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Sample records for human myogenic precursor

  1. Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo

    SciTech Connect

    Lafreniere, J.F.; Mills, P.; Bouchentouf, M.; Tremblay, J.P. . E-mail: Jacques-P.Tremblay@crchul.ulaval.ca

    2006-04-15

    Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation. Among the different mechanisms by which IL-4 might enhance myoblast migration processes, our results indicate that there are implications of some integrins and of three major components of the fibrinolytic system. Indeed, increases in the amount of active urokinase plasminogen activator and its receptor were observed following an IL-4 treatment, while the plasminogen activator inhibitor-1 decreased. Finally, IL-4 did not modify the amount of cell surface {alpha}5 integrin but increased the presence of {beta}3 and {beta}1 integrins. This integrin modulation might favor myogenic cell migration and its interaction with newly formed myotubes. Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.

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

    SciTech Connect

    Mills, Philippe; Lafreniere, Jean-Francois; Benabdallah, Basma Fattouma; El Fahime, El Mostafa; Tremblay, Jacques-P. . E-mail: Jacques-P.Tremblay@crchul.ulaval.ca

    2007-02-01

    Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferation while delaying the differentiation of C{sub 2}C{sub 12} cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.

  3. Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans.

    PubMed

    Soriano-Arroquia, Ana; Clegg, Peter D; Molloy, Andrew P; Goljanek-Whysall, Katarzyna

    2017-02-16

    Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs and their progeny - myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

  4. Myogenic potential of human alveolar mucosa derived cells.

    PubMed

    Zorin, Vadim L; Pulin, Andrey A; Eremin, Ilya I; Korsakov, Ivan N; Zorina, Alla I; Khromova, Natalia V; Sokova, Olga I; Kotenko, Konstantin V; Kopnin, Pavel B

    2017-03-19

    Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.

  5. The influence of anti-inflammatory medication on exercise-induced myogenic precursor cell responses in humans.

    PubMed

    Mackey, Abigail L; Kjaer, Michael; Dandanell, Sune; Mikkelsen, Kristian H; Holm, Lars; Døssing, Simon; Kadi, Fawzi; Koskinen, Satu O; Jensen, Charlotte H; Schrøder, Henrik D; Langberg, Henning

    2007-08-01

    The consumption of nonsteroidal anti-inflammatory drugs (NSAIDs) is widespread among athletes when faced with muscle soreness or injury, but the effects of NSAIDs on satellite cell activity in humans are unknown. To investigate this, 14 healthy male endurance athletes (mean peak oxygen consumption 62 ml x kg(-1) x min(-1)) volunteered for the study, which involved running 36 km. They were divided into two groups and received either 100 mg indomethacin per day or placebo. Muscle biopsies collected before the run and on days 1, 3, and 8 afterward were analyzed for satellite cells by immunohistochemistry with the aid of neural cell adhesion molecule (NCAM) and fetal antigen-1 (FA1) antibodies. Muscle biopsies were also collected from untrained individuals for comparison. Compared with preexercise levels, a 27% increase in the number of NCAM+ cells was observed on day 8 postexercise in the placebo group (P < 0.05), while levels remained similar at all time points in the NSAID group. No change was seen in the proportion of FA1+ cells, although lower levels were found in the muscle of endurance-trained athletes compared with untrained individuals (P < 0.05). These results suggest that ingestion of anti-inflammatory drugs attenuates the exercise-induced increase in satellite cell number, supporting the role of the cyclooxygenase pathway in satellite cell activity.

  6. Immortalization of human myogenic progenitor cell clone retaining multipotentiality

    SciTech Connect

    Hashimoto, Naohiro . E-mail: nao@nils.go.jp; Kiyono, Tohru; Wada, Michiko R.; Shimizu, Shirabe; Yasumoto, Shigeru; Inagawa, Masayo

    2006-10-06

    Human myogenic cells have limited ability to proliferate in culture. Although forced expression of telomerase can immortalize some cell types, telomerase alone delays senescence of human primary cultured myogenic cells, but fails to immortalize them. In contrast, constitutive expression of both telomerase and the E7 gene from human papillomavirus type 16 immortalizes primary human myogenic cells. We have established an immortalized primary human myogenic cell line preserving multipotentiality by ectopic expression of telomerase and E7. The immortalized human myogenic cells exhibit the phenotypic characteristics of their primary parent, including an ability to undergo myogenic, osteogenic, and adipogenic terminal differentiation under appropriate culture conditions. The immortalized cells will be useful for both basic and applied studies aimed at human muscle disorders. Furthermore, immortalization by transduction of telomerase and E7 represents a useful method by which to expand human myogenic cells in vitro without compromising their ability to differentiate.

  7. Smad1/5/8 are myogenic regulators of murine and human mesoangioblasts

    PubMed Central

    Costamagna, Domiziana; Quattrocelli, Mattia; van Tienen, Florence; Umans, Lieve; de Coo, Irineus F. M.; Zwijsen, An; Huylebroeck, Danny; Sampaolesi, Maurilio

    2016-01-01

    Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte marker genes and participate in skeletal muscle regeneration. Molecular circuits that regulate the myogenic commitment of MABs are still poorly characterized. The critical role of bone morphogenetic protein (BMP) signalling during proliferation and differentiation of adult myogenic precursors, such as satellite cells, has recently been established. We evaluated whether BMP signalling impacts on the myogenic potential of embryonic and adult MABs both in vitro and in vivo. Addition of BMP inhibited MAB myogenic differentiation, whereas interference with the interactions between BMPs and receptor complexes induced differentiation. Similarly, siRNA-mediated knockdown of Smad8 in Smad1/5-null MABs or inhibition of SMAD1/5/8 phosphorylation with Dorsomorphin (DM) also improved myogenic differentiation, demonstrating a novel role of SMAD8. Moreover, using a transgenic mouse model of Smad8 deletion, we demonstrated that the absence of SMAD8 protein improved MAB myogenic differentiation. Furthermore, once injected into α-Sarcoglycan (Sgca)-null muscles, DM-treated MABs were more efficacious to restore α-sarcoglycan (αSG) protein levels and re-establish functional muscle properties. Similarly, in acute muscle damage, DM-treated MABs displayed a better myogenic potential compared with BMP-treated and untreated cells. Finally, SMADs also control the myogenic commitment of human MABs (hMABs). BMP signalling antagonists are therefore novel candidates to improve the therapeutic effects of hMABs. PMID:26450990

  8. Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

    PubMed Central

    Froehlich, Jacob Michael; Seiliez, Iban; Gabillard, Jean-Charles; Biga, Peggy R.

    2014-01-01

    Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4. PMID:24835774

  9. Hierarchization of myogenic and adipogenic progenitors within human skeletal muscle.

    PubMed

    Pisani, Didier F; Clement, Noémie; Loubat, Agnès; Plaisant, Magali; Sacconi, Sabrina; Kurzenne, Jean-Yves; Desnuelle, Claude; Dani, Christian; Dechesne, Claude A

    2010-12-01

    Skeletal muscle cells constitute a heterogeneous population that maintains muscle integrity through a high myogenic regenerative capacity. More unexpectedly, this population is also endowed with an adipogenic potential, even in humans, and intramuscular adipocytes have been found to be present in several disorders. We tested the distribution of myogenic and adipogenic commitments in human muscle-derived cells to decipher the cellular basis of the myoadipogenic balance. Clonal analysis showed that adipogenic progenitors can be separated from myogenic progenitors and, interestingly, from myoadipogenic bipotent progenitors. These progenitors were isolated in the CD34(+) population on the basis of the expression of CD56 and CD15 cell surface markers. In vivo, these different cell types have been found in the interstitial compartment of human muscle. In vitro, we show that the proliferation of bipotent myoadipogenic CD56(+)CD15(+) progenitors gives rise to myogenic CD56(+)CD15(-) progenitors and adipogenic CD56(-)CD15(+) progenitors. A cellular hierarchy of muscle and fat progenitors thus occurs within human muscle. These results provide cellular bases for adipogenic differentiation in human skeletal muscle, which may explain the fat development encountered in different muscle pathological situations.

  10. Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

    PubMed Central

    Agley, Chibeza C.; Rowlerson, Anthea M.; Velloso, Cristiana P.; Lazarus, Norman L.; Harridge, Stephen D. R.

    2015-01-01

    The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56– and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 106 ± 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56– cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package. PMID:25650991

  11. Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis.

    PubMed

    Kallestad, Kristen M; Hebert, Sadie L; McDonald, Abby A; Daniel, Mark L; Cu, Sharon R; McLoon, Linda K

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin(-/-) (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation

  12. Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

    SciTech Connect

    Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.; Daniel, Mark L.; Cu, Sharon R.; McLoon, Linda K.

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a

  13. Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo.

    PubMed

    Ma, Xiaorong; Zhang, Shengli; Zhou, Junmei; Chen, Baisong; Shang, Yafeng; Gao, Tongbing; Wang, Xue; Xie, Hua; Chen, Fang

    2012-08-01

    Stem cell-based therapy may be the most promising method to cure skeletal muscle degenerative diseases such as Duchenne muscular dystrophy (DMD) and trauma in the future. Human amniotic fluid is enriched with early-stage stem cells from developing fetuses and these cells have cardiomyogenic potential both in vitro and in vivo. In the present study, we investigated the characteristics of human amniotic fluid-derived AF-type stem (HAF-AFS) cells by flow cytometry, immunofluorescence staining, reverse-transcription polymerase chain reaction, and osteogenic and adipogenic differentiation analysis. After confirming the stemness of HAF-AFS cells, we tested whether HAF-AFS cells could differentiate into skeletal myogenic cells in vitro and incorporate into regenerating skeletal muscle in vivo. By temporary exposure to the DNA demethylation agent 5-aza-2'-deoxycytidine (5-Aza dC) or co-cultured with C2C12 myoblasts, HAF-AFS cells differentiated into skeletal myogenic cells, expressing skeletal myogenic cell-specific markers such as Desmin, Troponin I (Tn I) and α-Actinin. Four weeks after transplantation into cardiotoxin-injured and X-ray-irradiated tibialis anterior (TA) muscles of NOD/SCID mice, HAF-AFS cells survived, differentiated into myogenic precursor cells and fused with host myofibres. The findings that HAF-AFS cells differentiate into myogenic cells in vitro and incorporate in skeletal muscle regeneration in vivo hold the promise of HAF-AFS cell-based therapy for skeletal muscle degenerative diseases.

  14. The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles.

    PubMed

    Hebert, Sadie L; Daniel, Mark L; McLoon, Linda K

    2013-01-01

    Many differences exist between extraocular muscles (EOM) and non-cranial skeletal muscles. One striking difference is the sparing of EOM in various muscular dystrophies compared to non-cranial skeletal muscles. EOM undergo continuous myonuclear remodeling in normal, uninjured adults, and distinct transcription factors are required for the early determination, development, and maintenance of EOM compared to limb skeletal muscle. Pitx2, a bicoid-like homeobox transcription factor, is required for the development of EOM and the maintenance of characteristic properties of the adult EOM phenotype, but is not required for the development of limb muscle. We hypothesize that these unique properties of EOM contribute to the constitutive differences between EOM and non-craniofacial skeletal muscles. Using flow cytometry, CD34(+)/Sca1(-/)CD45(-/)CD31(-) cells (EECD34 cells) were isolated from extraocular and limb skeletal muscle and in vitro, EOM EECD34 cells proliferated faster than limb muscle EECD34 cells. To further define these myogenic precursor cells from EOM and limb skeletal muscle, they were analyzed for their expression of Pitx2. Western blotting and immunohistochemical data demonstrated that EOM express higher levels of Pitx2 than limb muscle, and 80% of the EECD34 cells expressed Pitx2. siRNA knockdown of Pitx2 expression in EECD34 cells in vitro decreased proliferation rates and impaired the ability of EECD34 cells to fuse into multinucleated myotubes. High levels of Pitx2 were retained in dystrophic and aging mouse EOM and the EOM EECD34 cells compared to limb muscle. The differential expression of Pitx2 between EOM and limb skeletal muscle along with the functional changes in response to lower levels of Pitx2 expression in the myogenic precursor cells suggest a role for Pitx2 in the maintenance of constitutive differences between EOM and limb skeletal muscle that may contribute to the sparing of EOM in muscular dystrophies.

  15. In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study.

    PubMed

    Negroni, Elisa; Riederer, Ingo; Chaouch, Soraya; Belicchi, Marzia; Razini, Paola; Di Santo, James; Torrente, Yvan; Butler-Browne, Gillian S; Mouly, Vincent

    2009-10-01

    In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle-derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2(-/-) gammaC(-/-) C5(-/-) mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle-derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.

