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Negoro H.,Kyoto University | Kimura Y.,Institute for Frontier Medical science | Matsuoka R.,Nippon Shinyaku Co. | Tanaka M.,Nippon Shinyaku Co. | And 2 more authors.
Journal of Urology | Year: 2011

Purpose: Basic fibroblast growth factor is a candidate causative factor of detrusor overactivity in bladder outlet obstruction cases through up-regulation of the gap junction protein connexin 43. We addressed the transcriptional and behavioral implications of this axis. Materials and Methods: Cx43 and Cx45 mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction in the bladder of a rat bladder outlet obstruction model and in cultured rat bladder smooth muscle cells with and without basic fibroblast growth factor treatment. Involvement of the extracellular signal regulated kinase 1/2-activator protein-1 pathway was evaluated by immunofluorescence study and a promoter-reporter assay in bladder smooth muscle cells. The effect of basic fibroblast growth factor on micturition behavior was measured in unrestrained rats under a 12-hour light/dark cycle using a controlled release system from gelatin hydrogels fixed on the bladder. The expression of extracellular signal regulated kinase 1/2 and connexin 43 protein was assessed by Western blotting of rat bladder protein. Results: Cx43 but not Cx45 mRNA expression was increased in the bladder of the obstruction model and in bladder smooth muscle cells treated with basic fibroblast growth factor. The mitogen-activated and extracellular signal-regulated kinase kinase inhibitor PD98059 blocked the stimulatory effect of basic fibroblast growth factor on connexin 43 protein expression and promoter activity, which was also decreased by mutation or deletion of an activator protein-1 cis-element of the connexin 43 promoter. In vivo application of basic fibroblast growth factor on the bladder increased urinary frequency during the latter half of the dark phase, ie the late active phase of rats (F = 5.1, 2-way ANOVA p <0.05). The expression of phospho-extracellular signal regulated kinase 1/2 and connexin 43 protein was increased in the bladder. Conclusions: The extracellular signal regulated kinase 1/2-activator protein-1-connexin 43 axis could be a potential therapeutic target for increased urinary frequency. © 2011 American Urological Association Education and Research, Inc.


Kadonosono T.,Tokyo Institute of Technology | Kuchimaru T.,Tokyo Institute of Technology | Yamada S.,Akita University | Murakami A.,Kobe Biomedical Laboratory | And 17 more authors.
PLoS ONE | Year: 2011

An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg) mice that carry HRE/ODD-luciferase (HOL) gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential. © 2011 Kadonosono et al.


Fujiwara H.,Tohoku University | Saiki Y.,Tohoku University | Sato M.,Tohoku University | Sakamoto N.,Tohoku University | And 3 more authors.
Journal of Vascular Surgery | Year: 2010

Objectives: We investigated the outcomes of reinforcing anastomotic sites using (1) nonbiodegradable polytetrafluoroethylene (PTFE) felt, (2) biodegradable polyglycolic acid (PGA) felt, and (3) PGA felt with basic fibroblast growth factor (bFGF) in a canine descending thoracic aortic replacement model. Methods: Thirty-seven beagles underwent descending thoracic aorta replacement using a prosthetic graft with one of the above-mentioned reinforcements or no reinforcement for controls. Histologic evaluations were carried out 1 month and 3 months after surgery. The biomechanical strength of the anastomosis was assessed along the longitudinal axis of the aortic segments using a tensile tester. Local compliance at the anastomotic site was also evaluated in the circumferential direction. Results: The media was significantly thinner in the PTFE group than in the control group (65.8% ± 5.1% vs 95.0% ± 9.3% of normal thickness; P < .05). Relative to the control group, the adventitial layer was significantly thinner in the PTFE group (42.3% ± 8.2% of control; P < .05) but significantly thicker in the PGA and the PGA + bFGF groups (117.2% ± 11.3% and 134.1% ± 14.2% of control, respectively; P < .05). There were more vessels in the adventitial layer in the PGA + bFGF group than in the control, PTFE, and PGA groups (29.2 ± 2.1/mm2 vs 13.8 ± 0.8, 5.4 ± 0.7, 17.0 ± 1.3/mm2, respectively; P < .01). There were no significant differences between the four groups in the failure force at anastomotic sites. Local compliance at the anastomotic site was higher in the PGA group than that in the PTFE group (11.6 ± 1.6 10-6 m2/N vs 5.6 ± 1.9 10-6 m2/N; P < .05). Conclusion: Reinforcement of the experimental aortic wall with PTFE felt resulted in thinning of the media and adventitia and fewer vessels at the anastomotic site. These histologic changes were not observed when biodegradable felt was used. The bFGF failed to augment the modification of the aortic wall with the exception of increased adventitial vessel number. Biomechanical strength of the anastomosis along the longitudinal axis was comparable in all four groups; however, local vascular compliance was better in the biodegradable PGA felt group. © 2010 Society for Vascular Surgery.


