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Sample records for mdx mouse cells

  1. Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse

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    Morgan, J.E.; Hoffman, E.P.; Partridge, T.A.

    1990-01-01

    Dystrophin deficiency in skeletal muscle of the x-linked dystrophic (mdx) mouse can be partially remedied by implantation of normal muscle precursor cells (mpc). However, it is difficult to determine whether this biochemical rescue results in any improvement in the structure or function of the treated muscle, because the vigorous regeneration of mdx muscle more than compensates for the degeneration. By using x-ray irradiation to prevent mpc proliferation, it is possible to study loss of mdx muscle fibers without the complicating effect of simultaneous fiber regeneration. Thus, improvements in fiber survival resulting from any potential therapy can be detected easily. Here, we have implanted normal mpc, obtained from newborn mice, into such preirradiated mdx muscles, finding that it is far more extensively permeated and replaced by implanted mpc than is nonirradiated mdx muscle; this is evident both from analysis of glucose-6-phosphate isomerase isoenzyme markers and from immunoblots and immunostaining of dystrophin in the treated muscles. Incorporation of normal mpc markedly reduces the loss of muscle fibers and the deterioration of muscle structure which otherwise occurs in irradiated mdx muscles. Surprisingly, the regenerated fibers are largely peripherally nucleated, whereas regenerated mouse skeletal muscle fibers are normally centrally nucleated. We attribute this regeneration of apparently normal muscle to the tendency of newborn mouse mpc to recapitulate their neonatal ontogeny, even when grafted into 3-wk-old degenerating muscle

  2. Isolation and characterization of neural stem cells from dystrophic mdx mouse

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    Annese, Tiziana; Corsi, Patrizia; Ruggieri, Simona; Tamma, Roberto; Marinaccio, Christian; Picocci, Sabrina; Errede, Mariella; Specchia, Giorgina; De Luca, Annamaria; Frassanito, Maria Antonia; Desantis, Vanessa; Vacca, Angelo; Ribatti, Domenico; Nico, Beatrice

    2016-01-01

    The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier

  3. Isolation and characterization of neural stem cells from dystrophic mdx mouse

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    Annese, Tiziana [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Corsi, Patrizia [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari (Italy); Ruggieri, Simona; Tamma, Roberto; Marinaccio, Christian [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Picocci, Sabrina [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari (Italy); Errede, Mariella [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Specchia, Giorgina [Department of Emergency and Transplantation, Section of Hematology, University of Bari Medical School, Bari (Italy); De Luca, Annamaria [Department of Bioscience, Biotechnology and Pharmacological Sciences, Section of Pharmacology, University of Bari (Italy); Frassanito, Maria Antonia; Desantis, Vanessa; Vacca, Angelo [Department of Internal Medicine and Oncology, University of Bari Medical School, Bari (Italy); Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); National Cancer Institute “Giovanni Paolo II”, Bari (Italy); Nico, Beatrice, E-mail: beatrice.nico@uniba.it [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy)

    2016-05-01

    The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier

  4. Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

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    Chunli Zhao

    Full Text Available A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

  5. A new immuno- dystrophin-deficient model, the NSG-mdx4Cv mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation

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    Arpke, Robert W.; Darabi, Radbod; Mader, Tara L.; Zhang, Yu; Toyama, Akira; Lonetree, Cara-lin; Nash, Nardina; Lowe, Dawn A.; Perlingeiro, Rita C.R.; Kyba, Michael

    2013-01-01

    Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx4Cv mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx4Cv mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx4Cv recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function, and the utility of the NSG-mdx4Cv model for studies on muscle regeneration and Duchenne muscular dystrophy therapy. PMID:23606600

  6. Plantarflexion Contracture in the mdx Mouse

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    Garlich, Michael W.; Baltgalvis, Kristen A.; Call, Jarrod A.; Dorsey, Lisa L.; Lowe, Dawn A.

    2012-01-01

    Objective Contractures are a major clinical issue for patients with muscular dystrophies. However, it is unknown whether contractures are present in the widely used mdx mouse model of Duchenne muscular dystrophy. Therefore, the objectives of this study were to develop methods to measure muscle contractures in mice, to determine whether plantarflexion contractures are present in mdx mice, and to analyze the composition of the major muscles involved. Design Hindlimbs of eight wild type and six mdx mice were assessed every 2 wks during the course of a 12-wk study. Assessments included range of motion and in vivo torques about the ankle. At the end of the study, mice were euthanized, and muscles were analyzed for composition. Results The mdx mice had ~10 degrees less dorsiflexion, increased passive torque moving the ankle into dorsiflexion, and an increased passive-to-active torque ratio relative to wild type mice. Gastrocnemius muscle composition alterations included increased wet mass, decreased protein content, and increased collagen. Conclusions The results indicate that mdx mice have plantarflexion contractures similar to those seen in children with Duchenne muscular dystrophy. In future studies, these measures can be used to assess strategies to slow the progression of contractures that occur with muscular dystrophies. PMID:21403594

  7. Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophy via paracrine signaling

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    Ji-qing Cao

    2016-01-01

    Full Text Available Adipose-derived stem cells have been shown to promote peripheral nerve regeneration through the paracrine secretion of neurotrophic factors. However, it is unclear whether these cells can promote myogenic differentiation in muscular dystrophy. Adipose-derived stem cells (6 × 10 6 were injected into the gastrocnemius muscle of mdx mice at various sites. Dystrophin expression was found in the muscle fibers. Phosphorylation levels of Akt, mammalian target of rapamycin (mTOR, eIF-4E binding protein 1 and S6 kinase 1 were increased, and the Akt/mTOR pathway was activated. Simultaneously, myogenin levels were increased, whereas cleaved caspase 3 and vimentin levels were decreased. Necrosis and fibrosis were reduced in the muscle fibers. These findings suggest that adipose-derived stem cells promote the regeneration and survival of muscle cells by inhibiting apoptosis and fibrosis, thereby alleviating muscle damage in muscular dystrophy.

  8. Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

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    2013-01-01

    Introduction Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). Methods To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis. Results Surprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration. Conclusions Although WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs. PMID:23295128

  9. Caspase-12 ablation preserves muscle function in the mdx mouse

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    Moorwood, Catherine; Barton, Elisabeth R.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin. Several downstream consequences of dystrophin deficiency are triggers of endoplasmic reticulum (ER) stress, including loss of calcium homeostasis, hypoxia and oxidative stress. During ER stress, misfolded proteins accumulate in the ER lumen and the unfolded protein response (UPR) is triggered, leading to adaptation or apoptosis. We hypothesized that ER stress is heightened in dystrophic muscles and contributes to the pathology of DMD. We observed increases in the ER stress markers BiP and cleaved caspase-4 in DMD patient biopsies, compared with controls, and an increase in multiple UPR pathways in muscles of the dystrophin-deficient mdx mouse. We then crossed mdx mice with mice null for caspase-12, the murine equivalent of human caspase-4, which are resistant to ER stress. We found that deleting caspase-12 preserved mdx muscle function, resulting in a 75% recovery of both specific force generation and resistance to eccentric contractions. The compensatory hypertrophy normally found in mdx muscles was normalized in the absence of caspase-12; this was found to be due to decreased fibre sizes, and not to a fibre type shift or a decrease in fibrosis. Fibre central nucleation was not significantly altered in the absence of caspase-12, but muscle fibre degeneration found in the mdx mouse was reduced almost to wild-type levels. In conclusion, we have identified heightened ER stress and abnormal UPR signalling as novel contributors to the dystrophic phenotype. Caspase-4 is therefore a potential therapeutic target for DMD. PMID:24879640

  10. A Mathematical Model of Skeletal Muscle Disease and Immune Response in the mdx Mouse

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    Abdul Salam Jarrah

    2014-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is a genetic disease that results in the death of affected boys by early adulthood. The genetic defect responsible for DMD has been known for over 25 years, yet at present there is neither cure nor effective treatment for DMD. During early disease onset, the mdx mouse has been validated as an animal model for DMD and use of this model has led to valuable but incomplete insights into the disease process. For example, immune cells are thought to be responsible for a significant portion of muscle cell death in the mdx mouse; however, the role and time course of the immune response in the dystrophic process have not been well described. In this paper we constructed a simple mathematical model to investigate the role of the immune response in muscle degeneration and subsequent regeneration in the mdx mouse model of Duchenne muscular dystrophy. Our model suggests that the immune response contributes substantially to the muscle degeneration and regeneration processes. Furthermore, the analysis of the model predicts that the immune system response oscillates throughout the life of the mice, and the damaged fibers are never completely cleared.

  11. Klotho gene silencing promotes pathology in the mdx mouse model of Duchenne muscular dystrophy

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    Wehling-Henricks, Michelle; Li, Zhenzhi; Lindsey, Catherine; Wang, Ying; Welc, Steven S.; Ramos, Julian N.; Khanlou, Négar; Kuro-o, Makoto; Tidball, James G.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a lethal muscle disease involving progressive loss of muscle regenerative capacity and increased fibrosis. We tested whether epigenetic silencing of the klotho gene occurs in the mdx mouse model of DMD and whether klotho silencing is an important feature of the disease. Our findings show that klotho undergoes muscle-specific silencing at the acute onset of mdx pathology. Klotho experiences increased methylation of CpG sites in its promoter region, which is associated with gene silencing, and increases in a repressive histone mark, H3K9me2. Expression of a klotho transgene in mdx mice restored their longevity, reduced muscle wasting, improved function and greatly increased the pool of muscle-resident stem cells required for regeneration. Reductions of fibrosis in late, progressive stages of the mdx pathology achieved by transgene expression were paralleled by reduced expression of Wnt target genes (axin-2), transforming growth factor-beta (TGF-β1) and collagens types 1 and 3, indicating that Klotho inhibition of the profibrotic Wnt/TGFβ axis underlies its anti-fibrotic effect in aging, dystrophic muscle. Thus, epigenetic silencing of klotho during muscular dystrophy contributes substantially to lost regenerative capacity and increased fibrosis of dystrophic muscle during late progressive stages of the disease. PMID:27154199

  12. Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy.

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    Burns, David P; Roy, Arijit; Lucking, Eric F; McDonald, Fiona B; Gray, Sam; Wilson, Richard J; Edge, Deirdre; O'Halloran, Ken D

    2017-11-01

    Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8 weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO 2 production is equivalent in wild-type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild-type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles. Patients with Duchenne muscular dystrophy (DMD) hypoventilate with consequential arterial blood gas derangement relevant to disease progression. Whereas deficits in DMD diaphragm are recognized, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to perform an analysis of respiratory control in a model of DMD, the mdx mouse. In 8-week-old male wild-type and mdx mice, ventilation and metabolism, carotid body afferent activity, diaphragm muscle force-generating capacity, and muscle fibre size, distribution and centronucleation were determined. Diaphragm EMG activity and responsiveness to chemostimulation was determined. During normoxia, mdx mice hypoventilated, owing to a reduction in tidal volume. Basal CO 2 production was not different between wild-type and mdx mice. Carotid sinus nerve responses to hyperoxia were blunted in mdx, suggesting hypoactivity. However, carotid body, ventilatory and metabolic responses to hypoxia were equivalent in wild-type and

  13. X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD

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    Wakeford, S.; Watt, D.J.; Partridge, T.A.

    1991-01-01

    The mdx mouse, although a genetic and biochemical homologue of human Duchenne muscular dystrophy (DMD), presents a comparatively mild histopathological and clinical phenotype. These differences are partially attributable to the greater efficacy of regeneration in the mdx mouse than in DMD muscle. To lessen this disparity, we have used a single dose of X-irradiation (16 Gy) to inhibit regeneration in one leg of mdx mice. The result is an almost complete block of muscle fiber regeneration leading to progressive loss of muscle fibers and their replacement by loose connective tissue. Surviving fibers are mainly peripherally nucleated and, surprisingly, of large diameter. Thus, X-irradiation converts mdx muscle to a model system in which the degenerative process can be studied in isolation from the complicating effect of myofiber regeneration. This system should be of use for testing methods of alleviating the myofiber degeneration which is common to mdx and DMD

  14. X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD

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    Wakeford, S.; Watt, D.J.; Partridge, T.A. (Charing Cross and Westminster Medical School, London (England))

    1991-01-01

    The mdx mouse, although a genetic and biochemical homologue of human Duchenne muscular dystrophy (DMD), presents a comparatively mild histopathological and clinical phenotype. These differences are partially attributable to the greater efficacy of regeneration in the mdx mouse than in DMD muscle. To lessen this disparity, we have used a single dose of X-irradiation (16 Gy) to inhibit regeneration in one leg of mdx mice. The result is an almost complete block of muscle fiber regeneration leading to progressive loss of muscle fibers and their replacement by loose connective tissue. Surviving fibers are mainly peripherally nucleated and, surprisingly, of large diameter. Thus, X-irradiation converts mdx muscle to a model system in which the degenerative process can be studied in isolation from the complicating effect of myofiber regeneration. This system should be of use for testing methods of alleviating the myofiber degeneration which is common to mdx and DMD.

  15. Protein-Anchoring Therapy of Biglycan for Mdx Mouse Model of Duchenne Muscular Dystrophy.

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    Ito, Mikako; Ehara, Yuka; Li, Jin; Inada, Kosuke; Ohno, Kinji

    2017-05-01

    Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by loss-of-function mutations in DMD encoding dystrophin. No rational therapy is currently available. Utrophin is a paralog of dystrophin and is highly expressed at the neuromuscular junction. In mdx mice, utrophin is naturally upregulated throughout the muscle fibers, which mitigates muscular dystrophy. Protein-anchoring therapy was previously reported, in which a recombinant extracellular matrix (ECM) protein is delivered to and anchored to a specific target using its proprietary binding domains. Being prompted by a report that intramuscular and intraperitoneal injection of an ECM protein, biglycan, upregulates expression of utrophin and ameliorates muscle pathology in mdx mice, protein-anchoring therapy was applied to mdx mice. Recombinant adeno-associated virus serotype 8 (rAAV8) carrying hBGN encoding human biglycan was intravenously injected into 5-week-old mdx mice. The rAAV8-hBGN treatment improved motor deficits and decreased plasma creatine kinase activities. In muscle sections of treated mice, the number of central myonuclei and the distribution of myofiber sizes were improved. The treated mice increased gene expressions of utrophin and β1-syntrophin, as well as protein expressions of biglycan, utrophin, γ-sarcoglycan, dystrobrevin, and α1-syntrophin. The expression of hBGN in the skeletal muscle of the treated mice was 1.34-fold higher than that of the native mouse Bgn (mBgn). The low transduction efficiency and improved motor functions suggest that biglycan expressed in a small number of muscle fibers was likely to have been secreted and anchored to the cell surface throughout the whole muscular fibers. It is proposed that the protein-anchoring strategy can be applied not only to deficiency of an ECM protein as previously reported, but also to augmentation of a naturally induced ECM protein.

  16. Importância do camundongo mdx na fisiopatologia da distrofia muscular de Duchenne The importance of mdx mouse in the pathophysiology of Duchenne's muscular distrophy

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    Sandra Lopes Seixas

    1997-09-01

    Full Text Available O camundongo mdx desenvolve distrofia muscular recessiva ligada ao cromossoma X (locus Xp21.1 e não expressa distrofina. Embora não apresente intensa fibrose do tecido muscular e acúmulo de tecido adiposo, é considerado o modelo animal mais adequado da distrofia muscular de Duchenne. As alterações estruturais no tecido muscular associadas à mionecrose e presença do infiltrado inflamatório com predomínio de linfócitos e monócitos/macrófagos sugerem uma participação do sistema imunológico nesta miopatia. Além disso a modulação na expressão dos componentes da matriz extracelular no microambiente muscular nas várias fases da doença (início, mionecrose, regeneração indicam um papel importante do conjuntivo no direcionamento das células inflamatórias para o foco da lesão muscular. O camundongo mdx coloca-se como um excelente modelo para o estudo dos mecanismos patogenéticos da mionecrose e regeneração na distrofia muscular de Duchenne, possibilitando inclusive o desenvolvimento de estratégias terapêuticas mais adequadas.The mdx mouse develop an X-linked recessive muscular dystrophy (locus Xp21.1 and lack dystrophin expression. Despite showing less intense myofibrosis and scarce deposition of fatty tissue, mdx mice are considered an adequate animal model for studies on the pathogenesis of Duchenne-type muscular dystrophy. Marked histological alterations in the muscular tissues associated to myonecrosis and inflammatory mononuclear cell infiltrate (lymphocytes, monocytes/macrophages suggest a participation of the immune system in this myopathy. Modulation of the extracellular matrix (ECM components in the muscular tissue during all phases (onset, myonecrosis and regeneration of disease, indicate an important role for the ECM driving inflammatory cells to the foci of lesion. Therefore mdx mice should be regarded as an important tool for studies on pathogenetic mechanisms of Duchenne-type muscular dystrophy. Such

  17. IL-6 signaling blockade increases inflammation but does not affect muscle function in the mdx mouse

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    Kostek Matthew C

    2012-06-01

    Full Text Available Abstract Background IL-6 is a pleiotropic cytokine that modulates inflammatory responses and plays critical roles in muscle maintenance and remodeling. In the mouse model (mdx of Duchenne Muscular Dystrophy, IL-6 and muscle inflammation are elevated, which is believed to contribute to the chronic inflammation and failure of muscle regeneration in DMD. The purpose of the current study was to examine the effect of blocking IL-6 signaling on the muscle phenotype including muscle weakness and pathology in the mdx mouse. Methods A monoclonal antibody against the IL-6 receptor (IL-6r mAb that blocks local and systemic IL-6 signaling was administered to mdx and BL-10 mice for 5 weeks and muscle function, histology, and inflammation were examined. Results IL-6r mAb treatment increased mdx muscle inflammation including total inflammation score and ICAM-1 positive lumens in muscles. There was no significant improvement in muscle strength nor muscle pathology due to IL-6r mAb treatment in mdx mice. Conclusions These results showed that instead of reducing inflammation, IL-6 signaling blockade for 5 weeks caused an increase in muscle inflammation, with no significant change in indices related to muscle regeneration and muscle function. The results suggest a potential anti-inflammatory instead of the original hypothesized pro-inflammatory role of IL-6 signaling in the mdx mice.

  18. Skeletal muscle fibrosis in the mdx/utrn+/- mouse validates its suitability as a murine model of Duchenne muscular dystrophy.

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    Gutpell, Kelly M; Hrinivich, William T; Hoffman, Lisa M

    2015-01-01

    Various therapeutic approaches have been studied for the treatment of Duchenne muscular dystrophy (DMD), but none of these approaches have led to significant long-term effects in patients. One reason for this observed inefficacy may be the use of inappropriate animal models for the testing of therapeutic agents. The mdx mouse is the most widely used murine model of DMD, yet it does not model the fibrotic progression observed in patients. Other murine models of DMD are available that lack one or both alleles of utrophin, a functional analog of dystrophin. The aim of this study was to compare fibrosis and myofiber damage in the mdx, mdx/utrn+/- and double knockout (dko) mouse models. We used Masson's trichrome stain and percentage of centrally-nucleated myofibers as indicators of fibrosis and myofiber regeneration, respectively, to assess disease progression in diaphragm and gastrocnemius muscles harvested from young and aged wild-type, mdx, mdx/utrn+/- and dko mice. Our results indicated that eight week-old gastrocnemius muscles of both mdx/utrn+/- and dko hind limb developed fibrosis whereas age-matched mdx gastrocnemius muscle did not (p = 0.002). The amount of collagen found in the mdx/utrn+/- diaphragm was significantly higher than that found in the corresponding diaphragm muscles of wild-type animals, but not of mdx animals (p = 0.0003). Aged mdx/utrn+/- mice developed fibrosis in both diaphragm and gastrocnemius muscles compared to wild-type controls (p = 0.003). Mdx diaphragm was fibrotic in aged mice as well (p = 0.0235), whereas the gastrocnemius muscle in these animals was not fibrotic. We did not measure a significant difference in collagen staining between wild-type and mdx gastrocnemius muscles. The results of this study support previous reports that the moderately-affected mdx/utrn+/- mouse is a better model of DMD, and we show here that this difference is apparent by 2 months of age.

  19. Evaluation of the behavioral characteristics of the mdx mouse model of duchenne muscular dystrophy through operant conditioning procedures.

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    Lewon, Matthew; Peters, Christina M; Van Ry, Pam M; Burkin, Dean J; Hunter, Kenneth W; Hayes, Linda J

    2017-09-01

    The mdx mouse is an important nonhuman model for Duchenne muscular dystrophy (DMD) research. Characterizing the behavioral traits of the strain relative to congenic wild-type (WT) mice may enhance our understanding of the cognitive deficits observed in some humans with DMD and contribute to treatment development and evaluation. In this paper we report the results of a number of experiments comparing the behavior of mdx to WT mice in operant conditioning procedures designed to assess learning and memory. We found that mdx outperformed WT in all learning and memory tasks involving food reinforcement, and this appeared to be related to the differential effects of the food deprivation motivating operation on mdx mice. Conversely, WT outperformed mdx in an escape/avoidance learning task. These results suggest motivational differences between the strains and demonstrate the potential utility of operant conditioning procedures in the assessment of the behavioral characteristics of the mdx mouse. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Ventilatory chemosensory drive is blunted in the mdx mouse model of Duchenne Muscular Dystrophy (DMD.

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    Matias Mosqueira

    Full Text Available Duchenne Muscular Dystrophy (DMD is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure.

  1. Dystropathology increases energy expenditure and protein turnover in the Mdx mouse model of Duchenne muscular dystrophy

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    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the diet...

  2. Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice

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    Huxley Clare

    2005-03-01

    Full Text Available Abstract Background Sporadic and sometimes contradictory studies have indicated changes in satellite cell behaviour associated with the progressive nature of human Duchenne muscular dystrophy (DMD. Satellite cell proliferation and number are reportedly altered in DMD and the mdx mouse model. We recently found that satellite cells in MSVski transgenic mice, a muscle hypertrophy model showing progressive muscle degeneration, display a severe ageing-related differentiation defect in vitro. We tested the hypothesis that similar changes contribute to the gradual loss of muscle function with age in mdx and PMP22 mice, a model of human motor and sensory neuropathy type 1A (HMSN1A. Methods Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain. Results Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation. Conclusion Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals

  3. Dystropathology Increases Energy Expenditure and Protein Turnover in the Mdx Mouse Model of Duchenne Muscular Dystrophy

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    Radley-Crabb, Hannah G.; Marini, Juan C.; Sosa, Horacio A.; Castillo, Liliana I.; Grounds, Miranda D.; Fiorotto, Marta L.

    2014-01-01

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles. PMID:24586653

  4. Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy.

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    Percival, Justin M; Whitehead, Nicholas P; Adams, Marvin E; Adamo, Candace M; Beavo, Joseph A; Froehner, Stanley C

    2012-09-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO

  5. Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse.

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    Fletcher, Sue; Honeyman, Kaite; Fall, Abbie M; Harding, Penny L; Johnsen, Russell D; Steinhaus, Joshua P; Moulton, Hong M; Iversen, Patrick L; Wilton, Stephen D

    2007-09-01

    Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.

  6. Galectin-1 Protein Therapy Prevents Pathology and Improves Muscle Function in the mdx Mouse Model of Duchenne Muscular Dystrophy.

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    Van Ry, Pam M; Wuebbles, Ryan D; Key, Megan; Burkin, Dean J

    2015-08-01

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by mutations in the dystrophin gene, leading to the loss of a critical component of the sarcolemmal dystrophin glycoprotein complex. Galectin-1 is a small 14 kDa protein normally found in skeletal muscle and has been shown to be a modifier of immune response, muscle repair, and apoptosis. Galectin-1 levels are elevated in the muscle of mouse and dog models of DMD. Together, these findings led us to hypothesize that Galectin-1 may serve as a modifier of disease progression in DMD. To test this hypothesis, recombinant mouse Galectin-1 was produced and used to treat myogenic cells and the mdx mouse model of DMD. Here we show that intramuscular and intraperitoneal injections of Galectin-1 into mdx mice prevented pathology and improved muscle function in skeletal muscle. These improvements were a result of enhanced sarcolemmal stability mediated by elevated utrophin and α7β1 integrin protein levels. Together our results demonstrate for the first time that Galectin-1 may serve as an exciting new protein therapeutic for the treatment of DMD.

  7. Abnormal GABAA-mediated metabolic response in the MDX mouse - an explanation for the mental deficit in Duchenne muscular dystrophy?

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    Rae, C.; Bubb, W.A.; Maitland, A.; Head, S.I.

    2001-01-01

    Full text: Duchenne muscular dystrophy is an X-linked disorder associated with lack of the 728 kDa protein dystrophin. In addition to the well-known muscle wasting, sufferers also experience a 15 point downshift in IQ. Recently reduced clustering of GABA A receptors in cerebellar Purkinje and hippocampal CA1 neurons has been shown in the murine homologue of DMD, the mdx mouse. In this work, the functional efficacy of GABA A receptors in mdx mice (C57B1/10Sc-Sn-mdx) and control was tested by examining the metabolism of [1- 13 C]D-glucose under both normoxic and hypoxic conditions and also by examining the metabolic response to the GABA A agonist muscimol (5-aminomethyl-3-hydroxyisoxazole). Although total measured [ 13 C] was identical in mdx cf. control mice, the fractional enrichment of all metabolites was increased in mdx mice, suggesting decreased inhibitory input in these animals. Further, although flux into metabolites from [1- 13 C]D-glucose decreased as expected in control mice in the presence of muscimol, the GABA a agonist had weaker effect in mdx mice, consistent with weaker GABA A activation. Finally, the response of mdx mouse brain tissue slices to mild hypoxia (partially mediated by GABA A ) was altered cf. control mice, with increased production of lactate and decreased flux into Krebs cycle intermediates. These data are consistent with a functional lesion of a subset of GABA A receptors in DMD

  8. Cell surface and gene expression regulation molecules in dystrophinopathy: mdx vs. Duchenne

    Directory of Open Access Journals (Sweden)

    RICARDO FADIC

    2005-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is secondary to loss-of-function mutations in the dystrophin gene. The causes underlying the progression of DMD, differential muscle involvement, and the discrepancies in phenotypes among species with the same genetic defect are not understood. The mdx mouse, an animal model with dystrophin mutation, has a milder phenotype. This article reviews the available information on expression of signaling-related molecules in DMD and mdx. Extracellular matrix proteoglycans, growth factors, integrins, caveolin-3, and neuronal nitric oxide synthase expression do not show significant differences. Calcineurin is inconsistently activated in mdx, which is associated with lack of cardiomyopathy, compared to the permanent calcineurin activation in mdx/utrophin null mice that have a DMD-like cardiomyopathy. Levels of focal adhesion kinase (FAK and extracellular regulated kinases (ERKs differ among mdx and DMD. Further work is needed to identify the point of discrepancy in these signaling molecules' pathways in dystrophynopathies.

  9. Matrix metalloproteinase-9 inhibition improves proliferation and engraftment of myogenic cells in dystrophic muscle of mdx mice.

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    Sajedah M Hindi

    Full Text Available Duchenne muscular dystrophy (DMD caused by loss of cytoskeletal protein dystrophin is a devastating disorder of skeletal muscle. Primary deficiency of dystrophin leads to several secondary pathological changes including fiber degeneration and regeneration, extracellular matrix breakdown, inflammation, and fibrosis. Matrix metalloproteinases (MMPs are a group of extracellular proteases that are involved in tissue remodeling, inflammation, and development of interstitial fibrosis in many disease states. We have recently reported that the inhibition of MMP-9 improves myopathy and augments myofiber regeneration in mdx mice (a mouse model of DMD. However, the mechanisms by which MMP-9 regulates disease progression in mdx mice remain less understood. In this report, we demonstrate that the inhibition of MMP-9 augments the proliferation of satellite cells in dystrophic muscle. MMP-9 inhibition also causes significant reduction in percentage of M1 macrophages with concomitant increase in the proportion of promyogenic M2 macrophages in mdx mice. Moreover, inhibition of MMP-9 increases the expression of Notch ligands and receptors, and Notch target genes in skeletal muscle of mdx mice. Furthermore, our results show that while MMP-9 inhibition augments the expression of components of canonical Wnt signaling, it reduces the expression of genes whose products are involved in activation of non-canonical Wnt signaling in mdx mice. Finally, the inhibition of MMP-9 was found to dramatically improve the engraftment of transplanted myoblasts in skeletal muscle of mdx mice. Collectively, our study suggests that the inhibition of MMP-9 is a promising approach to stimulate myofiber regeneration and improving engraftment of muscle progenitor cells in dystrophic muscle.

  10. Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model

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    Jahnke Vanessa E

    2012-08-01

    Full Text Available Abstract Background Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice. Methods Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR, separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s' effects. Results We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage. Conclusions The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice.

  11. Expression patterns of regulatory RNAs, including lncRNAs and tRNAs, during postnatal growth of normal and dystrophic (mdx) mouse muscles, and their response to taurine treatment.

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    Butchart, Lauren C; Terrill, Jessica R; Rossetti, Giulia; White, Robert; Filipovska, Aleksandra; Grounds, Miranda D

    2018-06-01

    Post-natal skeletal muscle growth in mice is very rapid and involves complex changes in many cells types over the first 6 weeks of life. The acute onset of dystropathology also occurs around 3 weeks of age in the mdx mouse model of the human disease Duchenne Muscular Dystrophy (DMD). This study investigated (i) alterations in expression patterns of regulatory non-coding RNAs (ncRNAs) in vivo, including miRNAs, lncRNAs and tRNAs, during early growth of skeletal muscles in normal control C57Bl/10Scsn (C57) compared with dystrophic mdx mice from 2 to 6 weeks of postnatal age, and revealed inherent differences in vivo for levels of 3 ncRNAs between C57 and mdx muscles before the onset of dystropathology. Since the amino acid taurine has many benefits and reduces disease severity in mdx mice, this study also (ii) determined the impact of taurine treatment on these expression patterns in mdx muscles at the onset of dystropathology (3 weeks) and after several bouts of myonecrosis and regeneration (6 weeks). Taurine treatment of mdx mice only altered ncRNA levels when administered from 18 days to 6 weeks of age, but a deficiency in tRNA levels was rectified earlier in mdx skeletal muscles treated from 14 days to 3 weeks. Myogenesis in tissue culture was also used to (iii) compare ncRNA expression patterns for both strains, and (iv) the response to taurine treatment. These analyses revealed intrinsic differences in ncRNA expression patterns during myogenesis between strains, as well as increased sensitivity of mdx ncRNA levels to taurine treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Taurine deficiency, synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy.

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    Terrill, Jessica R; Grounds, Miranda D; Arthur, Peter G

    2015-09-01

    The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  13. Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.

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    Watanabe, M; Wallace, P K; Keler, T; Deo, Y M; Akewanlop, C; Hayes, D F

    1999-02-01

    MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcgammaRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcgammaRI is expressed on human monocytes, macrophages, and IFN-gamma activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions. Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit. Both BsAb MDX-210 (via FcgammaRI) and MoAb 520C9 (mouse IgG1, via FcgammaRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF. BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.

  14. Amitriptyline is efficacious in ameliorating muscle inflammation and depressive symptoms in the mdx mouse model of Duchenne muscular dystrophy.

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    Manning, Jennifer; Kulbida, Rebecca; Rai, Prerana; Jensen, Lindsay; Bouma, Judith; Singh, Sanjay P; O'Malley, Dervla; Yilmazer-Hanke, Deniz

    2014-10-01

    Mutations in the structural protein dystrophin underlie muscular dystrophies characterized by progressive deterioration of muscle function. Dystrophin-deficient mdx mice are considered a model for Duchenne muscular dystrophy (DMD). Individuals with DMD are also susceptible to mood disorders, such as depression and anxiety. Therefore, the study objectives were to investigate the effects of the tricyclic antidepressant amitriptyline on mood, learning, central cytokine expression and skeletal muscle inflammation in mdx mice. Amitriptyline-induced effects (10 mg kg(-1) daily s.c. injections, 25 days) on the behaviour of mdx mice were investigated using the open field arena and tail suspension tests. The effects of chronic amitriptyline treatment on inflammatory markers were studied in the muscle and plasma of mdx mice, and mood-associated monoamine and cytokine concentrations were measured in the amygdala, hippocampus, prefrontal cortex, striatum, hypothalamus and midbrain. The mdx mice exhibited increased levels of anxiety and depressive-like behaviour compared with wild-type mice. Amitriptyline treatment had anxiolytic and antidepressant effects in mdx mice associated with elevations in serotonin levels in the amygdala and hippocampus. Inflammation in mdx skeletal muscle tissue was also reduced following amitriptyline treatment as indicated by decreased immune cell infiltration of muscle and lower levels of the pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6 in the forelimb flexors. Interleukin-6 mRNA expression was remarkably reduced in the amygdala of mdx mice by chronic amitriptyline treatment. Positive effects of amitriptyline on mood, in addition to its anti-inflammatory effects in skeletal muscle, may make it an attractive therapeutic option for individuals with DMD. © 2014 The Authors. Experimental Physiology © 2014 The Physiological Society.

  15. Fetal microchimeric cells in a fetus-treats-its-mother paradigm do not contribute to dystrophin production in serially parous mdx females.

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    Seppanen, Elke Jane; Hodgson, Samantha Susan; Khosrotehrani, Kiarash; Bou-Gharios, George; Fisk, Nicholas M

    2012-10-10

    Throughout every pregnancy, genetically distinct fetal microchimeric stem/progenitor cells (FMCs) engraft in the mother, persist long after delivery, and may home to damaged maternal tissues. Phenotypically normal fetal lymphoid progenitors have been described to develop in immunodeficient mothers in a fetus-treats-its-mother paradigm. Since stem cells contribute to muscle repair, we assessed this paradigm in the mdx mouse model of Duchenne muscular dystrophy. mdx females were bred serially to either ROSAeGFP males or mdx males to obtain postpartum microchimeras that received either wild-type FMCs or dystrophin-deficient FMCs through serial gestations. To enhance regeneration, notexin was injected into the tibialis anterior of postpartum mice. FMCs were detected by qPCR at a higher frequency in injected compared to noninjected side muscle (P=0.02). However, the number of dystrophin-positive fibers was similar in mothers delivering wild-type compared to mdx pups. In addition, there was no correlation between FMC detection and percentage dystrophin, and no GFP+ve FMCs were identified that expressed dystrophin. In 10/11 animals, GFP+ve FMCs were detected by immunohistochemistry, of which 60% expressed CD45 with 96% outside the basal lamina defining myofiber contours. Finally we confirmed lack of FMC contribution to statellite cells in postpartum mdx females mated with Myf5-LacZ males. We conclude that the FMC contribution to regenerating muscles is insufficient to have a functional impact.