  16. In Vivo Myogenic Potential of Human CD133+ Muscle-derived Stem Cells: A Quantitative Study

    PubMed Central

    Negroni, Elisa; Riederer, Ingo; Chaouch, Soraya; Belicchi, Marzia; Razini, Paola; Di Santo, James; Torrente, Yvan; Butler-Browne, Gillian S; Mouly, Vincent

    2009-01-01

    In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2−/− γC−/− C5−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy. PMID:19623164

  17. Sphingosine-1-Phosphate Signaling Regulates Myogenic Responsiveness in Human Resistance Arteries

    PubMed Central

    Slack, Daniel L.; Burnstein, Marcus J.; Errett, Lee; Bonneau, Daniel; Latter, David; Rotstein, Ori D.; Bolz, Steffen-Sebastian; Lidington, Darcy; Voigtlaender-Bolz, Julia

    2015-01-01

    We recently identified sphingosine-1-phosphate (S1P) signaling and the cystic fibrosis transmembrane conductance regulator (CFTR) as prominent regulators of myogenic responsiveness in rodent resistance arteries. However, since rodent models frequently exhibit limitations with respect to human applicability, translation is necessary to validate the relevance of this signaling network for clinical application. We therefore investigated the significance of these regulatory elements in human mesenteric and skeletal muscle resistance arteries. Mesenteric and skeletal muscle resistance arteries were isolated from patient tissue specimens collected during colonic or cardiac bypass surgery. Pressure myography assessments confirmed endothelial integrity, as well as stable phenylephrine and myogenic responses. Both human mesenteric and skeletal muscle resistance arteries (i) express critical S1P signaling elements, (ii) constrict in response to S1P and (iii) lose myogenic responsiveness following S1P receptor antagonism (JTE013). However, while human mesenteric arteries express CFTR, human skeletal muscle resistance arteries do not express detectable levels of CFTR protein. Consequently, modulating CFTR activity enhances myogenic responsiveness only in human mesenteric resistance arteries. We conclude that human mesenteric and skeletal muscle resistance arteries are a reliable and consistent model for translational studies. We demonstrate that the core elements of an S1P-dependent signaling network translate to human mesenteric resistance arteries. Clear species and vascular bed variations are evident, reinforcing the critical need for further translational study. PMID:26367262

  18. Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation.

    PubMed

    Agley, Chibeza C; Rowlerson, Anthea M; Velloso, Cristiana P; Lazarus, Norman R; Harridge, Stephen D R

    2013-12-15

    We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells. The CD56(-) fraction obtained from the first sort was highly enriched in TE-7(+) fibroblasts. Using quantitative analysis of immunofluorescent staining for lipid content, lineage markers and transcription factors, we tested if the purified cell populations could differentiate into adipocytes in response to treatment with either fatty acids or adipocyte-inducing medium. Both treatments caused the fibroblasts to differentiate into adipocytes, as shown by loss of intracellular TE-7, upregulation of the adipogenic transcription factors PPARγ and C/EBPα, and adoption of a lipid-laden adipocyte morphology. By contrast, myogenic cells did not undergo adipogenesis and showed differential regulation of PPARγ and C/EBPα in response to these adipogenic treatments. Our results show that human skeletal muscle fibroblasts are at least bipotent progenitors that can remain as extracellular-matrix-producing cells or differentiate into adipocytes.

  19. Autophagy induction in the skeletal myogenic differentiation of human tonsil-derived mesenchymal stem cells

    PubMed Central

    Park, Saeyoung; Choi, Yoonyoung; Jung, Namhee; Kim, Jieun; Oh, Seiyoon; Yu, Yeonsil; Ahn, Jung-Hyuck; Jo, Inho; Choi, Byung-Ok; Jung, Sung-Chul

    2017-01-01

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs. PMID:28259927

  20. Autophagy induction in the skeletal myogenic differentiation of human tonsil-derived mesenchymal stem cells.

    PubMed

    Park, Saeyoung; Choi, Yoonyoung; Jung, Namhee; Kim, Jieun; Oh, Seiyoon; Yu, Yeonsil; Ahn, Jung-Hyuck; Jo, Inho; Choi, Byung-Ok; Jung, Sung-Chul

    2017-04-01

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.

  1. Induction of myogenic differentiation in a human rhabdomyosarcoma cell line by phenylacetate.

    PubMed

    Cinatl, J; Cinatl, J; Herneiz, P; Rabenau, H; Hovak, M; Benda, R; Gümbel, H O; Kornhuber, B; Doerr, H W

    1994-04-01

    Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 10 mM promoted myogenic differentiation of the human alveolar rhabdomyosarcoma cell line KFR. These concentrations inhibited DNA synthesis of the cells in a dose-dependent manner without significant effect on cell viability. The morphological differentiation of small mononuclear elements to terminal, elongated multinuclear structures resembling myotubes was accompanied by the expression of skeletal muscle myosin. The proportion of differentiated myosin-positive cells which was around 0.8-1.7% in control cultures 12 days after seeding was increased by NaPA treatment up to 47%. In the cytoplasm of differentiated cells, features of sarcomerogenesis were observed. These results suggest that NaPA is an effective inducer of rhabdomyosarcoma cell differentiation at concentrations that have been achieved in humans with no significant adverse effects.

  2. Myogenic response of human skeletal muscle to 12 weeks of resistance training at light loading intensity.

    PubMed

    Mackey, A L; Holm, L; Reitelseder, S; Pedersen, T G; Doessing, S; Kadi, F; Kjaer, M

    2011-12-01

    There is strong evidence for enhanced numbers of satellite cells with heavy resistance training. The satellite cell response to very light muscle loading is, however, unknown. We, therefore, designed a 12-week training protocol where volunteers trained one leg with a high load (H) and the other leg with a light load (L). Twelve young healthy men [mean age 25 ± 3 standard deviation (SD) years] volunteered for the study. Muscle biopsies were collected from the m. vastus lateralis of both legs before and after the training period and satellite cells were visualized by CD56 immunohistochemistry. A significant main effect of time was observed (P<0.001) for the number of CD56+ cells per fiber (L: from 0.11 ± 0.02 to 0.13 ± 0.03; H: from 0.12 ± 0.03 to 0.15 ± 0.05, mean ± SD). The finding that 12 weeks of training skeletal muscle even with very light loads can induce an increase in the number of satellite cells reveals a new aspect of myogenic precursor cell activation and suggests that satellite cells may play a role in skeletal muscle adaptation over a broad physiological range.

  3. Dose-dependent Effect of Boric Acid on Myogenic Differentiation of Human Adipose-derived Stem Cells (hADSCs).

    PubMed

    Apdik, Hüseyin; Doğan, Ayşegül; Demirci, Selami; Aydın, Safa; Şahin, Fikrettin

    2015-06-01

    Boron, a vital micronutrient for plant metabolism, is not fully elucidated for embryonic and adult body development, and tissue regeneration. Although optimized amount of boron supplement has been shown to be essential for normal gestational development in zebrafish and frog and beneficial for bone regeneration in higher animals, effects of boron on myogenesis and myo-regeneration remains to be solved. In the current study, we investigated dose-dependent activity of boric acid on myogenic differentiation of human adipose-derived stem cells (hADSCs) using immunocytochemical, gene, and protein expression analysis. The results revealed that while low- (81.9 μM) and high-dose (819.6 μM) boron treatment increased myogenic gene expression levels such as myosin heavy chain (MYH), MyoD, myogenin, and desmin at day 4 of differentiation, high-dose treatment decreased myogenic-related gene and protein levels at day 21 of differentiation, confirmed by immunocytochemical analysis. The findings of the study present not only an understanding of boron's effect on myogenic differentiation but also an opportunity for the development of scaffolds to be used in skeletal tissue engineering and supplements for embryonic muscle growth. However, fine dose tuning and treatment period arranging are highly warranted as boron treatment over required concentrations and time might result in detrimental outcomes to myogenesis and myo-regeneration.

  4. Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

    SciTech Connect

    Sung, Min Sun; Mun, Ji-Young; Kwon, Ohsuk; Kwon, Ki-Sun; Oh, Doo-Byoung

    2013-07-19

    Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

  5. Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

    PubMed

    Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

    2017-02-01

    Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

  6. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    PubMed Central

    Kim, Cy Hyun; Shin, Jin-Hong; Hwang, Sung Jun; Choi, Yung Hyun; Kim, Dae-Seong; Kim, Cheol Min

    2016-01-01

    Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy. PMID:27330287

  7. Human Myocardial Pericytes: Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity

    PubMed Central

    Chen, William C.W.; Baily, James E.; Corselli, Mirko; Diaz, Mary; Sun, Bin; Xiang, Guosheng; Gray, Gillian A.; Huard, Johnny; Péault, Bruno

    2015-01-01

    Perivascular mesenchymal precursor cells (i.e. pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, PDGFRβ, PDGFRα, αSMA, and SM-MHC, but not CD117, CD133 and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express MSC markers in situ and exhibited osteo- chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte co-culture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells. PMID:25336400

  8. Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation

    PubMed Central

    Schilbach, Karin; Alkhaled, Mohammed; Welker, Christian; Eckert, Franziska; Blank, Gregor; Ziegler, Hendrik; Sterk, Marco; Müller, Friederike; Sonntag, Katja; Wieder, Thomas; Braumüller, Heidi; Schmitt, Julia; Eyrich, Matthias; Schleicher, Sabine; Seitz, Christian; Erbacher, Annika; Pichler, Bernd J; Müller, Hartmut; Tighe, Robert; Lim, Annick; Gillies, Stephen D; Strittmatter, Wolfgang; Röcken, Martin; Handgretinger, Rupert

    2015-01-01

    Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo. PMID:26140238

  9. From supine to standing: in vivo segregation of myogenic and baroreceptor vasoconstriction in humans.

    PubMed

    Estañol, Bruno; Rivera, Ana Leonor; Martínez Memije, Raúl; Fossion, Ruben; Gómez, Fermín; Bernal, Katherine; Murúa Beltrán, Sofía; Delgado-García, Guillermo; Frank, Alejandro

    2016-12-01

    Myogenic vascular response is a form of systemic and regional vasoconstriction produced increasing the intra-arterial pressure by gravity. Here, the vasoconstriction due to the myogenic response, induced by the gravitational action in a dependent limb, is separated from that caused by the baroreceptor reflex. Regional changes of skin blood flow (SBF), total blood volume of the finger (TBVF), pulse pressure (PP), heart rate (HR), systolic, and diastolic blood pressure (BP) were analyzed in 10 healthy young subjects in supine and upright positions. By lowering the arm in supine position, SBF decreased compared to its basal measurement, PR increased, and PP contracted, indicating arterial vasoconstriction that rise BP TBVF increased, demonstrating an increment in venous volume. HR did not change, reflecting no action of the baroreceptor reflex. In upright position with lowered arm, there was an additional increase in BP variables, demonstrating vasoconstriction. Moreover, BP and HR showed oscillations at 0.1 Hz reflecting the entrance of the baroreceptor reflex. The action of gravity in a dependent limb in supine position induces a regional vasoconstriction and an increase of BP due to activation of the myogenic response, while the baroreceptor reflex or other neural factors do not appear to operate. In the upright position with the arm dependent, there is a further increase in regional vasoconstriction and BP with reciprocal changes in HR, indicating the entrance of the baroreceptor superimposed to the myogenic response. This study demonstrates that the myogenic and baroreceptor vasoconstriction can be separated in vivo.

  10. Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation.

    PubMed

    Seiler, Christof; Gazdhar, Amiq; Reyes, Mauricio; Benneker, Lorin M; Geiser, Thomas; Siebenrock, Klaus A; Gantenbein-Ritter, Benjamin

    2014-09-01

    Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of

  11. Adrenergic and myogenic regulation of viscoelasticity in the vascular bed of the human forearm.

    PubMed

    Frances, M F; Goswami, R; Rachinsky, M; Craen, R; Kiviniemi, A M; Fleischhauer, A; Steinback, C D; Zamir, M; Shoemaker, J K

    2011-11-01

    This study tested the hypothesis that the compliance (C) and viscoelasticity (K) of the forearm vascular bed are controlled by myogenic and/or α-adrenergic receptor (αAR) activation. Heart rate (HR) and waveforms of brachial artery blood pressure (Finometer) and forearm blood flow (Doppler ultrasound) were measured in baseline conditions and during infusion of noradrenaline (NA; αAR agonist), with and without phentolamine (αAR antagonist; n = 10; 6 men and 4 women). These baseline and αAR-agonist-based measures were repeated when the arm was positioned above or below the heart to modify the myogenic stimulus. A lumped Windkessel model was used to quantify the values of forearm C and K in each set of conditions. Baseline forearm C was inversely, and K directly, related to the myogenic load (P < 0.001). Compared with saline infusion, C was increased, but K was unaffected, with phentolanine, but only in the 'above' position. Compliance was reduced (P < 0.001) and K increased (P = 0.06) with NA infusion (main effects of NA) across arm positions; phentolamine minimized these NA-induced changes in C and K for both arm positions. Examination of conditions with and without NA infusion at similar forearm intravascular pressures indicated that the NA-induced changes in C and K were due largely to the concurrent changes in blood pressure. Therefore, within the range of arm positions used, it was concluded that vascular stiffness and vessel wall viscoelastic properties are acutely affected by myogenic stimuli. Additionally, forearm vascular compliance is sensitive to baseline levels of αAR activation when transmural pressure is low.