Nishimura H.,Institute for Integrated Cell Material science | Nishimura H.,Institute for Frontier Medical science | Ritchie K.,Purdue University | Kasai R.S.,Institute for Integrated Cell Material science | And 10 more authors.
Journal of Cell Biology | Year: 2013

Fluorescence microscopy is used extensively in cellbiological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for =10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 μm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. ©2013 Nishimura et al.


Nakayama R.,National Cancer Center Research Institute | Nakayama R.,Keio University | Mitani S.,National Cancer Center Research Institute | Nakagawa T.,National Cancer Center Research Institute | And 10 more authors.
American Journal of Surgical Pathology | Year: 2010

Poorly differentiated type synovial sarcoma (PDSS) is a variant of synovial sarcoma characterized by predominantly round or short-spindled cell morphology. Although accumulating evidence from clinicopathologic studies suggests a strong association between this variant of synovial sarcoma and poor prognosis, little has been reported on the molecular basis of PDSS. To gain insights into the mechanism(s) that underlie the emergence of PDSS, we analyzed the gene expression profiles of 34 synovial sarcoma clinical samples, including 5 cases of PDSS, using an oligonucleotide microarray. In an unsupervised analysis, the 34 samples fell into 3 groups that correlate closely with histologic subtypes: monophasic, biphasic, and poorly differentiated types. PDSS was characterized by down-regulation of genes associated with neuronal and skeletal development and cell adhesion. Moreover, upregulation of genes on a specific chromosomal locus, 8q21.11, was identified. This locus-specific transcriptional activation in PDSS was confirmed by reverse transcriptase-PCR analysis of 9 additional synovial sarcoma samples. Our results indicate that PDSS tumors constitute a distinct group based on expression profiles. © 2010 by Lippincott Williams & Wilkins.


Nishigaki T.,Kyoto University | Teramura Y.,Kyoto University | Suemori H.,Institute for Frontier Medical science | Iwata H.,Kyoto University
Cryobiology | Year: 2010

Human embryonic stem (hES) cells are expected to be useful in the fields of regenerative medicine and tissue engineering due to their pluripotency. Therefore, it is necessary to establish highly efficient and reliable methods for the cryopreservation of hES cells. We have cryopreserved cynomolgus and human ES cells by the vitrification method, using a chemically-defined dimethyl sulfoxide (Me2SO)-free and serum-free medium composed of Euro-Collins solution as a base medium and 40% (v/v) ethylene glycol (EG) and 10% (w/v) polyethylene glycol (PEG) as cryoprotectants. When the vitrification and the cryoprotectants were combined, the recovery ratio of hES cells was 22.9 ± 7.7%, compared to 0.4 ± 0.2% when the conventional slow-freezing method was used. After the cryopreservation and thawing cycle, hES cells were easily cultured and expressed undifferentiated cell markers such as Nanog, Oct-4, SSEA-4, and alkaline phosphatase activity after several subculturing steps. We also found that the pluripotency of hES cells was maintained, as demonstrated by teratoma formation of ES cells transplanted into severe combined immunodeficient (SCID) mice. Thus, we conclude that we have successfully cryopreserved primate ES cells with high efficiency using a Me2SO-free, chemically-defined medium. © 2009 Elsevier Inc. All rights reserved.


Nagae G.,Research Center for Advanced Science and Technology | Isagawa T.,Research Center for Advanced Science and Technology | Shiraki N.,Kumamoto University | Fujita T.,Research Center for Advanced Science and Technology | And 15 more authors.
Human Molecular Genetics | Year: 2011

