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Sample records for c2c12 cells up-take

  1. Investigation of interactions between poly-L-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation

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    G Ciofani

    2010-04-01

    Full Text Available G Ciofani1, L Ricotti1, S Danti2,3, S Moscato4, C Nesti2, D D’Alessandro2,4, D Dinucci5, F Chiellini5, A Pietrabissa3, M Petrini2,3, A Menciassi1,61Scuola Superiore Sant’Anna, Pisa, Italy; 2CUCCS-RRMR, Center for the Clinical Use of Stem Cells – Regional Network of Regenerative Medicine, 3Department of Oncology, Transplants and Advanced Technologies, 4Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy; 5Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab, UdR INSTM, Department of Chemistry and Industrial Chemistry, University of Pisa, San Piero a Grado, Italy; 6Italian Institute of Technology, Genova, ItalyAbstract: Boron nitride nanotubes (BNNTs have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-L-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription – polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.Keywords: boron nitride nanotubes, C2C12 cells, cytocompatibility, up-take, differentiation, MyoD, connexin 43

  2. Proliferative effect of Hachimijiogan, a Japanese herbal medicine, in C2C12 skeletal muscle cells

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    Takeda T

    2015-02-01

    Full Text Available Takashi Takeda,1,2 Kenji Tsuiji,2 Bin Li,2 Mari Tadakawa,2 Nobuo Yaegashi2 1Division of Women’s Health, Research Institute of Traditional Asian Medicine, Kinki University School of Medicine, Osaka, Japan; 2Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan Background: Hachimijiogan (HJG, Ba-Wei-Di-Huang-Wan in Chinese, is one of the most popular herbal medicines in Japanese Kampo. HJG is often prescribed for the prevention and treatment of age-related diseases. Muscle atrophy plays an important role in aging-related disabilities such as sarcopenia. The purpose of this study was to investigate the possible beneficial effect of HJG on skeletal muscle.Methods: Cells of murine skeletal muscle myoblast cell line C2C12 were used as an in vitro model of muscle cell proliferation and differentiation. The effect of HJG on C2C12 cell proliferation and differentiation was assessed. We counted the number of myotubes morphologically to assess the degree of differentiation.Results: HJG treatment (200 µg/mL for 3 days significantly increased C2C12 cell number by 1.23-fold compared with that of the control. HJG promoted the proliferation of C2C12 cells through activation of the ERK1/2 signaling pathway without affecting the Akt signaling pathway. HJG did not affect the differentiation of C2C12 cells. Conclusion: HJG had beneficial effects on skeletal muscle myoblast proliferation. These findings may provide a useful intervention for the prevention and treatment of sarcopenia. Keywords: ERK1/2 signaling pathway, herbal medicine, myoblast, proliferation, sarcopenia

  3. Cobalt triggers necrotic cell death and atrophy in skeletal C2C12 myotubes

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    Rovetta, Francesca [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy); Stacchiotti, Alessandra [Institute of Human Anatomy, Department of Clinical and Experimental Sciences, University of Brescia, Brescia I-25123 (Italy); Faggi, Fiorella [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy); Catalani, Simona; Apostoli, Pietro [Unit of Occupational Health and Industrial Hygiene, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Brescia I-25123 (Italy); Fanzani, Alessandro, E-mail: fanzani@med.unibs.it [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy); Aleo, Maria Francesca, E-mail: aleo@med.unibs.it [Unit of Biotechnologies, Department of Molecular and Translational Medicine, University of Brescia, Brescia I-25123 (Italy); Interuniversity Institute of Myology (IIM) (Italy)

    2013-09-01

    Severe poisoning has recently been diagnosed in humans having hip implants composed of cobalt–chrome alloys due to the release of particulate wear debris on polyethylene and ceramic implants which stimulates macrophagic infiltration and destroys bone and soft tissue, leading to neurological, sensorial and muscular impairments. Consistent with this premise, in this study, we focused on the mechanisms underlying the toxicity of Co(II) ions on skeletal muscle using mouse skeletal C2C12 myotubes as an in vitro model. As detected using propidium iodide incorporation, increasing CoCl{sub 2} doses (from 5 to 200 μM) affected the viability of C2C12 myotubes, mainly by cell necrosis, which was attenuated by necrostatin-1, an inhibitor of the necroptotic branch of the death domain receptor signaling pathway. On the other hand, apoptosis was hardly detectable as supported by the lack of caspase-3 and -8 activation, the latter resulting in only faint activation after exposure to higher CoCl{sub 2} doses for prolonged time points. Furthermore, CoCl{sub 2} treatment resulted in atrophy of the C2C12 myotubes which was characterized by the increased expression of HSP25 and GRP94 stress proteins and other typical 'pro-atrophic molecular hallmarks, such as early activation of the NF-kB pathway and down-regulation of AKT phosphorylation, followed by the activation of the proteasome and autophagy systems. Overall, these results suggested that cobalt may impact skeletal muscle homeostasis as an inducer of cell necrosis and myofiber atrophy. - Highlights: • The effects of cobalt on muscle myofibers in vitro were investigated. • Cobalt treatment mainly causes cell necrosis in skeletal C2C12 myotubes. • Cobalt impacts the PI3K/AKT and NFkB pathways and induces cell stress markers. • Cobalt induces atrophy of C2C12 myotubes through the activation of proteasome and autophagy systems. • Co treatment triggers NF-kB and PI3K/AKT pathways in C2C12 myotubes.

  4. Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom

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    da Silva, Aline; Vieira, Rodolfo Paula; Mesquita-Ferrari, Raquel Agnelli; Cogo, José Carlos; Zamuner, Stella Regina

    2016-01-01

    Background Snakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells. Methodology C2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation. Results In non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom. Conclusion LLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory

  5. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  6. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

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    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  7. Developmental Changes is Expression of Beta-Adrenergic Receptors in Cultures of C2C12 Skeletal Muscle Cells

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    Young, Ronald B.; Bridge, K. Y.; Vaughn, J. R.

    2000-01-01

    beta-Adrenergic receptor (bAR) agonists have been reported to modulate growth in several mammalian and avian species, and bAR agonists presumably exert their physiological action on skeletal muscle cells through this receptor. Because of the importance of bAR regulation on muscle protein metabolism in muscle cells, the objectives of this study were to determine the developmental expression pattern of the bAR population in C2C12 skeletal muscle cells, and to analyze changes in both the quantity and isoform expression of the major muscle protein, myosin. The number of bAR in mononucleated C2C12 cells was approximately 8,000 bAR per cell, which is comparable with the population reported in several other nonmuscle cell types. However, the bar population increased after myoblast fusion to greater than 50,000 bAR per muscle cell equivalent. The reasons for this apparent over-expression of bAR in C2C12 cells is not known. The quantity of myosin also increased after C2C12 myoblast fusion, but the quantity of myosin was less than that reported in primary muscle cell cultures. Finally, at least five different isoforms of myosin heavy chain could be resolved in C2C12 cells, and three of these exhibited either increased or decreased developmental regulation relative to the others. Thus, C2C12 myoblasts undergo developmental regulation of bAR population and myosin heavy chain isoform expression.

  8. Effects of myostatin propeptide gene tranfection on glucose metabolism in cultured C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    张莎莎

    2014-01-01

    Objective To investigate the effects of recombinant adeno-associated virus-mediated myostatin propeptide(MPRO)on uptake and oxidation of glucose,and glycogen synthesis in C2C12 myotubes,as well as the associated molecular mechanism.Methods Mature C2C12myotubes were assigned to the following 6 groups:control,insulin,green fluorescent protein(GFP),insulin+

  9. Effect of dehydroepiandrosterone on insulin action and development of insulin-induced resistance in C2C12 muscle cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Dehydroepiandrosterone (DHEA), a precursor of androgens and estrogens, has been demonstrated to have effect of preventing insulin resistance and development of diabetes mellitus. Administration of testosterone appears to induce a marked insulin resistance. How these two hormones affect insulin resistance through regulation of sensitivity of tissues to insulin deserves further studies. Here, the effects of DHEA and testosterone on response to insulin in C2C12 muscle cells are analyzed. After 24 h of DHEA (10-6 mol/L) treatment, C2C12 cells showed an increased insulin- stimulated glucose uptake and enhanced activities of glycogen synthase (GS), phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH), whereas testosterone gave the opposite effects. Incubation of C2C12 cells with high-dose insulin (5×10-7 mol/L) for 24 hours decreased their sensitivity to insulin and led to a state of resistance as assessed on insulin-stimulated glucose uptake and activities of GS, PFK and PDH. Addition of DHEA to insulin-resistant C2C12 cells could reverse the response of these cells to high-dose insulin, but testosterone could further impair insulin sensitivity in insulin-resistant C2C12 cells. These results suggest that the two hormones may influence the development or inhibition of insulin-resistance in type 2 diabetes through regulating glucose uptake, glycogenesis and glycolysis to some extent.

  10. Truncated Human LMP-1 Triggers Differentiation of C2C12 Cells to an Osteoblastic Phenotype in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    LIM mineralization protein-1 (LMP-1) is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in vivo. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 (hLMP-1 [t]), lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C2C12 cells to the osteoblast lineage. C2C12 cells were transiently transduced with Ad5-hLMP-1(t)-green fluorescent protein or viral vector control. The expression of hLMP-1 (t) RNA and the truncated protein were examined. The results showed that hLMP-1(t) blocked myotube formation in C2C12 cultures and significantly enhanced the alkaline phosphatase (ALP) activity. In addition, the expressions of ALP,osteocalcin, and bone morphogenetic protein (BMP)-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP-1(t) can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1 (t)enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage.Therefore, C2C12 cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP- 1 action.

  11. Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells

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    Zhenguo Dong

    2015-01-01

    Full Text Available Our previous study reported that muscle cell enhancement factor 2C (MEF2C was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors.

  12. Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure

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    Bloemberg, Darin; Quadrilatero, Joe

    2016-01-01

    Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpa...

  13. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

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    Kodaka, Manami [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yang, Zeyu [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang (China); Nakagawa, Kentaro; Maruyama, Junichi [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Xu, Xiaoyin [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Sarkar, Aradhan; Ichimura, Ayana [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Nasu, Yusuke [Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Ozawa, Takeaki [Department of Chemistry, School of Science, The University of Tokyo, Tokyo (Japan); Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Ishigami-Yuasa, Mari [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Shigeru [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); Kagechika, Hiroyuki [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); and others

    2015-08-15

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced.

  14. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    International Nuclear Information System (INIS)

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced

  15. Establishment and Identification of a Stable Human ASB12-Expressed C2C12 Cell Line%稳定表达人ASB12的C2C12细胞系的建立及鉴定

    Institute of Scientific and Technical Information of China (English)

    文斗斗; 周军媚; 赵明一; 胡维新; 吴秀山; 王跃群

    2012-01-01

    The human ASB12 (Homo sapiens ankyrin repeat and SOCS box containing 12) protein contains five ANK (ankyrin repeat sequence) domains and a SOCS (suppressor of cytokine signaling) box domain, belonging to the ASBs family. It was reported that ASB12 especially expressed in skeletal and cardiac muscles of adult tissues, which suggested that ASB12 closely associated with skeleton muscle development. To construct a stable ASB12-expressed C2C12 cell line, the fusion expression plasmid pCMV-tag2B-ASB12, which was identified by enzyme digestion and sequencing analysis, was transfected into C2C12 cell by cationic polymer. After screening culture by G418, the expression of ASB12 was detected by immunofluorescfence, RT-PCR and Western-blotting. The C2C12 cell line that expressing ASB12 stably was established successfully, which provide a cell model for studying the molecular function of ASB12 in skeleton muscle development.%ASB12 (homo sapiens ankyrin repeat and SOCS box containing 12)蛋白含有5个ANK (ankyrin repeat sequence)序列和一个保守的SOCS (suppressor of cytokine signaling)盒结构域,是ASBs (human ankyrin repeat and SOCS box containing protein family,ASB family)家族的成员.人类ASB12基因在成体心肌和骨骼肌组织中特异表达,是成肌分化的候选基因.利用阳离子聚合物转染技术将重组表达质粒pCMV-tag2B-ASB 12转染小鼠骨骼肌细胞系C2C12细胞,通过G418筛选、免疫荧光检测、RT-PCR分析、Western blotting检测建立了稳定表达ASB12的细胞系C2C12-ASB12,为研究ASB12在骨骼肌发育及其相关功能提供有用的细胞研究模型.

  16. Graphene oxide-stimulated myogenic differentiation of C2C12 cells on PLGA/RGD peptide nanofiber matrices

    Science.gov (United States)

    Shin, Y. C.; Lee, J. H.; Kim, M. J.; Hong, S. W.; Oh, J.-W.; Kim, C.-S.; Kim, B.; Hyun, J. K.; Kim, Y.-J.; Han, D.-W.

    2015-07-01

    During the last decade, much attention has been paid to graphene-based nanomaterials because they are considered as potential candidates for biomedical applications such as scaffolds for tissue engineering and substrates for the differentiation of stem cells. Until now, electrospun matrices composed of various biodegradable copolymers have been extensively developed for tissue engineering and regeneration; however, their use in combination with graphene oxide (GO) is novel and challenging. In this study, nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 phage with RGD peptide displayed on its surface (RGD peptide-M13 phage) were prepared as extracellular matrix (ECM)-mimicking substrates. RGD peptide is a tripeptide (Arg-Gly-Asp) found on ECM proteins that promotes various cellular behaviors. The physicochemical properties of PLGA and RGD peptide-M13 phage (PLGA/RGD peptide) nanofiber matrices were characterized by atomic force microscopy, Fourier-transform infrared spectroscopy and thermogravimetric analysis. In addition, the growth of C2C12 mouse myoblasts on the PLGA/RGD peptide matrices was examined by measuring the metabolic activity. Moreover, the differentiation of C2C12 mouse myoblasts on the matrices when treated with GO was evaluated. The cellular behaviors, including growth and differentiation of C2C12 mouse myoblasts, were substantially enhanced on the PLGA/RGD peptide nanofiber matrices when treated with GO. Overall, these findings suggest that the PLGA/RGD peptide nanofiber matrices can be used in combination with GO as a novel strategy for skeletal tissue regeneration.

  17. Protein O-fucosyltransferase 1 expression impacts myogenic C2C12 cell commitment via the Notch signaling pathway.

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    Der Vartanian, Audrey; Audfray, Aymeric; Al Jaam, Bilal; Janot, Mathilde; Legardinier, Sébastien; Maftah, Abderrahman; Germot, Agnès

    2015-01-01

    The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7(+)/MyoD(-) cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.

  18. Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells

    Institute of Scientific and Technical Information of China (English)

    Min YANG; Qing-jun MA; Geng-ting DANG; Kang-tao MA; Ping CHEN; Chun-yan ZHOU

    2005-01-01

    Aim: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. Methods: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7)with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAVBMP7 at multiplicities of infection of 1× 105 vector genomes per cell and subsequent culture, C2C12 cells were assessed qualitatively for BMP7 production, alkaline phosphatase activity, osteocalcin production and Cbfal and MyoD expression.Results: C2C 12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated.Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. Conclusion: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

  19. Biocompatibility of Sylgard184 coated with different matrix materials and C2C12 cells%不同基质材料修饰的Sylgard184与C2C12细胞的相容性

    Institute of Scientific and Technical Information of China (English)

    王齐; 廖华; 秦建强; 余磊; 邱小忠; 于巧莲; 艾鹤英

    2009-01-01

    目的 筛选能提高硅酮橡胶弹性体(Sylgard184)与C2C12相容性的理想基质材料. 方法Sylgard184双组分以10:1的比例均匀混合,倒入6孔板的其中4孔,室温下静置固化,其余2孔做为空白对照培养组(A组);固化后的Sylgard184表面依次经过以下处理:I型胶原包被(B组)、层黏连蛋白包被(C组)、多聚赖氨酸包被(D组);未经包被(E组),每组共6个样本.在不同基质材料修饰的Sylgard 184表面培养C2C12细胞,利用倒置显微镜观察5组C2C12细胞的增殖、分化状态,流式细胞术(FCM)检测增殖培养48h后C2C12细胞的分裂增殖情况,RT-PCR检测增殖和分化培养48h后C2C12细胞内MyoD、myogenin mRNA的表达.结果 Sylgard184材料存在细胞毒性,E组接种的C2C12细胞在24h内全部漂浮死亡;D组的大多数细胞出现死亡,仅少数贴壁存活;而B、C两组材料包被后明显减少Syhgard 184的毒性,增强其表面与C2C12细胞的相容性,且C组细胞处于合成期的百分比以及增殖期的MyoD和分化期Myogenin基因mRNA的表达水平均显著高于A、B两组(P<0.05). 结论 经层黏连蛋白包被后的Sylgard184表面更有利于C2C12细胞的增殖及分化活性的表达.

  20. Dexamethasone-Induced Skeletal Muscle Atrophy Increases O-GlcNAcylation in C2C12 Cells.

    Science.gov (United States)

    Massaccesi, Luca; Goi, Giancarlo; Tringali, Cristina; Barassi, Alessandra; Venerando, Bruno; Papini, Nadia

    2016-08-01

    Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc. PMID:26728070

  1. Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype

    Science.gov (United States)

    Warita, Katsuhiko; Oshima, Nana; Takeda-Okuda, Naoko; Tamura, Jun-ichi; Hosaka, Yoshinao Z.

    2016-01-01

    Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion. PMID:27775651

  2. Methylcobalamin promotes proliferation and migration and inhibits apoptosis of C2C12 cells via the Erk1/2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Okamoto, Michio [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Tanaka, Hiroyuki, E-mail: tanahiro-osk@umin.ac.jp [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Kiyoshi [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kuroda, Yusuke [Department of Orthopaedic Surgery, Kansai Rosai Hospital, 3-1-69 Inabaso, Amagasaki, Hyogo 660-8511 (Japan); Nishimoto, Shunsuke; Murase, Tsuyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2014-01-17

    Highlights: •Methylcobalamin activated the Erk1/2 signaling pathway in C2C12 cells. •Methylcobalamin promoted the proliferation and migration in C2C12 cells. •C2C12 cell apoptosis during differentiation was inhibited by methylcobalamin. -- Abstract: Methylcobalamin (MeCbl) is a vitamin B12 analog that has some positive effects on peripheral nervous disorders. Although some previous studies revealed the effects of MeCbl on neurons, its effect on the muscle, which is the final target of motoneuron axons, remains to be elucidated. This study aimed to determine the effect of MeCbl on the muscle. We found that MeCbl promoted the proliferation and migration of C2C12 myoblasts in vitro and that these effects are mediated by the Erk1/2 signaling pathway without affecting the activity of the Akt signaling pathway. We also demonstrated that MeCbl inhibits C2C12 cell apoptosis during differentiation. Our results suggest that MeCbl has beneficial effects on the muscle in vitro. MeCbl administration may provide a novel therapeutic approach for muscle injury or degenerating muscle after denervation.

  3. Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways.

    Science.gov (United States)

    Pijet, Maja; Pijet, Barbara; Litwiniuk, Anna; Pajak, Beata; Gajkowska, Barbara; Orzechowski, Arkadiusz

    2013-02-01

    Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T(202/)Y(204)P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y(705)P-STAT3) and MEK (T(202/)Y(204)P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S(473)) and GSK-3β (S(9)) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S(9)P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y(216)P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.

  4. Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways.

    Science.gov (United States)

    Pijet, Maja; Pijet, Barbara; Litwiniuk, Anna; Pajak, Beata; Gajkowska, Barbara; Orzechowski, Arkadiusz

    2013-02-01

    Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T(202/)Y(204)P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y(705)P-STAT3) and MEK (T(202/)Y(204)P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S(473)) and GSK-3β (S(9)) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S(9)P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y(216)P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost. PMID:23201486

  5. Effect of alkyl glycerophosphate on the activation of peroxisome proliferator-activated receptor gamma and glucose uptake in C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp [Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Haniu, Hisao [Department of Orthopedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan)

    2013-04-12

    Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

  6. Construction of highly organized three-dimensional muscle tissue induced by C2C12 cells in vitro%C2C12细胞诱导构建三维骨骼肌组织

    Institute of Scientific and Technical Information of China (English)

    王齐; 廖华; 秦建强; 余磊; 艾鹤英; 汪海仪; 邱小忠

    2010-01-01

    目的 利用修饰并铸型后的Sylgard 184凹槽与C2C12细胞复合培养、诱导分化,获取三维极性骨骼肌组织. 方法 Sylgard 184双组分以10∶1的比例均匀混合并倒板,室温下静置固化并对其表面压槽铸型,Hank液冲洗凹槽,Matrigel和胶原的混合液均匀铺被凹槽底部,置生物安全柜待细胞基质自然干燥、紫外线照射消毒1h以上时接种C2C12细胞悬液,细胞增殖约80%汇合时改用分化培养基进行分化诱导,倒置显微镜下观察肌管的分化状态, RT-PCR方法检测肌管内myogenin和desmin基因mRNAs的表达,免疫荧光检测生肌转录因子myogenin和desmin蛋白的表达,扫描电镜观察肌管形态和肌管间的连接. 结果 C2C12细胞在Sylgard 184弹性体铸型压槽中培养分化7d后,倒置显微镜下可见肌管呈极性分化,且肌管之间融合紧密;21d后,扫描电镜检测可见肌管之间排列紧密且相互重叠,形成膜样结构,厚度可达0.15mm,具有三维性;RT-PCR、免疫荧光检测证实极性分化肌管内具有myogenin和desmin的阳性表达. 结论 修饰并铸型的Sylgard 184凹槽具有一定的方向引导效应,能促进C2C12细胞分化形成多核肌管,且肌管呈极性重叠排列,形成三维极性骨骼肌组织结构.

  7. Berberine Hydrochloride Protects C2C12 Myoblast Cells Against Oxidative Stress-Induced Damage via Induction of Nrf-2-Mediated HO-1 Expression.

    Science.gov (United States)

    Choi, Yung Hyun

    2016-09-01

    Preclinical Research The aim of the present study was to evaluate the effects of berberine hydrochloride (BBH), an isoquinoline alkaloid that can be isolated from a variety of herbs, on hydrogen peroxide (H2 O2 )-induced oxidative stress in C2C12 myoblasts and to investigate the molecular mechanisms involved in this process, especially the expression of the Nrf2/HO-1 pathway. BBH preconditioning attenuated H2 O2 -induced growth inhibition and DNA damage as well as apoptosis in C2C12 cells via suppression of the accumulation of intracellular reactive oxygen species (ROS). Treatment with BBHride alone effectively upregulated the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and elevated HO-1 activity. However, the protective effects of BBH against H2 O2 -induced ROS generation and cell growth reduction were abolished by an HO-1 inhibitor. Moreover, BBH-mediated induction and activation of HO-1 were reduced by genetic silencing of Nrf2 using small interfering RNA (siRNA). In addition, the effects of BBH against H2 O2 -induced ROS accumulation and growth inhibition were abrogated in C2C12 cells transfected with Nrf2 siRNA. Therefore, the present study demonstrated that BBH could protect C2C12 cells against oxidative stress-induced injury and this effect involved activation of the Nrf2/HO-1 pathway. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc. PMID:27535021

  8. Sirtuin 1 promotes the proliferation of C2C12 myoblast cells via the myostatin signaling pathway.

    Science.gov (United States)

    Wang, Liang; Zhang, Ting; Xi, Yongyong; Yang, Cuili; Sun, Chengcao; Li, Dejia

    2016-08-01

    Accumulating evidence suggests that Sirtuin (Sirt)1 serves a significant role in proliferation and differentiation of myoblast cells; however the signaling mechanisms involved remain to be established. Myostatin (MSTN), a member of transforming growth factor‑β family, is an vital regulator of myoblast, fibroblast growth and differentiation. To determine if MSTN is involved in the regulation of myoblast cell proliferation by Sirt1, the present study administrated the Sirt1 activator resveratrol, inhibitor nicotinamide (NAM) and MSTN inhibitor SB431542 to C2C12 myoblast cells. It was demonstrated that the Sirt1 activator, resveratrol, repressed, whereas the Sirt1 inhibitor, NAM, enhanced C2C12 myoblast cells proliferation in a Sirt1‑dependent manner. SB431542 promoted the proliferation of C2C12 myoblast cells and reversed the inhibition effect of NAM on C2C12 myoblast cell proliferation. Additionally, resveratrol upregulated the mRNA expression of MyoD, but inhibited the expression of MSTN. Additionally, NAM significantly repressed the expression of MyoD and the phosphorylation of P107 (p‑P107), but enhanced the expression of MSTN and the protein expression of P107. SB431542 significantly mitigated the effect of NAM on the expression of MyoD, P107 and p‑P107. Taken together, these results indicated that Sirt1 promotes the proliferation of C2C12 myoblast cells via the MSTN signaling pathway. PMID:27279047

  9. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    International Nuclear Information System (INIS)

    Highlights: → hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. → Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. → hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  10. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes

    OpenAIRE

    Wang, Yanxin; Watford, Malcolm

    2006-01-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. C...

  11. Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Gia-Ming [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Present address: The University of Chicago, Section of Hematology/Oncology, 900 E. 57th Street, Room 7134, Chicago, IL 60637 (United States); Bain, Lisa J., E-mail: lbain@clemson.edu [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States)

    2012-05-01

    Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2

  12. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    Abstract Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation. A. Grossi, M. A. Lawson; Department of Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark The process of muscle...... development and growth is a complex sequence of events whereby muscle cells respond to a number of stimuli in order to form organised muscle tissue. Increase in muscle mass is greatly influenced by the rate of skeletal muscle protein synthesis and degradation, processes that can be altered by mechanical...... forces. Stretch- or load-induced signaling is now beginning to be understood as a factor which affects the mass and phenotype of muscles as well as the expression of a number of proteins within muscle cells. Use of magnetic field to produce mechanical forces to stimulate cell populations has been well...

  13. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways, inflammatory cytokines and myogenic markers in H22-treated C2C12 cells

    Indian Academy of Sciences (India)

    Allur Subramaniyan Sivakumar; Inho Hwang

    2015-03-01

    The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA expression of myogenic regulatory factors, such as MyoD and myogenin, inflammatory pathways, such as TNF- and NF-kB, as well as proteolytic enzymes, such as -calpain and m-calpain. The pre-treatment of Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) on H2O2-treated C2C12 cells significantly down-regulated the mRNA expression of myogenin and MyoD when compared to those treated with H2O2-induced alone. Additionally, the mRNA expression of -calpain and m-calpain were significantly ( < 0.05) increased in H2O2-treated C2C12 cells, whereas pre-treatment with Sunphenon/Polyphenon significantly down-regulated the above genes, namely -calpain and m-calpain. Furthermore, the mRNA expression of TNF- and NF-kB were significantly increased in H2O2-treated C2C12 cells, while pre-treatment with Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) significantly ( < 0.05) down-regulated it when compared to the untreated control group. Subsequent analysis of DNA degeneration and caspase activation revealed that Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) inhibited activation of caspase-3 and showed an inhibitory effect on DNA degradation. From this result, we know that, in stress conditions, -calpain may be involved in the muscle atrophy through the suppression of myogenin and MyoD. Moreover, Sunphenon may regulate the skeletal muscle genes/promote skeletal muscle recovery by the up-regulation of myogenin and MyoD and suppression of -calpain and inflammatory pathways and may regulate the apoptosis pathways. Our findings suggest that dietary supplementation of Sunphenon might reduce inflammatory events in muscle-associated diseases, such as myotube atrophy.

  14. Creatine Prevents the Structural and Functional Damage to Mitochondria in Myogenic, Oxidatively Stressed C2C12 Cells and Restores Their Differentiation Capacity.

    Science.gov (United States)

    Barbieri, Elena; Guescini, Michele; Calcabrini, Cinzia; Vallorani, Luciana; Diaz, Anna Rita; Fimognari, Carmela; Canonico, Barbara; Luchetti, Francesca; Papa, Stefano; Battistelli, Michela; Falcieri, Elisabetta; Romanello, Vanina; Sandri, Marco; Stocchi, Vilberto; Ciacci, Caterina; Sestili, Piero

    2016-01-01

    Creatine (Cr) is a nutritional supplement promoting a number of health benefits. Indeed Cr has been shown to be beneficial in disease-induced muscle atrophy, improve rehabilitation, and afford mild antioxidant activity. The beneficial effects are likely to derive from pleiotropic interactions. In accord with this notion, we previously demonstrated that multiple pleiotropic effects, including preservation of mitochondrial damage, account for the capacity of Cr to prevent the differentiation arrest caused by oxidative stress in C2C12 myoblasts. Given the importance of mitochondria in supporting the myogenic process, here we further explored the protective effects of Cr on the structure, function, and networking of these organelles in C2C12 cells differentiating under oxidative stressing conditions; the effects on the energy sensor AMPK, on PGC-1α, which is involved in mitochondrial biogenesis and its downstream effector Tfam were also investigated. Our results indicate that damage to mitochondria is crucial in the differentiation imbalance caused by oxidative stress and that the Cr-prevention of these injuries is invariably associated with the recovery of the normal myogenic capacity. We also found that Cr activates AMPK and induces an upregulation of PGC-1α expression, two events which are likely to contribute to the protection of mitochondrial quality and function. PMID:27610211

  15. Extracellular vesicles from a muscle cell line (C2C12 enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34

    Directory of Open Access Journals (Sweden)

    Roger D. Madison

    2014-02-01

    Full Text Available Introduction: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. Background: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. Methods: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Conclusion: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  16. Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion

    Directory of Open Access Journals (Sweden)

    Poussard S

    2015-02-01

    Full Text Available Sylvie Poussard,1,2 Marion Decossas,1,2 Olivier Le Bihan,1,2 Stéphane Mornet,3 Grégoire Naudin,1,2 Olivier Lambert1,2 1Institute of Chemistry and Biology of Membranes and Nanoobjects, University of Bordeaux, UMR5248, Pessac, France; 2Institute of Chemistry and Biology of Membranes and Nanoobjects, Centre National de la Recherche Scientifique, Institute of Chemistry and Biology of Membranes and Nanoobjects, UMR5248, Pessac, France; 3ICMCB, Institut de Chimie de la Matière Condensée de Bordeaux, CNRS UPR9048, Université de Bordeaux, Pessac, France Abstract: The use of silica nanoparticles for their cellular uptake capability opens up new fields in biomedical research. Among the toxicological effects associated with their internalization, silica nanoparticles induce apoptosis that has been recently reported as a biochemical cue required for muscle regeneration. To assess whether silica nanoparticles could affect muscle regeneration, we used the C2C12 muscle cell line to study the uptake of fluorescently labeled NPs and their cellular trafficking over a long period. Using inhibitors of endocytosis, we determined that the NP uptake was an energy-dependent process mainly involving macropinocytosis and clathrin-mediated pathway. NPs were eventually clustered in lysosomal structures. Myoblasts containing NPs were capable of differentiation into myotubes, and after 7 days, electron microscopy revealed that the NPs remained primarily within lysosomes. The presence of NPs stimulated the formation of myotubes in a dose-dependent manner. NP internalization induced an increase of apoptotic myoblasts required for myoblast fusion. At noncytotoxic doses, the NP uptake by skeletal muscle cells did not prevent their differentiation into myotubes but, instead, enhanced the cell fusion. Keywords: silica, nanoparticle, muscle, cell encapsulation, transmission electron microscopy, apoptosis

  17. Efeitos do ultra-som terapêutico contínuo sobre a proliferação e viabilidade de células musculares C2C12 Effects of continuous therapeutic ultrasound on proliferation and viability of C2C12 muscle cells

    Directory of Open Access Journals (Sweden)

    Paola Pelegrineli Artilheiro

    2010-06-01

    Full Text Available O ultra-som terapêutico (US é um recurso bioestimulante utilizado para propiciar reparo muscular de melhor qualidade e menor duração, mas o potencial terapêutico do US contínuo não está totalmente estabelecido. O objetivo deste trabalho foi avaliar o efeito do US contínuo sobre a proliferação e viabilidade de células musculares precursoras (mioblastos C2C12. Mioblastos C2C12 foram cultivados em meio de cultura contendo 10% de soro fetal bovino e irradiados com US contínuo nas freqüências de 1 e 3 MHz nas intensidades de 0,2 e 0,5 W/cm2, durante 2 e 5 minutos. A viabilidade e proliferação celular foram avaliadas após 24, 48 e 72 h de incubação. Grupos não-irradiados serviram como controle. Foram realizados experimentos independentes em cada condição acima, e os dados obtidos submetidos à análise estatística. Os resultados mostram que não houve diferença estatisticamente significativa na proliferação e viabilidade celular entre os mioblastos tratados com US e as culturas controles após os diferentes períodos de incubação, em todos os parâmetros avaliados. Conclui-se que o US contínuo, nos parâmetros avaliados, não foi capaz de alterar a proliferação e viabilidade dos mioblastos.Therapeutic ultrasound (US is a biophysical stimulation resource widely used in order to promote better, faster muscle repair, but the effectiveness of continuous US in treating injuries is not fully established. The aim of the present in vitro study was to assess the effects of continuous ultrasound on viability and proliferation of skeletal muscle precursor cells (C2C12 myoblasts. C2C12 myoblasts were cultured in a medium containing 10% foetal bovine serum and irradiated with continuous ultrasound at 1 and 3 MHz frequencies, at intensities of 0.2 and 0.5 W/cm² for 2 and 5 minutes. Cell viability and proliferation were assessed after different incubation periods (24, 48 and 72 h. Non-irradiated groups served as control and data were

  18. THE NUCLEOTIDE RECEPTORS ON MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; NELEMANS, A; VANDENAKKER, J; DENHERTOG, A

    1992-01-01

    1 The response of C2C12 mouse myotubes to stimulation with adenosine triphosphate (ATP) and other nucleotides was studied by measuring changes in membrane potential. 2 A transient hyperpolarization followed by a slowly declining depolarization of the cells was observed in the presence of ATP (10-mu-

  19. Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34)

    OpenAIRE

    Madison, Roger D.; McGee, Christopher; Rawson, Renee; Robinson, Grant A.

    2014-01-01

    Introduction: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy.Background: When a peripheral nerve is damaged, the communication between spinal cord ...

  20. 核仁素表达下调对C2C12细胞增殖与凋亡的影响%Effect of nucleolin down-regulation on the proliferation and apoptosis in C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    王慷慨; 蒋磊; 鄂顺梅; 刘可; 张玲莉; 刘梅冬; 肖献忠

    2005-01-01

    目的:探讨核仁素反义寡核苷酸对细胞增殖与凋亡的影响.方法:采用反义寡核苷酸技术以抑制C2C12细胞中核仁素的表达后,用MTT法检测细胞增殖状况,流式细胞术及DNA琼脂糖凝胶电泳检测细胞凋亡.结果:免疫印迹结果显示,反义寡核苷酸导入细胞后24 h,核仁素的表达受到明显抑制,同时反义寡核苷酸处理组细胞的增殖能力亦明显受到抑制,细胞凋亡百分率显著升高,并能检测到清晰的"梯状条带".而正义及随机核酸导入细胞后不能降低核仁素的表达,对细胞增殖及凋亡均无明显影响.结论:核仁素表达下调能抑制C2C12细胞增殖并能触发C2C12细胞凋亡.

  1. Inulin increases glucose transport in C2C12 myotubes and HepG2 cells via activation of AMP-activated protein kinase and phosphatidylinositol 3-kinase pathways.

    Science.gov (United States)

    Yun, Hee; Lee, Jong Hwa; Park, Chang Eun; Kim, Min-Jung; Min, Byung-Il; Bae, Hyunsu; Choe, Wonchae; Kang, Insug; Kim, Sung-Soo; Ha, Joohun

    2009-10-01

    Inulin, a naturally occurring, functional food ingredient found in various edible plants, has been reported to exert potential health benefits, including decreased risk of colonic diseases, non-insulin-dependent diabetes, obesity, osteoporosis, and cancer. However, the mechanism of the antidiabetic activity of inulin has not yet been elucidated. In this study, we showed that inulin increased the uptake of glucose in C2C12 myotubes, which was associated with both AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways, but both of these pathways appeared to transmit their signals in an independent manner. Moreover, we found that inulin was able to increase the uptake of glucose in C2C12 myotubes in which insulin resistance was induced by exposing cells to high glucose concentrations. The identical effects of inulin were also observed in HepG2 hepatoma cells. Collectively, we report the antidiabetic activity of inulin and further demonstrate for the first time that such activity is associated with AMPK and PI3-K activation.

  2. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

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    Yuanfei Zhou

    2016-10-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 integrates amino acid (AA availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2 activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser, arginine (Arg, threonine (Thr, alanine (Ala, methionine (Met, glutamine (Gln, and glycine (Gly, Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism.

  3. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    Science.gov (United States)

    Zhou, Yuanfei; Ren, Jiao; Song, Tongxing; Peng, Jian; Wei, Hongkui

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), methionine (Met), glutamine (Gln), and glycine (Gly), Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism. PMID:27727170

  4. 热休克蛋白70过表达对骨骼肌细胞内 ATP水平的影响%Effect of over-expression Hsp70 on the intracellular ATP level in C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    王磊; 王尊; 刘跃飞; 顾一煌

    2013-01-01

    Objective To examine the effect of over-expression Hsp70 on the intracellular ATP level in C2C12 cells. Methods Hsp70 gene was amplified from pAT153 plasmids and then cloned into pTRE2hyg vector. After the transfection of recombinant plasmids of pTRE2hyg-Hsp70 into the C2C12 cells, the expression of Hsp70 was examined by Western blot. Furthermore, the intracellular ATP level was evaluated in C2C12 cells at different time points (0 d, 3 d, 7 d) during cell culture. Results Compared with controls, the intracellular ATP level was significantly increased (P<0.05) at different time points,(14.5 ± 2.87)nmol/mg protein (3 d), (15.3 ± 3.12) nmol/mg protein(7 d) after transfection. Conclusion The C2C12 cells of over-expression Hsp70 can increase the intracellular ATP level, indicating that Hsp70 may play a role in the metabolism in skeletal muscle cells.%目的:探讨热休克蛋白70(heat shock protein 70,Hsp70)过表达对骨骼肌细胞(C2C12)内ATP水平的影响。方法通过构建重组pTRE2hyg-Hsp70质粒,稳定转染C2C12细胞系,建立Hsp70过表达的C2C12细胞系。分别在转染后细胞培养的不同时间点(0 d,3 d,7 d),检测细胞内ATP的水平。结果 Hsp70过表达的C2C12细胞系在培养的3 d,7 d,细胞内ATP的水平分别达到(14.5±2.87)nmol/mg蛋白质、(15.3±3.12)nmol/mg 蛋白质,与对照组相比明显增高(P<0.05)。结论 Hsp70过表达的骨骼肌细胞可以提高细胞内的ATP水平,提示Hsp70对骨骼肌细胞的能量代谢产生影响。

  5. Notch pathway activation contributes to inhibition of C2C12 myoblast differentiation by ethanol.

    Directory of Open Access Journals (Sweden)

    Michelle A Arya

    Full Text Available The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.

  6. Lrrc75b is a novel negative regulator of C2C12 myogenic differentiation

    Science.gov (United States)

    Zhong, Yuechun; Zou, Liyi; Wang, Zonggui; Pan, Yaqiong; Dai, Zhong; Liu, Xinguang; Cui, Liao; Zuo, Changqing

    2016-01-01

    Many transcription factors and signaling molecules involved in the guidance of myogenic differentiation have been investigated in previous studies. However, the precise molecular mechanisms of myogenic differentiation remain largely unknown. In the present study, by performing a meta-analysis of C2C12 myogenic differentiation microarray data, we found that leucine-rich repeat-containing 75B (Lrrc75b), also known as AI646023, a molecule of unknown biological function, was downregulated during C2C12 myogenic differentiation. The knockdown of Lrrc75b using specific siRNA in C2C12 myoblasts markedly enhanced the expression of muscle-specific myogenin and increased myoblast fusion and the myotube diameter. By contrast, the adenovirus-mediated overexpression of Lrrc75b in C2C12 cells markedly inhibited myoblast differentiation accompanied by a decrease in myogenin expression. In addition, the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) was suppressed in the cells in which Lrrc75b was silenced. Taken together, our results demonstrate that Lrrc75b is a novel suppressor of C2C12 myogenic differentiation by modulating myogenin and Erk1/2 signaling. PMID:27633041

  7. Effect of mitochondrial fission inhibition on C2C12 differentiation.

    Science.gov (United States)

    Bloemberg, Darin; Quadrilatero, Joe

    2016-06-01

    The differentiation of skeletal muscle is commonly examined in cell culture using the C2C12 line of mouse skeletal myoblasts. This process shares many similarities with that which occurs during embryonic development, such as the transient activation of caspases. Here, we examined the effect of inhibiting mitochondrial fission, using mdivi-1, on the ability of C2C12 cells to terminally differentiate. This was performed using immunofluorescent identification of cell morphology and myosin expression, as well as immunoblotting for markers of muscle differentiation. Furthermore, the effect of mdivi-1 administration on activation of caspase-2 and -3 was assessed using spectrofluorometric measurement of specific enzyme activity. PMID:27054170

  8. Autophagic flux data in differentiated C2C12 myotubes following exposure to acetylcholine and caffeine

    Directory of Open Access Journals (Sweden)

    Darin Bloemberg

    2016-06-01

    Full Text Available The C2C12 line of mouse myoblasts is a useful cell culture model in which to conduct in vitro analyses related to skeletal muscle. Here we present data regarding the autophagic response induced by two chemicals known to influence calcium release and contraction in skeletal muscles and C2C12 cells: acetylcholine and caffeine. More specifically, by concurrently administering acetylcholine or caffeine along with chloroquine to differentiated myotubes for various amounts of time and assessing the protein expression of LC3 and p62, we report data on the relative level of autophagic flux induced by these two calcium- and contraction-regulating chemicals.

  9. Characterization of an acute muscle contraction model using cultured C2C12 myotubes.

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    Yasuko Manabe

    Full Text Available A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion (Ca²⁺ transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt, 5' AMP-activated protein kinase (AMPK, p38 mitogen-activated protein kinase (p38, and c-Jun NH2-terminal kinase (JNK1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise invivo (exercise, in situ (hindlimb muscles in an anesthetized animal, and invitro (dissected muscle tissues in incubation buffer by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction.

  10. BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

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    Qiangling Zhang

    2015-08-01

    Full Text Available Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

  11. Lysophosphatidic Acid Stimulates MCP-1 Secretion from C2C12 Myoblast

    OpenAIRE

    Tamotsu Tsukahara; Hisao Haniu

    2012-01-01

    Chemokines are regulatory proteins that play an important role in muscle cell migration and proliferation. In this study, C2C12 cells treated with lysophosphatidic acid (LPA) showed an increase in endogenous monocyte chemotactic protein-1 (MCP-1) expression and secretion. LPA is a naturally occurring bioactive lysophospholipid with hormone- and growth-factor-like activities. LPA is produced by activated platelets, cytokine-stimulated leukocytes, and possibly by other cell types. However, the ...

  12. First intron of nestin gene regulates its expression during C2C12 myoblast ifferentiation

    Institute of Scientific and Technical Information of China (English)

    Hua Zhong; Zhigang Jin; Yongfeng Chen; Ting Zhang; Wei Bian; Xing Cui; Naihe Jing

    2008-01-01

    Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Nestin is an intermediate filament protein expressed in neural progenitor cells and in developing skeletal muscle. Nestin has been widely used as a neural progenitor cell marker. It is well established that the specific expression of the nestin gene in neural progenitor cells is conferred by the neural-specific enhancer located in the second intron of the nestin gene. However, the transcriptional mechanism of nestin expression in developing muscle is still unclear. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene in mouse myoblast C2C12 cells.We localized the core enhancer activity to the 291-661 region of the first intron, and showed that the two E-boxes in the core enhancer region were important for enhancer activity in differentiating C2C12 cells. We also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C12 cells.

