Takeshi Imamura

Ehime University, Matuyama, Ehime, Japan

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Publications (117)558.03 Total impact

  • Takeshi Imamura · Yusuke Oshima · Takashi Saitou
    Nippon rinsho. Japanese journal of clinical medicine 08/2015; 73(8):1246-9.
  • M Saitoh · K Endo · S Furuya · M Minami · A Fukasawa · T Imamura · K Miyazawa
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    ABSTRACT: The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor β (TGF-β) in certain kinds of cancer cells through the induction of Snail, a key regulator of EMT. We have previously found that TGF-β remarkably induces Snail expression in cooperation with Ras signals; however, the underlying mechanism of this synergism has not yet been determined. Here, we demonstrate that signal transducer and activator of transcription 3 (STAT3) acts as a mediator that synergizes TGF-β and Ras signals. The overexpression of STAT3 enhanced Snail induction, whereas siRNA-mediated knockdown of STAT3 inhibited it. The STAT3-YF mutant, which has Tyr 705 substituted with Phe, did not enhance Snail induction. Several STAT3 mutants lacking transcriptional activity also failed to enhance it; however, the putative STAT3-binding elements in the Snail promoter regions were not required for STAT3-mediated Snail induction. Protein inhibitor of activated STAT3 (PIAS3) inhibited the enhanced Snail promoter activity induced by TGF-β and Ras. The interaction between PIAS3 and STAT3 was reduced by TGF-β in cells harboring oncogenic Ras, whereas TGF-β promoted the binding of PIAS3 to Smad3, a crucial mediator of TGF-β signaling. Therefore, these findings suggest that STAT3 enhances Snail induction when it is dissociated from PIAS3 by TGF-β in cooperation with Ras signals.Oncogene advance online publication, 11 May 2015; doi:10.1038/onc.2015.161.
    Oncogene 05/2015; DOI:10.1038/onc.2015.161 · 8.56 Impact Factor
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    ABSTRACT: Bone modeling and remodeling are cellular events during which osteoblast lineage cells and osteoclasts interact. During these events, cells undergo drastic changes with time as they become differentiated. Their morphology, topology, and activity are affected by other cells and the extracellular matrix. Since the mechanisms underlying the cellular events of bone metabolism have not been elucidated, there is a need for systems to analyze these cellular networks and their microenvironments spatiotemporally at the cellular level. Here we report a novel in vitro system for reconstituting the bone cell network of osteoclasts, osteoblasts, and osteocytes in the mineralized nodule, allowing for observation of bone modeling and remodeling phenomena by 2-photon microscopy. Using this system, the change in morphology of osteoblasts from cuboidal to flat cells was observed and measured during the formation of mineralized nodules. Furthermore, the recruitment of osteoblasts to resorption pits and their replenishment by newly formed matrices were successfully observed, providing strong evidence for the coupling of bone resorption and bone formation at cellular level. During such remodeling cycle, flat osteoblasts that survived more than 7weeks were recruited to resorption pits, where they became cuboidal osteoblasts that express osteocalcin. This novel system permitted the elucidation of cellular behavior during bone modeling and remodeling, and can be used to analyze cellular events involved in bone metabolism. Copyright © 2015. Published by Elsevier Inc.
    Bone 03/2015; 76. DOI:10.1016/j.bone.2015.02.030 · 4.46 Impact Factor
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    ABSTRACT: Arkadia, a positive regulator of Smad-dependent signaling via the transforming growth factor-β (TGF-β) family, is an E3 ubiquitin ligase that induces ubiquitylation and proteasome-dependent degradation of TGF-β suppressors such as Smad7, c-Ski, and SnoN. In this study, we examined the effects of Arkadia on bone morphogenetic protein (BMP)-induced osteoblast differentiation. Knockdown of Arkadia reduced mineralization and expression of osteoblast differentiation markers. Furthermore, we showed that Smad6, a BMP-specific inhibitory Smad, is a target of Arkadia: wild-type (WT) Arkadia, but not the C937A (CA) mutant lacking E3 ubiquitin-ligase activity, induced ubiquitylation and proteasome-dependent degradation of Smad6. Accordingly, protein levels of Smad6, Smad7, and c-Ski were elevated in MEFs from Arkadia KO mice. Finally, expression of Arkadia attenuated blockade of BMP signaling by Smad6 in a transcriptional reporter assay. These results demonstrate that Smad6 is a novel target of Arkadia, and that Arkadia positively regulates BMP signaling via degradation of Smad6, Smad7, and c-Ski/SnoN. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
    Journal of Biochemistry 03/2015; 158(1). DOI:10.1093/jb/mvv024 · 3.07 Impact Factor
