Publications (19)51.16 Total impact
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Article: Electrical stimulation accelerates neuromuscular junction formation through ADAM19/neuregulin/ErbB signaling in vitro.
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ABSTRACT: The mechanism by which electrical stimulation affects formation of neuromuscular junctions (NMJ) remains unknown. NG108-15, a neural cell line, is commonly used in in vitro co-culture models of myotubes to observe synapse formation; therefore, we employed this model to observe the effects of electrical stimulation on NMJ formation. Initially, L6 cells were differentiated and NG108-15 cells were then added to the same culture dish. After 2 and 3 days of co-culture, the cells were electrically stimulated at 50V and 0.5Hz for 0, 5, 30, and 60min (C, ES5, ES30, and ES60 groups, respectively) and were analyzed after co-culture for 4 days. Immunofluorescence experiments showed significantly increased aggregation of acetylcholine receptors and inhibition of neural outgrowth in the ES30 and ES60 groups. Furthermore, ADAM19 and phospho-ErbB3 were found to be specifically localized in co-cultured NG108-15 cells. Immunoblotting demonstrated that synapsin 1, ADAM19 precursor and its activated form, phospho-ErbB3, and ERK1 protein levels had increased in an electrical stimulation period-dependent manner. Thus, we found that electrical stimulation accelerated NMJ formation, possibly through activation of ADAM19/neuregulin/ErbB signaling in NG108-15 cells.Neuroscience Letters 04/2013; · 2.11 Impact Factor -
Article: Simulated microgravity facilitates cell migration and neuroprotection after bone marrow stromal cell transplantation in spinal cord injury.
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ABSTRACT: INTRODUCTION: Recently, cell-based therapy has gained significant attention for the treatment of central nervous system diseases. Although bone marrow stromal cells (BMSCs) are considered to have good engraftment potential, challenges due to in vitro culturing, such as a decline in their functional potency, have been reported. Here, we investigated the efficacy of rat BMSCs (rBMSCs) cultured under simulated microgravity conditions, for transplantation into a rat model of spinal cord injury (SCI). METHODS: rBMSCs were cultured under two different conditions: standard gravity (1G) and simulated microgravity attained using the 3D-clinostat. After 7 days of culture, the rBMSCs were analyzed morphologically, by RT-PCR and immunostaining, and were used for grafting. Adult rats were employed for constructing SCI models using a weight-dropping method, and were grouped into three experimental groups for comparison. rBMSCs cultured under 1G and simulated microgravity were transplanted intravenously immediately after SCI. We evaluated the hindlimb functional improvement for three weeks. Tissue repair after SCI was examined by calculating the cavity area ratio and immunohistochemistry. RESULTS: rBMSCs cultured under simulated microgravity expressed Oct-4 and CXCR4, in contrast to those cultured under 1G conditions. Therefore, rBMSCs cultured under simulated microgravity were considered to be in an undifferentiated state and thus to possess high migration ability. After transplantation, grafted rBMSCs cultured under microgravity exhibited greater survival at the periphery of the lesion, and the motor function of the rats which received these grafts improved significantly compared to the rats which received rBMSCs cultured under 1G. In addition, rBMSCs cultured under microgravity were thought to have greater trophic effects on reestablishment and survival of host spinal neural tissues because cavity formations were reduced and apoptosis-inhibiting factor expression was high at the periphery of the SCI lesion. CONCLUSIONS: Here we show that transplantation of rBMSCs cultured under simulated microgravity facilitates functional recovery from SCI rather than those cultured under 1G condition.Stem Cell Research & Therapy 04/2013; 4(2):35. · 3.21 Impact Factor -
Article: Influence of dual-task performance on muscle and brain activity.
