103 results on '"Inoue, M."'
Search Results
2. Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia
- Author
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Hamanaka, Y., primary, Mukai, M., additional, Shimamura, M., additional, Kitagawa, T., additional, Nishida, T., additional, Isohashi, F., additional, Ito, T., additional, Nishizawa, Y., additional, Tatsuta, M., additional, Matsuda, H., additional, and Inoue, M., additional
- Published
- 2005
- Full Text
- View/download PDF
3. Roles of α1-Adrenoceptor Activity in the Release of Nitric Oxide during Ischemia of the Canine Heart
- Author
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Node, K., primary, Kitakaze, M., additional, Kosaka, H., additional, Komamura, K., additional, Minamino, T., additional, Tada, M., additional, Inoue, M., additional, Hori, M., additional, and Kamada, T., additional
- Published
- 1995
- Full Text
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4. Plasma Nitric Oxide End Products Are Increased in the Ischemic Canine Heart
- Author
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Node, K., primary, Kitakaze, M., additional, Kosaka, H., additional, Komamura, K., additional, Minamino, T., additional, Tada, M., additional, Inoue, M., additional, Hori, M., additional, and Kamada, T., additional
- Published
- 1995
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5. Antioxidant, Gallic Acid, Induces Apoptosis in HL-60RG Cells
- Author
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Inoue, M., primary, Suzuki, R., additional, Koide, T., additional, Sakaguchi, N., additional, Ogihara, Y., additional, and Yabu, Y., additional
- Published
- 1994
- Full Text
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6. Close Colocalization of CD4 and a Serine Esterase Tryptase TL2 on the Cell Surface of Human Monocytoid and CD4+ Lymphoid Cells
- Author
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Inoue, M., primary, Hoshino, T., additional, Fukuma, T., additional, Niwa, Y., additional, and Kido, H., additional
- Published
- 1994
- Full Text
- View/download PDF
7. Interleukin-1 Induction of Tumor Necrosis Factor-α mRNA and Bioactive Tumor Necrosis Factor-α in a Pancreatic β-Cell Line by a Mechanism Requiring No de Novo Protein Synthesis
- Author
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Takane, N., primary, Yamada, K., additional, Otabe, S., additional, Inoue, M., additional, and Nonaka, K., additional
- Published
- 1993
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8. Priming effect of 2,3-dibenzylbutane-1,4-diol (mammalian lignan) on superoxide production in human neutrophils
- Author
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Morikawa, M., primary, Abe, M., additional, Yamauchi, Y., additional, Inoue, M., additional, and Tsuboi, M., additional
- Published
- 1990
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9. The γ-glutamyl cycle serves as an amino acids supply system in colorectal cancer organoids under chronic hypoxia.
- Author
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Tabata S, Endo H, Makinoshima H, Soga T, and Inoue M
- Subjects
- Humans, Cell Hypoxia, Tumor Microenvironment, Glutathione metabolism, Hypoxia metabolism, Tumor Hypoxia, gamma-Glutamylcyclotransferase metabolism, gamma-Glutamylcyclotransferase genetics, Colorectal Neoplasms metabolism, Colorectal Neoplasms pathology, Organoids metabolism, Organoids pathology, gamma-Glutamyltransferase metabolism, Amino Acids metabolism
- Abstract
Malignant tumors are characterized by a hypoxic microenvironment, and metabolic reprogramming is necessary to ensure energy production and oxidative stress resistance. Although the microenvironmental properties of tumors vary under acute and chronic hypoxia, studies on chronic hypoxia-induced metabolic changes are limited. In the present study, we performed a comprehensive metabolic analysis in a chronic hypoxia model using colorectal cancer (CRC) organoids, and identified an amino acid supply system through the γ-glutamyl cycle, a glutathione recycling pathway. We analyzed the metabolic changes caused by hypoxia over time and observed that chronic hypoxia resulted in an increase in 5-oxoproline and a decrease in oxidized glutathione (GSSG) compared to acute hypoxia. These findings suggest that chronic hypoxia induces metabolic changes in the γ-glutamyl cycle. Moreover, inhibition of the γ-glutamyl cycle via γ-glutamyl cyclotransferase (GGCT) and γ-glutamyl transferase 1 (GGT1) knockdown significantly reversed chronic hypoxia-induced upregulation of 5-oxoproline and several amino acids. Notably, GGT1 knockdown downregulated the intracellular levels of γ-glutamyl amino acids. Conclusively, these results indicate that the γ-glutamyl cycle serves as an amino acid supply system in CRC under chronic hypoxia, which provides fresh insight into cancer metabolism under chronic hypoxia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
10. A pH imbalance is linked to autophagic dysregulation of inner ear hair cells in Atp6v1ba-deficient zebrafish.
- Author
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Ikeuchi M, Inoue M, Miyahara H, Sebastian WA, Miyazaki S, Takeno T, Kiyota K, Yano S, Shiraishi H, Shimizu N, Hanada R, Yoshimura A, Ihara K, and Hanada T
- Subjects
- Animals, Humans, Zebrafish metabolism, Mutation, Hair Cells, Auditory pathology, Hydrogen-Ion Concentration, Hair metabolism, Adenosine Triphosphate, Vacuolar Proton-Translocating ATPases genetics, Vacuolar Proton-Translocating ATPases metabolism, Hearing Loss, Sensorineural genetics, Hearing Loss, Sensorineural pathology, Acidosis, Renal Tubular genetics, Acidosis, Organometallic Compounds
- Abstract
V-ATPase is an ATP hydrolysis-driven proton pump involved in the acidification of intracellular organelles and systemic acid-base homeostasis through H
+ secretion in the renal collecting ducts. V-ATPase dysfunction is associated with hereditary distal renal tubular acidosis (dRTA). ATP6V1B1 encodes the B1 subunit of V-ATPase that is integral to ATP hydrolysis and subsequent H+ transport. Patients with pathogenic ATP6V1B1 mutations often exhibit an early onset of sensorineural hearing loss. However, the mechanisms underlying this association remain unclear. We employed morpholino oligonucleotide-mediated knockdown and CRISPR/Cas9 gene editing to generate Atp6v1ba-deficient (atp6v1ba-/- ) zebrafish as an ortholog model for ATP6V1B1. The atp6v1ba-/- zebrafish exhibited systemic acidosis and significantly smaller otoliths compared to wild-type siblings. Moreover, deficiency in Atp6v1ba led to degeneration of inner ear hair cells, with ultrastructural changes indicative of autophagy. Our findings indicate a critical role of ATP6V1B1 in regulating lysosomal pH and autophagy in hair cells, and the results provide insights into the pathophysiology of sensorineural hearing loss in dRTA. Furthermore, this study demonstrates that the atp6v1ba-/- zebrafish model is a valuable tool for further investigation into disease mechanisms and potential therapies for acidosis-related hearing impairment., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
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11. An immunocytokine consisting of a TNFR2 agonist and TNFR2 scFv enhances the expansion of regulatory T cells through TNFR2 clustering.