  16. A Reduction in Selenoprotein S Amplifies the Inflammatory Profile of Fast-Twitch Skeletal Muscle in the mdx Dystrophic Mouse

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    Craig Robert Wright

    2017-01-01

    Full Text Available Excessive inflammation is a hallmark of muscle myopathies, including Duchenne muscular dystrophy (DMD. There is interest in characterising novel genes that regulate inflammation due to their potential to modify disease progression. Gene polymorphisms in Selenoprotein S (Seps1 are associated with elevated proinflammatory cytokines, and in vitro SEPS1 is protective against inflammatory stress. Given that SEPS1 is highly expressed in skeletal muscle, we investigated whether the genetic reduction of Seps1 exacerbated inflammation in the mdx mouse. F1 male mdx mice with a heterozygous Seps1 deletion (mdx:Seps1−/+ were generated. The mdx:Seps1−/+ mice had a 50% reduction in SEPS1 protein expression in hindlimb muscles. In the extensor digitorum longus (EDL muscles, mRNA expression of monocyte chemoattractant protein 1 (Mcp-1 (P=0.034, macrophage marker F4/80 (P=0.030, and transforming growth factor-β1 (Tgf-β1 (P=0.056 were increased in mdx:Seps1−/+ mice. This was associated with a reduction in muscle fibre size; however, ex vivo EDL muscle strength and endurance were unaltered. In dystrophic slow twitch soleus muscles, SEPS1 reduction had no effect on the inflammatory profile nor function. In conclusion, the genetic reduction of Seps1 appears to specifically exacerbate the inflammatory profile of fast-twitch muscle fibres, which are typically more vulnerable to degeneration in dystrophy.

  17. A Reduction in Selenoprotein S Amplifies the Inflammatory Profile of Fast-Twitch Skeletal Muscle in the mdx Dystrophic Mouse.

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    Wright, Craig Robert; Allsopp, Giselle Larissa; Addinsall, Alex Bernard; McRae, Natasha Lee; Andrikopoulos, Sofianos; Stupka, Nicole

    2017-01-01

    Excessive inflammation is a hallmark of muscle myopathies, including Duchenne muscular dystrophy (DMD). There is interest in characterising novel genes that regulate inflammation due to their potential to modify disease progression. Gene polymorphisms in Selenoprotein S ( Seps1 ) are associated with elevated proinflammatory cytokines, and in vitro SEPS1 is protective against inflammatory stress. Given that SEPS1 is highly expressed in skeletal muscle, we investigated whether the genetic reduction of Seps1 exacerbated inflammation in the mdx mouse. F1 male mdx mice with a heterozygous Seps1 deletion ( mdx : Seps1 -/+ ) were generated. The mdx:Seps1 -/+ mice had a 50% reduction in SEPS1 protein expression in hindlimb muscles. In the extensor digitorum longus (EDL) muscles, mRNA expression of monocyte chemoattractant protein 1 ( Mcp-1 ) ( P = 0.034), macrophage marker F4/80 ( P = 0.030), and transforming growth factor-β1 ( Tgf-β1 ) ( P = 0.056) were increased in mdx:Seps1 -/+ mice. This was associated with a reduction in muscle fibre size; however, ex vivo EDL muscle strength and endurance were unaltered. In dystrophic slow twitch soleus muscles, SEPS1 reduction had no effect on the inflammatory profile nor function. In conclusion, the genetic reduction of Seps1 appears to specifically exacerbate the inflammatory profile of fast-twitch muscle fibres, which are typically more vulnerable to degeneration in dystrophy.

  18. Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy.

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    Terrill, Jessica R; Pinniger, Gavin J; Graves, Jamie A; Grounds, Miranda D; Arthur, Peter G

    2016-06-01

    Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N-acetyl cysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l-2-oxothiazolidine-4-carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex

  19. Mutation types and aging differently affect revertant fiber expansion in dystrophic mdx and mdx52 mice.

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    Yusuke Echigoya

    Full Text Available Duchenne muscular dystrophy (DMD, one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs, as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23 and mdx52 mice (containing deletion mutation of exon 52 with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.

  20. Levels of α7 integrin and laminin-α2 are increased following prednisone treatment in the mdx mouse and GRMD dog models of Duchenne muscular dystrophy

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    Ryan D. Wuebbles

    2013-09-01

    Duchenne muscular dystrophy (DMD is a fatal neuromuscular disease for which there is no cure and limited treatment options. Prednisone is currently the first line treatment option for DMD and studies have demonstrated that it improves muscle strength. Although prednisone has been used for the treatment of DMD for decades, the mechanism of action of this drug remains unclear. Recent studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD and is therefore a target for drug-based therapies. In this study we examined whether prednisone increased α7β1 integrin levels in mdx mouse and GRMD dog models and myogenic cells from humans with DMD. Our results show that prednisone promotes an increase in α7 integrin protein in cultured myogenic cells and in the muscle of mdx and GRMD animal models of DMD. The prednisone-mediated increase in α7 integrin was associated with increased laminin-α2 in prednisone-treated dystrophin-deficient muscle. Together, our results suggest that prednisone acts in part through increased merosin in the muscle basal lamina and through sarcolemmal stabilization of α7β1 integrin in dystrophin-deficient muscle. These results indicate that therapies that target an increase in muscle α7β1 integrin, its signaling pathways and/or laminin could be therapeutic in DMD.

  1. Pre-clinical evaluation of N-acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Pinniger, Gavin J; Terrill, Jessica R; Assan, Evanna B; Grounds, Miranda D; Arthur, Peter G

    2017-12-01

    Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a therapeutic intervention for DMD boys, but potential adverse effects of NAC have not been widely investigated. We used young (6 weeks old) growing mdx mice to investigate the capacity of NAC supplementation (2% in drinking water for 6 weeks) to improve dystrophic muscle function and to explore broader systemic effects of NAC treatment. NAC treatment improved normalised measures of muscle function, and decreased inflammation and oxidative stress, but significantly reduced body weight gain, muscle weight and liver weight. Unexpected significant adverse effects of NAC on body and muscle weights indicate that interpretation of muscle function based on normalised force measures should be made with caution and careful consideration is needed when proposing the use of NAC as a therapeutic treatment for young DMD boys. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 weeks old) mdx and non-dystrophic C57 mice receiving 2% NAC in drinking water for 6 weeks were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 week old mice were anaesthetised and extensor digitorum longus (EDL) muscles were excised for functional analysis and tissues were sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC-treated mdx [3.13 (0.58) vs 4.87 (0.78) g body weight (bw) -1 ; P muscles [9.80 (2.27) vs 13.07 (3.37) N cm -2 ; P = 0

  2. Increased plasma lipid levels exacerbate muscle pathology in the mdx mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Milad, Nadia; White, Zoe; Tehrani, Arash Y; Sellers, Stephanie; Rossi, Fabio M V; Bernatchez, Pascal

    2017-09-12

    Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin expression and leads to severe ambulatory and cardiac function decline. However, the dystrophin-deficient mdx murine model of DMD only develops a very mild form of the disease. Our group and others have shown vascular abnormalities in animal models of MD, a likely consequence of the fact that blood vessels express the same dystrophin-associated glycoprotein complex (DGC) proteins as skeletal muscles. To test the blood vessel contribution to muscle damage in DMD, mdx 4cv mice were given elevated lipid levels via apolipoprotein E (ApoE) gene knockout combined with normal chow or lipid-rich Western diets. Ambulatory function and heart function (via echocardiogram) were assessed at 4 and 7 months of age. After sacrifice, muscle histology and aortic staining were used to assess muscle pathology and atherosclerosis development, respectively. Plasma levels of total cholesterol, high-density lipoprotein (HDL), triglycerides, and creatine kinase (CK) were also measured. Although there was an increase in left ventricular heart volume in mdx-ApoE mice compared to that in mdx mice, parameters of heart function were not affected. Compared with wild-type and ApoE-null, only mdx-ApoE KO mice showed significant ambulatory dysfunction. Despite no significant difference in plasma CK, histological analyses revealed that elevated plasma lipids in chow- and Western diet-fed mdx-ApoE mice was associated with severe exacerbation of muscle pathology compared to mdx mice: significant increase in myofiber damage and fibrofatty replacement in the gastrocnemius and triceps brachii muscles, more reminiscent of human DMD pathology. Finally, although both ApoE and mdx-ApoE groups displayed increased plasma lipids, mdx-ApoE exhibited atherosclerotic plaque deposition equal to or less than that of ApoE mice. Since others have shown that lipid abnormalities correlate with DMD severity, our data suggest that plasma lipids could be

  3. Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

    Science.gov (United States)

    Gazzerro, Elisabetta; Baldassari, Simona; Assereto, Stefania; Fruscione, Floriana; Pistorio, Angela; Panicucci, Chiara; Volpi, Stefano; Perruzza, Lisa; Fiorillo, Chiara; Minetti, Carlo; Traggiai, Elisabetta; Grassi, Fabio; Bruno, Claudio

    2015-12-01

    Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-β and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. Identification of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography

    Science.gov (United States)

    Klyen, Blake R.; Shavlakadze, Thea; Radley-Crabb, Hannah G.; Grounds, Miranda D.; Sampson, David D.

    2011-07-01

    Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with co-registered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy.

  5. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse

    Energy Technology Data Exchange (ETDEWEB)

    Love, D.R.; Marsden, R.F.; Bloomfield, J.F.; Davies, K.E. (John Radcliffe Hospital, Oxford (England)); Morris, G.E.; Ellis, J.M. (North East Wales Inst., Deeside, Wales (England)); Fairbrother, U.; Edwards, Y.H. (Univ. College London (England)); Slater, C.P. (Newcastle General Hospital, Newcastle-upon-Tyne (England)); Parry, D.J. (Univ. of Ottawa, Ontario (Canada))

    1991-04-15

    The authors have previously reported a dystrophin-related locus (DMDL for Duchenne muscular dystrophy-like) on human chromosome 6 that maps close to the dy mutation on mouse chromosome 10. Here they show that this gene is expressed in a wide range of tissues at varying levels. The transcript is particularly abundant in several human fetal tissues, including heart, placenta, and intestine. Studies with antisera raised against a DMDL fusion protein identify a 400,000 M{sub r} protein in all mouse tissues tested, including those of mdx and dy mice. Unlike the dystrophin gene, the DMDL gene transcript is not differentially spliced at the 3{prime} end in either fetal muscle or brain.

  6. Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle

    DEFF Research Database (Denmark)

    Steinberger, Martin; Föller, Michael; Vogelgesang, Silke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety...... of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber...... size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30–50 %. Muscles from sgk1 -/- mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1...

  7. The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse

    Directory of Open Access Journals (Sweden)

    Aiping Zhang

    2015-01-01

    Full Text Available Phosphorodiamidate morpholino oligonucleotides (PMO are used as a promising exon-skipping gene therapy for Duchenne muscular dystrophy (DMD. One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23. We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10 mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg. Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI. The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at days 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO.

  8. Comparative proteomic profiling of soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscles from the mdx mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Carberry, Steven; Brinkmeier, Heinrich; Zhang, Yaxin; Winkler, Claudia K; Ohlendieck, Kay

    2013-09-01

    Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20-25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein

  9. The importance of mdx mouse in the pathophysiology of Duchenne's muscular distrophy

    OpenAIRE

    Seixas, Sandra Lopes; Lagrota-Cândido, Jussara; Savino, Wilson; Quirico-Santos, Thereza

    1997-01-01

    O camundongo mdx desenvolve distrofia muscular recessiva ligada ao cromossoma X (locus Xp21.1) e não expressa distrofina. Embora não apresente intensa fibrose do tecido muscular e acúmulo de tecido adiposo, é considerado o modelo animal mais adequado da distrofia muscular de Duchenne. As alterações estruturais no tecido muscular associadas à mionecrose e presença do infiltrado inflamatório com predomínio de linfócitos e monócitos/macrófagos sugerem uma participação do sistema imunológico nest...

  10. Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis.

    Science.gov (United States)

    Cabrera, Daniel; Gutiérrez, Jaime; Cabello-Verrugio, Claudio; Morales, Maria Gabriela; Mezzano, Sergio; Fadic, Ricardo; Casar, Juan Carlos; Hancke, Juan L; Brandan, Enrique

    2014-01-01

    Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy. mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice. mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells. These results suggest that andrographolide could be used to improve quality of life in individuals with DMD.

  11. A mouse anti-myostatin antibody increases muscle mass and improves muscle strength and contractility in the mdx mouse model of Duchenne muscular dystrophy and its humanized equivalent, domagrozumab (PF-06252616), increases muscle volume in cynomolgus monkeys.

    Science.gov (United States)

    St Andre, Michael; Johnson, Mark; Bansal, Prashant N; Wellen, Jeremy; Robertson, Andrew; Opsahl, Alan; Burch, Peter M; Bialek, Peter; Morris, Carl; Owens, Jane

    2017-11-09

    The treatments currently approved for Duchenne muscular dystrophy (DMD), a progressive skeletal muscle wasting disease, address the needs of only a small proportion of patients resulting in an urgent need for therapies that benefit all patients regardless of the underlying mutation. Myostatin is a member of the transforming growth factor-β (TGF-β) family of ligands and is a negative regulator of skeletal muscle mass. Loss of myostatin has been shown to increase muscle mass and improve muscle function in both normal and dystrophic mice. Therefore, myostatin blockade via a specific antibody could ameliorate the muscle weakness in DMD patients by increasing skeletal muscle mass and function, thereby reducing patients' functional decline. A murine anti-myostatin antibody, mRK35, and its humanized analog, domagrozumab, were developed and their ability to inhibit several TGB-β ligands was measured using a cell-based Smad-activity reporter system. Normal and mdx mice were treated with mRK35 to examine the antibody's effect on body weight, lean mass, muscle weights, grip strength, ex vivo force production, and fiber size. The humanized analog (domagrozumab) was tested in non-human primates (NHPs) for changes in skeletal muscle mass and volume as well as target engagement via modulation of circulating myostatin. Both the murine and human antibodies are specific and potent inhibitors of myostatin and GDF11. mRK35 is able to increase body weight, lean mass, and muscle weights in normal mice. In mdx mice, mRK35 significantly increased body weight, muscle weights, grip strength, and ex vivo force production in the extensor digitorum longus (EDL) muscle. Further, tibialis anterior (TA) fiber size was significantly increased. NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume and exhibited increased circulating levels of myostatin demonstrating target engagement. We demonstrated that the potent anti-myostatin antibody mRK35 and

  12. SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Sarathy, Apurva; Wuebbles, Ryan D; Fontelonga, Tatiana M; Tarchione, Ashley R; Mathews Griner, Lesley A; Heredia, Dante J; Nunes, Andreia M; Duan, Suzann; Brewer, Paul D; Van Ry, Tyler; Hennig, Grant W; Gould, Thomas W; Dulcey, Andrés E; Wang, Amy; Xu, Xin; Chen, Catherine Z; Hu, Xin; Zheng, Wei; Southall, Noel; Ferrer, Marc; Marugan, Juan; Burkin, Dean J

    2017-06-07

    Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD. Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.

  13. EPA protects against muscle damage in the mdx mouse model of Duchenne muscular dystrophy by promoting a shift from the M1 to M2 macrophage phenotype.

    Science.gov (United States)

    Carvalho, Samara Camaçari de; Apolinário, Leticia Montanholi; Matheus, Selma Maria Michelin; Santo Neto, Humberto; Marques, Maria Julia

    2013-11-15

    In dystrophic mdx mice and in Duchenne muscular dystrophy, inflammation contributes to myonecrosis. Previously, we demonstrated that eicosapentaenoic acid (EPA) decreased inflammation and necrosis in dystrophic muscle. In the present study, we examined the effects of EPA and the corticoid deflazacort (DFZ) as modulators of M1 (iNOS-expressing cells) and M2 (CD206-expressing cells) macrophages. Mdx mice (14 days old) received EPA or DFZ for 16 days. The diaphragm, biceps brachii and quadriceps muscles were studied. Immunofluorescence, immunoblotting and ELISA assays showed that EPA increased interleucin-10, reduced interferon-γ and was more effective than DFZ in promoting a shift from M1 to M2. © 2013.

  14. GLPG0492, a novel selective androgen receptor modulator, improves muscle performance in the exercised-mdx mouse model of muscular dystrophy.

    Science.gov (United States)

    Cozzoli, Anna; Capogrosso, Roberta Francesca; Sblendorio, Valeriana Teresa; Dinardo, Maria Maddalena; Jagerschmidt, Catherine; Namour, Florence; Camerino, Giulia Maria; De Luca, Annamaria

    2013-06-01

    Anabolic drugs may counteract muscle wasting and dysfunction in Duchenne muscular dystrophy (DMD); however, steroids have unwanted side effects. We focused on GLPG0492, a new non-steroidal selective androgen receptor modulator that is currently under development for musculo-skeletal diseases such as sarcopenia and cachexia. GLPG0492 was tested in the exercised mdx mouse model of DMD in a 4-week trial at a single high dose (30 mg/kg, 6 day/week s.c.), and the results were compared with those from the administration of α-methylprednisolone (PDN; 1 mg/kg, i.p.) and nandrolone (NAND, 5 mg/kg, s.c.). This assessment was followed by a 12-week dose-dependence study (0.3-30 mg/kg s.c.). The outcomes were evaluated in vivo and ex vivo on functional, histological and biochemical parameters. Similar to PDN and NAND, GLPG0492 significantly increased mouse strength. In acute exhaustion tests, a surrogate of the 6-min walking test used in DMD patients, GLPG0492 preserved running performance, whereas vehicle- or comparator-treated animals showed a significant increase in fatigue (30-50%). Ex vivo, all drugs resulted in a modest but significant increase of diaphragm force. In parallel, a decrease in the non-muscle area and markers of fibrosis was observed in GLPG0492- and NAND-treated mice. The drugs exerted minor effects on limb muscles; however, electrophysiological biomarkers were ameliorated in extensor digitorum longus muscle. The longer dose-dependence study confirmed the effect on mdx mouse strength and resistance to fatigue and demonstrated the efficacy of lower drug doses on in vivo and ex vivo functional parameters. These results support the interest of further studies of GLPG0492 as a potential treatment for DMD. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne Muscular Dystrophy.

    Directory of Open Access Journals (Sweden)

    Alfredo D Guerron

    2010-06-01

    Full Text Available The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin.In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy.These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity.

  16. Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Guerron, Alfredo D; Rawat, Rashmi; Sali, Arpana; Spurney, Christopher F; Pistilli, Emidio; Cha, Hee-Jae; Pandey, Gouri S; Gernapudi, Ramkishore; Francia, Dwight; Farajian, Viken; Escolar, Diana M; Bossi, Laura; Becker, Magali; Zerr, Patricia; de la Porte, Sabine; Gordish-Dressman, Heather; Partridge, Terence; Hoffman, Eric P; Nagaraju, Kanneboyina

    2010-06-21

    The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin. In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy. These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity.

  17. Polyethylenimine-modified Pluronics (PCMs) Improve Morpholino Oligomer Delivery in Cell Culture and Dystrophic mdx Mice

    OpenAIRE

    Wang, Mingxing; Wu, Bo; Lu, Peijuan; Cloer, Caryn; Tucker, Jay D; Lu, Qilong

    2012-01-01

    We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2–6 k, with hydrophilic-lipophilic balance (HLB) 7–23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system....

  18. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    Science.gov (United States)

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. PMID:20068555

  19. Prophylactic pamidronate partially protects from glucocorticoid-induced bone loss in the mdx mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Yoon, Sung-Hee; Chen, Jinghan; Grynpas, Marc D; Mitchell, Jane

    2016-09-01

    Glucocorticoids are extensively used to treat patients with Duchenne muscular dystrophy because of their ability to delay muscle damage, prolong ambulation and extend life. However, use of glucocorticoids significantly increases bone loss, fragility and fractures. To determine if antiresorptive bisphosphonates could prevent the effects of glucocorticoids on bone quality, we used dystrophic mdx mice treated with the glucocorticoid prednisone during 8weeks of rapid bone growth from 5 to 13weeks of age and treated some mice with the bisphosphonate pamidronate during the first two weeks of prednisone administration. Prednisone reduced long bone growth, decreased cortical bone thickness and area and decreased the strength of the femurs. Pamidronate treatment protected mice from cortical bone loss but did not increase bone strength. The combination of prednisone and pamidronate inhibited remodeling of metaphyseal trabecular bone with large numbers of trabeculae containing remnants of calcified cartilage. Prednisone improved muscle strength in the mdx mice and decreased serum creatine kinase with evidence of improved muscle histology and these effects were maintained in mice treated with pamidronate. Copyright © 2016. Published by Elsevier Inc.

  20. Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-06-01

    Full Text Available In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.

  1. Gene expression in mdx mouse muscle in relation to age and exercise: aberrant mechanical-metabolic coupling and implications for pre-clinical studies in Duchenne muscular dystrophy.

    Science.gov (United States)

    Camerino, Giulia Maria; Cannone, Maria; Giustino, Arcangela; Massari, Ada Maria; Capogrosso, Roberta Francesca; Cozzoli, Anna; De Luca, Annamaria

    2014-11-01

    Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wild-type (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor γ coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulated by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal overexpression of the slow Mhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational research. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Myogenin regulates exercise capacity but is dispensable for skeletal muscle regeneration in adult mdx mice.

    Directory of Open Access Journals (Sweden)

    Eric Meadows

    Full Text Available Duchenne muscular dystrophy (DMD is the most prevalent inherited childhood muscle disorder in humans. mdx mice exhibit a similar pathophysiology to the human disorder allowing for an in-depth investigation of DMD. Myogenin, a myogenic regulatory factor, is best known for its role in embryonic myogenesis, but its role in adult muscle maintenance and regeneration is still poorly understood. Here, we generated an mdx:Myog(flox/flox mouse harboring a tamoxifen-inducible Cre recombinase transgene, which was used to conditionally delete Myog during adult life. After tamoxifen treatment, three groups of mice were created to study the effects of Myog deletion: mdx:Myog(flox/flox mice (mdx, Myog(flox/flox mice (wild-type, and mdx:Myog(floxΔ/floxΔ:Cre-ER mice (mdx:Myog-deleted. mdx:Myog-deleted mice exhibited no adverse phenotype and behaved normally. When run to exhaustion, mdx:Myog-deleted mice demonstrated an enhanced capacity for exercise compared to mdx mice, running nearly as far as wild-type mice. Moreover, these mice showed the same signature characteristics of muscle regeneration as mdx mice. Unexpectedly, we found that myogenin was dispensable for muscle regeneration. Factors associated with muscle fatigue, metabolism, and proteolysis were significantly altered in mdx:Myog-deleted mice, and this might contribute to their increased exercise capacity. Our results reveal novel functions for myogenin in adult muscle and suggest that reducing Myog expression in other muscle disease models may partially restore muscle function.

  3. Enhancing translation: guidelines for standard pre-clinical experiments in mdx mice.

    Science.gov (United States)

    Willmann, Raffaella; De Luca, Annamaria; Benatar, Michael; Grounds, Miranda; Dubach, Judith; Raymackers, Jean-Marc; Nagaraju, Kanneboyina

    2012-01-01

    Duchenne Muscular Dystrophy is an X-linked disorder that affects boys and leads to muscle wasting and death due to cardiac involvement and respiratory complications. The cause is the absence of dystrophin, a large structural protein indispensable for muscle cell function and viability. The mdx mouse has become the standard animal model for pre-clinical evaluation of potential therapeutic treatments. Recent years have seen a rapid increase in the number of experimental compounds being evaluated in the mdx mouse. There is, however, much variability in the design of these pre-clinical experimental studies. This has made it difficult to interpret and compare published data from different laboratories and to evaluate the potential of a treatment for application to patients. The authors therefore propose the introduction of a standard study design for the mdx mouse model. Several aspects, including animal care, sampling times and choice of tissues, as well as recommended endpoints and methodologies are addressed and, for each aspect, a standard procedure is proposed. Testing of all new molecules/drugs using a widely accepted and agreed upon standard experimental protocol would greatly improve the power of pre-clinical experimentations and help identifying promising therapies for the translation into clinical trials for boys with Duchenne Muscular Dystrophy. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    Science.gov (United States)

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

  5. Macrophages improve survival, proliferation and migration of engrafted myogenic precursor cells into MDX skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Pierre-François Lesault

    Full Text Available Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i myoblast survival by limiting their massive death, ii myoblast expansion within the tissue and iii myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

  6. Evaluation of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse.

    Directory of Open Access Journals (Sweden)

    Christopher F Spurney

    2010-01-01

    Full Text Available Thymosin beta-4 (Tbeta4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tbeta4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ and mdx mice, 8-10 weeks old, were treated with 150 microg of Tbeta4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tbeta4 and amount of fibrosis were quantified using immunohistochemistry and Gomori's tri-chrome staining, respectively. Mdx mice treated with Tbeta4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tbeta4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tbeta4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

  7. MDX with SSAS 2012 cookbook

    CERN Document Server

    Li, Sherry

    2013-01-01

    This book is written in a recipe-based style packed full of practical tips and techniques to help you analyse multidimensional data stored in SSAS 2012 cubes. If you need to master MDX queries in SSAS, then this book is for you!If you are a Microsoft SQL Server Analysis Services developer and want to improve your solutions using MDX, then this book is for you. This book is also an essential resource for report developers who need to access the multidimensional cubes through the MDX language. The book assumes you have some basic working knowledge of MDX and a basic understanding of dimensional

  8. Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice.

    Directory of Open Access Journals (Sweden)

    Bruce E Markham

    Full Text Available Poloxamer 188 NF (national formulary (NF grade of P-188 improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c. injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone's effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.

  9. Effect of voluntary physical activity initiated at age 7 months on skeletal hindlimb and cardiac muscle function in mdx mice of both genders.

    Science.gov (United States)

    Ferry, Arnaud; Benchaouir, Rachid; Joanne, Pierre; Peat, Rachel A; Mougenot, Nathalie; Agbulut, Onnik; Butler-Browne, Gillian

    2015-11-01

    The effects of voluntary activity initiated in adult mdx (C57BL/10ScSc-DMD(mdx) /J) mice on skeletal and cardiac muscle function have not been studied extensively. We studied the effects of 3 months of voluntary wheel running initiated at age 7 months on hindlimb muscle weakness, increased susceptibility to muscle contraction-induced injury, and left ventricular function in mdx mice. We found that voluntary wheel running did not worsen the deficit in force-generating capacity and the force drop after lengthening contractions in either mdx mouse gender. It increased the absolute maximal force of skeletal muscle in female mdx mice. Moreover, it did not affect left ventricular function, structural heart dimensions, cardiac gene expression of inflammation, fibrosis, or remodeling markers. These results indicate that voluntary activity initiated at age 7 months had no detrimental effects on skeletal or cardiac muscles in either mdx mouse gender. © 2015 Wiley Periodicals, Inc.

  10. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    Science.gov (United States)

    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  11. Utrophin Compensates dystrophin Loss during Mouse Spermatogenesis

    OpenAIRE

    Chen, Hung-Chih; Chin, Yu-Feng; Lundy, David J.; Liang, Chung-Tiang; Chi, Ya-Hui; Kuo, Paolin; Hsieh, Patrick C. H.

    2017-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder resulting from mutations in the dystrophin gene. The mdx/utrn ?/? mouse, lacking in both dystrophin and its autosomal homologue utrophin, is commonly used to model the clinical symptoms of DMD. Interestingly, these mice are infertile but the mechanisms underlying this phenomenon remain unclear. Using dystrophin deficient mdx mouse and utrophin haplodeficient mdx/utrn +/? mouse models, we demonstrate the contribution of Dp427 (f...

  12. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

    Science.gov (United States)

    Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

    2010-10-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

  13. Radiosensitivity of mouse germ cells

    International Nuclear Information System (INIS)

    Matsuda, Yoichi; Takeuchi, Toyoko; Maemori, Mamiko; Seki, Naohiko; Tobari, Izuo

    1991-01-01

    To estimate radiosensitivity of mouse germ cells the analysis of chromosome aberrations was performed at diakinesis-metaphase I of spermatocytes and first-cleavage metaphase of one-cell embryos after exposure to radiations at various stages of primary spermatocytes and spermatids. The result provided evidence that there are two major types of DNA damage in X-irradiated sperm : (1) short-lived DNA lesions ; the lesions are subject to repair inhibition by agents added in G 1 , and are converted into chromosome-type aberrations during G 1 , and (2) long-lived DNA lesions ; the lesions persist until S phase and repair of the lesions is inhibited by caffeine, hydroxyurea and arabinofuranosyl cytosine in G 2 . The characteristic of X-ray damage induced in spermiogenic stage and repair mechanism for the damage in the fertilized egg were discussed comparing with the results with two chemicals, methyl methanesulfonate (MMS) and mitomycin C (MMC). (J.P.N.)

  14. Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice

    DEFF Research Database (Denmark)

    Jørgensen, Louise Helskov; Jensen, Charlotte Harken; Wewer, Ulla M

    2007-01-01

    mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired...... effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested...... overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic...

  15. Major alteration of the pathological phenotype in gamma irradiated mdx soleus muscles

    Energy Technology Data Exchange (ETDEWEB)

    Weller, B.; Karpati, G.; Lehnert, S.; Carpenter, S. (Montreal Neurological Institute, McGill University, Quebec (Canada))

    1991-07-01

    Two thousand rads of gamma irradiation delivered to the lower legs of ten day old normal and x-chromosome linked muscular dystrophy (mdx) mice caused significant inhibition of tibial bone and soleus muscle fiber growth. In the irradiated mdx solei, there was a major loss of muscle fibers, lack of central nucleation, and some endomysial fibrosis. These features were caused by a failure of regeneration of muscle fibers due to impaired proliferative capacity of satellite cells. Gamma irradiation transforms the late pathological phenotype of mdx muscles, so that in one major aspect (muscle fiber loss) they resemble muscles in Duchenne muscular dystrophy. However, extensive endomysial fibrosis which is another characteristic feature of Duchenne muscular dystrophy did not develop. This experimental model could be useful for the functional investigation of possible beneficial effects of therapeutic interventions in mdx dystrophy.

  16. Major alteration of the pathological phenotype in gamma irradiated mdx soleus muscles

    International Nuclear Information System (INIS)

    Weller, B.; Karpati, G.; Lehnert, S.; Carpenter, S.

    1991-01-01

    Two thousand rads of gamma irradiation delivered to the lower legs of ten day old normal and x-chromosome linked muscular dystrophy (mdx) mice caused significant inhibition of tibial bone and soleus muscle fiber growth. In the irradiated mdx solei, there was a major loss of muscle fibers, lack of central nucleation, and some endomysial fibrosis. These features were caused by a failure of regeneration of muscle fibers due to impaired proliferative capacity of satellite cells. Gamma irradiation transforms the late pathological phenotype of mdx muscles, so that in one major aspect (muscle fiber loss) they resemble muscles in Duchenne muscular dystrophy. However, extensive endomysial fibrosis which is another characteristic feature of Duchenne muscular dystrophy did not develop. This experimental model could be useful for the functional investigation of possible beneficial effects of therapeutic interventions in mdx dystrophy

  17. SERCA2a gene transfer improves electrocardiographic performance in aged mdx mice

    Directory of Open Access Journals (Sweden)

    Hajjar Roger

    2011-08-01

    Full Text Available Abstract Background Cardiomyocyte calcium overloading has been implicated in the pathogenesis of Duchenne muscular dystrophy (DMD heart disease. The cardiac isoform of sarcoplasmic reticulum calcium ATPase (SERCA2a plays a major role in removing cytosolic calcium during heart muscle relaxation. Here, we tested the hypothesis that SERCA2a over-expression may mitigate electrocardiography (ECG abnormalities in old female mdx mice, a murine model of DMD cardiomyopathy. Methods 1 × 1012 viral genome particles/mouse of adeno-associated virus serotype-9 (AAV-9 SERCA2a vector was delivered to 12-m-old female mdx mice (N = 5 via a single bolus tail vein injection. AAV transduction and the ECG profile were examined eight months later. Results The vector genome was detected in the hearts of all AAV-injected mdx mice. Immunofluorescence staining and western blot confirmed SERCA2a over-expression in the mdx heart. Untreated mdx mice showed characteristic tachycardia, PR interval reduction and QT interval prolongation. AAV-9 SERCA2a treatment corrected these ECG abnormalities. Conclusions Our results suggest that AAV SERCA2a therapy may hold great promise in treating dystrophin-deficient heart disease.

  18. PKC theta ablation improves healing in a mouse model of muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Luca Madaro

    Full Text Available Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-, where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/- mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.

  19. Correlation analysis of inorganic elements in biological tissue if DMD{sup mdx}/J mice using INAA

    Energy Technology Data Exchange (ETDEWEB)

    Metairon, Sabrina; Zamboni, Cibele B.; Suzuki, Miriam F., E-mail: metairon@usp.b, E-mail: czamboni@ipen.b, E-mail: mfsuzuki@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Bueno Junior, Carlos R. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Biociencias. Centro de Estudos do Genoma Humano; Sant' Anna, Osvaldo A., E-mail: gbrazil@usp.b [Instituto Butantan, Sao Paulo, SP (Brazil)

    2011-07-01

    Instrumental neutron activation analysis technique (INAA) has been used to determine Br, Ca, Cl, K, Mg, Na and S concentrations in bone and other organs samples from DMD{sup mdx}/J dystrophic mice as well as C57BL/6J control group mice. The DMD{sup mdx}/J mouse strain is relevant as an experimental model for Duchenne Muscular Dystrophy (DMD), which is the most severe and prevalent type of muscular dystrophy. Muscle weakness, premature death and instability of the membrane that involves the muscle fibers - causing functional/structural abnormalities and cell death - are main characteristics of this genetic disease. To show in more details the alterations that this disease may cause in bones (tibiae) and organs (quadriceps and heart), correlations matrixes were generated for both strains permitting a comparison between these groups. A significant change was observed in the analysis of the heart of dystrophic mice suggesting that this dysfunction affects severely the heart muscle. The results emphasize physiologic differences for Na, Ca and Mg and suggest that Br and S results are altered, emphasizing a constant monitoring needs. Other than that, these results may help the researchers to evaluate the efficiency of new treatments and to compare the advantages of different treatment approaches before performing tests in patients with muscular dystrophy. (author)

  20. AAV-based shRNA silencing of NF-κB ameliorates muscle pathologies in mdx mice.

    Science.gov (United States)

    Yang, Q; Tang, Y; Imbrogno, K; Lu, A; Proto, J D; Chen, A; Guo, F; Fu, F H; Huard, J; Wang, B

    2012-12-01

    Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

  1. the production of mouse embryonic stem cells

    Indian Academy of Sciences (India)

    MADU

    What history tells us VII. Twenty-five years ago: the production of mouse embryonic stem cells ... cells into the cavity of the blastocyst, it will be possible to test the effect of .... to the use of efficient immunosuppressive drugs like cyclosporin – was ...

  2. Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy.