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ABSTRACT: The simultaneous performance of multiple tasks is often required in daily life. The dual-task paradigm has been used extensively to evaluate the ability to perform simultaneous behavioral tasks. However, relatively few studies have been carried out to determine the muscle and brain activity underlying dual-task performance. This study determined the influence of single-task and dual-task conditions on muscle and brain activity by measuring ankle dorsiflexion, surface electromyography, and magnetoencephalography. Increased muscle activation and variability in the dorsiflexion interval was observed during dual-task performance compared with single-task performance, whereas brain activity in the contralateral motor and frontal areas was reduced. Incidental movement of the non-task-related ankle (contralateral ankle) showed a different pattern of brain activity during the dual-task performance in another experiment. These findings suggest that the activity decreased in several information-processing areas under dual-task conditions as a result of decentralization of attention. Activity in the right and left motor fields may play a role in the dual-task performance, as indicated by changes in brain activity during dual-task performance in two groups classified according to the activity level of the contralateral ankle.Im Alltag müssen oft mehrere Aufgaben gleichzeitig bewältigt werden. Das Dual-Task-Paradigma wurde im umfangreichen Maße zur Evaluation der Fähigkeit der gleichzeitigen Durchführung von verhaltensorientierten Aufgaben angewandt. Allerdings wurden bisher nur relativ wenige Studien zur Ermittlung der Muskel- und Hirnaktivität bei der Dual-Task-Leistung durchgeführt. Die vorliegende Studie maß anhand von Knöcheldorsalflexion, Oberflächen-Elektromyographie und Magnetoenzephalographie, welchen Einfluss die Single-Task- und Dual-Task-Bedingungen auf die Muskel- und Hirnaktivität haben. Bei der Dual-Task-Leistung wurde während des Dorsalflexionsintervalls im Gegensatz zur Single-Task-Leistung eine erhöhte Muskelaktivität und -variabilität beobachtet, während die Hirnaktivität in den kontralateralen motorischen und frontalen Zentren reduziert war. Die zufällige Bewegung des nicht-aufgabenbezogenen (kontralateralen) Knöchels wies bei einem anderen Experiment ein abweichendes Muster der Hirnaktivität bei der Dual-Task-Leistung auf. Diese Ergebnisse legen den Schluss nahe, dass die Aktivität in mehreren informationsbearbeitenden Zonen unter Dual-Task-Bedingungen infolge der Dezentralisierung der Aufmerksamkeit abnahm. Die Aktivität in den rechten und linken motorischen Zentren wirkt sich möglicherweise auf die Dual-Task-Leistung aus, wie durch Veränderungen der Hirnaktivität bei der Dual-Task-Leistung in beiden Gruppen angezeigt, die nach Aktivitätslevel des kontralateralen Knöchels klassifiziert sind.A menudo en nuestra vida diaria debemos realizar múltiples actividades de forma simultánea. El paradigma de tarea dual ha sido ampliamente utilizado para evaluar la capacidad de realizar tareas conductuales simultáneas. Sin embargo, es relativamente pequeño el número de estudios que se han llevado a cabo con el fin de determinar la actividad muscular y cerebral que forma la base de la ejecución de una tarea dual. Este estudio determinó la influencia de las condiciones de tarea simple y tarea dual en la actividad muscular y cerebral, gracias a la medición de la dorsiflexión del tobillo, la electromiografía de superficie y la magnetoencefalografía. Durante la ejecución de la tarea dual se observó un aumento de la activación muscular y la variabilidad en el intervalo de dorsiflexión en comparación con la tarea simple, mientras que se vio una disminución de la actividad cerebral en el área frontal y el área motora contralateral. El movimiento secundario del tobillo no implicado en la tarea (tobillo contralateral) presentó un patrón distinto de actividad cerebral durante la ejecución de la tarea dual en otro experimento. Estos hallazgos sugieren que la actividad disminuye en varias áreas de procesamiento de la información bajo las condiciones de tarea dual como resultado de la descentralización de la atención. La actividad de las áreas motoras izquierda y derecha puede desempeñar un papel importante en la realización de una tarea dual, tal y como indican los cambios producidos en la actividad cerebral durante la ejecución de la tarea dual en dos grupos clasificados en función del nivel de actividad del tobillo contralateral.L'exécution simultanée de plusieurs tâches est souvent nécessaire dans la vie quotidienne. Le paradigme des tâches doubles est très communément utilisé pour évaluer la capacité d'exécuter simultanément des tâches comportementales. Toutefois, relativement peu d'études ont été menées pour déterminer l'activité musculaire et cérébrale sous-jacente à l'exécution de tâches doubles. Cette étude a déterminé l'influence des conditions de tâches simples et doubles sur l'activité musculaire et cérébrale en mesurant la dorsiflexion de la cheville, l'électromyographie de surface et la magnétoencéphalographie. Une activation accrue des muscles et une plus grande variabilité dans l'intervalle ont été observées au cours de la flexion dorsale lors de l'exécution d'une double tâche par rapport à une tâche simple, tandis que l'activité du cerveau dans les zones frontale et motrice controlatérale a diminué. Le mouvement secondaire de la cheville non associée à la tâche (cheville controlatérale) a montré une tendance différente de l'activité cérébrale pendant l'exécution de la double tâche dans une autre expérience. Ces résultats suggèrent une diminution de l'activité dans plusieurs régions responsables du traitement de l'information dans des conditions de tâche double en raison de la décentralisation de l'attention. L'activité dans les champs moteurs droit et gauche peut jouer un rôle dans l'exécution des tâches doubles, comme indiqué par les changements observés dans l'activité du cerveau pendant l'exécution de tâches doubles chez deux groupes classés selon le niveau d'activité de la cheville controlatérale.International journal of rehabilitation research. Internationale Zeitschrift fur Rehabilitationsforschung. Revue internationale de recherches de readaptation 11/2012; · 0.36 Impact Factor -
Article: Electrical stimulation enhances neurogenin2 expression through β-catenin signaling pathway of mouse bone marrow stromal cells and intensifies the effect of cell transplantation on brain injury.