  13. Lysophosphatidic Acid Stimulates MCP-1 Secretion from C2C12 Myoblast.

    Science.gov (United States)

    Tsukahara, Tamotsu; Haniu, Hisao

    2012-01-01

    Chemokines are regulatory proteins that play an important role in muscle cell migration and proliferation. In this study, C2C12 cells treated with lysophosphatidic acid (LPA) showed an increase in endogenous monocyte chemotactic protein-1 (MCP-1) expression and secretion. LPA is a naturally occurring bioactive lysophospholipid with hormone- and growth-factor-like activities. LPA is produced by activated platelets, cytokine-stimulated leukocytes, and possibly by other cell types. However, the LPA analog cyclic phosphatidic acid (cPA) had no effect on the expression and secretion of MCP-1. LPA, although similar in structure to cPA, had potent inducing effects on MCP-1 expression in C2C12 cells. In this study, we showed that LPA enhanced MCP-1 mRNA expression and protein secretion in a dose-dependent manner. Taken together, these results suggest that LPA enhances MCP-1 secretion in C2C12 cells and thus may play an important role in cell proliferation. PMID:24049655

  14. Two-dimensional differential adherence and alignment of C2C12 myoblasts

    International Nuclear Information System (INIS)

    Surface microstructure is a critical parameter for scaffolds used in skeletal muscle tissue engineering. We have developed micromachined surfaces using matrix assisted pulsed laser evaporation-direct write (MAPLE-DW) that demonstrate differential adherence of C2C12 myoblast cells. The 60-400 μm diameter channels were micromachined onto 2% agarose surfaces using an ArF excimer laser and lined with Matrigel[reg] basement membrane matrix solution. Suspensions containing C2C12 myoblast cells were then placed on the surface of these micromachined channels. The C2C12 myoblast cells aligned themselves parallel to the 60-150 μm channels. Live/dead assays over 72 h demonstrated that cell number, cell size, and number of nuclei per cell increased within these channels. In addition, some of the myoblasts fused and differentiated into multinucleated myotubes. These results provide the basis for the development of direct-write scaffolds for skeletal muscle tissue engineering

  15. Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

    International Nuclear Information System (INIS)

    Highlights: ► Change in the epigenetic landscape during myogenesis was optically investigated. ► Mobility of nuclear proteins was used to state the epigenetic status of the cell. ► Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. ► Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

  16. Astragalus Polysaccharide Inhibits Autophagy and Apoptosis from Peroxide-Induced Injury in C2C12 Myoblasts.

    Science.gov (United States)

    Yin, Yi; Lu, Lu; Wang, Dongtao; Shi, Ying; Wang, Ming; Huang, Yanfeng; Chen, Dexiu; Deng, Cong; Chen, Jiebin; Lv, Peijia; Wang, Yanjing; Li, Chengjie; Wei, Lian-Bo

    2015-11-01

    The aim is to study the effects and underlying mechanisms of astragalus polysaccharide (APS) on the peroxide-induced injury in C2C12 myoblasts in vitro. Cell viability in the presence or absence of APS was detected by the methyl thiazolyl tetrazolium colorimetric assay. The autophagosomes were observed by electron microscopy to examine the influence of APS on autophagy caused by H2O2 in C2C12 cells, and the percentage of apoptosis cells was measured by flow cytometry. To further confirm the effect of H2O2 on C2C12 cells, the protein expression of LC3 and RARP, which are the markers of autophagy and apoptosis, respectively, was analyzed by Western blot, as well as the expression levels of p-p70S6K, p70S6K, Bcl-2, Bax, cyto-C, and Caspase-3, to reveal the underlying mechanisms. We observed multiple effects of APS on C2C12 functionality. APS treatment of C2C12 cells at 1 mg/mL reduced cell viability to less than 70 %, and analysis by electron microscopy revealed that APS also reduced the number of H2O2-induced autophagosome formation. Similarly, APS abated the H2O2-mediated increase in cell apoptosis, which was accompanied by the inhibition of LC3 II and RARP that are normally upregulated by H2O2. The expression of p-p70S6K and p70S6K, however, remained unchanged in C2C12 cells in the Control, H2O2 and H2O2 + APS groups. In addition, APS promoted the expression of protein Bcl-2 in H2O2-treated C2C12 cells, but did not change Bax, thus reducing the Bax/Bcl-2 ratio that in turn prevented the release of cytochrome c and the activation of caspase-3. APS inhibits the autophagy and apoptosis induced by peroxide injury in C2C12 myoblasts through two independent signaling pathways: the mTOR-independent pathway for the inhibition of autophagy, and the caspase-3-dependent pathway for the suppression of apoptosis. PMID:27352334

  17. Induced differentiation of C2C12 to osteoblast via adenovirus-mediated Cbfa1 in vitro%体外诱导C2C12细胞向成骨细胞的分化

    Institute of Scientific and Technical Information of China (English)

    张勇; 杨彤涛; 胡运生; 廖博; 文艳华; 范清宇

    2013-01-01

    目的 成骨细胞特异性转录因子a1(core binding factor a1,Cbfa1)通过调节生长因子和骨特异性细胞外基质蛋白的基因表达而参与成骨细胞的分化和骨发育过程.文中构建成Cbfa1,以腺病毒载体转染成肌细胞C2C12,为种子细胞构建组织工程化骨.方法 体外培养小鼠成肌细胞C2C12,用重组腺病毒质粒pAd-IL-31介导Cbfa1/Osf2基因瞬时转染小鼠成肌C2C12细胞,Western blot检测Cbfa1蛋白表达.结果 Cbfa1蛋白表达、碱性磷酸酶(alkaline phosphatase,ALP)活性测定、骨钙素(osteocalcin,OCN)分泌量以及茜素红染色感染组明显高于对照组.结论 成肌细胞C2C12可以作为种子细胞构建组织工程化骨.%Objective Osteoblast core binding factor a 1 ( Cbfal) plays a role in osteoblast differentiation and development by regulating the gene of growth factor and extracellular matrix proteins . Recombinant adenovirus vector mediated Cbfa 1 was transferred to myoblast C2C12 to construct the tissue-engineered bone. Methods The myoblast C2C12 was cultured in vitro, and then transiently transfected with recombinant adenovirus vector pAd -IL-31 mediated-Cbfal/Osf2. Western blot was used to detect the expression of Cbfal. Results Compared with the control group , the expression of Cbfal, activity of alkaline phosphtase (ALP) , secretory volume of osteocalcin (OCN) and staining via alizarin bordeaux were higher in the transfection group . Conclusion Myoblast C2C12 acts as a seed cell for constructing tissue -engineered bone.

  18. Cytoprotective Role of Nrf2 in Electrical Pulse Stimulated C2C12 Myotube.

    Directory of Open Access Journals (Sweden)

    Masaki Horie

    Full Text Available Regular physical exercise is central to a healthy lifestyle. However, exercise-related muscle contraction can induce reactive oxygen species and reactive nitrogen species (ROS/RNS production in skeletal muscle. The nuclear factor-E2-related factor-2 (Nrf2 transcription factor is a cellular sensor for oxidative stress. Regulation of nuclear Nrf2 signaling regulates antioxidant responses and protects organ structure and function. However, the role of Nrf2 in exercise- or contraction-induced ROS/RNS production in skeletal muscle is not clear. In this study, using differentiated C2C12 cells and electrical pulse stimulation (EPS of muscle contraction, we explored whether Nrf2 plays a role in the skeletal muscle response to muscle contraction-induced ROS/RNS. We found that EPS (40 V, 1 Hz, 2 ms stimulated ROS/RNS accumulation and Nrf2 activation. We also showed that expression of NQO1, HO-1 and GCLM increased after EPS-induced muscle contraction and was remarkably suppressed in cells with Nrf2 knockdown. We also found that the antioxidant N-acetylcysteine (NAC significantly attenuated Nrf2 activation after EPS, whereas the nitric oxide synthetase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME did not. Furthermore, Nrf2 knockdown after EPS markedly decreased ROS/RNS redox potential and cell viability and increased expression of the apoptosis marker Annexin V in C2C12 myotubes. These results indicate that Nrf2 activation and expression of Nrf2 regulated-genes protected muscle against the increased ROS caused by EPS-induced muscle contraction. Thus, our findings suggest that Nrf2 may be a key factor for preservation of muscle function during muscle contraction.

  19. JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Yuasa, Katsutoshi; Aoki, Natsumi; Hijikata, Takao, E-mail: hijikata@musashino-u.ac.jp

    2015-08-15

    Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes. AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis. - Highlights: • JAZF1 promotes cell cycle progression and proliferation of myoblasts. • JAZF1 retards myogenic differentiation and hypertrophy of myotubes. • JAZF1 transcriptionally represses Mef2C and Mrf4 expression. • JAZF1 has an impact on the phosphorylation of AMPK.

  20. Response of C2C12 Myoblasts to Hypoxia: The Relative Roles of Glucose and Oxygen in Adaptive Cellular Metabolism

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-01-01

    Full Text Available Background. Oxygen and glucose are two important nutrients for mammalian cell function. In this study, the effect of glucose and oxygen concentrations on C2C12 cellular metabolism was characterized with an emphasis on detecting whether cells show oxygen conformance (OC in response to hypoxia. Methods. After C2C12 cells being cultured in the levels of glucose at 0.6 mM (LG, 5.6 mM (MG, or 23.3 mM(HG under normoxic or hypoxic (1% oxygen condition, cellular oxygen consumption, glucose consumption, lactate production, and metabolic status were determined. Short-term oxygen consumption was measured with a novel oxygen biosensor technique. Longer-term measurements were performed with standard glucose, lactate, and cell metabolism assays. Results. It was found that oxygen depletion in normoxia is dependent on the glucose concentration in the medium. Cellular glucose uptake and lactate production increased significantly in hypoxia than those in normoxia. In hypoxia the cellular response to the level of glucose was different to that in normoxia. The metabolic activities decreased while glucose concentration increased in normoxia, while in hypoxia, metabolic activity was reduced in LG and MG, but unchanged in HG condition. The OC phenomenon was not observed in the present study. Conclusions. Our findings suggested that a combination of low oxygen and low glucose damages the viability of C2C12 cells more seriously than low oxygen alone. In addition, when there is sufficient glucose, C2C12 cells will respond to hypoxia by upregulating anaerobic respiration, as shown by lactate production.

  1. An adaptable stage perfusion incubator for the controlled cultivation of C2C12 myoblasts.

    Science.gov (United States)

    Kurth, Felix; Franco-Obregón, Alfredo; Bärtschi, Christoph A; Dittrich, Petra S

    2015-01-01

    Here we present a stage perfusion incubation system that allows for the cultivation of mammalian cells within PDMS microfluidic devices for long-term microscopic examination and analysis. The custom-built stage perfusion incubator is adaptable to any x-y microscope stage and is enabled for temperature, gas and humidity control as well as equipped with chip and tubing holder. The applied double-layered microfluidic chip allows the predetermined positioning and concentration of cells while the gas permeable PDMS material facilitates pH control via CO2 levels throughout the chip. We demonstrate the functionality of this system by culturing C2C12 murine myoblasts in buffer free medium within its confines for up to 26 hours. We moreover demonstrated the system's compatibility with various chip configurations, other cells lines (HEK-293 cells) and for longer-term culturing. The cost-efficient system are applicable for any type of PDMS-based cell culture system. Detailed technical drawings and specification to reproduce this perfusion incubation system is provided in the ESI.

  2. Nrf2-Mediated HO-1 Induction Contributes to Antioxidant Capacity of a Schisandrae Fructus Ethanol Extract in C2C12 Myoblasts

    Directory of Open Access Journals (Sweden)

    Ji Sook Kang

    2014-12-01

    Full Text Available This study was designed to confirm the protective effect of Schisandrae Fructus, which are the dried fruits of Schisandra chinensis (Turcz. Baill, against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms in C2C12 myoblasts. Preincubating C2C12 cells with a Schisandrae Fructus ethanol extract (SFEE significantly attenuated hydrogen peroxide (H2O2-induced inhibition of growth and induced scavenging activity against intracellular reactive oxygen species (ROS induced by H2O2. SFEE also inhibited comet tail formation and phospho-histone γH2A.X expression, suggesting that it prevents H2O2-induced cellular DNA damage. Furthermore, treating C2C12 cells with SFEE significantly induced heme oxygenase-1 (HO-1 and phosphorylation of nuclear factor-erythroid 2 related factor 2 (Nrf2. However, zinc protoporphyrin IX, a potent inhibitor of HO-1 activity, significantly reversed the protective effects of SFEE against H2O2-induced growth inhibition and ROS generation in C2C12 cells. Additional experiments revealed that the potential of the SFEE to induce HO-1 expression and protect against H2O2-mediated cellular damage was abrogated by transient transfection with Nrf2-specific small interfering RNA, suggesting that the SFEE protected C2C12 cells against oxidative stress-induced injury through the Nrf2/HO-1 pathway.

  3. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S., E-mail: bcssj@iacs.res.in

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  4. Over-expression of the transcription factor, ZBP-89, leads to enhancement of the C2C12 myogenic program

    OpenAIRE

    Salmon, Morgan; Owens, Gary K.; Zehner, Zendra E.

    2009-01-01

    Myogenesis involves the complex interplay between the down-regulation of non-muscle genes and the up-regulation of muscle-specific genes. This interplay is controlled by the myogenic regulatory factors Myf5, MRF4, MyoD and myogenin. To trigger the up-regulation of these muscle-specific factors, certain environmental cues, such as the removal of serum, signal C2C12 myoblast cells to withdraw from cell cycle, fuse and activate muscle-specific genes. Here, the level of ZBP-89 (zfp148), a Krüppel...

  5. Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

    Directory of Open Access Journals (Sweden)

    Dupuy Fabrice

    2009-10-01

    Full Text Available Abstract Background Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes. Results Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries, providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation. Conclusion For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.

  6. Metabolic profiling of heat or anoxic stress in mouse C2C12 myotubes using multinuclear magnetic resonance spectroscopy

    DEFF Research Database (Denmark)

    Straadt, Ida K; Young, Jette F; Petersen, Bent O;

    2010-01-01

    In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [(13)C]-glucose. Both the (13)C and the (1)H NMR......-energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in (13)C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift...

  7. Prevention of oxidative stress-induced apoptosis of C2C12 myoblasts by a Cichorium intybus root extract.

    Science.gov (United States)

    Lee, Yong-Hyeon; Kim, Dae-Hyun; Kim, Yoon Suk; Kim, Tack-Joong

    2013-01-01

    Cell injury associated with reactive oxygen species (ROS) has been reported in various muscular disorders. We found that a Cichorium intybus (Cii) extract reduced H(2)O(2)-induced viability loss in C2C12 myoblasts, inhibited oxidative stress-induced apoptosis and increased intracellular heat shock protein 70 (Hsp 70) expression. Cii also inhibited the level of intracellular ceramide. These results indicate that Cii may prevent skeletal muscle atrophy by inducing the expression of Hsp 70 and inhibiting the level of ceramide. PMID:23391909

  8. miR-143-3p促进C2C12成肌细胞分化%miR-143-3p Is Implicated in C2C12 Myoblasts Differentiation

    Institute of Scientific and Technical Information of China (English)

    云青; 吴国芳; 魏欢; 庞卫军; 杨公社; 沈清武

    2013-01-01

    MicroRNAs (miRNAs) are small non-coding RNA that play important roles in skeletal muscle development.To explore the function of miR-143-3p in the differentiation of C2C12 myoblasts,we detected miR-143-3p levels by real-time PCR in different mouse tissues,as well as C2C12 myoblasts during myogenesis.After the trasfection of miR-143-3p mimics and inhibitor in C2C12 myoblasts,the expression of myogenic regulatory factor MyoG and myogenic marker gene MyHC were detected by realtime PCR and Western blotting.The myotubule formation was detected by immunofluorescent staining.The results showed that miR-143-3p was ubiquitously expressed in various tissues and was upregulated during cell differentiation.The differentiation of C2C12 myoblasts was promoted with miR-143-3p overexpression as significant upregulation of MyoG and MyHC,and increased number of myotubules.The inhibitor of miR-143-3p significantly repressed cell differentiation.Interestingly,the transfection of miR-143-3p mimics had little effect on the expression of MyHCs.Our data suggested that miR-143-3p might be involved during the myogeneis of C2C12 myoblasts,but not directly impact MyHC expression.%MicroRNAs (miRNAs)是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用real-time PCR检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p的模拟物和特异性抑制剂分别处理细胞,采用real-time PCR和Western印迹分别检测成肌因子MyoG和成肌标志基因MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分

  9. Effects ShRNA-mediated Gene Silencing Smad4 on the Myofibrogenesis of C2C12 Myoblasts%ShRNA介导的Smad4基因沉默对C2C12成肌细胞纤维化的影响

    Institute of Scientific and Technical Information of China (English)

    陈始秋; 陈疾忤; 陈世益; 李宏云; 尚西亮; 蒋佳

    2011-01-01

    Objective To study the effect of Smad4-shRNA on the myofibrosis process. Methods (1) The most efficient silencing Smad4-shRNA was designed and used to produce lentivirus-Smad4 shRNA to infect C2C12 myoblasts. The Smad4 expression was examined. (2) According to the model that TGF-β1 induced the myofibrosis process of the C2C12 myoblasts, the lentivirus mediated cells were divided into four groups: group A, B, C, and D, respectively presenting the normal C2C12 cells group, the TGF-β1 induced C2C12 cells group, the lentivirus-mediated cells group and the TGF-β1 induced lentivirus-mediated cells group. Results (1) The mRNA and protein expression of Smad4 showed a lower level in Smad4-shRNAl Lentivirus-mediated C2C12 cells (P 0.05 ) . Conclusion Smad4 down-regulation could effectively suppress myofibrosis induced by TGF-β1, and Smad4 inhibition was a potential method for anti-fibrosis of the injured skeletal muscle.%目的:研究慢病毒介导的Smad4-shRNA对TGF-β1诱导的纤维化进程的影响.方法:(1)设计并选择抑制效能最高的Smad4-shRNA,包装生产慢病毒,转染C2C12成肌细胞,并检测转染后细胞的Smad4表达;(2)根据TGF-β1诱导C2C12成肌细胞向成肌纤维细胞分化的模型,以慢病毒介导的Smad4-shRNA转染细胞,将不同处理的细胞分为A、B、C、D四组,分别为C2C12细胞组、TGF-β1诱导组、C2C12细胞转染组和TGF-β1诱导后转染组.通过荧光Realtime-PCR和Westerblot检测各组collagen Ⅰ和α-SMA表达水平.结果:(1)慢病毒介导Smad4-shRNA1转染C2C12细胞后,其Smad4 mRNA和蛋白表达显著低于未转染组(P<0.05);(2) TGF-β1诱导组α-SMA和Collagen Ⅰ mRNA及蛋白表达均显著高于C2C12细胞组(P<0.05);(3) TGF-β1诱导后转染组α-SMA与collagen Ⅰ mRNA及蛋白表达显著低于TGF-β1诱导组(P<0.05),与C2C12细胞组和C2C12细胞转染组相比则无明显差异(P>0.05).结论:降低Smad4表达能有效抑制成肌细胞及受TGF-β1诱导成肌纤维

  10. 人参皂苷Rg1对体外培养C2C12成肌细胞凋亡的影响%Effect of ginsenoside Rg1 during serum-deprivation induced apoptosis in C2C12 myoblasts cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    叶东明; 余磊; 王乐禹; 邱小忠; 欧阳钧

    2011-01-01

    Objective: To investigate the effect and possible mechanism of ginsenoside Rgl during serum-deprivation induced apoptosis in C2C12 myoblasts cultured in vitro. Methods: The effect of different concentrations of ginsenoside Rgl during the cell apoptosis was assessed by MTT assay, Hoechst 33258-PI double staining and RT-PCR analysis. Results: After 48 h treatment, various doses of ginsenoside Rgl increased cell viability in serum-deprived C2C12 myoblasts using MTT assay. Hoechst 33258-PI double staining showed that the rate of apoptosis cells significantly decreased after being treated by ginsenoside Rgl. RT-PCR showed that ginsenoside Rgl caused the downregulation of pro-apoptotic caspase-3, Bax and AIF genes, while caused the up-regulation of anti-apoptotic Bcl-2 gene. Conclusion: Ginsenoside Rgl can protect the serum-deprived apoptosis in C2C12 myoblasts.%目的:研究人参皂苷Rg1对体外无血清诱导培养的C2C12成肌细胞凋亡的影响及其可能机制.方法:采用MTT法、人参皂苷Rg1处理48 h后hoechst 33258-PI染色,以及RT-PCR方法观察不同浓度人参皂苷Rg1对C2C12成肌细胞凋亡的影响.结果:MTT法结果显示人参皂苷Rg1处理48 h后可抑制C2C12成肌细胞凋亡;Hoechst 33258-PI染色可见C2C12成肌细胞凋亡率人参皂苷处理前后差异有统计学意义,人参皂苷处理后C2C12成肌细胞凋亡率显著下降;RT-PCR法结果显示人参皂苷Rg1可抑制Caspase-3、Bax和AIF mRNA表达,并能诱导Bcl-2 mRNA表达.结论:人参皂苷Rg1对C2C12成肌细胞凋亡具有保护作用.

  11. C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene.

    Science.gov (United States)

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli; Sun, Wenxing; Gao, Ying; Zhang, Lifan; Chen, Jie

    2016-03-25

    Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes.

  12. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Kamolrat, Torkamol [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom); Gray, Stuart R., E-mail: s.r.gray@abdn.ac.uk [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom)

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  13. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    International Nuclear Information System (INIS)

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using 3H-labelled phenylalanine. Protein breakdown was measured using 3H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion

  14. Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Jeffrey eKim

    2014-03-01

    Full Text Available Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25µM of arachidonate (AA or docosahexaenoate (DHA, 25µM of EC [anandamide (AEA, 2-arachidonoylglycerol (2-AG, docosahexaenoylethanolamide (DHEA], 1µM of CB1 antagonist NESS0327, and CB2 antagonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.Key Words: endocannabinoid system •C2C12 myoblasts cannabinoid receptors glucose uptake gene expression DHEA • polyunsaturated fatty acids

  15. Cyclic stretch induced miR-146a upregulation delays C2C12 myogenic differentiation through inhibition of Numb

    International Nuclear Information System (INIS)

    Proliferation and differentiation of muscle stem cells must be tightly regulated by intrinsic and extrinsic signals for effective regeneration and adaptive response. MicroRNAs have been implicated as potent regulators in diverse biological processes at the level of posttranscriptional repression. In this study, we found that miR-146a was significantly upregulated upon a 48-h cyclic stretch of 5% elongation/10cycles/min. Importantly, miR-146 was predicted to base-pair with sequences in the 3' UTR of Numb, which promotes satellite cell differentiation towards muscle cells by inhibiting Notch signaling. Through reporter assay and exogenous expression experiment, we confirmed Numb was inhibited by miR-146a. Inhibition of miR-146a by antago-miR-146a rescued the expression of Numb and facilitated the differentiation of C2C12 at a cost of compromised proliferation. Thus, for the first time, we propose a role of miR-146a in skewing the balance of muscle differentiation and proliferation through inhibiting the expression of Numb.

  16. The collagen derived dipeptide hydroxyprolyl-glycine promotes C2C12 myoblast differentiation and myotube hypertrophy.

    Science.gov (United States)

    Kitakaze, Tomoya; Sakamoto, Tomotaka; Kitano, Takehiro; Inoue, Naoki; Sugihara, Fumihito; Harada, Naoki; Yamaji, Ryoichi

    2016-09-23

    The majority of studies on possible roles for collagen hydrolysates in human health have focused on their effects on bone and skin. Hydroxyprolyl-glycine (Hyp-Gly) was recently identified as a novel collagen hydrolysate-derived dipeptide in human blood. However, any possible health benefits of Hyp-Gly remain unclear. Here, we report the effects of Hyp-Gly on differentiation and hypertrophy of murine skeletal muscle C2C12 cells. Hyp-Gly increased the fusion index, the myotube size, and the expression of the myotube-specific myosin heavy chain (MyHC) and tropomyosin structural proteins. Hyp-Gly increased the phosphorylation of Akt, mTOR, and p70S6K in myoblasts, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited their phosphorylation by Hyp-Gly. LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin repressed the enhancing effects of Hyp-Gly on MyHC and tropomyosin expression. The peptide/histidine transporter 1 (PHT1) was highly expressed in both myoblasts and myotubes, and co-administration of histidine inhibited Hyp-Gly-induced phosphorylation of p70S6K in myoblasts and myotubes. These results indicate that Hyp-Gly can induce myogenic differentiation and myotube hypertrophy and suggest that Hyp-Gly promotes myogenic differentiation by activating the PI3K/Akt/mTOR signaling pathway, perhaps depending on PHT1 for entry into cells. PMID:27553280

  17. Catalytic activity of nuclear PLC-beta(1) is required for its signalling function during C2C12 differentiation.

    Science.gov (United States)

    Ramazzotti, Giulia; Faenza, Irene; Gaboardi, Gian Carlo; Piazzi, Manuela; Bavelloni, Alberto; Fiume, Roberta; Manzoli, Lucia; Martelli, Alberto M; Cocco, Lucio

    2008-11-01

    Here we report that PLC-beta(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-beta(1) mutational analysis revealed the importance of His(331) and His(378) for the catalysis. The expression of PLC-beta(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-beta(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-beta(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-beta(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-beta(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-beta(1) and that c-Jun binding activity is significantly increased by insulin and PLC-beta(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-beta(1). These results hint at the fact that PLC-beta(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

  18. Palmitate increases musclin gene expression through activation of PERK signaling pathway in C2C12 myotubes.

    Science.gov (United States)

    Gu, Ning; Guo, Qian; Mao, Ke; Hu, Hailong; Jin, Sanli; Zhou, Ying; He, Hongjuan; Oh, Yuri; Liu, Chuanpeng; Wu, Qiong

    2015-11-20

    Musclin is a type of muscle-secreted cytokine and its increased gene expression induces insulin resistance in type 2 diabetes. However, the mechanism underlying increased musclin gene expression is currently unclear. Excessive saturated fatty acids (SFA) can activate the secretion of several muscle-secreted cytokines as well as endoplasmic reticulum (ER) stress pathway, thereby contributing to the development of type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the effect of palmitate, the most abundant SFA in the plasma, on the gene expression of musclin in C2C12 myotubes. Treatment of C2C12 myotubes with palmitate or tunicamycin significantly increased the expression of musclin as well as ER stress-related genes, but treatment with oleate did not. Pre-treatment of C2C12 myotubes with 4-phenyl butyrate suppressed the expression of ER stress-related genes, simultaneously, resulting in decreased expression of the musclin gene induced by palmitate or tunicamycin. These results indicate that ER stress is related to palmitate-induced musclin gene expression. Moreover, palmitate-induced musclin gene expression was significantly inhibited in C2C12 myotubes when PERK pathway signaling was suppressed by knockdown of the PERK gene or treatment with GSK2656157, a PERK autophosphorylation inhibitor. However, there was no difference in the palmitate-induced musclin gene expression when IRE1 and ATF6 signaling pathways were suppressed by knockdown of the IRE1 and ATF6 genes. These findings suggest that palmitate increases musclin gene expression via the activation of the PERK signaling pathway in C2C12 myotubes. PMID:26449458

  19. A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

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    Friedmann Theodore

    2009-08-01

    Full Text Available Abstract Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1, relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IκB kinase b (IκBKb and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

  20. Electrically induced contraction of C2C12 myotubes cultured on a porous membrane-based substrate with muscle tissue-like stiffness.

    Science.gov (United States)

    Kaji, Hirokazu; Ishibashi, Takeshi; Nagamine, Kuniaki; Kanzaki, Makoto; Nishizawa, Matsuhiko

    2010-09-01

    A porous membrane-based cell culture device was developed to electrically stimulate a confluent monolayer of C2C12 myotubes. The device's cell culture substrate is a microporous alumina membrane-modified by attaching an atelocollagen membrane on the upperside and a hole-spotted poly(dimethylsiloxane) (PDMS) film on the underside. When electric current is generated between the device's Pt ring electrodes--one of which is placed above the cells and the other below the PDMS layer--the focused current at the PDMS hole can electrically stimulate the cells. C2C12 myoblasts were cultured on the substrate and differentiated into myotubes. When the electrical pulses were applied, myotubes started to contract slightly in and near the hole, and that the continuous stimulation increased both the number of stimuli-responding myotubes and the magnitude of the contraction considerably owing to the underlying atelocollagen membrane with muscle tissue-like stiffness. Also, the generation of contractile myotubes on a wider region of the membrane substrate was possible by applying the electrical pulses through the array of holes in the PDMS film. Using the present system, the glucose uptake by contractile myotubes was examined with fluorescence-labeled glucose, 2-NBDG, which displayed a positive correlation between the contractile activity of myotubes and the uptake of 2-NBDG. PMID:20561677

  1. Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

    Institute of Scientific and Technical Information of China (English)

    ZHEN-MING HU; SEAN A. F. PEEL; STEPHEN K. C. HO; GEORGE K. B. SANDOR; CAMERON M. L. CLOKIE

    2009-01-01

    Objective To study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro. Methods Type I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the Ikaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating ostcoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type 1 collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

  2. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

    International Nuclear Information System (INIS)

    Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. WF decreased the viability of C2C12 myotubes, especially at concentrations of 20–25 μg.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model

  3. The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress.

    Science.gov (United States)

    Rodriguez-Ortiz, Carlos J; Flores, Julio C; Valenzuela, Joanna A; Rodriguez, Gema J; Zumkehr, Joannee; Tran, Diana N; Kimonis, Virginia E; Kitazawa, Masashi

    2016-06-01

    Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1-positive granules than wild-type in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCPR155H-knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2α unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration. PMID:27106764

  4. Infectious prions accumulate to high levels in non proliferative C2C12 myotubes.

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    Allen Herbst

    Full Text Available Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrP(C into a disease specific isoform PrP(Sc. Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrP(Sc and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrP(Sc and that the level of infectivity produced in these post-mitotic cells, 10(5.5 L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

  5. ACTIVATION OF THE PHOSPHOLIPASE-C PATHWAY BY ATP IS MEDIATED EXCLUSIVELY THROUGH NUCLEOTIDE TYPE P2-PURINOCEPTORS IN C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1993-01-01

    1 The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(

  6. Expression of Basic Fibroblast Growth Factor Results in the Decrease of Myostatin mRNA in Murine C2C12 Myoblasts

    Institute of Scientific and Technical Information of China (English)

    Hua-Zhong LIU; Qing LI; Xing-Yuan YANG; Lin LIU; Lei LIU; Xiao-Rong AN; Yong-Fu CHEN

    2006-01-01

    During the development and regeneration of skeletal muscle, many growth factors, such as basic fibroblast growth factor (bFGF, FGF-2) and myostatin, have been shown to play regulating roles.bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle, whereas myostatin plays a series of contrasting roles. In order to elucidate whether the expression of bFGF has any relationship with the expression of myostatin in skeletal muscle cells, we constructed a eukaryotic expression vector for the expression of exogenous bFGF in murine C2C12 myoblasts. Quantitative RT-PCR assays indicated that with the increase of the expression of exogenous bFGF gene, the expression of endogenous myostatin gene was suppressed at mRNA level and protein level.

  7. Dehydroepiandrosterone activates AMP kinase and regulates GLUT4 and PGC-1α expression in C2C12 myotubes

    International Nuclear Information System (INIS)

    Exercise and caloric restriction (CR) have been reported to have anti-ageing, anti-obesity, and health-promoting effects. Both interventions increase the level of dehydroepiandrosterone (DHEA) in muscle and blood, suggesting that DHEA might partially mediate these effects. In addition, it is thought that either 5′-adenosine monophosphate-activated protein kinase (AMPK) or peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mediates the beneficial effects of exercise and CR. However, the effects of DHEA on AMPK activity and PGC-1α expression remain unclear. Therefore, we explored whether DHEA in myotubes acts as an activator of AMPK and increases PGC-1α. DHEA exposure increased glucose uptake but not the phosphorylation levels of Akt and PKCζ/λ in C2C12 myotubes. In contrast, the phosphorylation levels of AMPK were elevated by DHEA exposure. Finally, we found that DHEA induced the expression of the genes PGC-1α and GLUT4. Our current results might reveal a previously unrecognized physiological role of DHEA; the activation of AMPK and the induction of PGC-1α by DHEA might mediate its anti-obesity and health-promoting effects in living organisms. - Highlights: • We assessed whether dehydroepiandrosterone (DHEA) activates AMPK and PGC-1α. • DHEA exposure increased glucose uptake in C2C12 myotubes. • The phosphorylation levels of AMPK were elevated by DHEA exposure. • DHEA induced the expression of the genes PGC-1α and GLUT4. • AMPK might mediate the anti-obesity and health-promoting effects of DHEA

  8. Dehydroepiandrosterone activates AMP kinase and regulates GLUT4 and PGC-1α expression in C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yokokawa, Takumi [Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (Japan); Sato, Koji [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Iwanaka, Nobumasa [The Graduate School of Science and Engineering, Ritsumeikan University, Shiga (Japan); Honda, Hiroki [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Higashida, Kazuhiko [Faculty of Sport Science, Waseda University, Saitama (Japan); Iemitsu, Motoyuki [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Hayashi, Tatsuya [Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (Japan); Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan)

    2015-07-17

    Exercise and caloric restriction (CR) have been reported to have anti-ageing, anti-obesity, and health-promoting effects. Both interventions increase the level of dehydroepiandrosterone (DHEA) in muscle and blood, suggesting that DHEA might partially mediate these effects. In addition, it is thought that either 5′-adenosine monophosphate-activated protein kinase (AMPK) or peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mediates the beneficial effects of exercise and CR. However, the effects of DHEA on AMPK activity and PGC-1α expression remain unclear. Therefore, we explored whether DHEA in myotubes acts as an activator of AMPK and increases PGC-1α. DHEA exposure increased glucose uptake but not the phosphorylation levels of Akt and PKCζ/λ in C2C12 myotubes. In contrast, the phosphorylation levels of AMPK were elevated by DHEA exposure. Finally, we found that DHEA induced the expression of the genes PGC-1α and GLUT4. Our current results might reveal a previously unrecognized physiological role of DHEA; the activation of AMPK and the induction of PGC-1α by DHEA might mediate its anti-obesity and health-promoting effects in living organisms. - Highlights: • We assessed whether dehydroepiandrosterone (DHEA) activates AMPK and PGC-1α. • DHEA exposure increased glucose uptake in C2C12 myotubes. • The phosphorylation levels of AMPK were elevated by DHEA exposure. • DHEA induced the expression of the genes PGC-1α and GLUT4. • AMPK might mediate the anti-obesity and health-promoting effects of DHEA.

  9. TNF-α and IFN-s-Dependent Muscle Decay Is Linked to NF-κB- and STAT-1α-Stimulated Atrogin1 and MuRF1 Genes in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    Barbara Pijet

    2013-01-01

    Full Text Available TNF-α was shown to stimulate mitogenicity in C2C12 myoblasts. Selected cytokines TNF-α, IFNα, or IFNγ reduced the expression of myosin heavy chain (MyHC IIa when given together. Molecular mechanisms of cytokine activities were controlled by NF-κB and JAK/STAT signaling pathways, as metabolic inhibitors, curcumin and AG490, inhibited some of TNF-α and IFNα/IFNγ effects. Insulin was hardly antagonistic to TNF-α- and IFNα/IFNγ-dependent decrease in MyHC IIa protein expression. Cytokines used individually or together also repressed myogenesis of C2C12 cells. Moreover, TNF-α- and IFNα/IFNγ-dependent effects on C2C12 myotubes were associated with increased activity of Atrogin1 and MuRF1 genes, which code ubiquitin ligases. MyHC IIa gene activity was unaltered by cytokines. Inhibition of NF-κB or JAK/STAT with specific metabolic inhibitors decreased activity of Atrogin1 and MuRF1 but not MyHC IIa gene. Overall, these results suggest cooperation between cytokines in the reduction of MyHC IIa protein expression level via NF-κB/JAK/STAT signaling pathways and activation of Atrogin1 and MuRF1 genes as their molecular targets. Insulin cotreatment or pretreatment does not protect against muscle decay induced by examined proinflammatory cytokines.

  10. Capric Acid Up-Regulates UCP3 Expression without PDK4 Induction in Mouse C2C12 Myotubes.

    Science.gov (United States)

    Abe, Tomoki; Hirasaka, Katsuya; Kohno, Shohei; Tomida, Chisato; Haruna, Marie; Uchida, Takayuki; Ohno, Ayako; Oarada, Motoko; Teshima-Kondo, Shigetada; Okumura, Yuushi; Choi, Inho; Aoyama, Toshiaki; Terao, Junji; Nikawa, Takeshi

    2016-01-01

    Uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle are key regulators of the glucose and lipid metabolic processes that are involved in insulin resistance. Medium-chain fatty acids (MCFAs) have anti-obesogenic effects in rodents and humans, while long-chain fatty acids (LCFAs) cause increases in body weight and insulin resistance. To clarify the beneficial effects of MCFAs, we examined UCP3 and PDK4 expression in skeletal muscles of mice fed a MCFA- or LCFA-enriched high-fat diet (HFD). Five-week feeding of the LCFA-enriched HFD caused high body weight gain and induced glucose intolerance in mice, compared with those in mice fed the MCFA-enriched HFD. However, the amounts of UCP3 and PDK4 transcripts in the skeletal muscle of mice fed the MCFA- or LCFA-enriched HFD were similar. To further elucidate the specific effects of MCFAs, such as capric acid (C10:0), on lipid metabolism in skeletal muscles, we examined the effects of various FAs on expression of UCP3 and PDK4, in mouse C2C12 myocytes. Although palmitic acid (C16:0) and lauric acid (C12:0) significantly induced expression of both UCP3 and PDK4, capric acid (C10:0) upregulated only UCP3 expression via activation of peroxisome proliferator-activated receptor-δ. Furthermore, palmitic acid (C16:0) disturbed the insulin-induced phosphorylation of Akt, while MCFAs, including lauric (C12:0), capric (C10:0), and caprylic acid (C12:0), did not. These results suggest that capric acid (C10:0) increases the capacity for fatty acid oxidation without inhibiting glycolysis in skeletal muscle. PMID:27117849

  11. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

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    Yan Shen

    Full Text Available We previously demonstrated that cinnamon extract (CE ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4 translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.

  12. Contracting C2C12 myotubes release CCL2 in an NF-κB-dependent manner to induce monocyte chemoattraction.

    Science.gov (United States)

    Miyatake, Shouta; Bilan, Philip J; Pillon, Nicolas J; Klip, Amira

    2016-01-15

    Muscle inflammation following exercise is characterized by expression of inflammatory cytokines and chemokines. Exercise also increases muscle macrophages derived from circulating monocytes. However, it is unknown whether muscle cells themselves attract circulating monocytes, or what is the underlying mechanism. We used an in vitro system of electrical stimulation (ES) causing C2C12 myotube contraction to explore whether monocyte chemoattraction ensues and investigated the mediating chemoattractants. Conditioned medium from ES-contracted myotubes caused robust chemoattraction of THP-1 monocytes across Boyden chambers. Following ES, expression of several known monocyte chemokines [C-C motif ligand 2 (CCL2) and C-X-C motif ligand (CXCL)1, -2, and -5] was elevated, but of these, only recombinant CCL2 effectively reproduced monocyte migration. Electrically stimulated myotubes secreted CCL2, and neutralization of CCL2 in conditioned medium or antagonizing the CCL2 receptor (CCR2) in THP-1 monocytes inhibited ES-induced monocyte migration. N-benzyl-p-toluene sulfonamide (BTS), a myosin II-ATPase inhibitor, prevented ES-induced myotube contraction but not CCL2 gene expression and secretion. The membrane-permeant calcium chelator BAPTA-AM reduced ES-induced CCL2 secretion. Hence, electrical depolarization, rather than mechanical contraction, drives the rise in CCL2, with partial calcium input. ES activated the NF-κB pathway; NF-κB inhibitors reduced ES-induced CCL2 gene expression and secretion and repressed ES-induced THP-1 chemoattraction. Thus, electrically stimulated myotubes chemoattract monocytes through NF-κB-regulated CCL2 secretion. PMID:26554595

  13. Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

    Science.gov (United States)

    Ochiai, Natsuki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2016-05-01

    Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level. PMID:26902224

  14. MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

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    Long Jia

    2013-12-01

    Full Text Available MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs of messenger RNAs (mRNAs. Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

  15. Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

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    Lomax Michael A

    2007-03-01

    Full Text Available Abstract Background The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis Results Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA, relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p Conclusion In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

  16. Effect of High Temperature- and High Pressure-Treated Red Ginseng on Lipolysis and Lipid Oxidation in C2C12 Myotubes.

    Science.gov (United States)

    Yu, Seok-Yeong; Lee, Jin-Ha; Cho, MyoungLae; Lee, Jong Seok; Hong, Hee-Do; Lee, Young-Chul; Kim, Young-Chan; Cho, Chang-Won; Kim, Kyung-Tack; Lee, Ok-Hwan

    2016-01-01

    Korean red ginseng (KRG), a highly valuable medicinal herb in oriental societies, has biological activity similar to that of Panax ginseng. Recently, it has been discovered that the biological activities of red ginseng can vary according to heating and steaming processes under different conditions that change the principal components of KRG and result in changes in biological activity. This study evaluated and compared the effects of high temperature- and high pressure-treated red ginseng (HRG) and commercial red ginseng (RG) on β-oxidation in C2C12 myotubes. HRG enhanced the phosphorylation levels of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), but RG did not affect the phosphorylation of AMPK in C2C12 myotubes. HRG also promoted the nuclear translocation of forkhead box protein O1 (FoxO1), and the translocation exerted an increase in the protein expression of adipose triglyceride lipase (ATGL). As a consequence, HRG increased the mRNA expression level of carnitine palmitoyltransferase 1 (CPT-1) compared to the control. Taken together, our results indicated that HRG promotes the lipolysis of triglycerides and mitochondrial β-oxidation of fatty acids in C2C12 myotubes, suggesting that alterations to the principal components by high temperature and pressure may positively influence the nutraceutical functions of HRG. PMID:26501225

  17. Effects of 1,25(OH)2 D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy.

    Science.gov (United States)

    van der Meijden, K; Bravenboer, N; Dirks, N F; Heijboer, A C; den Heijer, M; de Wit, G M J; Offringa, C; Lips, P; Jaspers, R T

    2016-11-01

    An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27018098

  18. Effects of 1,25(OH)2 D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy.

    Science.gov (United States)

    van der Meijden, K; Bravenboer, N; Dirks, N F; Heijboer, A C; den Heijer, M; de Wit, G M J; Offringa, C; Lips, P; Jaspers, R T

    2016-11-01

    An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  19. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Bauman, William A. [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Cardozo, Christopher, E-mail: chris.cardozo@va.gov [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  20. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    International Nuclear Information System (INIS)

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  1. The cytoprotective effect of isorhamnetin against oxidative stress is mediated by the upregulation of the Nrf2-dependent HO-1 expression in C2C12 myoblasts through scavenging reactive oxygen species and ERK inactivation.

    Science.gov (United States)

    Choi, Yung Hyun

    2016-04-01

    This study was designed to confirm the protective effects of isorhamnetin against oxidative stress-induced cellular damage. Our results indicated that isorhamnetin inhibited the hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against the intracellular reactive oxygen species (ROS) in mouse-derived C2C12 myoblasts. Isorhamnetin also significantly attenuated H2O2-induced DNA damage and apoptosis, and increased the levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) and its phosphorylation associated with the induction of heme oxygenase-1 (HO-1). However, the protective effects of isorhamnetin on H2O2-induced ROS and growth inhibition were significantly abolished by an HO-1 competitive inhibitor. Moreover, the potential of isorhamnetin to mediate HO-1 induction and protect against H2O2-mediated growth inhibition was abrogated by transient transfection with Nrf2-specific small interfering RNA. Additionally, isorhamnetin induced the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. However, the specific inhibitor of ERK, but not JNK and p38 MAPK, was able to abolish the HO-1 upregulation and the Nrf2 phosphorylation. Collectively, these results demonstrate that isorhamnetin augments the cellular antioxidant defense capacity by activating the Nrf2/HO-1 pathway involving the activation of the ERK pathway, thus protecting the C2C12 cells from H2O2-induced cytotoxicity. PMID:26830132

  2. Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants

    DEFF Research Database (Denmark)

    Poulsen, K A; Petersen, Stine Helene Falsig; Kolko, M;

    2007-01-01

    by specific short interfering (si)RNA knockdown of iPLA(2)-VIA. OGD was associated with an increase in iPLA(2)-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA(2)-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA(2)-VIA homolog...... to catalytically inactive 50-kDa iPLA(2)-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this approximately 50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA(2)-VIA seemed to predominantly reside at the endoplasmatic reticulum......, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA(2)-VIA activity that decreases cell viability. iPLA(2)-VIA activation may be modulated by changes in the levels of active and inactive iPLA(2)-VIA isoforms. Udgivelsesdato: 2007-Nov...

  3. DHA Inhibits Protein Degradation More Efficiently than EPA by Regulating the PPARγ/NFκB Pathway in C2C12 Myotubes

    OpenAIRE

    Yue Wang; Qiao-wei Lin; Pei-pei Zheng; Jian-song Zhang; Fei-ruo Huang

    2013-01-01

    This study was conducted to evaluate the mechanism by which n-3 PUFA regulated the protein degradation in C2C12 myotubes. Compared with the BSA control, EPA at concentrations from 400 to 600 µM decreased total protein degradation (P < 0.01). However, the total protein degradation was decreased when the concentrations of DHA ranged from 300 µM to 700 µM (P < 0.01). DHA (400 µM, 24 h) more efficiently decreased the I κ B α phosphorylation and increased in the I κ B α protein level than 400 µM E...

