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Takeshi Teramura,
Yuta Onodera,
Toshiyuki Takehara,
John Frampton, Toshiki Matsuoka,
Syunsuke Ito,
Koichi Nakagawa,
Yoshihisa Miki,
Yoshihiko Hosoi,
Chiaki Hamanishi,
Kanji Fukuda
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ABSTRACT: Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology, and can strongly influence stem cell fate. Here we demonstrate that severe-low O ₂ concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs), and that MSCs induced under severe hypoxic conditions function as normal MSCs; i.e., they repopulate after cloning, express specific markers (Vimentin, CD29, CD90, CD105 and CD140a) and differentiate into adipocytes, osteoblasts and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs.
Cell Transplantation 08/2012; · 5.13 Impact Factor
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Yuji Higashimoto,
Noritsugu Honda,
Toshiyuki Yamagata, Toshiki Matsuoka,
Kazushige Maeda,
Rhyuji Satoh,
Osamu Nishiyama,
Hiroyuki Sano,
Takashi Iwanaga,
Takayuki Miyara,
Masato Muraki,
Katsuyuki Tomita,
Hiroaki Kume,
Ichiro Miyai,
Yuji Tohda,
Kanji Fukuda
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ABSTRACT: Exertional dyspnea is the primary symptom that limits exercise in patients with chronic obstructive pulmonary disease (COPD). It is unknown which activated brain area is associated with this symptom in COPD patients.
To investigate the activation of cortical areas associated with dyspnea during exercise in COPD patients.
COPD patients (n = 10) and age-matched controls (n = 10) performed mild-intensity constant work rate cycle exercise (40% of their symptom-limited peak work rates) for 10 min, while cerebral hemodynamics and oxygenation were measured by near-infrared spectroscopy (NIRS). Ventilatory responses (breathing pattern and pulmonary gas exchange) and Borg scale ratings of dyspnea and leg fatigue were measured during exercise. Three NIRS probes were placed over the prefrontal and temporoparietal cortical regions of the subjects' heads. Changes in cortical oxyhemoglobin (oxy-Hb), deoxyhemoglobin (deoxy-Hb), and total hemoglobin (total Hb) concentrations from baseline recordings were measured. Increased oxy-Hb (oxygenation) was assumed to reflect cortical activation.
Oxy-Hb concentration was significantly increased in the prefrontal region during exercise in both groups but not in the temporoparietal regions. The change in prefrontal oxy-Hb concentration of COPD patients was not different from that of controls. Dyspnea scores were positively correlated with changes in oxy-Hb concentrations of the prefrontal regions in both groups. Multivariate analysis showed that oxy-Hb concentration in the prefrontal region was the best predictor of dyspnea in both groups.
Exertional dyspnea was related to activation (oxygenation) of the prefrontal cortex in COPD patients and control subjects.
Respiration 04/2011; 82(6):492-500. · 2.26 Impact Factor
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ABSTRACT: Abnormal mechanical stress loaded on the cartilage leads to the osteoarthritis (OA). Although intraarticular hyaluronan (HA) injection is an effective treatment for OA, the underlying mechanism has not been made clear.
Mechanical compression was loaded on the bovine cartilage using the Biopress system. Proteoglycan (PG) and reactive oxygen species (ROS) synthesis were measured with [(35)S] incorporation and fluorescent dye, respectively. Accumulation of peroxynitrite was determined with western blotting using nitrotyrosine antibody.
Mechanical compression inhibited PG synthesis and enhanced ROS. Externally added HA reversed stress-inhibited PG synthesis and attenuated ROS synthesis. HA also significantly decreased the generation of nitrotyrosine.
HA neutralized stress-enhanced ROS synthesis and resulted in the reversing of PG synthesis. These data suggest that HA plays an anabolic effect as an antioxidant.
Agents and Actions 12/2009; 59(6):471-7. · 1.59 Impact Factor
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Miyuri Kawasumi,
Yuichi Unno, Toshiki Matsuoka,
Megumi Nishiwaki,
Masayuki Anzai,
Tomoko Amano,
Tasuku Mitani,
Hiromi Kato,
Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Satoshi Kishigami,
Kazuya Matsumoto
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ABSTRACT: Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos--the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.
Molecular Reproduction and Development 11/2008; 76(4):342-50. · 2.53 Impact Factor
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Kazunobu Tsunemoto,
Masayuki Anzai, Toshiki Matsuoka,
Mikiko Tokoro,
Seung-Wook Shin,
Tomoko Amano,
Tasuku Mitani,
Hiromi Kato,
Yoshihiko Hosoi,
Kazuhiro Saeki,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre-implantation embryos, and examined the promoters for cis-acting elements of 5'-flanking region to obtain the best promoter for inducing oocyte-specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E-box region at position -72 of the H1oo promoter and at the -180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (-1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E-box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes.
Molecular Reproduction and Development 08/2008; 75(7):1104-8. · 2.53 Impact Factor
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Toshiki Matsuoka,
Manabu Sato,
Mikiko Tokoro,
Seung-Wook Shin,
Atsuto Uenoyama,
Kazunari Ito,
Syuji Hitomi,
Tomoko Amano,
Masayuki Anzai,
Hiromi Kato,
Tasuku Mitani,
Kazuhiro Saeki,
Yoshihiko Hosoi,
Akira Iritani,
Kazuya Matsumoto
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ABSTRACT: We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.
Journal of Reproduction and Development 07/2008; 54(3):192-7. · 1.46 Impact Factor
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ABSTRACT: We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.
Journal of Reproduction and Development 07/2006; 52(3):429-38. · 1.46 Impact Factor