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ABSTRACT: Bone marrow stromal cells (BMSCs) have received significant attention for its use in neural regeneration. However, neural replacement by transplanted BMSCs was not very effective. Recently, the gene transfection method has improved the capability of cell transplantation; however, this method results in canceration and immune rejection. We induced the differentiation of mouse BMSCs into neural cells using electrical stimulation and transplanted the cells into traumatic brain injury (TBI) model mice. We found that the electrically stimulated cells have good potential to differentiate into neural cells and contribute to recovery from TBI without differentiating into astrocytes. In addition, we found that electrical stimulation enhanced neurogenin2 (Ngn2) expression. Ngn2 is involved in neural differentiation and inhibits astrocytic differentiation during cell growth. Furthermore, we found that this enhancement of Ngn2 expression occurred through β-catenin signaling pathway. This study may contribute to the use of BMSCs for neural replacement in central nervous system diseases.Neuroscience Letters 11/2012; · 2.11 Impact Factor -
Article: Regulation of hematopoietic stem cells using protein transduction domain-fused Polycomb.
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ABSTRACT: The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.Experimental hematology 05/2012; 40(9):751-760.e1. · 3.11 Impact Factor -
Article: Simulated microgravity maintains the undifferentiated state and enhances the neural repair potential of bone marrow stromal cells.
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ABSTRACT: Recently, regenerative medicine with bone marrow stromal cells (BMSCs) has gained significant attention for the treatment of central nervous system diseases. Here, we investigated the activity of BMSCs under simulated microgravity conditions. Mouse BMSCs (mBMSCs) were isolated from C57BL/6 mice and harvested in 1G condition. Subjects were divided into 4 groups: cultured under simulated microgravity and 1G condition in growth medium and neural differentiation medium. After 7 days of culture, the mBMSCs were used for morphological analysis, reverse transcription (RT)-polymerase chain reaction, immunostaining analysis, and grafting. Neural-induced mBMSCs cultured under 1G conditions exhibited neural differentiation, whereas those cultured under simulated microgravity did not. Moreover, under simulated microgravity conditions, mBMSCs could be cultured in an undifferentiated state. Next, we intravenously injected cells into a mouse model of cerebral contusion. Graft mBMSCs cultured under simulated microgravity exhibited greater survival in the damaged region, and the motor function of the grafted mice improved significantly. mBMSCs cultured under simulated microgravity expressed CXCR4 on their cell membrane. Our study indicates that culturing cells under simulated microgravity enhances their survival rate by maintaining an undifferentiated state of cells, making this a potentially attractive method for culturing donor cells to be used in grafting.Stem cells and development 05/2011; 20(5):893-900. · 4.15 Impact Factor -
Article: Neuromagnetic beta oscillation changes during motor imagery and motor execution of skilled movements.
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ABSTRACT: We showed the differences in brain activities during motor imagery and motor execution when performing single skilled movements using magnetoencephalography. The tasks included finger tapping and chopstick usage with the dominant or nondominant hand. Chopstick usage with the nondominant hand was an unfamiliar task and required higher skill. Neuromagnetic data were processed by fast Fourier transformation, and β band event-related synchronization was evaluated. Beta oscillation changes were observed in the right and left sensorimotor cortices during both tasks; however, the ipsilateral changes were smaller during motor imagery than during motor execution. These results suggest that motor imagery of skilled movement tasks causes a smaller neuronal burden in the sensorimotor cortex.Neuroreport 03/2011; 22(5):217-22. · 1.66 Impact Factor -
Article: Effects of simulated microgravity on proliferation and chemosensitivity in malignant glioma cells.