    Science.gov (United States)

    Lozanoska-Ochser, Biliana; Benedetti, Anna; Rizzo, Giuseppe; Marrocco, Valeria; Di Maggio, Rosanna; Fiore, Piera; Bouche, Marina

    2018-03-01

    Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq -/- muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  3. Long-Term Blocking of Calcium Channels in mdx Mice Results in Differential Effects on Heart and Skeletal Muscle

    DEFF Research Database (Denmark)

    Jørgensen, Louise Helskov; Blain, Alison; Greally, Elizabeth

    2011-01-01

    in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight......The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing...... calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD). Treatment in utero onwards delayed onset...

  4. The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.

    Science.gov (United States)

    Di Certo, Maria Grazia; Corbi, Nicoletta; Strimpakos, Georgios; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Guglielmotti, Angelo; Batassa, Enrico Maria; Pisani, Cinzia; Floridi, Aristide; Benassi, Barbara; Fanciulli, Maurizio; Magrelli, Armando; Mattei, Elisabetta; Passananti, Claudio

    2010-03-01

    The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

  5. Nifedipine treatment reduces resting calcium concentration, oxidative and apoptotic gene expression, and improves muscle function in dystrophic mdx mice.

    Directory of Open Access Journals (Sweden)

    Francisco Altamirano

    Full Text Available Duchenne Muscular Dystrophy (DMD is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM decreased [Ca(2+]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox/p47(phox NOX2 subunits and pro-apoptotic (Bax genes in mdx diaphragm muscles and lowered serum creatine kinase (CK levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+]r in mdx skeletal muscle cells. The results in this work open new

  6. Instant MDX queries for SQL Server 2012

    CERN Document Server

    Emond, Nicholas

    2013-01-01

    Get to grips with a new technology, understand what it is and what it can do for you, and then get to work with the most important features and tasks. This short, focused guide is a great way to get stated with writing MDX queries. New developers can use this book as a reference for how to use functions and the syntax of a query as well as how to use Calculated Members and Named Sets.This book is great for new developers who want to learn the MDX query language from scratch and install SQL Server 2012 with Analysis Services

  7. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  8. Heregulin ameliorates the dystrophic phenotype in mdx mice

    DEFF Research Database (Denmark)

    Krag, Thomas O B; Bogdanovich, Sasha; Jensen, Claus J

    2004-01-01

    Duchenne's muscular dystrophy (DMD) is a fatal neuromuscular disease caused by absence of dystrophin. Utrophin is a chromosome 6-encoded dystrophin-related protein (DRP), sharing functional motifs with dystrophin. Utrophin's ability to compensate for dystrophin during development and when....... Therefore, this pathway offers a potential mechanism to modulate utrophin expression in muscle. We tested the ability of heregulin to improve the dystrophic phenotype in the mdx mouse model of DMD. Intraperitoneal injections of a small peptide encoding the epidermal growth factor-like region of heregulin...... ectodomain for 3 months in vivo resulted in up-regulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction mediated damage, and a reduction of muscle pathology. The amelioration of dystrophic phenotype by heregulin-mediated utrophin...

  9. Muscle Structure Influences Utrophin Expression in mdx Mice

    Science.gov (United States)

    Banks, Glen B.; Combs, Ariana C.; Odom, Guy L.; Bloch, Robert J.; Chamberlain, Jeffrey S.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin−/−, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD. PMID:24922526

  10. Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

    Energy Technology Data Exchange (ETDEWEB)

    Feder, D.; Rugollini, M.; Santomauro, A. Jr; Oliveira, L.P.; Lioi, V.P. [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Santos, R. dos; Ferreira, L.G.; Nunes, M.T.; Carvalho, M.H. [Universidade de São Paulo, Instituto de Ciências Biomédicas, São Paulo, SP (Brazil); Delgado, P.O.; Carvalho, A.A.S. [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Fonseca, F.L.A. [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Universidade Federal de São Paulo, Ambientais e Farmacêuticas, Instituto de Ciências Químicas, Diadema, SP (Brazil)

    2014-09-05

    Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.

  11. Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

    International Nuclear Information System (INIS)

    Feder, D.; Rugollini, M.; Santomauro, A. Jr; Oliveira, L.P.; Lioi, V.P.; Santos, R. dos; Ferreira, L.G.; Nunes, M.T.; Carvalho, M.H.; Delgado, P.O.; Carvalho, A.A.S.; Fonseca, F.L.A.

    2014-01-01

    Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle

  12. α-smooth muscle actin is not a marker of fibrogenic cell activity in skeletal muscle fibrosis.

    Directory of Open Access Journals (Sweden)

    Wanming Zhao

    Full Text Available α-Smooth muscle actin (α-SMA is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. We address whether α-SMA-expressing myofibroblasts are detectable in fibrotic muscles of mdx5cv mice, a mouse model for Duchenne muscular dystrophy (DMD, and whether the α-SMA expression correlates with the fibrogenic function of intramuscular fibrogenic cells. α-SMA immunostaining signal was not detected in collagen I (GFP-expressing cells in fibrotic muscles of ColI-GFP/mdx5cv mice, but it was readily detected in smooth muscle cells lining intramuscular blood vessel walls. α-SMA expression was detected by quantitative RT-PCR and Western blot in fibrogenic cells sorted from diaphragm and quadriceps muscles of the ColI-GFP/mdx5cv mice. Consistent with the more severe fibrosis in the ColI-GFP/mdx5cv diaphragm, the fibrogenic cells in the diaphragm exerted a stronger fibrogenic function than the fibrogenic cells in the quadriceps as gauged by their extracellular matrix gene expression. However, both gene and protein expression of α-SMA was lower in the diaphragm fibrogenic cells than in the quadriceps fibrogenic cells in the ColI-GFP/mdx5cv mice. We conclude that myofibroblasts are present in fibrotic skeletal muscles, but their expression of α-SMA is not detectable by immunostaining. The level of α-SMA expression by intramuscular fibrogenic cells does not correlate positively with the level of collagen gene expression or the severity of skeletal muscle fibrosis in the mdx5cv mice. α-SMA is not a functional marker of fibrogenic cells in skeletal muscle fibrosis associated with muscular dystrophy.

  13. Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

    Science.gov (United States)

    2013-01-01

    Background Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice. Methods We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles. Results Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function. Conclusions These data show that S1P is

  14. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells ...

  15. Differential requirement for utrophin in the induced pluripotent stem cell correction of muscle versus fat in muscular dystrophy mice.

    Directory of Open Access Journals (Sweden)

    Amanda J Beck

    Full Text Available Duchenne muscular dystrophy (DMD is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin. In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.

  16. Oocyte-like cells induced from mouse spermatogonial stem cells.

    Science.gov (United States)

    Wang, Lu; Cao, Jinping; Ji, Ping; Zhang, Di; Ma, Lianghong; Dym, Martin; Yu, Zhuo; Feng, Lixin

    2012-08-06

    During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

  17. Oocyte-like cells induced from mouse spermatogonial stem cells

    Directory of Open Access Journals (Sweden)

    Wang Lu

    2012-08-01

    Full Text Available Abstract Background During normal development primordial germ cells (PGCs derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs can also revert back to pluripotency as embryonic stem (ES-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. Results We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. Conclusions Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

  18. Restoration of pharyngeal dilator muscle force in dystrophin-deficient (mdx) mice following co-treatment with neutralizing interleukin-6 receptor antibodies and urocortin 2.

    Science.gov (United States)

    Burns, David P; Rowland, Jane; Canavan, Leonie; Murphy, Kevin H; Brannock, Molly; O'Malley, Dervla; O'Halloran, Ken D; Edge, Deirdre

    2017-09-01

    What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin-6 receptor signalling and stimulating corticotrophin-releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild-type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance. The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress-related factors are implicated in airway dilator muscle dysfunction. Six-week-old mdx (n = 26) and wild-type (WT; n = 26) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (0.2 mg kg -1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin 2; 30 μg kg -1 ) over 2 weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo. Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre-type distribution and fibre cross-sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100 Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx, and this was partly ameliorated after drug treatment. The areal

  19. Loss of cIAP1 attenuates soleus muscle pathology and improves diaphragm function in mdx mice

    Science.gov (United States)

    Enwere, Emeka K.; Boudreault, Louise; Holbrook, Janelle; Timusk, Kristen; Earl, Nathalie; LaCasse, Eric; Renaud, Jean-Marc; Korneluk, Robert G.

    2013-01-01

    The cellular inhibitor of apoptosis 1 (cIAP1) protein is an essential regulator of canonical and noncanonical nuclear factor κB (NF-κB) signaling pathways. NF-κB signaling is known to play important roles in myogenesis and degenerative muscle disorders such as Duchenne muscular dystrophy (DMD), but the involvement of cIAP1 in muscle disease has not been studied directly. Here, we asked whether the loss of cIAP1 would influence the pathology of skeletal muscle in the mdx mouse model of DMD. Double-mutant cIAP1−/−;mdx mice exhibited reduced muscle damage and decreased fiber centronucleation in the soleus, compared with single-mutant cIAP1+/+;mdx mice. This improvement in pathology was associated with a reduction in muscle infiltration by macrophages and diminished expression of inflammatory cytokines such as IL-6 and tumor necrosis factor-α. Furthermore, the cIAP1−/−;mdx mice exhibited reduced serum creatine kinase, and improved exercise endurance associated with improved exercise resilience by the diaphragm. Mechanistically, the loss of cIAP1 was sufficient to drive constitutive activation of the noncanonical NF-κB pathway, which led to increased myoblast fusion in vitro and in vivo. Collectively, these results show that the loss of cIAP1 protects skeletal muscle from the degenerative pathology resulting from systemic loss of dystrophin. PMID:23184147

  20. Skeletal muscle-specific overexpression of IGFBP-2 promotes a slower muscle phenotype in healthy but not dystrophic mdx mice and does not affect the dystrophic pathology.

    Science.gov (United States)

    Swiderski, Kristy; Martins, Karen Janet Bernice; Chee, Annabel; Trieu, Jennifer; Naim, Timur; Gehrig, Stefan Martin; Baum, Dale Michael; Brenmoehl, Julia; Chau, Luong; Koopman, René; Gregorevic, Paul; Metzger, Friedrich; Hoeflich, Andreas; Lynch, Gordon Stuart

    The insulin-like growth factor binding proteins (IGFBPs) are thought to modulate cell size and homeostasis via IGF-I-dependent and -independent pathways. There is a considerable dearth of information regarding the function of IGFBPs in skeletal muscle, particularly their role in the pathophysiology of Duchenne muscular dystrophy (DMD). In this study we tested the hypothesis that intramuscular IGFBP-2 overexpression would ameliorate the pathology in mdx dystrophic mice. 4week old male C57Bl/10 and mdx mice received a single intramuscular injection of AAV6-empty or AAV6-IGFBP-2 vector into the tibialis anterior muscle. At 8weeks post-injection the effect of IGFBP-2 overexpression on the structure and function of the injected muscle was assessed. AAV6-mediated IGFBP-2 overexpression in the tibialis anterior (TA) muscles of 4-week-old C57BL/10 and mdx mice reduced the mass of injected muscle after 8weeks, inducing a slower muscle phenotype in C57BL/10 but not mdx mice. Analysis of inflammatory and fibrotic gene expression revealed no changes between control and IGFBP-2 injected muscles in dystrophic (mdx) mice. Together these results indicate that the IGFBP-2-induced promotion of a slower muscle phenotype is impaired in muscles of dystrophin-deficient mdx mice, which contributes to the inability of IGFBP-2 to ameliorate the dystrophic pathology. The findings implicate the dystrophin-glycoprotein complex (DGC) in the signaling required for this adaptation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Rancourt, Ann; Dufresne, Sébastien S; St-Pierre, Guillaume; Lévesque, Julie-Christine; Nakamura, Haruka; Kikuchi, Yodai; Satoh, Masahiko S; Frenette, Jérôme; Sato, Sachiko

    2018-06-12

    The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD.-Rancourt, A., Dufresne, S. S., St-Pierre, G., Lévesque, J.-C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

  2. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

    Science.gov (United States)

    Nio, Yasunori; Tanaka, Masayuki; Hirozane, Yoshihiko; Muraki, Yo; Okawara, Mitsugi; Hazama, Masatoshi; Matsuo, Takanori

    2017-12-01

    Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy. © FASEB.

  3. Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers.

    Science.gov (United States)

    Miyazaki, Daigo; Nakamura, Akinori; Fukushima, Kazuhiro; Yoshida, Kunihiro; Takeda, Shin'ichi; Ikeda, Shu-ichi

    2011-05-01

    Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.

  4. Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration After Injury. Addendum

    Science.gov (United States)

    2012-03-01

    ged mdx mouse a n ovel therapeutic approach for Duchenne’s muscular dystrophy (DMD) based on the implantation of muscle -derived stem cells (MDSC), and...with limb ischemia. 15. SUBJECT TERMS Myostatin, muscle dystrophy , stem cells, myogenesis, Oct-4; Duchenne ; fibrosis 16. SECURITY CLASSIFICATION...derived stem cells (MDSC) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD

  5. Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

    International Nuclear Information System (INIS)

    Roblin, R.O.; Bell, T.E.; Young, P.L.

    1978-01-01

    A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radiommunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human and rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply. (Auth.)

  6. Mouse endometrial stromal cells produce basement-membrane components

    DEFF Research Database (Denmark)

    Wewer, U M; Damjanov, A; Weiss, J

    1986-01-01

    During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec......During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations....... Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells...... to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components....

  7. Myelination competent conditionally immortalized mouse Schwann cells

    NARCIS (Netherlands)

    Saavedra, José T.; Wolterman, Ruud A.; Baas, Frank; ten Asbroek, Anneloor L. M. A.

    2008-01-01

    Numerous mouse myelin mutants are available to analyze the biology of the peripheral nervous system related to health and disease in vivo. However, robust in vitro biochemical characterizations of players in peripheral nerve processes are still not possible due to the limited growth capacities of

  8. Role of Abcg2 During Mouse Embroyonic Stem Cell Diffferentiation

    Science.gov (United States)

    Role of Abcg2 During Mouse Embryonic Stem Cell Differentiation. Abcg2 is a multidrug resistance ATP-binding cassette (ABC) transporter whose activity may be considered a hallmark of stem cell plasticity. The role of Abcg2 during early embryogenesis, however, is unclear. Studies...

  9. Impact of 2-bromopropane on mouse embryonic stem cells and ...

    African Journals Online (AJOL)

    This study shows that 2-BP (5 to 10 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no effects at treatment dosages below 5 μM. In ESC-B5 cells, 2-BP directly increased the content of reactive oxygen species (ROS), significantly increased the cytoplasmic free calcium and nitric oxide ...

  10. Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

    International Nuclear Information System (INIS)

    Ota, Kimiko; Nakamura, Jiro; Li, Weiguo; Kozakae, Mika; Watarai, Atsuko; Nakamura, Nobuhisa; Yasuda, Yutaka; Nakashima, Eirtaro; Naruse, Keiko; Watabe, Kazuhiko; Kato, Koichi; Oiso, Yutaka; Hamada, Yoji

    2007-01-01

    Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS

  11. Voluntary wheel running in dystrophin-deficient (mdx) mice: Relationships between exercise parameters and exacerbation of the dystrophic phenotype.

    Science.gov (United States)

    Smythe, Gayle M; White, Jason D

    2011-12-18

    Voluntary wheel running can potentially be used to exacerbate the disease phenotype in dystrophin-deficient mdx mice. While it has been established that voluntary wheel running is highly variable between individuals, the key parameters of wheel running that impact the most on muscle pathology have not been examined in detail. We conducted a 2-week test of voluntary wheel running by mdx mice and the impact of wheel running on disease pathology. There was significant individual variation in the average daily distance (ranging from 0.003 ± 0.005 km to 4.48 ± 0.96 km), culminating in a wide range (0.040 km to 67.24 km) of total cumulative distances run by individuals. There was also variation in the number and length of run/rest cycles per night, and the average running rate. Correlation analyses demonstrated that in the quadriceps muscle, a low number of high distance run/rest cycles was the most consistent indicator for increased tissue damage. The amount of rest time between running bouts was a key factor associated with gastrocnemius damage. These data emphasize the need for detailed analysis of individual running performance, consideration of the length of wheel exposure time, and the selection of appropriate muscle groups for analysis, when applying the use of voluntary wheel running to disease exacerbation and/or pre-clinical testing of the efficacy of therapeutic agents in the mdx mouse.

  12. Beneficial effects of high dose taurine treatment in juvenile dystrophic mdx mice are offset by growth restriction.

    Science.gov (United States)

    Terrill, Jessica R; Pinniger, Gavin J; Nair, Keshav V; Grounds, Miranda D; Arthur, Peter G

    2017-01-01

    Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disease manifested in young boys, for which there is no current cure. We have shown that the amino acid taurine is safe and effective at preventing dystropathology in the mdx mouse model for DMD. This study aimed to establish if treating growing mdx mice with a higher dose of taurine was more effective at improving strength and reducing inflammation and oxidative stress. Mice were treated with a dose of taurine estimated to be 16 g/kg/day, in drinking water from 1-6 weeks of age, after which in vivo and ex vivo muscle strength was assessed, as were measures of inflammation, oxidative stress and taurine metabolism. While the dose did decrease inflammation and protein oxidation in dystrophic muscles, there was no improvement in muscle strength (in contrast with benefits observed with the lower dose) and growth of the young mice was significantly restricted. We present novel data that a high taurine dose increases the cysteine content of both mdx liver and plasma, a possible result of down regulation of the taurine synthesis pathway in the liver (which functions to dispose of excess cysteine, which is toxic). These data caution that a high dose of taurine can have adverse effects and may be less efficacious than lower taurine doses. Therefore, monitoring of taurine dosage needs to be considered in future pre-clinical trials, in anticipation of using taurine as a clinical therapy for growing DMD boys (and other conditions).

  13. Beneficial effects of high dose taurine treatment in juvenile dystrophic mdx mice are offset by growth restriction.

    Directory of Open Access Journals (Sweden)

    Jessica R Terrill

    Full Text Available Duchenne Muscular Dystrophy (DMD is a fatal muscle wasting disease manifested in young boys, for which there is no current cure. We have shown that the amino acid taurine is safe and effective at preventing dystropathology in the mdx mouse model for DMD. This study aimed to establish if treating growing mdx mice with a higher dose of taurine was more effective at improving strength and reducing inflammation and oxidative stress. Mice were treated with a dose of taurine estimated to be 16 g/kg/day, in drinking water from 1-6 weeks of age, after which in vivo and ex vivo muscle strength was assessed, as were measures of inflammation, oxidative stress and taurine metabolism. While the dose did decrease inflammation and protein oxidation in dystrophic muscles, there was no improvement in muscle strength (in contrast with benefits observed with the lower dose and growth of the young mice was significantly restricted. We present novel data that a high taurine dose increases the cysteine content of both mdx liver and plasma, a possible result of down regulation of the taurine synthesis pathway in the liver (which functions to dispose of excess cysteine, which is toxic. These data caution that a high dose of taurine can have adverse effects and may be less efficacious than lower taurine doses. Therefore, monitoring of taurine dosage needs to be considered in future pre-clinical trials, in anticipation of using taurine as a clinical therapy for growing DMD boys (and other conditions.

  14. Dysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice

    Science.gov (United States)

    Stapleton, David I.; Lau, Xianzhong; Flores, Marcelo; Trieu, Jennifer; Gehrig, Stefan M.; Chee, Annabel; Naim, Timur; Lynch, Gordon S.; Koopman, René

    2014-01-01

    Background Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice. Results Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (Pglycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (PGlycogen synthase activity was 12% higher (Pglycogen branching enzyme activity was 70% lower (Pglycogen breakdown, glycogen phosphorylase, had 62% lower activity (Pglycogen debranching enzyme expression was 50% higher (Pglycogen (Pglycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; Pglycogen but reduced amounts of liver glycogen. PMID:24626262

  15. Laryngeal Muscles Are Spared in the Dystrophin Deficient "mdx" Mouse

    Science.gov (United States)

    Thomas, Lisa B.; Joseph, Gayle L.; Adkins, Tracey D.; Andrade, Francisco H.; Stemple, Joseph C.

    2008-01-01

    Purpose: "Duchenne muscular dystrophy (DMD)" is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the…

  16. Whole body periodic acceleration is an effective therapy to ameliorate muscular dystrophy in mdx mice.

    Science.gov (United States)

    Altamirano, Francisco; Perez, Claudio F; Liu, Min; Widrick, Jeffrey; Barton, Elisabeth R; Allen, Paul D; Adams, Jose A; Lopez, Jose R

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca(2+) and Na(+) overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca(2+) and Na(+) overload, diminished abnormal sarcolemmal Ca(2+) entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.

  17. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  18. Interaction of X-irradiated mouse cells in heterokaryons

    International Nuclear Information System (INIS)

    Hofmanova, J.; Spurna, V.

    1985-01-01

    The frequency of heterokaryon formation and the ability of DNA synthesis in the system of mouse X-irradiated L fibroblasts and non-irradiated or irradiated LS/BL lymphosarcoma cells were studied. The frequency of heterokaryons after fusion of one or both irradiated parental cells was 3 to 6 times higher than in the non-irradiated cell cultures. In these heterokaryons we found 1.5 to 3 times more nuclei of irradiated L cells capable of DNA synthesis than in the population of non-fused irradiated cells. (author)

  19. Stepwise development of MAIT cells in mouse and human.

    Directory of Open Access Journals (Sweden)

    Emmanuel Martin

    2009-03-01

    Full Text Available Mucosal-associated invariant T (MAIT cells display two evolutionarily conserved features: an invariant T cell receptor (TCRalpha (iTCRalpha chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC molecule, MHC-related molecule 1 (MR1. MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanValpha7.2 antibody and MAIT cell-specific iTCRalpha and TCRbeta transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRalpha and TCRbeta transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%-4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the

  20. Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice

    Science.gov (United States)

    Froehner, Stanley C.; Reed, Sarah M.; Anderson, Kendra N.; Huang, Paul L.; Percival, Justin M.

    2015-01-01

    Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSβ, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSβ and use mdx mice engineered to lack nNOSμ and nNOSβ to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSβ was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSμ and nNOSβ is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSβ as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies. PMID:25214536

  1. Expression of GABAergic receptors in mouse taste receptor cells.

    Directory of Open Access Journals (Sweden)

    Margaret R Starostik

    Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

  2. Radiation response of spermatogonial stem cells in the mouse

    International Nuclear Information System (INIS)

    Bootsma, A.L.

    1978-01-01

    Spermatogonial stem cells are able to repopulate the testis by forming clones that elongate along the walls of the seminiferous tubules depleted of spermatogenetic cells as a result of an irradiation. The surviving number of stem cells after irradiation was estimated by determining the fraction of repopulated tubules in cross-sections of the testis 11 weeks after irradiation. This fraction, called the 'repopulation index', is assumed to be directly proportional to the number of surviving stem cells. The response of spermatogonial stem cells in the CBA mouse to 1-MeV fission neutrons was investigated. Radioresistant, colony forming stem cells in the mouse testis move into a much more radiosensitive phase of their cell cycle shortly after irradiation. This is demonstrated in publication II in experiments in which total doses of 300 rad of neutrons and 1200 rad of X-rays were split into two equal fractions. The radiation response of spermatogonial stem cells in the mouse which survived various doses of fission neutrons 24 hours before was studied in publication III. Twenty four hours after a dose of 150 rad of fission neutrons all first-dose survivors have moved from a radioresistant (D 0 89+-4 rad in this study) towards a radiosensitive phase of their cell cycle. Spermatogonial stem cells which survive a neutron dose of 150 rad all belong to a radioresistant stem cell population in the seminiferous epithelium. The data in publication IV show that during the first 26 days after a dose of 150 rad of neutrons the stem cell population first increases and then slowly decreases its radiosensitivity, to stay fixed at a relatively high level. (Auth.)

  3. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  4. Cell proliferation and ageing in mouse colon

    International Nuclear Information System (INIS)

    Hamilton, E.

    1978-01-01

    The descending colon of 4 month and 2 year old mice was exposed to 1250 rad X-rays. This killed most of the epithelial cells. The surviving cells formed new crypts and surface epithelium in animals of both ages. Not all of the crypts were replaced. The irradiated area contained not more than 80% of the control number of crypts per section for at least 6 weeks after irradiation. In the young mice new crypts were much larger and the labelling index (LI) was much higher than in unirradiated animals during the first week after irradiation. In the old mice the overshoot in LI and crypt size began later and continued longer than in young animals. This may be because the control of cell proliferation was much less precise in old than in young mice. The irradiation was repeated, in attempt to age prematurely the epithelial cells by increasing the number of divisions they underwent. The overshoot in LI and cells per crypt was smaller after a second dose than after the first in both young and old mice. There was almost no overshoot after a third dose was given to young mice. Increasing the number of divisions undergone by the surviving epithelial cells did not change the timing of repopulation in young mice compared to that found in old mice. Little evidence was found for the presence of a limited proliferative lifespan in colon epithelial cells. (author)

  5. Protection of dystrophic muscle cells with polyphenols from green tea correlates with improved glutathione balance and increased expression of 67LR, a receptor for (-)-epigallocatechin gallate

    OpenAIRE

    Dorchies OM Wagner S Buetler TM Ruegg UT

    2009-01-01

    Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by the absence of the protein dystrophin. Because oxidative stress contributes to the pathogenesis of DMD we investigated if a green tea polyphenol blend (GTP) and its major polyphenol ( ) epigallocatechin gallate (EGCg) could protect muscle cell primary cultures from oxidative damage induced by hydrogen peroxide (H(2)O(2)) in the widely used mdx mouse model. On line fluorimetric measurements using an H(2)O(2) sensitiv...

  6. Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs (miR-1, miR-133a, and miR-206 in C2C12 Myocytes and mdx Mice.

    Directory of Open Access Journals (Sweden)

    Yasunari Matsuzaka

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs, is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.

  7. Company profile: QuantuMDx group limited.

    Science.gov (United States)

    Burn, Jamie

    2013-07-01

    QuantuMDx Group Ltd (QMDx) is a group of companies based in the International Centre for Life, Newcastle upon Tyne, UK. The Group owns the founding patent and the exclusive worldwide license for, among others, the use of nanowires and nanotubes in DNA detection. It has further developed a patent estate around the functionalization of nanowires for DNA detection, and is working with commercial partners globally to produce the Q-POC™, a handheld point-of-care genetic testing device. This novel lab-on-a-chip technology integrates and automates the sample-to-result genetic testing process in a single microfluidic channel, incorporating novel lysis technologies, the filtration of cellular constituents to achieve DNA extraction and fractionation, a rapid, thermal PCR system, and nanowires functionalized with nucleic acid probes to capture targeted sequences of genetic material. The device further makes use of novel chemistries to boost the charge of nucleotides binding to the isolated material, increasing the sensitivity and read length of the device and making it capable of robust SNP and pathogen detection. Complete with a sophisticated software package, the Q-POC™ can detect binding events through changes in resistance and effectively convert genetic code into binary code, providing a simple display of the results, complete with treatment options. The competitive advantages of this system are the sensitivity and specificity of the nanowire detection system, the extremely low cost profile of the technology, and the speed of the process, which will allow the sample-to-result detection of targeted genetic material in less than 15 min.

  8. Cell proliferation and ageing in mouse colon

    International Nuclear Information System (INIS)

    Hamilton, E.; Franks, L.M.

    1980-01-01

    Cell kinetic parameters in the descending colon of unirradiated mice, 3-30-months-old were compared with those in mice irradiated repeatedly from the age of 6 or 24 months. The latter animals were given 1250 rad local X-irradiation to the colon every 6 weeks. Dose-survival curves showed the colon crypts of 6 and 24-months-old mice were similarly radiosensitive. In unirradiated mice the number of crypts per colon section decreased significantly at 30 months, but no significant age-related changes were seen in crypt size or labelling index (LI). Cell proliferation returned to control levels within 6 weeks of each X-ray dose and remained at this level for 20 weeks after the final dose. Later, cell proliferation in the irradiated colon fell significantly below control. A total of 6 or 7 doses each of 1250 rad produced only 1 colon carcinoma amongst 50 mice kept until they died. (author)

  9. Further characterization of protein kinase C in mouse mast cells

    International Nuclear Information System (INIS)

    White, J.R.; Ishizaka, T.

    1986-01-01

    Bridging of cell-bound IgE antibody molecules on colony stimulating factor dependent mouse mast cell line (PT-18) cells by multivalent antigen induces the mobilization and uptake of Ca 2+ monitored by Quin-2 and the production of diacylglycerol. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C (PKC) activity in the plasma membrane (340 units to 1375 units: 1 unit = 1 pmol of 32 P incorporated into Histone H-1/min/10 7 cells), within 30 seconds. There is also an increase in 3 H phorbol-12, 13-dibutyrate ( 3 H-PDB) binding which parallels the increase in PKC activity both in kinetics and antigen dose dependency. Determination of K/sub m/ and V/sub max/ for PKC revealed no difference between the cytosolic and membranous forms of PKC. Partial purification of PKC from the membrane of sensitized mast cells which had been labeled with 32 P and stimulated with DNP-HSA revealed a protein of 80-84,000 molecular weight, which migrated on polyacrylamide gel electrophoresis just above an authentic standard of PKC purified from rat brain. Treatment of the PKC from mouse mast cell membrane with alkaline phosphatase resulted in a reduction of phosphorylating activity and bindability of 3 H-PDB. In conclusion, the authors speculate that activation of mouse mast cells by cross-linking IgE results in the phosphorylation of a silent-pool of PKC converting it from an inactive state to an activated form

  10. Mechanism of testosterone deficiency in the transgenic sickle cell mouse.

    Directory of Open Access Journals (Sweden)

    Biljana Musicki

    Full Text Available Testosterone deficiency is associated with sickle cell disease (SCD, but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH levels compared with wild type (WT mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR, but not cholesterol side-chain cleavage enzyme (P450scc, in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

  11. The effect of the melatonin on cryopreserved mouse testicular cells

    Directory of Open Access Journals (Sweden)

    Ghasem Saki

    2016-01-01

    Full Text Available Background: After improvements in various cancer treatments, life expectancy has been raised, but success in treatment causes loss of fertility in many of the survived young men. Cryopreservation of immature testicular tissues or cells introduced as the only way to preserve fertility. However, freezing has some harmful effects. Melatonin, a pineal gland hormone, has receptors in reproductive systems of different species. It is assumed that melatonin has free radical scavenger properties. Objective: The aim of this study was to evaluate the effects of melatonin on the cryopreserved testicular cells in mouse. Materials and Methods: Cells from 7- 10 days old NMRI mice testes were isolated using two step enzymatic digestion. The testicular cells were divided into two groups randomly and cryopreserved in two different freezing media with and without the addition of 100 μm melatonin. Finally, apoptosis of the cells was assayed by flow cytometry. Also, lactate dehydrogenase activity test was performed to assess the cytotoxicity. Results: The results of lactate dehydrogenase showed the nearly cytotoxic effect of melatonin. The results of flow cytometry showed increase in apoptosis in the cryopreserved cells in the media containing melatonin compared to the control group. Conclusion: The present study shows that melatonin has an apoptotic effect on cryopreserved mouse testicular cells.

  12. Diseased muscles that lack dystrophin or laminin-α2 have altered compositions and proliferation of mononuclear cell populations

    Directory of Open Access Journals (Sweden)

    Miller Jeffrey

    2005-04-01

    Full Text Available Abstract Background Multiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-α2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells. Methods We used fluorescence-activated cell sorting combined with bromodeoxyuridine labeling to examine proliferation rates and compositions of mononuclear cells in diseased and healthy mouse skeletal muscle. We prepared mononucleate cells from muscles of mdx (dystrophin-deficient or Lama2-/- (laminin-α2-deficient mice and compared them to cells from healthy control muscles. We enumerated subsets of resident muscle cells based on Sca-1 and CD45 expression patterns and determined the proliferation of each cell subset in vivo by BrdU incorporation. Results We found that the proliferation and composition of the mononucleate cells in dystrophin-deficient and laminin-α2-deficient diseased muscles are different than in healthy muscle. The mdx and Lama2-/- muscles showed similar significant increases in CD45+ cells compared to healthy muscle. Changes in proliferation, however, differed between the two diseases with proliferation increased in mdx and decreased in Lama2-/- muscles compared to healthy muscles. In particular, the most abundant Sca-1-/CD45- subset, which contains muscle precursor cells, had increased proliferation in mdx muscle but decreased proliferation in Lama2-/- muscles. Conclusion The similar increases in CD45+ cells, but opposite changes in proliferation of muscle precursor cells, may underlie aspects of the distinct pathologies in the two diseases.

  13. Resistance of human and mouse myeloid leukemia cells to UV radiation

    International Nuclear Information System (INIS)

    Poljak-Blazi, M.; Osmak, M.; Hadzija, M.

    1989-01-01

    Sensitivity of mouse bone marrow and myeloid leukemia cells and sensitivity of human myeloid leukemia cells to UV light was tested. Criteria were the in vivo colony-forming ability of UV exposed cells and the inhibition of DNA synthesis during post-irradiation incubation for 24 h in vitro. Mouse bone marrow cells irradiated with a small dose of UV light (5 J/m 2 ) and injected into x-irradiated animals did not form hemopoietic colonies on recipient's spleens, and recipients died. However, mouse leukemia cells, after irradiation with higher doses of UV light, retained the ability to form colonies on the spleens, and all recipient mice died with typical symptoms of leukemia. In vitro, mouse bone marrow cells exhibited high sensitivity to UV light compared to mouse myeloid leukemia cells. Human leukemia cells were also resistant to UV light, but more sensitive than mouse leukemia cells. (author)

  14. Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

    Science.gov (United States)

    Borth, N; Heider, R; Assadian, A; Katinger, H

    1992-09-01

    The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.

  15. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

    Directory of Open Access Journals (Sweden)

    Diane Rebourcet

    Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

  16. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  17. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  18. CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

    Science.gov (United States)

    Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

    2012-03-01

    Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

  19. Discrimination of taste qualities among mouse fungiform taste bud cells.

    Science.gov (United States)

    Yoshida, Ryusuke; Miyauchi, Aya; Yasuo, Toshiaki; Jyotaki, Masafumi; Murata, Yoshihiro; Yasumatsu, Keiko; Shigemura, Noriatsu; Yanagawa, Yuchio; Obata, Kunihiko; Ueno, Hiroshi; Margolskee, Robert F; Ninomiya, Yuzo

    2009-09-15

    Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

  20. Radioprotection of mouse CNS endothelial cells in vivo

    International Nuclear Information System (INIS)

    Lyubimova, N.; Coultas, P.; Martin, R.

    1996-01-01

    Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy γ-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

  1. Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

    Science.gov (United States)

    Church, Jarrod E; Trieu, Jennifer; Chee, Annabel; Naim, Timur; Gehrig, Stefan M; Lamon, Séverine; Angelini, Corrado; Russell, Aaron P; Lynch, Gordon S

    2014-04-01

    New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the

  2. Sex-reversed somatic cell cloning in the mouse.

    Science.gov (United States)

    Inoue, Kimiko; Ogonuki, Narumi; Mekada, Kazuyuki; Yoshiki, Atsushi; Sado, Takashi; Ogura, Atsuo

    2009-10-01

    Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.