  4. CHARACTERIZATION OF P2-PURINOCEPTOR MEDIATED CYCLIC-AMP FORMATION IN MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1993-01-01

    1 The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2Cl2 myotubes. 2 ATP (100 muM) and ATPgammaS (100 muM) caused a sustained increase in cyclic AMP content of the cells,

  5. Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression

    DEFF Research Database (Denmark)

    Grossi, Alberto Blak; Lametsch, Rene; Karlsson, Anders H;

    2011-01-01

    to specific receptors on the cell surface. We showed that mechanical stimuli promote an increase of FAK phosphorylation. In order to further shed light in the process of myoblast induced differentiation by mechanical stimuli, we performed a proteomic analysis. Thirteen proteins were found to be affected...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

  6. Development of gas chromatography-flame ionization detection system with a single column and liquid nitrogen-free for measuring atmospheric C2-C12 hydrocarbons.

    Science.gov (United States)

    Liu, Chengtang; Mu, Yujing; Zhang, Chenglong; Zhang, Zhibo; Zhang, Yuanyuan; Liu, Junfeng; Sheng, Jiujiang; Quan, Jiannong

    2016-01-01

    A liquid nitrogen-free GC-FID system equipped with a single column has been developed for measuring atmospheric C2-C12 hydrocarbons. The system is consisted of a cooling unit, a sampling unit and a separation unit. The cooling unit is used to meet the temperature needs of the sampling unit and the separation unit. The sampling unit includes a dehydration tube and an enrichment tube. No breakthrough of the hydrocarbons was detected when the temperature of the enrichment tube was kept at -90 °C and sampling volume was 400 mL. The separation unit is a small round oven attached on the cooling column. A single capillary column (OV-1, 30 m × 0.32 mm I.D.) was used to separate the hydrocarbons. An optimal program temperature (-60 ∼ 170 °C) of the oven was achieved to efficiently separate C2-C12 hydrocarbons. There were good linear correlations (R(2)=0.993-0.999) between the signals of the hydrocarbons and the enrichment amount of hydrocarbons, and the relative standard deviation (RSD) was less than 5%, and the method detection limits (MDLs) for the hydrocarbons were in the range of 0.02-0.10 ppbv for sampling volume of 400 mL. Field measurements were also conducted and more than 50 hydrocarbons from C2 to C12 were detected in Beijing city.

  7. Development of gas chromatography-flame ionization detection system with a single column and liquid nitrogen-free for measuring atmospheric C2-C12 hydrocarbons.

    Science.gov (United States)

    Liu, Chengtang; Mu, Yujing; Zhang, Chenglong; Zhang, Zhibo; Zhang, Yuanyuan; Liu, Junfeng; Sheng, Jiujiang; Quan, Jiannong

    2016-01-01

    A liquid nitrogen-free GC-FID system equipped with a single column has been developed for measuring atmospheric C2-C12 hydrocarbons. The system is consisted of a cooling unit, a sampling unit and a separation unit. The cooling unit is used to meet the temperature needs of the sampling unit and the separation unit. The sampling unit includes a dehydration tube and an enrichment tube. No breakthrough of the hydrocarbons was detected when the temperature of the enrichment tube was kept at -90 °C and sampling volume was 400 mL. The separation unit is a small round oven attached on the cooling column. A single capillary column (OV-1, 30 m × 0.32 mm I.D.) was used to separate the hydrocarbons. An optimal program temperature (-60 ∼ 170 °C) of the oven was achieved to efficiently separate C2-C12 hydrocarbons. There were good linear correlations (R(2)=0.993-0.999) between the signals of the hydrocarbons and the enrichment amount of hydrocarbons, and the relative standard deviation (RSD) was less than 5%, and the method detection limits (MDLs) for the hydrocarbons were in the range of 0.02-0.10 ppbv for sampling volume of 400 mL. Field measurements were also conducted and more than 50 hydrocarbons from C2 to C12 were detected in Beijing city. PMID:26687163

  8. Oxidative stress-induced metabolic changes in mouse C2C12 myotubes studied with high-resolution 13C, 1H, and 31P NMR spectroscopy

    DEFF Research Database (Denmark)

    Straadt, Ida K; Young, Jette F; Petersen, Bent O;

    2010-01-01

    In this study, stress in relation to slaughter was investigated in a model system by the use of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H(2)O(2)-induced stress. Oxidative stress resulted in lower...... to lower levels of the unlabeled ((12)C) lactate were identified in the (1)H spectra after stress exposure. These data indicate an increase in de novo synthesis of alanine, concomitant with a release of lactate from the myotubes to the medium at oxidative stress conditions. The changes in the metabolite...... levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the (13)C spectra after incubation with [(13)C(1)]glucose. In the (13)C spectra [(13)C(3)]lactate tended to increase after exposure to increasing concentrations of H(2)O(2); conversely, a tendency...

  9. Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2

    Directory of Open Access Journals (Sweden)

    Lan eYe

    2012-09-01

    Full Text Available Rapamycin, an inhibitor of mTOR complex 1 (mTORC1, improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24-48 hours. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

  10. The Cytoprotective Effect of Petalonia binghamiae Methanol Extract against Oxidative Stress in C2C12 Myoblasts: Mediation by Upregulation of Heme Oxygenase-1 and Nuclear Factor-Erythroid 2 Related Factor 2

    Directory of Open Access Journals (Sweden)

    Ji Sook Kang

    2015-04-01

    Full Text Available This study was designed to examine the protective effects of the marine brown algae Petalonia binghamiae against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms. P. binghamiae methanol extract (PBME prevented hydrogen peroxide (H2O2-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS induced by H2O2 in mouse-derived C2C12 myoblasts. PBME also significantly attenuated H2O2-induced comet tail formation in a comet assay, histone γH2A.X phosphorylation, and annexin V-positive cells, suggesting that PBME prevented H2O2-induced cellular DNA damage and apoptotic cell death. Furthermore, PBME increased the levels of heme oxygenase-1 (HO-1, a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2 related factor 2 (Nrf2. However, zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of PBME on H2O2-induced ROS generation, growth inhibition, and apoptosis. Collectively, these results demonstrate that PBME augments the antioxidant defense capacity through activation of the Nrf2/HO-1 pathway.

  11. APS improves free fatty acid metabolism by activating AMPK and promoting translocation of FAT/CD36 in C2C12 myoblasts%黄芪多糖通过活化AMPK和促进骨骼肌FAT/CD36转位改善成肌细胞FFAs代谢

    Institute of Scientific and Technical Information of China (English)

    胡阳黔; 李静; 刘坚; 欧阳静萍; 宋杰

    2013-01-01

    AIM: To investigate the effect of Astragalus polysaccharides (APS) on the metabolism of free fatty acids (FFAs) in C2C12 myoblasts. METHODS: Cultured C2C12 myoblasts were used in the study. The viability of C2C12 myoblasts treated with FFAs at different concentrations for different time was observed by MTT assay. The concentrations of FFAs in the medium were detected by acetyl-CoA synthase (ACS) -acetyl-CoA oxidase (ACOD) method. The expression of fatty acid translocase (FAT/CD36), AMPK and p-AMPK protein was examined by Western blotting. RESULTS; FFAs decreased the viability of C2C12 myoblasts in a time- and concentration-dependent manner. Compared with FFAs group, the expression of cellular membrane FAT/CD36 and p-AMPK proteins increased in FFAs + APS group, but total AMPK and FAT/CD36 protein expression was not significantly changed. Meanwhile, the concentration of FFAs in the medium decreased and the cell viability increased in FFAs + APS group as compared with the group. CONCLUSION: APS improves the metabolism of FFAs by activating AMPK and promoting translocation of FAT/CD36 in C2C12 myoblasts.%目的:探讨黄芪多糖(Astragalus polysaccharides,APS)对骨骼肌游离脂肪酸(free fatty acids,FFAs)代谢的影响及其机制.方法:培养小鼠C2C12成肌细胞;MTT法检测不同浓度FFAs作用不同时间对细胞活性的影响.根据MTT结果选取FFAs最适浓度和时间处理细胞并用APS干预,采用乙酰辅酶A合成酶-乙酰辅酶A氧化酶法检测APS干预前后培养液FFAs浓度;Western blotting测APS干预前后细胞膜脂肪酸转位酶(FAT/CD36)、总FAT/CD36、磷酸化腺苷酸活化蛋白激酶(phosphorylated AMP-activated protein kinase,p-AMPK)和总AMPK蛋白表达.结果:FFAs对细胞的毒性呈浓度和时间依赖性.与FFAs组比较,FFAs+ APS组细胞膜FAT/CD36及p-AMPK蛋白表达增加(P<0.05),而总FAT/CD36及总AMPK蛋白表达无明显差异(P>0.05),同时培养液FFAs浓度降低,细胞活性增加(P<0.05).

  12. Irisin response to exercise training in adults and its effect on the regulation of C2C12 cell proliferation and differentiation

    OpenAIRE

    Qiu, Shanhu

    2015-01-01

    Irisin is now recognized as an exercise-induced hormone that is produced primarily by skeletal muscles and adipose tissue. However, its physiological characteristics in the response to acute or chronic exercise training still remains somewhat controversial. By using serum samples at different time points before and after an acute bout of exercise among trained and untrained healthy adults, this study showed that serum irisin transiently increases in response to acute exercise, which is indepe...

  13. Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Feveile Young, Jette; Oksbjerg, Niels;

    2003-01-01

    C2C12, calcium, cell volume regulation, 5-lipoxygenase, melittin, anoxia, secretory phospholipase A2......C2C12, calcium, cell volume regulation, 5-lipoxygenase, melittin, anoxia, secretory phospholipase A2...

  14. Ethanol extract of the Prunus mume fruits stimulates glucose uptake by regulating PPAR-γ in C2C12 myotubes and ameliorates glucose intolerance and fat accumulation in mice fed a high-fat diet.

    Science.gov (United States)

    Shin, Eun Ju; Hur, Haeng Jeon; Sung, Mi Jeong; Park, Jae Ho; Yang, Hye Jeong; Kim, Myung Sunny; Kwon, Dae Young; Hwang, Jin-Taek

    2013-12-15

    In this study, we performed in vitro and in vivo studies to examine whether a 70% ethanol extract of Prunus mume fruits (EMS) exhibits anti-diabetic effects. Treatment with EMS increased glucose uptake in C2C12 myotubes, and also increased PPAR-γ activity or PPAR-γ mRNA expression. To confirm these in vitro results, we next conducted an animal experiment. A high-fat diet significantly increased the body weight, fat accumulation, and glucose levels in mice. Under the same conditions, 5% EMS attenuated the high-fat diet-induced increase in body weight and fat accumulation and improved the impaired fasting glucose level and glucose tolerance. High performance liquid chromatography analysis demonstrated that EMS contained chlorogenic acid, caffeic acid, rutin, luteolin-7-glucoside, naringin, apigenin-7-glucoside, and hesperidin. Taken together, these findings suggest that EMS exerts an anti-diabetic effect both in vitro and in vivo, which is mediated, at least in part, by the activation of PPAR-γ. PMID:23993593

  15. Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

    International Nuclear Information System (INIS)

    Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process

  16. A Cistanches Herba Fraction/β-Sitosterol Causes a Redox-Sensitive Induction of Mitochondrial Uncoupling and Activation of Adenosine Monophosphate-Dependent Protein Kinase/Peroxisome Proliferator-Activated Receptor γ Coactivator-1 in C2C12 Myotubes: A Possible Mechanism Underlying the Weight Reduction Effect

    Directory of Open Access Journals (Sweden)

    Hoi Shan Wong

    2015-01-01

    Full Text Available Previous studies have demonstrated that HCF1, a semipurified fraction of Cistanches Herba, causes weight reduction in normal diet- and high fat diet-fed mice. The weight reduction was associated with the induction of mitochondrial uncoupling and changes in metabolic enzyme activities in mouse skeletal muscle. To further investigate the biochemical mechanism underlying the HCF1-induced weight reduction, the effect of HCF1 and its active component, β-sitosterol (BSS, on C2C12 myotubes was examined. Incubation with HCF1/BSS caused a transient increase in mitochondrial membrane potential (MMP, possibly by fluidizing the mitochondrial inner membrane. The increase in MMP was paralleled to an increase in mitochondrial reactive oxygen species (ROS production. Mitochondrial ROS, in turn, triggered a redox-sensitive induction of mitochondrial uncoupling by uncoupling protein 3 (UCP3. Biochemical analysis indicated that HCF1 was capable of activating an adenosine monophosphate-dependent protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 pathway and thereby increased the expression of cytochrome c oxidase and UCP3. Animal studies using mitochondrial recoupler also confirmed the role of mitochondrial uncoupling in the HCF1-induced weight reduction. In conclusion, a HCF1/BSS causes the redox-sensitive induction of mitochondrial uncoupling and activation of AMPK/PGC-1 in C2C12 myotubes, with resultant reductions in body weight and adiposity by increased energy consumption.

  17. Cure Behaviors and Water Up-take Evaluation of a New Waterborne Epoxy Resin

    Institute of Scientific and Technical Information of China (English)

    WAN Tao; ZANG Tianshun; ZHANG Rui; SUN Xianchang

    2012-01-01

    Cure behaviors and water up-take evaluation of a low cost,ecofriendly and water soluble epoxy resin prepared by reaction between epichlorohydrin and PEG400,PEG600 and PEG1000,respectively,were investigated using non-isothermal differential scanning calorimetry (DSC) and gravimetrical method,respectively.Factors affecting the cure behaviors as well as water up-take of waterborne epoxy resins,such as amount of triethylenetetramine (TETA) and triethylene diamine (TEDA),PEG molecular weight,curing temperature,were systematically investigated.The prepared water soluble epoxy resins can be cured under room temperature with the shape of the curing curves similar to that expected for an autocatalytic reaction.

  18. Selective processes for bioaccumulative up-take of persistent organic pollutants (POPs) in Arctic food webs

    OpenAIRE

    Carlsson, Pernilla Marianne

    2013-01-01

    The overall aim of the present study was to elucidate selective environmental up-take processes in Arctic food webs that lead to the bioaccumulation of persistent organic pollutants (POP) in food items consumed by Arctic indigenous people. In addition, this study aimed to increase the scientific understanding of the principles behind climate change related influences on transport processes of contaminants. Svalbard and Nuuk, Greenland were chosen as study areas since they represent Arctic con...

  19. Emissions of C2 - C12 hydrocarbons in the Hsuehshan tunnel, Taiwan

    Institute of Scientific and Technical Information of China (English)

    Chia-Hsiang Lai; Yen-Ping Peng

    2011-01-01

    The concentrations of 56 hydrocarbons from C2 to C12 were measured simultaneously in the southbound bore, the northbound bore and the exhaust air shafts of the Hsuehshan tunnel near Yilan, Taiwan for 12 days during 2007 and 2008.A total of 60 integrated air samples were collected using stainless steel canisters and analyzed using GC/FID and GC/MS.The five most abundant species in all samples were ethylene, acetylene, isopentane, propylene and toluene.The exit/entrance ratios of total non-methane hydrocarbons (NMHC) concentration were 7.8 and 4.8 for the southbound and northbound bores, respectively.Furthermore, the exhaust from the vertical shafts affects air quality in the neighborhood.The most abundant species of emission rate (ER) was toluene (21.93-42.89 mg/sec), followed by isopentane, ethylene, propylene and l~butene, with ER ranging from 2.50 to 9.31 mg/sec.The species in the three exhaust air shafts showed that the reacfivifies of these emissions are similar to those of vehicle emissions.Notably, the control of emissions in the vertical shafts of the vehicle tunnel will be important in the future.

  20. Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor

    OpenAIRE

    John, Scott A.; Ottolia, Michela; James N Weiss; Ribalet, Bernard

    2007-01-01

    To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600µM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (τ =5 to 15 s) in COS and C2C12 cells and slow (τ =20 to 40 s) in HEK and CHO cells. Upon ...

  1. Sequence-selective DNA recognition and enhanced cellular up-take by peptide-steroid conjugates.

    Science.gov (United States)

    Ruiz García, Yara; Iyer, Abhishek; Van Lysebetten, Dorien; Pabon, Y Vladimir; Louage, Benoit; Honcharenko, Malgorzata; De Geest, Bruno G; Smith, C I Edvard; Strömberg, Roger; Madder, Annemieke

    2015-12-25

    Several GCN4 bZIP TF models have previously been designed and synthesized. However, the synthetic routes towards these constructs are typically tedious and difficult. We here describe the substitution of the Leucine zipper domain of the protein by a deoxycholic acid derivative appending the two GCN4 binding region peptides through an optimized double azide-alkyne cycloaddition click reaction. In addition to achieving sequence specific dsDNA binding, we have investigated the potential of these compounds to enter cells. Confocal microscopy and flow cytometry show the beneficial influence of the steroid on cell uptake. This unique synthetic model of the bZIP TF thus combines sequence specific dsDNA binding properties with enhanced cell-uptake. Given the unique properties of deoxycholic acid and the convergent nature of the synthesis, we believe this work represents a key achievement in the field of TF mimicry.

  2. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  3. Differential regulation of iPLA2beta splice variants by in vitro ischemia in C2C12 myotubes

    DEFF Research Database (Denmark)

    Poulsen, K. A.; Kolko, M.; Lambert, I. H.

    2006-01-01

    in mice. Using PCR-cloning we identified a PCR-fragment that had a 29 bp insertion between exon 9 and 10. This sequence has high homology to the first part of the 53 bp human exon 9a. The 29 bp insertion induces a frame-shift and the introduction of a stop codon in exon 10. The protein product...

  4. Genome-wide examination of myoblast cell cycle withdrawal duringdifferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xun; Collier, John Michael; Hlaing, Myint; Zhang, Leanne; Delshad, Elizabeth H.; Bristow, James; Bernstein, Harold S.

    2002-12-02

    Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40 percent fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly

  5. Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Bolado-Carrancio, A. [Department of Molecular Biology, University of Cantabria, IDIVAL, Santander (Spain); Riancho, J.A. [Department of Internal Medicine, Hospital U.M. Valdecilla-IDIVAL, University of Cantabria, RETICEF, Santander (Spain); Sainz, J. [Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC-University of Cantabria, Santander (Spain); Rodríguez-Rey, J.C., E-mail: rodriguj@unican.es [Department of Molecular Biology, University of Cantabria, IDIVAL, Santander (Spain)

    2014-04-04

    Highlights: • NR5A2 expression in C2C12 is associated with myotube differentiation. • DLPC induces an increase in GLUT4 levels and glucose uptake in C2C12 myotubes. • In high glucose conditions the activation of NR5A2 inhibits fatty acids oxidation. - Abstract: NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Because skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity.

  6. Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

    International Nuclear Information System (INIS)

    Highlights: • NR5A2 expression in C2C12 is associated with myotube differentiation. • DLPC induces an increase in GLUT4 levels and glucose uptake in C2C12 myotubes. • In high glucose conditions the activation of NR5A2 inhibits fatty acids oxidation. - Abstract: NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Because skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity

  7. Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells.

    Directory of Open Access Journals (Sweden)

    Jerome E Lee

    Full Text Available BACKGROUND: Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. PRINCIPAL FINDINGS: We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE and AU-rich (ARE elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1

  8. Self-Healing Conductive Injectable Hydrogels with Antibacterial Activity as Cell Delivery Carrier for Cardiac Cell Therapy.

    Science.gov (United States)

    Dong, Ruonan; Zhao, Xin; Guo, Baolin; Ma, Peter X

    2016-07-13

    Cell therapy is a promising strategy to regenerate cardiac tissue for myocardial infarction. Injectable hydrogels with conductivity and self-healing ability are highly desirable as cell delivery vehicles for cardiac regeneration. Here, we developed self-healable conductive injectable hydrogels based on chitosan-graft-aniline tetramer (CS-AT) and dibenzaldehyde-terminated poly(ethylene glycol) (PEG-DA) as cell delivery vehicles for myocardial infarction. Self-healed electroactive hydrogels were obtained after mixing CS-AT and PEG-DA solutions at physiological conditions. Rapid self-healing behavior was investigated by rheometer. Swelling behavior, morphology, mechanical strength, electrochemistry, conductivity, adhesiveness to host tissue and antibacterial property of the injectable hydrogels were fully studied. Conductivity of the hydrogels is ∼10(-3) S·cm(-1), which is quite close to native cardiac tissue. Proliferation of C2C12 myoblasts in the hydrogel showed its good biocompatibility. After injection, viability of C2C12 cells in the hydrogels showed no significant difference with that before injection. Two different cell types were successfully encapsulated in the hydrogels by self-healing effect. Cell delivery profile of C2C12 myoblasts and H9c2 cardiac cells showed a tunable release rate, and in vivo cell retention in the conductive hydrogels was also studied. Subcutaneous injection and in vivo degradation of the hydrogels demonstrated their injectability and biodegradability. Together, these self-healing conductive biodegradable injectable hydrogels are excellent candidates as cell delivery vehicle for cardiac repair. PMID:27311127

  9. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Kazuya, E-mail: asuno10k@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Wada, Eiji, E-mail: gacchu1@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Zammit, Peter S., E-mail: peter.zammit@kcl.ac.uk [Randall Division of Cell and Molecular Biophysics, King' s College London, London SE1 1UL (United Kingdom); Shiozuka, Masataka, E-mail: cmuscle@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Matsuda, Ryoichi, E-mail: cmatsuda@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan)

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  10. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    International Nuclear Information System (INIS)

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells

  11. Application of 1H and 23Na magic angle spinning NMR spectroscopy to define the HRBC up-taking of MRI contrast agents

    Science.gov (United States)

    Calabi, Luisella; Paleari, Lino; Biondi, Luca; Linati, Laura; De Miranda, Mario; Ghelli, Stefano

    2003-09-01

    The up-take of Gd(III) complexes of BOPTA, DTPA, DOTA, EDTP, HPDO3A, and DOTP in HRBC has been evaluated by measuring the lanthanide induced shift (LIS) produced by the corresponding dysprosium complexes (DC) on the MAS-NMR resonances of water protons and free sodium ions. These complexes are important in their use as MRI contrast agents (MRI-CA) in diagnostics. 1H and 23Na MAS-NMR spectra of HRBC suspension, collected at 9.395 T, show only one signal due to extra- and intra-cellular water (or sodium). In MAS spectra, the presence of DC in a cellular compartment produces the LIS of only the nuclei (water proton or sodium) in that cellular compartment and this LIS can be related to the DC concentrations (by the experimental curves of LIS vs. DC concentrations) collected in the physiological solution. To obtain correct results about LIS, the use of MAS technique is mandatory, because it guarantees the only the nuclei staying in the same cellular compartment where the LC is present show the LIS. In all the cases considered, the addition of the DC to HRBC (100% hematocrit) produced a shift of only the extra-cellular water (or sodium) signal and the gradient of concentration ( GC) between extra- and intra-cellular compartments resulted greater than 100:1, when calculated by means of sodium signals. These high values of GC are direct proofs that none of the tested dysprosium complexes crosses the HRBC membrane. Since the DC are iso-structural to the gadolinium complexes the corresponding gadolinium ones (MRI-CA) do not cross the HRBC membrane and, consequently, they are not up-taken in HRBC. The GC values calculated by means of water proton signals resulted much lower than those obtained by sodium signals. This proves that the choice of the isotope is a crucial step in order to use this method in the best way. In fact, GC value depends on the lowest detectable LIS which, in turn, depends on the nature of the LC (lanthanide complex) and the observed isotopes.

  12. Bone marrow mesenchymal stromal cells stimulate skeletal myoblast proliferation through the paracrine release of VEGF.

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

    Full Text Available Mesenchymal stromal cells (MSCs are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.

  13. Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

    Science.gov (United States)

    Chellini, Flaminia; Mazzanti, Benedetta; Nistri, Silvia; Nosi, Daniele; Saccardi, Riccardo; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2012-01-01

    Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies. PMID:22815682

  14. Patterned three-dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography.

    Science.gov (United States)

    Bajaj, Piyush; Marchwiany, Daniel; Duarte, Carlos; Bashir, Rashid

    2013-03-01

    Controlling the assembly of cells in three dimensions is very important for engineering functional tissues, drug screening, probing cell-cell/cell-matrix interactions, and studying the emergent behavior of cellular systems. Although the current methods of cell encapsulation in hydrogels can distribute them in three dimensions, these methods typically lack spatial control of multi-cellular organization and do not allow for the possibility of cell-cell contacts as seen for the native tissue. Here, we report the integration of dielectrophoresis (DEP) with stereolithography (SL) apparatus for the spatial patterning of cells on custom made gold micro-electrodes. Afterwards, they are encapsulated in poly (ethylene glycol) diacrylate (PEGDA) hydrogels of different stiffnesses. This technique can mimic the in vivo microscale tissue architecture, where the cells have a high degree of three dimensional (3D) spatial control. As a proof of concept, we show the patterning and encapsulation of mouse embryonic stem cells (mESCs) and C2C12 skeletal muscle myoblasts. mESCs show high viability in both the DEP (91.79% ± 1.4%) and the no DEP (94.27% ± 0.5%) hydrogel samples. Furthermore, we also show the patterning of mouse embryoid bodies (mEBs) and C2C12 spheroids in the hydrogels, and verify their viability. This robust and flexible in vitro platform can enable various applications in stem cell differentiation and tissue engineering by mimicking elements of the native 3D in vivo cellular micro-environment.

  15. Yin-Yang Regulation of Adiponectin Signaling by APPL Isoforms in Muscle Cells*

    OpenAIRE

    Wang, Changhua; Xin, Xiaoban; Xiang, Ruihua; Ramos, Fresnida J.; Liu, Meilian; Lee, Hak Joo; Chen, Hongzhi; Mao, Xuming; Kikani, Chintan K.; Liu, Feng; Dong, Lily Q.

    2009-01-01

    APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 ...

  16. Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

    Science.gov (United States)

    Tani, Alessia; Anderloni, Giulia; Pierucci, Federica; Matteini, Francesca; Chellini, Flaminia; Zecchi Orlandini, Sandra; Meacci, Elisabetta

    2014-01-01

    Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration. PMID:25264785

  17. Mesenchymal stromal cell secreted sphingosine 1-phosphate (S1P exerts a stimulatory effect on skeletal myoblast proliferation.

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

    Full Text Available Bone-marrow-derived mesenchymal stromal cells (MSCs have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P, a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK, blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.

  18. An engineered approach to stem cell culture: automating the decision process for real-time adaptive subculture of stem cells.

    Science.gov (United States)

    Ker, Dai Fei Elmer; Weiss, Lee E; Junkers, Silvina N; Chen, Mei; Yin, Zhaozheng; Sandbothe, Michael F; Huh, Seung-il; Eom, Sungeun; Bise, Ryoma; Osuna-Highley, Elvira; Kanade, Takeo; Campbell, Phil G

    2011-01-01

    Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and developing robotic cell

  19. Experiment list: SRX956813 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available le|Tissue Diagnosis=NOS 45079115,96.0,22.7,572 GSM1633918: IgG parental C2C12, T24; Mus musculus; ChIP-Seq s...ource_name=C2C12 cells || cell line=parental C2C12 || antibody=normal rabbit IgG http://dbarchive.bioscience

  20. Experiment list: SRX956812 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available le|Tissue Diagnosis=NOS 49892791,96.2,19.4,629 GSM1633917: anti-Flag parental C2C12, T24; Mus musculus; ChIP...-Seq source_name=C2C12 cells || cell line=parental C2C12 || antibody=anti-Flag http://dbarchive.biosciencedb

  1. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  2. Expression of Lecithin: Cholesterol Acyltransferaseand/or apoA-I Mediated by Recombinant Adeno-as-sociated Virus in Myogenic Cell

    Institute of Scientific and Technical Information of China (English)

    王立峰; 范乐明; 陈丙莺; 刘宝瑞; 王若宁; 魏恩会

    2002-01-01

    Objective Lecithia: cholesterol acyltrmsfer ase (LCAT) is the major enzyme producing most plasma cholesterol esters( CE )and a key partiipant in the process of reverse cholesterol traansfer ( RCT). The aim of the study was to co-express LCAT and its nature activator apoA- I medi ated by recombinant adeno-associated virus vectors in the skeletal muscle cells, and open a new avenue of gene therapy touard the primary or secondary LCAT deficiency. Methods 293T cells were cotrans fected with pDG and rAAVAIL/rAAVL plasmid to produce infectious rAAV, and non-iouic iodixanol gradients centri f ngation followed by heparin affinity chromatography was per formed f or separation . pu rification and concentration of rAAV. The particle numbers of rAAV were assayed by dot-blot, then these vectors transduced C2C12 myoblasts. ELISA and Western Blot asasayed for human apoA- I and 3H-cholesterol labeled radiochemical methods for LCAT activity. Genomic DNA was extracted from transduced C2C12 and analyzed fo the presence of vector sequence by PCR amplifiations. Results The particle mumbers of rAAV were 7× 1014/L (rAAAIL) and 1 × 1014/L (rAAVL). The expres sion of human apoA- I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 3 0 d, even after myoblasts were differentiated into myotubes. PCR products for transgene indiated the long-term persistence of transduced vector sequences. Conclusion The result indicated that the meth ods used for production and purification of rAAV is an effiient and rAAV vector mediate the expres sion and secretion of LCAT and apoA- I gene in C2C12 myoblasts successfully. It suggested that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/ or apoA- I cDNA in skeletal muscle in vivo might be a safe and fessible strategy to the gene therapy of LCAT deficiency.

  3. Gene expression profiles and phosphorylation patterns of AMP-activated protein kinase subunits in various mesenchymal cell types

    Institute of Scientific and Technical Information of China (English)

    Wang Yugang; Fan Qiming; Ma Rui; Lin Wentao; Tang Tingting

    2014-01-01

    Background Recent studies on bone have shown an endocrine role of the skeleton,which could be impaired in various human diseases,including osteoporosis,obesity,and diabetes-associated bone diseases.As a sensor and regulator of energy metabolism,AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism.The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.Methods Reverse transcription-polymerase chain reaction (PCR) for relative quantification,real-time PCR for absolute quantification,and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types,including primary human mesenchymal stem cells (hMSCs) and hFOB,Saos-2,C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells.Results AMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types.AMPKY1 mRNA was abundantly expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 but not detected in human-derived cell types.AMPKY2 mRNA was mildly expressed in all cell types.AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells.AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2,in which AMPKβ2 protein overwhelmed AMPKβ1 expression.AMPKy1 and AMPKY2 proteins were expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells and only AMPKY2 protein was expressed in hMSCs,hFOB and Saos2 cells.AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.Conclusion The combination of AMPK α,β,and Y subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.

  4. A spectral k-means approach to bright-field cell image segmentation.

    Science.gov (United States)

    Bradbury, Laura; Wan, Justin W L

    2010-01-01

    Automatic segmentation of bright-field cell images is important to cell biologists, but difficult to complete due to the complex nature of the cells in bright-field images (poor contrast, broken halo, missing boundaries). Standard approaches such as level set segmentation and active contours work well for fluorescent images where cells appear as round shape, but become less effective when optical artifacts such as halo exist in bright-field images. In this paper, we present a robust segmentation method which combines the spectral and k-means clustering techniques to locate cells in bright-field images. This approach models an image as a matrix graph and segment different regions of the image by computing the appropriate eigenvectors of the matrix graph and using the k-means algorithm. We illustrate the effectiveness of the method by segmentation results of C2C12 (muscle) cells in bright-field images. PMID:21096019

  5. Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro

    DEFF Research Database (Denmark)

    Wewer, U M; Iba, K; Durkin, M E;

    1998-01-01

    cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced......Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell......, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates...

  6. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    Science.gov (United States)

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway. PMID:17588539

  7. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    Science.gov (United States)

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway.

  8. Cell entry of lymphocytic choriomeningitis virus is restricted in myotubes.

    Science.gov (United States)

    Iwasaki, Masaharu; Urata, Shuzo; Cho, Yoshitake; Ngo, Nhi; de la Torre, Juan C

    2014-06-01

    In mice persistently infected since birth with the prototypic arenavirus lymphocytic choriomeningitis viurs, viral antigen and RNA are readily detected in most organs and cell types but remarkably absent in skeletal muscle. Here we report that mouse C2C12 myoblasts that are readily infected by LCMV, become highly refractory to LCMV infection upon their differentiation into myotubes. Myotube's resistance to LCMV was not due to an intracellular restriction of virus replication but rather an impaired cell entry mediated by the LCMV surface glycoprotein. Our findings provide an explanation for the observation that in LCMV carrier mice myotubes, which are constantly exposed to blood-containing virus, remain free of viral antigen and RNA despite myotubes express high levels of the LCMV receptor alpha dystroglycan and do not pose an intracellular blockade to LCMV multiplication.

  9. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells.

    Science.gov (United States)

    Goutzourelas, Nikolaos; Stagos, Dimitrios; Spanidis, Ypatios; Liosi, Maria; Apostolou, Anna; Priftis, Alexandros; Haroutounian, Serko; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

    2015-10-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  10. The transcriptional repressor ZBP-89 and the lack of Sp1/Sp3, c-Jun, and Stat3 are important for the down-regulation of the vimentin gene during C2C12 myogenesis

    OpenAIRE

    Salmon, Morgan; Zehner, Zendra E.

    2009-01-01

    Currently, considerable information is available about how muscle-specific genes are activated during myogenesis, yet little is known about how non-muscle genes are down-regulated. The intermediate filament protein vimentin is known to be “turned off” during myogenesis to be replaced by desmin, the muscle-specific intermediate filament protein. Here, we demonstrate that vimentin down-regulation is the result of the combined effect of several transcription factors. Levels of the positive activ...

  11. Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

    Science.gov (United States)

    Whitson, Peggy A.; Stuart, Charles A.; Huls, M. H.; Sams, Clarence F.; Cintron, Nitza M.

    1989-01-01

    The effect of dexamethasone on the activity of creatine kinase (CK) and the insulin-like growth factor I (IGF-I) binding were investigated using skeletal- and cardiac-muscle-derived cultured cell lines (mouse, C2C12; rat, L6 and H9c2). It was found that, in skeletal muscle cells, dexamethasone treatment during differentiation of skeletal-muscle cells caused dose-dependent increases in CK activity and increases in the degree of myotube formation, whereas cardiac cells (H9c2) exhibited very low CK activity during culture or dexamethasone treatment. Results for IGF-I binding were similar in all three cell lines. The IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr on the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities.

  12. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  13. Robust fabrication of electrospun-like polymer mats to direct cell behaviour.

    Science.gov (United States)

    Ballester-Beltrán, José; Lebourg, Myriam; Capella, Hector; Diaz Lantada, Andres; Salmerón-Sánchez, Manuel

    2014-09-01

    Currently, cell culture systems that include nanoscale topography are widely used in order to provide cells additional cues closer to the in vivo environment, seeking to mimic the natural extracellular matrix. Electrospinning is one of the most common techniques to produce nanofiber mats. However, since many sensitive parameters play an important role in the process, a lack of reproducibility is a major drawback. Here we present a simple and robust methodology to prepare reproducible electrospun-like samples. It consists of a polydimethylsiloxane mold reproducing the fiber pattern to solvent-cast a polymer solution and obtain the final sample. To validate this methodology, poly(L-lactic) acid (PLLA) samples were obtained and, after characterisation, bioactivity and ability to direct cell response were assessed. C2C12 myoblasts developed focal adhesions on the electrospun-like fibers and, when cultured under myogenic differentiation conditions, similar differentiation levels to electrospun PLLA fibers were obtained.

  14. Network Analysis for the Identification of Differentially Expressed Hub Genes Using Myogenin Knock-down Muscle Satellite Cells.

    Directory of Open Access Journals (Sweden)

    Adeel Malik

    Full Text Available Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4. Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs. Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs using GeneMANIA, cadherin-associated protein (CTNNA2 was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM genes (type I collagen α1 and type I collagen α2 along with myostatin (MSTN, was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new

  15. Cell Stratification, Spheroid Formation and Bioscaffolds Used to Grow Cells in Three Dimensional Cultures

    Directory of Open Access Journals (Sweden)

    Hana Hrebíková

    2015-12-01

    Full Text Available The cell culture became an invaluable tool for studying cell behaviour, development, function, gene expression, toxicity of compounds and efficacy of novel drugs. Although most results were obtained from cell cultivation in two-dimensional (2D systems, in which cells are grown in a monolayer, three-dimensional (3D cultures are more promising as they correspond closely to the native arrangement of cells in living tissues. In our study, we focused on three types of 3D in vitro systems used for cultivation of one cell type. Cell morphology, their spatial distribution inside of resulting multicellular structures and changes in time were analysed with histological examination of samples harvested at different time periods. In multilayered cultures of WRL 68 hepatocytes grown on semipermeable membranes and non-passaged neurospheres generated by proliferation of neural progenitor cells, the cells were tightly apposed, showed features of cell differentiation but also cell death that was observable in short-term cultures. Biogenic scaffolds composed of extracellular matrix of the murine tibial anterior muscle were colonized with C2C12 myoblasts in vitro. The recellularized scaffolds did not reach high cell densities comparable with the former systems but supported well cell anchorage and migration without any signs of cell regression.

  16. Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry.

    Science.gov (United States)

    Davidenko, Natalia; Schuster, Carlos F; Bax, Daniel V; Farndale, Richard W; Hamaia, Samir; Best, Serena M; Cameron, Ruth E

    2016-10-01

    Studies of cell attachment to collagen-based materials often ignore details of the binding mechanisms-be they integrin-mediated or non-specific. In this work, we have used collagen and gelatin-based substrates with different dimensional characteristics (monolayers, thin films and porous scaffolds) in order to establish the influence of composition, crosslinking (using carbodiimide) treatment and 2D or 3D architecture on integrin-mediated cell adhesion. By varying receptor expression, using cells with collagen-binding integrins (HT1080 and C2C12 L3 cell lines, expressing α2β1, and Rugli expressing α1β1) and a parent cell line C2C12 with gelatin-binding receptors (αvβ3 and α5β1), the nature of integrin binding sites was studied in order to explain the bioactivity of different protein formulations. We have shown that alteration of the chemical identity, conformation and availability of free binding motifs (GxOGER and RGD), resulting from addition of gelatin to collagen and crosslinking, have a profound effect on the ability of cells to adhere to these formulations. Carbodiimide crosslinking ablates integrin-dependent cell activity on both two-dimensional and three-dimensional architectures while the three-dimensional scaffold structure also leads to a high level of non-specific interactions remaining on three-dimensional samples even after a rigorous washing regime. This phenomenon, promoted by crosslinking, and attributed to cell entrapment, should be considered in any assessment of the biological activity of three-dimensional substrates. Spreading data confirm the importance of integrin-mediated cell engagement for further cell activity on collagen-based compositions. In this work, we provide a simple, but effective, means of deconvoluting the effects of chemistry and dimensional characteristics of a substrate, on the cell activity of protein-derived materials, which should assist in tailoring their biological properties for specific tissue engineering

  17. Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells.

    Science.gov (United States)

    Doczi, Judit; Torocsik, Beata; Echaniz-Laguna, Andoni; Mousson de Camaret, Bénédicte; Starkov, Anatoly; Starkova, Natalia; Gál, Aniko; Molnár, Mária J; Kawamata, Hibiki; Manfredi, Giovanni; Adam-Vizi, Vera; Chinopoulos, Christos

    2016-01-01

    The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT's voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the 'thinness ratio' and the 'cobalt-calcein' technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca(2+) levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient. PMID:27221760

  18. Calcitriol enhances fat synthesis factors and calpain activity in co-cultured cells.

    Science.gov (United States)

    Choi, Hyuck; Myung, Kyuho

    2014-08-01

    We have conducted an in vitro experiment to determine whether calcitriol can act as a fat synthesizer and/or meat tenderizer when skeletal muscle cells, adipose tissue, and macrophages are co-cultured. When co-cultured, pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression increased, whereas decreased anti-inflammatory cytokine (IL-10 and IL-15) expression decreased in both C2C12 and 3T3-L1 cells. Calcitriol increased reactive oxygen species (ROS) production in the media. While adiponectin gene expression decreased, leptin, resistin, CCAAT-enhancer-binding protein-beta (C/EBP-β), and peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression was significantly (P meat tenderizer, in meat-producing animals. PMID:24687633

  19. Behavior of Cell on Vibrating Micro Ridges

    Directory of Open Access Journals (Sweden)

    Haruka Hino

    2015-06-01

    Full Text Available The effect of micro ridges on cells cultured at a vibrating scaffold has been studied in vitro. Several parallel lines of micro ridges have been made on a disk of transparent polydimethylsiloxane for a scaffold. To apply the vibration on the cultured cells, a piezoelectric element was attached on the outside surface of the bottom of the scaffold. The piezoelectric element was vibrated by the sinusoidal alternating voltage (Vp-p < 16 V at 1.0 MHz generated by a function generator. Four kinds of cells were used in the test: L929 (fibroblast connective tissue of C3H mouse, Hepa1-6 (mouse hepatoma, C2C12 (mouse myoblast, 3T3-L1 (mouse fat precursor cells. The cells were seeded on the micro pattern at the density of 2000 cells/cm2 in the medium containing 10% FBS (fetal bovine serum and 1% penicillin/ streptomycin. After the adhesion of cells in several hours, the cells are exposed to the ultrasonic vibration for several hours. The cells were observed with a phase contrast microscope. The experimental results show that the cells adhere, deform and migrate on the scaffold with micro patterns regardless of the ultrasonic vibration. The effects of the vibration and the micro pattern depend on the kind of cells.

  20. Nanonet Force Microscopy for Measuring Cell Forces.

    Science.gov (United States)

    Sheets, Kevin; Wang, Ji; Zhao, Wei; Kapania, Rakesh; Nain, Amrinder S

    2016-07-12

    The influence of physical forces exerted by or felt by cells on cell shape, migration, and cytoskeleton arrangement is now widely acknowledged and hypothesized to occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Our previous work using the non-electrospinning Spinneret-based Tunable Engineered Parameters' suspended fibers has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to focal adhesion cluster lengths. Here, we present the development and application of a suspended nanonet platform for measuring C2C12 mouse myoblast forces attached to fibers of three diameters (250, 400, and 800 nm) representing a wide range of structural stiffness (3-50 nN/μm). The nanonet force microscopy platform measures cell adhesion forces in response to symmetric and asymmetric external perturbation in single and cyclic modes. We find that contractility-based, inside-out forces are evenly distributed at the edges of the cell, and that forces are dependent on fiber structural stiffness. Additionally, external perturbation in symmetric and asymmetric modes biases cell-fiber failure location without affecting the outside-in forces of cell-fiber adhesion. We then extend the platform to measure forces of (1) cell-cell junctions, (2) single cells undergoing cyclic perturbation in the presence of drugs, and (3) cancerous single-cells transitioning from a blebbing to a pseudopodial morphology. PMID:27410747

  1. Tracking in real time the crawling dynamics of adherent living cells with a high resolution surface plasmon microscope

    Science.gov (United States)

    Streppa, L.; Berguiga, L.; Boyer Provera, E.; Ratti, F.; Goillot, E.; Martinez Torres, C.; Schaeffer, L.; Elezgaray, Juan; Arneodo, A.; Argoul, F.

    2016-03-01

    We introduce a high resolution scanning surface plasmon microscope for long term imaging of living adherent mouse myoblast cells. The coupling of a high numerical aperture objective lens with a fibered heterodyne interferometer provides both enhanced sensitivity and long term stability. This microscope takes advantage of the plasmon resonance excitation and the amplification of the electromagnetic field in near-field distance to the gold coated coverslip. This plasmon enhanced evanescent wave microscopy is particularly attractive for the study of cell adhesion and motility since it can be operated without staining of the biological sample. We show that this microscope allows very long-term imaging of living samples, and that it can capture and follow the temporal deformation of C2C12 myoblast cell protusions (lamellipodia), during their migration on a at surface.

  2. Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy.

    Science.gov (United States)

    Jaatinen, Leena; Young, Eleanore; Hyttinen, Jari; Vörös, János; Zambelli, Tomaso; Demkó, László

    2016-03-01

    This study presents the effect of external electric current on the cell adhesive and mechanical properties of the C2C12 mouse myoblast cell line. Changes in cell morphology, viability, cytoskeleton, and focal adhesion structure were studied by standard staining protocols, while single-cell force spectroscopy based on the fluidic force microscopy technology provided a rapid, serial quantification and detailed analysis of cell adhesion and its dynamics. The setup allowed measurements of adhesion forces up to the μN range, and total detachment distances over 40 μm. Force-distance curves have been fitted with a simple elastic model including a cell detachment protocol in order to estimate the Young's modulus of the cells, as well as to reveal changes in the dynamic properties as functions of the applied current dose. While the cell spreading area decreased monotonously with increasing current doses, small current doses resulted only in differences related to cell elasticity. Current doses above 11 As/m(2), however, initiated more drastic changes in cell morphology, viability, cellular structure, as well as in properties related to cell adhesion. The observed differences, eventually leading to cell death toward higher doses, might originate from both the decrease in pH and the generation of reactive oxygen species.