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ABSTRACT: A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(-3)G. We cultured human malignant glioma cell lines in a 3D-clinostat (CL group) and examined the growth properties and chemosensitivity of the cells compared to cells cultured under normal 1G conditions (C group). The growth rate was significantly inhibited in the CL group, but without cell cycle change. Mitochondrial activity was also inhibited in the CL group. Thus, inhibition of malignant glioma proliferation occurred that could be attributed to deceleration of mitosis. Chemosensitivity to cisplatin (cis-diamminedichloroplatinum(II), CDDP) in the CL group was significantly enhanced compared to the C group. This method has significant potential as a treatment of malignant gliomas and a tool for understanding developmental biology.Neuroscience Letters 08/2009; 463(1):54-9. · 2.11 Impact Factor -
Article: Low-intensity pulsed ultrasound accelerates osteoblast differentiation and promotes bone formation in an osteoporosis rat model.
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ABSTRACT: We examined the effects of low-intensity pulsed ultrasound (LIPUS) on cell differentiation, bone mineralized nodule formation and core-binding factor A1 (Cbfa1) expression in a normal human osteoblast (NHOst) cell line and bone formation in an osteoporosis animal model. NHOst cells were cultured in vitro in medium with or without LIPUS stimulation. The ultrasound stimulation frequency was 1.0 MHz at an intensity of 30 mW/cm(2) for 20 min. Rats were divided into a sham-operated group (Sham) and an ovariectomized group (OVX). The right femur was treated with LIPUS (Sham-LIPUS and OVX-LIPUS) and the left femur was left untreated (Sham-CON and OVX-CON). LIPUS stimulation accelerated bone nodule formation and enhanced alkaline phosphatase activity. The expression levels of Cbfa1 decreased and calcification occurred earlier and more frequently in the LIPUS than in the CON groups. The wet weight of the femur increased in OVX rats with LIPUS stimulation. Morphological images showed an increase in trabecular spongiosa in the OVX-LIPUS group. LIPUS accelerated osteogenesis. Moreover, since LIPUS prevents bone loss, it may be a promising treatment for osteoporosis.Pathobiology 06/2009; 76(3):99-107. · 1.18 Impact Factor -
Article: Reciprocal expression of Bmi1 and Mel-18 is associated with functioning of primitive hematopoietic cells.
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ABSTRACT: The Polycomb-group (PcG) genes regulate global gene expression in many biological processes, including hematopoiesis, by manipulating specific target genes. It is known that various PcG genes regulate self-renewal of hematopoietic stem cells (HSCs). Here we have shown that the reciprocal expression of PcG proteins regulates self-renewal and differentiation of HSCs. We used murine and human bone marrow cells and evaluated the reciprocal expression of PcG proteins on the basis of their respective intranuclear distributions. PcG-gene expression in HSCs was knocked down by small interfering RNAs. The function of each gene in HSCs was analyzed in vitro and in vivo. Cells were either Bmi1-positive or Mel-18-positive. The Bmi1-positive cells contained very little amounts of Mel-18 and vice versa. The bmi1-knockdown marrow cells did not show HSC function, while the mel-18-knockdown marrow cells showed increased stem cell function. Results of the analysis on human cells were similar to those observed in case of murine cells. In a clinical investigation, transplantation using sources with a low Bmi1 to Mel-18 ratio was associated with early hematopoietic recovery. Reciprocal expression of Bmi1 and Mel-18 regulated HSC function. Here, we observed that expression of the PcG genes-bmi1 and mel-18-is correlated with self-renewal and differentiation of HSCs. Thus, it was suggested that the balance between Bmi1 and Mel-18 regulates self-renewal of HSCs.Experimental hematology 05/2009; 37(7):857-866.e2. · 3.11 Impact Factor -
Article: LIF-free embryonic stem cell culture in simulated microgravity.
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ABSTRACT: Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.PLoS ONE 02/2009; 4(7):e6343. · 4.09 Impact Factor -
Article: Detrimental effects of microgravity on mouse preimplantation development in vitro.
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ABSTRACT: Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (microG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under microG. However, although we obtained 75 healthy offspring from microG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under microG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under microG environment in a mammal, but normal preimplantation embryo development might require 1G.PLoS ONE 02/2009; 4(8):e6753. · 4.09 Impact Factor -
Article: Neural stem cells improve learning and memory in rats with Alzheimer's disease.