  12. Spontaneous and specific myogenic differentiation of human mesenchymal stem cells on polyethylene glycol-linked multi-walled carbon nanotube films for skeletal muscle engineering.

    PubMed

    Zhao, Chunyan; Andersen, Henrik; Ozyilmaz, Barbaros; Ramaprabhu, Sundara; Pastorin, Giorgia; Ho, Han Kiat

    2015-11-21

    This study explored the influence of polyethylene glycol-linked multi-walled carbon nanotube (PEG-CNT) films on skeletal myogenic differentiation of human mesenchymal stem cells (hMSCs). PEG-CNT films were prepared with nanoscale surface roughness, orderly arrangement of PEG-CNTs, high hydrophilicity and high mechanical strength. Notably, PEG-CNT films alone could direct the skeletal myogenic differentiation of hMSCs in the absence of myogenic induction factors. The quantitative real-time polymerase chain reaction (RT-PCR) showed that the non-induced hMSCs plated on the PEG-CNT films, compared to the negative control, presented significant up-regulation of general myogenic markers including early commitment markers of myoblast differentiation protein-1 (MyoD) and desmin, as well as a late phase marker of myosin heavy chain-2 (MHC). Corresponding protein analysis by immunoblot assays corroborated these results. Skeletal muscle-specific markers, fast skeletal troponin-C (TnC) and ryanodine receptor-1 (Ryr) were also significantly increased in the non-induced hMSCs on PEG-CNT films by RT-PCR. For these cells, the commitment to specific skeletal myoblasts was further proved by the absence of enhanced adipogenic, chondrogenic and osteogenic markers. This study elucidated that PEG-CNT films supported a dedicated differentiation of hMSCs into a skeletal myogenic lineage and can work as a promising material towards skeletal muscle injury repair.

  13. Efficient delivery of human single fiber-derived muscle precursor cells via biocompatible scaffold.

    PubMed

    Boldrin, Luisa; Malerba, Alberto; Vitiello, Libero; Cimetta, Elisa; Piccoli, Martina; Messina, Chiara; Gamba, Pier Giorgio; Elvassore, Nicola; De Coppi, Paolo

    2008-01-01

    The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.

  14. Spontaneous and specific myogenic differentiation of human mesenchymal stem cells on polyethylene glycol-linked multi-walled carbon nanotube films for skeletal muscle engineering

    NASA Astrophysics Data System (ADS)

    Zhao, Chunyan; Andersen, Henrik; Ozyilmaz, Barbaros; Ramaprabhu, Sundara; Pastorin, Giorgia; Ho, Han Kiat

    2015-10-01

    This study explored the influence of polyethylene glycol-linked multi-walled carbon nanotube (PEG-CNT) films on skeletal myogenic differentiation of human mesenchymal stem cells (hMSCs). PEG-CNT films were prepared with nanoscale surface roughness, orderly arrangement of PEG-CNTs, high hydrophilicity and high mechanical strength. Notably, PEG-CNT films alone could direct the skeletal myogenic differentiation of hMSCs in the absence of myogenic induction factors. The quantitative real-time polymerase chain reaction (RT-PCR) showed that the non-induced hMSCs plated on the PEG-CNT films, compared to the negative control, presented significant up-regulation of general myogenic markers including early commitment markers of myoblast differentiation protein-1 (MyoD) and desmin, as well as a late phase marker of myosin heavy chain-2 (MHC). Corresponding protein analysis by immunoblot assays corroborated these results. Skeletal muscle-specific markers, fast skeletal troponin-C (TnC) and ryanodine receptor-1 (Ryr) were also significantly increased in the non-induced hMSCs on PEG-CNT films by RT-PCR. For these cells, the commitment to specific skeletal myoblasts was further proved by the absence of enhanced adipogenic, chondrogenic and osteogenic markers. This study elucidated that PEG-CNT films supported a dedicated differentiation of hMSCs into a skeletal myogenic lineage and can work as a promising material towards skeletal muscle injury repair.This study explored the influence of polyethylene glycol-linked multi-walled carbon nanotube (PEG-CNT) films on skeletal myogenic differentiation of human mesenchymal stem cells (hMSCs). PEG-CNT films were prepared with nanoscale surface roughness, orderly arrangement of PEG-CNTs, high hydrophilicity and high mechanical strength. Notably, PEG-CNT films alone could direct the skeletal myogenic differentiation of hMSCs in the absence of myogenic induction factors. The quantitative real-time polymerase chain reaction (RT-PCR) showed

  15. High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

    PubMed Central

    Lattanzi, L; Salvatori, G; Coletta, M; Sonnino, C; Cusella De Angelis, M G; Gioglio, L; Murry, C E; Kelly, R; Ferrari, G; Molinaro, M; Crescenzi, M; Mavilio, F; Cossu, G

    1998-01-01

    Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo. PMID:9593768

  16. Low-dose benzo(a)pyrene and its epoxide metabolite inhibit myogenic differentiation in human skeletal muscle-derived progenitor cells.

    PubMed

    Chiu, Chen-Yuan; Yen, Yuan-Peng; Tsai, Keh-Sung; Yang, Rong-Sen; Liu, Shing-Hwa

    2014-04-01

    The risk of low birth weights is elevated in prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants generated from combustion of organic compounds, including cigarette smoke. We hypothesized that benzo(a)pyrene (BaP), a member of PAHs existing in cigarette smoke, may affect the myogenesis to cause low birth weights. We investigated the effects of BaP and its main metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), on the myogenic differentiation of human skeletal muscle-derived progenitor cells (HSMPCs). HSMPCs were isolated by a modified preplate technique and cultured in myogenic differentiation media with or without BaP and BPDE (0.25 and 0.5 μM) for 4 days. The multinucleated myotube formation was morphologically analyzed by hematoxylin and eosin staining. The expressions of myogenic differentiation markers and related signaling proteins were determined by Western blotting. Both BaP and BPDE at the submicromolar concentrations (0.25 and 0.5 μM) dose-dependently repressed HSMPCs myogenic differentiation without obvious cell toxicity. Both BaP and BPDE inhibited the muscle-specific protein expressions (myogenin and myosin heavy chain) and phosphorylation of Akt (a known modulator in myogenesis), which could be significantly reversed by the inhibitors for aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and nuclear factor (NF)-κB. BaP- and BPDE-activated NF-κB-p65 protein phosphorylation could also be attenuated by both AhR and ER inhibitors. The inhibitory effects of BaP and BPDE on myogenesis were reversed after withdrawing BaP exposure, but not after BPDE withdrawal. These results suggest that both BaP and BPDE are capable of inhibiting myogenesis via an AhR- or/and ER-regulated NF-κB/Akt signaling pathway.

  17. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

    PubMed Central

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall; Jørgensen, Louise Helskov; Dhawan, Jyotsna; Nielsen, Michael Friberg Bruun; Schrøder, Henrik Daa

    2016-01-01

    Objectives Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. Materials and Methods Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. Results and Conclusions We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts. PMID:27442119

  18. Human embryonic epidermis contains a diverse Langerhans cell precursor pool.

    PubMed

    Schuster, Christopher; Mildner, Michael; Mairhofer, Mario; Bauer, Wolfgang; Fiala, Christian; Prior, Marion; Eppel, Wolfgang; Kolbus, Andrea; Tschachler, Erwin; Stingl, Georg; Elbe-Bürger, Adelheid

    2014-02-01

    Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207(-) cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14(+)CD16(-) blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14(-) haematopoietic precursors possess a much higher differentiation potential than CD14(+) precursor cells.

  19. Effect of Dexamethasone, Insulin and EGF on the Myogenic Potential on Human Endometrial Stem Cell

    PubMed Central

    Jalali Tehrani, Hora; Parivar, Kazem; Ai, Jafar; Kajbafzadeh, Abdolmohammad; Rahbarghazi, Reza; Hashemi, Mehrdad; Sadeghizadeh, Majid

    2014-01-01

    Human endometrium contains mesenchymal stem cells (eMSC) which have the ability to differentiate into three cell lineages and the potential in therapeutic applications. We hypothesize that using environmental induction in culture media such as dexamethasone, human recombinant insulin and human epidermal growth factor (hEGF) can differentiate endometrial stem cells into myoblast. These agents have a broad range of effects in myoblast differentiation in-vitro. We used immunohystochemistry analysis and RT –PCR to evaluate the presence of skeletal muscle - specific proteins some of which are expressed in the early stage of differentiation including myoD and Desmin which expressed at later stages of differentiation. In conclusion eMSC can differentiate in culture media which contains above mentioned factors and use for therapeutic purpose in muscular degenerative disease. PMID:25237362

  20. Human muscle precursor cells overexpressing PGC-1α enhance early skeletal muscle tissue formation.

    PubMed

    Haralampieva, Deana; Salemi, Souzan; Dinulovic, Ivana; Sulser, Tullio; M Ametamey, Simon; Handschin, Christoph; Eberli, Daniel

    2017-02-03

    Muscle precursor cells (MPCs) are activated satellite cells capable of muscle fiber reconstruction. Therefore, autologous MPC transplantation is envisioned for the treatment of muscle diseases. However, the density of MPCs, as well as their proliferation and differentiation potential gradually decline with age. The goal of this research was to genetically modify human MPCs (hMPCs) to overexpress the peroxisome proliferator-activated receptor gamma coactivator (PGC-1α), a key regulator of exercise-mediated adaptation, and thereby to enhance early skeletal muscle formation and quality. We were able to confirm the sustained myogenic phenotype of the genetically modified hMPCs. While maintaining their viability and proliferation potential, PGC-1α modified hMPCs showed an enhanced myofiber formation capacity in vitro. Engineered muscle tissues were harvested 1, 2 and 4 weeks after subcutaneous injection of cell-collagen suspensions and histological analysis confirmed the earlier myotube formation in PGC-1α modified samples, predominantly of slow twitch myofibers. Increased contractile protein levels were detected by Western Blot. In summary, by genetically modifying hMPCs to overexpress PGC-1α we were able to promote early muscle fiber formation in vitro and in vivo, with an initial switch to slow type myofibers. Therefore, overexpressing PGC-1α is novel strategy to further enhance skeletal muscle tissue engineering.

  1. MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

    PubMed Central

    Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

    2015-01-01

    The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

  2. Rb suppresses human cone-precursor-derived retinoblastoma tumours.

    PubMed

    Xu, Xiaoliang L; Singh, Hardeep P; Wang, Lu; Qi, Dong-Lai; Poulos, Bradford K; Abramson, David H; Jhanwar, Suresh C; Cobrinik, David

    2014-10-16

    Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

  3. Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

    PubMed

    Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

    2004-03-01

    Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

  4. Proliferation of myogenic stem cells in human skeletal muscle in response to low-load resistance training with blood flow restriction.

    PubMed

    Nielsen, Jakob Lindberg; Aagaard, Per; Bech, Rune Dueholm; Nygaard, Tobias; Hvid, Lars Grøndahl; Wernbom, Mathias; Suetta, Charlotte; Frandsen, Ulrik

    2012-09-01

    Low-load resistance training with blood flow restriction has been shown to elicit substantial increases in muscle mass and muscle strength; however, the effect on myogenic stem cells (MSCs) and myonuclei number remains unexplored. Ten male subjects (22.8 ± 2.3 years)performed four sets of knee extensor exercise (20% 1RM) to concentric failure during bloodflow restriction (BFR) of the proximal thigh (100 mmHg), while eight work-matched controls(21.9 ± 3.0 years) trained without BFR (control, CON). Twenty-three training sessions were performed within 19 days. Maximal isometric knee extensor strength (MVC) was examined pre- and post-training, while muscle biopsies were obtained at baseline (Pre), after 8 days intervention(Mid8) and 3 (Post3) and 10 days (Post10) post training to examine changes in myofibre area (MFA), MSC and myonuclei number. MVC increased by 7.1% (Post5) and 10.6% (Post12)(P <0.001) with BFR training, while type I and II MFA increased by 38% (Mid8), 35 – 37%(Post3) and 31 – 32% (Post10) (P <0.001). MSCs per myofibre increased with BFR training from 0.10 ± 0.01 (Pre) to 0.38 ± 0.02 (Mid8), 0.36 ± 0.04 (Post3) and 0.25 ± 0.02 (Post10) (P <0.001). Likewise, myonuclei per myofibre increased from 2.49 ± 0.07 (Pre) to 3.30 ± 0.22(Mid8), 3.20 ± 0.16 (Post3) and 3.11 ± 0.11 (Post10), (P<0.01). Although MFA increased in CON at Mid8, it returned to baseline at Post3. No changes in MSC or myonuclei number were observed in CON. This study is the first to show that short-term low-load resistance exercise performed with partial blood flow restriction leads to marked proliferation of myogenic stem cells and resulting myonuclei addition in human skeletal muscle, which is accompanied by substantial myofibre hypertrophy.

  5. The synergistic effect of surface topography and sustained release of TGF-β1 on myogenic differentiation of human mesenchymal stem cells.