  3. An engineered approach to stem cell culture: automating the decision process for real-time adaptive subculture of stem cells.

    Directory of Open Access Journals (Sweden)

    Dai Fei Elmer Ker

    Full Text Available Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and

  4. Micro Groove for Trapping of Flowing Cell

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    Yusuke Takahashi

    2015-06-01

    Full Text Available Micro grooves have been designed to trap a biological cell, which flows through a micro channel in vitro. Each micro groove of a rectangular shape (0.002 mm depth, 0.025 mm width and 0.2 mm length has been fabricated on the surface of the polydimethylsiloxane (PDMS disk with the photolithography technique. Variation has been made on the angle between the longitudinal direction of the groove and the flow direction: zero, 0.79, or 1.57 rad. A rectangular flow channel (0.1 mm depth x 5 mm width x 30 mm length has been constructed with a silicone film of 0.1 mm thick, which has been sandwiched by two transparent PDMS disks. Two types of biological cells were used in the test alternatively: C2C12 (mouse myoblast cell line originated with cross-striated muscle of C3H mouse, or 3T3-L1 (mouse fat precursor cells. A constant flow (2.8 x 10-11 m3/s of a suspension of cells was introduced with a syringe pump. The behavior of cells moving over the micro grooves was observed with an inverted phase contrast microscope. The results show that the cell is trapped with the micro grooves under the wall shear rate of 3 s-1 for a few seconds and that the trapped interval depends on the kind of cells.

  5. Antioxidant rich flavonoids from Oreocnide integrifolia enhance glucose uptake and insulin secretion and protects pancreatic β-cells from streptozotocin insult

    Directory of Open Access Journals (Sweden)

    Ansarullah

    2011-12-01

    Full Text Available Abstract Background Insulin deficiency is the prime basis of all diabetic manifestations and agents that can bring about insulin secretion would be of pivotal significance for cure of diabetes. To test this hypothesis, we carried out bioactivity guided fractionation of Oreocnide integrifolia (Urticaceae; a folklore plant consumed for ameliorating diabetic symptoms using experimental models. Methods We carried out bioassay guided fractionation using RINmF and C2C12 cell line for glucose stimulated insulin secretion (GSIS and glucose uptake potential of fractions. Further, the bioactive fraction was challenged for its GSIS in cultured mouse islets with basal (4.5 mM and stimulated (16.7 mM levels of glucose concentrations. The Flavonoid rich fraction (FRF was exposed to 2 mM streptozotocin stress and the anti-ROS/RNS potential was evaluated. Additionally, the bioactive fraction was assessed for its antidiabetic and anti-apoptotic property in-vivo using multidose streptozotocin induced diabetes in BALB/c mice. Results The results suggested FRF to be the most active fraction as assessed by GSIS in RINm5F cells and its ability for glucose uptake in C2C12 cells. FRF displayed significant potential in terms of increasing intracellular calcium and cAMP levels even in presence of a phosphodiesterase inhibitor, IBMX in cultured pancreatic islets. FRF depicted a dose-dependent reversal of all the cytotoxic manifestations except peroxynitrite and NO formation when subjected in-vitro along with STZ. Further scrutinization of FRF for its in-vivo antidiabetic property demonstrated improved glycemic indices and decreased pancreatic β-cell apoptosis. Conclusions Overall, the flavonoid mixture has shown to have significant insulin secretogogue, insulinomimetic and cytoprotective effects and can be evaluated for clinical trials as a therapeutant in the management of diabetic manifestations.

  6. Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Matthews, V B; Åström, Maj-Brit; Chan, M H S;

    2009-01-01

    AIMS/HYPOTHESIS: Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. METHODS: We first examined whether exercise induced BDNF expression in humans. Next, C2......C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr(172)) and acetyl coenzyme A carboxylase beta (ACCbeta) (Ser......(79)) were analysed, as was fatty acid oxidation (FAO). Finally, we electroporated a Bdnf vector into the tibialis cranialis muscle of mice. RESULTS: BDNF mRNA and protein expression were increased in human skeletal muscle after exercise, but muscle-derived BDNF appeared not to be released...

  7. β-agonists selectively modulate proinflammatory gene expression in skeletal muscle cells via non-canonical nuclear crosstalk mechanisms.

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    Krzysztof Kolmus

    Full Text Available The proinflammatory cytokine Tumour Necrosis Factor (TNF-α is implicated in a variety of skeletal muscle pathologies. Here, we have investigated how in vitro cotreatment of skeletal muscle C2C12 cells with β-agonists modulates the TNF-α-induced inflammatory program. We observed that C2C12 myotubes express functional TNF receptor 1 (TNF-R1 and β2-adrenoreceptors (β2-ARs. TNF-α activated the canonical Nuclear Factor-κB (NF-κB pathway and Mitogen-Activated Protein Kinases (MAPKs, culminating in potent induction of NF-κB-dependent proinflammatory genes. Cotreatment with the β-agonist isoproterenol potentiated the expression of inflammatory mediators, including Interleukin-6 (IL-6 and several chemokines. The enhanced production of chemotactic factors upon TNF-α/isoproterenol cotreatment was also suggested by the results from migrational analysis. Whereas we could not explain our observations by cytoplasmic crosstalk, we found that TNF-R1-and β2-AR-induced signalling cascades cooperate in the nucleus. Using the IL-6 promoter as a model, we demonstrated that TNF-α/isoproterenol cotreatment provoked phosphorylation of histone H3 at serine 10, concomitant with enhanced promoter accessibility and recruitment of the NF-κB p65 subunit, cAMP-response element-binding protein (CREB, CREB-binding protein (CBP and RNA polymerase II. In summary, we show that β-agonists potentiate TNF-α action, via nuclear crosstalk, that promotes chromatin relaxation at selected gene promoters. Our data warrant further study into the mode of action of β-agonists and urge for caution in their use as therapeutic agents for muscular disorders.

  8. NOV/CCN3 impairs muscle cell commitment and differentiation.

    Science.gov (United States)

    Calhabeu, Frederico; Lafont, Jérome; Le Dreau, Gwenvael; Laurent, Maryvonne; Kazazian, Chantal; Schaeffer, Laurent; Martinerie, Cécile; Dubois, Catherine

    2006-06-10

    NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

  9. Photovoltaic surfaces enable clonal myoblastic cell release using visible light as external stimulation.

    Science.gov (United States)

    Bhuyan, Mohammod Kabir; Rodriguez-Devora, Jorge; Tseng, Tzu-Liang Bill; Boland, Thomas

    2016-03-01

    Many new biomedical approaches to treating disease require the supply of cells delivered to an injured or diseased organ either individually, collectively as aggregates or sheets, or encapsulated with a scaffold. The collection of cells is accomplished by using enzymatic digestion witch suffer from the need to remove the enzymes after digestion. In addition, enzymatic methods are not applicable for all cells, cell aggregates, cell sheets or 3D structures. The objective of this study was to investigate the release of cultured cells from silicon based Photovoltaic (PV) surfaces using a light source as external stimulation. C2C12 myoblasts were cultured on the negative surface of a PV device and upon confluence they were exposed to light. The amount of released cells was quantified as a function light exposure. It was found that light exposure at 25,000 lux for one hour caused equivalent cell release from the PV surface than trypsination. The released cells are viable and can be re-cultured if needed. This mechanism may offer an alternative method to release excitable cells without using an enzymatic agent. This may be important for cell therapy if larger cell structures such as sheets need to be collected.

  10. Real-time, noninvasive optical coherence tomography of cross-sectional living cell-sheets in vitro and in vivo.

    Science.gov (United States)

    Kobayashi, Mari; Haraguchi, Yuji; Shimizu, Tatsuya; Mizuuchi, Kiminori; Iseki, Hiroshi

    2015-08-01

    Cell sheet technology has a history of application in regenerating various tissues, having successfully completed several clinical trials using autologous cell sheets. Tomographic analysis of living cell sheets is an important tool in the field of cell sheet-based regenerative medicine and tissue engineering to analyze the inner structure of layered living cells. Optical coherence tomography (OCT) is commonly used in ophthalmology to noninvasively analyze cross-sections of target tissues at high resolution. This study used OCT to conduct real-time, noninvasive analysis of living cell sheet cross sections. OCT showed the internal structure of cell sheets in tomographic images synthesized with backscatter signals from inside the living cell sheet without invasion or damage. OCT observations were used to analyze the static and dynamic behaviors of living cell sheets in vitro and in vivo including (1) the harvesting process of a C2C12 mouse skeletal myoblast sheet from a temperature-responsive culture surface; (2) cell-sheet adhesion onto various surfaces including a culture surface, a synthetic rubber glove, and the dorsal subcutaneous tissue of rats; and (3) the real-time propagation of beating rat cardiac cells within cardiac cell sheets. This study showed that OCT technology is a powerful tool in the field of cell sheet-based regenerative medicine and tissue engineering.

  11. Clones of ectopic stem cells in the regeneration of muscle defects in vivo.

    Directory of Open Access Journals (Sweden)

    Rujing Yang

    Full Text Available Little is known about whether clones of ectopic, non-muscle stem cells contribute to muscle regeneration. Stem/progenitor cells that are isolated for experimental research or therapeutics are typically heterogeneous. Non-myogenic lineages in a heterogeneous population conceptually may compromise tissue repair. In this study, we discovered that clones of mononucleated stem cells of human tooth pulp fused into multinucleated myotubes that robustly expressed myosin heavy chain in vitro with or without co-culture with mouse skeletal myoblasts (C2C12 cells. Cloned cells were sustainably Oct4+, Nanog+ and Stro1+. The fusion indices of myogenic clones were approximately 16-17 folds greater than their parent, heterogeneous stem cells. Upon infusion into cardio-toxin induced tibialis anterior muscle defects, undifferentiated clonal progenies not only engrafted and colonized host muscle, but also expressed human dystrophin and myosin heavy chain more efficaciously than their parent heterogeneous stem cell populations. Strikingly, clonal progenies yielded ∼9 times more human myosin heavy chain mRNA in regenerating muscles than those infused with their parent, heterogeneous stem cells. The number of human dystrophin positive cells in regenerating muscles infused with clonal progenies was more than ∼3 times greater than muscles infused with heterogeneous stem cells from which clonal progenies were derived. These findings suggest the therapeutic potential of ectopic myogenic clones in muscle regeneration.

  12. A combined watershed and level set method for segmentation of brightfield cell images

    Science.gov (United States)

    Tse, Shutong; Bradbury, Laura; Wan, Justin W. L.; Djambazian, Haig; Sladek, Robert; Hudson, Thomas

    2009-02-01

    Segmentation of brightfield cell images from microscopy is challenging in several ways. The contrast between cells and the background is low. Cells are usually surrounded by "halo", an optical artifact common in brightfield images. Also, cell divisions occur frequently, which raises the issue of topological change to segmentation. In this paper, we present a robust segmentation method based on the watershed and level set methods. Instead of heuristically locate where the initial markers for watershed should be, we apply a multiphase level set marker extraction to determine regions inside a cell. In contrast with the standard level set segmentation where only one level set function is used, we apply multiple level set functions (usually 3) to capture the different intensity levels in a cell image. This is particularly important to be able to distinguish regions of similar but different intensity levels in low contrast images. All the pixels obtained will be used as an initial marker for watershed. The region growing process of watershed will capture the rest of the cell until it hits the halo which serves as a "wall" to stop the expansion. By using these relatively large number of points as markers together with watershed, we show that the low contrast cell boundary can be captured correctly. Furthermore, we present a technique for watershed and level set to detect cell division automatically with no special human attention. Finally, we present segmentation results of C2C12 cells in brightfield images to illustrate the effectiveness of our method.

  13. Adult stem cells from the hyaluronic acid-rich node and duct system differentiate into neuronal cells and repair brain injury.

    Science.gov (United States)

    Lee, Seung J; Park, Sang H; Kim, Yu I; Hwang, Sunhee; Kwon, Patrick M; Han, In S; Kwon, Byoung S

    2014-12-01

    The existence of a hyaluronic acid-rich node and duct system (HAR-NDS) within the lymphatic and blood vessels was demonstrated previously. The HAR-NDS was enriched with small (3.0-5.0 μm in diameter), adult stem cells with properties similar to those of the very small embryonic-like stem cells (VSELs). Sca-1(+)Lin(-)CD45(-) cells were enriched approximately 100-fold in the intravascular HAR-NDS compared with the bone marrow. We named these adult stem cells "node and duct stem cells (NDSCs)." NDSCs formed colonies on C2C12 feeder layers, were positive for fetal alkaline phosphatase, and could be subcultured on the feeder layers. NDSCs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(+), while VSELs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(-). NDSCs had higher sphere-forming efficiency and proliferative potential than VSELs, and they were found to differentiate into neuronal cells in vitro. Injection of NDSCs into mice partially repaired ischemic brain damage. Thus, we report the discovery of potential adult stem cells that may be involved in tissue regeneration. The intravascular HAR-NDS may serve as a route that delivers these stem cells to their target tissues. PMID:25027245

  14. Individually programmable cell stretching microwell arrays actuated by a Braille display.

    Science.gov (United States)

    Kamotani, Yoko; Bersano-Begey, Tommaso; Kato, Nobuhiro; Tung, Yi-Chung; Huh, Dongeun; Song, Jonathan W; Takayama, Shuichi

    2008-06-01

    Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.

  15. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

    Science.gov (United States)

    Koh, T. J.; Tidball, J. G.

    2000-01-01

    We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

  16. Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

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    Victoria C. Foletta

    2016-10-01

    Full Text Available Deuterated water (2H2O, a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules, thus permitting the calculation of their synthesis rates. Here, we have combined 2H2O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation, protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines. Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both ‘self-made’ and exogenously-derived fatty acid.

  17. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

    Science.gov (United States)

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L

    2014-01-01

    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

  18. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Y., E-mail: yuta-n@mech.kumamoto-u.ac.jp [Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 096-8555 (Japan); Graduate School of Science and Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611 (Japan); Tsusu, K.; Minami, K. [Graduate School of Science and Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611 (Japan); Nakanishi, Y. [Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 096-8555 (Japan)

    2014-06-15

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  19. Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

    Directory of Open Access Journals (Sweden)

    Christopher L Dearth

    Full Text Available We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1 is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12 did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/- mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05 overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05 the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.

  20. The pesticide methoxychlor decreases myotube formation in cell culture by slowing myoblast proliferation.

    Science.gov (United States)

    Steffens, Bradley W; Batia, Lyn M; Baarson, Chad J; Choi, Chang-Kun Charles; Grow, Wade A

    2007-08-01

    We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported.

  1. Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle.

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    Nathalie Burch

    Full Text Available Adequate levels of physical activity are at the center of a healthy lifestyle. However, the molecular mechanisms that mediate the beneficial effects of exercise remain enigmatic. This gap in knowledge is caused by the lack of an amenable experimental model system. Therefore, we optimized electric pulse stimulation of muscle cells to closely recapitulate the plastic changes in gene expression observed in a trained skeletal muscle. The exact experimental conditions were established using the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha as a marker for an endurance-trained muscle fiber. We subsequently compared the changes in the relative expression of metabolic and myofibrillar genes in the muscle cell system with those observed in mouse muscle in vivo following either an acute or repeated bouts of treadmill exercise. Importantly, in electrically stimulated C2C12 mouse muscle cells, the qualitative transcriptional adaptations were almost identical to those in trained muscle, but differ from the acute effects of exercise on muscle gene expression. In addition, significant alterations in the expression of myofibrillar proteins indicate that this stimulation could be used to modulate the fiber-type of muscle cells in culture. Our data thus describe an experimental cell culture model for the study of at least some of the transcriptional aspects of skeletal muscle adaptation to physical activity. This system will be useful for the study of the molecular mechanisms that regulate exercise adaptation in muscle.

  2. Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.

    Directory of Open Access Journals (Sweden)

    Rachelle N Palchesko

    Full Text Available Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line and C2C12 (muscle cell line were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.

  3. Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.

    Science.gov (United States)

    Palchesko, Rachelle N; Zhang, Ling; Sun, Yan; Feinberg, Adam W

    2012-01-01

    Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.

  4. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    Science.gov (United States)

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells. PMID:26886589

  5. Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct Morquio skin fibroblasts in vitro.

    Science.gov (United States)

    Toietta, G; Severini, G M; Traversari, C; Tomatsu, S; Sukegawa, K; Fukuda, S; Kondo, N; Tortora, P; Bordignon, C

    2001-11-01

    Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA. PMID:11686941

  6. Omega-3 fatty acids differentially modulate enzymatic anti-oxidant systems in skeletal muscle cells.

    Science.gov (United States)

    da Silva, E P; Nachbar, R T; Levada-Pires, A C; Hirabara, S M; Lambertucci, R H

    2016-01-01

    During physical activity, increased reactive oxygen species production occurs, which can lead to cell damage and in a decline of individual's performance and health. The use of omega-3 polyunsaturated fatty acids as a supplement to protect the immune system has been increasing; however, their possible benefit to the anti-oxidant system is not well described. Thus, the aim of this study was to evaluate whether the omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) can be beneficial to the anti-oxidant system in cultured skeletal muscle cells. C2C12 myocytes were differentiated and treated with either eicosapentaenoic acid or docosahexaenoic acid for 24 h. Superoxide content was quantified using the dihydroethidine oxidation method and superoxide dismutase, catalase, and glutathione peroxidase activity, and expression was quantified. We observed that the docosahexaenoic fatty acids caused an increase in superoxide production. Eicosapentaenoic acid induced catalase activity, while docosahexaenoic acid suppressed superoxide dismutase activity. In addition, we found an increased protein expression of the total manganese superoxide dismutase and catalase enzymes when cells were treated with eicosapentaenoic acid. Taken together, these data indicate that the use of eicosapentaenoic acid may present both acute and chronic benefits; however, the treatment with DHA may not be beneficial to muscle cells. PMID:26386577

  7. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    International Nuclear Information System (INIS)

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7+ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration

  8. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Mazzanti, Benedetta [Dept. of Experimental and Clinical Medicine—Section of Haematology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Quercioli, Franco [CNR-National Institute of Optics (INO), Largo Enrico Fermi 6, 50125 Arcetri-Florence (Italy); Zecchi-Orlandini, Sandra [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Formigli, Lucia, E-mail: formigli@unifi.it [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy)

    2014-05-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.

  9. Amla Enhances Mitochondrial Spare Respiratory Capacity by Increasing Mitochondrial Biogenesis and Antioxidant Systems in a Murine Skeletal Muscle Cell Line

    Directory of Open Access Journals (Sweden)

    Hirotaka Yamamoto

    2016-01-01

    Full Text Available Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.

  10. PLLA/ZnO nanocomposites: Dynamic surfaces to harness cell differentiation.

    Science.gov (United States)

    Trujillo, Sara; Lizundia, Erlantz; Vilas, José Luis; Salmeron-Sanchez, Manuel

    2016-08-01

    This work investigates the effect of the sequential availability of ZnO nanoparticles, (nanorods of ∼40nm) loaded within a degradable poly(lactic acid) (PLLA) matrix, in cell differentiation. The system constitutes a dynamic surface, in which nanoparticles are exposed as the polymer matrix degrades. ZnO nanoparticles were loaded into PLLA and the system was measured at different time points to characterise the time evolution of the physicochemical properties, including wettability and thermal properties. The micro and nanostructure were also investigated using AFM, SEM and TEM images. Cellular experiments with C2C12 myoblasts show that cell differentiation was significantly enhanced on ZnO nanoparticles-loaded PLLA, as the polymer degrades and the availability of nanoparticles become more apparent, whereas the release of zinc within the culture medium was negligible. Our results suggest PLLA/ZnO nanocomposites can be used as a dynamic system where nanoparticles are exposed during degradation, activating the material surface and driving cell differentiation. PMID:27085047

  11. Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells

    Science.gov (United States)

    Pereyra, Andrea Soledad; Mykhaylyk, Olga; Lockhart, Eugenia Falomir; Taylor, Jackson Richard; Delbono, Osvaldo; Goya, Rodolfo Gustavo; Plank, Christian; Hereñu, Claudia Beatriz

    2016-01-01

    The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models. PMID:27274908

  12. Saffron (Crocus sativus L.) increases glucose uptake and insulin sensitivity in muscle cells via multipathway mechanisms.

    Science.gov (United States)

    Kang, Changkeun; Lee, Hyunkyoung; Jung, Eun-Sun; Seyedian, Ramin; Jo, MiNa; Kim, Jehein; Kim, Jong-Shu; Kim, Euikyung

    2012-12-15

    Saffron (Crocus sativus Linn.) has been an important subject of research in the past two decades because of its various biological properties, including anti-cancer, anti-inflammatory, and anti-atherosclerotic activities. On the other hand, the molecular bases of its actions have been scarcely understood. Here, we elucidated the mechanism of the hypoglycemic actions of saffron through investigating its signaling pathways associated with glucose metabolism in C(2)C(12) skeletal muscle cells. Saffron strongly enhanced glucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA carboxylase) and MAPKs (mitogen-activated protein kinases), but not PI 3-kinase (Phosphatidylinositol 3-kinase)/Akt. Interestingly, the co-treatment of saffron and insulin further improved the insulin sensitivity via both insulin-independent (AMPK/ACC and MAPKs) and insulin-dependent (PI 3-kinase/Akt and mTOR) pathways. It also suggested that there is a crosstalk between the two signaling pathways of glucose metabolism in skeletal muscle cells. These results could be confirmed from the findings of GLUT4 translocation. Taken together, AMPK plays a major role in the effects of saffron on glucose uptake and insulin sensitivity in skeletal muscle cells. Our study provides important insights for the possible mechanism of action of saffron and its potential as a therapeutic agent in diabetic patients.

  13. Proteomic profiling of glucocorticoid-exposed myogenic cells: Time series assessment of protein translocation and transcription of inactive mRNAs

    Directory of Open Access Journals (Sweden)

    Hoffman Eric P

    2009-07-01

    Full Text Available Abstract Background Prednisone, one of the most highly prescribed drugs, has well characterized effects on gene transcription mediated by the glucocorticoid receptor. These effects are typically occurring on the scale of hours. Prednisone also has a number of non-transcriptional effects (occurring on minutes scale on protein signaling, yet these are less well studied. We sought to expand the understanding of acute effects of prednisone action on cell signaling using a combination of SILAC strategy and subcellular fractionations from C2C12 myotubes. Results De novo translation of proteins was inhibited in both SILAC labeled and unlabeled C2C12 myotubes. Unlabeled cells were exposed to prednisone while SILAC labeled cells remained untreated. After 0, 5, 15, and 30 minutes of prednisone exposure, labeled and unlabeled cells were mixed at 1:1 ratios and fractionated into cytosolic and nuclear fractions. A total of 534 proteins in the cytosol and 626 proteins in the nucleus were identified and quantitated, using 3 or more peptides per protein with peptide based probability ≤ 0.001. We identified significant increases (1.7- to 3.1- fold in cytoplasmic abundance of 11 ribosomal proteins within 5 minutes of exposure, all of which returned to baseline by 30 min. We hypothesized that these drug-induced acute changes in the subcellular localization of the cell's protein translational machinery could lead to altered translation of quiescent RNAs. To test this, de novo protein synthesis was assayed after 15 minutes of drug exposure. Quantitative fluorography identified 16 2D gel spots showing rapid changes in translation; five of these were identified by MS/MS (pyruvate kinase, annexin A6 isoform A and isoform B, nasopharyngeal epithelium specific protein 1, and isoform 2 of Replication factor C subunit 1, and all showed the 5' terminal oligopyrimidine motifs associated with mRNA sequestration to and from inactive mRNA pools. Conclusion We describe novel

  14. M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic β-cells through modulation of respiratory chain activity.

    Directory of Open Access Journals (Sweden)

    Linda Cambier

    Full Text Available Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.

  15. TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells.

    Science.gov (United States)

    Malecova, Barbora; Dall'Agnese, Alessandra; Madaro, Luca; Gatto, Sole; Coutinho Toto, Paula; Albini, Sonia; Ryan, Tammy; Tora, Làszlò; Puri, Pier Lorenzo

    2016-01-01

    Change in the identity of the components of the transcription pre-initiation complex is proposed to control cell type-specific gene expression. Replacement of the canonical TFIID-TBP complex with TRF3/TBP2 was reported to be required for activation of muscle-gene expression. The lack of a developmental phenotype in TBP2 null mice prompted further analysis to determine whether TBP2 deficiency can compromise adult myogenesis. We show here that TBP2 null mice have an intact regeneration potential upon injury and that TBP2 is not expressed in established C2C12 muscle cell or in primary mouse MuSCs. While TFIID subunits and TBP are downregulated during myoblast differentiation, reduced amounts of these proteins form a complex that is detectable on promoters of muscle genes and is essential for their expression. This evidence demonstrates that TBP2 does not replace TBP during muscle differentiation, as previously proposed, with limiting amounts of TFIID-TBP being required to promote muscle-specific gene expression. PMID:26880551

  16. TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells

    Science.gov (United States)

    Malecova, Barbora; Dall'Agnese, Alessandra; Madaro, Luca; Gatto, Sole; Coutinho Toto, Paula; Albini, Sonia; Ryan, Tammy; Tora, Làszlò; Puri, Pier Lorenzo

    2016-01-01

    Change in the identity of the components of the transcription pre-initiation complex is proposed to control cell type-specific gene expression. Replacement of the canonical TFIID-TBP complex with TRF3/TBP2 was reported to be required for activation of muscle-gene expression. The lack of a developmental phenotype in TBP2 null mice prompted further analysis to determine whether TBP2 deficiency can compromise adult myogenesis. We show here that TBP2 null mice have an intact regeneration potential upon injury and that TBP2 is not expressed in established C2C12 muscle cell or in primary mouse MuSCs. While TFIID subunits and TBP are downregulated during myoblast differentiation, reduced amounts of these proteins form a complex that is detectable on promoters of muscle genes and is essential for their expression. This evidence demonstrates that TBP2 does not replace TBP during muscle differentiation, as previously proposed, with limiting amounts of TFIID-TBP being required to promote muscle-specific gene expression. DOI: http://dx.doi.org/10.7554/eLife.12534.001 PMID:26880551

  17. TNF inhibits Notch-1 in skeletal muscle cells by Ezh2 and DNA methylation mediated repression: implications in duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Swarnali Acharyya

    Full Text Available BACKGROUND: Classical NF-kappaB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFalpha on skeletal muscle differentiation are mediated in part through sustained NF-kappaB activity. In dystrophic muscles, NF-kappaB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFalpha that is also under IKKbeta and NF-kappaB control. METHODOLOGY/PRINCIPAL FINDINGS: Based on these findings we speculated that in DMD, TNFalpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFalpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappaB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFalpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2. CONCLUSIONS/SIGNIFICANCE: We propose that in dystrophic muscles, elevated levels of TNFalpha and NF-kappaB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.

  18. Brown fat determination and development from muscle precursor cells by novel action of bone morphogenetic protein 6.

    Directory of Open Access Journals (Sweden)

    Ankur Sharma

    Full Text Available Brown adipose tissue (BAT plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1 that differentiates BAT from its energy storing white adipose tissue (WAT counterpart. The clinical implication of "classical" BAT (originates from Myf5 positive myoblastic lineage or the "beige" fat (originates through trans-differentiation of WAT activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6 induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn and Cyclooxygenase-2 (Cox2. Furthermore, pathway analyses using the Causal Reasoning Engine (CRE identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R. Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat.

  19. New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

    Science.gov (United States)

    Sestili, Piero; Ambrogini, Patrizia; Barbieri, Elena; Sartini, Stefano; Fimognari, Carmela; Calcabrini, Cinzia; Diaz, Anna Rita; Guescini, Michele; Polidori, Emanuela; Luchetti, Francesca; Canonico, Barbara; Lattanzi, Davide; Cuppini, Riccardo; Papa, Stefano; Stocchi, Vilberto

    2016-08-01

    A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.

  20. Bright-field cell image segmentation by principal component pursuit with an Ncut penalization

    Science.gov (United States)

    Chen, Yuehuan; Wan, Justin W. L.

    2015-03-01

    Segmentation of cells in time-lapse bright-field microscopic images is crucial in understanding cell behaviours for oncological research. However, the complex nature of the cells makes it difficult to segment cells accurately. Furthermore, poor contrast, broken cell boundaries and the halo artifact pose additional challenges to this problem. Standard segmentation techniques such as edged-based methods, watershed, or active contours result in poor segmentation. Other existing methods for bright-field images cannot provide good results without localized segmentation steps. In this paper, we present two robust mathematical models to segment bright-field cells automatically for the entire image. These models treat cell image segmentation as a background subtraction problem, which can be formulated as a Principal Component Pursuit (PCP) problem. Our first segmentation model is formulated as a PCP with nonnegative constraints. We exploit the sparse component of the PCP solution for identifying the cell pixels. However, there is no control on the quality of the sparse component and the nonzero entries can scatter all over the image, resulting in a noisy segmentation. The second model is an improvement of the first model by combining PCP with spectral clustering. Seemingly unrelated approaches, we combine the two techniques by incorporating normalized-cut in the PCP as a measure for the quality of the segmentation. These two models have been applied to a set of C2C12 cells obtained from bright-field microscopy. Experimental results demonstrate that the proposed models are effective in segmenting cells from bright-field images.

  1. Combined immunotherapy and antiangiogenic therapy of cancer with microencapsulated cells.

    Science.gov (United States)

    Cirone, Pasquale; Bourgeois, Jacqueline M; Shen, Feng; Chang, Patricia L

    2004-10-01

    An alternative form of gene therapy involves immunoisolation of a nonautologous cell line engineered to secrete a therapeutic product. Encapsulation of these cells in a biocompatible polymer serves to protect these allogeneic cells from host-versus-graft rejection while recombinant products and nutrients are able to pass by diffusion. This strategy was applied to the treatment of cancer with some success by delivering either interleukin 2 or angiostatin. However, as cancer is a complex, multifactorial disease, a multipronged approach is now being developed to attack tumorigenesis via multiple pathways in order to improve treatment efficacy. A combination of immunotherapy with angiostatic therapy was investigated by treating B16-F0/neu melanoma-bearing mice with intraperitoneally implanted, microencapsulated mouse myoblasts (C2C12) genetically modified to deliver angiostatin and an interleukin 2 fusion protein (sFvIL-2). The combination treatment resulted in improved survival, delayed tumor growth, and increased histological indices of antitumor activity (apoptosis and necrosis). In addition to improved efficacy, the combination treatment also ameliorated some of the undesirable side effects from the individual treatments that have led to the previous failure of the single treatments, for example, inflammatory response to IL-2 or vascular mimicry due to angiostatin. In conclusion, the combination of immuno- and antiangiogenic therapies delivered by immunoisolated cells was superior to individual treatments for antitumorigenesis activity, not only because of their known mechanisms of action but also because of unexpected protection against the adverse side effects of the single treatments. Thus, the concept of a "cocktail" strategy, with microencapsulation delivering multiple antitumor recombinant molecules to improve efficacy, is validated. PMID:15585110

  2. Cell motility regulation on a stepped micro pillar array device (SMPAD) with a discrete stiffness gradient.

    Science.gov (United States)

    Lee, Sujin; Hong, Juhee; Lee, Junghoon

    2016-02-28

    Our tissues consist of individual cells that respond to the elasticity of their environment, which varies between and within tissues. To better understand mechanically driven cell migration, it is necessary to manipulate the stiffness gradient across a substrate. Here, we have demonstrated a new variant of the microfabricated polymeric pillar array platform that can decouple the stiffness gradient from the ECM protein area. This goal is achieved via a "stepped" micro pillar array device (SMPAD) in which the contact area with the cell was kept constant while the diameter of the pillar bodies was altered to attain the proper mechanical stiffness. Using double-step SU-8 mold fabrication, the diameter of the top of every pillar was kept uniform, whereas that of the bottom was changed, to achieve the desired substrate rigidity. Fibronectin was immobilized on the pillar tops, providing a focal adhesion site for cells. C2C12, HeLa and NIH3T3 cells were cultured on the SMPAD, and the motion of the cells was observed by time-lapse microscopy. Using this simple platform, which produces a purely physical stimulus, we observed that various types of cell behavior are affected by the mechanical stimulus of the environment. We also demonstrated directed cell migration guided by a discrete rigidity gradient by varying stiffness. Interestingly, cell velocity was highest at the highest stiffness. Our approach enables the regulation of the mechanical properties of the polymeric pillar array device and eliminates the effects of the size of the contact area. This technique is a unique tool for studying cellular motion and behavior relative to various stiffness gradients in the environment. PMID:26787193

  3. Optically transparent polymer devices for in situ assessment of cell electroporation.

    Science.gov (United States)

    Majhi, Amit Kumar; Thrivikraman, Greeshma; Basu, Bikramjit; Venkataraman, V

    2015-02-01

    In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 μl was quantified in situ as a function of applied voltage from 10 to 90 V in a series of ~2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be 45.6 ± 0.5 kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10% glycerol).

  4. Clozapine-induced mitochondria alterations and inflammation in brain and insulin-responsive cells.

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    Verόnica Contreras-Shannon

    Full Text Available BACKGROUND: Metabolic syndrome (MetS is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined. METHODOLOGY/PRINCIPAL FINDINGS: Cultured mouse myoblasts (C2C12, adipocytes (3T3-L1, hepatocytes (FL-83B, and monocytes (RAW 264.7 were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line. CONCLUSIONS/SIGNIFICANCE: Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated

  5. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

  6. IL-15 Mediates Mitochondrial Activity through a PPARδ-Dependent-PPARα-Independent Mechanism in Skeletal Muscle Cells

    Science.gov (United States)

    2016-01-01

    Molecular mediators of metabolic processes, to increase energy expenditure, have become a focus for therapies of obesity. The discovery of cytokines secreted from the skeletal muscle (SKM), termed “myokines,” has garnered attention due to their positive effects on metabolic processes. Interleukin-15 (IL-15) is a myokine that has numerous positive metabolic effects and is linked to the PPAR family of mitochondrial regulators. Here, we aimed to determine the importance of PPARα and/or PPARδ as targets of IL-15 signaling. C2C12 SKM cells were differentiated for 6 days and treated every other day with IL-15 (100 ng/mL), a PPARα inhibitor (GW-6471), a PPARδ inhibitor (GSK-3787), or both IL-15 and the inhibitors. IL-15 increased mitochondrial activity and induced PPARα, PPARδ, PGC1α, PGC1β, UCP2, and Nrf1 expression. There was no effect of inhibiting PPARα, in combination with IL-15, on the aforementioned mRNA levels except for PGC1β and Nrf1. However, with PPARδ inhibition, IL-15 failed to induce the expression levels of PGC1α, PGC1β, UCP2, and Nrf1. Further, inhibition of PPARδ abolished IL-15 induced increases in citrate synthase activity, ATP production, and overall mitochondrial activity. IL-15 had no effects on mitochondrial biogenesis. Our data indicates that PPARδ activity is required for the beneficial metabolic effects of IL-15 signaling in SKM.

  7. Antihyperglycemic Activity of Eucalyptus tereticornis in Insulin-Resistant Cells and a Nutritional Model of Diabetic Mice

    Science.gov (United States)

    Guillén, Alis; Granados, Sergio; Rivas, Kevin Eduardo; Estrada, Omar; Echeverri, Luis Fernando; Balcázar, Norman

    2015-01-01

    Eucalyptus tereticornis is a plant used in traditional medicine to control diabetes, but this effect has not been proved scientifically. Here, we demonstrated through in vitro assays that E. tereticornis extracts increase glucose uptake and inhibit their production in insulin-resistant C2C12 and HepG2 cells, respectively. Furthermore, in a nutritional model using diabetic mice, the administration of ethyl acetate extract of E. tereticornis reduced fasting glycaemia, improved tolerance to glucose, and reduced resistance to insulin. Likewise, this extract had anti-inflammatory effects in adipose tissue when compared to control diabetic mice. Via bioguided assays and sequential purification of the crude extract, a triterpenoid-rich fraction from ethyl acetate extracts was shown to be responsible for the biological activity. Similarly, we identified the main compound responsible for the antihyperglycemic activity in this extract. This study shows that triterpenes found in E. tereticornis extracts act as hypoglycemic/antidiabetic compounds and contribute to the understanding of their use in traditional medicine. PMID:26366171

  8. Antihyperglycemic Activity of Eucalyptus tereticornis in Insulin-Resistant Cells and a Nutritional Model of Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Alis Guillén

    2015-01-01

    Full Text Available Eucalyptus tereticornis is a plant used in traditional medicine to control diabetes, but this effect has not been proved scientifically. Here, we demonstrated through in vitro assays that E. tereticornis extracts increase glucose uptake and inhibit their production in insulin-resistant C2C12 and HepG2 cells, respectively. Furthermore, in a nutritional model using diabetic mice, the administration of ethyl acetate extract of E. tereticornis reduced fasting glycaemia, improved tolerance to glucose, and reduced resistance to insulin. Likewise, this extract had anti-inflammatory effects in adipose tissue when compared to control diabetic mice. Via bioguided assays and sequential purification of the crude extract, a triterpenoid-rich fraction from ethyl acetate extracts was shown to be responsible for the biological activity. Similarly, we identified the main compound responsible for the antihyperglycemic activity in this extract. This study shows that triterpenes found in E. tereticornis extracts act as hypoglycemic/antidiabetic compounds and contribute to the understanding of their use in traditional medicine.

  9. 1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells.

    Science.gov (United States)

    Valente, Sergio; Mellini, Paolo; Spallotta, Francesco; Carafa, Vincenzo; Nebbioso, Angela; Polletta, Lucia; Carnevale, Ilaria; Saladini, Serena; Trisciuoglio, Daniela; Gabellini, Chiara; Tardugno, Maria; Zwergel, Clemens; Cencioni, Chiara; Atlante, Sandra; Moniot, Sébastien; Steegborn, Clemens; Budriesi, Roberta; Tafani, Marco; Del Bufalo, Donatella; Altucci, Lucia; Gaetano, Carlo; Mai, Antonello

    2016-02-25

    Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy. PMID:26689352

  10. Migration of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells toward An Injured Spinal Cord

    Directory of Open Access Journals (Sweden)

    Zoleikha Golipoor

    2016-02-01

    Full Text Available Objective: Bone marrow (BM is one of the major hematopoietic organs in postnatal life that consists of a heterogeneous population of stem cells which have been previously described. Recently, a rare population of stem cells that are called very small embryonic-like (VSEL stem cells has been found in the BM. These cells express several developmental markers of pluripotent stem cells and can be mobilized into peripheral blood (PB in response to tissue injury. In this study we have attempted to investigate the ability of these cells to migrate toward an injured spinal cord after transplantation through the tail vein in a rat model. Materials and Methods: In this experimental study, VSELs were isolated from total BM cells using a fluorescent activated cell sorting (FACS system and sca1 and stage specific embryonic antigen (SSEA-1 antibodies. After isolation, VSELs were cultured for 7 days on C2C12 as the feeder layer. Then, VSELs were labeled with 1,1´-dioctadecyl-3,3,3´,3´- tetramethylindocarbocyanine perchlorate (DiI and transplanted into the rat spinal cord injury (SCI model via the tail vein. Finally, we sought to determine the presence of VSELs in the lesion site. Results: We isolated a high number of VSELs from the BM. After cultivation, the VSELs colonies were positive for SSEA-1, Oct4 and Sca1. At one month after transplantation, real-time polymerase chain reaction analysis confirmed a significantly increased expression level of Oct4 and SSEA-1 positive cells at the injury site. Conclusion: VSELs have the capability to migrate and localize in an injured spinal cord after transplantation.

  11. Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Eun Ju Lee

    Full Text Available BACKGROUND: Muscle satellite cells (MSCs represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs and adipocyte-like cells (ALCs, we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs. RESULTS: A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248 highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2 during myogenesis and hemoglobin subunit alpha2 (HBA2 during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay. CONCLUSION: In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to

  12. Effect of High Expression of Heat Shock Protein on 70 Skeletal Muscle Cells ATP%高表达热休克蛋白70对骨骼肌细胞ATP的影响

    Institute of Scientific and Technical Information of China (English)

    王磊; 王尊; 刘跃飞

    2013-01-01

    目的 探讨高表达热休克蛋白70(heat shock protein 70,Hsp70)对骨骼肌细胞(C2C12)内ATP水平的影响.方法 通过构建重组pTRE2hyg-Hsp70质粒,稳定转染C2C12细胞系,建立Hsp70过表达的C2C12细胞系.分别在转染后细胞培养的不同时间点(0d,3d,7 d),检测细胞内ATP的水平.结果 与对照组相比,过表达Hsp70的C2C12细胞系在培养的3d,7d,细胞内ATP的水平明显增高(P<0.05).结论 高表达Hsp70的骨骼肌细胞C2C12可以提高细胞内的ATP水平,提示Hsp70对骨骼肌细胞的能量代谢产生影响.

  13. A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor

    International Nuclear Information System (INIS)

    Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferation while delaying the differentiation of C2C12 cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine

  14. Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

    Science.gov (United States)

    van der Schaft, Daisy W J; van Spreeuwel, Ariane C C; Boonen, Kristel J M; Langelaan, Marloes L P; Bouten, Carlijn V C; Baaijens, Frank P T

    2013-03-19

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while

  15. Use of the methylcellulose gum for fat up-take absorption in coated products

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    Daniele Domingues Dill

    2008-08-01

    Full Text Available Even though coated meats have added value to products and gained consumers confidence, by improving these product’s appearance and taste, they go through a pre-frying stage during processing which releases the water and allows fat to enter the product, thus increasing fat content. The purpose of this work was to develop a coating system that absorbs less fat during the pre-frying stage of a chicken coated product. To do that, methylcellulose gum was added (MC in different concentrations in the covering systems, batter (coating liquid and breading (covering expresses. Five experiments were carried out, a standard experiment, without ingredient addition with technological function, formulations with addition of MC in the batter (0,5% and 2,0%, with addition of MC in the batter and in the breading (1% in each and with addition of MC in the breading (2,0%. Results showed that fat uptake reduction in formulations with 0,5 and 2,0% of MC in the batter were of 5,81% and 8,40%, respectively, and in experiments with 1% of MC in the batter and 1% of MC in the breading was of 7,66%. In experiments that received 2% of MC in the breading , fat uptake reduction was of 10,51%. Formulations with 2% of MC added to the breading (10,51% and 2% of MC added to the batter (8,40% presented the largest reductions in fat uptake during the pre-fried stage. Analysis of sensorial acceptance showed that all formulations had a superior acceptance index of 70%, with no significant difference among the other tested formulations acceptance results (p <0,05. These findings show the efficiency of MC as a fat uptake barrier during the pre-fried stage, increasing humidity content, and becoming an advantageous technological alternative for the food industry.

  16. New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

    Science.gov (United States)

    Sestili, Piero; Ambrogini, Patrizia; Barbieri, Elena; Sartini, Stefano; Fimognari, Carmela; Calcabrini, Cinzia; Diaz, Anna Rita; Guescini, Michele; Polidori, Emanuela; Luchetti, Francesca; Canonico, Barbara; Lattanzi, Davide; Cuppini, Riccardo; Papa, Stefano; Stocchi, Vilberto

    2016-08-01

    A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions. PMID:26724921

  17. Molecular imaging to target transplanted muscle progenitor cells.

    Science.gov (United States)

    Gutpell, Kelly; McGirr, Rebecca; Hoffman, Lisa

    2013-01-01

    Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse). Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living

  18. Mechano-growth factor peptide, the COOH terminus of unprocessed insulin-like growth factor 1, has no apparent effect on myoblasts or primary muscle stem cells.

    Science.gov (United States)

    Fornaro, Mara; Hinken, Aaron C; Needle, Saul; Hu, Erding; Trendelenburg, Anne-Ulrike; Mayer, Angelika; Rosenstiel, Antonia; Chang, Calvin; Meier, Viktor; Billin, Andrew N; Becherer, J David; Brace, Arthur D; Evans, William J; Glass, David J; Russell, Alan J

    2014-01-15

    A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed "mechano-growth factor" (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.

  19. l-glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF-α) Stress Via Reduced p38 MAPK Signal Transduction.

    Science.gov (United States)

    Girven, Matthew; Dugdale, Hannah F; Owens, Daniel J; Hughes, David C; Stewart, Claire E; Sharples, Adam P

    2016-12-01

    Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc.

  20. l-glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF-α) Stress Via Reduced p38 MAPK Signal Transduction.