  3. T cell progenitors in the mouse fetal liver

    International Nuclear Information System (INIS)

    Rabinowich, H.; Umiel, T.; Globerson, A.

    1983-01-01

    Fourteen-day mouse fetal liver was found to contain cells capable of giving rise to T as well as B cell functions. The experimental system consisted of congenic C3H/DiSn and (C3H/DiSn X C3H.SW)F1 lethally irradiated (900 R) mice reconstituted with C3H/DiSn fetal liver or bone marrow cells. Assays included thyroid allograft rejection as well as in vitro measurement of reactivity to phytohemagglutinin (PHA) and concanavalin A (Con A) and in a mixed lymphocyte culture (MLC) system in spleen, lymph node, and thymus cells. The fetal liver chimeras were found to become as capable as the bone marrow chimeras in responding in these various assays. The T cell responses lagged behind the responses to the B cell mitogens dextran sulfate (DXS) and lipopolysaccharide (LPS) (30 days after reconstitution, as compared with 14 days for DXS and 21 for LPS). The reacting cells were of the donor genotype, as revealed after treatment with C3H/DiSn (H-2k) anti-C3H.SW (H-2b) congenic sera. T cell responses were not manifest in thymectomized (TX) chimeras. Hence, the liver seems to contain cells capable of developing into T cell lineages in a thymus-dependent process

  4. Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

    2017-08-01

    Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

  5. Electroretinographic genotype-phenotype correlations for mouse and man at the dmd/DMD locus

    Energy Technology Data Exchange (ETDEWEB)

    Millers, D.M.; Weleber, R.G.; Woodward, W.R. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others

    1994-09-01

    Reduced or absent b-waves in the dark-adapted electroretinogram (ERG) of Duchenne and Becker muscular dystrophy (DMD/BMD) patients led to the identification of dystrophin in human retina and the proposal that it plays a role in retinal electrophysiology. Study of a large group of Duchenne and Becker muscular dystrophy males to determine their ocular characteristics indicated that there were position-specific effects of deletions, with 3{prime} defects associated with severe electroretinographic changes, whereas some 5{prime} patients demonstrated less severe, or even normal, ERGs. We studied the mdx mouse, a model with X-linked muscular dystrophy and defective full-length dystrophin, which failed to show any ERG abnormalities. Given the presence of alternate isoforms of dystrophin in retina, and the 5{prime} deletion DMD/BMD patients with normal ERGs, we studied mouse models with differing dystrophin mutations (mdx{sup Cv3}, mdx{sup Cv5}) to determine the usefulness of alternate strains as models for the visual effects of dystropin. Abnormal ERGs similar to those seen in DMD/BMS patients exist in the mdx{sup Cv3} strain of muscular dystrophy mice. Normal ERGs were found the mdx{sup Cv5} strain. The mutations in the mdx and mdx{sup Cv5} mice have been mapped to the 5{prime} end of the dmd gene, while the mutation in the mdx{sup Cv3} mouse is in the 3{prime} end. Thus, there are position effects of the gene defect on the ERG phenotype that are conserved in the mouse. Such genotype-phenotype correlations may reflect differential expression of shorter isoforms of dystrophin.

  6. Microsoft® SQL Server® 2008 MDX Step by Step

    CERN Document Server

    Smith, Bryan; Consulting, Hitachi

    2009-01-01

    Teach yourself the Multidimensional Expressions (MDX) query language-one step at a time. With this practical, learn-by-doing tutorial, you'll build the core techniques for using MDX with Analysis Services to deliver high-performance business intelligence solutions. Discover how to: Construct and execute MDX queriesWork with tuples, sets, and expressionsBuild complex sets to retrieve the exact data users needPerform aggregation functions and navigate data hierarchiesAssemble time-based business metricsCustomize an Analysis Services cube through the MDX scriptImplement dynamic security to cont

  7. Functional State of Haemopoietic Stem Cells in the Irradiated Mouse

    Energy Technology Data Exchange (ETDEWEB)

    Silini, G.; Pozzi, Laura V. [Laboratorio di Radiobiologica Animale, Centro Studi Nucleari, Casaccia, Rome (Italy)

    1968-08-15

    The repopulation kinetics of bone marrow in irradiated (C3H x C57BL) F{sub 1} hybrid mice were followed at different time intervals after a single whole-body dose of 150 rad X -rays. The changes in the number of total nucleated cells and of colony-forming cells were estimated and expressed as number of cells per femur shaft of fixed length. For the evaluation of the progenitor cell compartment an exogenous test of transplantation into heavily irradiated hosts followed by spleen colony counts was employed. In an attempt to distinguish between cycling and dormant cells in the progenitor pool, vinblastine was also administered under various schedules of treatment with respect to time and dosage to follow the changes induced by this drug in the irradiated recovering marrow. The depopulation of total bone-m arrow cells caused by vinblastine proceeded at a comparable rate in both the irradiated and the normal mouse. On the other hand, depopulation of the colony-formers is faster in animals irradiated 1 -2 days previously as compared with normal animals or mice irradiated 1 week or 2 weeks earlier. The data were interpreted to show that in the marrow of a newly-irradiated animal more cells are in a fast cycle than in a normal or a recovering animal. Data are finally presented and discussed concerning the use of vinblastine for studies of stem cell kinetics in haemopoietic tissues. (author)

  8. Establishment and characterization of a hypocatalasemic mouse cell strain

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi; Tano, Keizo; Hashimoto, Mitsumasa W.; Kodama, Seiji; Watanabe, Hiromitsu

    1998-01-01

    Contact-inhibited catalase-deficient fibroblast cell strain has been established from the homozygous hypocatalasemic C3H/Cs b mutant mouse. This cell strain has low level of catalase enzyme activity and has normal level of enzyme activities of both glutathione peroxidase and superoxide dismutase. Catalase-deficient C3H/Cs b mutant cell strain is markedly more sensitive to the toxicity of hydrogen peroxide compared to wild-type C3H/Cs a cell strain. In addition, mutant cell strain is sensitive to X-rays and near-UV compared to wild-type cell strain, but shows the same sensitivities to topoisomerase II inhibitors, adriamycin and 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA), and the DNA cross-linking agents, cis-diamminedichloroplatinum (II) (cis-Pt) and trans-diamminedichloroplatinum (II) (trans-Pt). These cell strains will be of use in the study of the roles which catalase plays in the intracellular prevention of DNA damage induced by oxidative stress. (author)

  9. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

    OpenAIRE

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2016-01-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However,...

  10. Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

    International Nuclear Information System (INIS)

    Bhagavati, Satyakam; Xu Weimin

    2005-01-01

    Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells

  11. Elements determination of clinical relevance in biological tissues Dmd{sup mdx}/J dystrophic mice strains investigated by NAA; Determinacao de elementos de relevancia clinica em tecidos biologicos de camundongos distroficos Dmd{sup mdx}/J por AAN

    Energy Technology Data Exchange (ETDEWEB)

    Metairon, Sabrina

    2012-07-01

    In this work the determination of chemistry elements in biological tissues (whole blood, bones and organs) of dystrophic mice, used as animal model of Duchenne Muscular Dystrophy (DMD), was performed using analytical nuclear technique. The aim of this work was to determine reference values of elements of clinical (Ca, Cl, K, Mg, Na) and nutritional (Br and S) relevance in whole blood, tibia, quadriceps and hearts from Dmdmdx/J (10 males and 10 females) dystrophic mice and C57BL/6J (10 males) control group mice, using Neutron Activation Analysis technique (NAA). To show in more details the alterations that this disease may cause in these biological tissues, correlations matrixes of the DMD{sup mdx}/J mouse strain were generated and compared with C57BL/6J control group. For this study 119 samples of biological tissue were irradiated in the IEA-R1 nuclear reactor at IPEN (Sao Paulo, Brazil). The concentrations of these elements in biological tissues of Dmd{sup mdx}/J and C57B/6J mice are the first indicative interval for reference values. Moreover, the alteration in some correlation coefficients data among the elements in the health status and in the diseased status indicates a connection between these elements in whole blood, tibia, quadriceps and heart. These results may help the researchers to evaluate the efficiency of new treatments and to compare the advantages of different treatment approaches before performing tests in patients with muscular dystrophy. (author)

  12. Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Siemionow, M; Cwykiel, J; Heydemann, A; Garcia-Martinez, J; Siemionow, K; Szilagyi, E

    2018-04-01

    Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 10 6 ) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

  13. Diffusion chamber culture of mouse bone marrow cells, (1)

    International Nuclear Information System (INIS)

    Sigeta, Chiharu; Tanaka, Kimio; Kawakami, Masahito; Takahashi, Hiroshi; Ohkita, Takeshi

    1980-01-01

    Mouse bone marrow cells were cultured in diffusion chambers (DC) implanted in the peritoneal cavity of host mice. Host mice were subjected to (1) irradiation ( 60 Co 800 rad) and/or (2) phenylhydrazine induced anemia and then receiving irradiation ( 60 Co 600 rad). After culture periods of 3-7 days, the total number of cells in DC was increased. A marked increase in DC is due to the proliferation of granulocyte series. When host mice were subjected to anemia and irradiation, the start of cell proliferation in DC was delay about two days. On the whole, anemia and irradiation host reduced a little cell growth in DC. The number of immature granulocytes grown in DC in irradiated hosts or anemia and irradiated hosts increased and reached a plateu at day 5. During the plateu period, the proportions between immature and mature granulocytes in DC were kept constantly. The number of macrophages showed a two-phase increasing. Erythroid cells and lymphocytes rapidly disappeared from the chambers during 3 days. The number of erythroid cells was not significantly influenced even in anemia and irradiation hosts. (author)

  14. Response of maternal immune cells of irradiation of mouse embryos

    International Nuclear Information System (INIS)

    Nicholls, E.M.; Markovic, B.

    1988-01-01

    This work began as an attempt to explain the paradox of pregnancy - the survival and growth of the semi-allogenic embryo in an immunologically hostile environment. In 1982 and 1983 we reported the tracing of quinacrine labelled maternal leukocytes (WBC) in maternal, placental and embryonic mouse tissues by fluorescence microscopy. We found that cells in the placenta phagocytose labelled WBC, so that after 1-2 hours the labelled nuclear DNA is found as brightly fluorescing particles in the cytoplasm of the phagocytes with no evidence of it in the nuclei. Identical cells were observed in slide preparations of embryos which had been carefully separated from their placentas. We also found a small population of intact labelled lymphocytes, clearly maternal in origin, in the embryos. This seems to be another paradox - placental phagocytes are observed to be phagocytosing maternal WBC in the placenta and embryo, but there are also free maternal cells in the placenta and embryo. A theoretical explanation is that maternal lymphocytes alloreactive against the embryo will attempt to react with placental cells and in the process be phagocytosed, while other maternal cells will be able to enter the embryo where they could have a surveillance function, removing dead or mutant embryonic cells. To test this theory a series of experiments were carried out and are reported

  15. Nuclear Reprogramming in Mouse Primordial Germ Cells: Epigenetic Contribution

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    Massimo De Felici

    2011-01-01

    Full Text Available The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occur in vivo under still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly defined in vitro culture conditions, when PGCs are transformed into embryonic germ (EG cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.

  16. Resveratrol Enhances Self-Renewal of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Li, Na; Du, Zhaoyu; Shen, Qiaoyan; Lei, Qijing; Zhang, Ying; Zhang, Mengfei; Hua, Jinlian

    2017-07-01

    Resveratrol (RSV) has been shown to affect the differentiation of several types of stem cells, while the detailed mechanism is elusive. Here, we aim to investigate the function of RSV in self-renewal of mouse embryonic stem cells (ESCs) and the related mechanisms. In contrast with its reported roles, we found unexpectedly that differentiated ESCs or iPSCs treated by RSV would not show further differentiation, but regained a naïve pluripotency state with higher expressions of core transcriptional factors and with the ability to differentiate into all three germ layers when transplanted in vivo. In accordance with these findings, RSV also enhanced cell cycle progression of ESCs via regulating cell cycle-related proteins. Finally, enhanced activation of JAK/STAT3 signaling pathway and suppressed activation of mTOR were found essential in enhancing the self-renewal of ESCs by RSV. Our finding discovered a novel function of RSV in enhancing the self-renewal of ESCs, and suggested that the timing of treatment and concentration of RSV determined the final effect of it. Our work may contribute to understanding of RSV in the self-renewal maintenance of pluripotent stem cells, and may also provide help to the generation and maintenance of iPSCs in vitro. J. Cell. Biochem. 118: 1928-1935, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

    Science.gov (United States)

    Murphy, Sandra; Zweyer, Margit; Mundegar, Rustam R.; Henry, Michael; Meleady, Paula; Swandulla, Dieter; Ohlendieck, Kay

    2015-01-01

    The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context. PMID:28248273

  18. Specialized mouse embryonic stem cells for studying vascular development.

    Science.gov (United States)

    Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

    2014-01-01

    Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

  19. Dual Innervation of Neonatal Merkel Cells in Mouse Touch Domes

    Science.gov (United States)

    Luo, Wenqin

    2014-01-01

    Merkel cell-neurite complexes are specialized mechanosensory end organs that mediate discriminative touch sensation. It is well established that type I slowly adapting (SAI) mechanoreceptors, which express neural filament heavy chain (NFH), innervate Merkel cells. It was previously shown that neurotrophic factor NT3 and its receptor TrkC play crucial roles in controlling touch dome Merkel cell innervation of NFH+ fibers. In addition, nerve fibers expressing another neurotrophic tyrosine receptor kinase (NTRK), Ret, innervate touch dome Merkel cells as well. However, the relationship between afferents responsive to NT3/TrkC signaling and those expressing Ret is unclear. It is also controversial if these Ret+ fibers belong to the early or late Ret+ DRG neurons, which are defined based on the co-expression and developmental dependence of TrkA. To address these questions, we genetically traced Ret+ and TrkC+ fibers and analyzed their developmental dependence on TrkA. We found that Merkel cells in neonatal mouse touch domes receive innervation of two types of fibers: one group is Ret+, while the other subset expresses TrkC and NFH. In addition, Ret+ fibers depend on TrkA for their survival and normal innervation whereas NFH+ Merkel cell innervating fibers are almost unaltered in TrkA mutant mice, supporting that Ret+ and NFH+/TrkC+ afferents are two distinct groups. Ret signaling, on the other hand, plays a minor role for the innervation of neonatal touch domes. In contrast, Merkel cells in the glabrous skin are mainly contacted by NFH+/TrkC+ afferents. Taken together, our results suggest that neonatal Merkel cells around hair follicles receive dual innervation while Merkel cells in the glabrous skin are mainly innervated by only SAI mechanoreceptors. In addition, our results suggest that neonatal Ret+ Merkel cell innervating fibers most likely belong to the late but not early Ret+ DRG neurons. PMID:24637732

  20. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  1. Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos

    OpenAIRE

    Guo, Jitong; Wu, Baojiang; Li, Shuyu; Bao, Siqin; Zhao, Lixia; Hu, Shuxiang; Sun, Wei; Su, Jie; Dai, Yanfeng; Li, Xihe

    2014-01-01

    Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cel...

  2. Tachykinins stimulate a subset of mouse taste cells.

    Directory of Open Access Journals (Sweden)

    Jeff Grant

    Full Text Available The tachykinins substance P (SP and neurokinin A (NKA are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1. These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+-imaging on isolated taste cells, it was observed that SP induces Ca(2+ -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like and umami-responsive Type II (Receptor cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+ responses evoked by umami stimuli in Type II (Receptor cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.

  3. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Yongping Yue

    2016-01-01

    Full Text Available Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity.

  4. N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis.

    Science.gov (United States)

    Terrill, Jessica R; Radley-Crabb, Hannah G; Grounds, Miranda D; Arthur, Peter G

    2012-05-01

    Oxidative stress is implicated as a factor that increases necrosis of skeletal muscles in Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse. Consequently, drugs that minimize oxidative stress are potential treatments for muscular dystrophy. This study examined the in vivo benefits to mdx mice of an antioxidant treatment with the cysteine precursor N-acetylcysteine (NAC), administered in drinking water. NAC was completely effective in preventing treadmill exercise-induced myofibre necrosis (assessed histologically) and the increased blood creatine kinase levels (a measure of sarcolemma leakiness) following exercise were significantly lower in the NAC treated mice. While NAC had no effect on malondialdehyde level or protein carbonylation (two indicators of irreversible oxidative damage), treatment with NAC for one week significantly decreased the oxidation of glutathione and protein thiols, and enhanced muscle protein thiol content. These data provide in vivo evidence for protective benefits of NAC treatment on dystropathology, potentially via protein thiol modifications. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Ginseng Berry Extract Promotes Maturation of Mouse Dendritic Cells.

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    Wei Zhang

    Full Text Available Ginseng extract has been shown to possess certain anti-virus, anti-tumor and immune-activating effects. However, the immunostimulatory effect of ginseng berry extract (GB has been less well characterized. In this study, we investigated the effect of GB on the activation of mouse dendritic cells (DCs in vitro and in vivo. GB treatment induced up-regulation of co-stimulatory molecules in bone marrow-derived DCs (BMDCs. Interestingly, GB induced a higher degree of co-stimulatory molecule up-regulation than ginseng root extract (GR at the same concentrations. Moreover, in vivo administration of GB promoted up-regulation of CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen DCs. GB also promoted the generation of Th1 and Tc1 cells. Furthermore, Toll like receptor 4 (TLR4 and myeloid differentiation primary response 88 (MyD88 signaling pathway were essential for DC activation induced by GB. In addition, GB strongly prompted the proliferation of ovalbumin (OVA-specific CD4 and CD8 T cells. Finally, GB induced DC activation in tumor-bearing mice and the combination of OVA and GB treatment inhibited B16-OVA tumor cell growth in C57BL/6 mice. These results demonstrate that GB is a novel tumor therapeutic vaccine adjuvant by promoting DC and T cell activation.

  6. Effects of hyperthermia and radiation on mouse testis stem cells

    International Nuclear Information System (INIS)

    Reid, B.O.; Mason, K.A.; Withers, H.R.; West, J.

    1981-01-01

    The response of mouse testis stem cells to hyperthermia and combined hyperthermia-radiation treatments was assayed by spermatogenic colony regrowth, sperm head counts, testis weight loss, and fertility. With the use of spermatogenic colony assay, thermal enhancement ratios at an isosurvival level of 0.1 were 1.27 at 41 degrees, 1.80 at 42 degrees, and 3.97 at 43 degrees for testes exposed to heat for 30 min prior to irradiation. Sperm head counts were reduced by heat alone from a surviving fraction of 0.58 at 41 degrees to 0.003 at 42.5-43.5 degrees. Curves for sperm head survival measured 56 days after the testes had been heated for 30 min prior to irradiation were biphasic and showed a progressive downward displacement to lower survival with increasing temperature. The 41, 42, and 43 degrees curves were displaced downward by factors of 2, 58, and 175, respectively. The proportion of animals remaining sterile after 30 min of heat (41-43 degrees) and the median sterility period in days increased with increasing temperature. The minimum sperm count necessary to regain fertility was 13% of the normal mouse level

  7. Comparative action spectra for pyrimidine dimer formation in Cloudman S91 mouse melanoma and EMT6 mouse mammary carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hill, H Z [New Jersey, Medical School, Newark (USA); Setlow, R B [Brookhaven National Lab., Upton, NY (USA)

    1982-05-01

    Pyrimidine dimer formation in melanotic mouse melanoma cells, Cloudman S91H-, and in mouse mammary carcinoma cells, EMT6, was compared as a function of wavelength by irradiating equal numbers of cells from the two cell lines simultaneously. More dimers were formed in EMT6 than in S91H- by light of wavelengths less than 289nm, while light of higher wavelengths caused equivalent dimer formation, as measured by the Micrococcus luteus UV-endonuclease assay. The cells of S91H- are lightly melanotic, yet shielding at lower wavelengths is considerable. It is speculated that melanin pigmentation arose by selection during an evolutionary period when UV-C light reaching the earth's surface was significantly greater than it is today.

  8. Regeneration of tracheal epithelium using mouse induced pluripotent stem cells.

    Science.gov (United States)

    Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Otsuki, Koshi; Miyake, Masao; Hazama, Akihiro; Wada, Ikuo; Omori, Koichi

    2016-01-01

    Conclusion The findings demonstrated the potential use of induced pluripotent stem cells for regeneration of tracheal epithelium. Objective Autologous tissue implantation techniques using skin or cartilage are often applied in cases of tracheal defects with laryngeal inflammatory lesions and malignant tumor invasion. However, these techniques are invasive with an unstable clinical outcome. The purpose of this study was to investigate regeneration in a tracheal defect site of nude rats after implantation of ciliated epithelium that was differentiated from induced pluripotent stem cells. Method Embryoid bodies were formed from mouse induced pluripotent stem cells. They were cultured with growth factors for 5 days, and then cultured at the air-liquid interface. The degree of differentiation achieved prior to implantation was determined by histological findings and the results of real-time polymerase chain reaction. Embryoid bodies including ciliated epithelium were embedded into collagen gel that served as an artificial scaffold, and then implanted into nude rats, creating an 'air-liquid interface model'. Histological evaluation was performed 7 days after implantation. Results The ciliated epithelial structure survived on the lumen side of regenerated tissue. It was demonstrated histologically that the structure was composed of ciliated epithelial cells.

  9. Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells.

    Science.gov (United States)

    Jin, Cheng-Yu; Zhu, Bang-Shang; Wang, Xue-Feng; Lu, Qing-Hua

    2008-09-01

    Nanotitanium dioxide (TiO2) is an important industrial material that is widely used as an additive in cosmetics, pharmaceuticals, and food colorants. Although the small size of the TiO2 nanoparticle is useful in various applications, the biosafety of this material needs to be evaluated. In this study, mouse fibroblast (L929) cells were used to evaluate the cytotoxicity of different concentrations (3-600 microg/mL) of homogeneous and weakly aggregated TiO2 nanoparticles in aqueous solution. The L929 cells became round and even shrank as the concentration of TiO2 nanoparticles increased. Moreover, TiO2 nanoparticle-treated cells had condensed fragmented chromatin or were directly necrosed, as observed by acridine orange (AO) staining. The transmission electron microscopy (TEM) analysis showed that in cells cultured in a medium containing 300 microg/mL TiO2, the number of lysosomes increased, and some cytoplasmic organelles were damaged. In addition, there was a significant increase in oxidative stress at higher TiO2 nanoparticle concentrations (>60 microg/mL). As the concentration of TiO2 nanoparticles increased in the culture medium, the levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) increased, while those of methyl tetrazolium cytotoxicity (MTT), glutathione (GSH), and superoxide dismutase (SOD) decreased. A possible mechanism for the cytotoxicity of TiO2 nanoparticles is also discussed.

  10. Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts

    Science.gov (United States)

    Krais, Annette M.; Mühlbauer, Karl-Rudolf; Kucab, Jill E.; Chinbuah, Helena; Cornelius, Michael G.; Wei, Quan-Xiang; Hollstein, Monica; Phillips, David H.; Arlt, Volker M.; Schmeiser, Heinz H.

    2015-01-01

    We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by 32P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells. PMID:25230394

  11. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available This presentation is about the photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses. It outlines the background on embryonic stem cells (ES) and phototransfection....

  12. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wen-Zhong; Ai, Zhi-Ying [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Wang, Zhi-Wei [School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chen, Lin-Lin [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Guo, Ze-Kun, E-mail: gzknwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Zhang, Yong, E-mail: zylabnwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China)

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  13. Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

    Directory of Open Access Journals (Sweden)

    Christa L Tanganyika-de Winter

    2012-01-01

    Full Text Available Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs. Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype. Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

  14. Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate.

    Science.gov (United States)

    Okubo, Tadashi; Clark, Cheryl; Hogan, Brigid L M

    2009-02-01

    The epithelium of the mouse tongue and soft palate consists of at least three distinct epithelial cell populations: basal cells, keratinized cells organized into filiform and fungiform papillae, and taste receptor cells present in tight clusters known as taste buds in the fungiform and circumvallate papillae and soft palate. All three cell types develop from the simple epithelium of the embryonic tongue and palate, and are continually replaced in the adult by cell turnover. Previous studies using pulse-chase tritiated thymidine labeling in the adult mouse provided evidence for a high rate of cell turnover in the keratinocytes (5-7 days) and taste buds (10 days). However, little is known about the localization and phenotype of the long-term stem or progenitor cells that give rise to the mature taste bud cells and surrounding keratinocytes in these gustatory tissues. Here, we make use of a tamoxifen-inducible K14-CreER transgene and the ROSA26 LacZ reporter allele to lineage trace the mature keratinocytes and taste bud cells of the early postnatal and adult mouse tongue and soft palate. Our results support the hypothesis that both the pore keratinocytes and receptor cells of the taste bud are derived from a common K14(+)K5(+)Trp63(+)Sox2(+) population of bipotential progenitor cells located outside the taste bud. The results are also compatible with models in which the keratinocytes of the filiform and fungiform papillae are derived from basal progenitor cells localized at the base of these structures.

  15. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  16. Control of cell division and radiation injury in mouse skin

    International Nuclear Information System (INIS)

    Yamaguchi, Takeo

    1974-01-01

    The method for determining the inhibitors of cell division (chalone-adrenalin system) in the irradiated epidermis and blood was developed using the epidermis of mouse ear conch during the cure of wounds (in vivo), and the epidermis cultured for a long period (in vitro). The whole body was irradiated with 200KV, 20 mA x-rays of 96 R/min filtered by 0.5 mmCu + 0.5 mmAl. Chalone, which is a physiologically intrinsic substance to control the proliferation, inhibits the DNA synthesis. From changes in cell division with time, chalone in the epidermis is considered to inhibit each process from G 2 to M, from G 2 to S, from G 1 to S. Adrenalin is indispensable when epidermal chalone acts the inhibition of cell division. Chalone activities in the epidermis irradiated with almost lethal doses were decreased. Factors to inhibit the proliferation of the epidermis by the potentiation of chalone and adrenalin are present in sera of animals irradiated to x-rays. (Serizawa, K.)

  17. Cell-specific cre recombinase expression allows selective ablation of glutamate receptors from mouse horizontal cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Ströh

    Full Text Available In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57, a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99% and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl. In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50% in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less

  18. Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

    Science.gov (United States)

    Janssen-Bienhold, Ulrike; Schultz, Konrad; Cimiotti, Kerstin; Weiler, Reto; Willecke, Klaus; Dedek, Karin

    2013-01-01

    In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input

  19. Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Marrocco, V; Fiore, P; Benedetti, A; Pisu, S; Rizzuto, E; Musarò, A; Madaro, L; Lozanoska-Ochser, B; Bouché, M

    2017-02-01

    Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease. Duchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a "cure" for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology

  20. An optimized method for mouse liver sinusoidal endothelial cell isolation

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

    2016-12-10

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  1. An optimized method for mouse liver sinusoidal endothelial cell isolation

    International Nuclear Information System (INIS)

    Meyer, Jeremy; Lacotte, Stéphanie; Morel, Philippe; Gonelle-Gispert, Carmen; Bühler, Léo

    2016-01-01

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  2. Development and function of human innate immune cells in a humanized mouse model.

    Science.gov (United States)

    Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V; Teichmann, Lino L; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A Karolina; Manz, Markus G; Flavell, Richard A

    2014-04-01

    Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

  3. Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research

    NARCIS (Netherlands)

    de Rooij, Dirk G.; Mizrak, S. Canan

    2008-01-01

    In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into

  4. Differentiation of Mouse Ovarian Stem Cells Toward Oocyte-Like Structure by Coculture with Granulosa Cells.

    Science.gov (United States)

    Parvari, Soraya; Yazdekhasti, Hossein; Rajabi, Zahra; Gerayeli Malek, Valliollah; Rastegar, Tayebeh; Abbasi, Mehdi

    2016-11-01

    An increasing body of evidence has confirmed existence and function of ovarian stem cells (OSCs). In this study, a novel approach on differentiation of OSCs into oocyte-like cells (OLCs) has been addressed. Recently, different methods have been recruited to isolate and describe aspects of OSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate OSCs. Cell suspension of mouse neonatal ovaries was cultured and formed colonies were harvested mechanically and cultivated on mouse embryonic fibroblasts. For differentiation induction, colonies transferred on inactive granulosa cells. Results showed that cells in colonies were positive for alkaline phosphatase activity and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the pluripotency characteristics of cells. Immunofluorescence revealed a positive signal for OCT4, DAZL, MVH, and SSEA1 in colonies as well. Results of RT-PCR and immunofluorescence confirmed that some OLCs were generated within the germ stem cell (GSCs) colonies. The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economic than other techniques. Our results demonstrate that granulosa cells were effective in inducing the differentiation of OSCs into OLCs through direct cell-to-cell contacts.

  5. Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

    Science.gov (United States)

    Lv, Lei; Zhang, Tianwei; Yi, Qiyi; Huang, Yun; Wang, Zheng; Hou, Heli; Zhang, Huan; Zheng, Wei; Hao, Qiaomei; Guo, Zongyou; Cooke, Howard J; Shi, Qinghua

    2012-08-01

    Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

  6. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Satoru Yoshida

    Full Text Available BACKGROUND: Application of induced pluripotent stem (iPS cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. METHODOLOGY/PRINCIPAL FINDINGS: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. CONCLUSIONS/SIGNIFICANCE: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.

  7. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    V.B. CÂRSTEA

    2007-05-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  8. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    CÂRSTEA V. B

    2007-01-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  9. Characterization of Bovine 5′-flanking Region during Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Hye-Jeong Jang

    2015-12-01

    Full Text Available Embryonic stem cells (ESCs have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181 bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181 promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

  10. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim

    2011-01-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

  11. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

    Science.gov (United States)

    Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

    2011-11-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

  12. Grafting of a Single Donor Myofibre Promotes Hypertrophy in Dystrophic Mouse Muscle

    Science.gov (United States)

    Boldrin, Luisa; Morgan, Jennifer E.

    2013-01-01

    Skeletal muscle has a remarkable capability of regeneration following injury. Satellite cells, the principal muscle stem cells, are responsible for this process. However, this regenerative capacity is reduced in muscular dystrophies or in old age: in both these situations, there is a net loss of muscle fibres. Promoting skeletal muscle muscle hypertrophy could therefore have potential applications for treating muscular dystrophies or sarcopenia. Here, we observed that muscles of dystrophic mdx nude host mice that had been acutely injured by myotoxin and grafted with a single myofibre derived from a normal donor mouse exhibited increased muscle area. Transplantation experiments revealed that the hypertrophic effect is mediated by the grafted fibre and does not require either an imposed injury to the host muscle, or the contribution of donor cells to the host muscle. These results suggest the presence of a crucial cross-talk between the donor fibre and the host muscle environment. PMID:23349935

  13. Impact of methoxyacetic acid on mouse Leydig cell gene expression

    Directory of Open Access Journals (Sweden)

    Waxman David J

    2010-06-01

    Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

  14. Generation of thalamic neurons from mouse embryonic stem cells.

    Science.gov (United States)

    Shiraishi, Atsushi; Muguruma, Keiko; Sasai, Yoshiki

    2017-04-01

    The thalamus is a diencephalic structure that plays crucial roles in relaying and modulating sensory and motor information to the neocortex. The thalamus develops in the dorsal part of the neural tube at the level of the caudal forebrain. However, the molecular mechanisms that are essential for thalamic differentiation are still unknown. Here, we have succeeded in generating thalamic neurons from mouse embryonic stem cells (mESCs) by modifying the default method that induces the most-anterior neural type in self-organizing culture. A low concentration of the caudalizing factor insulin and a MAPK/ERK kinase inhibitor enhanced the expression of the caudal forebrain markers Otx2 and Pax6. BMP7 promoted an increase in thalamic precursors such as Tcf7l2 + /Gbx2 + and Tcf7l2 + /Olig3 + cells. mESC thalamic precursors began to express the glutamate transporter vGlut2 and the axon-specific marker VGF, similar to mature projection neurons. The mESC thalamic neurons extended their axons to cortical layers in both organotypic culture and subcortical transplantation. Thus, we have identified the minimum elements sufficient for in vitro generation of thalamic neurons. These findings expand our knowledge of thalamic development. © 2017. Published by The Company of Biologists Ltd.

  15. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Jennifer A Mitchell

    Full Text Available In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  16. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  17. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

    Science.gov (United States)

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2017-03-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

  18. PPARalpha/gamma expression and activity in mouse and human melanocytes and melanoma cells.

    Science.gov (United States)

    Eastham, Linda L; Mills, Caroline N; Niles, Richard M

    2008-06-01

    We examined the expression of PPARs and the effects of PPARalpha and PPARgamma agonists on growth of mouse and human melanocytes and melanoma cells. PPARalpha,beta, and PPARgamma mRNA qualitative expression in melan-a mouse melanocytes, B16 mouse melanoma, human melanocytes, and A375 and SK-mel28 human melanoma cells was determined by RT-PCR, while quantitative PPARalpha mRNA levels were determined by QuantiGene assay. PPARalpha and PPARgamma protein was assessed by Western blotting. The effect of natural and synthetic PPAR ligands on cell growth was determined by either hemocytometer counting or crystal violet assay. PPAR transcriptional activity was determined by a PPRE-reporter gene assay, while knockdown of PPARalpha expression was achieved by transient transfection of siRNA. Both mouse and human melanoma cells produced more PPARalpha and PPARgamma protein compared to melanocytes. PPARalpha mRNA levels were elevated in human melanoma cells, but not in mouse melanoma cells relative to melanocytes. Silencing of PPARalpha in human melanoma cells did not alter cell proliferation or morphology. PPARgamma-selective agonists inhibited the growth of both mouse and human melanoma cells, while PPARalpha-selective agonists had limited effects. Increased expression of PPARalpha in melanoma relative to melanocytes may be a common occurrence, however its biologic significance remains to be determined. PPARgamma agonists may be useful for arresting the growth of some melanomas.

  19. Cell membrane and cell junctions in differentiation of preimplanted mouse embryos.

    Science.gov (United States)

    Izquierdo, L; Fernández, S; López, T

    1976-12-01

    Cell membrane and cell junctions in differentiation of preimplanted mouse embryos, (membrana celular y uniones celulares en la diferenciación del embrión de ratón antes de la implantación). Arch. Biol. Med. Exper. 10: 130-134, 1976. The development of cell junctions that seal the peripheral blastomeres could be a decisive step in the differentiation of morulae into blastocysts. The appearance of these junctions is studied by electron microscopy of late morulae and initial blastocysts. Zonulae occludentes as well as impermeability to lanthanum emulsion precedes the appearance of the blastocel and hence might be considered as one of its necessary causes.