- Author
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Inoue M, Tsuji Y, Kashiwada A, Yokoyama A, Iwata A, Abe Y, Kamada H, and Tsunoda SI
- Subjects
- Carrier Proteins metabolism, Mutant Proteins metabolism, Receptors, Tumor Necrosis Factor, Type II genetics, Receptors, Tumor Necrosis Factor, Type II agonists, Tumor Necrosis Factor-alpha metabolism, Humans, Animals, Mice, Single-Chain Antibodies genetics, Single-Chain Antibodies pharmacology, Single-Chain Antibodies metabolism, T-Lymphocytes, Regulatory
- Abstract
Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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- View/download PDF
12. Positive regulatory loop of platelet-derived growth factor DD-induced STAT3 activation is associated with poor prognosis in advanced urothelial carcinoma.
- Author
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Ando K, Kurashina R, Motoi N, Iizuka T, Inoue M, Maruyama R, Mitani K, Takenobu H, Haruta M, Onuki R, Matsuoka Y, Kamijo T, and Kageyama Y
- Abstract
Immune checkpoint inhibitor (ICI) therapy has been established for patients with advanced urothelial cancer (UC). The necessity of overcoming resistance to ICIs and identifying a predictive factor for the same has been highlighted, such as the assessment of combination therapy with other targeted drugs and the characterization of molecular signatures in the tumor microenvironment. Recently, we reported that low hemoglobin (Hb) levels and a high platelet-to-lymphocyte ratio (PLR) were significantly associated with overall survival in patients with UC who did not benefit from pembrolizumab treatment. In the present study, we identified a possible link between these unfavorable prognostic indicators and PDGF-DD-induced STAT3 activation in UC. Overlapping patients between the high STAT3- or phosphorylated STAT3-positive score group (as assessed by immunohistochemistry) and low Hb levels or high PLR group (as assessed by blood tests) showed significantly worse outcomes after pembrolizumab treatment. Additionally, using the bladder cancer JMSU1 cell line, we demonstrated a possible positive regulatory loop between autocrine/paracrine PDGF-DD and STAT3 signaling. Therefore, we suggest that STAT3 inhibition and PDGF-DD detection in the tumor microenvironment might represent a potential therapeutic strategy to overcome resistance to pembrolizumab. Moreover, this can help identify patients with UC who could benefit from combination treatment., 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 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
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13. Evaluation of the ability of human induced nephron progenitor cells to form chimeric renal organoids using mouse embryonic renal progenitor cells.
- Author
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Matsumoto N, Yamanaka S, Morimoto K, Matsui K, Nishimura S, Kinoshita Y, Inage Y, Fujimori K, Kuroda T, Saito Y, Takamura T, Fujimoto T, Tajiri S, Matsumoto K, Inoue M, Kobayashi E, and Yokoo T
- Subjects
- Humans, Mice, Animals, Embryonic Stem Cells, Cell Differentiation, Mice, Transgenic, Organoids, Kidney, Nephrons
- Abstract
The number of patients with end-stage renal failure is increasing annually worldwide and the problem is compounded by a shortage of renal transplantation donors. In our previous research, we have shown that transplantation of renal progenitor cells into the nephrogenic region of heterologous fetuses can induce the development of nephrons. We have also developed transgenic mice in which specific renal progenitor cells can be removed by drugs. By combining these two technologies, we have succeeded in generating human-mouse chimeric kidneys in fetal mice. We hope to apply these technologies to regenerative medicine. The quality of nephron progenitor cells (NPCs) derived from human pluripotent stem cells is important for the generation of chimeric kidneys, but there is currently no simple evaluation system for the chimerogenic potential of human NPCs. In this study, we focused on the fact that the re-aggregation of mouse renal progenitor cells can be used for nephron formation, even when merged into single cells. First, we examined the conditions under which nephron formation is likely to occur in mice during re-aggregation. Next, to improve the differentiation potential of human NPCs derived from pluripotent stem cells, NPCs were sorted using Integrin subunit alpha 8 (ITGA8). Finally, we demonstrated chimera formation between different species by mixing mouse cells with purified, selectively-induced human NPCs under optimum conditions. We observed these chimeric organoids at different time points to learn about these human-mouse chimeric structures at various stages of renal development. We found that the rate of chimera formation was affected by the purity of the human NPCs and the cell ratios used. We demonstrated that chimeric nephrons can be generated using a simple model, even between distant species. We believe that this admixture of human and mouse renal progenitor cells is a promising technology with potential application for the evaluation of the chimera formation abilities of NPCs., 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
- Full Text
- View/download PDF
14. Structural basis for the dual GTPase specificity of the DOCK10 guanine nucleotide exchange factor.
- Author
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Kukimoto-Niino M, Ihara K, Mishima-Tsumagari C, Inoue M, Fukui Y, Yokoyama S, and Shirouzu M
- Subjects
- Animals, Mice, Cytokinesis, Mutagenesis, cdc42 GTP-Binding Protein metabolism, Guanine Nucleotide Exchange Factors metabolism, rac1 GTP-Binding Protein metabolism
- Abstract
Dedicator of cytokinesis 10 (DOCK10), an evolutionarily conserved guanine nucleotide exchange factor (GEF) for Rho GTPases, has the unique specificity within the DOCK-D subfamily to activate both Cdc42 and Rac, but the structural bases for these activities remained unknown. Here we present the crystal structures of the catalytic DHR2 domain of mouse DOCK10, complexed with either Cdc42 or Rac1. The structures revealed that DOCK10
DHR2 binds to Cdc42 or Rac1 by slightly changing the arrangement of its two catalytic lobes. DOCK10 also has a flexible binding pocket for the 56th GTPase residue, allowing a novel interaction with Trp56Rac1 . The conserved residues in switch 1 of Cdc42 and Rac1 showed common interactions with the unique Lys-His sequence in the β5/β6 loop of DOCK10DHR2 . However, the interaction of switch 1 in Rac1 was less stable than that of switch 1 in Cdc42, due to amino acid differences at positions 27 and 30. Structure-based mutagenesis identified the DOCK10 residues that determine the Cdc42/Rac1 dual specificity., 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
- Full Text
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15. Expression and function of mitochondrial inhibitor factor-1 and TASK channels in adrenal cells.
