexpression system in mouse erythroleukemic (MEL) cells. Enforced AML1/ETO repressed the terminal erythroid differentiation. Furthermore, we performed representational difference analysis using this MEL cell system to clone the downstream targets of AML1 in erythroid cell differentiation. We cloned a novel transmembrane protein, Art-1 (AML1-regulated transmembrane protein 1), which is a member of tetramembrane spanning superfamily. Art-1 expression was restricted in hematopoietic cells. It was upregulated by AML1 and downregulated by AML1/ETO in both erythroid and myeloid cells, and increased during erythroid cell differentiation. Art-1 may play an important role in the differentiation of erythroid cells, possibly as a direct downstream target of AML1., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
37. Cyclic AMP inhibits the activity of c-Jun N-terminal kinase (JNKp46) but not JNKp55 and ERK2 in human helper T lymphocytes.
- Author
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Harada Y, Miyatake S, Arai K, and Watanabe S
- Subjects
- Antibodies immunology, Bucladesine pharmacology, Calcimycin pharmacology, Cell Line, Enzyme Activation drug effects, Humans, JNK Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinases chemistry, Mitogen-Activated Protein Kinases immunology, Molecular Weight, Myelin Basic Protein metabolism, Phosphorylation drug effects, Phosphoserine metabolism, Precipitin Tests, Proto-Oncogene Proteins c-jun metabolism, Recombinant Fusion Proteins metabolism, Signal Transduction drug effects, T-Lymphocytes, Helper-Inducer drug effects, T-Lymphocytes, Helper-Inducer metabolism, Tetradecanoylphorbol Acetate pharmacology, Cyclic AMP metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinases metabolism, T-Lymphocytes, Helper-Inducer enzymology
- Abstract
The cyclic AMP (cAMP) elevating agent PGE(2) and dibutyryl cyclic AMP (dBcAMP) affect T cell functions. Using human helper T cell clones, we examined effects of cAMP on c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), which are assumed to play a role in T cell regulation. When we analyzed the effects of dBcAMP on activities of mitogen-activated protein kinase (MAPK) family members ERK2, JNKp55 and JNKp46, dBcAMP did not inhibit the activities of ERK2 and JNKp55 induced by PMA/A23187 stimulation. JNKp46 activity was, however, inhibited by dBcAMP. JNK phosphorylates c-Jun on Ser-63 and Ser-73, the result being induction of its transcriptional activity. We found that dBcAMP inhibited the phosphorylation of c-Jun Ser-63 induced by PMA/A23187 stimulation. We suggest a different mechanism of regulation of JNKp55 and JNKp46 activities and that JNKp46 is a specific c-Jun kinase by which the activity of c-Jun is regulated in T lymphocytes., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
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38. Cloning and sequence analysis of mink growth hormone cDNA.
- Author
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Harada Y, Tatsumi H, Nakano E, and Umezu M
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cattle, Cloning, Molecular, DNA, Recombinant chemistry, Humans, Mink, Molecular Sequence Data, Molecular Weight, Open Reading Frames, Rats, Restriction Mapping, Sequence Homology, Nucleic Acid, Swine, DNA chemistry, Growth Hormone genetics
- Abstract
A cDNA clone for mink growth hormone (GH) was isolated from a mink pituitary cDNA library, employing a part of rat growth hormone cDNA sequence as a probe. According to the nucleotide sequence, mature mink GH consists of 190 amino acids with a calculated molecular weight of 21,720. The amino acid sequence homology between the mature region of mink GH and those of pig GH, rat GH, bovine GH and human GH was 98.4%, 93.7%, 89.0% and 66.7%, respectively.
- Published
- 1990
- Full Text
- View/download PDF
39. Identification and biological activity of dihydroleukotriene B4: a prominent metabolite of leukotriene B4 in the human lung.
- Author
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Kumlin M, Falck JR, Raud J, Harada Y, Dahlén SE, and Granström E
- Subjects
- Animals, Cricetinae, Guinea Pigs, Humans, Inflammation chemically induced, Leukotriene B4 pharmacology, Lung drug effects, Leukotriene B4 isolation & purification, Leukotriene B4 metabolism, Lung metabolism
- Abstract
Exogenous [3H]leukotriene B4 (LTB4) was converted into several polar and non-polar metabolites in the chopped human lung. One of the major metabolites was identified as 5(S),12-dihydroxy-6,8,14-eicosatrienoic acid (10,11-dihydro-LTB4) by means of co-chromatography with authentic standards, ultraviolet spectrometry and gas chromatography-mass spectrometry. Analysis of chiral straight phase HPLC revealed the presence of both the 12(S) and 12(R) epimers of dihydro-LTB4. Dihydro-LTB4 was also formed from endogenously generated LTB4 in ionophore A23187 stimulated incubations. The dihydro metabolites were approximately 100 times less potent than LTB4 in causing guinea pig lung strip contraction and leukocyte-dependent inflammation in the hamster cheek pouch in vivo.
- Published
- 1990
- Full Text
- View/download PDF
40. Fast-growing root nodule bacteria produce a novel polyamine, aminobutylhomospermidine.
- Author
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Fujihara S and Harada Y
- Subjects
- Fabaceae microbiology, Mass Spectrometry, Plants, Medicinal, Rhizobiaceae growth & development, Rhizobium growth & development, Species Specificity, Spermidine isolation & purification, Spermidine metabolism, Rhizobiaceae metabolism, Rhizobium metabolism, Spermidine analogs & derivatives
- Abstract
Polyamines in various root nodule bacteria including Bradyrhizobium japonicum, Rhizobium fredii, R. leguminosarum, R. meliloti and R. loti were identified by capillary gas chromatography. Homospermidine was the polyamine present in highest concentration in all the rhizobia tested. In addition to putrescine and homospermidine, fast-growing type of rhizobial cells contained a novel polyamine, aminobutylhomospermidine, NH2(CH2)4NH(CH2)4NH(CH2)4NH2. The unusual tetraamine was not found in the cells of slow-growing type of rhizobia throughout their growth period, indicating a difference in polyamine metabolism between fast-growing type and slow-growing type of root nodule bacteria.
- Published
- 1989
- Full Text
- View/download PDF
41. Amicyanin: an electron acceptor of methylamine dehydrogenase.
- Author
-
Tobari J and Harada Y
- Subjects
- Cytochrome c Group metabolism, Electron Transport, Metalloproteins isolation & purification, Methylamines metabolism, Molecular Weight, Spectrophotometry, Bacterial Proteins, Metalloproteins metabolism, Oxidoreductases Acting on CH-NH Group Donors metabolism, Pseudomonas enzymology
- Published
- 1981
- Full Text
- View/download PDF
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