  20. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  1. Morphometric studies with attached mouse C3H/10T 1/2 cells

    International Nuclear Information System (INIS)

    Geard, C.R.; Harding, T.

    1981-01-01

    Studies of in vitro transformation using the Syrian hamster embryo cell system and the mouse C3H/10T 1/2 cell system form an integral part of this laboratory's activities. As part of the studies with the mouse cell line we have monitored the behavior of these cells in culture in order to ascertain those variables which might influence the expression of transformation. The study of transformed cells versus normal cells could lead to insight into an earlier definition of transformation that the clonal morphological change currently in use. This present report details the changes in cellular morphology with time in culture of normal mouse C3H/10T 1/2 cells from early passages (9 to 13) and x-ray transformed cells which have been maintained in culture for three years

  2. Cell flux through S phase in the mouse duodenal epithelium determined by cell sorting and radioautography

    International Nuclear Information System (INIS)

    Bjerknes, M.; Cheng, H.

    1982-01-01

    An accumulation of cells in early S phase was observed in normal mouse duodenal epithelium studied with flow cytometry. To determine if this accumulation of cells was the result of a lower rate of DNA synthesis, animals were given a single injection of 3 H-thymidine and the epithelium collected one hour later. The epithelium was processed for flow cytometry. Seven sort windows were established in different portions of the DNA histogram. Cells from each window were sorted onto glass slides that were then processed for radioautography. The number of silver grains over the nuclei of each sorted population was counted. It was found that cells in early S phase had significantly fewer grains over their nuclei than did mid- or late-S phase cells. We conclude that the accumulation of cells in early S phase is due, at least in part, to a lower rate of DNA synthesis in early than in mid or late S phase

  3. Immunologic analyses of mouse cystathionase in normal and leukemic cells

    International Nuclear Information System (INIS)

    Bikel, I.; Faibes, D.; Uren, J.R.; Livingston, D.M.

    1978-01-01

    Rabbit antisera have been raised against mouse liver cystathionase and shown to possess enzyme neutralizing activity. Agar gel double immunodiffusion analyses demonstrated that both mouse liver cystathionase and rat liver cystathionase react with the antisera, the latter enzyme being completely cross-reactive with the former. Following radioiodination of the purified rat liver enzyme, a double antibody radioimmunoassay was developed in which greater than 90% of the labeled protein could be specifically precipitated with the anti-mouse cystathionase antibodies. In this test the purified rat liver and mouse liver enzymes were virtually indistinguishable, generating superimposable competition displacement curves on a protein mass basis. These results indicate that both enzymes are immunologically identical, thus validating the use of the rat in lieu of the murine liver enzyme as radiolabeled tracer in an assay for mouse cystathionase. In addition, competition radioimmunoassays demonstrated that the immunological reactivities of both the purified rat liver and mouse liver enzymes were equally heat sensitive. The sensitivity of the assay was determined to be 1 ng of enzyme protein/0.22 mL of assay mixture, and the assay could be used to detect the presence of enzyme protein in tissue homogenates of single mouse organs. Mouse or rat cross-reactivity with human liver cystathionase was incomplete; but, with the exception of heart and spleen, parallel radioimmunoassay competition displacement curves were obtained for cystathionase from different mouse organs including thymus. Extracts of 7-, 9-, and 10-month-old spontaneous AKR mouse thymomas were tested in the radioimmunoassay along with extracts of age-matched thymuses which were grossly tumor free. A reaction of nonidentity was observed for all of the tumor extracts while a reaction identical with that of the pure liver enzyme was found with all of the normal thymus extracts

  4. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  5. Repair and mutation induction in mouse germ cells: a summary and some thoughts

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    The various lines of evidence for repair of premutational damage in mouse germ cells are reviewed with the implications for future experiment planning. Relation between mutagenicity and carcinogenicity are discussed

  6. Characterization of mitomycin-C-sensitive mouse lymphoma L5178Y cell mutants

    International Nuclear Information System (INIS)

    Inaba, Hiroko; Shiomi, Naoko; Shiomi, Tadahiro; Sato, Koki; Yoshida, Michihiro.

    1985-01-01

    Twenty-six mutants showing high sensitivity to mytomicin-C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Twenty-five of the mutants were 5 - 10 times more sensitive to MMC than were parental cells, and showed normal sensitivity to U.V. light and x-rays. From a complementation analysis, 5 mutants (MC s ) isolated from independently mutagenized cell populations were classified into two groups. These mutants possessed recessive character for MMC-sensitivity and there were at least two genes involved in the MMC-sensitivity. As for DNA-damaging factors, such as photoadducts of 8-methoxypsoralen (8-MOP) and 3-carbethoxysoralen (3-CPs), MC s mutants showed higher sensitivity to photoadducts of 8-MOP than to (3-CPs). MC s mutants were also highly sensitive to a DNA cross-linking agent, cisplatin. Characterization of the sensitivity of mouse MC s mutants was analogous to that of Fanconi's anemia (FA)-derived cells. Low concentrations (10 ng/ml) of MMC induced chromosome aberration in a high incidence in mouse MC s cells, as well as in FA cells. The frequency of MMC-induced chromosome aberrations was normal in hybrid cells between normal human diploid somatic cells and mouse mutants and between FA cells and mouse wild cells, and hereditary deficiency became normal by hybrization. (Namekawa, K.)

  7. Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

    International Nuclear Information System (INIS)

    Gliddon, Briony L; Nguyen, Nhung V; Gunn, Priscilla A; Gleeson, Paul A; Driel, Ian R van

    2008-01-01

    Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H + /K + ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H + /K + ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H + /K + ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H + /K + ATPase which underpin the regulation of acid secretion

  8. Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells

    DEFF Research Database (Denmark)

    Dong, Guoyi; Shang, Zhouchun; Liu, Longqi

    2017-01-01

    Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse embryonic stem cells (ESCs) can directly differentiate into advanced male germ cell lineage in the same conditions. Here......, we showed rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by the activation of spermatogenesis-associated genes...... such as Mvh, Dazl, Prdm14, Stella, Scp1, Scp3, Stra8 and Rec8. In contrast, for cells cultured in control medium, the activation of the above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin-expressing cells and the positive ratio reached a peak at day 6. Our work thus...

  9. Is radiation-induced cell death in mouse testis apoptosis?

    International Nuclear Information System (INIS)

    Hasegawa, Masatoshi; Wilson, Gene; Yun Zhang; Russell, Lonnie D.; Meistrich, Marvin L.

    1996-01-01

    Purpose: Radiation-induced death of spermatogonia and other germ cells in the testis has been claimed to be by an apoptotic mechanism, but these processes have been incompletely characterized. We investigated irradiated mouse testis by multiple techniques to determine whether the mode of cell death of spermatogonia can be classified as apoptosis. Materials and Methods: Adult male C57BL/6 and p53 knockout mice were irradiated with single doses of 0.5, 2.5 or 5.0 Gy. Four, 6, 8, 12, 18 or 24 hours after irradiation, testes were fixed in Bouin's solution or in 10% formalin. Slides were stained with hematoxylin and eosin or TdT-mediated dUTP-biotin nick end labeling (TUNEL). Some testes were perfusion-fixed with 5% glutaraldehyde for electron microscopy. Gel electrophoresis of DNA was also performed to identify DNA fragmentation. The number of sperm heads was counted 29 days after irradiation to evaluate the effect of radiation on the eventual survival of the differentiated spermatogonia. Results: The earliest sign of histological damage was an increase in the numbers of abnormal spermatogonia in the seminiferous tubules, particularly in stage I-VI of the seminiferous epithelial cycle. The numbers of abnormal spermatogonia began to increase at 6 hours, reached a peak 12 hours after irradiation, and then declined. The total number of spermatogonia began to decrease at 12 hours after irradiation, resulting in a 60% decline in sperm produced 29 days after 0.5 Gy. Although changes were greatest following 5.0 Gy irradiation, even 0.5 Gy induced marked changes. However, these changes were not induced in p53 knockout mice. By both light and electron microscopy, spermatogonia showed some condensation of nuclear chromatin, but margination of chromatin with clear delineation and nuclear fragmentation was rare. Many of the abnormal spermatogonia showed a positive TUNEL reaction, which was also at a maximum at 12 hours after irradiation. In addition, some TUNEL-positive and

  10. Regulatory T cells suppress muscle inflammation and injury in muscular dystrophy

    Science.gov (United States)

    Villalta, S. Armando; Rosenthal, Wendy; Martinez, Leonel; Kaur, Amanjot; Sparwasser, Tim; Tidball, James G.; Margeta, Marta; Spencer, Melissa J.; Bluestone, Jeffrey A.

    2016-01-01

    We examined the hypothesis that regulatory T cells (Tregs) modulate muscle injury and inflammation in the mdx mouse model of Duchenne muscular dystrophy (DMD). Although Tregs were largely absent in the muscle of wildtype mice and normal human muscle, they were present in necrotic lesions, displayed an activated phenotype and showed increased expression of interleukin (IL)-10 in dystrophic muscle from mdx mice. Depletion of Tregs exacerbated muscle injury and the severity of muscle inflammation, which was characterized by an enhanced interferon-gamma (IFNγ) response and activation of M1 macrophages. To test the therapeutic value of targeting Tregs in muscular dystrophy, we treated mdx mice with IL-2/anti-IL-2 complexes (IL-2c), and found that Tregs and IL-10 concentrations were increased in muscle, resulting in reduced expression of cyclooygenase-2 and decreased myofiber injury. These findings suggest that Tregs modulate the progression of muscular dystrophy by suppressing type 1 inflammation in muscle associated with muscle fiber injury, and highlight the potential of Treg-modulating agents as therapeutics for DMD. PMID:25320234

  11. Cell of Origin and Cancer Stem Cells in Tumor Suppressor Mouse Models of Glioblastoma.

    Science.gov (United States)

    Alcantara Llaguno, Sheila R; Xie, Xuanhua; Parada, Luis F

    2016-01-01

    The cellular origins and the mechanisms of progression, maintenance of tumorigenicity, and therapeutic resistance are central questions in the glioblastoma multiforme (GBM) field. Using tumor suppressor mouse models, our group recently reported two independent populations of adult GBM-initiating central nervous system progenitors. We found different functional and molecular subtypes depending on the tumor-initiating cell lineage, indicating that the cell of origin is a driver of GBM subtype diversity. Using an in vivo model, we also showed that GBM cancer stem cells (CSCs) or glioma stem cells (GSCs) contribute to resistance to chemotherapeutic agents and that genetic ablation of GSCs leads to a delay in tumor progression. These studies are consistent with the cell of origin and CSCs as critical regulators of the pathogenesis of GBM. © 2016 Alcantara Llaguno et al; Published by Cold Spring Harbor Laboratory Press.

  12. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  13. Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

    2016-01-01

    Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

  14. Establishment of mouse neuron and microglial cell co-cultured models and its action mechanism.

    Science.gov (United States)

    Zhang, Bo; Yang, Yunfeng; Tang, Jun; Tao, Yihao; Jiang, Bing; Chen, Zhi; Feng, Hua; Yang, Liming; Zhu, Gang

    2017-06-27

    The objective of this study is to establish a co-culture model of mouse neurons and microglial cells, and to analyze the mechanism of action of oxygen glucose deprivation (OGD) and transient oxygen glucose deprivation (tOGD) preconditioning cell models. Mouse primary neurons and BV2 microglial cells were successfully cultured, and the OGD and tOGD models were also established. In the co-culture of mouse primary neurons and microglial cells, the cell number of tOGD mouse neurons and microglial cells was larger than the OGD cell number, observed by a microscope. CCK-8 assay result showed that at 1h after treatment, the OD value in the control group is lower compared to all the other three groups (P control group compared to other three groups (P neurons cells were cultured. In the meantime mouse BV2 microglia cells were cultured. Two types of cells were co-cultured, and OGD and tOGD cell models were established. There were four groups in the experiment: control group (OGD), treatment group (tOGD+OGD), placebo group (tOGD+OGD+saline) and minocycline intervention group (tOGD+OGD+minocycline). CCK-8 kit was used to detect cell viability and flow cytometry was used to detect apoptosis. In this study, mouse primary neurons and microglial cells were co-cultured. The OGD and tOGD models were established successfully. tOGD was able to effectively protect neurons and microglial cells from damage, and inhibit the apoptosis caused by oxygen glucose deprivation.

  15. Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhe-Hao Zhang

    Full Text Available Nicotinic acid adenine dinucleotide phosphate (NAADP is an endogenous Ca(2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca(2+ from acidic organelles through two pore channel 2 (TPC2 in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.

  16. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    International Nuclear Information System (INIS)

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-01-01

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development

  17. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    Energy Technology Data Exchange (ETDEWEB)

    Webb, Carol F., E-mail: carol-webb@omrf.org [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Ratliff, Michelle L., E-mail: michelle-ratliff@omrf.org [Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Powell, Rebecca, E-mail: rebeccapowell@gmail.com [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Wirsig-Wiechmann, Celeste R., E-mail: celeste-wirsig@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Lakiza, Olga, E-mail: olga-lakiza@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Obara, Tomoko, E-mail: tomoko-obara@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States)

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  18. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

    OpenAIRE

    Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

    2012-01-01

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomita...

  19. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    International Nuclear Information System (INIS)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-01-01

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1 + or nestin + stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU + cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU + cells, very few are mash1 + or nestin + stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1 + microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition

  20. Revertant fibers in the mdx murine model of Duchenne muscular dystrophy: an age- and muscle-related reappraisal.

    Directory of Open Access Journals (Sweden)

    Sarah R Pigozzo

    Full Text Available Muscles in Duchenne dystrophy patients are characterized by the absence of dystrophin, yet transverse sections show a small percentage of fibers (termed "revertant fibers" positive for dystrophin expression. This phenomenon, whose biological bases have not been fully elucidated, is present also in the murine and canine models of DMD and can confound the evaluation of therapeutic approaches. We analyzed 11 different muscles in a cohort of 40 mdx mice, the most commonly model used in pre-clinical studies, belonging to four age groups; such number of animals allowed us to perform solid ANOVA statistical analysis. We assessed the average number of dystrophin-positive fibers, both absolute and normalized for muscle size, and the correlation between their formation and the ageing process. Our results indicate that various muscles develop different numbers of revertant fibers, with different time trends; besides, they suggest that the biological mechanism(s behind dystrophin re-expression might not be limited to the early development phases but could actually continue during adulthood. Importantly, such finding was seen also in cardiac muscle, a fact that does not fit into the current hypothesis of the clonal origin of "revertant" myonuclei from satellite cells. This work represents the largest, statistically significant analysis of revertant fibers in mdx mice so far, which can now be used as a reference point for improving the evaluation of therapeutic approaches for DMD. At the same time, it provides new clues about the formation of revertant fibers/cardiomyocytes in dystrophic skeletal and cardiac muscle.

  1. [Regulatory T cells inhibit proliferation of mouse lymphoma cell line EL4 in vitro].

    Science.gov (United States)

    Zhang, Chen; Kong, Yan; Guo, Jun; Ying, Zhi-Tao; Yuan, Zhi-Hong; Zhang, Yun-Tao; Zheng, Wen; Song, Yu-Qin; Li, Ping-Ping; Zhu, Jun

    2010-10-01

    This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.

  2. Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice.

    Science.gov (United States)

    Divet, Alexandra; Huchet-Cadiou, Corinne

    2002-08-01

    The aim of the present study was to establish whether alterations in sarcoplasmic reticulum function are involved in the abnormal Ca(2+) homeostasis of skeletal muscle in mice with muscular dystrophy ( mdx). The properties of the sarcoplasmic reticulum and contractile proteins of fast- and slow-twitch muscles were therefore investigated in chemically skinned fibres isolated from the extensor digitorum longus (EDL) and soleus muscles of normal (C57BL/10) and mdx mice at 4 and 11 weeks of development. Sarcoplasmic reticulum Ca(2+) uptake, estimated by the Ca(2+) release following exposure to caffeine, was significantly slower in mdx mice, while the maximal Ca(2+) quantity did not differ in either type of skeletal muscle at either stage of development. In 4-week-old mice spontaneous sarcoplasmic reticulum Ca(2+) leakage was observed in EDL and soleus fibres and this was more pronounced in mdx mice. In addition, the maximal Ca(2+)-activated tension was smaller in mdx than in normal fibres, while the Ca(2+) sensitivity of the contractile apparatus was not significantly different. These results indicate that mdx hindlimb muscles are affected differently by the disease process and suggest that a reduced ability of the Ca(2+)-ATPase to load Ca(2+) and a leaky sarcoplasmic reticulum membrane may be involved in the altered intracellular Ca(2+) homeostasis.

  3. Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling

    NARCIS (Netherlands)

    Singh, Simar Pal; Pillai, Saravanan Y.; de Bruijn, Marjolein J. W.; Stadhouders, Ralph; Corneth, Odilia B. J.; van den Ham, Henk Jan; Muggen, Alice; van Ijcken, Wilfred; Slinger, Erik; Kuil, Annemieke; Spaargaren, Marcel; Kater, Arnon P.; Langerak, Anton W.; Hendriks, Rudi W.

    2017-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5(+) B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6,

  4. Mesenchymal and embryonic characteristics of stem cells obtained from mouse dental pulp

    DEFF Research Database (Denmark)

    Guimarães, Elisalva Teixeira; Cruz, Gabriela Silva; de Jesus, Alan Araújo

    2011-01-01

    abnormalities was evaluated by G banding. RESULTS: The mouse dental pulp stem cells (mDPSC) were highly proliferative, plastic-adherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were...

  5. EBI3 regulates the NK cell response to mouse cytomegalovirus infection

    DEFF Research Database (Denmark)

    Jensen, Helle; Chen, Shih-Yu; Folkersen, Lasse Westergaard

    2017-01-01

    Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection. The induc......Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection....... The induction of EBI3 protein expression in mouse NK cells is a late activation event. Thus, early activation events of NK cells, such as IFNγ production and CD69 expression, were not affected in EBI3-deficient (Ebi3-/-) C57BL/6 (B6) mice during MCMV infection. Furthermore, comparable levels of early viral...... replication in spleen and liver were observed in MCMV-infected Ebi3-/- and wild-type (WT) B6 mice. Interestingly, the viral load in salivary glands and oral lavage was strongly decreased in the MCMV-infected Ebi3-/- B6 mice, suggesting that EBI3 plays a role in the establishment of MCMV latency. We detected...

  6. Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse.

    Science.gov (United States)

    Arlt, Volker M; Gingerich, John; Schmeiser, Heinz H; Phillips, David H; Douglas, George R; White, Paul A

    2008-11-01

    FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

  7. Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

    OpenAIRE

    Sandra M. López-Heydeck

    2009-01-01

    Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be effi cient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain...

  8. Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Szu-Hsiu Liu

    2012-01-01

    Full Text Available Embryonic stem (ES cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs by a two-step differentiation protocol comprising of (i the formation of definitive endoderm in monolayer culture by activin A, and (ii this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

  9. Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

    2017-01-01

    New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

  10. Treatment with the cysteine precursor l-2-oxothiazolidine-4-carboxylate (OTC) implicates taurine deficiency in severity of dystropathology in mdx mice.

    Science.gov (United States)

    Terrill, Jessica R; Boyatzis, Amber; Grounds, Miranda D; Arthur, Peter G

    2013-09-01

    Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne muscular dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering l-2-oxothiazolidine-4-carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Transcription of a novel mouse semaphorin gene, M-semaH, correlates with the metastatic ability of mouse tumor cell lines

    DEFF Research Database (Denmark)

    Christensen, C R; Klingelhöfer, Jörg; Tarabykina, S

    1998-01-01

    identified a novel member of the semaphorin/collapsin family in the two metastatic cell lines. We have named it M-semaH. Northern hybridization to a panel of tumor cell lines revealed transcripts in 12 of 12 metastatic cell lines but in only 2 of 6 nonmetastatic cells and none in immortalized mouse...

  12. Allergen and Epitope Targets of Mouse-Specific T Cell Responses in Allergy and Asthma

    Directory of Open Access Journals (Sweden)

    Véronique Schulten

    2018-02-01

    Full Text Available Mouse allergy has become increasingly common, mainly affecting laboratory workers and inner-city households. To date, only one major allergen, namely Mus m 1, has been described. We sought to identify T cell targets in mouse allergic patients. PBMC from allergic donors were expanded with either murine urine or epithelial extract and subsequently screened for cytokine production (IL-5 and IFNγ in response to overlapping peptides spanning the entire Mus m 1 sequence, peptides from various Mus m 1 isoforms [major urinary proteins (MUPs], peptides from mouse orthologs of known allergens from other mammalian species and peptides from proteins identified by immunoproteomic analysis of IgE/IgG immunoblots of mouse urine and epithelial extracts. This approach let to the identification of 106 non-redundant T cell epitopes derived from 35 antigens. Three major T cell-activating regions were defined in Mus m 1 alone. Moreover, our data show that immunodominant epitopes were largely shared between Mus m 1 and other MUPs even from different species, suggesting that sequence conservation in different allergens is a determinant for immunodominance. We further identified several novel mouse T cell antigens based on their homology to known mammalian allergens. Analysis of cohort-specific T cell responses revealed that rhinitis and asthmatic patients recognized different epitope repertoires. Epitopes defined herein can be formulated into an epitope “megapool” used to diagnose mouse allergy and study mouse-specific T cell responses directly ex vivo. This analysis of T cell epitopes provides a good basis for future studies to increase our understanding of the immunopathology associated with MO-allergy and asthma.

  13. Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1

    International Nuclear Information System (INIS)

    Moon, Jai-Hee; Yoon, Byung Sun; Kim, Bona; Park, Gyuman; Jung, Hye-Youn; Maeng, Isaac; Jun, Eun Kyoung; Yoo, Seung Jun; Kim, Aeree; Oh, Sejong; Whang, Kwang Youn; Kim, Hyunggee; Kim, Dong-Wook; Kim, Ki Dong; You, Seungkwon

    2008-01-01

    Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1

  14. Cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bootsma, A.L. (Rijksuniversiteit Utrecht (Netherlands). Academisch Ziekenhuis); Davids, J.A.G. (Netherlands Energy Research Foundation, Petten (Netherlands))

    1988-03-01

    In the CBA mouse testis, about 10% of the stem cell population is highly resistant to neutron irradiation (D/sub 0/, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea, it appeared that in this cycle the S-phase is less radiosensitive (D/sub 0/, 0.43 Gy) than the other phases of the cell cycle (D/sub 0/, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation, the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most. (author).

  15. Single cell lineage analysis of mouse embryonic stem cells at the exit from pluripotency

    Directory of Open Access Journals (Sweden)

    Jamie Trott

    2013-08-01

    Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/β-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active β-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.

  16. Cyclin G1 inhibits the proliferation of mouse endometrial stromal cell in decidualization

    Directory of Open Access Journals (Sweden)

    Xu Qian

    2017-01-01

    Full Text Available Uterine stromal cell decidualization is a dynamic physiological process in which cell proliferation, differentiation and apoptosis are orchestrated and occur in a temporal and cell-specific manner. This process is important for successful embryo implantation. Many cell-cycle regulators are involved in decidualization. The protein cyclin G1 is a unique regulator of the cell cycle with dual functions in cell proliferation. It was reported that cyclin G1 is expressed in mouse uterine stromal cells during the period of peri-implantation. To prove the function of cyclin G1 in mouse uterine stromal cells during this period, immunohistochemistry was used to stain mouse uterine tissues on days 4-8 of pregnancy. The results showed obvious spatial and temporal expression of cyclin G1 in uterine stromal cells, and that it is expressed in the cells of the primary decidual zone (PDZ on day 5 and secondary decidual zone (SDZ on days 6 and 7, when the stromal cells experienced active proliferation and differentiation was initiated. Applying the decidualization model of cultured primary stromal cells in vitro, we further revealed that the expression of cyclin G1 is associated with decidualization of stromal cells induced by medroxyprogesterone acetate (MPA and estradiol-17β (E2. RNA interference was used for the knockdown of cyclin G1 in the induced decidual cells. Flow cytometry analysis indicated that the proportion of cells in the S stage was increased, and decreased in the G2/M phase. Our study indicates that cyclin G1, as a negative regulator of the cell cycle, plays an important role in the process of decidualization in mouse uterine stromal cells by inhibiting cell-cycle progression.

  17. Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

    Directory of Open Access Journals (Sweden)

    Cremer Thomas

    2005-12-01

    Full Text Available Abstract Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

  18. Ulex europaeus 1 lectin targets microspheres to mouse Peyer's patch M-cells in vivo.

    Science.gov (United States)

    Foster, N; Clark, M A; Jepson, M A; Hirst, B H

    1998-03-01

    The interaction of latex microspheres with mouse Peyer's patch membranous M-cells was studied in a mouse gut loop model after the microspheres were coated with a variety of agents. Carboxylated microspheres (diameter 0.5 micron) were covalently coated with lectins Ulex europaeus 1, Concanavalin A, Euonymus europaeus and Bandeiraea simplicifolia 1 isolectin-B4, human immunoglobulin A or bovine serum albumin. Of the treatments examined, only Ulex europaeus (UEA1) resulted in significant selective binding of microspheres to M-cells. UEA1-coated microspheres bound to M-cells at a level 100-fold greater than BSA-coated microspheres, but binding to enterocytes was unaffected. Incubation of UEA1-coated microspheres with alpha-L-fucose reduced M-cell binding to a level comparable with BSA-coated microspheres. This indicated that targeting by UEA1 was via a carbohydrate receptor on the M-cell surface. Adherence of UEA1-coated microspheres to M-cells occurred within 10 min of inoculation into mouse gut loops and UEA1-coated microspheres were transported to 10 microns below the apical surface of M-cells within 60 min of inoculation. UEA1-coated microspheres also targeted mouse Peyer's patch M-cells after intragastric administration. These results demonstrated that altering the surface chemistry of carboxylated polystyrene microspheres increased M-cell targeting, suggesting a strategy to enhance delivery of vaccine antigens to the mucosal immune system.

  19. E2F transcription factor-1 deficiency reduces pathophysiology in the mouse model of Duchenne muscular dystrophy through increased muscle oxidative metabolism.

    Science.gov (United States)

    Blanchet, Emilie; Annicotte, Jean-Sébastien; Pradelli, Ludivine A; Hugon, Gérald; Matecki, Stéfan; Mornet, Dominique; Rivier, François; Fajas, Lluis

    2012-09-01

    E2F1 deletion leads to increased mitochondrial number and function, increased body temperature in response to cold and increased resistance to fatigue with exercise. Since E2f1-/- mice show increased muscle performance, we examined the effect of E2f1 genetic inactivation in the mdx background, a mouse model of Duchenne muscular dystrophy (DMD). E2f1-/-;mdx mice demonstrated a strong reduction of physiopathological signs of DMD, including preservation of muscle structure, decreased inflammatory profile, increased utrophin expression, resulting in better endurance and muscle contractile parameters, comparable to normal mdx mice. E2f1 deficiency in the mdx genetic background increased the oxidative metabolic gene program, mitochondrial activity and improved muscle functions. Interestingly, we observed increased E2F1 protein levels in DMD patients, suggesting that E2F1 might represent a promising target for the treatment of DMD.

  20. Endogenous superoxide dismutase and catalase activities and radiation resistance in mouse cell lines

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Ostrand-Rosenberg, S.

    1988-01-01

    The relationship between the endogenous cytoplasmic levels of the enzymes superoxide dismutase and catalase and the inhibition of cell proliferation by γ-radiation has been studied in 11 mouse cell lines. The resistance of these mouse cell lines to radiation was found to vary by over 25-fold. No correlation was found between the cytoplasmic level of CuZn-superoxide dismutase or catalase and the resistance to radiation as measured by extrapolation number (EN), quasi-threshold dose (Dsub(q)), or Dsub(o). None of the cell lines had detectable cytoplasmic Mn-superoxide dismutase. The apparent Ksub(i) of potassium cyanide for mouse CuZn-superoxide dismutase was determined (Ksub(i) = 6.5 μmol dm -3 ). (author)

  1. Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Brandl, Julian

    2017-01-01

    , counting 801 different components in mouse. By employing our mouse RECON to the CHO-K1 genome in a comparative genomic approach, we could reconstruct the protein secretory pathway of CHO cells counting 764 CHO components. This RECON furthermore facilitated the development of three alternative methods...... to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional...... regulation of protein secretion than healthy mouse cells.  Conclusions: The RECON of the secretory pathway represents a strong tool for interpretation of data related to protein secretion as illustrated with transcriptomic data of Chinese Hamster Ovary (CHO) cells, the main platform for mammalian protein...

  2. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

    Science.gov (United States)

    Wang, Ruoxing; Guo, Yan-Lin

    2012-10-01

    Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Radiosensitization of mouse spermatogenic stem cells by Ro-07-0582

    International Nuclear Information System (INIS)

    Suzuki, N.; Withers, R.; Hunter, N.

    1977-01-01

    The hypoxic character of the spermatogenic stem cells of the mouse testis was investigated by measuring the effect on radiosensitivity of treatment with the hypoxic cell radiosensitizer, Ro-07-0582 or hyperbaric oxygen (30 psi). The D 0 values obtained were 181 (161-207) rad for irradiation alone, 140 (133-148) rad for irradiation after treatment with Ro-07-0582, and about 100 rad for irradiation in the presence of hyperbaric oxygen. Ro-07-0582 alone was slightly cytotoxic. The results demonstrate that mouse spermatogenic stem cells are radiosensitized by Ro-07-0582 or hyperbaric oxygen and are not as well oxygenated as other normal tissues

  4. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Hayashi, Katsuhiko; Saitou, Mitinori

    2013-08-01

    Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

  5. Cell cycle evaluation of granulosa cells in the {gamma}-irradiated mouse ovarian follicles

    Energy Technology Data Exchange (ETDEWEB)

    KIm, Jin Kyu; Lee, Chang Joo; Lee, Young Keun [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of); Song, Kang Won; Yoon, Yong Dal [Hanyang Univ., Seoul (Korea, Republic of)

    1999-03-01

    This study was carried out to evaluate the biochemical and morphological effects of ionizing radiation on mouse ovarian follicles. Immature mice (ICR, 3 week-old) were irradiated with a dose of LD{sub 80(30)} at KAERI. The ovaries were collected after 6 hours, 12 hours, 1 day, and 2 days post irradiation. With the morphological basis of the histological staining with hematoxylin-eosin, immunohistochemical preparation using in situ 3'-end labeling was evaluated. Flow cytometric evaluation of DNA extracted from the whole ovary was performed. The percentage of A{sub 0} (subpopulation of cells with degraded DNA and with lower DNA fluorescence than G{sub 0}/G{sub 1} cells), apoptotic, cells in the cell cycle was significantly higher in the irradiated group than in the control group. The number of in situ 3'-end labeled follicles increased at 6 hours post irradiation. All the analyses represented that the ionizing radiation-induced follicular atresia was taken place via an apoptotic degeneration. Such a degeneration underwent very fast and acutely. Therefore, it is concluded that the radiation-induced follicular degeneration is, like the spontaneous atresia, mediated by an acute apoptosis of follicular granulosa cells. Flow cytometric evaluation of cell cycles can make the role for quantifying the atretic follicles and understanding the mechanism of the radiation-induced cell death.

  6. The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell

    DEFF Research Database (Denmark)

    Møller, Morten; Rath, Martin F; Klein, David C

    2006-01-01

    The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal g...... for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

  7. Assessment of a 42 metal salts chemical library in mouse embryonic stem cells

    Science.gov (United States)

    The developmental effects of xenobiotics on differentiation can be profiled using mouse embryonic stem cells (mESCs). The adherent cell differentiation and cytotoxicity (ACDC) technique was used to evaluate a library of 42 metal and metaloid salts. Jl mESCs were allowed to prolif...

  8. The toxicity of silver and silica nanoparticles in comparable human and mouse cell lines

    DEFF Research Database (Denmark)

    Foldbjerg, Rasmus; Beer, Christiane; Sutherland, Duncan S

    The toxicity of silica (SiO2) and PVP-coated silver (Ag) nanoparticles (NPs) was investigated in two pairs of human or mouse cell lines originating from lung epithelium (A549 and ASB-XIV) and macrophages (THP-1 and J744A.1). Both NPs were characterized in H2O and cell media and demonstrated to be...

  9. In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

    International Nuclear Information System (INIS)

    Ishii, Takamichi; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Baba, Shinji; Naito, Masato; Machimoto, Takafumi; Kamo, Naoko; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao

    2005-01-01

    It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 ± 12.2% (means ± SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes

  10. Influence of Botulinumtoxin A on the Expression of Adult MyHC Isoforms in the Masticatory Muscles in Dystrophin-Deficient Mice (Mdx-Mice

    Directory of Open Access Journals (Sweden)

    Ute Ulrike Botzenhart

    2016-01-01

    Full Text Available The most widespread animal model to investigate Duchenne muscular dystrophy is the mdx-mouse. In contrast to humans, phases of muscle degeneration are replaced by regeneration processes; hence there is only a restricted time slot for research. The aim of the study was to investigate if an intramuscular injection of BTX-A is able to break down muscle regeneration and has direct implications on the gene expression of myosin heavy chains in the corresponding treated and untreated muscles. Therefore, paralysis of the right masseter muscle was induced in adult healthy and dystrophic mice by a specific intramuscular injection of BTX-A. After 21 days the mRNA expression and protein content of MyHC isoforms of the right and left masseter, temporal, and the tongue muscle were determined using quantitative RT-PCR and Western blot technique. MyHC-IIa and MyHC-I-mRNA expression significantly increased in the paralyzed masseter muscle of control-mice, whereas MyHC-IIb and MyHC-IIx/d-mRNA were decreased. In dystrophic muscles no effect of BTX-A could be detected at the level of MyHC. This study suggests that BTX-A injection is a suitable method to simulate DMD-pathogenesis in healthy mice but further investigations are necessary to fully analyse the BTX-A effect and to generate sustained muscular atrophy in mdx-mice.

  11. Stimulation of cytolytic T lymphocytes by azaguanine-resistant mouse tumor cells in selective hat medium

    International Nuclear Information System (INIS)

    Snick, J. van; Uyttenhove, C.; Pel, A. van; Boon, T.

    1981-01-01

    Primed syngeneic or umprimed allogeneic mouse spleen cells were stimulated with azaguanine-resistant P815 tumor cells that were killed by the addition of aminopterin to the stimulation medium. The recovery of lymphocytes and their cytolytic activity and specificity were similar to those obtained after stimulation with irradiated cells. This method conveniently replaces the inactivation of stimulatory cells by irradiation or mitomycin treatment. Moreover, it has the advantage of inactivating not only the stimulatory cells but also the tumor cells that often contaminate the spleens of tumor-bearing animals, provided these animals have been inoculated with azaguanine-resistant tumor cell mutants. (Auth.)

  12. Radiation response of mouse lymphoid and myeloid cell lines. Pt. 1

    International Nuclear Information System (INIS)

    Radford, I.R.