- Author
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Kim D, Harada K, and Inoue M
- Subjects
- Pregnancy, Female, Rats, Animals, Cell Hypoxia, Mitochondria metabolism, Hypoxia metabolism, Adenosine Triphosphate metabolism, Chromaffin Cells metabolism
- Abstract
Adrenal medullary chromaffin (AMC) cells in the perinatal period and carotid body glomus cells after birth respond to hypoxia with catecholamine secretion. The hypoxia detection mechanism in such O
2 -sensitive cells is still not well defined. One hypothesis is that a decrease in cellular ATP may be involved in the hypoxia detection. This idea is based on ATP dependence of TASK channel activity that regulates the resting membrane potential and is suppressed by hypoxia in glomus cells. Mitochondrial ATPase inhibitor factor-1 (IF1 ), a physiological regulator of ATP synthase, helps prevent ATP hydrolysis under hypoxic conditions. In cells where IF1 expression is high, exposure to hypoxia is expected to have no effect on TASK channel activity. This possibility was electrophysiologically and immunocytochemically explored. Single channel recordings revealed that 36-pS TASK3-like channels contribute to the resting membrane potential in young rat adrenal cortical (AC) cells. TASK3-like channel activity in a cell-attached patch was not affected by bath application of mitochondrial inhibitors. Consistent with this finding, IF1 -like immunoreactive material was well expressed in rat AC cells. In further support of our hypothesis, IF1 -like immunoreactive material was well expressed in adult rat AMC cells that are known to be hypoxia-insensitive and minimally expressed in newborn AMC cells that are hypoxia-sensitive. These results provide evidence for the functional relevance of IF1 expression in excitability in O2 -sensitive cells in response to mitochondrial inhibition., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)- Published
- 2023
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16. Miglustat, a glucosylceramide synthase inhibitor, mitigates liver fibrosis through TGF-β/Smad pathway suppression in hepatic stellate cells.
- Author
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Iwanaga T, Chiba T, Nakamura M, Kaneko T, Ao J, Qiang N, Ma Y, Zhang J, Kogure T, Yumita S, Ishino T, Ogawa K, Kan M, Nakagawa M, Fujiwara K, Fujita N, Sakuma T, Kanzaki H, Koroki K, Kusakabe Y, Inoue M, Kobayashi K, Kanogawa N, Kiyono S, Kondo T, Nakagawa R, Ogasawara S, Nakamoto S, Muroyama R, Kato J, Kanda T, Maruyama H, Mimura N, Honda T, Murayama T, Nakamura H, and Kato N
- Subjects
- Animals, Humans, Mice, Carbon Tetrachloride pharmacology, Hepatic Stellate Cells metabolism, Liver metabolism, Liver Cirrhosis chemically induced, Liver Cirrhosis drug therapy, Liver Cirrhosis metabolism, Mice, Inbred C57BL, Signal Transduction, Smad Proteins metabolism, Transforming Growth Factor beta metabolism, Transforming Growth Factor beta1 metabolism
- Abstract
Transforming growth factor (TGF)-β/Smad pathway is implicated in the pathogenesis of liver fibrosis, a condition characterized by excessive deposition of extracellular matrix (ECM) proteins such as collagen in response to chronic inflammation. It has been reported that ceramide regulates collagen production through TGF-β/Smad pathway activation. In this study, we examined whether miglustat, an inhibitor of glucosylceramide synthase, can suppress liver fibrosis by reducing TGF-β/Smad pathway activity. Human hepatic stellate cells (HHSteCs) were cultured with TGF-β and multiple miglustat concentrations to examine dose-dependent effects on the expression levels of ECM-related genes and Smad proteins. To evaluate the efficacy of miglustat for fibrosis mitigation, C57BL/6 mice were treated with carbon tetrachloride (CCl
4 ) for 4 weeks to induce liver fibrosis, followed by combined CCl4 plus miglustat for a further 2 weeks. To examine if miglustat can also prevent fibrosis, mice were treated with CCl4 for 2 weeks, followed by CCl4 plus miglustat for 2 weeks. Miglustat dose-dependently downregulated expression of α-smooth muscle actin and ECM components in TGF-β-treated HHSteCs. Both phosphorylation and nuclear translocation of Smad2 and Smad3 were also suppressed by miglustat treatment. Sirius-Red staining and hydroxyproline assays of model mouse liver samples revealed that miglustat reduced fibrosis, an effect accompanied by decreased expression of ECM. Our findings suggest that miglustat can both prevent and reverse liver fibrosis by inhibiting TGF-β/Smad pathway., 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 © 2022 Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
17. Eribulin induces tumor vascular remodeling through intussusceptive angiogenesis in a sarcoma xenograft model.
- Author
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Taguchi E, Horiuchi K, Senoo A, Susa M, Inoue M, Ishizaka T, Rikitake H, Matsuhashi Y, and Chiba K
- Subjects
- Animals, Bevacizumab pharmacology, Bevacizumab therapeutic use, Cell Line, Tumor, Cell Proliferation drug effects, Cell Shape drug effects, Endothelial Cells drug effects, Endothelial Cells ultrastructure, Furans administration & dosage, Furans pharmacology, Intussusception drug therapy, Ketones administration & dosage, Ketones pharmacology, Mice, Inbred BALB C, Mice, Nude, Neovascularization, Pathologic complications, Pericytes drug effects, Pericytes pathology, Pericytes ultrastructure, Sarcoma complications, Sarcoma ultrastructure, Tumor Hypoxia drug effects, Vascular Endothelial Growth Factor A metabolism, Xenograft Model Antitumor Assays, Mice, Furans therapeutic use, Intussusception complications, Ketones therapeutic use, Neovascularization, Pathologic drug therapy, Sarcoma blood supply, Sarcoma drug therapy, Vascular Remodeling drug effects
- Abstract
Eribulin is a novel microtubule inhibitor that, similar to other types of microtubule inhibitors, induces apoptosis by inhibiting the mitotic division of cells. Besides this direct effect on tumor cells, previous studies have shown that eribulin has the potential to induce tumor vascular remodeling in several different cancers; however, the mechanisms underlying this phenomenon remain unclear. In the present study, we aimed to elucidate whether eribulin is effective against synovial sarcoma, a relatively rare sarcoma that often affects adolescents and young adults, and to histologically investigate the microstructure of tumor vessels after the administration of eribulin. We found that eribulin exhibits potent antitumor activity against synovial sarcoma in a tumor xenograft model and that tumor vessels frequently have intervascular pillars, a hallmark of intussusceptive angiogenesis (IA), after the administration of eribulin. IA is a distinct form of angiogenesis that is involved in normal developmental processes as well as pathological conditions. Our data indicate that IA is potentially involved in eribulin-induced vascular remodeling and thereby suggest previously unacknowledged role of IA in regulating the tumor vasculature after eribulin administration., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
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18. Tyrosine pre-transfer RNA fragments are linked to p53-dependent neuronal cell death via PKM2.