    Science.gov (United States)

    Girven, Matthew; Dugdale, Hannah F; Owens, Daniel J; Hughes, David C; Stewart, Claire E; Sharples, Adam P

    2016-12-01

    Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991744

  1. Expression of sarcomeric tropomyosin in striated muscles in axolotl treated with shz-1, a small cardiogenic molecule.

    Science.gov (United States)

    Nan, Changlong; Dube, Syamalima; Matoq, Amr; Mikesell, Lauren; Abbott, Lynn; Alshiekh-Nasany, Ruham; Chionuma, Henry; Huang, Xupei; Poiesz, Bernard J; Dube, Dipak K

    2015-01-01

    We evaluated the effect of shz-1, a cardiogenic molecule, on the expression of various tropomyosin (TM) isoforms in the Mexican axolotl (Ambystoma mexicanum) hearts. qRT-PCR data show a ~1.5-fold increase in cardiac transcripts of the Nkx2.5 gene, which plays a crucial role in cardiogenesis in vertebrates. Shz-1 augments the expression of transcripts of the total sarcomeric TPM1 (both TPM1α & TPM1κ) and sarcomeric TPM4α. In order to understand the mechanism by which shz-1 augments the expression of sarcomeric TPM transcription in axolotl hearts, we transfected C2C12 cells with pGL3.axolotl. We transfected C2C12 cells with pGL3-axolotl TPM4 promoter constructs containing the firefly luciferase reporter gene. The transfected C2C12 cells were grown in the absence or presence of shz-1 (5 μM). Subsequently, we determined the firefly luciferase activity in the extracts of transfected cells. The results suggest that shz-1 activates the axolotl TPM4 promoter-driven ectopic expression in C2C12 cells. Also, we transfected C2C12 cells with a pGL3.1 vector containing the promoter of the mouse skeletal muscle troponin-I and observed a similar increase in the luciferase activity in shz-1-treated cells. We conclude that shz-1 activates the promoters of a variety of genes including axolotl TPM4. We have quantified the expression of the total sarcomeric TPM1 and observed a 1.5-fold increase in treated cells. Western blot analyses with CH1 monoclonal antibody specific for sarcomeric isoforms show that shz-1 does not increase the expression of TM protein in axolotl hearts, whereas it does in C2C12 cells. These findings support our hypothesis that cardiac TM expression in axolotl undergoes translational control. PMID:24958154

  2. Expression of sarcomeric tropomyosin in striated muscles in axolotl treated with shz-1, a small cardiogenic molecule.

    Science.gov (United States)

    Nan, Changlong; Dube, Syamalima; Matoq, Amr; Mikesell, Lauren; Abbott, Lynn; Alshiekh-Nasany, Ruham; Chionuma, Henry; Huang, Xupei; Poiesz, Bernard J; Dube, Dipak K

    2015-01-01

    We evaluated the effect of shz-1, a cardiogenic molecule, on the expression of various tropomyosin (TM) isoforms in the Mexican axolotl (Ambystoma mexicanum) hearts. qRT-PCR data show a ~1.5-fold increase in cardiac transcripts of the Nkx2.5 gene, which plays a crucial role in cardiogenesis in vertebrates. Shz-1 augments the expression of transcripts of the total sarcomeric TPM1 (both TPM1α & TPM1κ) and sarcomeric TPM4α. In order to understand the mechanism by which shz-1 augments the expression of sarcomeric TPM transcription in axolotl hearts, we transfected C2C12 cells with pGL3.axolotl. We transfected C2C12 cells with pGL3-axolotl TPM4 promoter constructs containing the firefly luciferase reporter gene. The transfected C2C12 cells were grown in the absence or presence of shz-1 (5 μM). Subsequently, we determined the firefly luciferase activity in the extracts of transfected cells. The results suggest that shz-1 activates the axolotl TPM4 promoter-driven ectopic expression in C2C12 cells. Also, we transfected C2C12 cells with a pGL3.1 vector containing the promoter of the mouse skeletal muscle troponin-I and observed a similar increase in the luciferase activity in shz-1-treated cells. We conclude that shz-1 activates the promoters of a variety of genes including axolotl TPM4. We have quantified the expression of the total sarcomeric TPM1 and observed a 1.5-fold increase in treated cells. Western blot analyses with CH1 monoclonal antibody specific for sarcomeric isoforms show that shz-1 does not increase the expression of TM protein in axolotl hearts, whereas it does in C2C12 cells. These findings support our hypothesis that cardiac TM expression in axolotl undergoes translational control.

  3. Experiment list: SRX142511 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  4. Experiment list: SRX143622 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available of C3H mice after crush injury || cell sex=F || antibody=Input || antibody description=Control signal which ...ncing || cell=C2C12 || cell organism=mouse || cell description=Myoblast cell line derived from thigh muscle

  5. Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE.

    Science.gov (United States)

    Hage Hassan, Rima; Pacheco de Sousa, Ana Catarina; Mahfouz, Rana; Hainault, Isabelle; Blachnio-Zabielska, Agnieszka; Bourron, Olivier; Koskas, Fabien; Górski, Jan; Ferré, Pascal; Foufelle, Fabienne; Hajduch, Eric

    2016-02-01

    In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells. PMID:26698173

  6. Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE.

    Science.gov (United States)

    Hage Hassan, Rima; Pacheco de Sousa, Ana Catarina; Mahfouz, Rana; Hainault, Isabelle; Blachnio-Zabielska, Agnieszka; Bourron, Olivier; Koskas, Fabien; Górski, Jan; Ferré, Pascal; Foufelle, Fabienne; Hajduch, Eric

    2016-02-01

    In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells.

  7. Role of miR-181a-5p and endoplasmic reticulum stress in the regulation of myogenic differentiation.

    Science.gov (United States)

    Wei, Yingying; Tao, Xuelian; Xu, Huaming; Chen, Yan; Zhu, Li; Tang, Guoqing; Li, Mingzhou; Jiang, Anan; Shuai, Surong; Ma, Jideng; Jin, Long; Wen, Anxiang; Wang, Qin; Zhu, Guangxiang; Xie, Meng; Wu, Jiayun; He, Tao; Jiang, Yanzhi; Li, Xuewei

    2016-10-30

    Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway.

  8. Nafion and modified-Nafion membranes for polymer electrolyte fuel cells: An overview

    Indian Academy of Sciences (India)

    A K Sahu; S Pitchumani; P Sridhar; A K Shukla

    2009-06-01

    Polymer electrolyte fuel cells (PEFCs) employ membrane electrolytes for proton transport during the cell reaction. The membrane forms a key component of the PEFC and its performance is controlled by several physical parameters, viz. water up-take, ion-exchange capacity, proton conductivity and humidity. The article presents an overview on Nafion membranes highlighting their merits and demerits with efforts on modified-Nafion membranes.

  9. Emergent behavior of cells on microfabricated soft polymeric substrates

    Science.gov (United States)

    Anand, Sandeep Venkit

    ) with a single cell scale (micro scale) cross section. We plate C2C12 cells on the platform and characterize their migration, proliferation, and differentiation patterns in contrast to 2D culture. We find that the cells land on the 2D surface, and then migrate to the filament only when the 2D surface has become nearly confluent. Individual and isolated cells randomly approaching the filament always retract away towards the 2D surface. Once on the filament, their differentiation to myotubes is expedited compared to that on 2D substrate. The myotubes generate periodic twitching forces that deform the filament producing more than 17 um displacement at the tip. Such flagellar motion can be used to develop autonomous micro scale bio-bots. Finally, the design and fabrication of a polymeric micro-pillar based force sensor capable of measuring cellular focal-adhesion forces under externally applied stretch is discussed. The force sensor consists of arrays of uniformly spaced PDMS micro-pillars of 1-2 um diameter and 2-3 um spacing on a macroscale PDMS substrate. The tips of the micro-pillars are selectively patterned with fluorescently labeled ECM proteins using micro-contact printing to promote cell adhesion while simultaneously acting as markers for strain measurements. Cells adhere and spread on top of the pillars causing them to deform. When stretched, the cells reorganize their internal structure and modulate their traction forces in response to the applied stretch. The dynamically varying cellular forces in response to the stretch are computed by measuring the cell induced displacements estimated by isolating the displacements caused by the applied stretch from the net displacements of the tips.

  10. ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth

    DEFF Research Database (Denmark)

    Markljung, Ellen; Jiang, Lin; Jaffe, Jacob D;

    2009-01-01

    and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (Ch......IP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications...

  11. Palmitate activates mTOR/p70S6K through AMPK inhibition and hypophosphorylation of raptor in skeletal muscle cells: Reversal by oleate is similar to metformin.

    Science.gov (United States)

    Kwon, Bumsup; Querfurth, Henry W

    2015-11-01

    Excessive saturated free fatty acids (SFFAs; e.g. palmitate) in blood are a pathogenic factor in diabetes, obesity, cardiovascular disease and liver failure. In contrast, monounsaturated free fatty acids (e.g. oleate) prevent the toxic effect of SFFAs in various types of cells. The mechanism is poorly understood and involvement of the mTOR complex is untested. In the present study, we demonstrate that oleate preconditioning, as well as coincubation, completely prevented palmitate-induced markers of inflammatory signaling, insulin resistance and cytotoxicity in C2C12 myotubes. We then examined the effect of palmitate and/or oleate on the mammalian target of rapamycin (mTOR) signal path and whether their link is mediated by AMP-activated protein kinase (AMPK). Palmitate decreased the phosphorylation of raptor and 4E-BP1 while increasing the phosphorylation of p70S6K. Palmitate also inhibited phosphorylation of AMPK, but did not change the phosphorylated levels of mTOR or rictor. Oleate completely prevented the palmitate-induced dysregulation of mTOR components and restored pAMPK whereas alone it produced no signaling changes. To understand this more, we show activation of AMPK by metformin also prevented palmitate-induced changes in the phosphorylations of raptor and p70S6K, confirming that the mTORC1/p70S6K signaling pathway is responsive to AMPK activity. By contrast, inhibition of AMPK phosphorylation by Compound C worsened palmitate-induced changes and correspondingly blocked the protective effect of oleate. Finally, metformin modestly attenuated palmitate-induced insulin resistance and cytotoxicity, as did oleate. Our findings indicate that palmitate activates mTORC1/p70S6K signaling by AMPK inhibition and phosphorylation of raptor. Oleate reverses these effects through a metformin-like facilitation of AMPK. PMID:26344902

  12. Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

    OpenAIRE

    Yeilding, N M; W.M. Lee

    1997-01-01

    Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the h...

  13. CaMK Ⅱ在收缩促进骨骼肌细胞GLUT4转位中的作用%The Role of CaMKⅡ in Contraction-Stimulated GLUT4 Translocation in Skeletal Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    于俊娜; 牛文彦

    2011-01-01

    Objective: To study the role of calcium-/calmodulin- dependent protein kinase Ⅱ (CaMK Ⅱ) in the mechanism of contraction-stimulated translocation of glucose transporter 4(GLUT4) in skeletal muscle cells. Methods:C2C12GLUT4myc myotubes were divided into two groups treated with or without carbachol (Cch). CaMK Ⅱ inhibitor KN93or KN62 was added to the medium prior to treatment, respectively. Then cell surface levels of GLUT4myc were measured by enzyme linked immunosorbent assay(ELISA). The phosphorylation of CaMK Ⅱ was detected by immunoblotting after pre-incubation with KN93. Results: Carbachol increased phosphorylation of CaMK Ⅱ which was reduced by KN93. KN93 and KN62 inhibited the gain of cell surface GLUT4myc induced by Cch. KN93 did not affect the phosphorylation of CaMK Ⅱ but it inhibited the CaMK Ⅱ phosphorylation stimulated by Cch. Conclusion: CaMK Ⅱ is the downstream signal of CaZ+ and mediates contraction-stimulated GLUT4myc traffic in skeletal muscle cells.%目的:探讨钙一钙调蛋白依赖性蛋白激酶Ⅱ(CaMK Ⅱ)在收缩促进骨骼肌细胞葡萄糖转运子4(GLUT4)转位机制中的作用.方法:将接种在培养板中的C2C12 GLUT4myc小鼠肌管随机分为对照组和氨乙酰胆碱(Cch)组,各组分为抑制剂亚组和对照亚组.分别用10 μmol/L CaMK Ⅱ的特异性抑制剂KN93或KN62预孵育,用酶联免疫吸附法(ELISA)测定细胞膜上GLUT4myc的含量(转位);用KN93预孵育后,免疫印迹法测定蛋白激酶CaMKⅡ的磷酸化.结果:氨乙酰胆碱使细胞膜上GLUT4myc的水平显著增加.KN93和KN62抑制氨乙酰胆碱刺激的GLUT4myc转位.氨乙酰胆碱可增加CaMKⅡ的磷酸化,KN93不影响对照组CaMK Ⅱ的磷酸化水平,但可以抑制Cch刺激的CaMK Ⅱ磷酸化.结论:CaMK Ⅱ位于Ca2+下游并有介导收缩促进骨骼肌细胞GLUT4myc转位的作用.

  14. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    the cells are able to to regulate Myo-D and myogenin is poorly understood. In the present work, we investigate the role of mechanical loading, through specific receptors to intracellular matrix proteins such as laminin and fibronectin, in both Myo-D and myogenin expression in C(2)C(12) cells. We propose...... recepotors plays a role in myoblast differentiation and fusion....

  15. Experiment list: SRX344977 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gged ING1 || cell type=Growing C1C12 myoblasts || chromatin preparation method=So...ource_name=Input_ING1_flag_myoblasts || cell line=C2C12 || genotype/variation=ectopically expressing Flag-ta

  16. Experiment list: SRX344978 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available urce_name=Input_ING1_flag_myoblasts || cell line=C2C12 || genotype/variation=ectopically expressing Flag-tagged ING1 || cell type=Gro...wing C1C12 myoblasts || chromatin preparation method=Son

  17. Experiment list: SRX451714 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ue Diagnosis=NOS 81447171,94.3,36.2,943 GSM1316961: MLL3 MB; Mus musculus; ChIP-Seq source_name=Growing C1C1...2 myoblasts || cell line=C2C12 || cell type=growing myoblasts (MB) || chromatin preparation method=Sonicatio

  18. Experiment list: SRX451712 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ue Diagnosis=NOS 2016249,43.5,11.1,312 GSM1316959: LSD1 MB; Mus musculus; ChIP-Seq source_name=Growing C1C12... myoblasts || cell line=C2C12 || cell type=growing myoblasts (MB) || chromatin preparation method=Sonication

  19. Matrix-immobilized BMP-2 on microcontact printed fibronectin as in vitro tool to study BMP-mediated signaling and cell migration

    Directory of Open Access Journals (Sweden)

    Kristin eHauff

    2015-05-01

    Full Text Available During development, bone morphogenetic proteins (BMPs exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo the extracellular matrix (ECM not only provides a support for adherent cells, but also presents a reservoir of growth factors (GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell transmembrane receptors, such as integrins, which convey adhesion-mediated signaling to the intracellular compartment. Integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors, in regulating cell responses to extracellular signals. To this, we present here the immobilization of BMP-2 onto cellular fibronectin (cFN, a key protein of the ECM, to investigate their impact on GF-mediated signaling and migration.Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin (NA as cross-linker. Characterization with QCM-D and ELISA confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h.To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2 we investigated short- and long-term responses of C2C12 myoblasts in comparison to soluble BMP-2 (sBMP-2 or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation into the nucleus corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after six days.We next implemented this approach to fabricate cFN micro patterned stripes by soft lithography. These stripes only allowed cell-surface interaction on the pattern due to passivation of the surface in between, thus serving as platform for studies on directed cell migration. During a 10 h-period, cells showed an increased migratory activity upon BMP-2 exposure.Thus, this versatile tool retains the GF's bioactivity and allows the presentation of ECM

  20. In vitro profiling of epigenetic modifications underlying heavy metal toxicity of tungsten-alloy and its components

    International Nuclear Information System (INIS)

    Tungsten-alloy has carcinogenic potential as demonstrated by cancer development in rats with intramuscular implanted tungsten-alloy pellets. This suggests a potential involvement of epigenetic events previously implicated as environmental triggers of cancer. Here, we tested metal induced cytotoxicity and epigenetic modifications including H3 acetylation, H3-Ser10 phosphorylation and H3-K4 trimethylation. We exposed human embryonic kidney (HEK293), human neuroepithelioma (SKNMC), and mouse myoblast (C2C12) cultures for 1-day and hippocampal primary neuronal cultures for 1-week to 50-200 μg/ml of tungsten-alloy (91% tungsten/6% nickel/3% cobalt), tungsten, nickel, and cobalt. We also examined the potential role of intracellular calcium in metal mediated histone modifications by addition of calcium channel blockers/chelators to the metal solutions. Tungsten and its alloy showed cytotoxicity at concentrations > 50 μg/ml, while we found significant toxicity with cobalt and nickel for most tested concentrations. Diverse cell-specific toxic effects were observed, with C2C12 being relatively resistant to tungsten-alloy mediated toxic impact. Tungsten-alloy, but not tungsten, caused almost complete dephosphorylation of H3-Ser10 in C2C12 and hippocampal primary neuronal cultures with H3-hypoacetylation in C2C12. Dramatic H3-Ser10 dephosphorylation was found in all cobalt treated cultures with a decrease in H3 pan-acetylation in C2C12, SKNMC and HEK293. Trimethylation of H3-K4 was not affected. Both tungsten-alloy and cobalt mediated H3-Ser10 dephosphorylation were reversed with BAPTA-AM, highlighting the role of intracellular calcium, confirmed with 2-photon calcium imaging. In summary, our results for the first time reveal epigenetic modifications triggered by tungsten-alloy exposure in C2C12 and hippocampal primary neuronal cultures suggesting the underlying synergistic effects of tungsten, nickel and cobalt mediated by changes in intracellular calcium homeostasis and

  1. Phenotypic nutrient up-take differences in an alley cropping system in semi-arid Machakos, Kenya

    NARCIS (Netherlands)

    Mungai, D.N.; Coulson, C.L.; Stigter, C.J.; Ng'ang'a, J.K.; Mugendi, D.N.

    2001-01-01

    Alley cropping of Cassia siamea and maize was studied in semi-arid Kenya for soil fertility improvement. Katumani composite maize was planted except in the short rains of 1988 (SR88) when a hybrid variety was sown. Therefore the grain yield per row increased differently in the alley cropped maize (C

  2. Effect of Arbuscular Mycorrhiza Fungi Inoculation on Growth and Up take of Mineral Nutrition in Ipomoea Aquatica.

    Directory of Open Access Journals (Sweden)

    Milton Halder

    2015-04-01

    Full Text Available A green house experiment was conducted to investigate the effect of arbuscular mycorrhiza inoculation on plant growth and uptake of mineral nutrition in Ipomoea aquatica considering the objective of using environmental friendly biofertilizer instead of chemical fertilizer. A common leafy vegetable plant Ipomoea aquatica was grown with mycorrhiza and without mycorrhiza for 42 days. After harvest the plants were analyzed for mineral nutrition concentration. Plant fresh weight, dry weight, macronutrient (P, K, Mg, Na, micronutrient (Fe, Mn, Zn concentration was higher in arbuscular mycorrhiza inoculated plant than non-mycorrhiza inoculated plant. For sustainable agriculture, introducing biofertilizer by using arbuscular mycorrhiza inoculation would be one of the most efficient techniques for replacing chemical fertilizer to meet the nutrient deficiency in nutrient deficient soils of Bangladesh.

  3. Phenotypic nutrient up-take differences in an alley cropping system in semi-arid Machakos, Kenya

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Alley cropping of Cassia siamea and maize was studied insemi-arid Kenya for soil fertility improvement. Katumani composite maize was planted except in the short rains of 1988 (SR88) when a hybrid variety was sown. Therefore the grain yield per row increased differently in the alley cropped maize (CM). Sole maize (SM) and CM yields were higher in SR88 than in the long rains of 1988 (LR88) by 62% and 38%, while yields from the same treatments in LR89 were only 21% and 45% of those in SR88. These differences in relative maize yields are attributed to differences between the two maize varieties in competition under nutrient stress conditions.

  4. Prolonged activation of S6K1 does not suppress IRS or PI-3 kinase signaling during muscle cell differentiation

    Directory of Open Access Journals (Sweden)

    MacKenzie Matthew G

    2010-05-01

    Full Text Available Abstract Background Myogenesis in C2C12 cells requires the activation of the PI3K/mTOR signaling pathways. Since mTOR signaling can feedback through S6K1 to inhibit the activation of PI3K, the aim of this work was to assess whether feedback from S6K1 played a role in myogenesis and determine whether siRNA mediated knockdown of S6K1 would lead to an increased rate of myotube formation. Results S6K1 activity increased in a linear fashion following plating and was more than 3-fold higher after Day 3 of differentiation (subconfluent = 11.09 ± 3.05, Day 3 = 29.34 ± 3.58. IRS-1 levels tended to increase upon serum withdrawal but decreased approximately 2-fold (subconfluent = 0.88 ± 0.10, Day 3 = 0.42 ± 0.06 3 days following differentiation whereas IRS-2 protein remained stable. IRS-1 associated p85 was significantly reduced upon serum withdrawal (subconfluent = 0.86 ± 0.07, Day 0 = 0.31 ± 0.05, remaining low through day 1. IRS-2 associated p85 decreased following serum withdrawal (subconfluent = 0.96 ± 0.05, Day 1 = 0.56 ± 0.08 and remained suppressed up to Day 3 following differentiation (0.56 ± 0.05. Phospho-tyrosine associated p85 increased significantly from subconfluent to Day 0 and remained elevated throughout differentiation. siRNA directed against S6K1 and S6K2 did not result in changes in IRS-1 levels after either 48 or 96 hrs. Furthermore, neither 48 nor 96 hrs of S6K1 knockdown caused a change in myotube formation. Conclusions Even though S6K1 activity increases throughout muscle cell differentiation and IRS-1 levels decrease over this period, siRNA suggests that S6K1 is not mediating the decrease in IRS-1. The decrease in IRS-1/2 associated p85 together with the increase in phospho-tyrosine associated p85 suggests that PI3K associates primarily with scaffolds other than IRS-1/2 during muscle cell differentiation.

  5. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways and transcription factors

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Murgia, Marta; Nagaraja, Nagarjuna;

    2015-01-01

    spectrometric (MS) workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins...

  6. Adipophilin protein expression in muscle - a possible protective role against insulin resistance

    NARCIS (Netherlands)

    Wilde, de J.; Smit, E.; Snepvangers, F.J.M.; Wit, de N.J.W.; Mohren, R.; Hulshof, M.F.M.; Mariman, E.C.M.

    2010-01-01

    Adipophilin is a 50 kDa protein that belongs to the PAT family (perilipin, adipophilin, TIP47, S3-12 and OXPAT), which comprises proteins involved in the coating of lipid droplets. Little is known about the functional role of adipophilin in muscle. Using the C2C12 cell line as a model, we demonstrat

  7. Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Koh, Ho-Jin; Toyoda, Taro; Fujii, Nobuharu;

    2010-01-01

    . Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated glucose transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated glucose transport. SNARK is activated by muscle contraction and is a unique mediator of contraction...

  8. Reconstruction of spatially orientated myotubes in vitro using electrospun, parallel microfibre arrays

    Directory of Open Access Journals (Sweden)

    A Huber

    2007-10-01

    Full Text Available The stable culture of myogenic cells and their differentiation into myotubes in vitro is often hindered by the mechanical destabilisation of the spontaneously contractile neotissue formed, resulting in the complete loss of differentiating myotubes. Electrospun, parallel aligned nylon 6/6 microfibre arrays were use successfully for the culture of C2C12 myoblasts and their differentiation to form mechanically stable, orientated myotubes in vitro. Myoblasts adhered strongly to the parallel fibre array, forming a compact cell sheath across the entire array, aligning individual cells in parallel to the direction of the fibrous substratum. The myogenic potential of C2C12 myoblasts was not impaired and resulted in the formation of elongated myotubes expressing alpha-actinin, adult myosin heavy chain and nicotinic acetylcholine receptors as muscle-specific marker proteins. Newly formed C2C12 myotubes were themselves orientated in parallel to the direction of the underlying fibrous substratum and exhibited a high level of structural integration with the surrounding cells. In contrast, non-woven, non-orientated nylon 6/6 meshes, produced by conventional electrospinning, exhibited greatly reduced levels of C2C12 myoblast attachment and adherent myoblasts did not differentiate into myotubes. In conclusion, parallel microfibre arrays provided a superior microscale topography for the stable maintenance and differentiation of myotubes in vitro.

  9. MicroRNA-138 regulates hypoxia-induced endothelial cell dysfunction by targeting S100A1.

    Directory of Open Access Journals (Sweden)

    Anagha Sen

    Full Text Available The Ca(2+ sensor S100A1 is essential for proper endothelial cell (EC nitric oxide (NO synthase (eNOS activation. S100A1 levels are greatly reduced in primary human microvascular ECs subjected to hypoxia, rendering them dysfunctional. However mechanisms that regulate S100A1 levels in ECs are unknown. Here we show that ECs transfected with a S100A1-3' untranslated region (UTR luciferase reporter construct display significantly reduced gene expression when subjected to low oxygen levels or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (MiR-138 could target the 3'UTR of S100A1. Patients with critical limb ischemia (CLI or mice subjected to femoral artery resection (FAR displayed increased MiR-138 levels and decreased S100A1 protein expression. Consistent with this finding, hypoxia greatly increased MiR-138 levels in ECs, but not in skeletal muscle C2C12 myoblasts or differentiated myotubes or primary human vascular smooth muscle cells. Transfection of a MiR-138 mimic into ECs reduced S100A1-3 'UTR reporter gene expression, while transfection of an anti MiR-138 prevented the hypoxia-induced downregulation of the reporter gene. Deletion of the 22 nucleotide putative MiR-138 target site abolished the hypoxia-induced loss of reporter gene expression. Knockdown of Hif1-α mediated by siRNA prevented loss of hypoxia-induced reporter gene expression. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2 reduced reporter gene expression even in the absence of hypoxia. Finally, primary ECs transfected with a MiR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. We conclude that hypoxia-induced MiR-138 is an essential mediator of EC dysfunction via its ability to target the 3'UTR of S100A1.

  10. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-{beta} in myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan); Yamaguchi, Takahiro, E-mail: ty1010@bios.tohoku.ac.jp [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan)

    2010-03-19

    Myostatin and TGF-{beta} negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-{beta} signaling remains unclear. TGF-{beta} inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-{beta} signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-{beta} signaling using C2C12 myoblasts. Myostatin and TGF-{beta} induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-{beta} enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-{beta} in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-{beta}. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-{beta} that prevents excess action in myoblasts.

  11. Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells.

    Science.gov (United States)

    Calhabeu, F; Hayashi, S; Morgan, J E; Relaix, F; Zammit, P S

    2013-01-31

    Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and myogenin. There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect myogenesis in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) Pax3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/FOXO1A in C2C12 myogenic cells again as seen with Pax3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via

  12. Skeletal muscle cells possess a 'memory' of acute early life TNF-α exposure: role of epigenetic adaptation.

    Science.gov (United States)

    Sharples, Adam P; Polydorou, Ioanna; Hughes, David C; Owens, Daniel J; Hughes, Thomas M; Stewart, Claire E

    2016-06-01

    Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that

  13. Skeletal muscle cells possess a 'memory' of acute early life TNF-α exposure: role of epigenetic adaptation.

    Science.gov (United States)

    Sharples, Adam P; Polydorou, Ioanna; Hughes, David C; Owens, Daniel J; Hughes, Thomas M; Stewart, Claire E

    2016-06-01

    Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that

  14. Self-Assembling Peptide Amphiphiles for Therapeutic Delivery of Proteins, Drugs, and Stem Cells

    Science.gov (United States)

    Lee, Sungsoo Seth

    Biomaterials are used to help regenerate or replace the structure and function of damaged tissues. In order to elicit desired therapeutic responses in vivo, biomaterials are often functionalized with bioactive agents, such as growth factors, small molecule drugs, or even stem cells. Therefore, the strategies used to incorporate these bioactive agents in the microstructures and nanostructures of biomaterials can strongly influence the their therapeutic efficacy. Using self-assembling peptide amphiphiles (PAs), this work has investigated supramolecular nanostructures with improved interaction with three types of therapeutic agents: bone morphogenetic protein 2 (BMP-2) which promotes osteogenic differentiation and bone growth, anti-inflammatory drug naproxen which is used to treat osteo- and rheumatoid arthritis, and neural stem cells that could differentiate into neurons to treat neurodegenerative diseases. For BMP-2 delivery, two specific systems were investigated with affinity for BMP-2: 1) heparin-binding nanofibers that display the natural ligand of the osteogenic protein, and 2) nanofibers that display a synthetic peptide ligand discovered in our laboratory through phage display to directly bind BMP-2. Both systems promoted enhanced osteoblast differentiation of pluripotent C2C12 cells and augmented bone regeneration in two in vivo models, a rat critical-size femur defect model and spinal arthrodesis model. The thesis also describes the use of PA nanofibers to improve the delivery of the anti-inflammatory drug naproxen. To promote a controlled release, naproxen was chemically conjugated to the nanofiber surface via an ester bond that would only be cleaved by esterases, which are enzymes found naturally in the body. In the absence of esterases, the naproxen remained conjugated to the nanofibers and was non-bioactive. On the other hand, in the presence of esterases, naproxen was slowly released and inhibited cyclooxygenase-2 (COX-2) activity, an enzyme responsible

  15. Development of a Neuromuscular Junction Model on Surfaces Modified by Plasma Polymerization

    OpenAIRE

    E. Zuñiga-Aguilar; R. Godínez; O. Ramírez-Fernández; Morales, J.; R. Olayo

    2013-01-01

    The aim of this work is to implement a biological model of neuromuscular junctions to study the mechanisms involved in intra and inter cellular processes using cell co-cultures. To optimize growth and development of the neuromuscular junction, cells were seeded on plasma polymerized pyrrole which has proven suitable for other types of cell cultures. The cell lines used were motor neuron NG108-15 and skeletal muscle C2C12. Cells were evaluated according to their morphology and elect...

  16. Modulation of Cell Cycle Progression by 5-Azacytidine Is Associated with Early Myogenesis Induction in Murine Myoblasts

    Science.gov (United States)

    Montesano, Anna; Luzi, Livio; Senesi, Pamela; Terruzzi, Ileana

    2013-01-01

    Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein

  17. Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.

    Science.gov (United States)

    Montesano, Anna; Luzi, Livio; Senesi, Pamela; Terruzzi, Ileana

    2013-01-01

    Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein

  18. Insulin Resistance Is Correlated with Palmitic Acid Uptake in Skeletal Muscle Cells%棕榈酸的组织吸收分布及对骨骼肌胰岛素抵抗的影响

    Institute of Scientific and Technical Information of China (English)

    彭恭; 刘延波; 李凌海; 刘平生

    2012-01-01

    Retinoids (vitamin A and its derivatives) play important roles in the maintenance of various tissues in the adult vertebrate and are essential for diverse embryological processes. As a member of retinoids (vitamin A and its derivatives), retinoic acid (RA) has been extensively investigated in embryopathology. However, the mechanisms by which RA influences these processes are not completely understood. In the present study, we found that embryonic RA exposure via maternal treatment with gavage-fed 3 successive doses of RA on day 8 of gestation led to a high incidence (96.77%, 30/31) of rachischisis with myeloschisis, I.e., spina bifida aperta, among the surviving day 18 fetuses. Using microarray technology, we identified 134 genes in the spinal cords of mice that exhibit at least a 1.5-fold change between mice with spina bifida and control samples. Several downstream genes of RA signaling involved in lipid metabolism were regulated at the transcriptional level after maternal RA exposure. Furthermore, a gene set enrichment analysis (GSEA) implicate many altered expression of genes, involved in pro- or anti-apoptosis, cell proliferation, migration, cytoskeleton components, and cell or focal adhesion, which are associated which the spina bifida induced by the maternal RA exposure. This indicates that defective functions of these cell components and biological processes preceded the abnormal development of neural tube. Our study provides a global analysis of gene expression patterns in spina bifida and will help the understanding of the etiology and pathology of neural tube defects.%脂肪酸代谢紊乱是Ⅱ型糖尿病的主要致病因素之一.棕榈酸是血液中含量最高的游离脂肪酸.我们建立了大鼠颈静脉置管输注棕榈酸的模型,发现血液中的大部分棕榈酸被骨骼肌组织所吸收.以棕榈酸处理的C2C12骨骼肌细胞为实验模型发现,棕榈酸进入骨骼肌细胞后的中间代谢产物(磷脂和甘油二酯)的累

  19. The interplay between physical and chemical properties of protein films affects their bioactivity.

    Science.gov (United States)

    Grover, Chloe N; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

    2012-09-01

    Although mechanical properties, roughness, and receptor molecule expression have all been shown to influence the cellular reactivity of collagen-based biomaterials, their relative contribution, in a given system remains unclear. Here, we study films containing combinations of collagen, gelatin, and soluble and insoluble elastin, crosslinking of which results in altered film stiffness and roughness. Collagen and gelatin have similar amino acid sequences but altered cell-binding sites. We studied cell response with both C2C12 myoblast cells (which possess RGD-recognizing integrins α(V)β(3) and α(5)β(1)) and C2C12-α2+ cells (which, in addition, express the collagen-binding integrin α(2)β(1)) to establish the effect of altering the available binding sites on cell adhesion and spreading on films. Systematically altering the composition, crosslinking and cell type, allows us to deconvolute the effects of physical parameters and available binding sites on the cell reactivity of films in this system. Collagen-based films were rougher and stiffer and supported lower cell surface coverage than gelatin-based films. Additionally, C2C12-α2+ cells showed preferential attachment to collagen-based films compared with C2C12 cells, but no significant difference was seen using gelatin-based films. The cell count and surface coverage were found to decrease significantly on all films after crosslinking (Coll XL coverage = 2-6%, Gel XL coverage = 20-32%), but cell area and aspect ratio on collagen films were affected to a greater extent than on gelatin films. The results show that, in this system, the composition, and more significantly, crosslinking, of films affects the cell reactivity to a greater extent than their stiffness or roughness.

  20. Bio-inspired Hybrid Carbon Nanotube Muscles

    OpenAIRE

    Tae Hyeob Kim; Cheong Hoon Kwon; Changsun Lee; Jieun An; Tam Thi Thanh Phuong; Sun Hwa Park; Lima, Márcio D.; Baughman, Ray H.; Tong Mook Kang; Seon Jeong Kim

    2016-01-01

    There has been continuous progress in the development for biomedical engineering systems of hybrid muscle generated by combining skeletal muscle and artificial structure. The main factor affecting the actuation performance of hybrid muscle relies on the compatibility between living cells and their muscle scaffolds during cell culture. Here, we developed a hybrid muscle powered by C2C12 skeletal muscle cells based on the functionalized multi-walled carbon nanotubes (MWCNT) sheets coated with p...

  1. Antisnake Venom Activity of Hibiscus aethiopicus L. against Echis ocellatus and Naja n. nigricollis

    OpenAIRE

    Hasson, S. S.; Al-Jabri, A. A.; Sallam, T. A.; Al-balushi, M. S.; Mothana, R. A. A.

    2010-01-01

    The objective of the study is to investigate whether the Hibiscus aethiopicus L. plant has neutralization activity against venoms of two clinically important snakes. The H. aethiopicus was dried and extracted with water. Different assays were performed to evaluate the plant's acute toxicity and its anti-snake venom activities. The results showed that H. aethiopicus extract alone had no effect on the viability of C2C12 muscle cells, but significantly (P < .05) protected muscle cells against th...

  2. Vindogentianine, a hypoglycemic alkaloid from Catharanthus roseus (L.) G. Don (Apocynaceae).

    Science.gov (United States)

    Tiong, Soon Huat; Looi, Chung Yeng; Arya, Aditya; Wong, Won Fen; Hazni, Hazrina; Mustafa, Mohd Rais; Awang, Khalijah

    2015-04-01

    Vindogentianine, a new indole alkaloid together with six known alkaloids, vindoline, vindolidine, vindolicine, vindolinine, perivine and serpentine were isolated from leaf extract (DA) of Catharanthus roseus (L.) G. Don. Their structures were elucidated by spectroscopic methods; NMR, MS, UV and IR. Vindogentianine is a dimer containing a vindoline moiety coupled to a gentianine moiety. After 24h incubation, vindogentianine exhibited no cytotoxic effect in C2C12 mouse myoblast and β-TC6 mouse pancreatic cells (IC50>50μg/mL). Real-time cell proliferation monitoring also indicated vindogentianine had little or no effect on C2C12 mouse myoblast cell growth at the highest dose tested (200μg/mL), without inducing cell death. Vindogentianine exhibited potential hypoglycemic activity in β-TC6 and C2C12 cells by inducing higher glucose uptake and significant in vitro PTP-1B inhibition. However, in vitro α-amylase and α-glucosidase inhibition assay showed low inhibition under treatment of vindogentianine. This suggests that hypoglycemic activity of vindogentianine may be due to the enhancement of glucose uptake and PTP-1B inhibition, implying its therapeutic potential against type 2 diabetes. PMID:25665941

  3. Enhancement of energy production by black ginger extract containing polymethoxy flavonoids in myocytes through improving glucose, lactic acid and lipid metabolism.

    Science.gov (United States)

    Toda, Kazuya; Takeda, Shogo; Hitoe, Shoketsu; Nakamura, Seikou; Matsuda, Hisashi; Shimoda, Hiroshi

    2016-04-01

    Enhancement of muscular energy production is thought to improve locomotive functions and prevent metabolic syndromes including diabetes and lipidemia. Black ginger (Kaempferia parviflora) has been cultivated for traditional medicine in Thailand. Recent studies have shown that black ginger extract (KPE) activated brown adipocytes and lipolysis in white adipose tissue, which may cure obesity-related dysfunction of lipid metabolism. However, the effect of KPE on glucose and lipid utilization in muscle cells has not been examined yet. Hence, we evaluated the effect of KPE and its constituents on energy metabolism in pre-differentiated (p) and differentiated (d) C2C12 myoblasts. KPE (0.1-10 μg/ml) was added to pC2C12 cells in the differentiation process for a week or used to treat dC2C12 cells for 24 h. After culturing, parameters of glucose and lipid metabolism and mitochondrial biogenesis were assessed. In terms of the results, KPE enhanced the uptake of 2-deoxyglucose and lactic acid as well as the mRNA expression of glucose transporter (GLUT) 4 and monocarboxylate transporter (MCT) 1 in both types of cells. The expression of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α was enhanced in pC2C12 cells. In addition, KPE enhanced the production of ATP and mitochondrial biogenesis. Polymethoxy flavonoids in KPE including 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone enhanced the expression of GLUT4 and PGC-1α. Moreover, KPE and 5,7-dimethoxyflavone enhanced the phosphorylation of 5'AMP-activated protein kinase (AMPK). In conclusion, KPE and its polymethoxy flavonoids were found to enhance energy metabolism in myocytes. KPE may improve the dysfunction of muscle metabolism that leads to metabolic syndrome and locomotive dysfunction. PMID:26581843

  4. Indirect Low-Intensity Ultrasonic Stimulation for Tissue Engineering

    OpenAIRE

    Hyoungshin Park; Michael C. Yip; Beata Chertok; Joseph Kost; James B. Kobler; Robert Langer; Zeitels, Steven M.

    2010-01-01

    Low-intensity ultrasound (LIUS) treatment has been shown to increase mass transport, which could benefit tissue grafts during the immediate postimplant period, when blood supply to the implanted tissue is suboptimal. In this in vitro study, we investigated effects of LIUS stimulation on dye diffusion, proliferation, metabolism, and tropomyosin expression of muscle cells (C2C12) and on tissue viability and gene expression of human adipose tissue organoids. We found that LIUS increased dye diff...

  5. Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

    OpenAIRE

    ZHANG, Zong-Kang; Li, Jie; Liu, Jin; Baosheng GUO; Leung, Albert; Zhang, Ge; Zhang, Bao-Ting

    2016-01-01

    Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment...

  6. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    OpenAIRE

    A Deshmukh; Murgia, M.; Nagaraj, N; Treebak, J.; Cox, J; Mann, M

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and...

  7. Inhibitory Effect of High Temperature- and High Pressure-Treated Red Ginseng on Exercise-Induced Oxidative Stress in ICR Mouse

    OpenAIRE

    Seok-Yeong Yu; Bo-Ra Yoon; Young-Jun Lee; Jong Seok Lee; Hee-Do Hong; Young-Chul Lee; Young-Chan Kim; Chang-Won Cho; Kyung-Tack Kim; Ok-Hwan Lee

    2014-01-01

    As previously reported, high temperature- and high pressure-treated red ginseng (HRG) contain higher contents of phenolic compounds and protect C2C12 muscle cells and 3T3-L1 adipocytes against oxidative stress. This study investigated the effect of HRG on oxidative stress using a mouse model. Our results show that the levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, hepatic malondialdehyde in the HRG group were significantly lower than those of the exercise grou...

  8. Astragalus Polysaccharide Suppresses Skeletal Muscle Myostatin Expression in Diabetes: Involvement of ROS-ERK and NF-κB Pathways

    Directory of Open Access Journals (Sweden)

    Min Liu

    2013-01-01

    Full Text Available Objective. The antidiabetes drug astragalus polysaccharide (APS is capable of increasing insulin sensitivity in skeletal muscle and improving whole-body glucose homeostasis. Recent studies suggest that skeletal muscle secreted growth factor myostatin plays an important role in regulating insulin signaling and insulin resistance. We hypothesized that regulation of skeletal muscle myostatin expression may be involved in the improvement of insulin sensitivity by APS. Methods. APS was administered to 13-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Complementary studies examined APS effects on the saturated acid palmitate-induced insulin resistance and myostatin expression in C2C12 cells. Results. APS treatment ameliorated hyperglycemia, hyperlipidemia, and insulin resistance and decreased the elevation of myostatin expression and malondialdehyde production in skeletal muscle of noninsulin-dependent diabetic KKAy mice. In C2C12 cells in vitro, saturated acid palmitate-induced impaired glucose uptake, overproduction of ROS, activation of extracellular regulated protein kinases (ERK, and NF-κB were partially restored by APS treatment. The protective effects of APS were mimicked by ERK and NF-κB inhibitors, respectively. Conclusion. Our study demonstrates elevated myostatin expression in skeletal muscle of type 2 diabetic KKAy mice and in cultured C2C12 cells exposed to palmitate. APS is capable of improving insulin sensitivity and decreasing myostatin expression in skeletal muscle through downregulating ROS-ERK-NF-κB pathway.

  9. Delta-like 1/fetal antigen 1(DLK1/FA1) inhibits BMP2 induced osteoblast differentiation through modulation of NFκB signaling pathway

    DEFF Research Database (Denmark)

    Qiu, Weimin; Abdallah, Basem; Kassem, Moustapha

    as assessed by reduced Alp activity and osteogenic gene expression including Alp, Col1a1, Runx2 and Bglap. In addition, DLK1/FA1 inhibited BMP signaling as demonstrated by reduced gene expression of BMP-responsive genes: Junb and Id1, reduced BMP2 induced luciferase activity in C2C12 BMP luciferase reporter....... Besides, we observed that DLK1/FA1 induced strong NFκB activity evidenced by NFκB responsive luciferase reporter assay and real-time RT-PCR analysis of NFκB target genes. The inhibitory effect of NFκB signaling on BMP signaling was confirmed by luciferase assay in C2C12 BMP luciferase reporter cells...

  10. Peanut cultivar selection for BLSS in terms of the biomass productivity, nutritional quality, photosynthetic character and mineral ions up-take by PTNDS cultivation

    Science.gov (United States)

    Liu, Hui; Wang, Minjuan; Fu, Yuming; Liu, Hong

    2016-11-01

    Peanut (Arachis hypogaea L.) has been selected as one of the crop candidates for BLSS, because its seeds have high nutritional value, being rich in vegetable oil and protein. Porous-Tube Nutrient Delivery System (PTNDS) has been successfully used for crop cultivation in controllable environments. In this paper, four peanut cultivars ('HY25', 'HY28', 'HY31' and 'BS1016') were evaluated in terms of yield, photosynthetic efficiency, insoluble fiber and ions uptake efficiency. Besides protein, total oil content and fatty acid composition were monitored in the seeds. 'HY25' plants showed much higher yield and harvest index, in addition to the lower lignin content of inedible biomass. Data showed that 'HY25' had the higher photosynthetic capacity of peanut leaves with regard to highest photosynthetic rate, qP and ΦPSII, lowest energy dissipation (qN) values, whereas instantaneous carboxylation efficiency and water use efficiency carotenoids content were no difference with the other cultivars. 'BS1016‧ showed the lowest photosynthetic capacity contrarily. These suggested that 'HY25‧ could be the most suitable for the cultivation in a closed controlled environment with PTNDS. While, both cations and anions except NH4+ and H2PO4-, were accumulated excessively compared to controls, especially with anions in PTNDS. Hence, further studies are needed in order to improve the nutritional quality of seeds and modify the fertilization strategy of this cultivar in the growth environment feasible during a closed environment and space mission.