    1994-01-01

    The sensitivity of 10 mouse lymphoid or myeloid cell lines to γ-ray- and DNA-associated 125 I-decay-induced clonogenic cell killing have been compared with their rate of loss of viability (membrane integrity) and with their putative cell type of origin. The increased sensitivity of haematopoietic cell lines to killing by DNA dsb may be related to their mode of death (apoptosis versus necrosis). Mode of cell death may thus be an important factor in determining the 'inherent radiosensitivity' of normal cells/tissues. Haematopoietic cell lines that undergo rapid interphase apoptotic death showed extreme sensitivity to DNA dsb. (author)

  13. Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: Importance of stem cell composition in competitor cells.

    Science.gov (United States)

    Ema, Hideo; Uchinomiya, Kouki; Morita, Yohei; Suda, Toshio; Iwasa, Yoh

    2016-04-07

    The transplantation of blood tissues from bone marrow into a lethally irradiated animal is an experimental procedure that is used to study how the blood system is reconstituted by haematopoietic stem cells (HSC). In a competitive repopulation experiment, a lethally irradiated mouse was transplanted with a single HSC as a test cell together with a number of bone marrow cells as competitor cells, and the fraction of the test cell progeny (percentage of chimerism) was traced over time. In this paper, we studied the stem cell kinetics in this experimental procedure. The balance between symmetric self-renewal and differentiation divisions in HSC determined the number of cells which HSC produce and the length of time for which HSC live after transplantation. The percentage of chimerism depended on the type of test cell (long-, intermediate-, or short-term HSC), as well as the type and number of HSC included in competitor cells. We next examined two alternative HSC differentiation models, one-step and multi-step differentiation models. Although these models differed in blood cell production, the percentage of chimerism appeared very similar. We also estimated the numbers of different types of HSC in competitor cells. Based on these results, we concluded that the experimental results inevitably include stochasticity with regard to the number and the type of HSC in competitor cells, and that, in order to detect different types of HSC, an appropriate number of competitor cells needs to be used in transplantation experiments. Copyright © 2016. Published by Elsevier Ltd.

  14. Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system.

    Directory of Open Access Journals (Sweden)

    Yuen Fei Wong

    2011-02-01

    Full Text Available Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle's loop and distal tubules.Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the

  15. 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.

  16. Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells

    NARCIS (Netherlands)

    van den Berg, T. K.; Hasbold, J.; Renardel de Lavalette, C.; Döpp, E. A.; Dijkstra, C. D.; Klaus, G. G.

    1996-01-01

    In this study we describe the tissue distribution of mouse CD40 using two monoclonal antibodies (mAb) against different epitopes of the molecule. In lymphoid tissues CD40 was expressed by B lymphocytes. Most B cells in typical B-cell compartments were CD40-positive, including germinal centre B

  17. A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

    Directory of Open Access Journals (Sweden)

    Reza Ebrahimzadeh-Vesal

    2014-08-01

    Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

  18. Nanopatterned muscle cell patches for enhanced myogenesis and dystrophin expression in a mouse model of muscular dystrophy.

    Science.gov (United States)

    Yang, Hee Seok; Ieronimakis, Nicholas; Tsui, Jonathan H; Kim, Hong Nam; Suh, Kahp-Yang; Reyes, Morayma; Kim, Deok-Ho

    2014-02-01

    Skeletal muscle is a highly organized tissue in which the extracellular matrix (ECM) is composed of highly-aligned cables of collagen with nanoscale feature sizes, and provides structural and functional support to muscle fibers. As such, the transplantation of disorganized tissues or the direct injection of cells into muscles for regenerative therapy often results in suboptimal functional improvement due to a failure to integrate with native tissue properly. Here, we present a simple method in which biodegradable, biomimetic substrates with precisely controlled nanotopography were fabricated using solvent-assisted capillary force lithography (CFL) and were able to induce the proper development and differentiation of primary mononucleated cells to form mature muscle patches. Cells cultured on these nanopatterned substrates were highly-aligned and elongated, and formed more mature myotubes as evidenced by up-regulated expression of the myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). When transplanted into mdx mice models for Duchenne muscular dystrophy (DMD), the proposed muscle patches led to the formation of a significantly greater number of dystrophin-positive muscle fibers, indicating that dystrophin replacement and myogenesis is achievable in vivo with this approach. These results demonstrate the feasibility of utilizing biomimetic substrates not only as platforms for studying the influences of the ECM on skeletal muscle function and maturation, but also to create transplantable muscle cell patches for the treatment of chronic and acute muscle diseases or injuries. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruoxing [Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive 5018, Hattiesburg, MS 39406 (United States); Guo, Yan-Lin, E-mail: yanlin.guo@usm.edu [Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive 5018, Hattiesburg, MS 39406 (United States)

    2012-10-01

    Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. -- Highlights: Black-Right-Pointing-Pointer Inhibition of Cdks slows down mESCs proliferation. Black-Right-Pointing-Pointer mESCs display remarkable recovery capacity from short-term cell cycle interruption. Black-Right-Pointing-Pointer Short-term cell cycle interruption does not compromise mESC self-renewal. Black

  20. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Wang, Ruoxing; Guo, Yan-Lin

    2012-01-01

    Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. -- Highlights: ► Inhibition of Cdks slows down mESCs proliferation. ► mESCs display remarkable recovery capacity from short-term cell cycle interruption. ► Short-term cell cycle interruption does not compromise mESC self-renewal. ► Oct4 and Nanog are up-regulated via de novo synthesis by cell cycle interruption.

  1. Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions during Mouse Early Gastrulation

    Directory of Open Access Journals (Sweden)

    Hisham Mohammed

    2017-08-01

    Full Text Available The mouse inner cell mass (ICM segregates into the epiblast and primitive endoderm (PrE lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation. To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing (seq of conceptuses from E3.5 to E6.5. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6.5, the transition from epiblast to primitive streak is linked with decreased expression of polycomb subunits, suggesting a key regulatory role. Notably, our analyses suggest elevated transcriptional noise at E3.5 and within the non-committed epiblast at E6.5, coinciding with exit from pluripotency. By contrast, E6.5 primitive streak cells became highly synchronized and exhibit a shortened G1 cell-cycle phase, consistent with accelerated proliferation. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions.

  2. Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

    Science.gov (United States)

    Ströh, Sebastian; Puller, Christian; Swirski, Sebastian; Hölzel, Maj-Britt; van der Linde, Lea I S; Segelken, Jasmin; Schultz, Konrad; Block, Christoph; Monyer, Hannah; Willecke, Klaus; Weiler, Reto; Greschner, Martin; Janssen-Bienhold, Ulrike; Dedek, Karin

    2018-02-21

    In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light

  3. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    Energy Technology Data Exchange (ETDEWEB)

    Huimin, Zhou [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Li, Jia [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Shujing, Wang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Hongmei, Wang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Department of Medical Microbiology and Parasitology, School of Medicine, Liaodong College, Dandong 118000 (China); Haiying, Chu [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Yichuan, Hu [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Jun, Cao [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Jianing, Zhang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China)

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  4. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    International Nuclear Information System (INIS)

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing

    2006-01-01

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression

  5. Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

    Science.gov (United States)

    Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

    2014-09-01

    Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

  6. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

    Science.gov (United States)

    Schatten, Heide; Schatten, Gerald; Balczon, Ron; Simerly, Calvin; Mazia, Daniel

    1986-01-01

    The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

  7. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, ... from embryonic stem (ES) cell or induced pluripotent.

  8. Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

    Science.gov (United States)

    Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

    2015-12-01

    TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

  9. Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

    OpenAIRE

    Van Schravendijk, C F; Hooghe-Peters, E L; De Meyts, P; Pipeleers, D G

    1984-01-01

    The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insu...

  10. Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes

    DEFF Research Database (Denmark)

    Owens, T; Kaplan, J G

    1980-01-01

    Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation...... cells. Attempts to demonstrate a diffusible factor in the supernatants of stimulated T cells were unsuccessful. The measured interaction is sufficient to explain our previous paradoxical findings that enrichment of T cells as measured by membrane markers did not cause a corresponding enrichment...

  11. Molecular mechanisms involved in casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Lee, E.Y.H.P.; Lee, W.H.; Parry, G.; Bissell, M.J.

    1985-01-01

    Mouse mammary epithelial cells (MMEC) secrete a group of milk-specific proteins including various caseins and whey proteins. Dissociated mammary epithelial cells maintain expression of most of their differentiated functions only if cells are plated on a suitable substratum. Casein production and section, cell morphology, and production of α-lactalbumin have been used as markers to assess the degree of differentiation of mammary cells in culture. The general consensus is that cells express their differentiated properties at high levels and for longer periods of time on such substrata. In this paper, the authors demonstrate that modulation of the expression of caseins by floating collagen gels is manifested at several regulatory points

  12. Immunologic glycosphingolipidomics and NKT cell development in mouse thymus

    DEFF Research Database (Denmark)

    Li, Yunsen; Thapa, Prakash; Hawke, David

    2009-01-01

    Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting...

  13. Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.

    Science.gov (United States)

    Kawamoto, Kohei; Izumikawa, Masahiko; Beyer, Lisa A; Atkin, Graham M; Raphael, Yehoash

    2009-01-01

    Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be

  14. Anti-cancer potential of MAPK pathway inhibition in paragangliomas-effect of different statins on mouse pheochromocytoma cells

    NARCIS (Netherlands)

    Fliedner, S.M.; Engel, T.G.P.; Lendvai, N.K.; Shankavaram, U.; Nolting, S.; Wesley, R.; Elkahloun, A.G.; Ungefroren, H.; Oldoerp, A.; Lampert, G.; Lehnert, H.; Timmers, H.J.; Pacak, K.

    2014-01-01

    To date, malignant pheochromocytomas and paragangliomas (PHEOs/PGLs) cannot be effectively cured and thus novel treatment strategies are urgently needed. Lovastatin has been shown to effectively induce apoptosis in mouse PHEO cells (MPC) and the more aggressive mouse tumor tissue-derived cells

  15. Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells

    International Nuclear Information System (INIS)

    Wang Enxiu; Obeng-Adjei, Nyamekye; Ying Qihua; Meertens, Laurent; Dragic, Tanya; Davey, Robert A.; Ross, Susan R.

    2008-01-01

    Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment

  16. Inference of Transcriptional Network for Pluripotency in Mouse Embryonic Stem Cells

    International Nuclear Information System (INIS)

    Aburatani, S

    2015-01-01

    In embryonic stem cells, various transcription factors (TFs) maintain pluripotency. To gain insights into the regulatory system controlling pluripotency, I inferred the regulatory relationships between the TFs expressed in ES cells. In this study, I applied a method based on structural equation modeling (SEM), combined with factor analysis, to 649 expression profiles of 19 TF genes measured in mouse Embryonic Stem Cells (ESCs). The factor analysis identified 19 TF genes that were regulated by several unmeasured factors. Since the known cell reprogramming TF genes (Pou5f1, Sox2 and Nanog) are regulated by different factors, each estimated factor is considered to be an input for signal transduction to control pluripotency in mouse ESCs. In the inferred network model, TF proteins were also arranged as unmeasured factors that control other TFs. The interpretation of the inferred network model revealed the regulatory mechanism for controlling pluripotency in ES cells

  17. Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

    Science.gov (United States)

    Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

    2014-05-01

    We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

    Science.gov (United States)

    Clark, M A; Jepson, M A; Simmons, N L; Hirst, B H

    1995-12-01

    The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

  19. β-catenin functions pleiotropically in differentiation and tumorigenesis in mouse embryo-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Noriko Okumura

    Full Text Available The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-cat(Δ/Δ mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs. However, teratomas formed from embryo-derived β-cat(Δ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

  20. Intraocular Injection of ES Cell-Derived Neural Progenitors Improve Visual Function in Retinal Ganglion Cell-Depleted Mouse Models

    Directory of Open Access Journals (Sweden)

    Mundackal S. Divya

    2017-09-01

    Full Text Available Retinal ganglion cell (RGC transplantation is a promising strategy to restore visual function resulting from irreversible RGC degeneration occurring in glaucoma or inherited optic neuropathies. We previously demonstrated FGF2 induced differentiation of mouse embryonic stem cells (ESC to RGC lineage, capable of retinal ganglion cell layer (GCL integration upon transplantation. Here, we evaluated possible improvement of visual function by transplantation of ES cell derived neural progenitors in RGC depleted glaucoma mice models. ESC derived neural progenitors (ES-NP were transplanted into N-Methyl-D-Aspartate (NMDA injected, RGC-ablated mouse models and a pre-clinical glaucoma mouse model (DBA/2J having sustained higher intra ocular pressure (IOP. Visual acuity and functional integration was evaluated by behavioral experiments and immunohistochemistry, respectively. GFP-expressing ES-NPs transplanted in NMDA-injected RGC-depleted mice differentiated into RGC lineage and possibly integrating into GCL. An improvement in visual acuity was observed after 2 months of transplantation, when compared to the pre-transplantation values. Expression of c-Fos in the transplanted cells, upon light induction, further suggests functional integration into the host retinal circuitry. However, the transplanted cells did not send axonal projections into optic nerve. Transplantation experiments in DBA/2J mouse showed no significant improvement in visual functions, possibly due to both host and transplanted retinal cell death which could be due to an inherent high IOP. We showed that, ES NPs transplanted into the retina of RGC-ablated mouse models could survive, differentiate to RGC lineage, and possibly integrate into GCL to improve visual function. However, for the survival of transplanted cells in glaucoma, strategies to control the IOP are warranted.

  1. Oxidized CaMKII (Ca2+/Calmodulin-Dependent Protein Kinase II) Is Essential for Ventricular Arrhythmia in a Mouse Model of Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Wang, Qiongling; Quick, Ann P; Cao, Shuyi; Reynolds, Julia; Chiang, David Y; Beavers, David; Li, Na; Wang, Guoliang; Rodney, George G; Anderson, Mark E; Wehrens, Xander H T

    2018-04-01

    Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca 2+ ) homeostasis and elevated reactive oxygen species. CaMKII (Ca 2+ /calmodulin-dependent protein kinase II) is vital for normal Ca 2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca 2+ homeostasis and arrhythmias in cardiomyocytes. Reactive oxygen species induce CaMKII to become autonomously active. We hypothesized that genetic inhibition of CaMKII oxidation (ox-CaMKII) in a mouse model of Duchenne muscular dystrophy can alleviate abnormal Ca 2+ homeostasis, thus, preventing ventricular arrhythmia. The objective of this study was to test if selective loss of ox-CaMKII affects ventricular arrhythmias in the mdx mouse model of Duchenne muscular dystrophy. 5-(6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate staining revealed increased reactive oxygen species production in ventricular myocytes isolated from mdx mice, which coincides with elevated ventricular ox-CaMKII demonstrated by Western blotting. Genetic inhibition of ox-CaMKII by knockin replacement of the regulatory domain methionines with valines (MM-VV [CaMKII M281/282V]) prevented ventricular tachycardia in mdx mice. Confocal calcium imaging of ventricular myocytes isolated from mdx :MM-VV mice revealed normalization of intracellular Ca 2+ release events compared with cardiomyocytes from mdx mice. Abnormal action potentials assessed by optical mapping in mdx mice were also alleviated by genetic inhibition of ox-CaMKII. Knockout of the NADPH oxidase regulatory subunit p47 phox normalized elevated ox-CaMKII, repaired intracellular Ca 2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice. Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca 2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy. © 2018 American Heart Association, Inc.

  2. In vivo transplantation of enteric neural crest cells into mouse gut; Engraftment, functional integration and long-term safety

    NARCIS (Netherlands)

    J.E. Cooper (Julie E.); C. Mccann; D. Natarajan (Dipa); S. Choudhury; W. Boesmans (Werend); J.-M. Delalande (Jean-Marie); P.V. Berghe (Pieter Vanden); A.J. Burns (Alan); N. Thapar (Nikhil)

    2016-01-01

    textabstractObjectives: Enteric neuropathies are severe gastrointestinal disorders with unsatisfactory outcomes. We aimed to investigate the potential of enteric neural stem cell therapy approaches for such disorders by transplanting mouse enteric neural crest cells (ENCCs) into ganglionic and

  3. Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

    International Nuclear Information System (INIS)

    Androlewicz, M.J.; Straus, D.S.; Brandenburg, D.F.

    1989-01-01

    Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125 I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degree C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation

  4. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Marica Grskovic

    2007-08-01

    Full Text Available Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.

  5. Toxicity of silver nanoparticles in mouse embryonic stem cells and chemical based reprogramming of somatic cells to sphere cells

    Science.gov (United States)

    Rajanahalli Krishnamurthy, Pavan

    Abstract 1: Silver nanoparticles (Ag Np's) have an interesting surface chemistry and unique plasmonic properties. They are used in a wide variety of applications ranging from consumer products like socks, medical dressing, computer chips and it is also shown to have antimicrobial, anti bacterial activity and wound healing. Ag Np toxicity studies have been limited to date which needs to be critically addressed due to its wide applications. Mouse embryonic stem (MES) cells represent a unique cell population with the ability to undergo both self renewal and differentiation. They exhibit very stringent and tightly regulated mechanisms to circumvent DNA damage and stress response. We used 10 nm coated (polysaccharide) and uncoated Ag Np's to test its toxic effects on MES cells. MES cells and embryoid bodies (EB's) were treated with two concentrations of Ag Np's: 5 microg/ml and 50 ug/ml and exposed for 24, 48 and 72 hours. Increased cell death, ROS production and loss of mitochondrial membrane potential and alkaline phosphatase (AP) occur in a time and a concentration dependant manner. Due to increased cell death, there is a progressive increase in Annexin V (apoptosis) and Propidium Iodide (PI) staining (necrosis). Oct4 and Nanog undergo ubiquitination and dephosphorylation post-translational modifications in MES cells thereby altering gene expression of pluripotency factors and differentiation of EB's into all the three embryonic germ layers with specific growth factors were also inhibited after Ag Np exposure. Flow cytometry analysis revealed Ag Np's treated cells had altered cell cycle phases correlating with altered self renewal capacity. Our results suggest that Ag Np's effect MES cell self renewal, pluripotency and differentiation and serves as a perfect model system for studying toxicity induced by engineered Ag Np's. Abstract 2: The reprogramming of fibroblasts to pluripotent stem cells and the direct conversion of fibroblasts to functional neurons has been

  6. Characterization of calcium responses and electrical activity in differentiating mouse neural progenitor cells in vitro

    NARCIS (Netherlands)

    de Groot, Martje W G D M; Dingemans, Milou M L; Rus, Katinka H; de Groot, Aart; Westerink, Remco H S

    In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs)

  7. Redox Disrupting Potential of ToxCast™Chemicals Ranked by Activity in Mouse Embryonic Stem Cells

    Science.gov (United States)

    Little is known regarding the adverse outcome pathways responsible for developmental toxicity following exposure to chemicals. An evaluation of Toxoast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay revealed a redox sensitive pathway that correlated with...

  8. REDOX DISRUPTING POTENTIAL OF TOXCAST CHEMICALS RANKED BY ACTIVITY IN MOUSE EMBRYONIC STEM CELLS

    Science.gov (United States)

    To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...

  9. Quantitative transcriptional profiling of ATDC5 mouse progenitor cells during chondrogenesis

    DEFF Research Database (Denmark)

    Chen, Li; Fink, Trine; Zhang, Xiao-Yan

    2005-01-01

    During the differentiation of a mouse chondroprogenitor cell line, ATDC5, an analysis of the transcription cartilage-related genes was carried out using real-time RT-PCR in a semiquantitative fashion. A total number of 104 genes both previously linked to chondrogenesis and hitherto not associated...

  10. Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

    NARCIS (Netherlands)

    Colaianna, M.; Ilmjärv, S.; Peterson, H.; Ilse Kern, I.; Julien, S.; Baquié, M.; allocca, G.; Bosgra, S.; Sachinidis, A.; Hengstler, J.G.; Leist, M.; Krause, K.H.

    2017-01-01

    Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell

  11. Differential effects of fractionated X irradiation on mouse spermatogonial stem cells

    NARCIS (Netherlands)

    van der Meer, Y.; Huiskamp, R.; Davids, J. A.; de rooij, D. G.

    1993-01-01

    The response of spermatogonial stem cells to fractionated X irradiation was studied in the various stages of the spermatogenic cycle of the CBA mouse. Fractionated doses of 2 + 2, 1 + 3, and 3 + 1 Gy with a 24-h interval between the doses were compared with a single dose of 4 Gy. The numbers of

  12. Mast cells trigger epithelial barrier dysfunction, bacterial translocation and postoperative ileus in a mouse model

    NARCIS (Netherlands)

    Snoek, S. A.; Dhawan, S.; van Bree, S. H.; Cailotto, C.; van Diest, S. A.; Duarte, J. M.; Stanisor, O. I.; Hilbers, F. W.; Nijhuis, L.; Koeman, A.; van den Wijngaard, R. M.; Zuurbier, C. J.; Boeckxstaens, G. E.; de Jonge, W. J.

    2012-01-01

    Background Abdominal surgery involving bowel manipulation commonly results in inflammation of the bowel wall, which leads to impaired intestinal motility and postoperative ileus (POI). Mast cells have shown to play a key role in the pathogenesis of POI in mouse models and human studies. We studied

  13. Basement membrane components secreted by mouse yolk sac carcinoma cell lines

    DEFF Research Database (Denmark)

    Damjanov, A; Wewer, U M; Tuma, B

    1990-01-01

    Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac...

  14. Nuclear hormone receptor expression in mouse kidney and renal cell lines.

    Directory of Open Access Journals (Sweden)

    Daisuke Ogawa

    Full Text Available Nuclear hormone receptors (NHRs are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN, the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m, and cell lines of mesangial (MES13, podocyte (MPC, proximal tubular epithelial (mProx24 and collecting duct (mIMCD3 origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77, nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN.

  15. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    Science.gov (United States)

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Coppé

    2010-02-01

    Full Text Available Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP, which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that "senescent" mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen, do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3% oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-alpha. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

  17. Mouse neuroblastoma cell based model and the effect of epileptic events on calcium oscillations and neural spikes

    Science.gov (United States)

    Kim, Suhwan; Baek, Juyeong; Jung, Unsang; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-05-01

    Recently, Mouse neuroblastoma cells are considered as an attractive model for the study of human neurological and prion diseases, and intensively used as a model system in different areas. Among those areas, differentiation of neuro2a (N2A) cells, receptor mediated ion current, and glutamate induced physiological response are actively investigated. The reason for the interest to mouse neuroblastoma N2A cells is that they have a fast growing rate than other cells in neural origin with a few another advantages. This study evaluated the calcium oscillations and neural spikes recording of mouse neuroblastoma N2A cells in an epileptic condition. Based on our observation of neural spikes in mouse N2A cell with our proposed imaging modality, we report that mouse neuroblastoma N2A cells can be an important model related to epileptic activity studies. It is concluded that the mouse neuroblastoma N2A cells produce the epileptic spikes in vitro in the same way as produced by the neurons or the astrocytes. This evidence advocates the increased and strong level of neurotransmitters release by enhancement in free calcium using the 4-aminopyridine which causes the mouse neuroblastoma N2A cells to produce the epileptic spikes and calcium oscillation.

  18. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells

    DEFF Research Database (Denmark)

    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe

    2009-01-01

    We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell......-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However......, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell...

  19. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  20. Patch clamp study of mouse glomus cells using a whole carotid body.

    Science.gov (United States)

    Yamaguchi, Shigeki; Lande, Boris; Kitajima, Toshimitsu; Hori, Yuichi; Shirahata, Machiko

    2004-03-04

    Some electrophysiological characteristics of mouse glomus cells (DBA/2J strain) were investigated using an undissociated carotid body. The carotid body with major carotid arteries was placed in a recording chamber, and glomus cells were visualized with a water immersion lens combined with an infrared differential interference video camera. Patch clamp experiments revealed that voltage-gated outward current, but not inward current, was easily observed in glomus cells. Pharmacological experiments and the kinetics of the current suggest that outward current is via delayed rectifier, A type, and large conductance calcium-activated K channels. Furthermore, K current was reversibly attenuated by mild hypoxia. The results suggest electrophysiological similarities of glomus cells among the cat, the rat, and the DBA/2J mouse. The method appears useful for physiological experiments.

  1. Ras Signaling Regulates Stem Cells and Amelogenesis in the Mouse Incisor.

    Science.gov (United States)

    Zheng, X; Goodwin, A F; Tian, H; Jheon, A H; Klein, O D

    2017-11-01

    The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

  2. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    International Nuclear Information System (INIS)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

    2007-01-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

  3. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    International Nuclear Information System (INIS)

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  4. Micronuclei induced by municipal landfill leachate in mouse bone marrow cells in vivo

    International Nuclear Information System (INIS)

    Li Guangke; Sang Nan; Zhao Youcai

    2004-01-01

    The induction of micronuclei (MN) in polychromatic erythrocytes (PCE) of mouse bone marrow by municipal landfill leachate was studied in vivo. Results showed that mouse exposure via drinking water containing various concentrations of leachate caused a significant increase of MN frequencies in a concentration (Chemical oxygen demand measured with potassium dichromate oxidation, COD Cr )-dependent manner. MN induction in female and male mice was different at higher concentrations. This implies that leachate is a genotoxic agent in mammalian cells and that exposure to leachate in an aquatic environment may pose a potential genotoxic risk to human beings

  5. Lipid profiling of in vitro cell models of adipogenic differentiation: relationships with mouse adipose tissues

    OpenAIRE

    Liaw, Lucy; Prudovsky, Igor; Koza, Robert A.; Anunciado-Koza, Rea V.; Siviski, Matthew E.; Lindner, Volkhard; Friesel, Robert E.; Rosen, Clifford J.; Baker, Paul R.S.; Simons, Brigitte; Vary, Calvin P.H.

    2016-01-01

    Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MSALL. Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-...

  6. The phenotype of FancB-mutant mouse embryonic stem cells

    OpenAIRE

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu, Lingchuan; Hasty, Paul

    2011-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslink...

  7. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

  8. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  9. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    International Nuclear Information System (INIS)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-01-01

    Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

  10. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan); Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  11. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    International Nuclear Information System (INIS)

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2012-01-01

    Highlights: ► Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. ► Angiotensin II may enhance the DNA synthesis via induction of superoxide. ► Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. ► Angiotensin II enhanced differentiation into mesodermal progenitor cells. ► Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT 1 R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation

  12. Generation of stomach tissue from mouse embryonic stem cells.

    Science.gov (United States)

    Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

    2015-08-01

    Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

  13. Isolation and characterization of mouse innate lymphoid cells.

    Science.gov (United States)

    Halim, Timotheus Y F; Takei, Fumio

    2014-08-01

    Innate lymphoid cells (ILCs) are rare populations of cytokine-producing lymphocytes and are divided into three groups, namely ILC1, ILC2, and ILC3, based on the cytokines that they produce. They comprise less than 1% of lymphocytes in mucosal tissues and express no unique cell surface markers. Therefore, they can only be identified by combinations of multiple cell surface markers and further characterized by cytokine production in vitro. Thus, multicolor flow cytometry is the only reliable method to purify and characterize ILCs. Here we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILC2 and ILC3. Copyright © 2014 John Wiley & Sons, Inc.

  14. A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

    International Nuclear Information System (INIS)

    Shikanai, Mima; Asahina, Kinji; Iseki, Sachiko; Teramoto, Kenichi; Nishida, Tomohiro; Shimizu-Saito, Keiko; Ota, Masato; Eto, Kazuhiro; Teraoka, Hirobumi

    2009-01-01

    Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

  15. Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow

    International Nuclear Information System (INIS)

    Park, Y.-H.; Osmond, D.G.

    1989-01-01

    To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, (iii) μ heavy chains in the cytoplasm (cμ) and at the cell surface (sμ). Three populations of μ - cells (TdT + 14.8 - ; TdT + 14.8 + ; TdT - 14.8 + ) have been proposed to be early B-cell precursors which would give rise to cμ + sμ - pre-B cells and to sμ + B lymphocytes. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT + 14.8 - , TdT + 14.8 + and TdT - 14.8 + cells form three successive stages in B-cell differentiation before the expression of μ chains, presumptively including the stage of μ chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow. (author)

  16. Dose-related estrogen effects on gene expression in fetal mouse prostate mesenchymal cells.

    Directory of Open Access Journals (Sweden)

    Julia A Taylor

    Full Text Available Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2 in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM were enriched in the glycolytic pathway. At the highest dose (100 nM, E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high.

  17. Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

    DEFF Research Database (Denmark)

    Schneider, H R; Reichert, G H; Issinger, O G

    1986-01-01

    Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation. Together with the CKII activity...... mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme...

  18. Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

    International Nuclear Information System (INIS)

    Yamamoto, Ryohei; Ohshiro, Yukari; Shimotani, Tatsuhiko; Yamamoto, Mizuki; Matsuyama, Satoshi; Ide, Hiroshi; Kubo, Kihei

    2014-01-01

    Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H 2 O 2 ) sensitivity. Hypersensitivity to H 2 O 2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1

  19. Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea.

    Science.gov (United States)

    Zhang, Zhi-Jian; Guan, Hong-Xia; Yang, Kun; Xiao, Bo-Kui; Liao, Hua; Jiang, Yang; Zhou, Tao; Hua, Qing-Quan

    2017-10-01

    This study aimed in fully investigating the toxicities of ouabain to mouse cochlea and the related cellular environment, and providing an optimal animal model system for cell transplantation in the treatment of auditory neuropathy (AN) and sensorineural hearing loss (SNHL). Different dosages of ouabain were applied to mouse round window. The auditory brainstem responses and distortion product otoacoustic emissions were used to evaluate the cochlear function. The immunohistochemical staining and cochlea surface preparation were performed to detect the spiral ganglion neurons (SGNs), Schwann cells and hair cells. Ouabain at the dosages of 0.5 mM, 1 mM and 3 mM selectively and permanently destroyed SGNs and their functions, while leaving the hair cells relatively intact. Ouabain at 3 mM resulted in the most severe SGNs loss and induced significant loss of Schwann cells started as early as 7 days and with further damages at 14 and 30 days after ouabain exposure. The application of ouabain to mouse round window induces damages of SGNs and Schwann cells in a dose- and time-dependent manner, this study established a reliable and accurate animal model system of AN and SNHL.

  20. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

    Science.gov (United States)

    van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

    2014-11-01

    Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'. © 2014. Published by The Company of Biologists Ltd.

  1. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    International Nuclear Information System (INIS)

    Dou, Q.P.; Chen, K.Y.

    1987-01-01

    An 18,000-dalton protein can be metabolically labeled by [ 3 H]putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0

  2. Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

    Science.gov (United States)

    Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

    2008-03-01

    Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

  3. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform

    Directory of Open Access Journals (Sweden)

    Kenji Kozuka

    2017-12-01

    Full Text Available Summary: We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound. : Siegel and colleagues describe their development of a human and mouse intestinal epithelial cell monolayer platform that maintains the cellular, molecular, and functional characteristics of tissue for each intestinal segment. They demonstrate the platform's application to drug discovery by screening a library of over 2,000 compounds to identify an inhibitor of potassium ion transport in the mouse distal colon. Keywords: intestinal epithelium, organoids, monolayer, colon, small intestine, phenotype screening assays, enteroid, colonoid

  4. Immunocompromised and immunocompetent mouse models for head and neck squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Lei ZG

    2016-01-01

    Full Text Available Zhen-ge Lei,1,* Xiao-hua Ren,2,* Sha-sha Wang,3 Xin-hua Liang,3,4 Ya-ling Tang3,5 1Department of Oral and Maxillofacial Surgery, Stomatological Hospital Affiliated to Nanchang University, Nanchang, Jiangxi, 2Department of Stomatology, Sichuan Medical Science Academy and Sichuan Provincial People’s Hospital, 3State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, 4Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, 5Department of Oral Pathology, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, People’s Republic of China *These authors contributed equally to this work Abstract: Mouse models can closely mimic human oral squamous epithelial carcinogenesis, greatly expand the in vivo research possibilities, and play a critical role in the development of diagnosis, monitoring, and treatment of head and neck squamous cell carcinoma. With the development of the recent research on the contribution of immunity/inflammation to cancer initiation and progression, mouse models have been divided into two categories, namely, immunocompromised and immunocompetent mouse models. And thus, this paper will review these two kinds of models applied in head and neck squamous cell carcinoma to provide a platform to understand the complicated histological, molecular, and genetic changes of oral squamous epithelial tumorigenesis. Keywords: head and neck squamous cell carcinoma, HNSCC, mouse models, immunocompromised models, immunocompetent models, transgenic models

  5. Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hideki Uosaki

    Full Text Available RATIONALE: Pluripotent stem cell-derived cardiac progenitor cells (CPCs have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations. OBJECTIVE: Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells. METHOD AND RESULT: To test the hypothesis, we cocultured mouse embryonic stem (ES cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1(+ PDGFRa(+ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5(+ and Isl1(+ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR(+ PDGFRa(+ CPCs from human ES cells. CONCLUSIONS: Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.

  6. Mouse Incisor Stem Cell Niche and Myb Transcription Factors

    Czech Academy of Sciences Publication Activity Database

    Švandová, Eva; Veselá, Barbora; Šmarda, J.; Hampl, A.; Radlanski, R.J.; Matalová, Eva

    2015-01-01

    Roč. 44, č. 5 (2015), s. 338-344 ISSN 0340-2096 R&D Projects: GA ČR GAP304/11/1418; GA ČR GCP302/12/J059 Institutional support: RVO:67985904 Keywords : c-Myb * stem cell niches Subject RIV: EA - Cell Biology Impact factor: 0.615, year: 2015

  7. Expression of tyrosine kinase gene in mouse thymic stromal cells

    NARCIS (Netherlands)

    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  8. Dynamic changes in mouse hematopoietic stem cell numbers during aging

    NARCIS (Netherlands)

    de Haan, G; Van Zant, G

    1999-01-01

    To address the fundamental question of whether or not stem cell populations age, we performed quantitative measurements of the cycling status and frequency of hematopoietic stem cells in long-lived C57BL/6 (B6) and short-lived DBA/2 (DBA) mice at different developmental and aging stages. The

  9. Neovascular niche for human myeloma cells in immunodeficient mouse bone.

    Directory of Open Access Journals (Sweden)

    Hirono Iriuchishima

    Full Text Available The interaction with bone marrow (BM plays a crucial role in pathophysiological features of multiple myeloma (MM, including cell proliferation, chemoresistance, and bone lesion progression. To characterize the MM-BM interactions, we utilized an in vivo experimental model for human MM in which a GFP-expressing human MM cell line is transplanted into NOG mice (the NOG-hMM model. Transplanted MM cells preferentially engrafted at the metaphyseal region of the BM endosteum and formed a complex with osteoblasts and osteoclasts. A subpopulation of MM cells expressed VE-cadherin after transplantation and formed endothelial-like structures in the BM. CD138(+ myeloma cells in the BM were reduced by p53-dependent apoptosis following administration of the nitrogen mustard derivative bendamustine to mice in the NOG-hMM model. Bendamustine maintained the osteoblast lining on the bone surface and protected extracellular matrix structures. Furthermore, bendamustine suppressed the growth of osteoclasts and mesenchymal cells in the NOG-hMM model. Since VE-cadherin(+ MM cells were chemoresistant, hypoxic, and HIF-2α-positive compared to the VE-cadherin(- population, VE-cadherin induction might depend on the oxygenation status. The NOG-hMM model described here is a useful system to analyze the dynamics of MM pathophysiology, interactions of MM cells with other cellular compartments, and the utility of novel anti-MM therapies.