- Author
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Inoue M, Hada K, Shiraishi H, Yatsuka H, Fujinami H, Morisaki I, Nishida Y, Matsubara E, Ishitani T, Hanada R, Matsumoto M, Penninger JM, Ihara K, and Hanada T
- Subjects
- Animals, Cell Death, Cell Differentiation, Cell Line, Embryo, Nonmammalian metabolism, Humans, Zebrafish embryology, Neurons enzymology, Neurons pathology, Pyruvate Kinase metabolism, RNA Precursors metabolism, Tumor Suppressor Protein p53 metabolism, Tyrosine metabolism
- Abstract
Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5' tRNA fragments derived from tyrosine pre-tRNA (5' Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5' Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5' Tyr-tRF. Our findings partially uncovered a mechanistic link between 5' Tyr-tRF and neuronal cell death that is regulated by PKM2., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
19. Dedifferentiation of neuroendocrine carcinoma of the uterine cervix in hypoxia.
- Author
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Kubota S, Tanaka M, Endo H, Ito Y, Onuma K, Ueda Y, Kamiura S, Yoshino K, Kimura T, Kondo J, and Inoue M
- Subjects
- Animals, Cell Dedifferentiation, Female, Humans, Mice, Tumor Cells, Cultured, Carcinoma, Neuroendocrine pathology, Carcinoma, Small Cell pathology, Cervix Uteri pathology, Tumor Hypoxia, Uterine Cervical Neoplasms pathology
- Abstract
Neuroendocrine carcinoma of small cell type (SCNEC) is a rare pathological subtype in cervical cancer, which has a worse prognosis than other histological cell types. Due to its low incidence and the lack of experimental platforms, the molecular characteristics of SCNEC in the cervix remain largely unknown. Using the cancer tissue-originated spheroid (CTOS) method-an ex vivo 3D culture system that preserves the differentiation status of the original tumors-we established a panel of CTOS lines of SCNEC. We demonstrated that xenograft tumors and CTOSs, respectively, exhibited substantial intra-tumor and intra-CTOS variation in the expression levels of chromogranin A (CHGA), a neuroendocrine tumor marker. Since hypoxia affects differentiation in various tumors and in stem cells, we also investigated how hypoxia affected neuroendocrine differentiation of SCNEC of the uterine cervix. In the CTOS line cerv21, hypoxia suppressed expression of the neuroendocrine markers CHGA and synaptophysin (SYP). Flow cytometry analysis using CD99 (a membrane protein marker of SCNEC) revealed decreased CD99 expression in a subset of cells under hypoxic conditions. These expression changes were attenuated by HIF-1α knockdown, and by a Notch inhibitor, suggesting that these molecules played a role in the regulation of neuroendocrine differentiation. The examined SCNEC markers were suppressed under hypoxia in multiple CTOS lines. Overall, our present results indicated that neuroendocrine differentiation in SCNEC of the uterus is a variable phenotype, and that hypoxia may be one of the factors regulating the differentiation status., Competing Interests: Declaration of competing interest J.K. and M.I. belong to the Department of Clinical Bio-resource Research and Development in at Kyoto University, which is sponsored by KBBM, Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
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20. CLP1 acts as the main RNA kinase in mice.
- Author
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Fujinami H, Shiraishi H, Hada K, Inoue M, Morisaki I, Higa R, Shin T, Kobayashi T, Hanada R, Penninger JM, Mimata H, and Hanada T
- Abstract
CLP1 plays an essential role in the protein complex involved in mRNA 3'-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5' end of RNAs. From the evidence that phosphorylation of the 5' end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1
K/K ) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5' end of RNAs in the siRNA pathway, with no apparent involvement of NOL9., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
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21. A Prdm8 target gene Ebf3 regulates multipolar-to-bipolar transition in migrating neocortical cells.
- Author
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Iwai R, Tabata H, Inoue M, Nomura KI, Okamoto T, Ichihashi M, Nagata KI, and Mizutani KI
- Subjects
- Animals, DNA-Binding Proteins, Gene Expression Regulation, Developmental physiology, Histone Methyltransferases, Mice, Mice, Inbred ICR, Neocortex cytology, Neurogenesis physiology, Neurons cytology, Cell Movement physiology, Embryonic Development physiology, Histone-Lysine N-Methyltransferase metabolism, Neocortex embryology, Neocortex physiology, Neurons physiology, Transcription Factors metabolism
- Abstract
Precise control of neuronal migration is essential for the development of the neocortex. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here we identified helix-loop-helix transcription factor Ebf3 as a Prdm8 target gene, and found that Ebf3 is a key regulator of neuronal migration via multipolar-to-bipolar transition. Ebf3 knockdown cells exhibited severe defects in the formation of leading processes and an inhibited shift to the locomotion mode. Moreover, we found that Ebf3 knockdown represses NeuroD1 transcription, and NeuroD1 overexpression partially rescued migration defects in Ebf3 knockdown cells. Our findings highlight the critical role of Ebf3 in multipolar-to-bipolar transition via positive feedback regulation of NeuroD1 in the developing neocortex., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
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22. Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2).
- Author
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Frykman S, Inoue M, Ikeda A, Teranishi Y, Kihara T, Lundgren JL, Yamamoto NG, Bogdanovic N, Winblad B, Schedin-Weiss S, and Tjernberg LO
- Subjects
- Alzheimer Disease metabolism, Animals, Brain metabolism, Epilepsy metabolism, Female, Gene Silencing, Glycosylation, Humans, Male, Mice, Mice, Inbred C57BL, Neurons metabolism, Pyrimidines chemistry, Rats, Rats, Sprague-Dawley, Amyloid beta-Peptides metabolism, Amyloid beta-Protein Precursor metabolism, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels metabolism, Potassium Channels metabolism
- Abstract
The toxic amyloid β-peptide (Aβ) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase (to yield APP-C-terminal fragment β (APP-CTFβ) and soluble APPβ (sAPPβ)) and γ-secretase (to yield Aβ). We reasoned that proteins that associate with γ-secretase are likely to regulate Aβ production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aβ levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aβ, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aβ levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by β-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2017
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23. Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors.
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Toki H, Minowa O, Inoue M, Motegi H, Karashima Y, Ikeda A, Kaneda H, Sakuraba Y, Saiki Y, Wakana S, Suzuki H, Gondo Y, Shiroishi T, and Noda T
- Subjects
- Alleles, Animals, Carcinoma, Squamous Cell metabolism, Gene Expression Regulation, Neoplastic, Loss of Heterozygosity, Male, Mice, Inbred C57BL, Mice, Knockout, Models, Molecular, Protein Conformation, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Carcinoma, Squamous Cell genetics, Carcinoma, Squamous Cell pathology, Epithelial Cells pathology, Mutation, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics
- Abstract
Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1-4] and humans to Darier disease (DD) [14-17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca(2+)-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway., (Copyright © 2016. Published by Elsevier Inc.)
- Published
- 2016
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24. Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins.
- Author
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Yuasa K, Ota R, Matsuda S, Isshiki K, Inoue M, and Tsuji A
- Subjects
- 14-3-3 Proteins metabolism, Amino Acid Motifs, Animals, Binding Sites, Biomarkers, Tumor metabolism, COS Cells, Calcineurin genetics, Calcineurin metabolism, Chlorocebus aethiops, Death-Associated Protein Kinases metabolism, Exoribonucleases metabolism, Gene Expression Regulation, Humans, MCF-7 Cells, Molecular Sequence Data, Phosphorylation, Plasmids chemistry, Plasmids metabolism, Protein Binding, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction, Threonine metabolism, Transfection, 14-3-3 Proteins genetics, Apoptosis genetics, Biomarkers, Tumor genetics, Death-Associated Protein Kinases genetics, Exoribonucleases genetics, Proto-Oncogene Proteins c-akt genetics
- Abstract
Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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25. Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3.