    PubMed

    Moghadasi Boroujeni, Samaneh; Mashayekhan, Shohreh; Vakilian, Saeid; Ardeshirylajimi, Abdolreza; Soleimani, Masoud

    2016-07-01

    A combination of topographical cues and controlled release of biochemical factors is a potential platform in controlling stem cells differentiation. In this study the synergistic effect of nanotopography and sustained release of biofunctional transforming growth factor beta 1 (TGF-β1) on differentiation of human Wharton's Jelly-derived mesenchymal stem cell (hWJ-derived UC-MSCs) toward myogenic lineage was investigated. In order to achieve a sustained release of TGF-β1, this factor was encapsulated within chitosan nanoparticles. Afterwards the aligned composite mats were fabricated using poly-ɛ-caprolacton (PCL) containing TGF-β1-loaded chitosan nanoparticles and poly-L-lactic acid (PLLA). The nanofiber topography notably up-regulated the expressions of calponin1 and SM22α compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and sustained TGF-β1release resulted in more significant enhancement of SMC marker, in particular smooth muscle α-actin (ASMA) expression, compared with bolus delivery despite lower amounts of TGF-β1 (>10 times lower). Additionally, immunofluorescence staining showed that ASMA and desmin were expressed at higher intensity in cells exposed to controlled TGF-β1 delivery rather than bolus delivery. These results demonstrated the importance of combined effect of topography and drug delivery in directing stem cell fate and the potential of such biofunctional scaffolds for cell transplantation applications in bladder tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1610-1621, 2016.

  6. SESAME Opens: A Precursor to Human Asteroid Missions

    NASA Astrophysics Data System (ADS)

    Maiwald, Volker; Marchand, Emilien

    2013-09-01

    A common path for human spaceflight is currently often envisioned to lead to Near Earth Asteroids (NEA) within the next decades. While the goal is clear this is not so true for the targets. Just as unmanned probes investigated the lunar surface before humans ever set foot on our natural satellite, it is advisable - considering the current lack of knowledge about NEAs even mandatory - to send an unmanned mission ahead to conduct measurements in preparation of a human mission. Just as with the moon more than one target area should be investigated, i.e. more than one target asteroid. While many designs for the actual human mission already exist, scenarios for a precursor mission are scarcer. In this paper we present a feasible design for a multi-rendezvous mission to targets suitable for human missions, able to reach up to 5 asteroids with one launch. We will propose a system that will be able to measure various properties of each asteroid, e.g. chemical composition and topography and describe it on a subsystem level, providing mass and power budgets for the whole system. The results show that a spacecraft of about 1,600 kg launch mass and utilizing solar electric propulsion can fly a 5 target mission within 10 years. With a sensitivity analysis we will show the robustness of the design and generally establish the feasibility of such a mission.

  7. Defining the cellular precursors to human breast cancer

    PubMed Central

    Keller, Patricia J.; Arendt, Lisa M.; Skibinski, Adam; Logvinenko, Tanya; Klebba, Ina; Dong, Shumin; Smith, Avi E.; Prat, Aleix; Perou, Charles M.; Gilmore, Hannah; Schnitt, Stuart; Naber, Stephen P.; Garlick, Jonathan A.; Kuperwasser, Charlotte

    2012-01-01

    Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues. PMID:21940501

  8. Opposing effects of shear-mediated dilation and myogenic constriction on artery diameter in response to handgrip exercise in humans.

    PubMed

    Atkinson, Ceri L; Carter, Howard H; Naylor, Louise H; Dawson, Ellen A; Marusic, Petra; Hering, Dagmara; Schlaich, Markus P; Thijssen, Dick H J; Green, Daniel J

    2015-10-15

    While the impact of changes in blood flow and shear stress on artery function are well documented, the acute effects of increases in arterial pressure are less well described in humans. The aim of this study was to assess the effect of 30 min of elevated blood pressure, in the absence of changes in shear stress or sympathetic nervous system (SNS) activation, on conduit artery diameter. Ten healthy male subjects undertook three sessions of 30 min unilateral handgrip exercise at 5, 10, and 15% of maximal voluntary contractile (MVC) strength. Brachial artery shear rate and blood flow profiles were measured simultaneously during exercise in the active and contralateral resting arms. Bilateral brachial artery diameter was simultaneously assessed before and immediately postexercise. In a second experiment, six subjects repeated the 15% MVC condition while continuous vascular measurements were collected during muscle sympathetic nerve activity (MSNA) assessment using peroneal microneurography. We found that unilateral handgrip exercise at 5, 10, and 15% MVC strength induced stepwise elevations in blood pressure (P < 0.01, Δmean arterial pressure: 7.06 ± 2.44, 8.50 ± 2.80, and 18.35 ± 3.52 mmHg, P < 0.01). Whereas stepwise increases were evident in shear rate in the exercising arm (P < 0.001), no changes were apparent in the nonexercising limb (P = 0.42). Brachial artery diameter increased in the exercising arm (P = 0.02), but significantly decreased in the nonexercising arm (P = 0.03). At 15% MVC, changes in diameter were significantly different between arms (interaction effect: P = 0.01), whereas this level of exertion produced no significant changes in MSNA. We conclude that acute increases in transmural pressure, independent of shear rate and changes in SNS activation, reduce arterial caliber in normotensive humans in vivo. These changes in diameter were mitigated by exercise-induced elevations in shear rate in the active limb.

  9. MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation

    PubMed Central

    Alexander, M S; Kawahara, G; Motohashi, N; Casar, J C; Eisenberg, I; Myers, J A; Gasperini, M J; Estrella, E A; Kho, A T; Mitsuhashi, S; Shapiro, F; Kang, P B; Kunkel, L M

    2013-01-01

    In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx5cv mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation. PMID:23764775

  10. MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation.

    PubMed

    Alexander, M S; Kawahara, G; Motohashi, N; Casar, J C; Eisenberg, I; Myers, J A; Gasperini, M J; Estrella, E A; Kho, A T; Mitsuhashi, S; Shapiro, F; Kang, P B; Kunkel, L M

    2013-09-01

    In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx(5cv) mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.

  11. Modeling Stem Cell Myogenic Differentiation

    PubMed Central

    Deshpande, Rajiv S.; Spector, Alexander A.

    2017-01-01

    The process of stem cell myogenesis (transformation into skeletal muscle cells) includes several stages characterized by the expression of certain combinations of myogenic factors. The first part of this process is accompanied by cell division, while the second part is mainly associated with direct differentiation. The mechanical cues are known to enhance stem cell myogenesis, and the paper focuses on the stem cell differentiation under the condition of externally applied strain. The process of stem cell myogenic differentiation is interpreted as the interplay among transcription factors, targeted proteins and strain-generated signaling molecule, and it is described by a kinetic multi-stage model. The model parameters are optimally adjusted by using the available data from the experiment with adipose-derived stem cells subjected to the application of cyclic uniaxial strains of the magnitude of 10%. The modeling results predict the kinetics of the process of myogenic differentiation, including the number of cells in each stage of differentiation and the rates of differentiation from one stage to another for different strains from 4% to 16%. The developed model can help better understand the process of myogenic differentiation and the effects of mechanical cues on stem cell use in muscle therapies. PMID:28106095

  12. Affordable Precursor Missions to Search for Life and Pave the Way for Human Exploration of Mars

    NASA Astrophysics Data System (ADS)

    Stoker, C. R.

    2017-02-01

    The goal of landing humans on Mars in the 2030s requires a precursor program to assess modern life on Mars and assess the water resource of near surface ground ice. Missions that address these issues are presented.

  13. Inhibitory action of amyloid precursor protein against human Hageman factor (factor XII).

    PubMed

    Niwano, H; Embury, P B; Greenberg, B D; Ratnoff, O D

    1995-02-01

    Amyloid precursor protein forms that contain Kunitz protease inhibitor domains are released from activated platelets, T-lymphocytes, and leukocytes and inhibit trypsin, plasmin, and activated factor XI. We investigated the effects of amyloid precursor protein isoforms on activated Hageman factor (factor XII), activated factor X (Stuart factor), and thrombin. Recombinant amyloid precursor proteins with or without the Kunitz domain, 770 and 695 amino acids, respectively, were produced in insect cells by Baculovirus expression (BAC770 and BAC695). Neither BAC695 nor BAC770 inhibited human alpha-thrombin or activated factor X. The partial thromboplastin time was prolonged by both amyloid precursor proteins, only one of which, BAC770, contains the Kunitz protease inhibitor domain. Both forms of amyloid precursor proteins inhibited ellagic acid-induced activation of Hageman factor but did not inhibit activated Hageman factor. Bismuth subgallate, which is an insoluble analog of ellagic acid, lost its ability to activate Hageman factor on being exposed to BAC770. Inhibition of ellagic acid-induced activation of Hageman factor by both forms of amyloid precursor protein was enhanced by heparin. These findings suggested that the heparin-binding domain of amyloid precursor proteins is not in the Kunitz domain. This heparin-binding domain may block the activation of Hageman factor by negatively charged agents. Thus, amyloid precursor proteins may be involved in the control of hemostasis, properties not all dependent on the Kunitz domain.

  14. Backbone resonance assignments of the micro-RNA precursor binding region of human TRBP.

    PubMed

    Benoit, Matthieu P M H; Plevin, Michael J

    2013-10-01

    TAR-RNA binding protein (TRBP) is a multidomain human protein involved in micro-RNA (miRNA) biogenesis. TRBP is a component of both the Dicer complex, which processes precursor miRNAs, and the RNA-induced silencing complex-loading complex. In addition, TRBP is implicated in the human immunodeficiency virus replication cycle and interferon-protein kinase R activity. TRBP contains 3 double-stranded RNA binding domains the first two of which have been shown to interact with miRNA precursors. Here we present the backbone resonance assignments and secondary structure of residues 19-228 of human TRBP2.

  15. Mars scientific investigations as a precursor for human exploration.

    PubMed

    Ahlf, P; Cantwell, E; Ostrach, L; Pline, A

    2000-01-01

    In the past two years, NASA has begun to develop and implement plans for investigations on robotic Mars missions which are focused toward returning data critical for planning human missions to Mars. The Mars Surveyor Program 2001 Orbiter and Lander missions will mark the first time that experiments dedicated to preparation for human exploration will be carried out. Investigations on these missions and future missions range from characterization of the physical and chemical environment of Mars, to predicting the response of biology to the Mars environment. Planning for such missions must take into account existing data from previous Mars missions which were not necessarily focused on human exploration preparation. At the same time, plans for near term missions by the international community must be considered to avoid duplication of effort. This paper reviews data requirements for human exploration and applicability of existing data. It will also describe current plans for investigations and place them within the context of related international activities.

  16. Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

    PubMed Central

    2011-01-01

    Background A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy. Results We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, in vitro. Conclusions These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells. PMID:21798090

  17. Assessing Influences of Ozone Precursor Emissions on Human Health & Ecosystems"

    EPA Science Inventory

    The Clean Air Act supports the establishment of a national standard for ambient concentrations of atmospheric pollutants to protect human health and public welfare (CAA, 1990). The primary standard has been viewed as sucient for also protecting public welfare. We seek to explore ...

  18. Precursors of hexoneogenesis within the human mammary gland

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The human mammary gland is capable of de novo synthesis of glucose and galactose (hexoneogenesis); however, the carbon source is incompletely understood. In this study, we investigated the role of acetate, glutamine, lactate and glycerol as potential carbon sources for hexoneogenesis. Healthy breast...

  19. Mars scientific investigations as a precursor for human exploration

    NASA Technical Reports Server (NTRS)

    Ahlf, P.; Cantwell, E.; Ostrach, L.; Pline, A.

    2000-01-01

    In the past two years, NASA has begun to develop and implement plans for investigations on robotic Mars missions which are focused toward returning data critical for planning human missions to Mars. The Mars Surveyor Program 2001 Orbiter and Lander missions will mark the first time that experiments dedicated to preparation for human exploration will be carried out. Investigations on these missions and future missions range from characterization of the physical and chemical environment of Mars, to predicting the response of biology to the Mars environment. Planning for such missions must take into account existing data from previous Mars missions which were not necessarily focused on human exploration preparation. At the same time, plans for near term missions by the international community must be considered to avoid duplication of effort. This paper reviews data requirements for human exploration and applicability of existing data. It will also describe current plans for investigations and place them within the context of related international activities. c 2000 International Astronautical Federation. Published by Elsevier Science Ltd. All rights reserved.

  20. Primary structure of the human follistatin precursor and its genomic organization

    SciTech Connect

    Shimasaki, Shunichi; Koga, Makoto; Esch, F.; Cooksey, K.; Mercado, M.; Koba, A.; Ueno, Naoto; Ying, Shaoyao; Ling, N.; Guillemin, R. )

    1988-06-01

    Follistatin is a single-chain gonadal protein that specifically inhibits follicle-stimulating hormone release. By use of the recently characterized porcine follistatin cDNA as a probe to screen a human testis cDNA library and a genomic library, the structure of the complete human follistatin precursor as well as its genomic organization have been determined. Three of eight cDNA clones that were sequenced predicted a precursor with 344 amino acids, whereas the remaining five cDNA clones encoded a 317 amino acid precursor, resulting from alternative splicing of the precursor mRNA. Mature follistatins contain four contiguous domains that are encoded by precisely separated exons; three of the domains are highly similar to each other, as well as to human epidermal growth factor and human pancreatic secretory trypsin inhibitor. The genomic organization of the human follistatin is similar to that of the human epidermal growth factor gene and thus supports the notion of exon shuffling during evolution.