  10. Estrogen action in the mouse uterus: differential nuclear localization of estradiol in uterine cell types

    International Nuclear Information System (INIS)

    Korach, K.S.; Lamb, J.C.

    1981-01-01

    Autoradiographic studies of labeled steroid uptake in mouse uterine tissue indicated that labeled estradiol was predominantly sequestered in the nuclei of stromal and glandular epithelial cells at 1 h. Luminal epithelial cells did not show appreciable nuclear accumulation of labeled estradiol until 7-8 h after hormone injection. Studies using non-target tissues and unlabeled steroids indicated that the nuclear uptake events were tissue and estrogen steroid specific. The temporal pattern of steroid hormone uptake in the uterus would suggest that an initial interaction in stromal and glandular epithelial cells may be required prior to nuclear stimulation in the luminal epithelial target cell

  11. Data on the potential impact of food supplements on the growth of mouse embryonic stem cells.

    Science.gov (United States)

    Correia, Marcelo; Sousa, Maria I; Rodrigues, Ana S; Perestrelo, Tânia; Pereira, Sandro L; Ribeiro, Marcelo F; Ramalho-Santos, João

    2016-06-01

    The use of new compounds as dietary supplements is increasing, but little is known in terms of possible consequences of their use. Pluripotent stem cells are a promising research tool for citotoxicological research for evaluation of proliferation, cell death, pluripotency and differentiation. Using the mouse embryonic stem cell (mESC) model, we present data on three different compounds that have been proposed as new potential supplements for co-adjuvant disease treatments: kaempferol, berberine and Tauroursodeoxycholic acid (TUDCA). Cell number and viability were monitored following treatment with increased concentrations of each drug in pluripotent culture conditions.

  12. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus

    Directory of Open Access Journals (Sweden)

    Farrell Regina M

    2004-09-01

    Full Text Available Abstract Background Human infections with Sin Nombre virus (SNV and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS, a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC from deer mouse bone marrow using commercially-available house mouse (Mus musculus granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

  13. Poly (ADP-ribose) metabolism in alkylated mouse L5178Y cells

    International Nuclear Information System (INIS)

    Boyle, J.M.

    1985-01-01

    Poly ADP-ribosylation of two mouse lymphoma cell lines, L5178Y (LS) and the radiation and alkylating agent resistant derivative AII, was investigated by uptake of [ 3 H]NAD by permeabilised cells into acid-precipitable material that was sensitive to phosphodiesterase but insensitive to DNase and RNase. Basal activities in both lymphoma lines were 3-4-fold greater than in mouse L1210 leukaemia cells. However, total endogenous poly (ADP-R) polymerase activity in both L5178Y cell lines, stimulated by a large excess of DNase in the presence of Triton X-100, was less than half the activity in L1210 cells. Doses of N-methyl-N-nitrosourea (MNU) that produced 20-50% survival of colony-forming units increased poly (ADP-R) in both lymphoma lines by only 25% compared with 377% in L1210 cells when synthesis was measured immediately after a 30-min exposure of MNU. Concentrations of 3-aminobenzamide (3AB) above 2.5 mM inhibited colony-forming ability of lymphoma cells and equally inhibited uptake of [ 14 C]formate into protein, RNA and DNA indicating that 3AB behaves as a general metabolic poison. Non-toxic concentrations of 3AB potentiated cell killing by MNU to a similar degree in both lymphoma cell lines. In conclusion, the authors have found little evidence to support the hypothesis that the differential sensitivity of LS and AII is related to poly ADP-ribosylation. Compared with other mouse cells, L5178Y cells appear deficient in poly (ADP-R) polymerase and poly (ADP-R) glycohydrolase activities

  14. In Vitro Chondrogenesis Transformation Study of Mouse Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2012-01-01

    Full Text Available A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.. Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P<0.05. Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P<0.05 in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

  15. Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18

    Directory of Open Access Journals (Sweden)

    Biliang Hu

    2017-09-01

    Full Text Available The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors.

  16. Mouse DRG Cell Line with Properties of Nociceptors.

    Science.gov (United States)

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

  17. Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

    International Nuclear Information System (INIS)

    Spangrude, G.J.; Scollay, R.

    1990-01-01

    To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes

  18. Uma análise comparativa de funções MDX nos servidores Analysis Services e Mondrian

    OpenAIRE

    ALBUQUERQUE, Erivam Anselmo de

    2013-01-01

    A MultiDimensional eXpression (MDX) é uma linguagem de consulta para processamento analítico de dados ou On-line Analytical Processing (OLAP). Apesar de esta linguagem ser usada pela maioria dos servidores OLAP, esta não é um padrão de direito. Portanto, tem-se pouca (ou nenhuma) garantia de que as funções MDX usadas por um servidor OLAP também possam ser usadas em outros servidores. Neste contexto, de forma a comparar as funções MDX de um servidor OLAP de código aberto e outro de código fech...

  19. Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice.

    Science.gov (United States)

    Marques, Maria Julia; Taniguti, Ana Paula Tiemi; Minatel, Elaine; Neto, Humberto Santo

    2007-02-01

    Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1-month-old) and adult (6-month-old) mdx mice was injected with 60 microl lidocaine hydrochloride to induce muscle degeneration-regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4-Di-2-ASP and rhodamine-alpha-bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated-regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated-regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve-dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution.

  20. The chondrogenic response to exercise in the proximal femur of normal and mdx mice

    Directory of Open Access Journals (Sweden)

    Nye David J

    2010-09-01

    Full Text Available Abstract Background Submaximal exercise is used in the management of muscular dystrophy. The effects of mechanical stimulation on skeletal development are well understood, although its effects on cartilage growth have yet to be investigated in the dystrophic condition. The objective of this study was to investigate the chondrogenic response to voluntary exercise in dystrophin-deficient mice. Methods Control and dystrophin-deficient (mdx mice were divided into sedentary and exercise-treated groups and tested for chondral histomorphometric differences at the proximal femur. Results Control mice ran 7 km/week further than mdx mice on average, but this difference was not statistically significant (P > 0.05. However, exercised control mice exhibited significantly enlarged femur head diameter, articular cartilage thickness, articular cartilage tissue area, and area of calcified cartilage relative to sedentary controls and exercised mdx mice (P Conclusions Mdx mice exhibit a reduced chondrogenic response to increased mechanical stimulation relative to controls. However, no significant reduction in articular dimensions was found, indicating loss of chondral tissue may not be a clinical concern with dystrophinopathy.

  1. Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

    2009-01-01

    Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study...... in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep...... into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP...

  2. [Activation of nucleolar organizers during in vitro cultivation of mouse R1 embryonic stem cells].

    Science.gov (United States)

    Kunafina, E R; Chaplina, M V; Filiasova, E I; Gibanova, N V; Khodarovich, Iu M; Larionov, O A; Zatsepina, O V

    2005-01-01

    We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

  3. Growth retardation of paramecium and mouse cells by shielding them from background radiation

    International Nuclear Information System (INIS)

    Kawanishi, Masanobu; Okuyama, Katsuyuki; Shiraishi, Kazunori; Matsuda, Yatsuka; Taniguchi, Ryoichi; Shiomi, Nobuyuki; Yonezawa, Morio; Yagi, Takashi

    2012-01-01

    In the 1970s and 1980s, Planel et al. reported that the growth of paramecia was decreased by shielding them from background radiation. In the 1990s, Takizawa et al. found that mouse cells displayed a decreased growth rate under shielded conditions. The purpose of the present study was to confirm that growth is impaired in organisms that have been shielded from background radiation. Radioprotection was produced with a shielding chamber surrounded by a 15 cm thick iron wall and a 10 cm thick paraffin wall that reduced the γ ray and neutron levels in the chamber to 2% and 25% of the background levels, respectively. Although the growth of Paramecium tetraurelia was not impaired by short-term radioprotection (around 10 days), which disagreed with the findings of Planel et al., decreased growth was observed after long-term (40-50 days) radiation shielding. When mouse lymphoma L5178Y cells were incubated inside or outside of the shielding chamber for 7 days, the number of cells present on the 6th and 7th days under the shielding conditions was significantly lower than that present under the non-shielding conditions. These inhibitory effects on cell growth were abrogated by the addition of a 137 Cs γ-ray source disk to the chamber. Furthermore, no growth retardation was observed in XRCC4-deficient mouse M10 cells, which display impaired DNA double strand break repair. (author)

  4. Control of RFM strain endogenous retrovirus in RFM mouse cells

    International Nuclear Information System (INIS)

    Tennant, R.W.; Otten, J.A.; Wang, T.W.; Liou, R.S.; Brown, A.; Yang, W.K.

    1983-01-01

    RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1/sup n/ allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission

  5. Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yinxiang Wang

    2018-01-01

    Full Text Available Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

  6. Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice

    Directory of Open Access Journals (Sweden)

    Sirsi Shashank R

    2008-04-01

    Full Text Available Abstract Background Exon skipping oligonucleotides (ESOs of 2'O-Methyl (2'OMe and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD. However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine (PEI and poly(ethylene glycol (PEG are regarded as effective agents for enhanced delivery of nucleic acids in various applications. Results We examined whether PEG-PEI copolymers can facilitate ESO-mediated dystrophin expression after intramuscular injections into tibialis anterior (TA muscles of mdx mice. We utilized a set of PEG-PEI copolymers containing 2 kDa PEI and either 550 Da or 5 kDa PEG, both of which bind 2'OMe ESOs with high affinity and form stable nanoparticulates with a relatively low surface charge. Three weekly intramuscular injections of 5 μg of ESO complexed with PEI2K-PEG550 copolymers resulted in about 500 dystrophin-positive fibers and about 12% of normal levels of dystrophin expression at 3 weeks after the initial injection, which is significantly greater than for injections of ESO alone, which are known to be almost completely ineffective. In an effort to enhance biocompatibility and cellular uptake, the PEI2K-PEG550 and PEI2K-PEG5K copolymers were functionalized by covalent conjugation with nanogold (NG or adsorbtion of colloidal gold (CG, respectively. Surprisingly, using the same injection and dosing regimen, we found no significant difference in dystrophin expression by Western blot between the NG-PEI2K-PEG550, CG-PEI2K-PEG5K, and non-functionalized PEI2K-PEG550 copolymers. Dose-response experiments using the CG-PEI2K-PEG5K copolymer with total ESO ranging from 3–60 μg yielded a maximum of about 15% dystrophin expression. Further improvements in dystrophin expression up to 20% of normal

  7. Structural and functional development of rat and mouse gastric mucous cells in relation to their proliferative activity

    International Nuclear Information System (INIS)

    Wattel, W.

    1978-01-01

    An investigation has been carried out to find a relation between the differentiation and the mitotic activity of gastric mucous cells of the rat and the mouse. It is shown that the bulk mucous production is carried out by the older, non-proliferative, surface mucous cells that line the foveolae and the gastric surface. One experiment describes the renewal of mouse gastric mucous cells following fast neutron irradiation. (C.F.)

  8. Developmental immunolocalization of the Klotho protein in mouse kidney epithelial cells

    Directory of Open Access Journals (Sweden)

    J.H. Song

    2014-01-01

    Full Text Available A defect in Klotho gene expression in the mouse results in a syndrome that resembles rapid human aging. In this study, we investigated the detailed distribution and the time of the first appearance of Klotho in developing and adult mouse kidney. Kidneys from 16-(F16, 18-(F18 and 20-day-old (F20 fetuses, 1- (P1, 4- (P4, 7- (P7, 14- (P14, and 21-day-old (P21 pups and adults were processed for immunohistochemistry and immunoblot analyses. In the developing mouse kidney, Klotho immunoreactivity was initially observed in a few cells of the connecting tubules (CNT of 18-day-old fetus (F and in the medullary collecting duct (MCD and distal nephron of the F16 developing kidney. In F20, Klotho immunoreactivity was increased in CNT and additionally observed in the outer portion of MCD and tip of the renal papilla. During the first 3 weeks after birth, Klotho-positive cells gradually disappeared from the MCD due to apoptosis, but remained in the CNT and cortical collecting ducts (CCD. In the adult mouse, the Klotho protein was expressed only in a few cells of the CNT and CCD in cortical area. Also, Klotho immunoreactivity was observed in the aquaporin 2-positive CNT, CCD, and NaCl co-transporter-positive distal convoluted tubule (DCT cells and type B and nonA-nonB intercalated cells of CNT, DCT, and CCD. Collectively, our data indicate that immunolocalization of Klotho is closely correlated with proliferation in the intercalated cells of CNT and CCD from aging, and may be involved in the regulation of tubular proliferation.

  9. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    International Nuclear Information System (INIS)

    Winkler, Sandra; Borkham-Kamphorst, Erawan; Stock, Peggy; Brückner, Sandra; Dollinger, Matthias; Weiskirchen, Ralf; Christ, Bruno

    2014-01-01

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH

  10. TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

    Science.gov (United States)

    Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

    2014-05-15

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

  11. Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis

    International Nuclear Information System (INIS)

    Hicks, Sarah M.; Vassallo, Jeffrey D.; Dieter, Matthew Z.; Lewis, Cindy L.; Whiteley, Laurence O.; Fix, Andrew S.; Lehman-McKeeman, Lois D.

    2003-01-01

    Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression

  12. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Borkham-Kamphorst, Erawan, E-mail: ekamphorst@ukaachen.de [Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [Department for Internal Medicine I, University Hospital Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Weiskirchen, Ralf, E-mail: rweiskirchen@ukaachen.de [Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig (Germany)

    2014-08-15

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH.

  13. Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage.

    Science.gov (United States)

    Denissova, Natalia G; Nasello, Cara M; Yeung, Percy L; Tischfield, Jay A; Brenneman, Mark A

    2012-01-01

    Resveratrol has elicited many provocative anticancer effects in laboratory animals and cultured cells, including reduced levels of oxidative DNA damage, inhibition of tumor initiation and progression and induction of apoptosis in tumor cells. Use of resveratrol as a cancer-preventive agent in humans will require that its anticancer effects not be accompanied by damage to normal tissue stem or progenitor cells. In mouse embryonic stem cells (mESC) or early mouse embryos exposed to ethanol, resveratrol has been shown to suppress apoptosis and promote survival. However, in cells exposed to genotoxic stress, survival may come at the expense of genome stability. To learn whether resveratrol can protect stem cells from DNA damage and to study its effects on genomic integrity, we exposed mESC pretreated with resveratrol to ionizing radiation (IR). Forty-eight hours pretreatment with a comparatively low concentration of resveratrol (10 μM) improved survival of mESC >2-fold after exposure to 5 Gy of X-rays. Cells pretreated with resveratrol sustained the same levels of reactive oxygen species and DNA strand breakage after IR as mock-treated controls, but repaired DNA damage more rapidly and resumed cell division sooner. Frequencies of IR-induced mutation at a chromosomal reporter locus were not increased in cells pretreated with resveratrol as compared with controls, indicating that resveratrol can improve viability in mESC after DNA damage without compromising genomic integrity.

  14. Leptin Suppresses Mouse Taste Cell Responses to Sweet Compounds.

    Science.gov (United States)

    Yoshida, Ryusuke; Noguchi, Kenshi; Shigemura, Noriatsu; Jyotaki, Masafumi; Takahashi, Ichiro; Margolskee, Robert F; Ninomiya, Yuzo

    2015-11-01

    Leptin is known to selectively suppress neural and behavioral responses to sweet-tasting compounds. However, the molecular basis for the effect of leptin on sweet taste is not known. Here, we report that leptin suppresses sweet taste via leptin receptors (Ob-Rb) and KATP channels expressed selectively in sweet-sensitive taste cells. Ob-Rb was more often expressed in taste cells that expressed T1R3 (a sweet receptor component) than in those that expressed glutamate-aspartate transporter (a marker for Type I taste cells) or GAD67 (a marker for Type III taste cells). Systemically administered leptin suppressed taste cell responses to sweet but not to bitter or sour compounds. This effect was blocked by a leptin antagonist and was absent in leptin receptor-deficient db/db mice and mice with diet-induced obesity. Blocking the KATP channel subunit sulfonylurea receptor 1, which was frequently coexpressed with Ob-Rb in T1R3-expressing taste cells, eliminated the effect of leptin on sweet taste. In contrast, activating the KATP channel with diazoxide mimicked the sweet-suppressing effect of leptin. These results indicate that leptin acts via Ob-Rb and KATP channels that are present in T1R3-expressing taste cells to selectively suppress their responses to sweet compounds. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  15. Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

    Science.gov (United States)

    Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

    2015-04-01

    Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

    Science.gov (United States)

    Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

    2016-06-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

  17. Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

    Directory of Open Access Journals (Sweden)

    Enders Greg H

    2010-06-01

    Full Text Available Abstract Background Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF, that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts. Results We show that mouse embryo fibroblasts (MEFs and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells. Conclusions In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types to become immortalized and transformed, compared to human cells.

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

    Directory of Open Access Journals (Sweden)

    Mingxing Wang

    2016-01-01

    Full Text Available A series of poly(esteramines (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 found in the order of PEA 01, PEA 04 > PEA 05 > others. The in vivo study in mdx mice demonstrated enhanced exon-skipping of 2′-OMePS with the order of PEA 06 > PEA 04, PEA 07 > PEA 03 > PEA 01 > others, and much higher than PEI 25k formulated 2′-OMePS. Exon-skipping efficiency of PMO in formulation with the PEAs were significantly enhanced in the order of PEA 02 > PEA 10 > PEA 01, PEA 03 > PEA 05, PEA 07, PEA 08 > others, with PEA 02 reaching fourfold of Endo-porter formulated PMO. PEAs improve PMO delivery more effectively than 2′-OMePS delivery in vivo, and the systemic delivery evaluation further highlight the efficiency of PEA for PMO delivery in all skeletal muscle. The results suggest that the flexibility of PEA polymers could be explored for delivery of different AO chemistries, especially for antisense therapy.

  19. Sensitivity of mitochondria of the mouse liver cells to radiation

    International Nuclear Information System (INIS)

    Shima, Akihiro

    1974-01-01

    In order to study the sensitivity of mitochondria (Mt) of the liver cells to radiation, 0.4 mg of riboflavine (RF) was intraperitoneally injected into mice which had been fed RF deficient food for 13 weeks. Three hours later 400 R of X-ray (190 KVP, 25 mA, 0.5 mmCu, 0.5 mmAl filter, FSD 61.5 cm, and HVL 0.80 mmCu) were irradiated to the whole body, and giant Mt of the liver cells were observed. When the liver cells were observed 24 hours after injection, neither giant Mt nor mitotic findings of Mt were found. All Mt observed were small (1.2 μ), although mice received 400 R of X-ray. (Serizawa, K.)

  20. Sensitivity of mitochondria of the mouse liver cells to radiation

    Energy Technology Data Exchange (ETDEWEB)

    Shima, A [Tokyo Univ. (Japan). Faculty of Science

    1974-06-01

    In order to study the sensitivity of mitochondria (Mt) of the liver cells to radiation, 0.4 mg of riboflavine (RF) was intraperitoneally injected into mice which had been fed RF deficient food for 13 weeks. Three hours later 400 R of X-ray (190 KVP, 25 mA, 0.5 mmCu, 0.5 mmAl filter, FSD 61.5 cm, and HVL 0.80 mmCu) were irradiated to the whole body, and giant Mt of the liver cells were observed. When the liver cells were observed 24 hours after injection, neither giant Mt nor mitotic findings of Mt were found. All Mt observed were small (1.2 ..mu..), although mice received 400 R of X-ray.

  1. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy); Campagnolo, Luisa, E-mail: campagno@med.uniroma2.it [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy)

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75{sup NTR}), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75{sup NTR} and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75{sup NTR} and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75{sup NTR}/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75{sup NTR} or TrkA. Interestingly, immunoreactivity to anti-p75{sup NTR} antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75{sup NTR}, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75{sup NTR} is turned on.

  2. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio; Campagnolo, Luisa

    2009-01-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75 NTR ), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75 NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75 NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75 NTR /TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75 NTR or TrkA. Interestingly, immunoreactivity to anti-p75 NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75 NTR , when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75 NTR is turned on.

  3. Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

    Science.gov (United States)

    Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

    2017-09-01

    The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

  4. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

    Directory of Open Access Journals (Sweden)

    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

  5. Interferon synthesis in mouse peritoneal cells damaged by x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Szolgay, E; T' alas, M

    1976-01-01

    NDV-induced interferon of peritoneal cells of irradiated (x-rays, 400 R) and control mice was investigated in vitro. Irradiation or treatment with hydroxyurea (10(-5) M) and mitomycin C (25 microng/ml) did not change interferon synthesis in spite of an 80 to 90% inhibition of 3H-thymidine incorporation. Increased doses of mitomycin C and treatment with actinomycin D and puromycin blocked interferon production. De novo interferon synthesis occurred in cells with damaged replicative activity of DNA caused by irradiation or by treatment with antimetabolites.

  6. Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development.

    Science.gov (United States)

    McCann, Matthew R; Tamplin, Owen J; Rossant, Janet; Séguin, Cheryle A

    2012-01-01

    Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration.

  7. Identification and characterization of adult mouse meniscus stem/progenitor cells.

    Science.gov (United States)

    Gamer, Laura W; Shi, Rui Rui; Gendelman, Ashira; Mathewson, Dylan; Gamer, Jackson; Rosen, Vicki

    Meniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

  8. Radiation and thermal characteristics of mouse lymphoma cells and their radiation-sensitive mutant

    International Nuclear Information System (INIS)

    Baba, Yuji; Yasunaga, Tadamasa; Uozumi, Hideaki; Takahashi, Mutsumasa; Sawada, Shozo.

    1988-01-01

    Radiation and thermal characteristics of L5178Y cells and their radiation-sensitive mutant M10 cells were studied by the colony-forming method and the dye-exclusion method using eosin-Y. Although M10 cells were remarkably radiation-sensitive compared with L5178Y cells, it was diffcult to cause interphase death of M10 after a large dose of irradiation. After heat treatments, L5178Y cells revealed more cell destruction and were stained well by eosin-Y, but it was relatively difficult to produce cell destruction of M10 cells, which showed poor staining by eosin-Y. When assayed by the colony-forming method, M10 cells were also heat-resistant compared to L5178Y. The dye-exclusion rate was closely correlated with cell survival after hyperthermia of L5178Y cells, suggesting that this is a simple method of detecting the thermosensitivity and thermotolerance of cancer cells. The difference in survival of L5178Y cells and M10 cells after combined treatment with gamma irradiation and hyperthermia was smaller than with gamma irradiation alone. It was also found that there was a relationship between radiation-induced interphase death and hyperthermia-induced interphase death, and that interphase death accounted for a major part of cell death caused by hyperthermia in mouse leukemia cells. (author)

  9. Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate.

    Directory of Open Access Journals (Sweden)

    Karen G Dowell

    Full Text Available Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation.

  10. Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

    Science.gov (United States)

    Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

    2012-07-01

    Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma

  11. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    John J Vincent

    Full Text Available The cell intrinsic programming that regulates mammalian primordial germ cell (PGC development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs from mouse embryonic stem cells (ESCs. Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

  12. Cultivation of mouse mammary tumor cells derived from DD/Tbr, 3

    International Nuclear Information System (INIS)

    Iwai, Mineko; Iwai, Yoshiaki; Takamori, Yasuhiko; Okumoto, Masaaki; Nishikawa, Ryosuke

    1981-01-01

    The factors affecting production of MuMTV by DD-762 cells, an established cell line from a spontaneous mammary tumor in a DD/Tbr mouse, were examined. When the cells were seeded and cultures medium were refreshed at every 3 - 4 day intervals without passage of cells, virus production began after exponential pase of cell growth and attained to peaks at every 10 - 12 days intervals up to approximately 60 days after seeding. MuMTV production was dependent on cell seeding density. Seeding at higher cell density, virus release occurred earlier. Maximum amount of MuMTV was observed with the medium containing 10 μg INS, 5 μg DXM and 10% FCS. The RDDP activities in the culture fluid were rapidly inactivated by incubation at 37 0 C. (author)

  13. DNA replication and post-replication repair in U.V.-sensitive mouse neuroblastoma cells

    International Nuclear Information System (INIS)

    Lavin, M.F.; McCombe, P.; Kidson, C.

    1976-01-01

    Mouse neuroblastoma cells differentiated when grown in the absence of serum; differentiation was reversed on the addition of serum. Differentiated cells were more sensitive to U.V.-radiation than proliferating cells. Whereas addition of serum to differentiated neuroblastoma cells normally resulted in immediate, synchronous entry into S phase, irradiation just before the addition of serum resulted in a long delay in the onset of DNA replication. During this lag period, incorporated 3 H-thymidine appeared in the light density region of CsCl gradients, reflecting either repair synthesis or abortive replication. Post-replication repair (gap-filling) was found to be present in proliferating cells and at certain times in differentiated cells. It is suggested that the sensitivity of differentiated neuroblastoma cells to U.V.-radiation may have been due to ineffective post-replication repair or to deficiencies in more than one repair mechanism, with reduction in repair capacity beyond a critical threshold. (author)

  14. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    Science.gov (United States)

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  15. Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.

    Science.gov (United States)

    Dodds, E; Dunckley, M G; Naujoks, K; Michaelis, U; Dickson, G

    1998-04-01

    Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

  16. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

    Directory of Open Access Journals (Sweden)

    Satoshi Ohtsuka

    Full Text Available Mouse epiblast stem cells (mEpiSCs are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

  17. Titanium dioxide nanoparticle-induced cytotoxicity and the underlying mechanism in mouse myocardial cells

    Science.gov (United States)

    Zhou, Yingjun; Hong, Fashui; Wang, Ling

    2017-11-01

    Exposure to fine particulate matter (PM) is known to cause cardiovascular disease. While extensive research has focused on the risk of atmospheric PM to public health, particularly heart disease, limited studies to date have attempted to clarify the molecular mechanisms underlying myocardial cell damage caused by exposure to titanium dioxide nanoparticles (TiO2 NPs). Data from the current investigation showed that TiO2 NPs are deposited in myocardial mitochondria via the blood circulation accompanied by obvious ultrastructural changes and impairment of mitochondrial structure and function in mouse myocardial cells, including reduction in mitochondrial membrane potential and ATP production, aggravation of oxidative stress along with increased levels of reactive oxygen species, malondialdehyde and protein carbonyl, and decreased glutathione content and enzymatic activities, including superoxide dismutase and glutathione peroxidase. Furthermore, TiO2 NPs induced a significant decrease in the activities of complex I, complex II, complex III, complex IV, succinate dehydrogenase, NADH oxidase, Ca2+-ATPase, Na+/K+-ATPase, and Ca2+/Mg2+-ATPase, and upregulation of cytokine expression (including cytochrome c, caspase-3, and p-JNK) in mitochondria-mediated apoptosis while downregulating Bcl-2 expression in mouse myocardial cells. Our results collectively indicate that chronic exposure to TiO2 NPs induces damage in mitochondrial structure and function as well as mitochondria-mediated apoptosis in mouse myocardial cells, which may be closely associated with heart disease in animals and humans.

  18. Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

    Science.gov (United States)

    Bodel, P

    1978-05-01

    Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.

  19. Tumour-cell killing by X-rays and immunity quantitated in a mouse model system

    International Nuclear Information System (INIS)

    Porteous, D.D.; Porteous, K.M.; Hughes, M.J.

    1979-01-01

    As part of an investigation of the interaction of X-rays and immune cytotoxicity in tumour control, an experimental mouse model system has been used in which quantitative anti-tumour immunity was raised in prospective recipients of tumour-cell suspensions exposed to varying doses of X-rays in vitro before injection. Findings reported here indicate that, whilst X-rays kill a proportion of cells, induced immunity deals with a fixed number dependent upon the immune status of the host, and that X-rays and anti-tumour immunity do not act synergistically in tumour-cell killing. The tumour used was the ascites sarcoma BP8. (author)

  20. Fundus Autofluorescence and Photoreceptor Cell Rosettes in Mouse Models

    Science.gov (United States)

    Flynn, Erin; Ueda, Keiko; Auran, Emily; Sullivan, Jack M.; Sparrow, Janet R.

    2014-01-01

    Purpose. This study was conducted to study correlations among fundus autofluorescence (AF), RPE lipofuscin accumulation, and photoreceptor cell degeneration and to investigate the structural basis of fundus AF spots. Methods. Fundus AF images (55° lens; 488-nm excitation) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired in pigmented Rdh8−/−/Abca4−/− mice (ages 1–9 months) with a confocal scanning laser ophthalmoscope (cSLO). For quantitative fundus AF (qAF), gray levels (GLs) were calibrated to an internal fluorescence reference. Retinal bisretinoids were measured by quantitative HPLC. Histometric analysis of outer nuclear layer (ONL) thicknesses was performed, and cryostat sections of retina were examined by fluorescence microscopy. Results. Quantified A2E and qAF intensities increased until age 4 months in the Rdh8−/−/Abca4−/− mice. The A2E levels declined after 4 months of age, but qAF intensity values continued to rise. The decline in A2E levels in the Rdh8−/−/Abca4−/− mice paralleled reduced photoreceptor cell viability as reflected in ONL thinning. Hyperautofluorescent puncta in fundus AF images corresponded to photoreceptor cell rosettes in SD-OCT images and histological sections stained with hematoxylin and eosin. The inner segment/outer segment–containing core of the rosette emitted an autofluorescence detected by fluorescence microscopy. Conclusions. When neural retina is disordered, AF from photoreceptor cells can contribute to noninvasive fundus AF images. Hyperautofluorescent puncta in fundus AF images are attributable, in at least some cases, to photoreceptor cell rosettes. PMID:25015357

  1. Fetal progenitor cell transplantation treats methylmalonic aciduria in a mouse model

    International Nuclear Information System (INIS)

    Buck, Nicole E.; Pennell, Samuel D.; Wood, Leonie R.; Pitt, James J.; Allen, Katrina J.; Peters, Heidi L.

    2012-01-01

    Highlights: ► Fetal cells were transplanted into a methylmalonic acid mouse model. ► Cell engraftment was detected in liver, spleen and bone marrow. ► Biochemical disease correction was measured in blood samples. ► A double dose of 5 million cells (1 week apart) proved more effective. ► Higher levels of engraftment may be required for greater disease correction. -- Abstract: Methylmalonic aciduria is a rare disorder caused by an inborn error of organic acid metabolism. Current treatment options are limited and generally focus on disease management. We aimed to investigate the use of fetal progenitor cells to treat this disorder using a mouse model with an intermediate form of methylmalonic aciduria. Fetal liver cells were isolated from healthy fetuses at embryonic day 15–17 and intravenously transplanted into sub-lethally irradiated mice. Liver donor cell engraftment was determined by PCR. Disease correction was monitored by urine and blood methylmalonic acid concentration and weight change. Initial studies indicated that pre-transplantation sub-lethal irradiation followed by transplantation with 5 million cells were suitable. We found that a double dose of 5 million cells (1 week apart) provided a more effective treatment. Donor cell liver engraftment of up to 5% was measured. Disease correction, as defined by a decrease in blood methylmalonic acid concentration, was effected in methylmalonic acid mice transplanted with a double dose of cells and who showed donor cell liver engraftment. Mean plasma methylmalonic acid concentration decreased from 810 ± 156 (sham transplanted) to 338 ± 157 μmol/L (double dose of 5 million cells) while mean blood C3 carnitine concentration decreased from 20.5 ± 4 (sham transplanted) to 5.3 ± 1.9 μmol/L (double dose of 5 million cells). In conclusion, higher levels of engraftment may be required for greater disease correction; however these studies show promising results for cell transplantation biochemical

  2. Isolation of sphere-forming stem cells from the mouse inner ear.

    Science.gov (United States)

    Oshima, Kazuo; Senn, Pascal; Heller, Stefan

    2009-01-01

    The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

  3. [Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

    Science.gov (United States)

    Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

    2012-07-01

    To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

  4. Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation

    NARCIS (Netherlands)

    van Gisbergen, Klaas P. J. M.; Kragten, Natasja A. M.; Hertoghs, Kirsten M. L.; Wensveen, Felix M.; Jonjic, Stipan; Hamann, Jörg; Nolte, Martijn A.; van Lier, Rene A. W.

    2012-01-01

    The transcriptional repressor Blimp-1 mediates the terminal differentiation of many cell types, including T cells. Here we identified Hobit (Znf683) as a previously unrecognized homolog of Blimp-1 that was specifically expressed in mouse natural killer T cells (NKT cells). Through studies of

  5. Hyperthermia studies using inductive and ultrasound methods on E. coli bacteria and mouse glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Cabral–Prieto, A., E-mail: agustin.cabral@inin.gob.mx; López-Callejas, R., E-mail: regulo.lopez@inin.gob.mx; Rodríguez-Méndez, B. G., E-mail: benjamin.rodriguez@inin.gob.mx; Santos-Cuevas, C. L., E-mail: clara.cuevas@inin.gob.mx [Carretera México-Toluca s/n, La Marquesa, Instituto Nacional de Investigaciones Nucleares (Mexico); Celis-Almazán, J., E-mail: jony-jac-5@hotmail.com; Olea-Mejía, O., E-mail: oleaoscar@yahoo.com.mx [Universidad Autónoma del Estado de México, Centro Conjunto de Investigación en Química Sustentable (Mexico); Gómez-Morales, J. L. [Universidad Autónoma del Estado de México, Campus El Cerrillo, Facultad de Ciencias (Mexico); Peña-Eguiluz, R., E-mail: rosendo.eguiluz@inin.gob.mx; Valencia-Alvarado, R., E-mail: raul.valencia@inin.gob.mx; Mercado-Cabrera, A., E-mail: antonio.mercado@inin.gob.mx; Muñoz-Castro, A. E., E-mail: arturo.munoz@inin.gob.mx [Carretera México-Toluca s/n, La Marquesa, Instituto Nacional de Investigaciones Nucleares (Mexico); García-Santibañez, F., E-mail: fegasa2@yahoo.com.mx [Universidad Autónoma del Estado de México, Campus El Cerrillo, Facultad de Ciencias (Mexico)

    2017-11-15

    The survival of Escherichia coli bacteria and mouse glioma cells were studied under different temperatures using direct heating in water, ultrasound, and magnetic fluid hyperthermia. The survival of these microorganisms depended on whether the heating mode was continuous or discontinuous, surviving more in the former than in the discontinuous heating mode. Whereas Escherichia coli bacteria did not survive at temperatures ≥50{sup ∘}C, the mouse glioma cells did not survive at temperatures ≥48{sup ∘}C. The survival of both these microorganisms was independent of the presence or absence of the magnetic nanoparticles of magnetite, suggesting that these, having mean particle sizes of 9.5, 8.5 and 5, did not show any apparent cytotoxicity effect. Present results also showed that the inductive heating system which used a radiofrequency of 13.56 MHz, providing a maximum magnetic field strength of 160 A/m, the electric rather than magnetic heating predominated.