- Author
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Kamada H, Taki S, Nagano K, Inoue M, Ando D, Mukai Y, Higashisaka K, Yoshioka Y, Tsutsumi Y, and Tsunoda S
- Subjects
- Animals, Cell Line, Tumor, Cytotoxicity, Immunologic, Humans, Leukocytes, Mononuclear metabolism, Mice, Protein Binding, Transfection, Antibodies, Bispecific biosynthesis, Antibodies, Bispecific immunology, CD3 Complex immunology, Receptors, Eph Family immunology
- Abstract
The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2015
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26. Eph receptor A10 has a potential as a target for a prostate cancer therapy.
- Author
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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
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27. Role of ATF3 in synergistic cancer cell killing by a combination of HDAC inhibitors and agonistic anti-DR5 antibody through ER stress in human colon cancer cells.
- Author
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Liu J, Edagawa M, Goshima H, Inoue M, Yagita H, Liu Z, and Kitajima S
- Subjects
- Activating Transcription Factor 3 genetics, Antibodies, Monoclonal immunology, Apoptosis, Cell Line, Tumor, Colon drug effects, Colon metabolism, Colon pathology, Colonic Neoplasms genetics, Colonic Neoplasms metabolism, Colonic Neoplasms pathology, Drug Synergism, Gene Expression Regulation, Neoplastic drug effects, Gene Knockdown Techniques, Histone Deacetylase Inhibitors chemistry, Humans, Receptors, TNF-Related Apoptosis-Inducing Ligand immunology, Signal Transduction drug effects, Transcription Factor CHOP genetics, Transcription Factor CHOP metabolism, Tumor Suppressor Protein p53 genetics, Up-Regulation drug effects, eIF-2 Kinase metabolism, Activating Transcription Factor 3 metabolism, Antibodies, Monoclonal pharmacology, Colonic Neoplasms drug therapy, Endoplasmic Reticulum Stress drug effects, Histone Deacetylase Inhibitors pharmacology, Receptors, TNF-Related Apoptosis-Inducing Ligand genetics
- Abstract
Histone deacetylase inhibitors (HDACIs) are promising agents for cancer therapy. However, the mechanism(s) responsible for the efficacy of HDACIs have not yet to be fully elucidated. Death receptor 5 (DR5) is a transmembrane receptor containing death domain that triggers cell death upon binding to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or agonistic anti-DR5 monoclonal antibody, and the combination of TRAIL/agonistic anti-DR5 monoclonal antibody and agents that increase the expression of DR5 is expected as a novel anticancer therapeutic strategy. Here we report that six different HDACIs activated endoplasmic reticulum (ER) stress sensor PERK and eIF2α and induced the ATF4/ATF3/CHOP pathway in p53-deficient human colon cancer cells. This resulted in an increased expression of DR5 on the cell surface and sensitized cells to apoptosis by agonistic anti-DR5 monoclonal antibody. Stress response gene ATF3 was required for efficient DR5 induction by HDACIs, and DR5 reporter assay showed that ATF3 play crucial role for the HDACIs-induced activation of DR5 gene transcription. These provide important mechanistic insight into how HDACIs exhibit pro-apoptotic activity in clinical anti-cancer treatments when they are used in combination with other therapeutic strategies., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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28. DDX3 RNA helicase is required for HIV-1 Tat function.
- Author
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Yasuda-Inoue M, Kuroki M, and Ariumi Y
- Subjects
- Cell Line, Cytoplasm metabolism, DEAD-box RNA Helicases genetics, Humans, rev Gene Products, Human Immunodeficiency Virus metabolism, tat Gene Products, Human Immunodeficiency Virus genetics, DEAD-box RNA Helicases metabolism, HIV-1 metabolism, tat Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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29. Distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev function.
- Author
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Yasuda-Inoue M, Kuroki M, and Ariumi Y
- Subjects
- Blotting, Western, Cell Nucleolus metabolism, Cell Nucleolus virology, DEAD-box RNA Helicases genetics, HEK293 Cells, HIV-1 genetics, Humans, Luciferases genetics, Luciferases metabolism, Microscopy, Confocal, Protein Binding, RNA Transport, RNA, Viral genetics, RNA, Viral metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transfection, rev Gene Products, Human Immunodeficiency Virus genetics, DEAD-box RNA Helicases metabolism, HIV-1 metabolism, rev Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
RNA helicase plays an important role in host mRNA and viral mRNA transcription, transport, and translation. Many viruses utilize RNA helicases in their life cycle, while human immunodeficiency virus type 1 (HIV-1) does not encode an RNA helicase. Thus, host RNA helicase has been involved in HIV-1 replication. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether distinct DDX RNA helicases cross-talk and cooperate to modulate the HIV-1 Rev function. In this study, we noticed that distinct DDX RNA helicases, including DDX1, DDX3, DDX5, DDX17, DDX21, DDX56, except DDX6, bound to the Rev protein and they colocalized with Rev in nucleolus or nucleus. In this context, these DEAD-box RNA helicases except DDX6 markedly enhanced the HIV-1 Rev-dependent RNA export. Furthermore, DDX3 interacted with DDX5 and synergistically enhanced the Rev function. As well, combination of other distinct DDX RNA helicases cooperated to stimulate the Rev function. Altogether, these results suggest that distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev function., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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30. Annexin A4 is a possible biomarker for cisplatin susceptibility of malignant mesothelioma cells.
- Author
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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
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31. Expression of prohibitins on the surface of activated T cells.
- Author
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Yurugi H, Tanida S, Ishida A, Akita K, Toda M, Inoue M, and Nakada H
- Subjects
- Animals, Antibodies, Female, Humans, Jurkat Cells, Mice, Mice, Inbred BALB C, Prohibitins, Repressor Proteins antagonists & inhibitors, Repressor Proteins immunology, Cell Membrane immunology, Lymphocyte Activation, Repressor Proteins biosynthesis, T-Lymphocytes immunology
- Abstract
Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
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32. Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi.
- Author
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Nara T, Hashimoto M, Hirawake H, Liao CW, Fukai Y, Suzuki S, Tsubouchi A, Morales J, Takamiya S, Fujimura T, Taka H, Mineki R, Fan CK, Inaoka DK, Inoue M, Tanaka A, Harada S, Kita K, and Aoki T
- Subjects
- Immunoprecipitation, Aspartate Carbamoyltransferase metabolism, Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) metabolism, Dihydroorotase metabolism, Pyrimidines biosynthesis, Trypanosoma cruzi enzymology
- Abstract
The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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33. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm.
- Author
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Hashimoto M, Morales J, Fukai Y, Suzuki S, Takamiya S, Tsubouchi A, Inoue S, Inoue M, Kita K, Harada S, Tanaka A, Aoki T, and Nara T
- Subjects
- Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) genetics, Cytoplasm metabolism, Gene Knockout Techniques, HeLa Cells, Humans, Trypanosoma cruzi genetics, Chagas Disease metabolism, Chagas Disease parasitology, Cytoplasm parasitology, Pyrimidines biosynthesis, Trypanosoma cruzi growth & development
- Abstract
The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
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34. Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge.