  1. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  2. Formation and Human Risk of Carcinogenic Heterocyclic Amines Formed from Natural Precursors in Meat

    SciTech Connect

    Knize, M G; Felton, J S

    2004-11-22

    A group of heterocyclic amines that are mutagens and rodent carcinogens form when meat is cooked to medium and well-done states. The precursors of these compounds are natural meat components: creatinine, amino acids and sugars. Defined model systems of dry-heated precursors mimic the amounts and proportions of heterocyclic amines found in meat. Results from model systems and cooking experiments suggest ways to reduce their formation and, thus, to reduce human intake. Human cancer epidemiology studies related to consumption of well-done meat products are listed and compared.

  3. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  4. Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells

    PubMed Central

    Wei, Min; Zhao, Xia; Liu, Mi; Niu, Meijuan; Seif, Elias; Kleiman, Lawrence

    2016-01-01

    In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5’ leader and 3’ trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNAIle was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNAIle within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNAIle also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNAIle with long 5’ leader and long 3’ trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNAIle could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus. PMID:27101286

  5. SLS-Derived Lab: Precursor to Deep Space Human Exploration

    NASA Technical Reports Server (NTRS)

    Griffin, Brand; Lewis, Ruthan; Eppler, Dean; Smitherman, David

    2014-01-01

    Plans to send humans to Mars are in work and the launch system is being built. Are we ready? Robotic missions have successfully demonstrated transportation, entry, landing and surface operations but for human missions there are significant, potentially show-stopping issues. These issues, called Strategic Knowledge Gaps (SKGs) are the unanswered questions concerning long-duration exploration beyond low-earth-orbit. The gaps represent a risk of loss of life or mission and because they require extended exposure to the weightless environment outside earth's protective geo-magnetic field they cannot be resolved on the earth or on the International Space Station (ISS). Placing a laboratory at the relatively close and stable lunar Distant Retrograde Orbit (DRO) provides an accessible location with the requisite environmental conditions for conducting SKG research and testing mitigation solutions. Configurations comprised of multiple 3 meter and 4.3 meter diameter modules have been studied but the most attractive solution uses elements of the human Mars launch vehicle or Space Launch System (SLS) for a Mars proving ground laboratory. A shortened version of an SLS hydrogen propellant tank creates a Skylab-like pressure vessel that flies fully outfitted on a single launch. This not only offers significant savings by incorporating SLS pressure vessel development costs but avoids the expensive ISS approach using many launches with substantial on-orbit assembly before becoming operational. One of the most challenging SKGs is crew radiation protection; this is why SKG laboratory research is combined with Mars transit Habitat systems development. Fundamentally, the two cannot be divorced because using the habitat systems for protection requires actual hardware geometry and material properties intended to contribute to shielding effectiveness. The SKGs are difficult problems, solutions are not obvious, and require integrated, iterative, and multi-disciplinary development. A lunar

  6. TRBP alters human precursor microRNA processing in vitro.

    PubMed

    Lee, Ho Young; Doudna, Jennifer A

    2012-11-01

    MicroRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms that can vary in length at their 5' or 3' ends, but the biogenesis and functional significance of these RNAs are largely unknown. We show here that the human trans-activation response (TAR) RNA binding protein (TRBP), a known molecular partner of the miRNA processing enzyme Dicer, changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. Furthermore, TRBP can trigger the generation of iso-miRNAs (isomiRs) that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. These results implicate TRBP as a key regulator of miRNA processing and targeting in humans.

  7. SLS-Derived Lab- Precursor to Deep Space Human Exploration

    NASA Technical Reports Server (NTRS)

    Griffin, Brand M.; Lewis, Ruthan; Eppler, Dean; Smitherman, David

    2015-01-01

    Plans to send humans to Mars are in the works and the launch system is being built. Are we ready? Transportation, entry, landing, and surface operations have been successfully demonstrated for robotic missions. However, for human missions, there are significant, potentially show-stopping issues. These issues, called Strategic Knowledge Gaps (SKGs), are the unanswered questions concerning long duration exploration Beyond low Earth Orbit (BEO). The gaps represent a risk of loss of life or mission and because they require extended exposure to the weightless environment outside of earth's protective geo-magnetic field, they cannot be resolved on Earth or on the International Space Station (ISS). Placing a laboratory at a relatively close and stable lunar Distant Retrograde Orbit (DRO) provides an accessible location with the requisite environmental conditions for conducting SKG research and testing mitigation solutions. Configurations comprised of multiple 3 m and 4.3 m diameter modules have been studied but the most attractive solution uses elements of the human Mars launch vehicle or Space Launch System (SLS) for a Mars proving ground laboratory. A shortened version of an SLS hydrogen propellant tank creates a Skylab-like pressure vessel that flies fully outfitted on a single launch. This not only offers significant savings by incorporating SLS pressure vessel development costs but avoids the expensive ISS approach using many launches with substantial on-orbit assembly before becoming operational. One of the most challenging SKGs is crew radiation protection; this is why SKG laboratory research is combined with Mars transit habitat systems development. Fundamentally, the two cannot be divorced because using the habitat systems for protection requires actual hardware geometry and material properties intended to contribute to shielding effectiveness. The SKGs are difficult problems. The solutions to these problems are not obvious; they require integrated, iterative

  8. Muscle-Bound Primordial Stem Cells Give Rise to Myofiber-Associated Myogenic and Non-Myogenic Progenitors

    PubMed Central

    Chapal-Ilani, Noa; Itzkovitz, Shalev; Horovitz, Inna; Reizel, Yitzhak; Benayahu, Dafna; Shapiro, Ehud

    2011-01-01

    Myofiber cultures give rise to myogenic as well as to non-myogenic cells. Whether these myofiber-associated non-myogenic cells develop from resident stem cells that possess mesenchymal plasticity or from other stem cells such as mesenchymal stem cells (MSCs) remain unsolved. To address this question, we applied a method for reconstructing cell lineage trees from somatic mutations to MSCs and myogenic and non-myogenic cells from individual myofibers that were cultured at clonal density. Our analyses show that (i) in addition to myogenic progenitors, myofibers also harbor non-myogenic progenitors of a distinct, yet close, lineage; (ii) myofiber-associated non-myogenic and myogenic cells share the same muscle-bound primordial stem cells of a lineage distinct from bone marrow MSCs; (iii) these muscle-bound primordial stem-cells first part to individual muscles and then differentiate into myogenic and non-myogenic stem cells. PMID:22022423

  9. Circulating connective tissue precursors: extreme rarity in humans and chondrogenic potential in guinea pigs.

    PubMed

    Kuznetsov, Sergei A; Mankani, Mahesh H; Leet, Arabella I; Ziran, Navid; Gronthos, Stan; Robey, Pamela Gehron

    2007-07-01

    Using a variety of cell separation techniques and cultivation conditions, circulating, adherent, connective tissue, clonogenic cells were found in just 3 donors out of 66, demonstrating that these precursors are extremely rare in postnatal human blood. Contrary to humans, guinea pig blood shows much more reproducible connective tissue colony formation; it was therefore chosen to study the differentiation potential of adherent blood-derived clonogenic cells. Out of 22 single colony-derived strains of various morphologies, only 5 spindle-shaped strains showed extensive proliferative capacity in vitro. None of these strains formed bone upon in vivo transplantation, whereas two strains formed cartilage in high-density pellet cultures in vitro. Both chondrogenic strains included cells expressing aggrecan, whereas nonchondrogenic strains did not. Out of four polyclonal strains studied, one formed both cartilage and abundant bone accompanied by hematopoiesis-supporting stroma. Evidently, there are cells in adult guinea pig blood capable of both extensive proliferation and differentiation toward cartilage: circulating chondrogenic precursors. Although some of these cells lack osteogenic potential and therefore represent committed chondrogenic precursors, others may be multipotential and consequently belong to the family of skeletal stem cells. This is the first demonstration of postnatal circulating chondrogenic precursors, as well as of precursor cells with chondrogenic but not osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.

  10. Communication and Empathy as Precursors to Burnout among Human Service Workers.

    ERIC Educational Resources Information Center

    Miller, Katherine I.; And Others

    1988-01-01

    Examines the role of communicative responsiveness, empathic concern, and emotional contagion as precursors to burnout among human service workers. Concludes that empathic concern leads to communicative responsiveness, emotional contagion decreases responsiveness, and responsiveness predicts three dimensions of burnout and occupational commitment.…

  11. Precursors of hexoneogenesis within the human mammary gland.

    PubMed

    Mohammad, Mahmoud A; Maningat, Patricia; Sunehag, Agneta L; Haymond, Morey W

    2015-04-15

    The human mammary gland is capable of de novo synthesis of glucose and galactose (hexoneogenesis); however, the carbon source is incompletely understood. In this study, we investigated the role of acetate, glutamine, lactate and glycerol as potential carbon sources for hexoneogenesis. Healthy breastfeeding women were studied following a 24-h fast on two occasions separated by 1-3 wk. Five women were infused with [U-¹³C]lactate or [1,2-¹³C₂]glutamine and five women with [U-¹³C]glycerol or [1,2-¹³C₂]acetate. Enrichments of ¹³C in plasma and milk substrates were analyzed using GC-MS. Infusion of labeled lactate, glycerol, glutamine, and acetate resulted in plasma glucose being 22.0±3.7, 11.2±1.0, 2.5±0.5, and 1.3±0.2% labeled, respectively. Lactate, glutamine, or acetate did not contribute to milk glucose or galactose (0-2%). In milk, ¹³C-free glycerol enrichment was one-fourth that in plasma but free glycerol concentration in milk was fourfold higher than in plasma. Using [U-¹³C]glycerol and by accounting for tracer dilution, glycerol alone contributed to 10±2 and 69±11% of the hexoneogenesis of milk glucose and galactose, respectively. During [U-¹³C]glycerol infusion, the ratio of M₃ enrichment on 4-6 carbons/M₃ on 1-3 carbons of galactose was higher (P<0.05, 1.22±0.05) than those of glucose in plasma (1.05±0.03) and milk (1.07±0.02). Reanalysis of samples from a previous study involving [U-¹³C]glucose infusion alone suggested labeling a portion of galactose consistent with pentose phosphate pathway (PPP) activity. We conclude that, although lactate contributed significantly to gluconeogenesis, glycerol alone provides the vast majority of substrate for hexoneogenesis. The relative contribution of the PPP vs. the reversal Embden-Meyerhof pathway to hexoneogenesis within the human mammary gland remains to be determined.

  12. Myogenic cytodifferentiation of the precardiac mesoderm in the rat.

    PubMed

    Baldwin, H S; Jensen, K L; Solursh, M

    1991-08-01

    The contractile cells of the primitive heart are derived from a subpopulation of the lateral plate splanchnic mesoderm. While the formation of the cardiac primordia has been studied in the avian embryo, little is known about this cell population in the mammal. To investigate the distribution and cellular differentiation of the myocardial precursors in the early mammalian embryo, we studied the sequential immunohistochemical appearance of desmin and myosin in whole mounts of rat embryos from the presomite (gestational day 9) through the 6-8 somite, straight heart tube (gestational day 10) stages of early cardiac morphogenesis. In contrast to the chicken, and previous reports in the mouse, our results show that myogenic differentiation of the muscle precursor cells of the heart begins in the presomite embryo prior to formation of the anterior intestinal portal or foregut. In addition, this cell population of the precardiac mesoderm appears as a single crescent-shaped population of cells in continuity across the midline which extends caudally during development and then fuses in the midline to form the primitive heart tube. Unlike skeletal myogenesis, desmin and myosin appear simultaneously and are codistributed throughout this initial period of heart development. These results suggest that myocardial differentiation in the rat is precocious when compared to the chicken and precedes the morphogenetic processes involved in formation of the primitive heart tube. Furthermore, this study provides the first description in the mammal of the spatial distribution of the myogenic precardiac mesoderm.

  13. Diploid and tetraploid precursors of megakaryocytes in normal human bone marrow detected by immunofluorescence.

    PubMed

    Renner, D; Propp, H; Queisser, W

    1987-11-01

    A sequential preparation method is described which allows immunological identification, morphological characterization, cytophotometric determination of relative DNA content of the megakaryocyte lineage as well as quantitation of megakaryocyte precursors in human bone marrow aspirates. We compared several monoclonal (anti-GP IIIa and HD 19) and polyclonal (A225, RAHPS) antiplatelet antibodies for immunofluorescent staining. Among the identified cells, a small number of cells showing a diploid and tetraploid DNA content were found which must be regarded as promegakaryoblasts, representing 2.5-4.7% of all megakaryocytes. The heterogenous morphology of these precursors in panoptically stained smears is described.