  11. 重组小鼠白介素-2基因在真核细胞中的转染表达%THE EXPRESSION OF RECOMBINANT mIL-2 GENE IN TRANSFECTED EUKARYOTIC CELLS

    Institute of Scientific and Technical Information of China (English)

    路晓辉; 杜军; 曾军; 李晓玲; 徐珍霞; 蔡绍晖

    2006-01-01

    目的:在构建重组真核表达载体pIRESneo 2/mIL-2的基础上,建立能够持续稳定表达mIL-2的哺乳类工程细胞.方法:运用分子克隆技术,将由RT-PCR获得的mIL-2 cDNA片断插入真核表达质粒pIRESneo2构建成mIL-2重组表达载体pIRESneo2/mIL-2.通过脂质体转染法将pIRESneo2/mIL-2导入C2C12细胞.转染后第30天,用Western blots检测mIL-2表达情况.结果:经DNA测序证明mIL-2 cDNA片断插入方向和碱基组成顺序均准确无误,Western blots检测转染真核重组表达载体pIRESneo 2/mIL-2的C2C12细胞系表达mIL-2.结论:利用pIRESneo2/mIL-2构建的真核表达载体在C2C12细胞系中能够持续稳定表达mIL-2.

  12. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  13. Roles of extracellular signal-regulated kinase 1/2 on the suppression of myostatin gene expression induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    Huazhoag Liu; Xiaorong An; Yongfu Chen; Jieping Zhong

    2008-01-01

    Basic fibmblast growth factor (bFGF, FG F-2 ) has an inhibitory effect on the expression of the myostatin gene in murine C2C12 myoblasts, as shown in our recent investigation. To further verify the regulatory effects of bFGF on the myostalin gene and to better understand its mechanism in skeletal muscle, and to promote clinical applications of bFGF to treat skeletal muscle diseases correlated to muscular dystrophy or AIDS and so on, recombinant human bFGF (rh-bFGF) was added into media and stimulated murine C2C12 myoblasts to investigate the dose-dependent effect ofbFGF on suppression of myostatin gene expression and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulatory mechanism. Simultaneously, complete coding sequence of ovine 18 kDa-bFGF gene was inserted into eukaryotic vector pCMV-neo (originated from pEGFP-N1 vector, from which the EGFP gene has been removed), the recombinant plasmid pCMV-neo-bFGF was harvested and injected into the mouse skeletal muscle of posterior limb. Expression levels of bFGF,myostatin, and ERKI/2 genes in murine C2C12 myoblasts and the skeletal muscle were analyzed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. The results showed that bFGFimpaired the expression ofmyostatin gene in a dose-dependent manner in C2C12 cells, with increasing concentration of rh-bFGF,myostatin mRNA declined gradually. In addition, results in skeletal muscle indicated that bFGF also suppressed the expression of the myostatin gene in vivo. Furthermore, we found ERKI/2 participated in the regulatory mechanism of bFGF on the expression of the myostatin gene.

  14. Identification of retinoic acid in a high content screen for agents that overcome the anti-myogenic effect of TGF-beta-1.

    Directory of Open Access Journals (Sweden)

    Chateen Krueger

    Full Text Available BACKGROUND: Transforming growth factor beta 1 (TGF-β1 is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1. METHODOLOGY/PRINCIPAL FINDINGS: A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be

  15. The omega-3 fatty acid, eicosapentaenoic acid (EPA, prevents the damaging effects of tumour necrosis factor (TNF-alpha during murine skeletal muscle cell differentiation

    Directory of Open Access Journals (Sweden)

    Pearson Stephen

    2008-07-01

    Full Text Available Abstract Background Eicosapentaenoic acid (EPA is a ώ-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α. Results The deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (p p p p p p Conclusion In conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury.

  16. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells

    Science.gov (United States)

    Baharara, Javad; Ramezani, Tayebe; Divsalar, Adeleh; Mousavi, Marzieh; Seyedarabi, Arefeh

    2016-01-01

    Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose-dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. PMID:27141266

  17. Stem Cells

    Science.gov (United States)

    Stem cells are cells with the potential to develop into many different types of cells in the body. They serve as a repair ... body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  18. Highly fluorescent semiconductor core-shell CdTe-CdS nanocrystals for monitoring living yeast cells activity

    Energy Technology Data Exchange (ETDEWEB)

    Farias, P.M.A. de; Motta, M.A.; Castro-Neto, A.G.; Vieira, A.A.S.; Silva, D.C.N.; Fontes, A. [Universidade Federal de Pernambuco, Departamento de Biofisica e Radiobiologia, Recife, PE (Brazil); Santos, B.S.; Brasil, A.G. Jr. [Universidade Federal de Pernambuco, Departamento de Ciencias Farmaceuticas, Recife, PE (Brazil); Menezes, F.D.; Ferreira, R. [Universidade Federal de Pernambuco, Departamento de Quimica Fundamental, Recife, PE (Brazil); Cesar, C.L. [Universidade Estadual de Campinas, Departamento de Eletronica Quantica, Campinas, SP (Brazil)

    2007-12-15

    Fluorescent semiconductor nanocrystals in quantum confinement regime (quantum dots) present several well-known features which make them very useful tools for biological labeling purposes. Low photobleaching rates, high chemical stability and active surface allowing conjugation to living cells explain the success of this labeling procedure over the commonly used fluorescent dyes. In this paper we report the results obtained with highly fluorescent core-shell CdTe-CdS (diameter=3-7 nm) colloidal nanocrystals synthesized in aqueous medium and conjugated to glucose molecules. The conjugated nanocrystals were incubated with living yeast cells, in order to investigate their glucose up-take activity in real time, by confocal microscopy analysis. (orig.)

  19. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R;

    2000-01-01

    Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins...... blocking antibodies for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers....

  20. Poly(ester amine) Composed of Polyethylenimine and Pluronic Enhance Delivery of Antisense Oligonucleotides In Vitro and in Dystrophic mdx Mice

    OpenAIRE

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Bollinger, Lauren E; Lu, Peijuan; Lu, Qilong

    2016-01-01

    A series of poly(esteramine)s (PEAs) constructed from low molecular weight polyethyleneimine (LPEI) and Pluronic were evaluated for the delivery of antisense oligonuclotides (AOs), 2′-O-methyl phosphorothioate RNA (2′-OMePS) and phosphorodiamidate morpholino oligomer (PMO) in cell culture and dystrophic mdx mice. Improved exon-skipping efficiency of both 2′-OMePS and PMO was observed in the C2C12E50 cell line with all PEA polymers compared with PEI 25k or LF-2k. The degree of efficiency was f...

  1. METTL21C is a potential pleiotropic gene for osteoporosis and sarcopenia acting through the modulation of the NFκB signaling pathway

    OpenAIRE

    Huang, Jian; Hsu, Yi-Hsiang; Mo, Chenglin; Abreu, Eduardo; Kiel, Douglas P; Bonewald, Lynda F.; Brotto, Maxrco; Karasik, David

    2014-01-01

    Sarcopenia and osteoporosis are important public health problems that occur concurrently. A bivariate genome-wide association study (GWAS) identified METTL21c as a suggestive pleiotropic gene for both bone and muscle. METTL21 family of proteins methylates chaperones involved in the etiology of both Inclusion Body Myositis with Paget's disease. To validate these GWAS results, Mettl21c mRNA expression was reduced with siRNA in a mouse myogenic C2C12 cell line and the mouse osteocyte-like cell l...

  2. Biocompatibility of fluorescent nanoparticles NaYF4:Yb,Er as imaging media%荧光纳米颗粒NaYF4:Yb,Er作为显像介质的生物相容性

    Institute of Scientific and Technical Information of China (English)

    虞永江; 马晓荣; 于国鹏; 高同斌; 齐隽; 陈方

    2011-01-01

    Objective To investigate the biocompatibility of upconversion fluorescent nanoparticles in vivo and in vitro, and verify its safety as imaging media.Methods Mouse bone mesenchymal stem cells (BMSC), mouse embryonic fibroblasts (NIH/3T3) and primary myoblasts (C2C12) were incubated with different concentrations of NaYF4: Yb, Er (0, 10, 50, 100 and 200 μg/mL).Cell proliferation was determined by MTT assay, and the formation of myotube cells from C2C12 myoblasts was detected.DMEM with NaYF4: Yb, Er nanoparticles were injected into C57BL/6 mice, and liver function and renal function were examined.HE staining was performed for main body organs, and toxicity was detected.Results MTT assay revealed that the cytotoxicity of NaYF4: Yb, Er on NIH/3T3 and C2C12 was positively correlated with incubation dose and time ( NIH/3T3: r =0.974, P <0.05; C2C12: r =0.996, P <0.05), while the same result was not found for BMSC ( r = - 0.218, P > 0.05).The formation of myotube cells from C2C12 myoblasts was not significantly affected by incubation with NaYF4: Yb, Er for 48 h.No obvious damage of liver and renal function and main body organs was observed after injection of DMEM with NaYF4: Yb, Er nanoparticles in mice.Conclusion As biological luminescent labels with strong intensity, NaYF4: Yb, Er has less toxicity both in vivo and in vitro to the requirement of imaging, and is an ideal biological imaging media.%目的 检测上转频荧光纳米颗粒的生物学体内、外相容性,证实其作为显像介质的生物安全性.方法 将培育后的小鼠骨髓间充质干细胞(BMSC)、胚胎成纤维细胞(NIH/3T3)及成肌细胞(C2C12)分别与不同浓度(0、10、50、100、200μg/mL)的NaYF4:Yb,Er共孵育,采用MTT法检测细胞的增殖活性,并测定C2C12成肌细胞形成肌管细胞的功能.将NaYF4:Yb,Er纳米颗粒DMEM混悬液注射入C57BL/6小鼠,行小鼠肝肾功能测定;并对重要脏器行HE组织学染色,检测小鼠的体内毒性.结果 MTT法细

  3. Wdr68 requires nuclear access for craniofacial development.

    Directory of Open Access Journals (Sweden)

    Bingyan Wang

    Full Text Available Wdr68 is a highly conserved scaffolding protein required for craniofacial development and left-right asymmetry. A Ras-Map3k-Wdr68-Dyrk1 signaling relay may mediate these and other diverse signaling events important in development and disease. While the sub-cellular localization of Wdr68 has been shown to be dependent on that of its interaction partners, it is not clear where Wdr68 activity is required during development. Here we show that while a GFP-Wdr68 fusion functionally substituted for craniofacial development in the zebrafish, that a Nuclear Export Signal (NES fusion protein (GFPNESWdr68 failed to support craniofacial development. As control for NES activity, we show that while GFP-Wdr68 exhibited a pan-cellular distribution in C2C12 cells, the GFPNESWdr68 fusion predominantly localized to the cell cytoplasm, as expected. Interestingly, while GFP-Wdr68 and RFP-Dyrk1a co-localized to the cell nucleus as expected based on the known sub-cellular localization for Dyrk1a, we found that the GFPNESWdr68 fusion redistributed RFP-Dyrk1a to the cell cytoplasm potentially disconnecting the Ras/Dyrk1 signal relay from further downstream targets. Consistent with a nuclear role in gene regulation, we also found that while a transcriptional activation domain fusion, CebpFlagWdr68, functionally substituted for endogenous Wdr68 for craniofacial development, that a transcriptional repression domain fusion, MadFlagWdr68, failed to support craniofacial development. Dyrk1b is required for myogenin (myog expression in differentiating mouse C2C12 cells and here we report that wdr68 is also important for myog expression in differentiating C2C12 cells. Using a C2C12 cell myog promoter-reporter system, we found that Wdr68 overexpression increased reporter activity while moderate expression levels of MadFlagWdr68 interfered with reporter activity. Taken together, these findings support a nuclear role for Wdr68-containing complexes.

  4. T Cells

    Science.gov (United States)

    T Cells - National Multiple Sclerosis Society Skip to navigation Skip to content Menu Navigation National Multiple Sclerosis Society Sign ... Is MS? Definition of MS T Cells T Cells Share Smaller Text Larger Text Print In this ...

  5. Cell counting.

    Science.gov (United States)

    Phelan, M C; Lawler, G

    2001-05-01

    This unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live-dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells. PMID:18770655

  6. Growth factor array fabrication using a color ink jet printer.

    Science.gov (United States)

    Watanabe, Kohei; Miyazaki, Takeshi; Matsuda, Ryoichi

    2003-04-01

    We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.

  7. Molecular Mechanisms of Microcystin Toxicity in Animal Cells

    Directory of Open Access Journals (Sweden)

    Alexandre Campos

    2010-01-01

    Full Text Available Microcystins (MC are potent hepatotoxins produced by the cyanobacteria of the genera Planktothrix, Microcystis, Aphanizomenon, Nostoc and Anabaena. These cyclic heptapeptides have strong affinity to serine/threonine protein phosphatases (PPs thereby acting as an inhibitor of this group of enzymes. Through this interaction a cascade of events responsible for the MC cytotoxic and genotoxic effects in animal cells may take place. Moreover MC induces oxidative stress in animal cells and together with the inhibition of PPs, this pathway is considered to be one of the main mechanisms of MC toxicity. In recent years new insights on the key enzymes involved in the signal-transduction and toxicity have been reported demonstrating the complexity of the interaction of these toxins with animal cells. Key proteins involved in MC up-take, biotransformation and excretion have been identified, demonstrating the ability of aquatic animals to metabolize and excrete the toxin. MC have shown to interact with the mitochondria. The consequences are the dysfunction of the organelle, induction of reactive oxygen species (ROS and cell apoptosis. MC activity leads to the differential expression/activity of transcriptional factors and protein kinases involved in the pathways of cellular differentiation, proliferation and tumor promotion activity. This activity may result from the direct inhibition of the protein phosphatases PP1 and PP2A. This review aims to summarize the increasing data regarding the molecular mechanisms of MC toxicity in animal systems, reporting for direct MC interacting proteins and key enzymes in the process of toxicity biotransformation/excretion of these cyclic peptides.

  8. Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

    Science.gov (United States)

    Vaes, Bart L T; Lute, Carolien; van der Woning, Sebastian P; Piek, Ester; Vermeer, Jenny; Blom, Henk J; Mathers, John C; Müller, Michael; de Groot, Lisette C P G M; Steegenga, Wilma T

    2010-02-01

    S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

  9. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  10. Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis

    Science.gov (United States)

    Yasa, I. Ceren; Gunduz, Nuray; Kilinc, Murat; Guler, Mustafa O.; Tekinay, Ayse B.

    2015-11-01

    Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.

  11. Galvanic Cells

    Science.gov (United States)

    Young, I. G.

    1973-01-01

    Many standard physical chemistry textbooks contain ambiguities which lead to confusion about standard electrode potentials, calculating cell voltages, and writing reactions for galvanic cells. This article shows how standard electrode potentials can be used to calculate cell voltages and deduce cell reactions. (Author/RH)

  12. Cell Biochips

    Science.gov (United States)

    Pioufle, B. Le; Picollet-D'Hahan, N.

    A cell biochip is a microsystem, equipped with electronic and microfluidic functions, designed to manipulate or analyse living cells. The first publications in this emerging area of research appeared toward the end of the 1980s. In 1989 Washizu described a biochip designed to fuse two cells by electropermeabilisation of the cytoplasmic membrane [1]. Research centers have devised a whole range of cell chip structures, for simultaneous or sequential analysis of single cells, cell groups, or cell tissues reconstituted on the chip. The cells are arranged in a square array on a parallel cell chip for parallel analysis, while they are examined and processed one by one in a microchannel in the case of a series cell chip. In contrast to these biochips for high-throughput analysis of a large number of cells, single-cell chips focus on the analysis of a single isolated cell. As in DNA microarrays, where a large number of oligonucleotides are ordered in a matrix array, parallel cell chips order living cells in a similar way. At each point of the array, the cells can be isolated, provided that the cell type allows this, e.g., blood cells, or cultivated in groups (most adhesion cells can only survive in groups). The aim is to allow massively parallel analysis or processing. Le Pioufle et al. describe a microdevice for the culture of single cells or small groups of cells in a micropit array [2]. Each pit is equipped to stimulate the cell or group of cells either electrically or fluidically. Among the applications envisaged are gene transfer, cell sorting, and screening in pharmacology. A complementary approach, combining the DNA microarray and cell biochip ideas, has been put forward by Bailey et al. [3]. Genes previously arrayed on the chip transfect the cultured cells on the substrate depending on their position in the array (see Fig. 19.1). This way of achieving differential lipofection on a chip was then taken up again by Yoshikawa et al. [4] with primary cells, more

  13. Functional and Activity Analysis of Cattle UCP3 Promoter with MRFs-Related Factors

    OpenAIRE

    Wei Chen; Houqiang Xu; Xiang Chen; Zhongwei Liu; Wen Zhang; Dan Xia

    2016-01-01

    Uncoupling protein 3 (UCP3) is mainly expressed in muscle. It plays an important role in muscle, but less research on the regulation of cattle UCP3 has been performed. In order to elucidate whether cattle UCP3 can be regulated by muscle-related factors, deletion of cattle UCP3 promoter was amplified and cloned into pGL3-basic, pGL3-promoter and PEGFP-N3 vector, respectively, then transfected into C2C12 myoblasts cells and UCP3 promoter activity was measured using the dual-Luciferase reporter ...

  14. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    OpenAIRE

    Huang, Yong-Zhen; Liang-zhi ZHANG; Lai, Xin-Sheng; Li, Ming-xun; Sun, Yu-Jia; Li, Cong-jun; Lan, Xian-yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-01-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter...

  15. Phosphatidic acid enhances mTOR signaling and resistance exercise induced hypertrophy

    OpenAIRE

    Joy, Jordan M; Gundermann, David M.; Ryan P. Lowery; Jäger, Ralf; McCleary, Sean A; Purpura, Martin; Roberts, Michael D.; Wilson, Stephanie MC; Hornberger, Troy A.; Wilson, Jacob M.

    2014-01-01

    Introduction The lipid messenger phosphatidic acid (PA) plays a critical role in the stimulation of mTOR signaling. However, the mechanism by which PA stimulates mTOR is currently unknown. Therefore, the purpose of this study was to compare the effects of various PA precursors and phospholipids on their ability to stimulate mTOR signaling and its ability to augment resistance training-induced changes in body composition and performance. Methods In phase one, C2C12 myoblasts cells were stimula...

  16. Ethanol Extract of Ganoderma lucidum Augments Cellular Anti-oxidant Defense through Activation of Nrf2/HO-1

    Directory of Open Access Journals (Sweden)

    Yoo-hwan Lee

    2016-03-01

    Full Text Available Objectives: The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL against oxidative stress (hydrogen peroxide, H2O2 and elucidated the underlying mechanisms in a C2C12 myoblast cell line. Methods: Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS generation and deoxyribonucleic acid (DNA damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and H2O2 in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2-specific small interfering ribonucleic acid (siRNA was conducted to understand the relationship between Nrf2 expression and H2O2-induced growth inhibition. Results: The results showed that EGL effectively inhibited H2O2-induced growth and the generation of ROS. EGL markedly suppressed H2O2-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 (p-γH2AX, a widely used marker of DNA damage, suggesting that EGL prevented H2O2-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1, with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against H2O2-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner. Conclusion: Collectively, these results demonstrate that EGL

  17. The construction of three-dimensional composite fibrous macrostructures with nanotextures for biomedical applications.

    Science.gov (United States)

    Song, Juqing; Gao, Huichang; Zhu, Guanglin; Cao, Xiaodong; Shi, Xuetao; Wang, Yingjun

    2016-01-01

    The development of modern biomedical nanotechnology requires three-dimensional macrostructures with nanotextures to meet the requirements for practical applications in intricate biological systems. Additionally, the restoration and regeneration of some specific body tissues and organs rely on the function of conductive polymers, which can provide electrical cues for cells. In this study, we fabricated three-dimensional composite nanofibre macrostructures of polycaprolactone (PCL) with different concentrations of polyaniline (PANi) by employing an improved electrospinning technology with a specially designed collector. The 3D structures possessed cap-like macrostructures with centimetre-scale thickness and interconnected pore nanotextures with nanometre-scale nanofibres. To estimate the biocompatibility of the 3D PCL/PANi composite nanofibre macrostructures, mouse myoblasts (C2C12 cells) were cultured as model cells. The initial responses of C2C12 cells to the 3D PCL/PANi composite macrostructures were significantly superior to those to pure PCL, that is, the cells exhibited typical myoblast-like morphologies with obvious pseudopodia and the moderate incorporation (less than 2.0 wt%) of conductive PANi facilitated cell proliferation, which indicated that PANi has appreciable cell affinity. Moreover, the addition of conductive PANi to the 3D composite nanofibre macrostructures considerably enhanced myoblast differentiation and myotube maturation. These results suggest that electrospun 3D PCL/PANi composite nanofibre macrostructures would have promising applications in tissue engineering. PMID:27563025

  18. AlphaB-crystallin is involved in oxidative stress protection determined by VEGF in skeletal myoblasts.

    Science.gov (United States)

    Mercatelli, Neri; Dimauro, Ivan; Ciafré, Silvia Anna; Farace, Maria Giulia; Caporossi, Daniela

    2010-08-01

    Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin. PMID:20441791

  19. Cell Wall

    OpenAIRE

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Albenne, Cécile; Pont-Lezica, Rafael F

    2008-01-01

    This chapter covers our present knowledge of cell wall proteomics highlighting the distinctive features of cell walls and cell wall proteins in relation to problems encountered for protein extraction, separation and identification. It provides clues to design strategies for efficient cell wall proteomic studies. It gives an overview of the kinds of proteins that have yet been identified: the expected proteins vs the identified proteins. Finally, the new vision of the cell wall proteome, and t...

  20. Stem Cells

    OpenAIRE

    Madhukar Thakur

    2009-01-01

    Objective: The objective of this presentation is to create awareness of stem cell applications in the ISORBE community and to foster a strategy of how the ISORBE community can disseminate information and promote the use of radiolabeled stem cells in biomedical applications. Methods: The continued excitement in Stem Cells, in many branches of basic and applied biomedical science, stems from the remarkable ability of stem cells to divide and develop into different types of cells in ...

  1. Engineering cell-cell signaling.

    Science.gov (United States)

    Blagovic, Katarina; Gong, Emily S; Milano, Daniel F; Natividad, Robert J; Asthagiri, Anand R

    2013-10-01

    Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling on the basis of quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilize synthetic cells, advanced 'chassis' and predictive modeling to engineer the form and function of living tissues.

  2. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zirong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Jin, Guorong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Lin, Shuibin [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Lin, Xiumei [Department of Hematology, Guangzhou First Municipal People' s Hospital, Guangzhou 510180 (China); Gu, Yumei [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Wu, Lizi [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Shen, Huangxuan, E-mail: shenhx@mail.sysu.edu.cn [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CDA-II inhibits myogenic differentiation in a dose-dependent manner. Black-Right-Pointing-Pointer CDA-II repressed expression of muscle transcription factors and structural proteins. Black-Right-Pointing-Pointer CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  3. Cell Motility

    CERN Document Server

    Lenz, Peter

    2008-01-01

    Cell motility is a fascinating example of cell behavior which is fundamentally important to a number of biological and pathological processes. It is based on a complex self-organized mechano-chemical machine consisting of cytoskeletal filaments and molecular motors. In general, the cytoskeleton is responsible for the movement of the entire cell and for movements within the cell. The main challenge in the field of cell motility is to develop a complete physical description on how and why cells move. For this purpose new ways of modeling the properties of biological cells have to be found. This long term goal can only be achieved if new experimental techniques are developed to extract physical information from these living systems and if theoretical models are found which bridge the gap between molecular and mesoscopic length scales. Cell Motility gives an authoritative overview of the fundamental biological facts, theoretical models, and current experimental developments in this fascinating area.

  4. Prostaglandin A2 enhances cellular insulin sensitivity via a mechanism that involves the orphan nuclear receptor NR4A3.

    Science.gov (United States)

    Zhu, X; Walton, R G; Tian, L; Luo, N; Ho, S-R; Fu, Y; Garvey, W T

    2013-03-01

    We have previously reported that members of the NR4A family of orphan nuclear receptors can augment insulin's ability to stimulate glucose transport in adipocytes. In the current study, we endeavored to test for an insulin-sensitizing effect in muscle cells and to identify a potential transactivator. Lentiviral constructs were used to engineer both hyperexpression and shRNA silencing of NR4A3 in C2C12 myocytes. The NR4A3 hyper-expression construct led to a significant increase in glucose transport rates in the presence of maximal insulin while the NR4A3 knock-down exhibited a significant reduction in insulin-stimulated glucose transport rates. Consistently, insulin-mediated AKT phosphorylation was increased by NR4A3 hyperexpression and decreased following shRNA NR4A3 suppression. Then, we examined effects of prostaglandin A2 (PGA2) on insulin action and NR4A3 transactivation. PGA2 augmented insulin-stimulated glucose uptake in C2C12 myocytes and AKT phosphorylation after 12-h treatment, without significant effects on basal transport or basal AKT phosphorylation. More importantly, we demonstrated that PGA2 led to a greater improvement in insulin-stimulated glucose rates in NR4A3 overexpressing C2C12 myocytes, when compared with Lac-Z controls stimulated with insulin and PGA2. Moreover, the sensitizing effect of PGA2 was significantly diminished in NR4A3 knockdown myocytes compared to scramble controls. These results show for the first time that: (i) PGA2 augments insulin action in myocytes as manifested by enhanced stimulation of glucose transport and AKT phosphorylation; and (ii) the insulin sensitizing effect is dependent upon the orphan nuclear receptor NR4A3. PMID:23104421

  5. Photovoltaic Cells

    Directory of Open Access Journals (Sweden)

    Karolis Kiela

    2012-04-01

    Full Text Available The article deals with an overview of photovoltaic cells that are currently manufactured and those being developed, including one or several p-n junction, organic and dye-sensitized cells using quantum dots. The paper describes the advantages and disadvantages of various photovoltaic cells, identifies the main parameters, explains the main reasons for the losses that may occur in photovoltaic cells and looks at the ways to minimize them.Article in Lithuanian

  6. Solar cells

    Science.gov (United States)

    Cuquel, A.; Roussel, M.

    The physical and electronic characteristics of solar cells are discussed in terms of space applications. The principles underlying the photovoltaic effect are reviewed, including an analytic model for predicting the performance of individual cells and arrays of cells. Attention is given to the effects of electromagnetic and ionizing radiation, micrometeors, thermal and mechanical stresses, pollution and degassing encountered in space. The responses of different types of solar cells to the various performance-degrading agents are examined, with emphasis on techniques for quality assurance in the manufacture and mounting of Si cells.

  7. Engineering Cell-Cell Signaling

    OpenAIRE

    Blagovic, Katarina; Gong, Emily S.; Milano, Daniel F.; Natividad, Robert J.; Asthagiri, Anand R

    2013-01-01

    Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cel...

  8. An in vitro assessment of the effect of Athrixia phylicoides DC. aqueous extract on glucose metabolism.

    Science.gov (United States)

    Chellan, N; Muller, C J F; de Beer, D; Joubert, E; Page, B J; Louw, J

    2012-06-15

    Athrixia phylicoides DC. is an aromatic shrub indigenous to the eastern parts of Southern Africa. Indigenous communities brew "bush tea" from dried twigs and leaves of A. phylicoides, which is consumed as a beverage and used for its medicinal properties. Plant polyphenols have been shown to be beneficial to Type 2 diabetes mellitus (T2D) and obesity. Aqueous extracts of the plant have been shown to be rich in polyphenols, in particular phenolic acids, which may enhance glucose uptake and metabolism. The aim of this study was to determine the phenolic composition of a hot water A. phylicoides extract and assess its in vitro effect on cellular glucose utilisation. The most abundant phenolic compounds in the extract were 6-hydroxyluteolin-7-O-glucoside, chlorogenic acid, protocatechuic acid, a di-caffeoylquinic acid and a methoxy-flavonol derivative. The extract increased glucose uptake in C2C12, Chang and 3T3-L1 cells, respectively. Intracellular glucose was utilised by both oxidation (C2C12 myocytes and Chang cells; p < 0.01 and p < 0.05, respectively) and by increased glycogen storage (Chang cells; p < 0.05). No cytotoxicity was observed in Chang cells at the concentrations tested. The effects of the extract were not dose-dependent. A. phylicoides aqueous extract stimulated in vitro glucose uptake and metabolism, suggesting that consumption of this phenolic-rich extract could potentially ameliorate metabolic disorders related to obesity and T2D. PMID:22516895

  9. Glucose-induced Ca2 + signals in rat pancreatic β cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using microfluorometry to assay intracellular Ca2+ , the influences of varied factors on glucose induced Ca22+ signals, such as glucose-induced initial decline phase (GIDP), Ca2+ oscillation, and Ca2+ release from internal stores, were investigated in single rat pancreatic β cells. Glucose was able to evoke GIDP even at non-stimulus concentration (5 mol/L), which is insufficient to induce Ca2+ spikes. GIDP was dependent on neither membrane depo larization nor extraeellular Ca2+ . However, GIDP was inhibited by thapsigargin, indicating a dependence on Ca2+ up take by Ca22+ stores. The glucose-induced calcium oscillation was inhibited when external Ca2+ was removed. However, thapsigargin could not block the Ca2+ oscillation. These results suggest that maintenance of Ca22+ oscillation requires ex tracellular Ca2+ but not Ca2+ stores. Glucose was able to evoke Ca2+ signals even in the absence of external Ca2+ . The glucose-induced Ca2+ release from intracellular Ca2+ stores was blocked by TTX. However, TTX had no effect on high K--induced Ca2+ store release, suggesting that membrane depolarization can directly release Ca2+ from some internal Ca2+ stores in β cells.

  10. Stem Cells

    Directory of Open Access Journals (Sweden)

    Madhukar Thakur

    2015-02-01

    Full Text Available Objective: The objective of this presentation is to create awareness of stem cell applications in the ISORBE community and to foster a strategy of how the ISORBE community can disseminate information and promote the use of radiolabeled stem cells in biomedical applications. Methods: The continued excitement in Stem Cells, in many branches of basic and applied biomedical science, stems from the remarkable ability of stem cells to divide and develop into different types of cells in the body. Often called as Magic Seeds, stem cells are produced in bone marrow and circulate in blood, albeit at a relatively low concentration. These virtues together with the ability of stem cells to grow in tissue culture have paved the way for their applications to generate new and healthy tissues and to replace diseased or injured human organs. Although possibilities of stem cell applications are many, much remains yet to be understood of these remarkable magic seeds. Conclusion: This presentation shall briefly cover the origin of stem cells, the pros and cons of their growth and division, their potential application, and shall outline some examples of the contributions of radiolabeled stem cells, in this rapidly growing branch of biomedical science

  11. Types of Stem Cells

    Science.gov (United States)

    ... PDF) Download an introduction to stem cells and stem cell research. Stem Cell Glossary Stem cell terms to know. ... stem cells blog from the International Society for Stem Cell Research. Learn About Stem Cells From Lab to You ...

  12. Spatial Geometries of Self-Assembled Chitohexaose Monolayers Regulate Myoblast Fusion

    Directory of Open Access Journals (Sweden)

    Pornthida Poosala

    2016-05-01

    Full Text Available Myoblast fusion into functionally-distinct myotubes to form in vitro skeletal muscle constructs under differentiation serum-free conditions still remains a challenge. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexa-N-acetyl-d-glucosamine (GlcNAc6 modulated the efficiency of myoblast fusion without requiring horse serum or any differentiation medium during cell culture. Promotion of the differentiation of dissociated mononucleated skeletal myoblasts (C2C12; a mouse myoblast cell line into robust myotubes was found only on GlcNAc6 micropatterns, whereas the myoblasts on control, non-patterned GlcNAc6 substrates or GlcNAc6-free patterns exhibited an undifferentiated form. We also examined the possible role of GlcNAc6 micropatterns with various widths in the behavior of C2C12 cells in early and late stages of myogenesis through mRNA expression of myosin heavy chain (MyHC isoforms. The spontaneous contraction of myotubes was investigated via the regulation of glucose transporter type 4 (GLUT4, which is involved in stimulating glucose uptake during cellular contraction. Narrow patterns demonstrated enhanced glucose uptake rate and generated a fast-twitch muscle fiber type, whereas the slow-twitch muscle fiber type was dominant on wider patterns. Our findings indicated that GlcNAc6-mediated integrin interactions are responsible for guiding myoblast fusion forward along with myotube formation.

  13. Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

    Science.gov (United States)

    Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

    2010-10-15

    Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

  14. Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle.

    Science.gov (United States)

    Duffy, Rebecca M; Sun, Yan; Feinberg, Adam W

    2016-06-01

    Skeletal muscle lost through trauma or disease has proven difficult to regenerate due to the challenge of differentiating human myoblasts into aligned, contractile tissue. To address this, we investigated microenvironmental cues that drive myoblast differentiation into aligned myotubes for potential applications in skeletal muscle repair, organ-on-chip disease models and actuators for soft robotics. We used a 2D in vitro system to systematically evaluate the role of extracellular matrix (ECM) protein composition and geometric patterning for controlling the formation of highly aligned myotubes. Specifically, we analyzed myotubes differentiated from murine C2C12 cells and human skeletal muscle derived cells (SkMDCs) on micropatterned lines of laminin compared to fibronectin, collagen type I, and collagen type IV. Results showed that laminin supported significantly greater myotube formation from both cells types, resulting in greater than twofold increase in myotube area on these surfaces compared to the other ECM proteins. Species specific differences revealed that human SkMDCs uniaxially aligned over a wide range of micropatterned line dimensions, while C2C12s required specific line widths and spacings to do the same. Future work will incorporate these results to engineer aligned human skeletal muscle tissue in 2D for in vitro applications in disease modeling, drug discovery and toxicity screening. PMID:26983843

  15. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Institute of Scientific and Technical Information of China (English)

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

  16. Promoting osteoblast differentiation by the flavanes from Huangshan Maofeng tea is linked to a reduction of oxidative stress.

    Science.gov (United States)

    Zeng, Xiaobin; Tian, Jun; Cai, Kangyong; Wu, Xin; Wang, Yang; Zheng, Yayuan; Su, Yanjie; Cui, Liao

    2014-02-15

    Epidemiological evidence has shown an association between tea consumption and the prevention of bone loss in the elderly. Previous studies indicated that green tea exerted osteoprotective effect in vivo. This study aims to investigate the constituents in Huangshan Maofeng tea and systemically evaluate their antioxidative and osteogenic effects in vitro. Five flavanes, isolated from Huangshan Maofeng tea, showed effects on proliferation of osteoblastic cells and ameliorated H2O2-induced C2C12 mouse myoblast cell apoptosis at 3.125-50 μg/ml. (-)-Epicatechin observably increased alkaline phosphatase (ALP) activity and hydroxyproline content. (-)-Epiafzelechin at 25 μg/ml significantly increased the area of mineralized bone nodules. The activities of flavanes in promoting osteblastic proliferation and differentiation are positively correlated with activities in protecting against apoptosis in C2C12 cells. It indicates that anti-osteoporosis effect of these flavanes may be linked to their antioxidative activity. The observed effects of these flavanes suggest that these flavanes may have beneficial effects on bone health.

  17. Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

    International Nuclear Information System (INIS)

    Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants - Highlights: • Demineralized bone matrix (DBM) was gamma-irradiated for sterilization. • Irradiated DBM had higher alkaline phosphatase and osteocalcin production. • It was reasoned the more released bone morphogenetic proteins by irradiation. • This result supports the application of radiation sterilization for bone implants

  18. Fuel Cells

    DEFF Research Database (Denmark)

    Smith, Anders; Pedersen, Allan Schrøder

    2014-01-01

    Fuel cells have been the subject of intense research and development efforts for the past decades. Even so, the technology has not had its commercial breakthrough yet. This entry gives an overview of the technological challenges and status of fuel cells and discusses the most promising applications...... of the different types of fuel cells. Finally, their role in a future energy supply with a large share of fluctuating sustainable power sources, e.g., solar or wind, is surveyed....

  19. Cell suicide

    International Nuclear Information System (INIS)

    In the fight of the cell against the damages caused to its DNA by genotoxic agents and specially by ionizing radiations, the p53 protein plays a central part. It intervenes in the proliferation control and the differentiation but also in the keeping of genome integrity. It can direct the damages cells toward suicide, or apoptosis, to avoid the risk of tumor appearance that would be fatal to the whole organism. That is by the disordered state of cells suicide programs that the tumor cells are going to develop. The knowledge of apoptosis mechanisms, to eventually start them on demand, rises up broad hopes in the cancer therapy. (N.C.)

  20. Higher prevalence of sexual transmitted diseases and correlates of genital warts among heterosexual males attending sexually transmitted infection clinics (MSCs in Jiangmen, China: implication for the up-taking of STD related service.

    Directory of Open Access Journals (Sweden)

    Shujie Huang

    Full Text Available Increasing burden of STDs is one of China's major public health concerns. However, only a limited number of studies have ever investigated the prevalence of these STDs, particular for genital warts and its correlates among heterosexual males attending STD clinics in China. In order to fill this gap, we conducted a cross-sectional study among MSCs in Jiangmen, China, between the years of 2009 and 2010.The eligible participants were recruited from several STD-clinics in public hospitals. We collected demographic information and behaviors of the participants. After HIV and syphilis testing, we further checked whether the participants had genital warts and genital herpes. In addition, urine samples were collected from part of the participants for CT and NG testing.Of the 533 eligible participants, over three-fifths were aged 35 or below, nearly three quarters had no college degree, over three-fifths were residence of Jiangmen. The prevalence of HIV, syphilis, genital warts, genital herpes, CT and NG were 0.19%, 7.50%, 7.32%, 5.25%, 9.73% and 6.19%, respectively. Living with family members (versus living alone, no STD-related service in past year, experiencing STDs related symptoms in past year, and sex with FSWs in last three months were positively associated with genital warts, with adjusted ORs of 5.54 (95% CI 1.94-15.81, 2.26 (95% CI 1.08-4.74, 1.99 (95% CI 1.00-3.99 and 2.01 (95% CI 1.00-4.04, respectively.Our study indicates that the prevalence of STDs among MSCs in Jiangmen was high, which may further spread HIV among MSCs. Targeted interventions that focused on STDs related services uptake should be implemented urgently.

  1. Reprogrammed Pluripotent Stem Cells from Somatic Cells

    OpenAIRE

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-01-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-li...

  2. Fuel Cells

    Science.gov (United States)

    Hawkins, M. D.

    1973-01-01

    Discusses the theories, construction, operation, types, and advantages of fuel cells developed by the American space programs. Indicates that the cell is an ideal small-scale power source characterized by its compactness, high efficiency, reliability, and freedom from polluting fumes. (CC)

  3. Syringaresinol induces mitochondrial biogenesis through activation of PPARβ pathway in skeletal muscle cells.

    Science.gov (United States)

    Thach, Trung Thanh; Lee, Chan-Kyu; Park, Hyun Woo; Lee, Sang-Jun; Lee, Sung-Joon

    2016-08-15

    Activation of peroxisome proliferator-activated receptors (PPARs) plays a crucial role in cellular energy metabolism that directly impacts mitochondrial biogenesis. In this study, we demonstrate that syringaresinol, a pharmacological lignan extracted from Panax ginseng berry, moderately binds to and activates PPARβ with KD and EC50 values of 27.62±15.76μM and 18.11±4.77μM, respectively. Subsequently, the expression of peroxisome proliferator-activated receptor γ coactivator-1α together with PPARβ transcriptional targets, mitochondrial carnitine palmitoyltransferase 1 and uncoupling protein 2, was also enhanced in terms of both mRNA and protein levels. The activation of these proteins induced mitochondrial biogenesis by enrichment of mitochondrial replication and density within C2C12 myotubes. Importantly, knockdown of PPARβ reduced the syringaresinol-induced protein expression followed by the significant reduction of mitochondrial biogenesis. Taken together, our results indicate that syringaresinol induces mitochondrial biogenesis by activating PPARβ pathway. PMID:27450788

  4. Nrf2 Protects Against TWEAK-mediated Skeletal Muscle Wasting

    Science.gov (United States)

    Al-Sawaf, Othman; Fragoulis, Athanassios; Rosen, Christian; Kan, Yuet Wai; Sönmez, Tolga Taha; Pufe, Thomas; Wruck, Christoph Jan

    2014-01-01

    Skeletal muscle (SM) regeneration after injury is impaired by excessive inflammation. Particularly, the inflammatory cytokine tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a potent inducer of skeletal muscle wasting and fibrosis. In this study we investigated the role of Nrf2, a major regulator of oxidative stress defence, in SM ischemia/reperfusion (I/R) injury and TWEAK induced atrophy. We explored the time-dependent expression of TWEAK after I/R in SM of Nrf2-wildtype (WT) and knockout (KO) mice. Nrf2-KO mice expressed significant higher levels of TWEAK as compared to WT mice. Consequently, Nrf2-KO mice present an insufficient regeneration as compared to Nrf2-WT mice. Moreover, TWEAK stimulation activates Nrf2 in the mouse myoblast cell line C2C12. This Nrf2 activation inhibits TWEAK induced atrophy in C2C12 differentiated myotubes. In summary, we show that Nrf2 protects SM from TWEAK-induced cell death in vitro and that Nrf2-deficient mice therefore have poorer muscle regeneration.

  5. Lamin A/C mutants disturb sumo1 localization and sumoylation in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Émilie Boudreau

    Full Text Available A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna(H222P/H222P mouse model. In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.

  6. Virulence diversity among bacteremic Aeromonas isolates: ex vivo, animal, and clinical evidences.

    Directory of Open Access Journals (Sweden)

    Po-Lin Chen

    Full Text Available BACKGROUND: The objective of this study was to compare virulence among different Aeromonas species causing bloodstream infections. METHODOLOGY/PRINCIPAL FINDINGS: Nine of four species of Aeromonas blood isolates, including A. dhakensis, A. hydrophila, A. veronii and A. caviae were randomly selected for analysis. The species was identified by the DNA sequence matching of rpoD. Clinically, the patients with A. dhakensis bacteremia had a higher sepsis-related mortality rate than those with other species (37.5% vs. 0%, P = 0.028. Virulence of different Aeromonas species were tested in C. elegans, mouse fibroblast C2C12 cell line and BALB/c mice models. C. elegans fed with A. dhakensis and A. caviae had the lowest and highest survival rates compared with other species, respectively (all P values <0.0001. A. dhakensis isolates also exhibited more cytotoxicity in C2C12 cell line (all P values <0.0001. Fourteen-day survival rate of mice intramuscularly inoculated with A. dhakensis was lower than that of other species (all P values <0.0001. Hemolytic activity and several virulence factor genes were rarely detected in the A. caviae isolates. CONCLUSIONS/SIGNIFICANCE: Clinical data, ex vivo experiments, and animal studies suggest there is virulence variation among clinically important Aeromonas species.

  7. Long-Term Consumption of Platycodi Radix Ameliorates Obesity and Insulin Resistance via the Activation of AMPK Pathways

    Directory of Open Access Journals (Sweden)

    Chae Eun Lee

    2012-01-01

    Full Text Available This study was designed to evaluate the effects and mechanism of Platycodi radix, having white balloon flower (Platycodon grandiflorum for. albiflorum (Honda H. Hara on obesity and insulin resistance. The extracts of Platycodi radix with white balloon flower were tested in cultured cells and administered into mice on a high-fat diet. The Platycodi radix activated the AMPK/ACC phosphorylation in C2C12 myotubes and also suppressed adipocyte differentiation in 3T3-L1 cells. In experimental animal, it suppressed the weight gain of obese mice and ameliorated obesity-induced insulin resistance. It also reduced the elevated circulating mediators, including triglyceride (TG, T-CHO, leptin, resistin, and monocyte chemotactic protein (MCP-1 in obesity. As shown in C2C12 myotubes, the administration of Platycodi radix extracts also recovered the AMPK/ACC phosphorylation in the muscle of obese mice. These results suggest that Platycodi radix with white balloon flower ameliorates obesity and insulin resistance in obese mice via the activation of AMPK/ACC pathways and reductions of adipocyte differentiation.

  8. Myogenic factors that regulate expression of muscle-specific microRNAs.

    Science.gov (United States)

    Rao, Prakash K; Kumar, Roshan M; Farkhondeh, Mina; Baskerville, Scott; Lodish, Harvey F

    2006-06-01

    Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.

  9. [Cell cultures].

    Science.gov (United States)

    Cipro, Simon; Groh, Tomáš

    2014-01-01

    Cell or tissue cultures (both terms are interchangeable) represent a complex process by which eukaryotic cells are maintained in vitro outside their natural environment. They have a broad usage covering not only scientific field but also diagnostic one since they represent the most important way of monoclonal antibodies production which are used for both diagnostic and therapeutic purposes. Cell cultures are also used as a "cultivation medium" in virology and for establishing proliferating cells in cytodiagnostics. They are well-established and easy-to-handle models in the area of research, e.g. as a precious source of nucleic acids or proteins. This paper briefly summarizes their importance and methods as well as the pitfalls of the cultivation and new trends in this field. PMID:24624984

  10. Fuel cells

    Directory of Open Access Journals (Sweden)

    D. N. Srivastava

    1962-05-01

    Full Text Available The current state of development of fuel cells as potential power sources is reviewed. Applications in special fields with particular reference to military requirements are pointed out.