- Author
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Takahara Y, Matsuoka S, Kuwano T, Tsukamoto T, Yamamoto H, Ishii H, Nakasone T, Takeda A, Inoue M, Iida A, Hara H, Shu T, Hasegawa M, Sakawaki H, Horiike M, Miura T, Igarashi T, Naruse TK, Kimura A, and Matano T
- Subjects
- AIDS Vaccines therapeutic use, Acquired Immunodeficiency Syndrome prevention & control, Animals, Humans, Macaca mulatta, SAIDS Vaccines therapeutic use, Simian Acquired Immunodeficiency Syndrome immunology, AIDS Vaccines immunology, Antigens, Viral immunology, SAIDS Vaccines immunology, Simian Acquired Immunodeficiency Syndrome prevention & control, Simian Immunodeficiency Virus immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
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35. PIH1D1, a subunit of R2TP complex, inhibits doxorubicin-induced apoptosis.
- Author
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Inoue M, Saeki M, Egusa H, Niwa H, and Kamisaki Y
- Subjects
- ATPases Associated with Diverse Cellular Activities, Apoptosis Regulatory Proteins genetics, Cell Line, Cell Line, Tumor, Doxorubicin pharmacology, Humans, Tissue Distribution, Apoptosis, Apoptosis Regulatory Proteins metabolism, Carrier Proteins metabolism, DNA Helicases metabolism
- Abstract
We have previously reported that the two components of R2TP complex, RNA polymerase II-associated protein 3 (RPAP3), and Reptin, regulate apoptosis. Here we characterize another component of the complex, PIH1 domain containing protein 1 (PIH1D1). PIH1D1 interacts with both RPAP3 and Monad in HEK293 or U2OS cells. PIH1D1 transcripts were abundant in lung, leukocyte, and placenta. The reduction in endogenous PIH1D1 by siRNA enhanced apoptosis and caspase-3 activation induced by doxorubicin in U2OS cells. These results suggest that PIH1D1 may also function as a novel modulator of apoptosis pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
36. Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9.
- Author
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Ohta M, Ishida A, Toda M, Akita K, Inoue M, Yamashita K, Watanabe M, Murata T, Usui T, and Nakada H
- Subjects
- Cell Line, Tumor, Humans, Immunomodulation, Mucins blood, Sialic Acid Binding Immunoglobulin-like Lectins, Antigens, CD metabolism, Carcinoma immunology, Dendritic Cells immunology, Lectins metabolism, Monocytes immunology, Mucins metabolism, Neoplasms immunology, Tumor Escape
- Abstract
Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
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37. 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
38. Normal islet vascularization is dispensable for expansion of beta-cell mass in response to high-fat diet induced insulin resistance.
- Author
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Toyofuku Y, Uchida T, Nakayama S, Hirose T, Kawamori R, Fujitani Y, Inoue M, and Watada H
- Subjects
- Animals, Diet adverse effects, Mice, Mice, Transgenic, Vascular Endothelial Growth Factor A genetics, Dietary Fats administration & dosage, Insulin Resistance, Insulin-Secreting Cells drug effects, Insulin-Secreting Cells pathology, Neovascularization, Pathologic chemically induced
- Abstract
The inability to increase of islet mass adequately to compensate for the demand of insulin due to insulin resistance is an important pathophysiological feature of type 2 diabetes. Previous studies suggested a relationship between pancreatic beta-cell mass and islet vascularization, although no evidence has confirmed this association in response to insulin resistance. Vascular endothelial growth factor-A (VEGF-A) in islets is essential for maintaining normal islet blood vessels. Here, insulin resistance was induced in mice carrying a beta-cell-specific VEGF-A gene mutation (RIP-Cre:Vegf(fl/fl)) by 20-week feeding of high-fat diet as a model of impaired islet vascularization. These mice showed only a modest decrease in glucose tolerance, compared with control mice. In addition, although the endothelial cell area in the islets of high-fat-fed RIP-Cre:Vegf(fl/fl) mice remained diminished, the pancreatic beta-cell area was modestly more than in high-fat-fed control mice. Thus, normal islet vascularization does not seem to be essential for expansion of beta cell mass in response to insulin resistance.
- Published
- 2009
- Full Text
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39. Distribution, gene expression, and functional role of EphA4 during ossification.
- Author
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Kuroda C, Kubota S, Kawata K, Aoyama E, Sumiyoshi K, Oka M, Inoue M, Minagi S, and Takigawa M
- Subjects
- Alkaline Phosphatase genetics, Animals, Cell Line, Cell Membrane enzymology, Cell Nucleus enzymology, Cells, Cultured, Gene Expression, Growth Plate growth & development, Humans, Mice, Osteocalcin genetics, RNA Interference, RNA, Small Interfering genetics, Receptor, EphA4 genetics, Chondrocytes enzymology, Growth Plate enzymology, Osteoblasts enzymology, Osteogenesis, Receptor, EphA4 metabolism
- Abstract
EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles.
- Published
- 2008
- Full Text
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40. Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism.
- Author
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Baba M, Inoue M, Itoh K, and Nishizawa Y
- Subjects
- Adenosine Triphosphate metabolism, Animals, Antibodies, Blocking pharmacology, Antibodies, Monoclonal pharmacology, Basigin drug effects, Basigin genetics, Cell Line, Tumor, Energy Metabolism, Humans, Mice, Monocarboxylic Acid Transporters metabolism, RNA, Small Interfering genetics, Symporters metabolism, Apoptosis, Basigin metabolism, Colonic Neoplasms metabolism, Glycolysis drug effects, Melanoma metabolism
- Abstract
CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.
- Published
- 2008
- Full Text
- View/download PDF
41. Down-modulation of B cell signal transduction by ligation of mucins to CD22.
- Author
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Toda M, Akita K, Inoue M, Taketani S, and Nakada H
- Subjects
- Animals, Cattle, Cell Line, Tumor, Down-Regulation, Humans, Immune Tolerance, Immunoglobulin Fab Fragments immunology, Immunoglobulin M immunology, Mice, Mice, Inbred Strains, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Phosphorylation, Protein Tyrosine Phosphatase, Non-Receptor Type 6 metabolism, Receptors, Antigen, B-Cell metabolism, Sialic Acid Binding Ig-like Lectin 2 genetics, Spleen immunology, B-Lymphocytes immunology, Colonic Neoplasms immunology, Mucins metabolism, Sialic Acid Binding Ig-like Lectin 2 metabolism, Signal Transduction
- Abstract
Epithelial cancer cells secrete mucins carrying carbohydrate antigens such as a sialyl-Tn antigen into cancer tissues and/or the bloodstream, in which mucins may interact with CD22 (Siglec-2). Mucins isolated from colon cancer cells and bovine submaxillary mucins bound to CD22 cDNA transfectants and a human B cell line, Daudi cell, and the binding of soluble recombinant CD22 to the mucins was confirmed by means of a plate assay. The binding specificity was demonstrated by the fact that the mucins bound to the recombinant CD22 with an intact ectodomain but not to that with a mutated ectodomain. Daudi cells were stimulated with anti-IgM F(ab')(2) in the presence or absence of mucins. Ligation of mucins to CD22 decreased the phosphorylation of CD22 and SHP-1 recruitment, and the phosphorylation of ERK-1/2 prominently. The in vivo effect of mucins on splenic B cells in the tumor-bearing state was investigated using mucin-producing (TA3-Ha) and non-producing (TA3-St) mammary adenocarcinoma-bearing mice. When fluorescence-labeled epiglycanins were administered to normal mice, a portion of them was taken up by the spleen and became associated with splenic B cells. We found that splenic B cells were reduced in TA3-Ha-bearing mice but not in TA3-St-bearing ones. These results suggest that in the tumor-bearing state a portion of the mucins in the bloodstream was taken up by the spleen and ligated to CD22 expressed on splenic B cells, which may have led to down-regulation of signal transduction.