  14. Spontaneous myogenic differentiation of Flk-1-positive cells from adult pancreas and other nonmuscle tissues.

    PubMed

    Di Rocco, Giuliana; Tritarelli, Alessandra; Toietta, Gabriele; Gatto, Ilaria; Iachininoto, Maria Grazia; Pagani, Francesca; Mangoni, Antonella; Straino, Stefania; Capogrossi, Maurizio C

    2008-02-01

    At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1(+) progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1(+) compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

  15. Frequency tuning of the cervical vestibular-evoked myogenic potential (cVEMP) recorded from multiple sites along the sternocleidomastoid muscle in normal human subjects.

    PubMed

    Wei, Wei; Jeffcoat, Ben; Mustain, William; Zhu, Hong; Eby, Thomas; Zhou, Wu

    2013-02-01

    Frequency tuning of tone burst-evoked myogenic potentials recorded from the sternocleidomastoid muscle (cervical VEMP or cVEMP) is used clinically to assess vestibular function. Understanding the characteristics of cVEMP is important for improving the specificity of cVEMP testing in diagnosing vestibular deficits. In the present study, we analyzed the frequency tuning properties of the cVEMPs by constructing detailed tuning curves and examining their morphology and dependence on SCM tonic level, sound intensity, and recording site along the SCM. Here we report two main findings. First, by employing nine tone frequencies between 125 and 4,000 Hz, some tuning curves exhibited two distinct peaks, which cannot be modeled by a single mass spring system as previously suggested. Instead, the observed tuning is better modeled as linear summation of two mass spring systems, with resonance frequencies at ~300 and ~1,000 Hz. Peak frequency of cVEMP tuning curves was not affected by SCM tonic level, sound intensity, and location of recording site on the SCM. However, sharpness of cVEMP tuning was increased at lower sound intensities. Second, polarity of cVEMP responses recorded from the lower quarter of the SCM was reversed as compared to that at the two upper sites. While more studies are needed, these results suggest that cVEMP tuning is mediated through multiple generators with different resonance frequencies. Future studies are needed to explore implications of these results on development of selective VEMP tests and determine the nature of polarity inversion at the lower quarter of SCM.

  16. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

    PubMed

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

    2015-11-06

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.

  17. Promotion of human adipocyte precursor replication by 17beta-estradiol in culture.

    PubMed Central

    Roncari, D A; Van, R L

    1978-01-01

    The influence of 17beta-estradiol and 17alpha-estradiol on adult human omental adipocyte precursors grown in a propagating culture system was studied. Cells were grown in subculture in the presence or absence of hormone. 17beta-estradiol resulted in significant promotion of adipocyte precursor replication, as determined by cell counting and incorporation of radioactive thymidine into DNA. The hormone stimulated cell multiplication in the concentration range 0.5--500 ng/ml growth medium. The highest level tested was 500 ng/ml. The maximal effects were obtained at 50 ng/ml (P less than 0.001 by paired t test, 48 h after hormone addition). All 10 cell strains (five were derived from men and five from women) that were tested responded similarly to the hormone. 17beta-estradiol did not affect cell size. 17alpha-estradiol did not promote the replication of adipocyte precursors, nor did it influence cell size. Thus, 17beta-estradiol, which is the active isomer in known target tissues, stimulates the multiplication of human adipocyte precursors in culture. Images PMID:690182

  18. The Cervical Vestibular-Evoked Myogenic Potentials (cVEMPs) Recorded Along the Sternocleidomastoid Muscles During Head Rotation and Flexion in Normal Human Subjects.

    PubMed

    Ashford, Alexander; Huang, Jun; Zhang, Chunming; Wei, Wei; Mustain, William; Eby, Thomas; Zhu, Hong; Zhou, Wu

    2016-08-01

    Tone burst-evoked myogenic potentials recorded from tonically contracted sternocleidomastoid muscles (SCM) (cervical VEMP or cVEMP) are widely used to assess the vestibular function. Since the cVEMP response is mediated by the vestibulo-collic reflex (VCR) pathways, it is important to understand how the cVEMPs are determined by factors related to either the sensory components (vestibular end organs) or the motor components (SCM) of the VCR pathways. Compared to the numerous studies that have investigated effects of sound parameters on the cVEMPs, there are few studies that have examined effects of SCM-related factors on the cVEMPs. The goal of the present study is to fill this knowledge gap by testing three SCM-related hypotheses. The first hypothesis is that contrary to the current view, the cVEMP response is only present in the SCM ipsilateral to the stimulated ear. The second hypothesis is that the cVEMP response is not only dependent on tonic level of the SCM, but also on how the tonic level is achieved, i.e., by head rotation or head flexion. The third hypothesis is that the SCM is compartmented and the polarity of the cVEMP response is dependent on the recording site. Seven surface electrodes were positioned along the left SCMs in 12 healthy adult subjects, and tone bursts were delivered to the ipsilateral or contralateral ear (8 ms plateau, 1 ms rise/fall, 130 dB SPL, 50-4000 Hz) while subjects activated their SCMs by head rotation (HR condition) or chin downward head flexion (CD condition). The first hypothesis was confirmed by the finding that the contralateral cVEMPs were minimal at all recording sites for all the tested tones during both HR and CD conditions. The second hypothesis was confirmed by the finding that the ipsilateral cVEMPs were larger in HR condition than in CD condition at recording sites above and below the SCM midpoint. Finally, the third hypothesis was confirmed by the finding that the cVEMPs exhibit reversed polarities at the sites

  19. Anatomical Location of LPA1 Activation and LPA Phospholipid Precursors in Rodent and Human Brain

    PubMed Central

    González de San Román, E; Manuel, I; Giralt, MT; Chun, J; Estivill-Torrús, G; Rodriguez de Fonseca, F; Santín, LJ; Ferrer, I; Rodriguez-Puertas, R

    2016-01-01

    Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCRs): LPA1–LPA6. LPA evokes several responses in the CNS including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [35S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1-null mice (a variant of LPA1-null) lack [35S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides (PA) and phosphatidylcholines (PC). Both PA and PC species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. PMID:25857358

  20. Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

    PubMed Central

    Piper, David R.; Mujtaba, Tahmina; Keyoung, Hansoo; Roy, Neeta S.; Goldman, Steven A.; Rao, Mahendra S.; Lucero, Mary T.

    2010-01-01

    We have examined primary human neuronal precursors (HNPs) from 18–22-week-old fetuses. We showed that E-NCAM/MAP2/β-III tubulin-immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament-associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage-gated ion channels, and ligand-gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Tα1 tubulin promoter was E-NCAM/β-III tubulin-immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population. PMID:11746353

  1. Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain.

    PubMed

    González de San Román, Estibaliz; Manuel, Iván; Giralt, María Teresa; Chun, Jerold; Estivill-Torrús, Guillermo; Rodríguez de Fonseca, Fernando; Santín, Luis Javier; Ferrer, Isidro; Rodríguez-Puertas, Rafael

    2015-08-01

    Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors: LPA1 -LPA6 . LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1 -null mice (a variant of LPA1 -null) lack [(35) S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCR), LPA1 to LPA6 . LPA evokes several responses in the central nervous system (CNS), including cortical development and folding, growth of the axonal cone and its retraction process. We used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 -binding sites in adult rodent and human brain. The distribution of LPA1 receptors in rat, mouse and human brains show the highest activity in white matter myelinated areas. The basal and

  2. Involvement of Epithelial Na+ Channel in the Elevated Myogenic Response in Posterior Cerebral Arteries from Spontaneously Hypertensive Rats

    PubMed Central

    Choi, Soo-Kyoung; Yeon, Soo-In; Kwon, Youngin; Byeon, Seonhee; Lee, Young-Ho

    2017-01-01

    Hypertension is characterized by increased peripheral vascular resistance which is related with elevated myogenic response. Recent findings have indicated that epithelial sodium channel (ENaC) is involved in mechanotransduction of the myogenic response. The purpose of this study was to investigate the involvement of ENaC in the elevated myogenic response of posterior cerebral arteries (PCAs) from spontaneously hypertensive rats (SHRs). Sixteen to eighteen weeks old male wistar kyoto rats (WKYs) and SHRs were used in this study. We found that wall to lumen (W/L) ratio was increased in the PCAs from SHRs compared with WKYs at the resting state. Interestingly, amiloride significantly inhibited myogenic response in the PCAs from SHRs and WKYs, however, the magnitude of the blockade was greater in SHRs. The transfection of γENaC-siRNA significantly reduced the expression of γENaC protein and inhibited myogenic response in the PCAs from SHRs. Furthermore, these data were supported by the findings that serum/glucocorticoid-induced kinase (Sgk1) and neural precursor cell-expressed developmentally downregulated gene 4-2 (Nedd4-2) were increased in SHRs compared with WKYs. Our results suggest that γENaC may play an important role in the elevated myogenic response in PCAs from SHRs. PMID:28383056

  3. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    ERIC Educational Resources Information Center

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  4. Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs

    PubMed Central

    Witt, Kristen C.; Castillo-Menendez, Luis; Ding, Haitao; Espy, Nicole; Zhang, Shijian; Kappes, John C.; Sodroski, Joseph

    2017-01-01

    The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context

  5. The influence of platelet-rich plasma on myogenic differentiation.

    PubMed

    McClure, Michael J; Garg, Koyal; Simpson, David G; Ryan, John J; Sell, Scott A; Bowlin, Gary L; Ericksen, Jeffery J

    2016-04-01

    The ability to expand and direct both precursor and stem cells towards a differential fate is considered extremely advantageous in tissue engineering. Platelet-rich plasma (PRP) possesses a milieu of growth factors and cytokines, which have the potential to have either a differentiative or proliferative influence on the cell type tested. Here, we investigated the effect of PRP on C2C12 myoblasts. A range of PRP concentrations in differentiation media was used to determine whether a concentration dependence existed, while PRP embedded in fibres of aligned electrospun polydioxanone and polycaprolactone was used to determine whether this presence of fibres would cause any differences in response. In both cases, it was found that late myogenic markers were suppressed after 7 days in culture. However, an early differentiation marker, MyoD, was upregulated during this same time period. The results from this study represent the ability of PRP to have an influence over both myogenic proliferation and differentiation, a factor which could prove useful in future studies involved with skeletal muscle tissue engineering.

  6. Human placental transfer of perfluoroalkyl acid precursors: Levels and profiles in paired maternal and cord serum.

    PubMed

    Yang, Lin; Wang, Zhen; Shi, Yu; Li, Jingguang; Wang, Yuxin; Zhao, Yunfeng; Wu, Yongning; Cai, Zongwei

    2016-02-01

    Perfluoroalkyl acids (PFAAs) precursors, the indirect source of PFAA exposure, have been observed in environmental and human samples. However, the maternal-fetal transfer of these chemicals has not been well examined. In this study, 50 paired maternal and cord serum samples collected in Jiangsu province of China were analyzed for fifteen PFAA precursors. Among the detected PFAAs, 6:2 fluorotelomer sulfonate (6:2 FTS), N-methyl- and N-ethyl-perfluorooctanesulfonamidoacetates had comparable detection rate in both maternal and cord sera, while the mean concentrations and detection rates of 8:2 FTS and perfluorooctane sulfonamide (PFOSA) were higher in maternal sera compared to cord sera (Mann-Whitney U test, P < 0.05). Analysis of variance and least significant difference tests showed that the youngest maternal age group (21-24 years old) had the highest concentration of 6:2 FTS in cord sera. Maternal serum PFOSA was found significantly correlated with the cord serum perfluorooctanesulfonate (PFOS) (Spearman test, r = 0.361, P = 0.010), indicating that maternal serum PFOSA might be an indirect source of PFOS in fetuses. The obtained results suggested the potential prenatal exposure and human placental transfer of perfluoroalkyl acid precursors.

  7. Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

    PubMed Central

    Rettenmier, C W; Roussel, M F

    1988-01-01

    The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo. Images PMID:3264877

  8. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

    PubMed

    Bogan, Katrina L; Brenner, Charles

    2008-01-01

    Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research.

  9. Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay

    SciTech Connect

    Crosby, S.R.; Stewart, M.F.; Ratcliffe, J.G.; White, A.

    1988-12-01

    An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful.

  10. A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein.

    PubMed

    Lee, Geun-Shik; Jeong, Yeon Woo; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Moon, Changjong; Hyun, Sang Hwan; Hwang, Kyu-Chan; Kim, Nam-Hyung; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

    2014-04-01

    Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and β-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.

  11. Deficiency of the myogenic factor MyoD causes a perinatally lethal fetal akinesia

    PubMed Central

    Crinnion, Laura A; Murphy, Helen; Newbould, Melanie; Harrison, Sally M; Lascelles, Carolina; Antanaviciute, Agne; Carr, Ian M; Sheridan, Eamonn; Bonthron, David T; Smith, Audrey

    2016-01-01

    Background Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. Methods Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. Results Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. Conclusions We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme. PMID:26733463

  12. Non-invasive Imaging and Tracking of Engineered Human Muscle Precursor Cells for Skeletal Muscle Tissue Engineering Using Positron Emission Tomography

    PubMed Central

    Haralampieva, Deana; Betzel, Thomas; Dinulovic, Ivana; Salemi, Souzan; Stoelting, Meline; Kraemer, Stefanie; Schibli, Roger; Sulser, Tullio; Handschin, Christoph; Eberli, Daniel; Ametamey, Simon M.