  11. Dry cell battery poisoning

    Science.gov (United States)

    Batteries - dry cell ... Acidic dry cell batteries contain: Manganese dioxide Ammonium chloride Alkaline dry cell batteries contain: Sodium hydroxide Potassium hydroxide Lithium dioxide dry cell batteries ...

  12. Creatine supplementation with methylglyoxal: a potent therapy for cancer in experimental models.

    Science.gov (United States)

    Pal, Aparajita; Roy, Anirban; Ray, Manju

    2016-08-01

    The anti-cancer effect of methylglyoxal (MG) is now well established in the literature. The main aim of this study was to investigate the effect of creatine as a supplement in combination with MG both in vitro and in vivo. In case of the in vitro studies, two different cell lines, namely MCF-7 (human breast cancer cell line) and C2C12 (mouse myoblast cell line) were chosen. MG in combination with creatine showed enhanced apoptosis as well as higher cytotoxicity in the breast cancer MCF-7 cell line, compared to MG alone. Pre-treatment of well-differentiated C2C12 myotubes with cancerogenic 3-methylcholanthrene (3MC) induced a dedifferentiation of these myotubes towards cancerous cells (that mimic the effect of 3MC observed in solid fibro-sarcoma animal models) and subsequent exposure of these induced cancer cells with MG proved to be cytotoxic. Thus, creatine plus ascorbic acid enhanced the anti-cancer effects of MG. In contrast, when normal C2C12 muscle cells or myotubes (mouse normal myoblast cell line) were treated with MG or MG plus creatine and ascorbic acid, no detrimental effects were seen. This indicated that cytotoxic effects of MG are specifically limited towards cancer cells and are further enhanced when MG is used in combination with creatine and ascorbic acid. For the in vivo studies, tumors were induced by injecting Sarcoma-180 cells (2 × 10(6) cells/mouse) in the left hind leg. After 7 days of tumor inoculation, treatments were started with MG (20 mg/kg body wt/day, via the intravenous route), with or without creatine (150 mg/kg body wt/day, fed orally) and ascorbic acid (50 mg/kg body wt/day, fed orally) and continued for 10 consecutive days. Significant regression of tumor size was observed when Sarcoma-180 tumor-bearing mice were treated with MG and even more so with the aforesaid combination. The creatine-supplemented group demonstrated better overall survival in comparison with tumor-bearing mice without creatine. In conclusion, it may be

  13. Cell sorting by deterministic cell rolling

    OpenAIRE

    Choi, Sungyoung; Karp, Jeffrey M.; Karnik, Rohit

    2011-01-01

    This communication presents the concept of “deterministic cell rolling”, which leverages transient cell-surface molecular interactions that mediate cell rolling to sort cells with high purity and efficiency in a single step.

  14. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

    Directory of Open Access Journals (Sweden)

    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  15. Effect of Oenothera odorata Root Extract on Microgravity and Disuse-Induced Muscle Atrophy

    Directory of Open Access Journals (Sweden)

    Yong-Hyeon Lee

    2015-01-01

    Full Text Available Muscle atrophy, a reduction of muscle mass, strength, and volume, results from reduced muscle use and plays a key role in various muscular diseases. In the microgravity environment of space especially, muscle atrophy is induced by muscle inactivity. Exposure to microgravity induces muscle atrophy through several biological effects, including associations with reactive oxygen species (ROS. This study used 3D-clinostat to investigate muscle atrophy caused by oxidative stress in vitro, and sciatic denervation was used to investigate muscle atrophy in vivo. We assessed the effect of Oenothera odorata root extract (EVP on muscle atrophy. EVP helped recover cell viability in C2C12 myoblasts exposed to microgravity for 24 h and delayed muscle atrophy in sciatic denervated mice. However, the expressions of HSP70, SOD1, and ceramide in microgravity-exposed C2C12 myoblasts and in sciatic denervated mice were either decreased or completely inhibited. These results suggested that EVP can be expected to have a positive effect on muscle atrophy by disuse and microgravity. In addition, EVP helped characterize the antioxidant function in muscle atrophy.

  16. Validation of skeletal muscle cis-regulatory module predictions reveals nucleotide composition bias in functional enhancers.

    Directory of Open Access Journals (Sweden)

    Andrew T Kwon

    2011-12-01

    Full Text Available We performed a genome-wide scan for muscle-specific cis-regulatory modules (CRMs using three computational prediction programs. Based on the predictions, 339 candidate CRMs were tested in cell culture with NIH3T3 fibroblasts and C2C12 myoblasts for capacity to direct selective reporter gene expression to differentiated C2C12 myotubes. A subset of 19 CRMs validated as functional in the assay. The rate of predictive success reveals striking limitations of computational regulatory sequence analysis methods for CRM discovery. Motif-based methods performed no better than predictions based only on sequence conservation. Analysis of the properties of the functional sequences relative to inactive sequences identifies nucleotide sequence composition can be an important characteristic to incorporate in future methods for improved predictive specificity. Muscle-related TFBSs predicted within the functional sequences display greater sequence conservation than non-TFBS flanking regions. Comparison with recent MyoD and histone modification ChIP-Seq data supports the validity of the functional regions.

  17. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Cambier, Linda [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France); Pomies, Pascal, E-mail: pascal.pomies@crbm.cnrs.fr [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)

    2011-06-17

    Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  18. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    Science.gov (United States)

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  19. A Novel Synaptobrevin/VAMP Homologous Protein (VAMP5) Is Increased during In Vitro Myogenesis and Present in the Plasma Membrane

    Science.gov (United States)

    Zeng, Qi; Subramaniam, V. Nathan; Wong, Siew Heng; Tang, Bor Luen; Parton, Robert G.; Rea, Shane; James, David E.; Hong, Wanjin

    1998-01-01

    cDNA clones encoding a novel protein (VAMP5) homologous to synaptobrevins/VAMPs are detected during database searches. The predicted 102–amino acid VAMP5 harbors a 23-residue hydrophobic region near the carboxyl terminus and exhibits an overall amino acid identity of 33% with synaptobrevin/VAMP1 and 2 and cellubrevin. Northern blot analysis reveals that the mRNA for VAMP5 is preferentially expressed in the skeletal muscle and heart, whereas significantly lower levels are detected in several other tissues but not in the brain. During in vitro differentiation (myogenesis) of C2C12 myoblasts into myotubes, the mRNA level for VAMP5 is increased ∼8- to 10-fold. Immunoblot analysis using antibodies specific for VAMP5 shows that the protein levels are also elevated ∼6-fold during in vitro myogenesis of C2C12 cells. Indirect immunofluorescence microscopy and immunoelectron microscopy reveal that VAMP5 is associated with the plasma membrane as well as intracellular perinuclear and peripheral vesicular structures of myotubes. Epitope-tagged versions of VAMP5 are similarly targeted to the plasma membrane. PMID:9725904

  20. Electrochemical cell

    Science.gov (United States)

    Nagy, Zoltan; Yonco, Robert M.; You, Hoydoo; Melendres, Carlos A.

    1992-01-01

    An electrochemical cell has a layer-type or sandwich configuration with a Teflon center section that houses working, reference and counter electrodes and defines a relatively narrow electrolyte cavity. The center section is surrounded on both sides with thin Teflon membranes. The membranes are pressed in place by a pair of Teflon inner frames which are in turn supported by a pair of outer metal frames. The pair of inner and outer frames are provided with corresponding, appropriately shaped slits that are in plane generally transverse to the plane of the working electrode and permit X-ray beams to enter and exit the cell through the Teflon membranes that cover the slits so that the interface between the working electrode and the electrolyte within the cell may be analyzed by transmission geometry. In one embodiment, the center section consists of two parts, one on top of the other. Alternatively, the center section of the electrochemical cell may consist of two intersliding pieces or may be made of a single piece of Teflon sheet material. The electrolyte cavity is shaped so that the electrochemical cell can be rotated 90.degree. in either direction while maintaining the working and counter electrodes submerged in the electrolyte.

  1. Solar cells

    Science.gov (United States)

    Treble, F. C.

    1980-11-01

    The history, state of the art, and future prospects of solar cells are reviewed. Solar cells are already competitive in a wide range of low-power applications, and during the 1980's they are expected to become cheaper to run than diesel or gasoline generators, the present mainstay of isolated communities. At this stage they will become attractive for water pumping, irrigation, and rural electrification, particularly in developing countries. With further cost reduction, they may be used to augment grid supplies in domestic, commercial, institutional, and industrial premises. Cost reduction to the stage where photovoltaics becomes economic for large-scale power generation in central stations depends on a technological breakthrough in the development of thin-film cells. DOE aims to reach this goal by 1990, so that by the end of the century about 20% of the estimated annual additions to their electrical generating capacity will be photovoltaic.

  2. Stem Cells

    DEFF Research Database (Denmark)

    Sommerlund, Julie

    2004-01-01

    In his influential essay on markets, An essay on framing and overflowing (1998), Michel Callon writes that `the growing complexity of industrialized societies [is] due in large part to the movements of the technosciences, which are causing connections and interdependencies to proliferate'. This p...... and tantalizing than stem cells, in research, in medicine, or as products.......'. This paper is about tech-noscience, and about the proliferation of connections and interdependencies created by it.More specifically, the paper is about stem cells. Biotechnology in general has the power to capture the imagination. Within the field of biotechnology nothing seems more provocative...

  3. Cell Libraries

    Science.gov (United States)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  4. Solar cells

    International Nuclear Information System (INIS)

    A method of producing solar cells is described which consists of producing a substantially monocrystalline tubular body of silicon or other suitable semiconductor material, treating this body to form an annular rectifying junction and then cutting it longitudinally to form a number of nearly flat ribbons from which the solar cells are fabricated. The P=N rectifying junction produced by the formation of silicon dioxide on the layers at the inner and outer surfaces of the body can be formed by ion-implantation or diffusion. (U.K.)

  5. Learn About Stem Cells

    Science.gov (United States)

    ... PDF) Download an introduction to stem cells and stem cell research. Stem Cell Glossary Stem cell terms to know. ... ISSCR Get Involved Media © 2015 International Society for Stem Cell Research Terms of Use Disclaimer Privacy Policy

  6. Stem Cell Basics

    Science.gov (United States)

    ... Information Stem Cell Basics Stem Cell Basics: Introduction Stem Cell Information General Information Clinical Trials Funding Information Current Research Policy Glossary Site Map Stem Cell Basics Introduction: What are stem cells, and why ...

  7. Stem Cell Information: Glossary

    Science.gov (United States)

    ... Neurons Oligodendrocyte Parthenogenesis Passage Pluripotent Polar body Preimplantation Proliferation Regenerative medicine Reproductive cloning Signals Somatic cell Somatic cell nuclear transfer (SCNT) Somatic (adult) stem cell Stem cells Stromal cells Subculturing Surface markers ...

  8. Photovoltaic cell

    Science.gov (United States)

    Gordon, Roy G.; Kurtz, Sarah

    1984-11-27

    In a photovoltaic cell structure containing a visibly transparent, electrically conductive first layer of metal oxide, and a light-absorbing semiconductive photovoltaic second layer, the improvement comprising a thin layer of transition metal nitride, carbide or boride interposed between said first and second layers.

  9. Fuel cells:

    DEFF Research Database (Denmark)

    Sørensen, Bent

    2013-01-01

    A brief overview of the progress in fuel cell applications and basic technology development is presented, as a backdrop for discussing readiness for penetration into the marketplace as a solution to problems of depletion, safety, climate or environmental impact from currently used fossil...... and nuclear fuel-based energy technologies....

  10. Potent Cells

    Science.gov (United States)

    Liu, Dennis

    2007-01-01

    It seems hard to believe that Dolly the cloned sheep was born 10 years ago, kindling furious arguments over the prospects and ethics of cloning a human. Today, the controversy over cloning is entwined, often confused, with concerns over the use of human embryonic stem cells. Most people are unclear what cloning is, and they know even less when it…

  11. Electrochemical Cell

    DEFF Research Database (Denmark)

    1999-01-01

    The invention relates to a rechargeable electrochemical cell comprising a negative electrode, an electrolyte and a positive electrode in which the positive electrode structure comprises a lithium cobalt manganese oxide of the composition Li¿2?Co¿y?Mn¿2-y?O¿4? where 0

  12. Cell Docking, Movement and Cell-Cell Interactions of Heterogeneous Cell Suspensions in a Cell Manipulation Microdevice

    OpenAIRE

    Long-Sun Huang; Yu-Hung Wang; Yu-Wei Chung; Fei-Lung Lai; Shiaw-Min Hwang

    2011-01-01

    This study demonstrates a novel cell manipulation microdevice for cell docking, culturing, cell-cell contact and interaction by microfluidic manipulation of heterogeneous cell suspensions. Heterogeneous cell suspensions include disparate blood cells of natural killer cells and leukemia cancer cells for immune cell transplantation therapy. However, NK cell alloreactivity from different healthy donors present various recovery response levels. Little is still known about the interactions and cyt...

  13. Isolation and maintenance-free culture of contractile myotubes from Manduca sexta embryos.

    Directory of Open Access Journals (Sweden)

    Amanda L Baryshyan

    Full Text Available Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment

  14. Reprogrammed pluripotent stem cells from somatic cells.

    Science.gov (United States)

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-06-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction. PMID:24298328

  15. Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expression in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A series of adeno-associated viral vectors conraining a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338Awere obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338Awas prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFIXR338A viral stocks on a large scale and higher fiter was established,which can be used for industrial purpose. The titer of rAAV-hFIXR338A was more than 1.25x1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IXknock-out mice were transfected with the rAAV-hFIXR338Ain vitro and in vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng@ (106cells)-1 @ (24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76±64.36) ng@ (106 cells)-1@ (24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFIX.``

  16. Ghost cell lesions

    Directory of Open Access Journals (Sweden)

    E Rajesh

    2015-01-01

    Full Text Available Ghost cells have been a controversy for a long time. Ghost cell is a swollen/enlarged epithelial cell with eosnophilic cytoplasm, but without a nucleus. In routine H and E staining these cells give a shadowy appearance. Hence these cells are also called as shadow cells or translucent cells. The appearance of these cells varies from lesion to lesion involving odontogenic and nonodontogenic lesions. This article review about the origin, nature and significance of ghost cells in different neoplasms.

  17. Experimental investigation of air relative humidity (RH) cycling tests on MEA/cell aging in PEMFC. Pt. II. Study of low RH cycling test with air RH at 62%/0%

    Energy Technology Data Exchange (ETDEWEB)

    Huang, B.T.; Chatillon, Y.; Bonnet, C.; Lapicque, F. [Laboratoire Reactions et Genie des Procedes, CNRS-Nancy University, Nancy (France); Leclerc, S. [Laboratoire d' Energetique et de Mecanique Theorique et Appliquee, CNRS-Nancy University, Vandoeuvre-les-Nancy (France); Hinaje, M.; Rael, S. [Groupe de Recherche en Electrotechnique et Electronique de Nancy, Nancy University, Vandoeuvre-les-Nancy (France)

    2012-06-15

    The effect of low relative humidity (RH) cycling (RH{sub C} 62%/0%) on the degradation mechanisms of a single proton exchange membrane fuel cell (5 x 5 cm{sup 2}) was investigated and compared to a cell operated at constant humidification (RH{sub C} = 62%). The overall cell performance loss was near 33 {mu}V h{sup -1}, which is greater than the voltage decay under constant RH condition near 3 {mu}V h{sup -1}. The electroactive surface was reduced but to an acceptable level. Impedance spectroscopy revealed that the ohmic and charge transfer resistances were reduced by the likely improved hydration of the ionomeric layer at the catalyst due to hydrogen crossover. This was so important that H{sub 2} starvation was finally responsible for the collapse of the cell after 650 h. Transmission electron microscopy showed occurrence of various phenomena, e.g., bubbles and pinholes formation in the membrane due to local overheat from hydrogen combustion at the cathode, and thickness reduction of catalytic layers. The water up take obtained by {sup 1}H NMR within the membrane electrode assembly (MEA) after low RH cycling reduced by 24% compared to a fresh MEA. Observations are also compared to those obtained at high RH cycling (RH{sub C} 62%/100%) presented in Part I of this study [1]. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo.

    Science.gov (United States)

    Knight, Matthew I; Tester, Angus M; McDonagh, Matthew B; Brown, Andrew; Cottrell, Jeremy; Wang, Jianghui; Hobman, Peter; Cocks, Benjamin G

    2014-12-01

    Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.

  19. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    International Nuclear Information System (INIS)

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture

  20. Sickle Cell Information Center

    Science.gov (United States)

    ... Around the Web Google Custom Search – sickle cell Sickle Cell Anemia - In-Depth Report - NY Times Health January 18, 1970 Sickle cell disease (also called sickle cell anemia) is an inherited blood disorder that affects red ...

  1. Electrorefining cell evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Bronson, M.C.; Thomas, R.L. (ed.)

    1989-04-14

    Operational characteristics of the LANL electrorefining cell, a modified LANL electrorefining cell, and an advanced electrorefining cell (known as the CRAC cell) were determined. Average process yields achieved were: 75% for the LANL cell, 82% for the modified LANL cell, and 86% for the CRAC cell. All product metal from the LANL and modified LANL cells was within foundry specifications. Metal from one run in the CRAC cell exceeded foundry specifications for tantalum. The LANL and modified LANL cells were simple in design and operation, but product separation was more labor intensive than with the CRAC cell. The CRAC cell was more complicated in design but remained relatively simple in operation. A decision analysis concluded that the modified LANL cell was the preferred cell. It was recommended that the modified LANL cell be implemented by the Plutonium Recovery Project at Rocky Flats and that development of the CRAC cell continue. 8 refs., 22 figs., 12 tabs.

  2. Preparation, characterization, and cytotoxicity of CPT/Fe2O3-embedded PLGA ultrafine composite fibers: a synergistic approach to develop promising anticancer material

    Directory of Open Access Journals (Sweden)

    Amna T

    2012-03-01

    Full Text Available Touseef Amna1, M Shamshi Hassan2, Ki-Taek Nam2, Yang You Bing3, Nasser AM Barakat2, Myung-Seob Khil2, Hak Yong Kim1,21Center for Healthcare Technology Development, 2Department of Organic Materials and Fiber Engineering, Chonbuk National University, Jeonju, Korea; 3Animal Science and Technology College, Henan University of Science and Technology, Luoyang, ChinaAbstract: The aim of this study was to fabricate camptothecin/iron(III oxide (CPT/Fe2O3-loaded poly(D,L-lactide-co-glycolide (PLGA composite mats to modulate the CPT release and to improve the structural integrity and antitumor activity of the released drug. The CPT/ Fe2O3-loaded PLGA ultrafine fibers were prepared for the first time by electrospinning a composite solution of CPT/Fe2O3 and neat PLGA (4 weight percent. The physicochemical characterization of the electrospun composite mat was carried out by scanning electron microscopy, energy dispersive X-ray spectroscopy, electron probe microanalysis, thermogravimetry, transmission electron microscopy, ultraviolet-visible spectroscopy, and X-ray diffraction pattern. The medicated composite fibers were evaluated for their cytotoxicity on C2C12 cells using Cell Counting Kit-8 assay (Sigma-Aldrich Corporation, St Louis, MO. The in vitro studies indicated a slow and prolonged release over a period of 96 hours with mild initial burst. Scanning electron microscopy, thermogravimetry, and X-ray diffraction studies confirmed the interaction of CPT/Fe2O3 with the PLGA matrix and showed that the crystallinity of CPT decreased after loading. Incorporation of CPT in the polymer media affected both the morphology and the size of the CPT/Fe2O3-loaded PLGA composite fibers. Electron probe microanalysis and energy dispersive X-ray spectroscopy results confirmed well-oriented composite ultrafine fibers with good incorporation of CPT/Fe2O3. The cytotoxicity results illustrate that the pristine PLGA did not exhibit noteworthy cytotoxicity; conversely, the CPT

  3. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  4. What are Stem Cells?

    OpenAIRE

    Ahmadshah Farhat; Ashraf Mohammadzadeh; M. Rezaie

    2014-01-01

      Stem cells are undifferentiated self regenerating multi potential cells. There are three types of stem cells categories by the ability to form after cells and correlated with the body’s development process. Totipotent: these stem cells can form an entire organism such as fertilized egg. Ploripotent: ploripotent cells are those that can form any cell in the body but cannot form an entire organism such as developing embryo’s totipotent cells become ploripotent  Multipotent: Multi potent stem ...

  5. Pluripotent stem cell lines

    OpenAIRE

    Yu, Junying; Thomson, James A.

    2008-01-01

    The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while ma...

  6. Protein kinase D2 is an essential regulator of murine myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Alexander Kleger

    Full Text Available Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC family. The protein kinase D (PKD isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12 as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

  7. DNA-cell conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  8. Non-invasive monitoring of osteogenic differentiation on microtissue arrays under physiological conditions using scanning electrochemical microscopy

    NARCIS (Netherlands)

    Sridhar, Adithya; Berg, van den Albert; Le Gac, Séverine

    2014-01-01

    In this paper, we present a non-invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP-2), C2C12 microtissues differentiate and exp

  9. Molecular Mechanisms of Cell-cell Recognition

    Institute of Scientific and Technical Information of China (English)

    WANG Jia-Huai

    2004-01-01

    Cell-cell recognition is the key for multicellular organisms to survive. This recognition critically depends on protein-protein interactions from opposing cell surfaces. Recent structural investigations reveal unique features of these cell surface receptors and how they interact. These interactions are specific, but usually relatively weak, with more hydrophilic forces involved in binding. The receptors appear to have specialized ways to present their key interacting elements for ligand-binding from the cell surface. Cell-cell contacts are multivalent. A large group of cell surface molecules are engaged in interactions. Characteristic weak interactions make possible for each individual molecule pair within the group to constantly associate-dissociate-reassociate, such that the cell-cell recognition becomes a dynamic process. The immunological synapse is a good example for immune receptors to be orchestrated in performing immunological function in a collective fashion.

  10. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    Science.gov (United States)

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  11. Dynamics of the skeletal muscle secretome during myoblast differentiation

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Rigbolt, Kristoffer T G; Blagoev, Blagoy;

    2010-01-01

    During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative...... proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics...... of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188...

  12. nduced pluripotent stem cells and cell therapy

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2013-12-01

    Full Text Available Human embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo. They hold a huge promise for cell therapy with their self-renewing ability and pluripotency, which is known as the potential to differentiate into all cell types originating from three embryonic germ layers. However, their unique pluripotent feature could not be utilised for therapeutic purposes due to the ethical and legal problems during derivation. Recently, it was shown that the cells from adult tissues could be reverted into embryonic state, thereby restoring their pluripotent feature. This has strenghtened the possiblity of directed differentition of the reprogrammed somatic cells into the desired cell types in vitro and their use in regenerative medicine. Although these cells were termed as induced pluripotent cells, the mechanism of pluripotency has yet to be understood. Still, induced pluripotent stem cell technology is considered to be significant by proposing novel approaches in disease modelling, drug screening and cell therapy. Besides their self-renewing ability and their potential to differentiate into all cell types in a human body, they arouse a great interest in scientific world by being far from the ethical concerns regarding their embryonic counterparts and their unique feature of being patient-specific in prospective cell therapies. In this review, induced pluripotent stem cell technology and its role in cell-based therapies from past to present will be discussed. J Clin Exp Invest 2013; 4 (4: 550-561

  13. Modeling cell-in-cell structure into its biological significance

    OpenAIRE

    He, M-f; S. Wang; Wang, Y.; Wang, X-N.

    2013-01-01

    Although cell-in-cell structure was noted 100 years ago, the molecular mechanisms of ‘entering' and the destination of cell-in-cell remain largely unclear. It takes place among the same type of cells (homotypic cell-in-cell) or different types of cells (heterotypic cell-in-cell). Cell-in-cell formation affects both effector cells and their host cells in multiple aspects, while cell-in-cell death is under more intensive investigation. Given that cell-in-cell has an important role in maintainin...

  14. Monitoring cell growth.

    Science.gov (United States)

    Strober, W

    2001-05-01

    This appendix provides two protocols for monitoring cell growth. Counting cells using a hemacytometer is tedious but it allows one to effectively distinguish live cells from dead cells (using Trypan Blue exclusion). In addition, this procedure is less subject to errors due to cell clumping or heterogeneity of cell size. The use of an electronic cell counter is quicker and easier than counting cells using a hemacytometer. However, an electronic cell counter as currently constructed does not distinguish live from dead cells in a reliable fashion and is subject to error due to the presence of cell clumps. Overall, the electronic cell counter is best reserved for repetitive and rapid counting of fresh peripheral blood cells and should be used with caution when counting cell populations derived from tissues. PMID:18432653

  15. Automated Cell-Cutting for Cell Cloning

    Science.gov (United States)

    Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

    We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

  16. Skeletal muscle secreted factors prevent glucocorticoid-induced osteocyte apoptosis through activation of β-catenin.

    Science.gov (United States)

    Jähn, K; Lara-Castillo, N; Brotto, L; Mo, C L; Johnson, M L; Brotto, M; Bonewald, L F

    2012-01-01

    It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability. PMID:22972510

  17. Skeletal muscle secreted factors prevent glucocorticoid-induced osteocyte apoptosis through activation of β-catenin

    Directory of Open Access Journals (Sweden)

    K Jähn

    2012-09-01

    Full Text Available It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex, a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s appears to protect osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s that preserve osteocyte viability.

  18. Sickle Cell Anemia

    Science.gov (United States)

    Sickle cell anemia is a disease in which your body produces abnormally shaped red blood cells. The cells are shaped like a crescent or sickle. They ... last as long as normal, round red blood cells. This leads to anemia. The sickle cells also ...

  19. Efficacy of supermacroporous poly(ethylene glycol)–gelatin cryogel matrix for soft tissue engineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Archana [Department of Biological Sciences, Birla Institute of Technology and Science, Pilani-K.K Birla Goa Campus, 403726 Goa (India); Bhat, Sumrita [Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, 208016 UP (India); Nayak, Vijayashree, E-mail: vijayashree@goa.bits-pilani.ac.in [Department of Biological Sciences, Birla Institute of Technology and Science, Pilani-K.K Birla Goa Campus, 403726 Goa (India); Kumar, Ashok, E-mail: ashokkum@iitk.ac.in [Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, 208016 UP (India)

    2015-02-01

    Three dimensional scaffolds synthesized using natural or synthetic polymers act as an artificial niche for cell adherence and proliferation. In this study, we have fabricated cryogels employing blend of poly (ethylene glycol) (PEG) and gelatin using two different crosslinkers like, glutaraldehyde and EDC-NHS by cryogelation technique. Synthesized matrices possessed interconnected porous structure in the range of 60–100 μm diameter and regained their original length after 90% compression without deformation. Visco-elastic behavior was studied by rheology and unconfined compression analysis, elastic modulus of these cryogels was observed to be > 10{sup 5} Pa which showed their elasticity and mechanical strength. TGA and DSC also showed the stability of these cryogels at different temperatures. In vitro degradation capacity was analyzed for 4 weeks at 37 °C. IMR-32, C2C12 and Cos-7 cells proliferation and ECM secretion on PEG–gelatin cryogels were observed by SEM and fluorescent analysis. In vitro biocompatibility was analyzed by MTT assay for the period of 15 days. Furthermore, cell proliferation efficiency, metabolic activity and functionality of IMR-32 cells were analyzed by neurotransmitter assay and DNA quantification. The cell–matrix interaction, elasticity, mechanical strength, stability at different temperatures, biocompatible, degradable nature showed the potentiality of these cryogels towards soft tissue engineering such as neural, cardiac and skin. - Highlights: • PEG–gelatin cryogel matrices were produced by cryogelation technology. • Matrices showed suitable properties for tissue engineering applications. • Polymeric cryogels supported growth of IMR-32, C2C12 and Cos-7 cells in vitro.

  20. Identification of intracellular signaling pathways that induce myotonic dystrophy protein kinase expression during myogenesis.

    Science.gov (United States)

    Carrasco, Marta; Canicio, Judith; Palacín, Manuel; Zorzano, Antonio; Kaliman, Perla

    2002-08-01

    Myotonic dystrophy (DM) is the most common inherited adult neuromuscular disorder. DM is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Decreased DMPK protein levels may contribute to the pathology of DM, as revealed by gene target studies. However, the postnatal regulation of DMPK expression and its pathophysiological role remain undefined. We studied the regulation of DMPK protein and mRNA expression during myogenesis in rat L6E9 myoblasts, mouse C2C12 myoblasts, and 10T1/2 fibroblasts stably expressing the myogenic transcription factor MyoD (10T1/2-MyoD). We detected DMPK as an 80-kDa protein mainly localized to the cytosolic fraction of skeletal muscle cells. DMPK expression and protein kinase activity were enhanced in IGF-II-differentiated cells. In L6E9 and C2C12 cells, DMPK expression was regulated through the same signaling pathways (i.e. phosphatidylinositol 3-kinase, nuclear factor-kappaB, nitric oxide synthase, and p38 mitogen-activated protein kinase) that had been described as being crucial for the myogenesis induced by either low serum or IGF-II. However, in 10T1/2-MyoD cells, p38 MAPK inhibition blocked cell fusion and caveolin-3 expression without affecting DMPK up-regulation. These results suggest that although DMPK is induced during myogenesis, its expression cannot be totally associated with the development of a fully differentiated phenotype. PMID:12130568

  1. CELL RESEARCH

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    REVIEWSInducible resistance to Fas-mediated apoptosis in B cells…………………………………ROTHSTEIN Thomas L (245)Executionary pathway for apoptosis: lessons from mutant mice………………………………………WOO Minna, Razqallah Hakem, Tak W Mak (267)The SHP-2 tyrosine phosphatase: Signaling mechanisms and biological functions…………………………………QU Cheng Kui (279)REGULAR ARTICLESTemperature dependent expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis…………………………KONG Wei Hua, Zheng GU, Jining LU, Jiake TSO (289)Transgenic mice overexpressing γ-aminobutyric acid transporter subtype I develop obesity…………………………………MA Ying Hua, Jia Hua HU, Xiao Gang ZHOU, Ruo Wang ZENG, Zhen Tong MEI, Jian FEI, Li He GUO (303)Genetic aberration in primary hepatocellular carcinoma: correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21-q23 and 9p21-p23………………………………………WANG Gang, Chang Hui HUANG, Yan ZHAO, Ling CAI, Ying WANG, Shi Jin XIU, Zheng Wen JIANG, Shuang YANG, Xin Tai ZHAO, Wei HUANG, Jian Ren GU (311)Identification and genetic mapping of four novel genes that regulate leaf deve- lopment in Arabidopsis………………………………………………SUN Yue, Wei ZHANG, Feng Ling LI, Ying Li GUO, Tian Lei LIU, Hai HUANG (325)NOTICE FOR CONTRIBUTORS…………………………………(337)CONTENTS of Vol. 10, 2000…………………………………………………(338)

  2. Construction and Myogenic Differentiation of 3D Myoblast Tissues Fabricated by Fibronectin-Gelatin Nanofilm Coating

    Science.gov (United States)

    Gribova, Varvara; Liu, Chen Yun; Nishiguchi, Akihiro; Matsusaki, Michiya; Boudou, Thomas; Picart, Catherine; Akashi, Mitsuru

    2016-01-01

    In this study, we used a recently developed approach of coating the cells with fibronectin-gelatin nanofilms to build 3D skeletal muscle tissue models. We constructed the microtissues from C2C12 myoblasts and subsequently differentiated them to form muscle-like tissue. The thickness of the constructs could be successfully controlled by altering the number of seeded cells. We were able to build up to ~ 76 µm thick 3D constructs that formed multinucleated myotubes. We also found that Rho-kinase inhibitor Y27632 improved myotube formation in thick constructs. Our approach makes it possible to rapidly form 3D muscle tissues and is promising for the in vitro construction of physiologically relevant human skeletal muscle tissue models. PMID:27125461

  3. Theophylline is able to partially revert cachexia in tumour-bearing rats

    Directory of Open Access Journals (Sweden)

    Olivan Mireia

    2012-08-01

    Full Text Available Abstract Background and aims The aim of the present investigation was to examine the anti-wasting effects of theophylline (a methylxantine present in tea leaves on a rat model of cancer cachexia. Methods The in vitro effects of the nutraceuticals on proteolysis were examined on muscle cell cultures submitted to hyperthermia. Individual muscle weights, muscle gene expression, body composition and cardiac function were measured in rats bearing the Yoshida AH-130 ascites hepatoma, following theophylline treatment. Results Theophylline treatment inhibited proteolysis in C2C12 cell line and resulted in an anti-proteolytic effect on muscle tissue (soleus and heart, which was associated with a decrease in circulating TNF-alpha levels and with a decreased proteolytic systems gene expression. Treatment with the nutraceutical also resulted in an improvement in body composition and cardiac function. Conclusion Theophylline - alone or in combination with drugs - may be a candidate molecule for the treatment of cancer cachexia.

  4. Cell Membrane Softening in Cancer Cells

    Science.gov (United States)

    Schmidt, Sebastian; Händel, Chris; Käs, Josef

    Biomechanical properties are useful characteristics and regulators of the cell's state. Current research connects mechanical properties of the cytoskeleton to many cellular processes but does not investigate the biomechanics of the plasma membrane. We evaluated thermal fluctuations of giant plasma membrane vesicles, directly derived from the plasma membranes of primary breast and cervical cells and observed a lowered rigidity in the plasma membrane of malignant cells compared to non-malignant cells. To investigate the specific role of membrane rigidity changes, we treated two cell lines with the Acetyl-CoA carboxylase inhibitor Soraphen A. It changed the lipidome of cells and drastically increased membrane stiffness by up regulating short chained membrane lipids. These altered cells had a decreased motility in Boyden chamber assays. Our results indicate that the thermal fluctuations of the membrane, which are much smaller than the fluctuations driven by the cytoskeleton, can be modulated by the cell and have an impact on adhesion and motility.

  5. High-Density Spot Seeding for Tissue Model Formation

    Science.gov (United States)

    Marquette, Michele L. (Inventor); Sognier, Marguerite A. (Inventor)

    2016-01-01

    A model of tissue is produced by steps comprising seeding cells at a selected concentration on a support to form a cell spot, incubating the cells to allow the cells to partially attach, rinsing the cells to remove any cells that have not partially attached, adding culture medium to enable the cells to proliferate at a periphery of the cell spot and to differentiate toward a center of the cell spot, and further incubating the cells to form the tissue. The cells may be C2C12 cells or other subclones of the C2 cell line, H9c2(2-1) cells, L6 cells, L8 cells, QM7 cells, Sol8 cells, G-7 cells, G-8 cells, other myoblast cells, cells from other tissues, or stem cells. The selected concentration is in a range from about 1 x 10(exp 5) cells/ml to about 1 x 10(exp 6) cells/ml. The tissue formed may be a muscle tissue or other tissue depending on the cells seeded.

  6. Insulin-like growth factor-1 receptor is regulated by microRNA-133 during skeletal myogenesis.

    Directory of Open Access Journals (Sweden)

    Mian-Bo Huang

    Full Text Available BACKGROUND: The insulin-like growth factor (IGF signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs, has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133. A conserved and functional binding site for miR-133 was identified in the 3'untranslated region (3'UTR of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation. CONCLUSION/SIGNIFICANCE: Our results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases.

  7. Sickle cell anemia - resources

    Science.gov (United States)

    Resources - sickle cell anemia ... The following organizations are good resources for information on sickle cell anemia : American Sickle Cell Anemia Association -- www.ascaa.org National Heart, Blood, and Lung Institute -- www. ...

  8. Sickle Cell Disease

    Science.gov (United States)

    ... in Sickle Cell Disease New supplement from the American Journal of Preventive Medicine describes the state of sickle cell disease related care in the United States. Read Supplement » ... are affected by sickle cell disease. More WEBINAR ...

  9. Sickle Cell Disease

    Science.gov (United States)

    ... from the NHLBI on Twitter. What Is Sickle Cell Disease? Español The term sickle cell disease (SCD) ... common forms of SCD. Some Forms of Sickle Cell Disease Hemoglobin SS Hemoglobin SC Hemoglobin Sβ 0 thalassemia ...

  10. Squamous cell skin cancer

    Science.gov (United States)

    ... earliest form of squamous cell cancer is called Bowen disease (or squamous cell carcinoma in situ). This type ... cancer; Squamous cell carcinoma of the skin Images Bowen's disease on the hand Keratoacanthoma Keratoacanthoma Skin cancer, squamous ...

  11. Lung cancer - small cell

    Science.gov (United States)

    Cancer - lung - small cell; Small cell lung cancer; SCLC ... About 15% of all lung cancer cases are SCLC. Small cell lung cancer is slightly more common in men than women. Almost all cases of SCLC ...

  12. GSPEL - Fuel Cell Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The Fuel Cell Lab (FCL) Provides testing for technology readiness of fuel cell systems The FCL investigates, tests and verifies the performance of fuel-cell systems...

  13. Lung cancer - small cell

    Science.gov (United States)

    Cancer - lung - small cell; Small cell lung cancer; SCLC ... About 15% of all lung cancer cases are SCLC. Small cell lung cancer is slightly more common in men than women. Almost all cases of SCLC are ...

  14. Basal Cell Carcinoma (BCC)

    Science.gov (United States)

    ... epithelioma, is the most common form of skin cancer. Basal cell carcinoma usually occurs on sun-damaged skin, especially ... other health issues. Infiltrating or morpheaform basal cell carcinomas: Infiltrating basal cell carcinomas can be more aggressive and locally destructive ...

  15. Immobilization of cells via activated cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Markt, M.; Kas, J.; Valentova, O.; Demnerova, K.; Vodrazka, Z.

    1986-10-01

    Cell walls of Saccharomyces cerevisiae and S. uvarum were activated by periodate oxidation of vicinal diol groups in cell wall polysaccharides. The aldehyde groups thus generated allow the yeast cells to be covalently bound to modified bead cellulose or macroporous glycidyl methacrylate supports, or to enzymes such as glucose oxidase and catalase. 6 references.

  16. Snail modulates cell metabolism in MDCK cells

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Misako, E-mail: haraguci@m3.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Indo, Hiroko P. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Iwasaki, Yasumasa [Health Care Center, Kochi University, Kochi 780-8520 (Japan); Iwashita, Yoichiro [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Fukushige, Tomoko [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Majima, Hideyuki J. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Izumo, Kimiko; Horiuchi, Masahisa [Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Kanekura, Takuro [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Furukawa, Tatsuhiko [Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Ozawa, Masayuki [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan)

    2013-03-22

    Highlights: ► MDCK/snail cells were more sensitive to glucose deprivation than MDCK/neo cells. ► MDCK/snail cells had decreased oxidative phosphorylation, O{sub 2} consumption and ATP content. ► TCA cycle enzyme activity, but not expression, was lower in MDCK/snail cells. ► MDCK/snail cells showed reduced PDH activity and increased PDK1 expression. ► MDCK/snail cells showed reduced expression of GLS2 and ACLY. -- Abstract: Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of

  17. Cell aggregation and sedimentation.

    Science.gov (United States)

    Davis, R H

    1995-01-01

    The aggregation of cells into clumps or flocs has been exploited for decades in such applications as biological wastewater treatment, beer brewing, antibiotic fermentation, and enhanced sedimentation to aid in cell recovery or retention. More recent research has included the use of cell aggregation and sedimentation to selectively separate subpopulations of cells. Potential biotechnological applications include overcoming contamination, maintaining plasmid-bearing cells in continuous fermentors, and selectively removing nonviable hybridoma cells from perfusion cultures.

  18. Artificial Stem Cell Niches

    OpenAIRE

    Lutolf, Matthias P.; Blau, Helen M.

    2009-01-01

    Stem cells are characterized by their dual ability to reproduce themselves (self-renew) and specialize (differentiate), yielding a plethora of daughter cells that maintain and regenerate tissues. In contrast to their embryonic counterparts, adult stem cells retain their unique functions only if they are in intimate contact with an instructive microenvironment, termed stem cell niche. In these niches, stem cells integrate a complex array of molecular signals that, in concert with induced cell-...

  19. Fish Stem Cell Cultures

    OpenAIRE

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  20. Stem Cell Separation Technologies

    OpenAIRE

    Zhu, Beili; Murthy, Shashi K

    2013-01-01

    Stem cell therapy and translational stem cell research require large-scale supply of stem cells at high purity and viability, thus leading to the development of stem cell separation technologies. This review covers key technologies being applied to stem cell separation, and also highlights exciting new approaches in this field. First, we will cover conventional separation methods that are commercially available and have been widely adapted. These methods include Fluorescence-activated cell so...

  1. Cell control report

    CERN Document Server

    2013-01-01

    Please note this is a Short Discount publication. This extensive report provides an essential overview of cells and their use as factory automation building blocks. The following issues are discussed in depth: Cell integration Cell software and standards Future technologies applied to cells Plus Cell control applications including: - rotary parts manufacturing - diesel engine component development - general cell control development at the General Electric Corporation - a vendor list.

  2. Resveratrol ameliorates muscular pathology in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Hori, Yusuke S; Kuno, Atsushi; Hosoda, Ryusuke; Tanno, Masaya; Miura, Tetsuji; Shimamoto, Kazuaki; Horio, Yoshiyuki

    2011-09-01

    Muscular dystrophies are inherited myogenic disorders accompanied by progressive skeletal muscle weakness and degeneration. We previously showed that resveratrol (3,5,4'-trihydroxy-trans-stilbene), an antioxidant and activator of the NAD(+)-dependent protein deacetylase SIRT1, delays the progression of heart failure and prolongs the lifespan of δ-sarcoglycan-deficient hamsters. Because a defect of dystroglycan complex causes muscular dystrophies, and δ-sarcoglycan is a component of this complex, we hypothesized that resveratrol might be a new therapeutic tool for muscular dystrophies. Here, we examined resveratrol's effect in mdx mice, an animal model of Duchenne muscular dystrophy. mdx mice that received resveratrol in the diet for 32 weeks (4 g/kg diet) showed significantly less muscle mass loss and nonmuscle interstitial tissue in the biceps femoris compared with mdx mice fed a control diet. In the muscles of these mice, resveratrol significantly decreased oxidative damage shown by the immunostaining of nitrotyrosine and 8-hydroxy-2'-deoxyguanosine and suppressed the up-regulation of NADPH oxidase subunits Nox4, Duox1, and p47(phox). Resveratrol also reduced the number of α-smooth muscle actin (α-SMA)(+) myofibroblast cells and endomysial fibrosis in the biceps femoris, although the infiltration of CD45(+) inflammatory cells and increase in transforming growth factor-β1 (TGF-β1) were still observed. In C2C12 myoblast cells, resveratrol pretreatment suppressed the TGF-β1-induced increase in reactive oxygen species, fibronectin production, and expression of α-SMA, and SIRT1 knockdown blocked these inhibitory effects. SIRT1 small interfering RNA also increased the expression of Nox4, p47(phox), and α-SMA in C2C12 cells. Taken together, these findings indicate that SIRT1 activation may be a useful strategy for treating muscular dystrophies. PMID:21652783

  3. Hydrogen improves glycemic control in type1 diabetic animal model by promoting glucose uptake into skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Haruka Amitani

    Full Text Available Hydrogen (H(2 acts as a therapeutic antioxidant. However, there are few reports on H(2 function in other capacities in diabetes mellitus (DM. Therefore, in this study, we investigated the role of H(2 in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H(2 promoted 2-[(14C]-deoxy-d-glucose (2-DG uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K, protein kinase C (PKC, and AMP-activated protein kinase (AMPK, although it did not stimulate the translocation of Glut2 in Hep G2 cells. H(2 significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p. and oral (p.o. administration. However, long-term p.o. administration of H(2 had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H(2 exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

  4. Fusion with stem cell makes the hepatocellular carcinoma cells similar to liver tumor-initiating cells

    OpenAIRE

    Wang, Ran; Chen, Shuxun; Li, Changxian; Ng, Kevin Tak Pan; Kong, Chi-Wing; Cheng, Jinping; Cheng, Shuk Han; Li, Ronald A.; Lo, Chung Mau; Man, Kwan; Sun, Dong

    2016-01-01

    Background Cell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus, tumor-initiating cell-like cells are generated. Methods We employed laser-induced single-cell fusion technique to fuse the hepatocellular carci...