- Published
- 2008
- Full Text
- View/download PDF
42. Antigen-specific T-cell induction by vaccination with a recombinant Sendai virus vector even in the presence of vector-specific neutralizing antibodies in rhesus macaques.
- Author
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Moriya C, Horiba S, Inoue M, Iida A, Hara H, Shu T, Hasegawa M, and Matano T
- Subjects
- Animals, Antigens immunology, Genetic Vectors genetics, Lymphocyte Activation, Macaca mulatta, Sendai virus genetics, T-Lymphocytes, Cytotoxic immunology, Antibodies, Viral blood, Genetic Vectors immunology, Neutralization Tests methods, Sendai virus immunology, Vaccination
- Abstract
Recombinant viral vectors are promising vaccine tools for eliciting potent cellular immune responses against immunodeficiency virus infection, but pre-existing anti-vector antibodies can be an obstacle to their clinical use in humans. We have previously vaccinated rhesus macaques with a recombinant Sendai virus (SeV) vector twice at an interval of more than 1 year and have shown efficient antigen-specific T-cell induction by the second as well as the first vaccination. Here, we have established the method for measurement of SeV-specific neutralizing titers and have found efficient SeV-specific neutralizing antibody responses just before the second SeV vaccination in these macaques. This suggests the feasibility of inducing antigen-specific T-cell responses by SeV vaccination even in the host with pre-existing anti-SeV neutralizing antibodies.
- Published
- 2008
- Full Text
- View/download PDF
43. In vivo repopulation of cytoplasmically gene transferred hematopoietic cells by temperature-sensitive mutant of recombinant Sendai viral vector.
- Author
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Yoshida K, Yonemitsu Y, Tanaka S, Yoshida S, Shibata S, Kondo H, Okano S, Ishikawa F, Akashi K, Inoue M, Hasegawa M, and Sueishi K
- Subjects
- Animals, Cell Differentiation, Cell Lineage, Cytoplasm metabolism, Female, Flow Cytometry, Gene Transfer Techniques, Mice, Mice, Inbred C57BL, Temperature, Viral Envelope Proteins genetics, Viral Envelope Proteins metabolism, Bone Marrow Transplantation, Genetic Therapy methods, Genetic Vectors, Hematopoietic Stem Cells metabolism, Mutation, Sendai virus genetics
- Abstract
Recent clinical studies revealed 'proof of concept' of gene therapy targeting hematopoietic stem cells (HSCs) to treat hematopoietic disorders. However, vector integration-related adverse events of retroviral vectors have slowed progress in this field. As an initial step to overcoming this hurdle, we examined the potential of an improved cytoplasmic RNA vector, temperature-sensitive mutant non-transmissible recombinant Sendai virus (ts-rSeV/dF), for gene transfer to murine HSCs and progenitors. Both conventional vector and ts-rSeV/dF-GFP showed efficient gene transfer to T-lymphocyte-depleted syngeneic bone marrow cells (BMCs) (>85%), but only BMCs treated with ts-rSeV/dF-GFP but not with conventional vector efficiently repopulated in the recipient mice, associated with multilineage differentiation in vitro and in vivo. To our knowledge, this is the first demonstration of the in vivo reconstruction of hematopoietic series by cytoplasmically gene transferred BMCs, that warrants further investigation to realize this strategy in clinical settings.
- Published
- 2007
- Full Text
- View/download PDF
44. RhoC is essential for TGF-beta1-induced invasive capacity of rat ascites hepatoma cells.
- Author
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Mukai M, Endo H, Iwasaki T, Tatsuta M, Togawa A, Nakamura H, and Inoue M
- Subjects
- Animals, Cells, Cultured, Gene Expression Profiling, Liver Neoplasms pathology, Neoplasm Invasiveness, Rats, Transforming Growth Factor beta1, rho GTP-Binding Proteins genetics, rhoA GTP-Binding Protein physiology, Liver Neoplasms, Experimental pathology, Transforming Growth Factor beta physiology, rho GTP-Binding Proteins physiology
- Abstract
Transforming growth factor-beta1 (TGF-beta1) is a multifunctional growth factor that plays a role in cell proliferation, differentiation, extracellular matrix production, apoptosis, and cell motility. We show here that TGF-beta1 increased the invasiveness of MM1 cells, which are a highly invasive clone of rat ascites hepatoma cells. Both mRNA and protein levels of RhoC but not RhoA in TGF-beta1-treated MM1 cells increased. In parallel with this increase in expression, RhoC activity was induced by TGF-beta1 treatment. When RhoC was overexpressed in MM1 cells, the invasive capacity increased. The RhoC-overexpressing cells formed more nodules than did mock cells when injected into rat peritoneum. Furthermore, when RhoC expression was reduced by transfection with shRNA/RhoC, the invasiveness of MM1 cells decreased with concomitant suppression of RhoC expression. Thus, the induced expression of RhoC by TGF-beta1 in MM1 cells plays a critical role in TGF-beta1-induced cell migration.
- Published
- 2006
- Full Text
- View/download PDF
45. Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet.
- Author
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Motomura W, Inoue M, Ohtake T, Takahashi N, Nagamine M, Tanno S, Kohgo Y, and Okumura T
- Subjects
- Animals, Cell Line, Fatty Liver, Humans, Male, Mice, Mice, Inbred C57BL, Organ Specificity, Perilipin-2, Species Specificity, Tissue Distribution, Up-Regulation, Dietary Fats metabolism, Hepatocytes metabolism, Liver metabolism, Membrane Proteins metabolism, PPAR gamma metabolism
- Abstract
The present study was performed to examine a role of adipose differentiation-related protein (ADRP) in the process of liver steatosis. Immunohistochemical findings indicated that ADRP expression is increased in the hepatocytes in patients with fatty liver when compared with normal liver. ADRP expression is localized in the surface of lipid droplets in the hepatocytes. Increased expression of ADRP mRNA and protein was similarly observed in fatty liver in ob/ob mice and the liver steatosis induced by high fat diet in mice. The up-regulation of ADRP mRNA and protein in the liver by high fat diet was identified in the surface of lipid droplets in a time-dependent manner. Recent studies demonstrated that up-regulation of PPARgamma in the hepatocytes is deeply involved in liver steatosis. To clarify whether ADRP expression is increased by PPARgamma activation in hepatocytes, we examined the effect of a PPARgamma ligand, troglitazone, on ADRP mRNA expression in HepG2 cells. ADRP mRNA expression was increased by troglitazone in dose- and time-dependent manners. All these results suggest that ADRP is up-regulated in liver steatosis in human and mice, and that high fat diet increases expression of ADRP through PPARgamma activation, followed by induction of liver steatosis.