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ABSTRACT: We investigated whether neural stem cells (NSC) with transgenic expression of human nerve growth factor (hNGF) transplanted into the brain could offer a therapeutic option for the treatment of Alzheimer's disease (AD). We infused okadaic acid into rat lateral ventricles to establish a chronic AD animal model. In addition, NSC were stably transduced with hNGF and enhanced green fluorescent protein (eGFP) genes (NSC-hNGF-eGFP) by using a recombination adeno-associated virus serotype 2 (rAAV2) vector. These genetically modified stem cells were grafted into the cerebral cortex of AD rats. AD model rats showed significant damage in learning and memory function, with the formation of senile plaques and neurofibrillary tangles in the cerebral cortex. The transferred hNGF gene conferred stable and high levels of protein expression in NSC in vitro. Moreover, the NSC-hNGF-eGFP, but not the NSC, survived, integrating into the host brain and enhancing cognitive performance after transplantation. The injection of okadaic acid into rat lateral ventricles constitutes a promising animal model for investigating selective aspects of AD. rAAV2-mediated hNGF delivery can render long-term and stable transduction of hNGF in NSC. NSC-hNGF-eGFP transplantation may offer a viable therapeutic approach for treatment of AD.Pathobiology 02/2008; 75(3):186-94. · 1.18 Impact Factor -
Article: Floating culture promotes the maintenance of hematopoietic stem cells.
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ABSTRACT: In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine.FEBS Letters 11/2007; 581(24):4645-50. · 3.54 Impact Factor -
Article: Microgravity potentiates stem cell proliferation while sustaining the capability of differentiation.
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ABSTRACT: A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(3) G. Here we report that human mesenchymal stem cells (hMSCs) cultured in a 3D-clinostat (group CL) showed marked proliferation (13-fold in a week) compared with cells cultured under normal conditions of 1 G (group C) (4-fold in a week). Flow cytometry revealed a 6-fold increase in the number of hMSCs double-positive for CD44/CD29 or CD90/CD29 in group CL after 7 days in culture, compared with group C. Telomere length remained the same in cells from both groups during culturing. Group C cells showed increasing expression levels of type II collagen and aggrecan over the culture period, whereas group CL cells showed a decrease to undetectable levels. Pellets of hMSCs from each group were explanted into cartilagedefective mice. The transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed only noncartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat possess the strong proliferative characteristic of stem cells and retain their ability to differentiate into hyaline cartilage after transplantation. On the contrary, cells cultured in a 1-G environment do not maintain these features. Simulated microgravity may thus provide an environment to successfully expand stem cell populations in vitro without culture supplements that can adversely affect stem cell-derived transplantations. This method has significant potential for regenerative medicine and developmental biology.Stem Cells and Development 01/2007; 15(6):921-9. · 4.46 Impact Factor -
Article: Novel electrical stimulation sets the cultured myoblast contractile function to 'on'.
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ABSTRACT: In the present study, the effect of electrical stimulation was examined for the ability to induce morphological, physiological, and molecular biological effects on myoblasts during cell differentiation. L6 rat myoblasts were electrically stimulated by newly developed methods on culture days 6, 8, 10 and 12. This electrical stimulation accelerated the appearance of myotubes, and subsequently produced spontaneously contracting muscle fibers. Measurement of membrane potential showed that the contracting cell had functional ion channels and gap junctional intercellular communication. In the electrically stimulated cells, an enhanced expression of MyoD family and M-cadherin was also observed. Expression of connexin 43 was increased and maintained at a high level in the electrically stimulated cells. This is the first demonstration of in vitro induction of myoblasts in spontaneously contractile muscle fibers by intermittent stimulation. This novel method for induction of myoblast differentiation represents an important advance in cell therapy.Pathobiology 02/2006; 73(6):288-94. · 1.18 Impact Factor -
Article: Self-protection mechanism in D-cycloserine-producing Streptomyces lavendulae. Gene cloning, characterization, and kinetics of its alanine racemase and D-alanyl-D-alanine ligase, which are target enzymes of D-cycloserine.
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ABSTRACT: An antibiotic, D-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K(i) value (920 microM) was obviously higher (40 approximately 100-fold) than those for E. coli DdlA (9 microM) or DdlB (27 microM). The high K(i) value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.Journal of Biological Chemistry 11/2004; 279(44):46143-52. · 4.77 Impact Factor -
Article: Self-protection Mechanism in d-Cycloserine-producing Streptomyces lavendulae
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ABSTRACT: An antibiotic, d-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the Ki value (920 μm) was obviously higher (40∼100-fold) than those for E. coli DdlA (9 μm) or DdlB (27 μm). The high Ki value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.Journal of Biological Chemistry 10/2004; 279(44):46143-46152. · 4.77 Impact Factor -
Article: Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells
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ABSTRACT: Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen αI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of β3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.Cell Biology International.
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Top Journals
- Neuroscience Letters (3)
- Journal of Biological Chemistry (2)
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- Experimental hematology (2)
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Institutions
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2006–2013
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Hiroshima University
- • Department of Bio-Environmental Adaptation Sciences
- • Department of Pediatrics
Hiroshima-shi, Hiroshima-ken, Japan
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