Epigenetic regulation is essential in determining cellular phenotypes during differentiation. Although tissuespecific DNA methylation has been studied, the significance of methylation variance for tissue phenotypes remains unresolved, especially for CpG-poor promoters. Here, we comprehensively studied methylation levels of 27 578 CpG sites among 21 human normal tissues from 12 anatomically different regions using an epigenotyping beadarray system. Remarkable changes in tissue-specific DNA methylation were observed within CpG-poor promoters but not CpG-rich promoters. Of note, tissue-specific hypomethylation is accompanied by an increase in gene expression, which gives rise to specialized cellular functions. The hypomethylated regions were significantly enriched with recognition motifs for transcription factors that regulate cell-type-specific differentiation. To investigate the dynamics of hypomethylation events, we analyzed methylation levels of the entire APOA1 gene locus during in vitro differentiation of embryonic stem cells toward the hepatic lineage. A decrease in methylation was observed after day 13, coinciding with alpha-feto- protein detection, in the vicinity of its transcription start sites (TSSs), and extends up to ~200 bp region encompassing the TSS at day 21, equivalent to the hepatoblastic stage. This decrease is even more pro- nounced in the adult liver, where the entire APOA1 gene locus is hypomethylated. Furthermore, when we compared the methylation status of induced pluripotent stem (iPS) cells with their parental cell, IMR-90, we found that fibroblast-specific hypomethylation is restored to a fully methylated state in iPS cells after reprogramming. These results illuminate tissue-specific methylation dynamics in CpG-poor promoters and provide more comprehensive views on spatiotemporal gene regulation in terminal differentiation. © The Author 2011. Published by Oxford University Press. All rights reserved.


Toguchida J.,Institute for Frontier Medical science
Clinical calcium | Year: 2014

The application of iPS cells for diseases in the locomotive system includes the cell therapy using bone or cartilage cells and the understanding of pathogenesis and drug discovery for intractable diseases using patients' derived iPS cells. To promote these researches, the proper knowledge concerning the process from iPS cells to multipotential mesenchymal stromal cell (MSC) and terminally differentiated cells is inevitable. In addition, because there are several different ways to induce terminally differentiation, the biological quality of cells induced by each method should be compared in future.


Yamamizu K.,Institute for Frontier Medical science | Yamashita J.K.,Institute for Frontier Medical science
Circulation Journal | Year: 2011

Cyclic adenosine monophosphate (cAMP) is an important second messenger mediating physiological functions, including metabolism, gene expression, cell growth and differentiation. Recently, we demonstrated novel roles of cAMP pathway in endothelial cell (EC) differentiation and arterial_venous specification using an embryonic stem cell differentiation system. These studies offered a concept that vascular formation is accomplished by a 2-layered mechanism: (1) a basal mechanism for common EC differentiation, whereby vascular endothelial growth factor (VEGF) signaling plays a central role in the basal mechanism, and (2) a vascular diversification mechanism working on the basis of common EC differentiation. Vascular diversification, such as artery and vein formation, can be only achieved by enacting specific machineries in the presence of the basal EC machinery. cAMP/protein kinase A signaling contributes to common EC differentiation through upregulation of the VEGF-A receptors, Flk1 and neuropilin1. On the other hand, cAMP can activate phosphatidylinositol-3 kinase, which induces an arterial fate in vascular progenitors via dual activation of Notch and β-catenin signaling as an arterial-specific machinery. cAMP signaling thus plays a pivotal role in both the basal and diversification machinery during vascular development.


Watanabe M.,Children's Hospital of Philadelphia | Watanabe M.,University of Tsukuba | Li H.,Children's Hospital of Philadelphia | Roybal J.,Children's Hospital of Philadelphia | And 6 more authors.
Tissue Engineering - Part A | Year: 2011

Myelomeningocele (MMC) is a common and devastating malformation. As an alternative to fetal surgical repair, tissue engineering has the potential to provide a less invasive approach for tissue coverage applicable at an earlier stage of gestation. We have previously evaluated the use of gelatin hydrogel composites composed of gelatin sponges and sheets as a platform for tissue coverage of the MMC defect in the retinoic acid induced fetal rat model of MMC. In the current study, we compare our previous composite with gelatin microspheres as a scaffold for tissue ingrowth and cellular adhesion within the amniotic fluid environment. We also examine the relative efficacy of various bioactive protein coatings on the adhesion of amniotic fluid cells to the construct within the amniotic cavity. We conclude from this study that gelatin microspheres are as effective as gelatin sponges as a scaffold for cellular ingrowth and amniotic fluid cell adhesion and that collagen type I and fibronectin coatings enhance amniotic fluid cell adhesion to the gelatin-based scaffolds. These findings support the potential for the development of a tissue-engineered injectable scaffold that could be applied by ultrasound-guided injection, much earlier and less invasively than sponge or sheet-based composites. © 2011 Mary Ann Liebert, Inc.

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