- Published
- 2006
- Full Text
- View/download PDF
46. Molecular characterization of ENU mouse mutagenesis and archives.
- Author
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Sakuraba Y, Sezutsu H, Takahasi KR, Tsuchihashi K, Ichikawa R, Fujimoto N, Kaneko S, Nakai Y, Uchiyama M, Goda N, Motoi R, Ikeda A, Karashima Y, Inoue M, Kaneda H, Masuya H, Minowa O, Noguchi H, Toyoda A, Sakaki Y, Wakana S, Noda T, Shiroishi T, and Gondo Y
- Subjects
- Animals, Base Sequence, Male, Mice, Inbred C57BL, Molecular Sequence Data, Mutagens pharmacology, Chromosome Mapping methods, DNA Mutational Analysis methods, Ethylnitrosourea pharmacology, Mice genetics, Spermatozoa drug effects
- Abstract
The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.
- Published
- 2005
- Full Text
- View/download PDF
47. Increased expression of PPARgamma in high fat diet-induced liver steatosis in mice.
- Author
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Inoue M, Ohtake T, Motomura W, Takahashi N, Hosoki Y, Miyoshi S, Suzuki Y, Saito H, Kohgo Y, and Okumura T
- Subjects
- Animals, Base Sequence, Body Weight, Cyclic AMP Response Element-Binding Protein metabolism, DNA Primers, Fatty Liver etiology, Fatty Liver genetics, Gene Expression Profiling, Liver metabolism, Male, Mice, Mice, Inbred C57BL, PPAR gamma genetics, RNA, Messenger genetics, Dietary Fats administration & dosage, Fatty Liver metabolism, PPAR gamma metabolism
- Abstract
The present study was performed to examine a hypothesis that peroxisome proliferator-activated receptor gamma (PPARgamma) is implicated in high fat diet-induced liver steatosis. Mice were fed with control or high fat diet containing approximately 10% or 80% cholesterol, respectively. Macroscopic and microscopic findings demonstrated that lipid accumulation in the liver was observed as early as 2 weeks after high fat diet and that high fat diet for 12 weeks developed a fatty liver phenotype, establishing a novel model of diet-induced liver steatosis. Gene profiling with microarray and real-time PCR studies demonstrated that among genes involved in lipid metabolism, adipogenesis-related genes, PPARgamma and its targeted gene, CD36 mRNA expression was specifically up-regulated in the liver by high fat diet for 2 weeks. Immunohistochemical study revealed that PPARgamma protein expression is increased in the nuclei of hepatocytes by high fat diet. It was also shown that protein expression of cAMP response element-binding protein (CREB), an upstream molecule of PPARgamma, in the liver was drastically suppressed by high fat diet. All these results suggest for the first time that the CREB-PPARgamma signaling pathway may be involved in the high fat diet-induced liver steatosis.
- Published
- 2005
- Full Text
- View/download PDF
48. A potential pro-angiogenic cell therapy with human placenta-derived mesenchymal cells.
- Author
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Nishishita T, Ouchi K, Zhang X, Inoue M, Inazawa T, Yoshiura K, Kuwabara K, Nakaoka T, Watanabe N, Igura K, Takahashi TA, and Yamashita N
- Subjects
- Animals, Blood Vessels cytology, Blood Vessels growth & development, Cell Transplantation, Cells, Cultured, Female, HeLa Cells, Hindlimb blood supply, Hindlimb metabolism, Hindlimb pathology, Humans, Mesoderm cytology, Mice, Mice, Inbred NOD, Mice, SCID, Pregnancy, Vascular Endothelial Growth Factor A metabolism, Ischemia therapy, Mesoderm metabolism, Neovascularization, Physiologic, Placenta cytology
- Abstract
Recently several strategies to treat ischemic diseases have been proposed but the ideal way has to be determined. We explored whether human placenta-derived mesenchymal cells (hPDMCs) can be used for this purpose because placenta is very rich in vessels. First, production of human vascular endothelial growth factor (hVEGF) from hPDMCs was examined. The amount of hVEGF secreted by hPDMCs was similar to the amount produced by HeLa cells. hVEGF was barely detected in human umbilical vein endothelial cells (hUVECs) or human peripheral blood mononuclear cells. hVEGF secreted from hPDMCs stimulated the proliferation of hUVECs, indicating its biological activity. Transplantation of hPDMCs to the ischemic limbs of NOD/Shi-scid mice significantly improved the blood flow of the affected limbs. Blood vessel formation was more prominently observed in the limbs of treated mice as compared to the control mice. Real-time RT-PCR revealed that hPDMCs produced hVEGF for at least 7 days after transplantation. Thus, transplantation of hPDMCs could potentially be a promising treatment for human ischemic diseases.
- Published
- 2004
- Full Text
- View/download PDF
49. Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin.
- Author
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Ito HO, Soutome S, Nokihara K, and Inoue M
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Epitope Mapping, Female, Fibronectins chemistry, Fibronectins immunology, Mice, Mice, Inbred BALB C, Fibronectins metabolism, Lactobacillaceae metabolism, Repetitive Sequences, Amino Acid
- Abstract
To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.
- Published
- 2004
- Full Text
- View/download PDF
50. Immunodominance of conformation-dependent B-cell epitopes of protein antigens.
- Author
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Ito HO, Nakashima T, So T, Hirata M, and Inoue M
- Subjects
- Animals, Antibodies, Monoclonal chemistry, B-Lymphocytes chemistry, Chickens, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Epitopes chemistry, Fimbriae, Bacterial metabolism, Hybridomas, Immunoblotting, Lymphocytes metabolism, Male, Mice, Mice, Inbred BALB C, Muramidase chemistry, Porphyromonas gingivalis metabolism, Protein Conformation, Protein Denaturation, Protein Structure, Quaternary, Protein Structure, Tertiary, Serratia marcescens metabolism, Streptococcus mutans metabolism, B-Lymphocytes immunology
- Abstract
Immunodominance of conformational epitopes over linear ones in four proteins was quantified making use of the B-cell hybridoma technology. The proteins were immunized in their native forms into BALB/c mice, and clonal frequencies of B-cell hybridomas that produce antibodies to the native and denatured forms were determined, using ELISA and immunoblotting. All 16 monoclonal antibodies (mAbs) to Porphyromonas gingivalis fimbria were suggested to recognize conformational epitopes expressed by the oligomer. Ten out of 14 mAbs to Serratia marcescens fimbria and 13 of 15 mAbs to hen lysozyme were also specific to their conformational epitopes. In contrast, all 18 mAbs to a surface protein of Streptococcus mutans, termed PAc, reacted to both the native and denatured forms, thereby indicating the immunodominance of linear epitopes in this protein. The results suggest that B-cell epitopes of proteins possessing stable tertiary or quaternary structures are predominantly expressed by the higher-order structures.
- Published
- 2003
- Full Text
- View/download PDF
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