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    ABSTRACT: Osteoarthritis (OA) restricts the daily activities of patients and significantly decreases their quality of life. The development of non-invasive quantitative methods for properly diagnosing and evaluating the process of degeneration of articular cartilage due to OA is essential. Second harmonic generation (SHG) imaging enables the observation of collagen fibrils in live tissues or organs without staining. In the present study, we employed SHG imaging of the articular cartilage in OA model mice ex vivo. Consequently, three-dimensional SHG imaging with successive image processing and statistical analyses allowed us to successfully characterize histopathological changes in the articular cartilage consistently confirmed on histological analyses. The quantitative SHG imaging technique presented in this study constitutes a diagnostic application of this technology in the setting of OA.
    Biomedical Optics Express 02/2015; 6(2):405. DOI:10.1364/BOE.6.000405 · 3.50 Impact Factor
  • Takeshi Imamura · Yusuke Oshima
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    ABSTRACT: Intravital fluorescence imaging technique is a method that enables to visualize behavior and function of molecules and cells in vivo . Recently, the intravital fluorescence imaging technique has developed rapidly. In particular, development of functional fluorescent proteins and fluorescent dyes in conjunction with appropriate equipment including two-photon fluorescence microscope and image processing software enables to investigate bone and cartilage tissues in vivo . Here we show recent technological development of the intravital fluorescence imaging in biology. Moreover, we would discuss about issues of the intravital fluorescence imaging and the application of the fluorescent imaging approaches to bone and cartilage biology.
    Clinical calcium 01/2015; 25(8):1175-81.
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    ABSTRACT: In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.
    PLoS Computational Biology 12/2014; 10(12):e1003957. DOI:10.1371/journal.pcbi.1003957 · 4.87 Impact Factor
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    ABSTRACT: We have previously reported that transforming growth factor β (TGF-β) plays an essential role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde-assisted isolation of regulatory elements and chromatin immunoprecipitation followed by sequencing (FAIRE-seq and ChIP-seq) analyses indicated the cooperation of Smad2/3 with c-Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c-Fos is required for their nuclear translocation. The gene expression of Nfatc1, a key regulator of osteoclastogenesis, was regulated by RANKL and TGF-β, and c-Fos binding to open chromatin sites was suppressed by inhibition of TGF-β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c-Fos deficiency. These results suggest that TGF-β regulates RANKL-induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c-Fos. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 11/2014; 30(5). DOI:10.1002/jbmr.2418 · 6.59 Impact Factor
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    ABSTRACT: Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.
    International Journal of Molecular Sciences 11/2014; 15(11):19971-86. DOI:10.3390/ijms151119971 · 2.86 Impact Factor
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    ABSTRACT: Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon–excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon–excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high–pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low–numerical aperture (NA) objective lens and high–peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec.
    Biomedical Optics Express 10/2014; 5(10):3311-3325. DOI:10.1364/BOE.5.003311 · 3.50 Impact Factor
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    ABSTRACT: No abstract is available for this article.
    Cancer Science 10/2014; 105(10). DOI:10.1111/cas.12528 · 3.53 Impact Factor
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    ABSTRACT: Objective Mechanical loading on the bone is sensed by osteocytes. Sclerostin is a molecule secreted by osteocytes that is downregulated by mechanical loading; therefore, its expression level is a potent sensor that indicates the spatial transduction of biomechanical properties in bone. This study applied macroconfocal microscopy to observe the spatial response of alveolar bone to orthodontic forces after immunofluorescence using anti-sclerostin antibodies. Design Orthodontic tooth movement with the Ni-Ti closed-coil spring was applied between the upper bilateral incisors and the left first molar of mice. Four days after this application, the animals were subjected to multimodal confocal fluorescence imaging analyses. Results Obvious downregulation of sclerotin in the osteocytic lacuna-canalicular system (LCS) was observed specifically in tensile sites of alveolar bone. Confocal-based three-dimensional fluorescence morphometry further quantitatively demonstrated that the distribution and expression of sclerostin in the tensile sites was significantly reduced compared to that observed in the corresponding control sites. Interestingly, the levels of sclerotin signals in the compression sites were significantly higher than those observed in the control sites, although the distribution of sclerotin was not significantly different. Conclusions Our observations suggest that spatial changes in the level and distribution of sclerostin in the alveolar LCS trigger successive bone remodeling due to orthodontic tooth movement. The multimodal confocal imaging analyses applied in this work will enhance comprehensive understanding regarding the spatial regulation of molecules of interest from the tissue to the cellular level.