  5. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  6. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    International Nuclear Information System (INIS)

    Research highlights: → Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). → Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. → Monoclonal cell lines showed reduced sensitivity for Paclitaxel. → In situ CD133+ cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. → CD133+ and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133+ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  7. Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    Xingxing Kong

    Full Text Available BACKGROUND: Sirtuin 3 (SIRT3 is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS-detoxifying enzymes, but the molecular mechanism underlying this is not well understood. RESULTS: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR binding element (ERRE (-407/-399 mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2C(12 myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2C(12 myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2C(12 myotubes. CONCLUSION: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus

  8. 脂蛋白脂酶缺失症基因治疗载体的构建及功能验证%Construction and Verification of Gene Therapy Vector for Lipoprotein Lipase Deficiency Disease

    Institute of Scientific and Technical Information of China (English)

    王恺龙; 郑李彬; 张帆; 沈良才; Libby Andrew; 李旭丽; 张瑾

    2013-01-01

    脂蛋白脂酶(lipoprotein lipase,LPL)是甘油三酯分解的限速酶,LPL基因缺失会引起高血脂症,虽然发病率低,但到目前为止,尚无有效治疗手段.该文构建了用于纠正LPL缺失基因型的逆转录病毒载体MSCV-hLPL,结果表明,MSCV-hLPL可以高效侵染体外培养的细胞系C2C12、HEK293和3T3-L1,并且都可以产生具有活性的脂蛋白脂酶.利用MSCV-hLPL侵染后的C2C12、HEK293和3T3-L1,分别注射到裸鼠皮下组织,发现C2C12和3T3-L1可以分泌脂蛋白脂酶到临近的肌肉组织中,显著提高LPL活性.以上工作证明,基因治疗载体可以纠正脂蛋白脂酶缺失的基因型,而脂肪细胞和肌肉细胞移植入裸鼠体内后,均可以作为生物反应器产生具有活性的LPL.这是该领域中的一次开拓性尝试,为脂蛋白脂酶缺失症治疗方法的开发打下了坚实的基础.%Lipoprotein lipase (LPL) is the rate limiting enzyme for triglycerides hydrolysis,which catalyses the hydrolysis of the triacylglycerol component of chylomicrons and very low density lipoproteins,thereby providing fatty acids and monoacylglycerol for tissue utilization.LPL gene mutation or deletion may affect the activity of LPL,and result in lipid metabolism disorder.Although the LPL deficiency disease is rare,no cure method is developed till now.In this study,the gene therapy construct MSCV-hLPL was made,which could infect muscle cell line (C2C12),kidney cell line (HEK293T) and pre-adipocyte cell line (3T3-L1) with over 80% efficiency.Nevertheless,active LPL could be detected at the surface of all these three kinds of cells.Then,three types of cells were injected into nude mice,LPL activity increased significantly in the muscle tissues under the injection sites of the 3T3-L1 line.Our results show that MSCV-hLPL could correct the LPL-/-genotype and the adipose tissue may be the best tissue for transplantation in the future.This is a ground-breaking test in LPL deficiency treatment field

  9. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  10. SMOOTH MUSCLE STEM CELLS

    Science.gov (United States)

    Vascular smooth muscle cells (SMCs) originate from multiple types of progenitor cells. In the embryo, the most well-studied SMC progenitor is the cardiac neural crest stem cell. Smooth muscle differentiation in the neural crest lineage is controlled by a combination of cell intrinsic factors, includ...

  11. Sickle Cell Disease

    Science.gov (United States)

    ... sickle cell disease? Sickle cell disease, also called sickle cell anemia, is a hereditary condition (which means it runs ... or blocks blood and oxygen reaching nearby tissues. Sickle cell disease ... the whites of the eyes) Anemia (the decreased ability of the blood to carry ...

  12. Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux.

    Science.gov (United States)

    Liemburg-Apers, Dania C; Schirris, Tom J J; Russel, Frans G M; Willems, Peter H G M; Koopman, Werner J H

    2015-10-01

    ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate. The latter can be metabolized into lactate, which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer. To gain insight into the kinetic properties of this adaptive mechanism we determined here how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII(12)Pglu-700μδ6 (FLII). Following in situ FLII calibration, the kinetic properties of GLC uptake (V1) and GLC consumption (V2) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics. After validating the model, it was applied to quantitatively predict V1 and V2 at steady-state (i.e., when V1 = V2 = Vsteady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on lactate production, cell volume, and O2 consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a twofold increase in Vsteady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions. Taken together, we demonstrate here that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts. This activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand. PMID:26445438

  13. The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease.

    Science.gov (United States)

    Davignon, Laurianne; Chauveau, Claire; Julien, Cédric; Dill, Corinne; Duband-Goulet, Isabelle; Cabet, Eva; Buendia, Brigitte; Lilienbaum, Alain; Rendu, John; Minot, Marie Christine; Guichet, Agnès; Allamand, Valérie; Vadrot, Nathalie; Fauré, Julien; Odent, Sylvie; Lazaro, Leïla; Leroy, Jean Paul; Marcorelles, Pascale; Dubourg, Odile; Ferreiro, Ana

    2016-04-15

    Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation inTRIP4encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted inTRIP4mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and inTrip4knocked-down C2C12 led to a significant reduction in myotube diameterex vivoandin vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the firstTRIP4mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism. PMID:27008887

  14. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

    International Nuclear Information System (INIS)

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Cavβ1a NH2-terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. - Highlights: • Previously, we demonstrated that Cavβ1a is a gene transcription regulator. • Here, we show that TnT3 interacts with Cavβ1a. • We mapped TnT3 and Cavβ1a interaction domain. • TnT3 facilitates Cavβ1a nuclear enrichment. • The two proteins play a heretofore unknown role during early muscle differentiation

  15. In vitro haematic proteins adsorption and cytocompatibility study on acrylic copolymer to realise coatings for drug-eluting stents

    International Nuclear Information System (INIS)

    In the present paper, a preliminary in vitro analysis of biocompatibility of newly-synthesised acrylic copolymers is reported. In particular, with the aim to obtain coatings for drug-eluting stents, blood protein absorption and cytocompatibility were studied. For protein absorption tests, bovine serum albumin and bovine plasma fibrinogen were considered. Cytocompatibility was tested using C2C12 cell line as model, analysing the behaviour of polymeric matrices and of drug-eluting systems, obtained loading polymeric matrices with paclitaxel, an anti-mitotic drug, in order to evaluate the efficacy of a pharmacological treatment locally administered from these materials. Results showed that the amount of albumin absorbed was greater than the amount of fibrinogen (comprised in the range of 70%–85% and 10%–22% respectively) and it is a good behaviour in terms of haemocompatibility. Cell culture tests showed good adhesion properties and a relative poor proliferation. In addition, a strong effect related to drug elution and a correlation with the macromolecular composition were detected. In this preliminary analysis, tested materials showed good characteristics and can be considered possible candidates to obtain coatings for drug-eluting stents. Highlights: ► Preliminary evaluation of haemo- and cytocompatibility of newly-synthesised acrylic copolymers ► Materials adsorb higher amounts of albumin and with a faster rate than fibrinogen. ► Protein adsorption depended on the macromolecular composition and surface properties. ► Cell viability on pure samples and efficacy of paclitaxel release were verified in C2C12 cultures.

  16. Delta-like 1 homolog (dlk1: a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Louise H Jørgensen

    Full Text Available Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the

  17. In vitro haematic proteins adsorption and cytocompatibility study on acrylic copolymer to realise coatings for drug-eluting stents

    Energy Technology Data Exchange (ETDEWEB)

    Gagliardi, Mariacristina, E-mail: mariacristina.gagliardi@iit.it

    2012-12-01

    In the present paper, a preliminary in vitro analysis of biocompatibility of newly-synthesised acrylic copolymers is reported. In particular, with the aim to obtain coatings for drug-eluting stents, blood protein absorption and cytocompatibility were studied. For protein absorption tests, bovine serum albumin and bovine plasma fibrinogen were considered. Cytocompatibility was tested using C2C12 cell line as model, analysing the behaviour of polymeric matrices and of drug-eluting systems, obtained loading polymeric matrices with paclitaxel, an anti-mitotic drug, in order to evaluate the efficacy of a pharmacological treatment locally administered from these materials. Results showed that the amount of albumin absorbed was greater than the amount of fibrinogen (comprised in the range of 70%-85% and 10%-22% respectively) and it is a good behaviour in terms of haemocompatibility. Cell culture tests showed good adhesion properties and a relative poor proliferation. In addition, a strong effect related to drug elution and a correlation with the macromolecular composition were detected. In this preliminary analysis, tested materials showed good characteristics and can be considered possible candidates to obtain coatings for drug-eluting stents. Highlights: Black-Right-Pointing-Pointer Preliminary evaluation of haemo- and cytocompatibility of newly-synthesised acrylic copolymers Black-Right-Pointing-Pointer Materials adsorb higher amounts of albumin and with a faster rate than fibrinogen. Black-Right-Pointing-Pointer Protein adsorption depended on the macromolecular composition and surface properties. Black-Right-Pointing-Pointer Cell viability on pure samples and efficacy of paclitaxel release were verified in C2C12 cultures.

  18. Prostaglandin E2: from clinical applications to its potential role in bone- muscle crosstalk and myogenic differentiation.

    Science.gov (United States)

    Mo, Chenglin; Romero-Suarez, Sandra; Bonewald, Lynda; Johnson, Mark; Brotto, Marco

    2012-12-01

    Prostaglandin E(2) (PGE(2)), a prostanoid synthesized from arachidonic acid via the cyclooxygenase pathway, is a modulator of physiological responses including inflammation, fever, and muscle regeneration. Several patents have been filed that are related to PGE(2), one of them being directly related to skeletal muscles. In this report, we first summarize the key patents describing inventions for the utilization of PGE(2) for either diagnostic or therapeutic purposes, including skeletal muscle. In the second part of our work we present new and exciting data that demonstrates that PGE(2) accelerates skeletal muscle myogenic differentiation. Our discovery resulted from our recent and novel concept of bone-muscle crosstalk. Bone and muscle are anatomically intimate endocrine organs and we aimed to determine whether this anatomical intimacy also translates into a biochemical communication from bone cells to muscle cells at the in vitro level. The effects of MLOY4 osteocyte-like cell conditioned medium (CM) and three osteocyte-secreted factors, PGE(2), sclerostin and monocyte chemotactic protein (MCP-3), on C2C12 myogenic differentiation were evaluated using morphological analyses, a customized 96-gene PCR array, and measurements of intracellular calcium levels. MLO-Y4 CM and PGE(2), but not sclerostin and MCP-3, induced acceleration of myogenesis of C2C12 myoblasts that was linked with significant modifications in intracellular calcium homeostasis. This finding should further stimulate the pursuit of new patents to explore the use of PGE(2) and the new concept of bone-muscle crosstalk for the development and application of inventions designed to treat muscle diseases characterized by enhanced muscle wasting, such as sarcopenia. PMID:23092433

  19. YB-1 gene expression is kept constant during myocyte differentiation through replacement of different transcription factors and then falls gradually under the control of neural activity.

    Science.gov (United States)

    Kobayashi, Shunsuke; Tanaka, Toru; Moue, Masamitsu; Ohashi, Sachiyo; Nishikawa, Taishi

    2015-11-01

    We have previously reported that translation of acetylcholine receptor α-subunit (AChR α) mRNA in skeletal muscle cells is regulated by Y-box binding protein 1 (YB-1) in response to neural activity, and that in the postnatal mouse developmental changes in the amount of YB-1 mRNA are similar to those of AChR α mRNA, which is known to be regulated by myogenic transcription factors. Here, we examined transcriptional regulation of the YB-1 gene in mouse skeletal muscle and differentiating C2C12 myocytes. Although neither YB-1 nor AChR α was detected at either the mRNA or protein level in adult hind limb muscle, YB-1 expression was transiently activated in response to denervation of the sciatic nerve and completely paralleled that of AChR α, suggesting that these genes are regulated by the same transcription factors. However, during differentiation of C2C12 cells to myotubes, the level of YB-1 remained constant even though the level of AChR α increased markedly. Reporter gene, gel mobility shift and ChIP assays revealed that in the initial stage of myocyte differentiation, transcription of the YB-1 gene was regulated by E2F1 and Sp1, and was then gradually replaced under the control of both MyoD and myogenin through an E-box sequence in the proximal region of the YB-1 gene promoter. These results suggest that transcription factors for the YB-1 gene are exchanged during skeletal muscle cell differentiation, perhaps playing a role in translational control of mRNAs by YB-1 in both myotube formation and the response of skeletal muscle tissues to neural stimulation.

  20. When Blood Cells Bend: Understanding Sickle Cell Disease

    Science.gov (United States)

    ... please review our exit disclaimer . Subscribe When Blood Cells Bend Understanding Sickle Cell Disease For people who don’t suspect they ... Cells Bend Wise Choices Links Living with Sickle Cell Disease See a sickle cell disease expert regularly. ...

  1. Ganglion cell like cells, diagnostic dilemma

    Directory of Open Access Journals (Sweden)

    Anand Shankar Ammanagi

    2013-01-01

    Full Text Available We report a case of cutaneous swelling found on the left anterior axillary fold of a 41-year-old man. Gross examination of specimen excised from the dermis showed a well-circumscribed nodule histologically composed of spindle cells with interspersed ganglion cell like cells. On hematoxylin and eosine (H and E staining it was diagnosed as ganglioneuroma. Ganglioneuromas are rare, benign, fully differentiated tumors that contain mature schwann cells, ganglion cells, fibrous tissue, and nerve fibers. They are commonly found along the paravertebral sympathetic ganglia and sometimes in the adrenal medulla. However primary cutaneous ganglioneuroma is an extremely rare tumor. Immunohistochemical workup revealed a fibroblastic origin and hence the case was diagnosed as fibromatosis with ganglion cell like fibroblasts. This case report suggests that the features considered diagnostic of ganglioneuromas can occur in other cutaneous lesions and, therefore, this diagnosis cannot be offered only on the basis of H and E.

  2. Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells

    OpenAIRE

    Bonde, Sabrina; Pedram, Mehrdad; Stultz, Ryan; Zavazava, Nicholas

    2010-01-01

    Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1+, a myeloid cell mar...

  3. Hepatic stem cell niches

    OpenAIRE

    Kordes, Claus; Häussinger, Dieter

    2013-01-01

    Stem cell niches are special microenvironments that maintain stem cells and control their behavior to ensure tissue homeostasis and regeneration throughout life. The liver has a high regenerative capacity that involves stem/progenitor cells when the proliferation of hepatocytes is impaired. In recent years progress has been made in the identification of potential hepatic stem cell niches. There is evidence that hepatic progenitor cells can originate from niches in the canals...

  4. Stem Cell Networks

    OpenAIRE

    Werner, Eric

    2016-01-01

    We present a general computational theory of stem cell networks and their developmental dynamics. Stem cell networks are special cases of developmental control networks. Our theory generates a natural classification of all possible stem cell networks based on their network architecture. Each stem cell network has a unique topology and semantics and developmental dynamics that result in distinct phenotypes. We show that the ideal growth dynamics of multicellular systems generated by stem cell ...

  5. Stem cell mechanobiology

    OpenAIRE

    David A. Lee; Knight, Martin M.; Jonathan J Campbell; Bader, Dan L.

    2010-01-01

    Stem cells are undifferentiated cells that are capable of proliferation, self-maintenance and differentiation towards specific cell phenotypes. These processes are controlled by a variety of cues including physicochemical factors associated with the specific mechanical environment in which the cells reside. The control of stem cell biology through mechanical factors remains poorly understood and is the focus of the developing field of mechanobiology. This review provides an insight into the c...

  6. Embryonic Stem Cell Markers

    OpenAIRE

    Lan Ma; Liang Li; Wenxiu Zhao; Xiang Ji; Fangfang Zhang

    2012-01-01

    Embryonic stem cell (ESC) markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal, therefore helping to accelerate the clinical application of ES cells. Unfortunately, different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other type...

  7. Limbal stem cell transplantation

    OpenAIRE

    Fernandes Merle; Sangwan Virender; Rao Srinivas; Basti Surendra; Sridhar Mittanamalli; Bansal Aashish; Dua Harminder

    2004-01-01

    The past two decades have witnessed remarkable progress in limbal stem cell transplantation. In addition to harvesting stem cells from a cadaver or a live related donor, it is now possible to cultivate limbal stem cells in vitro and then transplant them onto the recipient bed. A clear understanding of the basic disease pathology and a correct assessment of the extent of stem cell deficiency are essential. A holistic approach towards management of limbal stem cell deficiency is needed. This ...

  8. Intraoperative Stem Cell Therapy

    OpenAIRE

    Coelho, Mónica Beato; Cabral, Joaquim M. S.; Karp, Jeffrey M.

    2012-01-01

    Stem cells hold significant promise for regeneration of tissue defects and disease-modifying therapies. Although numerous promising stem cell approaches are advancing in clinical trials, intraoperative stem cell therapies offer more immediate hope by integrating an autologous cell source with a well-established surgical intervention in a single procedure. Herein, the major developments in intraoperative stem cell approaches, from in vivo models to clinical studies, are reviewed, and the poten...

  9. The leukemic stem cell

    OpenAIRE

    Jordan, Craig T.

    2007-01-01

    Malignant stem cells have recently been described as the source of several types of human cancer. These unique cell types are typically rare and possess properties that are distinct from most other tumor cells. The properties of leukemic stem cells indicate that current chemotherapy drugs will not be effective. The use of current cytotoxic agents is not effective in leukemia because the agents target both the leukemic and normal stem cell populations. Consequently, new strategies are required...

  10. Cancer Stem Cells

    OpenAIRE

    Katarzyna Wieczorek; Jolanta Niewiarowska

    2008-01-01

    Cancer stem cell theory gains increasingly greater significance in the world of medicine. Numerous findings of scientific research in vivo and in vitro indicate that it is the population of undifferentiated, self-renewing cells which is responsible for recurrence of cancer and metastasis. Similarly to normal stem cells, cancer stem cells (CSC) function in the environment of the other cells of the organism, called the niche, where they receive signals for differentiation and proliferation proc...

  11. The cell cycle as a brake for β-cell regeneration from embryonic stem cells

    OpenAIRE

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-01

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle ...

  12. Mechanically facilitated cell-cell electrofusion.

    OpenAIRE

    Jaroszeski, M. J.; Gilbert, R.; Fallon, P.G.; Heller, R

    1994-01-01

    Apparatus and methods were developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. The key component of this fusion system was a newly developed fusion chamber. The chamber was composed of two functionally identical electrodes that were housed in a multi-layer structure. The layers functioned as support for the electrodes. They also allowed adjustment of the distance between opposing ele...

  13. FOXO1 delays skeletal muscle regeneration and suppresses myoblast proliferation.

    Science.gov (United States)

    Yamashita, Atsushi; Hatazawa, Yukino; Hirose, Yuma; Ono, Yusuke; Kamei, Yasutomi

    2016-08-01

    Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading. PMID:27010781

  14. Circulating micrornas as potential biomarkers of muscle atrophy

    Science.gov (United States)

    Wang, Fei

    2016-07-01

    Noninvasive biomarkers with diagnostic value and prognostic applications have long been desired to replace muscle biopsy for muscle atrophy patients. Growing evidence indicates that circulating microRNAs are biomarkers to assess pathophysiological status. Here, we show that the medium levels of six muscle-specific miRNAs (miR-1/23a/206/133/499/208b, also known as myomiRs) were all elevated in the medium of starved C2C12 cell (P muscle mass and muscle fiber cross section area in muscle atrophy patients, indicating that they might represent the degree of muscle atrophy. Collectively, our data indicated that circulating myomiRs could serve as promising biomarkers for muscle atrophy.

  15. ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation.

    Science.gov (United States)

    Dao, Trong-Tuan; Lee, Kwang-Youl; Jeong, Hyung-Min; Nguyen, Phi-Hung; Tran, Tien Lam; Thuong, Phuong-Thien; Nguyen, Bich-Thu; Oh, Won-Keun

    2011-12-27

    Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), ent-14β-hydroxy-18-acetoxykaur-16-ene (3), and ent-7α-hydroxy-18-acetoxykaur-16-ene (4). Compounds 1-4 significantly increased alkaline phosphatase activity and osteoblastic gene promoter activity. Compounds 1-3 also increased the levels of ALP and collagen type I alpha mRNA in C2C12 cells in a dose-dependent manner. These results suggest that ent-kaurane diterpenoids from C. tonkinensis have a direct stimulatory effect on osteoblast differentiation and may be potential therapeutic molecules against bone diseases such as osteoporosis. PMID:22085418

  16. Inhibitory Effect of High Temperature- and High Pressure-Treated Red Ginseng on Exercise-Induced Oxidative Stress in ICR Mouse

    Directory of Open Access Journals (Sweden)

    Seok-Yeong Yu

    2014-03-01

    Full Text Available As previously reported, high temperature- and high pressure-treated red ginseng (HRG contain higher contents of phenolic compounds and protect C2C12 muscle cells and 3T3-L1 adipocytes against oxidative stress. This study investigated the effect of HRG on oxidative stress using a mouse model. Our results show that the levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, hepatic malondialdehyde in the HRG group were significantly lower than those of the exercise groups supplemented with commercial red ginseng (CRG or not supplemented. The muscular glycogen level, glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities of the HGR group were higher than that of the CGR group. Furthermore, the HRG treatment group displayed upregulated mRNA expression of Cu/Zn-SOD and muscle regulatory factor 4. These results indicate that HRG may protect oxidative stress induced by exercise as well as improve exercise performance capacity.

  17. Chromium Picolinate did not Effect on the Proliferation and Differentiation of Myoblasts

    Directory of Open Access Journals (Sweden)

    M. C. Tsa

    2007-01-01

    Full Text Available This experiment is conducted in vitro to investigate trivalent chromium picolinate affects the proliferation and differentiation of myoblasts. A myoblasts cell line (C2C12 from rats was used in the experiment. These were randomly divided into the control group, the Pic group (50ppb picolinate and the CrPic group (50ppb chromium picolinate. The differentiation of myoblasts reveals that the number of differentiated myotubes, creatine kinase (CK activity and the aldolase (ALB activity do not differ among the three groups (P > 0.05. The activity of hexokinase in the CrPic and Pic groups clearly exceeds that in the control group (P 0.05. Myoblast proliferation was the same across the three groups (P > 0.05, and the quantity of DNA in the control group exceeded that in the Pic group (P < 0.05. The experiment indicated that 200ppb chromium picolinate did not influence the proliferation and differentiation of myoblasts.

  18. Optimizing stem cell culture.

    Science.gov (United States)

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  19. Living with Sickle Cell Disease

    Science.gov (United States)

    ... sickle cell disease, go to the Health Topics Sickle Cell Anemia article. Living With and Managing Sickle Cell Disease ( ... the most severe form of sickle cell disease, sickle cell anemia, Tiffany has lived with the symptoms and complications ...

  20. What Causes Sickle Cell Disease?

    Science.gov (United States)

    ... sickle cell disease, go to the Health Topics Sickle Cell Anemia article. Living With and Managing Sickle Cell Disease ( ... the most severe form of sickle cell disease, sickle cell anemia, Tiffany has lived with the symptoms and complications ...

  1. Plant stem cell niches.

    Science.gov (United States)

    Aichinger, Ernst; Kornet, Noortje; Friedrich, Thomas; Laux, Thomas

    2012-01-01

    Multicellular organisms possess pluripotent stem cells to form new organs, replenish the daily loss of cells, or regenerate organs after injury. Stem cells are maintained in specific environments, the stem cell niches, that provide signals to block differentiation. In plants, stem cell niches are situated in the shoot, root, and vascular meristems-self-perpetuating units of organ formation. Plants' lifelong activity-which, as in the case of trees, can extend over more than a thousand years-requires that a robust regulatory network keep the balance between pluripotent stem cells and differentiating descendants. In this review, we focus on current models in plant stem cell research elaborated during the past two decades, mainly in the model plant Arabidopsis thaliana. We address the roles of mobile signals on transcriptional modules involved in balancing cell fates. In addition, we discuss shared features of and differences between the distinct stem cell niches of Arabidopsis.

  2. What are Stem Cells?

    Directory of Open Access Journals (Sweden)

    Ahmadshah Farhat

    2014-05-01

    Full Text Available   Stem cells are undifferentiated self regenerating multi potential cells. There are three types of stem cells categories by the ability to form after cells and correlated with the body’s development process. Totipotent: these stem cells can form an entire organism such as fertilized egg. Ploripotent: ploripotent cells are those that can form any cell in the body but cannot form an entire organism such as developing embryo’s totipotent cells become ploripotent  Multipotent: Multi potent stem cells are those that can only form specific cells in the body such as blood cells based. Based on the sources of stem cells we have three types of these cells: Autologous: Sources of the patient own cells are (Autologous either the cells from patient own body or his or her cord blood. For this type of transplant the physician now usually collects the periphery rather than morrow because the procedure is easier on like a bane morrow harvest it take place outside of an operating room, and the patient does not to be under general unsetting . Allogenic: Sources of stem cells from another donore are primarily relatives (familial allogenic or completely unrelated donors. Xenogenic: In these stem cells from different species are transplanted e .g striatal porcine fetal mesan cephalic (FVM xenotransplants for Parkinson’s disease. On sites of isolation such as embryo, umbilical cord and other body tissues stem cells are named embnyonic, cord blood, and adult stem cells. The scope of results and clinical application of stem cells are such as: Neurodegenerative conditions (MS,ALS, Parkinson’s, Stroke, Ocular disorders- Glaucoma, retinitis Pigmentosa (RP, Auto Immune Conditions (Lupus, MS,R. arthritis, Diabetes, etc, Viral Conditions (Hepatitis C and AIDS, Heart Disease, Adrenal Disorders, Injury(Nerve, Brain, etc, Anti aging (hair, skin, weight control, overall well being/preventive, Emotional disorders, Organ / Tissue Cancers, Blood cancers, Blood diseases

  3. Lung Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Sharon R. Pine

    2008-01-01

    Full Text Available Lung cancer remains a major cause of cancer-related lethality because of high incidence and recurrence in spite of significant advances in staging and therapies. Recent data indicates that stem cells situated throughout the airways may initiate cancer formation. These putative stem cells maintain protumorigenic characteristics including high proliferative capacity, multipotent differentiation, drug resistance and long lifespan relative to other cells. Stem cell signaling and differentiation pathways are maintained within distinct cancer types, and destabilization of this machinery may participate in maintenance of cancer stem cells. Characterization of lung cancer stem cells is an area of active research and is critical for developing novel therapies. This review summarizes the current knowledge on stem cell signaling pathways and cell markers used to identify the lung cancer stem cells.

  4. Stem cells in urology.

    Science.gov (United States)

    Aboushwareb, Tamer; Atala, Anthony

    2008-11-01

    The shortage of donors for organ transplantation has stimulated research on stem cells as a potential resource for cell-based therapy in all human tissues. Stem cells have been used for regenerative medicine applications in many organ systems, including the genitourinary system. The potential applications for stem cell therapy have, however, been restricted by the ethical issues associated with embryonic stem cell research. Instead, scientists have explored other cell sources, including progenitor and stem cells derived from adult tissues and stem cells derived from the amniotic fluid and placenta. In addition, novel techniques for generating stem cells in the laboratory are being developed. These techniques include somatic cell nuclear transfer, in which the nucleus of an adult somatic cell is placed into an oocyte, and reprogramming of adult cells to induce stem-cell-like behavior. Such techniques are now being used in tissue engineering applications, and some of the most successful experiments have been in the field of urology. Techniques to regenerate bladder tissue have reached the clinic, and exciting progress is being made in other areas, such as regeneration of the kidney and urethra. Cell therapy as a treatment for incontinence and infertility might soon become a reality. Physicians should be optimistic that regenerative medicine and tissue engineering will one day provide mainstream treatment options for urologic disorders.

  5. Effect of Exercise Intensity on Isoform-Specific Expressions of NT-PGC-1α mRNA in Mouse Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Xingyuan Wen

    2014-01-01

    Full Text Available PGC-1α is an inducible transcriptional coactivator that regulates mitochondrial biogenesis and cellular energy metabolism in skeletal muscle. Recent studies have identified two additional PGC-1α transcripts that are derived from an alternative exon 1 (exon 1b and induced by exercise. Given that the PGC-1α gene also produces NT-PGC-1α transcript by alternative 3′ splicing between exon 6 and exon 7, we have investigated isoform-specific expression of NT-PGC-1α mRNA in mouse skeletal muscle during physical exercise with different intensities. We report here that NT-PGC-1α-a mRNA expression derived from a canonical exon 1 (exon 1a is increased by high-intensity exercise and AMPK activator AICAR in mouse skeletal muscle but not altered by low- and medium-intensity exercise and β2-adrenergic receptor agonist clenbuterol. In contrast, the alternative exon 1b-driven NT-PGC-1α-b (PGC-1α4 and NT-PGC-1α-c are highly induced by low-, medium-, and high-intensity exercise, AICAR, and clenbuterol. Ectopic expression of NT-PGC-1α-a in C2C12 myotube cells upregulates myosin heavy chain (MHC I, MHC II a and Glut4, which represent oxidative fibers, and promotes the expression of mitochondrial genes (Cyc1, COX5B, and ATP5B. In line with gene expression data, citrate synthase activity was significantly increased by NT-PGC-1α-a in C2C12 myotube cells. Our results indicate the regulatory role for NT-PGC-1α-a in mitochondrial biogenesis and adaptation of skeletal muscle to endurance exercise.

  6. Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK.

    Science.gov (United States)

    Murase, Takatoshi; Misawa, Koichi; Haramizu, Satoshi; Minegishi, Yoshihiko; Hase, Tadashi

    2010-08-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is implicated in the control of energy metabolism and is considered to be a molecular target for the suppression of obesity and the treatment of metabolic syndrome. Here, we identified and characterized nootkatone, a constituent of grapefruit, as a naturally occurring AMPK activator. Nootkatone induced an increase in AMPKalpha1 and -alpha2 activity along with an increase in the AMP/ATP ratio and an increase the phosphorylation of AMPKalpha and the downstream target acetyl-CoA carboxylase (ACC), in C(2)C(12) cells. Nootkatone-induced activation of AMPK was possibly mediated both by LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase. Nootkatone also upregulated PPARgamma coactivator-1alpha in C(2)C(12) cells and C57BL/6J mouse muscle. In addition, administration of nootkatone (200 mg/kg body wt) significantly enhanced AMPK activity, accompanied by LKB1, AMPK, and ACC phosphorylation in the liver and muscle of mice. Whole body energy expenditure evaluated by indirect calorimetry was also increased by nootkatone administration. Long-term intake of diets containing 0.1% to 0.3% (wt/wt) nootkatone significantly reduced high-fat and high-sucrose diet-induced body weight gain, abdominal fat accumulation, and the development of hyperglycemia, hyperinsulinemia, and hyperleptinemia in C57BL/6J mice. Furthermore, endurance capacity, evaluated as swimming time to exhaustion in BALB/c mice, was 21% longer in mice fed 0.2% nootkatone than in control mice. These findings indicate that long-term intake of nootkatone is beneficial toward preventing obesity and improving physical performance and that these effects are due, at least in part, to enhanced energy metabolism through AMPK activation in skeletal muscle and liver. PMID:20501876

  7. Interdigitated array of Pt electrodes for electrical stimulation and engineering of aligned muscle tissue.

    Science.gov (United States)

    Ahadian, Samad; Ramón-Azcón, Javier; Ostrovidov, Serge; Camci-Unal, Gulden; Hosseini, Vahid; Kaji, Hirokazu; Ino, Kosuke; Shiku, Hitoshi; Khademhosseini, Ali; Matsue, Tomokazu

    2012-09-21

    Engineered skeletal muscle tissues could be useful for applications in tissue engineering, drug screening, and bio-robotics. It is well-known that skeletal muscle cells are able to differentiate under electrical stimulation (ES), with an increase in myosin production, along with the formation of myofibers and contractile proteins. In this study, we describe the use of an interdigitated array of electrodes as a novel platform to electrically stimulate engineered muscle tissues. The resulting muscle myofibers were analyzed and quantified in terms of their myotube characteristics and gene expression. The engineered muscle tissues stimulated through the interdigitated array of electrodes demonstrated superior performance and maturation compared to the corresponding tissues stimulated through a conventional setup (i.e., through Pt wires in close proximity to the muscle tissue). In particular, the ES of muscle tissue (voltage 6 V, frequency 1 Hz and duration 10 ms for 1 day) through the interdigitated array of electrodes resulted in a higher degree of C2C12 myotube alignment (∼80%) as compared to ES using Pt wires (∼65%). In addition, higher amounts of C2C12 myotube coverage area, myotube length, muscle transcription factors and protein biomarkers were found for myotubes stimulated through the interdigitated array of electrodes compared to those stimulated using the Pt wires. Due to the wide array of potential applications of ES for two- and three-dimensional (2D and 3D) engineered tissues, the suggested platform could be employed for a variety of cell and tissue structures to more efficiently investigate their response to electrical fields.

  8. Effects of isoleucine on glucose uptake through the enhancement of muscular membrane concentrations of GLUT1 and GLUT4 and intestinal membrane concentrations of Na+/glucose co-transporter 1 (SGLT-1) and GLUT2.

    Science.gov (United States)

    Zhang, Shihai; Yang, Qing; Ren, Man; Qiao, Shiyan; He, Pingli; Li, Defa; Zeng, Xiangfang

    2016-08-01

    Knowledge of regulation of glucose transport contributes to our understanding of whole-body glucose homoeostasis and human metabolic diseases. Isoleucine has been reported to participate in regulation of glucose levels in many studies; therefore, this study was designed to examine the effect of isoleucine on intestinal and muscular GLUT expressions. In an animal experiment, muscular GLUT and intestinal GLUT were determined in weaning pigs fed control or isoleucine-supplemented diets. Supplementation of isoleucine in the diet significantly increased piglet average daily gain, enhanced GLUT1 expression in red muscle and GLUT4 expression in red muscle, white muscle and intermediate muscle (P<0·05). In additional, expressions of Na+/glucose co-transporter 1 and GLUT2 were up-regulated in the small intestine when pigs were fed isoleucine-supplemented diets (P<0·05). C2C12 cells were used to examine the expressions of muscular GLUT and glucose uptake in vitro. In C2C12 cells supplemented with isoleucine in the medium, cellular 2-deoxyglucose uptake was increased (P<0·05) through enhancement of the expressions of GLUT4 and GLUT1 (P<0·05). The effect of isoleucine was greater than that of leucine on glucose uptake (P<0·05). Compared with newborn piglets, 35-d-old piglets have comparatively higher GLUT4, GLUT2 and GLUT5 expressions. The results of this study demonstrated that isoleucine supplementation enhanced the intestinal and muscular GLUT expressions, which have important implications that suggest that isoleucine could potentially increase muscle growth and intestinal development by enhancing local glucose uptake in animals and human beings. PMID:27464458

  9. Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Quanwen Liu

    2016-01-01

    Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

  10. Pluripotent Stem Cells for Schwann Cell Engineering

    NARCIS (Netherlands)

    Ma, Ming-San; Boddeke, Erik; Copray, Sjef

    2015-01-01

    Tissue engineering of Schwann cells (SCs) can serve a number of purposes, such as in vitro SC-related disease modeling, treatment of peripheral nerve diseases or peripheral nerve injury, and, potentially, treatment of CNS diseases. SCs can be generated from autologous stem cells in vitro by recapitu

  11. Assessment of pancreas cells

    Science.gov (United States)

    Vanoss, C. J.

    1978-01-01

    Pancreatic islets were obtained from guinea pig pancreas by the collagenase method and kept alive in tissue culture prior to further studies. Pancreas cell morphology was studied by standard histochemical techniques using light microscopy. Preparative vertical electrophoresis-levitation of dispersed fetal guinea pig pancreas cells was conducted in phosphate buffer containing a heavy water (D20) gradient which does not cause clumping of cells or alter the osmolarity of the buffers. The faster migrating fractions tended to be enriched in beta-cell content. Alpha and delta cells were found to some degree in most fractions. A histogram showing the cell count distribution is included.

  12. Resident Peritoneal NK cells

    OpenAIRE

    Gonzaga, Rosemary; Matzinger, Polly; Perez-Diez, Ainhoa

    2011-01-01

    Here we describe a new population of NK cells that reside in the normal, un-inflamed peritoneal cavity. Phenotypically, they share some similarities with the small population of CD49b negative, CD27 positive immature splenic NK cells, and liver NK cells but differ in their expression of CD62L, TRAIL and EOMES. Functionally, the peritoneal NK cells resemble the immature splenic NK cells in their production of IFN-γ, GM-CSF and TNF-α and in the killing of YAC-1 target cells. We also found that ...

  13. Multipotent adult progenitor cell and stem cell plasticity

    OpenAIRE

    Jahagirdar, Balkrishna N; Verfaillie, Catherine

    2005-01-01

    Stem cells are defined by their biological function. A stem cell is an undifferentiated cell that self-renews to maintain the stem cell pool and at the single-cell level differentiates into more than one mature, functional cell. In addition, when transplanted, a stem cell should be capable of replacing a damaged organ or tissue for the lifetime of the recipient. Some would argue that stem cells should also be capable of functionally integrating into nondamaged tissues. Stem cells are critical...

  14. Epidermal Stem Cells

    Directory of Open Access Journals (Sweden)

    Osman Köse

    2015-03-01

    Full Text Available The epidermis is the outermost layer of the human skin and comprises a multilayered epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands, and eccrine sweat glands. There are many origins of stem cells in the skin and skin appendages. These stem cells are localized in different part of the pilosebaseous units and also express many different genes. Epidermal stem cells in the pilosebaseous units not only ensure the maintenance of epidermal homeostasis and hair regeneration, but also contribute to repair of the epidermis after injury. In recent years, human induced pluripotent skin stem cells are produced from the epidermal cells such as keratinocytes, fibroblasts and melanocytes. These cells can be transdifferentiated to embriyonic stem cells. Human induced pluripotent stem cells have potential applications in cell replacement therapy and regenerative medicine. These cells provide a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. In this review, we aimed an overview of epidermal stem cells for better understanding their functions in the skin. Skin will be main organ for using the epidermal cells for regenerative medicine in near future.

  15. The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

    Science.gov (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-13

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  16. Regulatory T cells and B cells: implication on autoimmune diseases

    OpenAIRE

    Wang, Ping; Zheng, Song Guo

    2013-01-01

    The regulatory T (Treg) cells play an important role in the maintenance of homeostasis and the prevention of autoimmune diseases. Although most studies are focusing on the role of Treg cells in T cells and T cells-mediated diseases, these cells also directly affect B cells and other non-T cells. This manuscript updates the role of Treg cells on the B cells and B cell-mediated diseases. In addition, the mechanisms whereby Treg cells suppress B cell responses have been discussed.

  17. Toward 'SMART' stem cells.

    Science.gov (United States)

    Cheng, T

    2008-01-01

    Stem cell research is at the heart of regenerative medicine, which holds great promise for the treatment of many devastating disorders. However, in addition to hurdles posed by well-publicized ethical issues, this emerging field presents many biological challenges. What is a stem cell? How are embryonic stem cells different from adult stem cells? What are the physiological bases for therapeutically acceptable stem cells? In this editorial review, I will briefly discuss these superficially simple but actually rather complex issues that surround this fascinating cell type. The goal of this special issue on stem cells in Gene Therapy is to review some fundamental and critical aspects of current stem cell research that have translational potential. PMID:18046429

  18. Cell signaling review series

    Institute of Scientific and Technical Information of China (English)

    Aiming Lin; Zhenggang Liu

    2008-01-01

    @@ Signal transduction is pivotal for many, if not all, fundamental cellular functions including proliferation, differentiation, transformation and programmed cell death. Deregulation of cell signaling may result in certain types of cancers and other human diseases.

  19. Giant Cell Arteritis

    Science.gov (United States)

    Giant cell arteritis is a disorder that causes inflammation of your arteries, usually in the scalp, neck, and arms. ... arteries, which keeps blood from flowing well. Giant cell arteritis often occurs with another disorder called polymyalgia ...

  20. Sickle cell anemia.

    OpenAIRE

    ŘÍHOVÁ, Tereza

    2013-01-01

    This thesis is about the disease called sickle cell anemia, or drepanocytosis. In this thesis is described the history of the disease, pathophysiology, laboratory features, various clinical features, diferencial diagnosis, quality of life in sickle cell anemia and therapy.

  1. Sickle Cell Trait

    Science.gov (United States)

    ... About Us Information For... Media Policy Makers Sickle Cell Trait Language: English Español (Spanish) Recommend on Facebook ... the trait on to their children. How Sickle Cell Trait is Inherited If both parents have SCT, ...

  2. Sickle Cell Disease Quiz

    Science.gov (United States)

    ... About Us Information For... Media Policy Makers Sickle Cell Disease Quiz Language: English Español (Spanish) Recommend on ... True or False: Only African Americans get sickle cell disease. A True B False 2. True or ...

  3. Mammalian cell biology

    International Nuclear Information System (INIS)

    This section contains summaries of research on mechanisms of lethality and radioinduced changes in mammalian cell properties, new cell systems for the study of the biology of mutation and neoplastic transformation, and comparative properties of ionizing radiations

  4. Anaplastic Large Cell Lymphoma

    Science.gov (United States)

    Anaplastic Large Cell Lymphoma Overview Lymphoma is the most common blood cancer. The two main forms of lymphoma are ... organs, and can accumulate to form tumors. Anaplastic large cell lymphoma (ALCL) is arare type of NHL, ...

  5. What Are Islet Cells?

    Science.gov (United States)

    ... Video Be Part of the Cure Commitment to Stem Cell Research Exercise + Drug Therapy Tibi Creates Garment to Benefit ... Video Be Part of the Cure Commitment to Stem Cell Research Exercise + Drug Therapy Tibi Creates Garment to Benefit ...

  6. NIA Aging Cell Repository

    Data.gov (United States)

    Federal Laboratory Consortium — To facilitate aging research on cells in culture, the NIA provides support for the NIA Aging Cell Repository, located at the Coriell Institute for Medical Research...

  7. Sickle cell anemia

    Science.gov (United States)

    ... for avascular necrosis of the hip Surgery for eye problems Treatment for overuse or abuse of narcotic pain medicines Wound care for leg ulcers Bone marrow or stem cell transplants can cure sickle cell anemia, but this treatment ...

  8. NBD peptide promotes osteoblast differentiation%NBD多肽促进成骨细胞分化的实验研究

    Institute of Scientific and Technical Information of China (English)

    李文锋; 侯树勋; 张伟佳; 洪磊

    2011-01-01

    [ Objective ] To investigate the effect and molecular mechanism of NBD peptide on osteoblast differentiation. [ Method ] The C2C12 cells stimulated with BMP - 2 were used as an in vitro model for studying osteoblast differentiation. Transient transfection, luciferase reporter activity assay and real-time RT-PCR were performed to study the effect of NBD peptide on osteoblast differentiation. [ Result] TNF-a significantly reduced the ALP activities in a dose dependent manner in BMP2-induced C2C12 cells. TNF-a decreased the BMP-2 activity from 7. 12 to 1. 31 fold,and NBD peptide restored it back to 6. 7 fold, while mNBD peptide left it at 1.4 fold. [Conclusion]TNF-a inhibits osteoblast differentiation through the activation of NF-kB. Application of NBD peptide ameliorates this inhibitory effect.%[目的]探讨核因子κB必需分子(NF-κB essential modulator,NEMO)结合的小分子多肽(NEMO binding domain,NBD)通过阻断肿瘤坏死因子-α信号通路影响成骨细胞分化的作用及其分子机制.[方法]应用BMP -2体外诱导鼠肌源细胞C2C12向成骨细胞分化模型,外源添加TNF-α和/或BMP -2细胞因子培养,通过碱性磷酸酶(ALP)活性检测,瞬时转染和基因测定,研究NBD多肽对抗NF-κB活性和改善TNF-α抑制成骨细胞分化的过程.[结果]ALP染色显示NBD多肽能明显阻断TNF-α对C2C12向成骨细胞分化的抑制而促进其分化,荧光素酶活性测定显示TNF-α降低BMP -2活性从7.12倍到1.31倍,而NBD多肽使其恢复到6.7倍和mNBD肽恢复到1.4倍.[结论]TNF-α抑制成骨细胞分化的分子生物机制是通过激活NF-kB阻碍成骨细胞的分化.NBD多肽具有对抗NF-κB活性和改善TNF-α抑制成骨细胞分化过程的作用.

  9. Diagram of Cell to Cell Communication

    Science.gov (United States)

    2002-01-01

    Diagram depicts the importance of cell-cell communication as central to the understanding of cancer growth and progression, the focus of the NASA bioreactor demonstration system (BDS-05) investigation. Microgravity studies will allow us to unravel the signaling and communication between these cells with the host and potential development of therapies for the treatment of cancer metastasis. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: Emory University.

  10. STEM CELLS AND PROTEOMICS

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yong-ming; GUO Tian-nan; HUANG Shi-ang

    2006-01-01

    The distinctive features of proteomics are large-scale and high throughput. The ke