    2016-01-01

    Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible non-invasive tool to monitor cell survival, migration and integration into the host tissue is still missing. Methods In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of a human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by Reverse Transcriptase Polymerase Chain Reaction (RTPCR) and infection efficiency was visualized by fluorescent microscopy. Viability, proliferation and differentiation capacity of the transduced cells were confirmed and their sustained myogenic phenotype was shown by flow cytometry analysis and fluorescent microscopy. 18F-Fallypride and 18F-FMISO, two well-established PET radioligands, were successfully synthesized and evaluated for their potential to image engineered hMPCs in a mouse model. Furthermore, biodistribution studies and autoradiography were also performed to determine the extent of signal specificity. Results To address the feasibility of the presented approach for tracking of hMPCs in an in vivo model, we first evaluated the safety of the adenoviral gene-delivery, which showed no detrimental effects on the primary human cells. Specific binding of 18F-Fallypride to hD2R_hMPCs was demonstrated in vitro, as well as in vivo, by performing autoradiography, biodistribution and PET experiments, respectively. Furthermore, 18F-FMISO uptake was evaluated at different time-points after cell inoculation in vivo, showing high signal only at the early stages. Finally, histological assessment of the harvested tissues confirmed the sustained survival of the transplanted cells at different time-points with formation of muscle tissue at the site of injection. Conclusion We here propose a signaling-deficient human D2R as a potent reporter for in vivo hMPCs PET tracking by 18F-Fallypride. This approach

  13. Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells

    PubMed Central

    Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

    2016-01-01

    The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders. PMID:27992514

  14. Role of PUFAs, the precursors of endocannabinoids, in human obesity and type 2 diabetes.

    PubMed

    Dain, Alejandro; Repossi, Gaston; Das, Undurti N; Eynard, Aldo Renato

    2010-06-01

    Polyunsaturated fatty acids (PUFAs) serve as precursors of the endocannabinoids (ECs) that are bioactive lipids molecules. Recent studies revealed that ECs participate in several physiological and pathological processes including obesity and type 2 diabetes mellitus. Here we review the experimental and clinical aspects of the role of endocannabinoids in obesity and type 2 diabetes mellitus and the modification of the endocannabinoids by exogenously administered PUFAs. Based on these evidences, we propose that the endocannabinoid system (ECS) can be modulated by exogenous manipulation of PUFAs that could help in the prevention and management of human diseases such as obesity, metabolic syndrome and type 2 diabetes mellitus.

  15. A potential regulatory network underlying distinct fate commitment of myogenic and adipogenic cells in skeletal muscle

    PubMed Central

    Sun, Wenjuan; He, Ting; Qin, Chunfu; Qiu, Kai; Zhang, Xin; Luo, Yanhong; Li, Defa; Yin, Jingdong

    2017-01-01

    Mechanism controlling myo-adipogenic balance in skeletal muscle is of great significance for human skeletal muscle dysfunction and myopathies as well as livestock meat quality. In the present study, two cell subpopulations with particular potency of adipogenic or myogenic differentiation were isolated from neonatal porcine longissimus dorsi using the preplate method to detect mechanisms underlying distinct fate commitment of myogenic and adipogenic cells in skeletal muscle. Both cells share a common surface expression profile of CD29+CD31−CD34−CD90+CD105+, verifying their mesenchymal origin. A total of 448 differentially expressed genes (DEGs) (FDR < 0.05 and |log2 FC| ≥ 1) between two distinct cells were identified via RNA-seq, including 358 up-regulated and 90 down-regulated genes in myogenic cells compared with adipogenic cells. The results of functional annotation and enrichment showed that 42 DEGs were implicated in cell differentiation, among them PDGFRα, ITGA3, ITGB6, MLCK and MLC acted as hubs between environment information processing and cellular process, indicating that the interaction of the two categories exerts an important role in distinct fate commitment of myogenic and adipogenic cells. Particularly, we are first to show that up-regulation of intracellular Ca2+-MLCK and Rho-DMPK, and subsequently elevated MLC, may contribute to the distinct commitment of myogenic and adipogenic lineages via mediating cytoskeleton dynamics. PMID:28276486

  16. A potential regulatory network underlying distinct fate commitment of myogenic and adipogenic cells in skeletal muscle.

    PubMed

    Sun, Wenjuan; He, Ting; Qin, Chunfu; Qiu, Kai; Zhang, Xin; Luo, Yanhong; Li, Defa; Yin, Jingdong

    2017-03-09

    Mechanism controlling myo-adipogenic balance in skeletal muscle is of great significance for human skeletal muscle dysfunction and myopathies as well as livestock meat quality. In the present study, two cell subpopulations with particular potency of adipogenic or myogenic differentiation were isolated from neonatal porcine longissimus dorsi using the preplate method to detect mechanisms underlying distinct fate commitment of myogenic and adipogenic cells in skeletal muscle. Both cells share a common surface expression profile of CD29(+)CD31(-)CD34(-)CD90(+)CD105(+), verifying their mesenchymal origin. A total of 448 differentially expressed genes (DEGs) (FDR < 0.05 and |log2 FC| ≥ 1) between two distinct cells were identified via RNA-seq, including 358 up-regulated and 90 down-regulated genes in myogenic cells compared with adipogenic cells. The results of functional annotation and enrichment showed that 42 DEGs were implicated in cell differentiation, among them PDGFRα, ITGA3, ITGB6, MLCK and MLC acted as hubs between environment information processing and cellular process, indicating that the interaction of the two categories exerts an important role in distinct fate commitment of myogenic and adipogenic cells. Particularly, we are first to show that up-regulation of intracellular Ca(2+)-MLCK and Rho-DMPK, and subsequently elevated MLC, may contribute to the distinct commitment of myogenic and adipogenic lineages via mediating cytoskeleton dynamics.

  17. Evidence for a Direct Effect of the NAD+ Precursor Acipimox on Muscle Mitochondrial Function in Humans

    PubMed Central

    van de Weijer, Tineke; Phielix, Esther; Bilet, Lena; Williams, Evan G.; Ropelle, Eduardo R.; Bierwagen, Alessandra; Livingstone, Roshan; Nowotny, Peter; Sparks, Lauren M.; Paglialunga, Sabina; Szendroedi, Julia; Havekes, Bas; Moullan, Norman; Pirinen, Eija; Hwang, Jong-Hee; Schrauwen-Hinderling, Vera B.; Hesselink, Matthijs K.C.; Auwerx, Johan

    2015-01-01

    Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD+) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD+ precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m2) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD+ levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD+ boosters can also directly affect skeletal muscle mitochondrial function in humans. PMID:25352640

  18. Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors

    SciTech Connect

    Colleoni, Silvia; Giannelli, Serena G.; Armentero, Marie-Therese; Blandini, Fabio; Broccoli, Vania; Lazzari, Giovanna

    2010-04-15

    In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation. In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.

  19. Precursor Asteroid Missions and Synergies to Human Exploration of Phobos and Deimos

    NASA Technical Reports Server (NTRS)

    Abell, Paul

    2013-01-01

    U.S. President Obama stated on April 15, 2010 that the next goal for human spaceflight will be to send human beings to a near-Earth asteroid by 2025 and then on to the Martian system in the 2030s. Given this direction from the White House, NASA has been involved in studying various strategies for near-Earth object (NEO) exploration in order to follow U.S. space exploration policy. These missions would be the first human expeditions to interplanetary bodies beyond the Earth-Moon system and would prove useful for testing technologies required for human missions to Mars and its moons, as well as other Solar System destinations. Robotic precursor missions to NEOs would undoubtedly provide a great deal of technical and engineering data on spacecraft operations for future human space exploration while conducting in-depth scientific investigations of these primitive objects. In addition, the resulting scientific investigations would refine designs for future extraterrestrial resource extraction and utilization, which may play a vital role in leveraging potential resources from the Martian moons that in turn could enable robotic and human exploration of Mars.

  20. Kilowatt-Class Fission Power Systems for Science and Human Precursor Missions

    NASA Technical Reports Server (NTRS)

    Mason, Lee S.; Gibson, Marc Andrew; Poston, Dave

    2013-01-01

    Nuclear power provides an enabling capability for NASA missions that might otherwise be constrained by power availability, mission duration, or operational robustness. NASA and the Department of Energy (DOE) are developing fission power technology to serve a wide range of future space uses. Advantages include lower mass, longer life, and greater mission flexibility than competing power system options. Kilowatt-class fission systems, designated "Kilopower," were conceived to address the need for systems to fill the gap above the current 100-W-class radioisotope power systems being developed for science missions and below the typical 100-k We-class reactor power systems being developed for human exploration missions. This paper reviews the current fission technology project and examines some Kilopower concepts that could be used to support future science missions or human precursors.

  1. Kilowatt-Class Fission Power Systems for Science and Human Precursor Missions

    NASA Technical Reports Server (NTRS)

    Mason, Lee; Gibson, Marc; Poston, Dave

    2013-01-01

    Nuclear power provides an enabling capability for NASA missions that might otherwise be constrained by power availability, mission duration, or operational robustness. NASA and the Department of Energy (DOE) are developing fission power technology to serve a wide range of future space uses. Advantages include lower mass, longer life, and greater mission flexibility than competing power system options. Kilowatt-class fission systems, designated "Kilopower," were conceived to address the need for systems to fill the gap above the current 100-Wclass radioisotope power systems being developed for science missions and below the typical 100-kWe-class reactor power systems being developed for human exploration missions. This paper reviews the current fission technology project and examines some Kilopower concepts that could be used to support future science missions or human precursors.

  2. Malignant Precursor Cells Pre-Exist in Human Breast DCIS and Require Autophagy for Survival

    PubMed Central

    Espina, Virginia; Mariani, Brian D.; Gallagher, Rosa I.; Tran, Khoa; Banks, Stacey; Wiedemann, Joy; Huryk, Heather; Mueller, Claudius; Adamo, Luana; Deng, Jianghong; Petricoin, Emanuel F.; Pastore, Lucia; Zaman, Syed; Menezes, Geetha; Mize, James; Johal, Jasbir; Edmiston, Kirsten; Liotta, Lance A.

    2010-01-01

    Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from

  3. Langerhans cell precursors acquire RANK/CD265 in prenatal human skin.

    PubMed

    Schöppl, Alice; Botta, Albert; Prior, Marion; Akgün, Johnnie; Schuster, Christopher; Elbe-Bürger, Adelheid

    2015-01-01

    The skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10-11 weeks of estimated gestational age (EGA)] or only faintly (13-15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation - a phenomenon previously observed also for other markers on LCs in prenatal human skin.

  4. Differential localization of TGF-beta-precursor isotypes in normal human skin.

    PubMed

    Wataya-Kaneda, M; Hashimoto, K; Kato, M; Miyazono, K; Yoshikawa, K

    1994-08-01

    Transforming growth factor-beta (TGF-beta) can act as a multi-functional regulator of both cell growth and differentiation. Three isotypes of TGF-beta s namely TGF-beta 1, TGF-beta 2 and TGF-beta 3, have been found in human tissues. Up to now, little is known about the distribution patterns of the TGF-beta isotypes in human skin. Using the TGF-beta-precursor (latency-associated peptides) specific antibodies to confirm the specificity, we studied the immunohistochemical distribution of TGF-beta 1-3 in human skin. TGF-beta 2 was found mainly in the intercellular space of all the layers of the epidermis as well as in the cytoplasm with a weak staining. In contrast, TGF-beta 3 was present in the subepidermal area of the dermis. TGF-beta 1 was observed obviously in neither epidermis nor dermis. These results showed the differential localization of TGF-beta isotypes in human skin, suggesting that the TGF-beta 2 and TGF-beta 3 may regulate the human skin function in an epithelial autocrine or mesenchymal-epithelial interaction manner.

  5. Role of JAK3 in myogenic differentiation.

    PubMed

    Jang, You-Na; Lee, Il Jae; Park, Myong Chul; Baik, Eun Joo

    2012-03-01

    Skeletal muscle differentiation is regulated by transcription factors, including members of the myogenic regulatory factor (MRF) family and many signaling pathways. The JAK1 and JAK2 pathways are known to each have different effects on myoblast proliferation and differentiation; however, the role of JAK3 in myoblast differentiation remains unclear. In this study, we investigated the effect of JAK3 inhibition on myogenic differentiation in the C2C12 mouse myoblast cell line. During myogenic differentiation, treatment with the JAK3 inhibitor WHIp154 significantly increased the number of MHC-positive multinucleated myotubes and the expressions of myosin heavy chain (MHC), myogenin (MGN), MyoD, and myogenic enhancer factor 2 (MEF2). Knockdown of the JAK3 gene using siJAK3 also significantly increased MHC, MGN and MyoD mRNA expressions as well as insulin-like growth factor-II (IGF-II) gene expression. During differentiation, JAK3 was initially activated and later decreased. Differentiation decreased STAT1, which was further decreased by WHIp154. In contrast, STAT3 gradually was elevated during differentiation, and was increased by JAK3 inhibition. Moreover, we found that up-regulation of AKT activity and down-regulation of ERK activity cooperated to accelerate myogenic differentiation. Taken together, these data indicate that JAK3 inhibition potently facilitates myoblast differentiation through antagonistic STAT1/STAT3 activities. Additionally, JAK3 inhibition induced precocious differentiation and played important roles for terminal differentiation, including fusion, which is involved with regulation of AKT and ERK pathways.