    Archives of Oral Biology 09/2014; 60(1). DOI:10.1016/j.archoralbio.2014.08.013 · 1.88 Impact Factor
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    ABSTRACT: Background and Objective: Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. Materials and Methods: In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. Results: For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. Conclusion: The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future.(C) 2014 Wiley Periodicals, Inc.
    Lasers in Surgery and Medicine 09/2014; 46(7). DOI:10.1002/lsm.22266 · 2.61 Impact Factor
  • Y. Oshima · H. Horiuchi · T. Ogata · A. Hikita · H. Miura · T. Imamura
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    ABSTRACT: Fluorescent imaging technique is a promising method and has been developed for in vivo applications in cellular biology. In particular, nonlinear optical imaging technique, multi-photon microscopy has make it possible to analyze deep portion of tissues in living animals such as axons of spinal code. Traumatic spinal cord injuries (SCIs) are usually caused by contusion damages. Therefore, observation of spinal cord tissue after the contusion injury is necessary for understanding cellular dynamics in response to traumatic SCI and development of the treatment for traumatic SCI. Our goal is elucidation of mechanism for degeneration of axons after contusion injuries by establishing SCI model and chronic observation of injured axons in the living animals. Firstly we generated and observed acute SCI model by contusion injury. By using a multi-photon microscope, axons in dorsal cord were visualized approximately 140 micron in depth from the surface. Immediately after injury, minimal morphological change of spinal cord was observed. At 3 days after injury, spinal cord was swelling and the axons seem to be fragmented. At 7 days after injury, increased degradation of axons could be observed, although the image was blurred due to accumulation of the connective tissue. In the present study, we successfully observed axon degeneration after the contusion SCI in a living animal in vivo. Our final goal is to understand molecular mechanisms and cellular dynamics in response to traumatic SCIs in acute and chronic stage.
    Proceedings of SPIE - The International Society for Optical Engineering 02/2014; DOI:10.1117/12.2041042 · 0.20 Impact Factor
  • Takeshi Imamura · Yusuke Oshima · Atsuhiko Hikita
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    ABSTRACT: Members of the transforming growth factor-β (TGF-β) family, including TGF-βs, activin, and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration, and apoptosis. TGF-β family signaling is mainly mediated by membranous serine/threonine kinase receptors and intracellular Smad proteins. This signaling is tightly regulated by various posttranslational modifications including ubiquitination. Several E3 ubiquitin ligases play a crucial role in the recognition and ubiquitin-dependent degradation of TGF-β family receptors, Smad proteins and their interacted proteins to regulate positively and negatively TGF-β family signaling. In contrast, non-degradative ubiquitin modifications also regulate TGF-β family signaling. Recently, in addition to protein ubiquitination, deubiquitination by deubiquitinating enzymes has been reported to control TGF-β family signaling pathways. Interestingly, more recent studies suggest that TGF-β signaling is not only regulated via ubiquitination and/or deubiquitination, but also it relies on ubiquitination for its effect on other pathways. Thus, ubiquitin modifications play key roles in TGF-β family signal transduction and cross-talk between TGF-β family signaling and other signaling pathways. Here, we review the current understandings of the positive and negative regulatory mechanisms by ubiquitin modifications that control TGF-β family signaling.
    Journal of Biochemistry 10/2013; 154(6). DOI:10.1093/jb/mvt097 · 3.07 Impact Factor