  6. The Effect of Combination of Radiation with 5-Fluorouracil on Mouse Jejunal Crypt Cells

    International Nuclear Information System (INIS)

    Huh, Seung Jae; Park, Charn Il

    1985-01-01

    The interaction of radiation and 5-Fluorouracil (5-FU) on mouse jejunal crypt cells was studied using the microcolony survival assay. 150mg/kg of 5-FU was injected intraperitoneally 15 minutes before irradiation and 6 hours after irradiation. Jejunal crypt cells of mouse survived more when 5-FU was given 15 minutes before irradiation than giving it 6 hours after irradiation. The mean lethal doses (Do) of each of irradiation alone group, 5-FU injection group of 15 minutes preceding irradiation, and 5-FU injection group of 6 hours post irradiation were 135, 135, and 114 rad respectively. The dose effect factor (DEF) of each of 5-FU injection groups of 15 minutes preceding irradiation and of 6 hours post irradiation were 1.13 and 1.27

  7. In vitro effects of triiodothyronine on gene expression in mouse trophoblast cells.

    Science.gov (United States)

    Silva, J F; Ocarino, N M; Serakides, R

    2015-01-01

    The objective of the present study was to evaluate the effects of different doses of T3 (10(-4) M, 10(-7) M, 10(-9) M) on the in vitro gene expression of Tpbp, Prl3b1, VEGF, PGF, PL-1, and INFy in mouse trophoblast cells by real-time RT-PCR. Doses of 10(-7) and 10(-9) M T3 increased the mRNA levels of Tpbp, Pl3b1, VEGF, PGF, INFy and PL-1. In contrast, the dose of 10(-4) M reduced the gene expression of PL-1 and VEGF. T3 affected the gene expression of differentiation, hormonal, immune and angiogenic factors in mouse trophoblast cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Hyperthermia studies using inductive and ultrasound methods on E. coli bacteria and mouse glioma cells

    International Nuclear Information System (INIS)

    Cabral–Prieto, A.; López-Callejas, R.; Rodríguez-Méndez, B. G.; Santos-Cuevas, C. L.; Celis-Almazán, J.; Olea-Mejía, O.; Gómez-Morales, J. L.; Peña-Eguiluz, R.; Valencia-Alvarado, R.; Mercado-Cabrera, A.; Muñoz-Castro, A. E.; García-Santibañez, F.

    2017-01-01

    The survival of Escherichia coli bacteria and mouse glioma cells were studied under different temperatures using direct heating in water, ultrasound, and magnetic fluid hyperthermia. The survival of these microorganisms depended on whether the heating mode was continuous or discontinuous, surviving more in the former than in the discontinuous heating mode. Whereas Escherichia coli bacteria did not survive at temperatures ≥50"∘C, the mouse glioma cells did not survive at temperatures ≥48"∘C. The survival of both these microorganisms was independent of the presence or absence of the magnetic nanoparticles of magnetite, suggesting that these, having mean particle sizes of 9.5, 8.5 and 5, did not show any apparent cytotoxicity effect. Present results also showed that the inductive heating system which used a radiofrequency of 13.56 MHz, providing a maximum magnetic field strength of 160 A/m, the electric rather than magnetic heating predominated.

  9. Revealing new mouse epicardial cell markers through transcriptomics.

    Directory of Open Access Journals (Sweden)

    Lars Bochmann

    2010-06-01

    Full Text Available The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1 that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.

  10. Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

    Directory of Open Access Journals (Sweden)

    Elena Bassi

    2012-01-01

    Full Text Available In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage. In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

  11. Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract

    Directory of Open Access Journals (Sweden)

    Burkhard eSchütz

    2015-03-01

    Full Text Available The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP under the control of the ChAT promoter (EGFPChAT and by using in situ hybridization and immunohistochemistry we found that EGFPChAT cells were clustered in the epithelium lining the gastric groove. EGFPChAT cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFPChAT cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1 was never detected. Except for the proximal colon, EGFPChAT cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT. EGFPChAT cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18, transient receptor potential melastatin-like subtype 5 channel (TRPM5, and for cyclooxygenases 1 (COX1 and 2 (COX2. The ex vivo stimulation of colonic EGFPChAT cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFPChAT brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

  12. Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract.

    Science.gov (United States)

    Schütz, Burkhard; Jurastow, Innokentij; Bader, Sandra; Ringer, Cornelia; von Engelhardt, Jakob; Chubanov, Vladimir; Gudermann, Thomas; Diener, Martin; Kummer, Wolfgang; Krasteva-Christ, Gabriela; Weihe, Eberhard

    2015-01-01

    The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT)-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP) under the control of the ChAT promoter (EGFP (ChAT) ) and by using in situ hybridization and immunohistochemistry we found that EGFP (ChAT) cells were clustered in the epithelium lining the gastric groove. EGFP (ChAT) cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFP (ChAT) cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1) was never detected. Except for the proximal colon, EGFP (ChAT) cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT). EGFP (ChAT) cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18), transient receptor potential melastatin-like subtype 5 channel (TRPM5), and for cyclooxygenases 1 (COX1) and 2 (COX2). The ex vivo stimulation of colonic EGFP (ChAT) cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFP (ChAT) brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

  13. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

    Science.gov (United States)

    Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F.; Mistretta, Charlotte M.; Mishina, Yuji; Liu, Hong-Xiang

    2016-01-01

    Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC. PMID:26741369

  14. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

    Directory of Open Access Journals (Sweden)

    Kristin Boggs

    Full Text Available Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC. Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5 and young postnatal (P1-10 mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1 P0-Cre/R26-tdTomato (RFP to label NC, NC derived Schwann cells and derivatives; (2 Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3 Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

  15. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

    Science.gov (United States)

    Boggs, Kristin; Venkatesan, Nandakumar; Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F; Mistretta, Charlotte M; Mishina, Yuji; Liu, Hong-Xiang

    2016-01-01

    Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

  16. NaCl responsive taste cells in the mouse fungiform taste buds.

    Science.gov (United States)

    Yoshida, R; Horio, N; Murata, Y; Yasumatsu, K; Shigemura, N; Ninomiya, Y

    2009-03-17

    Previous studies have demonstrated that rodents' chorda tympani (CT) nerve fibers responding to NaCl can be classified according to their sensitivities to the epithelial sodium channel (ENaC) blocker amiloride into two groups: amiloride-sensitive (AS) and -insensitive (AI). The AS fibers were shown to respond specifically to NaCl, whereas AI fibers broadly respond to various electrolytes, including NaCl. These data suggest that salt taste transduction in taste cells may be composed of at least two different systems; AS and AI ones. To further address this issue, we investigated the responses to NaCl, KCl and HCl and the amiloride sensitivity of mouse fungiform papilla taste bud cells which are innervated by the CT nerve. Comparable with the CT data, the results indicated that 56 NaCl-responsive cells tested were classified into two groups; 25 cells ( approximately 44%) narrowly responded to NaCl and their NaCl response were inhibited by amiloride (AS cells), whereas the remaining 31 cells ( approximately 56%) responded not only to NaCl, but to KCl and/or HCl and showed no amiloride inhibition of NaCl responses (AI cells). Amiloride applied to the basolateral side of taste cells had no effect on NaCl responses in the AS and AI cells. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that ENaC subunit mRNA was expressed in a subset of AS cells. These findings suggest that the mouse fungiform taste bud is composed of AS and AI cells that can transmit taste information differently to their corresponding types of CT fibers, and apical ENaCs may be involved in the NaCl responses of AS cells.

  17. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells.

    Science.gov (United States)

    Shabani, R; Ashtari, K; Behnam, B; Izadyar, F; Asgari, H; Asghari Jafarabadi, M; Ashjari, M; Asadi, E; Koruji, M

    2016-06-01

    Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture. © 2015 Blackwell Verlag GmbH.

  18. The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells.

    Science.gov (United States)

    Huang, Bo Yuan; Zhan, Yi Ping; Zong, Wen Jing; Yu, Chun Jiang; Li, Jun Fa; Qu, Yan Ming; Han, Song

    2015-01-01

    Glioblastoma multiforme (GBM) is the most malignant primary type of brain tumor in adults. There has been increased focus on the immunotherapies to treat GBM patients, the therapeutic value of natural killer (NK) cells is still unknown. Programmed death-1 (PD-1) is a major immunological checkpoint that can negatively regulate the T-cell-mediated immune response. We tested the combination of the inhibiting the PD-1/B7H1 pathway with a NK-cell mediated immune response in an orthotopic mouse model of GBM. Mouse glioma stem cells (GL261GSCs) and mouse NK cells were isolated and identified. A lactate dehydrogenase (LDH) assay was perfomed to detect the cytotoxicity of NK cells against GL261GSCs. GL261GSCs were intracranially implanted into mice, and the mice were stratified into 3 treatment groups: 1) control, 2) NK cells treatment, and 3) PD-1 inhibited NK cells treatment group. Overall survival was quantified, and animal magnetic resonance imaging (MRI) was performed to determine tumor growth. The brains were harvested after the mice were euthanized, and immunohistochemistry against CD45 and PCNA was performed. The mouse NK cells were identified as 90% CD3- NK1.1+CD335+ by flow cytometric analysis. In the LDH assay, the ratios of the damaged GL261GSCs, with the E:T ratios of 2.5:1, 5:1, and 10:1, were as follows: 1) non-inhibited group: 7.42%, 11.31%, and 15.1%, 2) B7H1 inhibited group: 14.75%, 18.25% and 29.1%, 3) PD-1 inhibited group: 15.53%, 19.21% and 29.93%, 4) double inhibited group: 33.24%, 42.86% and 54.91%. In the in vivo experiments, the mice in the PD-1 inhibited NK cells treatment group and IL-2-stimulated-NK cells treatment group displayed a slowest tumor growth (F = 308.5, Pmouse NK cells to kill the GL261GSCs, and the PD-1-inhibited NK cells could be a feasible immune therapeutic approach against GBM.

  19. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Gao, Xiugong; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-01-01

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds

  20. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  1. An investigation of the shedding of macromolecules from the Ehrlich mouse ascites tumor cell

    International Nuclear Information System (INIS)

    Edwards, E.H.

    1984-01-01

    The spontaneous release, or shedding, of cell surface components into the extracellular medium may be important in the determination of several features of the cancer cell phenotype. The release of macromolecules from the Erhlich mouse ascites tumor cell was studied under a variety of experimental conditions to elucidate the origin and the underlying mechanisms of release. The extrinsic macromolecules are a diverse group with apparent molecular weights ranging from 13,500 to 400,000 daltons. External labeling of the cell surface with tritiated 4,4'-diisothiocyano-1,2-diphenylethane-2,2-disulfonic acid ([ 3 H]H 2 DIDS) reveals a slow loss of labeled components at 4 degrees C, while at 21 degrees C and 37 degrees C an initial rapid loss is followed by a slower release. In vitro metabolic labeling with [1- 14 C]-D-glucosamine hydrochloride, D-[2- 3 H]-mannose and various [ 3 H]-L-amino acids results in the appearance of labeled macromolecules in the medium suggesting tumor, not mouse, origin. These data suggest that the extrinsic macromolecules originate from the cell surface. Macromolecules are shed by a temperature and pH sensitive process. These results suggest that a limited proteolytic digestion, or sublethal autolysis, of the cell surface may occur in this system. The macromolecules shed by the Ehrlich cell originate from the surface and are probably released by sublethal autolysis, direct secretion and a passive process

  2. Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

    International Nuclear Information System (INIS)

    Yu, Song-Hee; Jang, Yu-Jin; Lee, Eun-Shil; Hwang, Dong-Youn; Jeon, Chang-Jin

    2010-01-01

    Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell

  3. Inhibition of Cell Division and DNA Replication Impair Mouse-Naïve Pluripotency Exit.

    Science.gov (United States)

    Waisman, Ariel; Vazquez Echegaray, Camila; Solari, Claudia; Cosentino, María Soledad; Martyn, Iain; Deglincerti, Alessia; Ozair, Mohammad Zeeshan; Ruzo, Albert; Barañao, Lino; Miriuka, Santiago; Brivanlou, Ali; Guberman, Alejandra

    2017-09-01

    The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.

    Science.gov (United States)

    Choi, Hannah; Zhang, Lei; Cembrowski, Mark S; Sabottke, Carl F; Markowitz, Alexander L; Butts, Daniel A; Kath, William L; Singer, Joshua H; Riecke, Hermann

    2014-09-15

    In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell. Copyright © 2014 the American Physiological Society.

  5. Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

    Directory of Open Access Journals (Sweden)

    Haiying Guo

    2018-01-01

    Full Text Available Dermal papilla (DP plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

  6. Chaotic electrical activity of living β-cells in the mouse pancreatic islet

    Science.gov (United States)

    Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

    2007-02-01

    To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

  7. Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Zhengpin Wang

    Full Text Available In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β family member activin (ACT contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST, during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.

  8. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie

    2014-01-01

    nanoparticles could be effective for stem cell differentiation in vitro. Materials & methods: We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells......Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...... was evaluated by expression of MN-specific transcription factors monitored by quantitative real-time PCR reactions and immunocytochemistry. Results: Mesoporous nanoparticles have strong affiliation to the embryoid bodies, penetrate inside the embryoid bodies and come in contact with differentiating cells...

  9. A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Marcel Veltrop

    Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

  10. Cryo-imaging of fluorescently labeled single cells in a mouse

    Science.gov (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  11. MicroRNA modulation induced by AICA ribonucleotide in J1 mouse ES cells.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Shi

    Full Text Available ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells. It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells. AICA ribonucleotide (AICAR is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for

  12. LASP-01: Distribution of Mouse Embryonic Stem Cells Expressing MicroRNAs | Frederick National Laboratory for Cancer Research

    Science.gov (United States)

    The Laboratory Animal Sciences Program manages the expansion, processing, and distribution of1,501 genetically engineered mouse embryonic stem cell (mESC) linesharboring conditional microRNA transgenes. The Laboratory Animal Sciences Prog

  13. Impact of 2-bromopropane on mouse embryonic stem cells and ...

    African Journals Online (AJOL)

    EWOMAZINO

    medium containing various concentrations of 2-BP at 37°C in a CO2 incubator for 24 h. Cells were then washed twice with ice-cold PBS and lysed on ice for 10 min in 400 μl lysis buffer (20 mM Tris-HCl,. pH 7.4, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, ...

  14. Voluntary wheel running in dystrophin-deficient (mdx) mice: Relationships between exercise parameters and exacerbation of the dystrophic phenotype

    OpenAIRE

    Smythe, Gayle M; White, Jason D

    2012-01-01

    Voluntary wheel running can potentially be used to exacerbate the disease phenotype in dystrophin-deficient mdx mice. While it has been established that voluntary wheel running is highly variable between individuals, the key parameters of wheel running that impact the most on muscle pathology have not been examined in detail. We conducted a 2-week test of voluntary wheel running by mdx mice and the impact of wheel running on disease pathology. There was significant individual variation in the...

  15. A novel approach for amplification and purification of mouse oligodendrocyte progenitor cells

    Directory of Open Access Journals (Sweden)

    Junlin Yang

    2016-08-01

    Full Text Available Although transgenic and knockout mice are widely used to study the specification and differentiation of oligodendrocyte precursor cells (OPCs, mouse primary OPCs are difficult to be purified and maintained, and many in vitro studies have to resort to rat OPCs as substitutes. In this study, we reported that mouse O4 negative early-stage OPCs can be obtained by culturing cortical tissue blocks, and the simultaneous treatment of OPCs with PDGFaa, bFGF and EGF is the key for the propagation of mouse OPCs in culture. Epidermal growth factor (EGF was found to be a potent mitogen for OPCs and cooperate with Platelet Derived Growth Factor-AA (PDGFaa to extend cell division and inhibit their differentiation. EGF also collaborates with PDGFaa and basic fibroblast growth factor (bFGF to convert bipolar or tripolar OPCs to more vital fibroblast-like OPCs without compromising their oligodendrocyte differentiation potential. In addition, EGF promoted the survival and proliferation of glial progenitor cells (GPCs derived from primary OPC cultures, and a mixture of GPCs and OPCs can be obtained and propagated in the presence of EGF, bFGF and PDGFaa. Once EGF is withdrawn, GPC population decreased sharply and fibroblast-like OPCs changed into typical OPCs morphology, then homogeneous OPCs were obtained subsequently.

  16. Epithelial cell kinetics in mouse and rat skin irradiated with electrons

    International Nuclear Information System (INIS)

    McMaster-Schuyler, L.

    1984-02-01

    Experiments were performed to examine the kinetic responses of mouse and rat epidermal cells in vivo after single doses of ionizing radiation including responses of hair follicles at times after irradiation. The labeling indices in both species were reduced to 30 to 50% of control values immediately following irradiation at all the doses. In the rat, the labeling indices recovered and overshot control values within the first three days after 300 to 1200 rads. The mouse labeling indices continued to be suppressed for up to 10 days after 300 to 2400 rads. This indicated that rat G 1 phase epidermal cells recovered three times faster than those of the mouse with respect to the ability to maintain or increase control level cell proliferation after irradiation. After 1800 and 2400 rads, doses which produce skin ulceration, both species showed a reduction in their labeling indices for up to 7 days, indicating that a dose-dependent mechanism of recovery may be operable in the rat. 99 refs., 15 figs., 6 tabs

  17. Cell-type-dependent action potentials and voltage-gated currents in mouse fungiform taste buds.

    Science.gov (United States)

    Kimura, Kenji; Ohtubo, Yoshitaka; Tateno, Katsumi; Takeuchi, Keita; Kumazawa, Takashi; Yoshii, Kiyonori

    2014-01-01

    Taste receptor cells fire action potentials in response to taste substances to trigger non-exocytotic neurotransmitter release in type II cells and exocytotic release in type III cells. We investigated possible differences between these action potentials fired by mouse taste receptor cells using in situ whole-cell recordings, and subsequently we identified their cell types immunologically with cell-type markers, an IP3 receptor (IP3 R3) for type II cells and a SNARE protein (SNAP-25) for type III cells. Cells not immunoreactive to these antibodies were examined as non-IRCs. Here, we show that type II cells and type III cells fire action potentials using different ionic mechanisms, and that non-IRCs also fire action potentials with either of the ionic mechanisms. The width of action potentials was significantly narrower and their afterhyperpolarization was deeper in type III cells than in type II cells. Na(+) current density was similar in type II cells and type III cells, but it was significantly smaller in non-IRCs than in the others. Although outwardly rectifying current density was similar between type II cells and type III cells, tetraethylammonium (TEA) preferentially suppressed the density in type III cells and the majority of non-IRCs. Our mathematical model revealed that the shape of action potentials depended on the ratio of TEA-sensitive current density and TEA-insensitive current one. The action potentials of type II cells and type III cells under physiological conditions are discussed. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  18. Comparative study of the organisation and phenotypes of bladder interstitial cells in human, mouse and rat.

    Science.gov (United States)

    Gevaert, Thomas; Neuhaus, Jochen; Vanstreels, Els; Daelemans, Dirk; Everaerts, Wouter; Der Aa, Frank Van; Timmermans, Jean-Pierre; Roskams, Tania; Steiner, Clara; Pintelon, Isabel; De Ridder, Dirk

    2017-12-01

    With most research on interstitial cells (IC) in the bladder being conducted on animal models, it remains unclear whether all structural and functional data on IC from animal models can be translated to the human context. This prompted us to compare the structural and immunohistochemical properties of IC in bladders from mouse, rat and human. Tissue samples were obtained from the bladder dome and subsequently processed for immunohistochemistry and electron microscopy. The ultrastructural properties of IC were compared by means of electron microscopy and IC were additionally characterized with single/double immunohistochemistry/immunofluorescence. Our results reveal a similar organization of the IC network in the upper lamina propria (ULP), the deep lamina propria (DLP) and the detrusor muscle in human, rat and mouse bladders. Furthermore, despite several similarities in IC phenotypes, we also found several obvious inter-species differences in IC, especially in the ULP. Most remarkably in this respect, ULP IC in human bladder predominantly displayed a myoid phenotype with abundant presence of contractile micro-filaments, while those in rat and mouse bladders showed a fibroblast phenotype. In conclusion, the organization of ULP IC, DLP IC and detrusor IC is comparable in human, rat and mouse bladders, although several obvious inter-species differences in IC phenotypes were found. The present data show that translating research data on IC in laboratory animals to the human setting should be carried out with caution.

  19. Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Hongran Wang

    2016-03-01

    Full Text Available Induced pluripotent stem (iPS cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc, without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs, iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin, cardiac alpha myosin heavy chain (α-MHC, cardiac troponin T (cTnT, and connexin 43 (CX43, as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5 and gata binding protein 4 (gata4. The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI in the future.

  20. CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Marco L Davila

    Full Text Available Although many adults with B cell acute lymphoblastic leukemia (B-ALL are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.

  1. Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

    Science.gov (United States)

    Piprek, Rafal P; Kolasa, Michal; Podkowa, Dagmara; Kloc, Malgorzata; Kubiak, Jacek Z

    2017-10-01

    Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Foxp3 regulates ratio of Treg and NKT cells in a mouse model of asthma.

    Science.gov (United States)

    Lu, Yanming; Guo, Yinshi; Xu, Linyun; Li, Yaqin; Cao, Lanfang

    2015-05-01

    Chronic inflammatory disorder of the airways causes asthma. Regulatory T cells (Treg cells) and Natural killer T cells (NKT cells) both play critical roles in the pathogenesis of asthma. Activation of Treg cells requires Foxp3, whereas whether Foxp3 may regulate the ratio of Treg and NKT cells to affect asthma is uncertain. In an ovalbumin (OVA)-induced mouse model of asthma, we either increased Treg cells by lentivirus-mediated forced expression of exogenous Foxp3, or increased NKT cells by stimulation with its activator α-GalCer. We found that the CD4+CD25+ Treg cells increased by forced Foxp3 expression, and decreased by α-GalCer, while the CD3+CD161+ NKT cells decreased by forced Foxp3 expression, and increased by α-GalCer. Moreover, forced Foxp3 expression, but not α-GalCer, significantly alleviated the hallmarks of asthma. Furthermore, forced Foxp3 increased levels of IL_10 and TGFβ1, and α-GalCer increased levels of IL_4 and INFγ in the OVA-treated lung. Taken together, our study suggests that Foxp3 may activate Treg cells and suppress NKT cells in asthma. Treg and NKT cells may antagonize the effects of each other in asthma.

  3. Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

    International Nuclear Information System (INIS)

    Rutz, H. Peter; Little, John B.

    1995-01-01

    Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO 2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G 2 /M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

  4. Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds.

    Science.gov (United States)

    Ohtubo, Yoshitaka; Iwamoto, Masafumi; Yoshii, Kiyonori

    2012-06-01

    Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ∼60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  5. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo.

    Science.gov (United States)

    Zou, Zhiyuan; Sun, Zhaolin; Li, Pan; Feng, Tao; Wu, Sen

    2016-12-14

    Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter) is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB) and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26 lacZ , we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases.

  6. Cell and tissue kinetics of the subependymal layer in mouse brain following heavy charged particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Manley, N.B.; Fabrikant, J.I.; Alpen, E.L.

    1988-12-01

    The following studies investigate the cellular response and cell population kinetics of the subependymal layer in the mouse brain exposed to heavy charged particle irradiation. Partial brain irradiation with helium and neon ions was confined to one cortex of the brain. Both the irradiated and the unirradiated contralateral cortex showed similar disturbances of the cell and tissue kinetics in the subependymal layers. The irradiated hemisphere exhibited histological damage, whereas the unirradiated side appeared normal histologically. This study concerns the cell population and cell cycle kinetics of the subependymal layer in the mouse brain, and the effects of charged particle irradiations on this cell population. Quantitative high resolution autoradiography was used to study the kinetic parameters in this cell layer. This study should help in understanding the effects of these high-energy heavy ions on normal mammalian brain tissue. The response of the mammalian brain exposure to charged particle ionizing radiation may be extremely variable. It varies from minimal physiological changes to overt tissue necrosis depending on a number of factors such as: the administered dose, dose-rate, the volume of the irradiated tissue, and the biological end-point being examined.

  7. The Knockout of Secretin in Cerebellar Purkinje Cells Impairs Mouse Motor Coordination and Motor Learning

    Science.gov (United States)

    Zhang, Li; Chung, Sookja Kim; Chow, Billy Kwok Chong

    2014-01-01

    Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells. PMID:24356714

  8. Immunohistochemical Examination for the Distribution of Podoplanin-Expressing Cells in Developing Mouse Molar Tooth Germs

    Science.gov (United States)

    Imaizumi, Yuri; Amano, Ikuko; Tsuruga, Eichi; Kojima, Hiroshi; Sawa, Yoshihiko

    2010-01-01

    We recently reported the expression of podoplanin in the apical bud of adult mouse incisal tooth. This study was aimed to investigate the distribution of podoplanin-expressing cells in mouse tooth germs at several developing stages. At the bud stage podoplanin was expressed in oral mucous epithelia and in a tooth bud. At the cap stage podoplanin was expressed on inner and outer enamel epithelia but not in mesenchymal cells expressing the neural crest stem cell marker nestin. At the early bell stage nestin and podoplanin were expressed in cervical loop and odontoblasts. At the root formation stage both nestin and podoplanin were weakly expressed in odontoblasts generating radicular dentin. Podoplanin expression was also found in the Hertwig epithelial sheath. These results suggest that epithelial cells of developing tooth germ acquire the ability to express nestin, and that tooth germ epithelial cells maintain the ability to express podoplanin in oral mucous epithelia. The expression of podoplanin in odontoblasts was induced as tooth germ development advanced, but was suppressed with the completion of the primary dentin, suggesting that podoplanin may be involved in the cell growth of odontoblasts. Nestin may function as an intermediate filament that binds podoplanin in odontoblasts. PMID:21060740

  9. Radiosensitivity and cell kinetics of the human solid cancer transplanted to nude mouse

    International Nuclear Information System (INIS)

    Ikeuchi, Shunji

    1983-01-01

    This study was undertaken to analyse the relationship between radiosensitivity and cell kinetics of human solid cancer in experimental nude mouse system. Four strains of tumors used for the experiment were poorly differentiated squamous cell carcinoma of the lung (Lu-9), oat cell carcinoma of the lung (Lu-24), well differentiated squamous cell carcinoma of the tongue (To-1) and moderately differentiated squamous cell carcinoma of the esophagus (Es-4) which were serially transplantable to BALB/c nude mice. Radiosensitivity was evaluated by tumor growth in terms of inhibition rate, histological change and host reaction after irradiation. Cell kinetics were studied by autoradiography with pulse administration of 3 H-thymidine to mice. Although Lu-24 was most radiosensitive, followed by To-1, Es-4 and Lu-9 in the order of sensitivity, it was suggested that they might be more radioresistant in nude mice without T-cell function than in human. Regarding squamous cell carcinomas, well differentiated type was more radiosensitive than poorly differentiated one. All of these tumors in nude mouse revealed distinct percent labeled mitosis curves with two clear peaks which were quite different from those in human body. Lu-24 showed a characteristic pattern with a long time lag before visible growth, short G 1 , and low growth fraction, compared to other three tumors. Three strains of squamous cell carcinoma demonstrated similar cell kinetic factors which were almost the same as those in human body reported previously. The differences in volume doubling time of tumor, growth fraction and cell loss factor were partially related to those of radiosensitivities among tumors except for Lu-24. The theoretical volume doubling time was proved to be most reliable for estimation of effectiveness of irradiation, but the labeling index was not a valuable indicator for it. (author)

  10. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

    Science.gov (United States)

    Tichy, Elisia D; Pillai, Resmi; Deng, Li; Liang, Li; Tischfield, Jay; Schwemberger, Sandy J; Babcock, George F; Stambrook, Peter J

    2010-11-01

    Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

  11. Arginine metabolism by macrophages promotes cardiac and muscle fibrosis in mdx muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Michelle Wehling-Henricks

    2010-05-01

    Full Text Available Duchenne muscular dystrophy (DMD is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD.We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial

  12. Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

    Science.gov (United States)

    Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

    2015-11-01

    Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

  13. Induction of Functional 3D Ciliary Epithelium-Like Structure From Mouse Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kinoshita, Hirofumi; Suzuma, Kiyoshi; Kaneko, Jun; Mandai, Michiko; Kitaoka, Takashi; Takahashi, Masayo

    2016-01-01

    To generate ciliary epithelium (CE) from mouse induced pluripotent stem (iPS) cells. Recently, a protocol for self-organizing optic cup morphogenesis in three-dimensional culture was reported, and it was suggested that ocular tissue derived from neural ectoderm could be differentiated. We demonstrated that a CE-like double-layered structure could be induced in simple culture by using a modified Eiraku differentiation protocol. Differentiation of a CE-like double-layered structure could be promoted by glycogen synthase kinase 3β (GSK-3β) inhibitor. Connexin43 and aquaporin1 were expressed in both thin layers, and induced CE-like cells expressed ciliary marker genes, such as cyclinD2, zic1, tgfb2, aldh1a3, wfdc1, otx1, BMP4, and BMP7. Increases in cytoplasmic and nuclear β-catenin in aggregates of the CE-like double-layered structure were confirmed by Western blot analysis. In addition, tankyrase inhibitor prevented the induction of the CE-like double-layered structure by GSK-3β inhibitor. Dye movement from pigmented cells to nonpigmented cells in the mouse iPS cell-derived CE-like structure was observed in a fluid movement experiment, consistent with the physiological function of CE in vivo. We could differentiate CE from mouse iPS cells in the present study. In the future, we hope that this CE-like complex will become useful as a graft for transplantation therapy in pathologic ocular hypotension due to CE dysfunction, and as a screening tool for the development of drugs for diseases associated with CE function.

  14. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    International Nuclear Information System (INIS)

    Kawahara, Takeshi

    2012-01-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine–aminopterin–thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, β, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D 2 and leukotriene C 4 in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.

  15. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, Takeshi, E-mail: tkawafb@shinshu-u.ac.jp [Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Nagano (Japan)

    2012-11-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  16. Efficient derivation of functional hepatocytes from mouse induced pluripotent stem cells by a combination of cytokines and sodium butyrate

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; YANG Yang; ZHANG Jian; WANG Guo-ying; LIU Wei; QIU Dong-bo; HEI Zi-qing; YING Qi-long; CHEN Gui-hua

    2011-01-01

    Background Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency.Unfortunately,the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies.Therefore,it is urgent to find new ways to provide ample hepatocytes.Induced pluripotent stem (iPS) cells,a breakthrough in stem cell research,may terminate these hinders for cell transplantation.For the promise of iPS cells to be realized in liver diseases,it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.Methods In this study,we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches:conditions via embryonic body (EB) formation plus cytokines,conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined,serum free monolayer conditions.Among these three induction conditions,more homogenous populations can be promoted under chemically defined,serum free conditions.The cells generated under these conditions exhibited hepatic functions in vitro,including glycogen storage,indocynine green (ICG) uptake and release as well as urea secretion.Although efficient hepatocytes differentiation from mouse iPS cells were observed,mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.Results Mouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro,which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.Conclusion We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

  17. Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

    Science.gov (United States)

    Kim, Suhwan; Jung, Unsang; Baek, Juyoung; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-01-01

    Recently, mouse neuroblastoma cells have been considered as an attractive model for the study of human neurological and prion diseases, and they have been intensively used as a model system in different areas. For example, the differentiation of neuro2a (N2A) cells, receptor-mediated ion current, and glutamate-induced physiological responses have been actively investigated with these cells. These mouse neuroblastoma N2A cells are of interest because they grow faster than other cells of neural origin and have a number of other advantages. The calcium oscillations and neural spikes of mouse neuroblastoma N2A cells in epileptic conditions are evaluated. Based on our observations of neural spikes in these cells with our proposed imaging modality, we reported that they can be an important model in epileptic activity studies. We concluded that mouse neuroblastoma N2A cells produce epileptic spikes in vitro in the same way as those produced by neurons or astrocytes. This evidence suggests that increased levels of neurotransmitter release due to the enhancement of free calcium from 4-aminopyridine causes the mouse neuroblastoma N2A cells to produce epileptic spikes and calcium oscillations.

  18. Single cell analysis of caspase-3 in apoptotic and non-apoptotic cells during mouse limb development

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Klepárník, Karel; Matalová, E.

    2014-01-01

    Roč. 3, - (2014), PP58 ISSN 2052-1219. [European Calcified Tissue Society Congress /41./. 17.05.2014-20.05.2014, Praha] R&D Projects: GA ČR GAP206/11/2377; GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : single cell analysis * caspase-3 * mouse limb development Subject RIV: CB - Analytical Chemistry, Separation

  19. Extract of mouse embryonic stem cells induces the expression of pluripotency genes in human adipose tissue-derived stem cells.

    Science.gov (United States)

    Salehi, Paria Motamen; Foroutan, Tahereh; Javeri, Arash; Taha, Masoumeh Fakhr

    2017-11-01

    In some previous studies, the extract of embryonic carcinoma cells (ECCs) and embryonic stem cells (ESCs) have been used to reprogram somatic cells to more dedifferentiated state. The aim of this study was to investigate the effect of mouse ESCs extract on the expression of some pluripotency markers in human adipose tissue-derived stem cells (ADSCs). Human ADSCs were isolated from subcutaneous abdominal adipose tissue and characterized by flow cytometric analysis for the expression of some mesenchymal stem cell markers and adipogenic and osteogenic differentiation. Frequent freeze-thaw technique was used to prepare cytoplasmic extract of ESCs. Plasma membranes of the ADSCs were reversibly permeabilized by streptolysin-O (SLO). Then the permeabilized ADSCs were incubated with the ESC extract and cultured in resealing medium. After reprogramming, the expression of some pluripotency genes was evaluated by RT-PCR and quantitative real-time PCR (qPCR) analyses. Third-passaged ADSCs showed a fibroblast-like morphology and expressed mesenchymal stem cell markers. They also showed adipogenic and osteogenic differentiation potential. QPCR analysis revealed a significant upregulation in the expression of some pluripotency genes including OCT4 , SOX2 , NANOG , REX1 and ESG1 in the reprogrammed ADSCs compared to the control group. These findings showed that mouse ESC extract can be used to induce reprogramming of human ADSCs. In fact, this method is applicable for reprogramming of human adult stem cells to a more pluripotent sate and may have a potential in regenerative medicine.

  20. Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

    Science.gov (United States)

    Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

    2018-03-03

    Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for