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    ABSTRACT: Remodeling of collagen fibrils is involved in a variety of physiological and pathological processes including development, tissue repair, and metastasis. Fibroblast-populated collagen gel contraction has been employed as a model system to investigate the collagen fibril remodeling within three-dimensional collagen matrices. Research on collagen gel contraction is also important for understanding the mechanism underlying connective tissue repair, and for design considerations for engineered tissues in regenerative medicine. Second harmonic generation (SHG) is a non-linier optical effect by which well-ordered protein assemblies, including collagen fibrils, can be visualized without any labeling, and used for a noninvasive imaging of collagen fibrils in the skin. Here we demonstrate that the remodeling of collagen fibrils in the fibroblast-populated collagen gel can be analyzed by SHG imaging with a multiphoton microscope. Two models of collagen gel contraction (freely versus restrained contraction) were prepared, and orientation of fibroblasts, density, diameter, and distribution of collagen fibrils were examined by multiphoton fluorescent and SHG microscopy. Three-dimensional construction images revealed vertical and horizontal orientation of fibroblasts in freely and restrained gel contraction, respectively. Quantitative analysis indicated that collagen fibrils were accumulated within the gel and assembled into the thicker bundles in freely but not restrained collagen gel contraction. We also found that actomyosin contractility was involved in collagen fibril remodeling. This study elucidates how collagen fibrils are remodeled by fibroblasts in collagen gel contraction, and also proves that SHG microscopy can be used for the investigation of the fibroblast-populated collagen gel.
    Cell Structure and Function 10/2013; 38. DOI:10.1247/csf.13017 · 2.35 Impact Factor
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    ABSTRACT: Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.
    Scientific Reports 07/2013; 3:2231. DOI:10.1038/srep02231 · 5.58 Impact Factor
  • Takeshi Imamura · Atsuhiko Hikita · Yusuke Oshima · Shigehiro Koga
    Japanese Journal of Thrombosis and Hemostasis 01/2013; 24(6):582-587. DOI:10.2491/jjsth.24.582
  • Takeshi Imamura · Atsuhiko Hikita · Yusuke Oshima · Tadahiro Iimura
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    ABSTRACT: Intravital optical imaging technique is a promising method that allows us to investigate complex vital phenomena in vivo . In particular, discovery and development of unique fluorescent proteins and smart fluorescent dyes in conjunction with appropriate equipments such as two-photon microscopy and image processing software allow visualization of the behavior and function of bone and cartilage-related cells as well as the microenvironment of the cells in bone and cartilage in living animals. Here we show recent technological development and issues of the intravital optical imaging and the application of the fluorescent imaging approaches to bone and cartilage biology.
    Clinical calcium 01/2013; 23(12):1767-73.
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    ABSTRACT: Smad7 is an inhibitory molecule induced by members of the transforming growth factor-β (TGF-β) family, including TGF-β, activin, nodal and bone morphogenetic proteins (BMPs). To elucidate the in vivo functions of Smad7, we generated conditional Smad7-knockout mice in which the Mad homology 2 (MH2) domain and the poly (A) signal sequence were flanked with loxP sites (floxed). The Smad7-floxed mice exhibited no obvious phenotype. Smad7 total-null mice on a C57BL/6 background died within a few days of birth, whereas mice with an ICR background developed to adulthood but were significantly smaller than wild-type mice. Unexpectedly, phospho-Smad2 and phospho-Smad3 were decreased in Smad7-deficient mouse embryonic fibroblast (MEF) cells, whereas phospho-Smad1/5/8 was similarly expressed in wild-type and Smad7-deficient MEF cells. Moreover, expression levels of TGF-β type I receptor (ALK5) were higher in Smad7-deficient MEF cells than in wild-type MEF cells. Plasminogen activator inhibitor-1 (PAI-1) and inhibitor of differentiation-1 (Id-1) mRNA were similarly expressed in wild-type and Smad7-deficient MEF cells. Some differences were observed in mitogen-activated protein kinase (MAPK)-signalling between wild-type and Smad7-deficient MEF cells. We demonstrated that Smad7 plays an important role in normal mouse growth and provide a useful tool for analysing Smad7 functions in vivo.
    Journal of Biochemistry 02/2012; 151(6):621-31. DOI:10.1093/jb/mvs022 · 3.07 Impact Factor

Publication Stats

6k Citations
558.03 Total Impact Points

Institutions

  • 2011–2015
    • Ehime University
      • Proteo-Science Center
      Matuyama, Ehime, Japan
    • Japan Science and Technology Agency (JST)
      Edo, Tōkyō, Japan
  • 1997–2011
    • Japanese Foundation for Cancer Research
      • Department of Urology
      Edo, Tōkyō, Japan
  • 2005–2008
    • The University of Tokyo
      • • Research Center for Advanced Science and Technology
      • • Faculty & Graduate School of Medicine
      Edo, Tōkyō, Japan
  • 1998–2008
    • Kagoshima University
      • Department of Orthopaedic Surgery
      Kagosima, Kagoshima, Japan
  • 2006
    • Tokyo Medical and Dental University
      • Division of Advanced Therapeutic Sciences
      Edo, Tōkyō, Japan
  • 2004
    • Tokyo Institute of Technology
      • Department of Biological Sciences
      Edo, Tōkyō, Japan