  6. Insulin acts as a myogenic differentiation signal for neural stem cells with multilineage differentiation potential.

    PubMed

    Bani-Yaghoub, Mahmud; Kendall, Stephen E; Moore, Daniel P; Bellum, Stephen; Cowling, Rebecca A; Nikopoulos, George N; Kubu, Chris J; Vary, Calvin; Verdi, Joseph M

    2004-09-01

    Reports of non-neural differentiation of neural stem cells (NSCs) have been challenged by alternative explanations for expanded differentiation potentials. In an attempt to demonstrate the plasticity of NSC, neurospheres were generated from single retrovirally labeled embryonic cortical precursors. In a defined serum-free insulin-containing media, 40% of the neurospheres contained both myogenic and neurogenic differentiated progeny. The number of NSCs displaying multilineage differentiation potential declines through gestation but does exist in the adult animal. In this system, insulin appears to function as a survival and dose-dependent myogenic differentiation signal for multilineage NSCs (MLNSC). MLNSC-derived cardiomyocytes contract synchronously, respond to sympathetic and parasympathetic stimulation, and regenerate injured heart tissues. These studies provide support for the hypothesis that MLNSCs exist throughout the lifetime of the animal, and potentially provide a population of stem cells for cell-based regenerative medicine strategies inside and outside of the nervous system.

  7. Measurement of precursor miRNA in exosomes from human ESC-derived mesenchymal stem cells.

    PubMed

    Chen, Tian Sheng; Lim, Sai Kiang

    2013-01-01

    Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

  8. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    SciTech Connect

    Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  9. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    SciTech Connect

    Zhu, Liang; Dong, Chuanming; Sun, Chenxi; Ma, Rongjie; Yang, Danjing; Zhu, Hongwen; Xu, Jun

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  10. Lunar Precursor Missions for Human Exploration of Mars - II. Studies of Mission Operations

    NASA Astrophysics Data System (ADS)

    Mendell, W. W.; Griffith, A. D.

    necessary precursor to human missions to Mars. He observed that mission parameters for Mars expeditions far exceed current and projected near-term space operations experience in categories such as duration, scale, logistics, required system reliability, time delay for communications, crew exposure to the space environment (particularly reduced gravity), lack of abort-to-Earth options, degree of crew isolation, and long-term political commitment. He demonstrated how a program of lunar exploration could be structured to expand the experience base, test operations approaches, and validate proposed technologies. In this paper, we plan to expand the discussion on the topic of mission operations, including flight and trajectory design, crew activity planning, procedure development and validation, and initialization load development. contemplating the nature of the challenges posed by a mission with a single crew lasting 3 years with no possibility of abort to Earth and at a distance where the light-time precludes conversation between with the astronauts. The brief durations of Apollo or Space Shuttle missions mandates strict scheduling of in-space tasks to maximize the productivity. On a mission to Mars, the opposite obtains. Transit times are long (~160 days), and crew time may be principally devoted to physical conditioning and repeated simulations of the landing sequence. While the physical exercise parallels the experience on the International Space Station (ISS), the remote refresher training is new. The extensive surface stay time (~500 days) implies that later phases of the surface missions will have to be planned in consultation with the crew to a large extent than is currently the case. resolve concerns over the form of new methodologies and philosophies needed. Recent proposed reductions in scope and crew size for ISS exacerbate this problem. One unknown aspect is whether any sociological pathologies will develop in the relationship of the crew to Mission

  11. Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac----liver transition.

    PubMed Central

    Migliaccio, G; Migliaccio, A R; Petti, S; Mavilio, F; Russo, G; Lazzaro, D; Testa, U; Marinucci, M; Peschle, C

    1986-01-01

    Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS----L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts----macrocytes) and globin expression (zeta----alpha, epsilon----gamma). Images PMID:3722384

  12. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

    PubMed

    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2016-12-05

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 9999: 1-16, 2016. © 2016 Wiley Periodicals, Inc.

  13. Next Gen NEAR: Near Earth Asteroid Human Robotic Precursor Mission Concept

    NASA Technical Reports Server (NTRS)

    Rivkin, Andrew S.; Kirby, Karen; Cheng, Andrew F.; Gold, Robert; Kelly, Daniel; Reed, Cheryl; Abell, Paul; Garvin, James; Landis, Rob

    2012-01-01

    Asteroids have long held the attention of the planetary science community. In particular, asteroids that evolve into orbits near that of Earth, called near-Earth objects (NEO), are of high interest as potential targets for exploration due to the relative ease (in terms of delta V) to reach them. NASA's Flexible Path calls for missions and experiments to be conducted as intermediate steps towards the eventual goal of human exploration of Mars; piloted missions to NEOs are such example. A human NEO mission is a valuable exploratory step beyond the Earth-Moon system enhancing capabilities that surpass our current experience, while also developing infrastructure for future mars exploration capabilities. To prepare for a human rendezvous with an NEO, NASA is interested in pursuing a responsible program of robotic NEO precursor missions. Next Gen NEAR is such a mission, building on the NEAR Shoemaker mission experience at the JHU/APL Space Department, to provide an affordable, low risk solution with quick data return. Next Gen NEAR proposes to make measurements needed for human exploration to asteroids: to demonstrate proximity operations, to quantify hazards for human exploration and to characterize an environment at a near-Earth asteroid representative of those that may be future human destinations. The Johns Hopkins University Applied Physics Laboratory has demonstrated exploration-driven mission feasibility by developing a versatile spacecraft design concept using conventional technologies that satisfies a set of science, exploration and mission objectives defined by a concept development team in the summer of 2010. We will describe the mission concept and spacecraft architecture in detail. Configuration options were compared with the mission goals and objectives in order to select the spacecraft design concept that provides the lowest cost, lowest implementation risk, simplest operation and the most benefit for the mission implementation. The Next Gen NEAR

  14. Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells.

    PubMed

    Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

    Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol's effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

  15. Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

    PubMed Central

    Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

    2016-01-01

    Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

  16. Melatonin and its precursors in Y79 human retinoblastoma cells - Effect of sodium butyrate

    NASA Technical Reports Server (NTRS)

    Deng, Mei H.; Lopez G.-Coviella, Ignacio; Lynch, Harry J.; Wurtman, Richard J.

    1991-01-01

    We studied the release of melatonin and the production of its precursors, 5-hydroxytryptophan and serotonin, in cultured Y79 human retinoblastoma cells. This biosynthetic capability was found to be dependent on cell differentiation, which was initiated by culturing Y79 cells for 7 days in dishes coated with poly-D-lysine to promote cell adhesion to the surface of the culture dishes. Differentiation was further induced by exposing the cell monolayer to sodium butyrate (3 mM) for three days. This protocol dramatically increased the release of melatonin, and the syntheses of 5-hydroxytryptophan and serotonin in response to forskolin stimulation. Exposure to dopamine or L-DOPA markedly diminished the forskolin-stimulated release of melatonin, as well as the production of 5-hydroxytryptophan and serotonin. These observations indicate that Y79 cells represent a primitive cell line which, following appropriate differentiation can display biochemical characteristics similar to those of the human retina. Moreover, serotonin synthesis and melatonin release appear to be coupled in Y79 ceils.

  17. Melatonin and its precursors in Y79 human retinoblastoma cells: Effect of sodium butyrate

    NASA Technical Reports Server (NTRS)

    Deng, Mei Hua; Coviella, Ignacio Lopez G.; Lynch, Harry J.; Wurtman, Richard J.

    1991-01-01

    The release of melatonin and the production of its precursors, S-hydroxytryptophan and serotonin, in cultured Y79 human retinoblastoma cells were studied. This biosynthetic capability was found to be dependent on cell differentiation, which was initiated by culturing Y79 cells for 7 days in dishes coated with poly-D-lysine to promote cell adhesion to the surface of the culture dishes. Differentiation was further induced by exposing the cell monolayer to sodium butyrate (3 mM) for 3 days. This protocol dramatically increased the release of melatonin, and the syntheses of 5-hydroxytryptophan and serotonin in response to forskolin stimulation. Exposure to dopamine (10 micro-M) or L-DOPA (100 micro-M) markedly diminished the forskolin-stimulated release of melatonin, as well as the production of 5-hydroxytryptophan and serotonin. These observations indicate that Y79 cells represent a primitive cell line which, following appropriate differentiation (e.g. treatment with sodium butyrate) can display biochemical characteristics similar to those of the human retina. Moreover, serotonin synthesis and melatonin release appear to be coupled in Y79 cells. The inhibition of melatonin release by dopamine supports the hypothesis that in these cells, melatonin and dopamine are components of a retinal feedback loop.

  18. Identification of myocardial and vascular precursor cells in human and mouse epicardium.

    PubMed

    Limana, Federica; Zacheo, Antonella; Mocini, David; Mangoni, Antonella; Borsellino, Giovanna; Diamantini, Adamo; De Mori, Roberta; Battistini, Luca; Vigna, Elisa; Santini, Massimo; Loiaconi, Vincenzo; Pompilio, Giulio; Germani, Antonia; Capogrossi, Maurizio C

    2007-12-07

    During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit(+) and CD34(+) cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit(+) and CD34(+) cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit(+) cells 3 days after coronary ligation; at this time point, epicardial c-kit(+) cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit(+), together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage.

  19. The role of donor age and gender in the success of human muscle precursor cell transplantation.

    PubMed

    Stölting, Meline N L; Hefermehl, Lukas J; Tremp, Mathias; Azzabi, Fahd; Sulser, Tullio; Eberli, Daniel

    2017-02-01

    Autologous cell transplantation for the treatment of muscle damage is envisioned to involve the application of muscle precursor cells (MPCs) isolated from adult skeletal muscle. At the onset of trauma, these cells are recruited to proliferate and rebuild injured muscle fibres. However, a variety of donor-specific cues may directly influence the yield and quality of cells isolated from a muscle biopsy. In this study, we isolated human MPCs and assessed the role of donor gender and age on the ability of these MPCs to form functional bioengineered muscle. We analysed the cell yield, growth and molecular expression in vitro, and the muscle tissue formation and contractility of the bioengineered muscle, from cells isolated from men and women in three different age groups: young (20-39 years), adult (40-59 years) and elderly (60-80 years). Our results suggest that human MPCs can be successfully isolated and grown from patients of all ages and both genders. However, young female donors provide fast-growing cells in vitro with an optimum contractile output in vivo and are therefore an ideal cell source for muscle reconstruction. Taken together, these findings describe the donor-related limitations of MPC transplantation and provide insights for a straightforward and unbiased clinical application of these cells for muscle reconstruction. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Recombinant human CD19L-sTRAIL effectively targets B cell precursor acute lymphoblastic leukemia

    PubMed Central

    Uckun, Fatih M.; Myers, Dorothea E.; Qazi, Sanjive; Ozer, Zahide; Rose, Rebecca; D’Cruz, Osmond J.; Ma, Hong

    2015-01-01

    Patients with B cell precursor acute lymphoblastic leukemia (BPL) respond well to chemotherapy at initial diagnosis; however, therapeutic options are limited for individuals with BPL who relapse. Almost all BPL cells express CD19, and we recently cloned the gene encoding a natural ligand of the human CD19 receptor (CD19L). We hypothesized that fusion of CD19L to the soluble extracellular domain of proapoptotic TNF-related apoptosis-inducing ligand (sTRAIL) would markedly enhance the potency of sTRAIL and specifically induce BPL cell apoptosis due to membrane anchoring of sTRAIL and simultaneous activation of the CD19 and TRAIL receptor (TRAIL-R) apoptosis signaling pathways. Here, we demonstrate that recombinant human CD19L-sTRAIL was substantially more potent than sTRAIL and induced apoptosis in primary leukemia cells taken directly from BPL patients. CD19L-sTRAIL effectively targeted and eliminated in vivo clonogenic BPL xenograft cells, even at femtomolar-picomolar concentrations. In mice, CD19L-sTRAIL exhibited a more favorable pharmacokinetic (PK) profile than sTRAIL and was nontoxic at doses ranging from 32 fmol/kg to 3.2 pmol/kg. CD19L-sTRAIL showed potent in vivo antileukemic activity in NOD/SCID mouse xenograft models of relapsed and chemotherapy-resistant BPL at nontoxic fmol/kg dose levels. Together, these results suggest that recombinant human CD19L-sTRAIL has clinical potential as a biotherapeutic agent against BPL. PMID:25621496

  1. Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein

    PubMed Central

    1988-01-01

    A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2- terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2- terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic. PMID:3199069

  2. Derivation of Neural Precursor Cells from Human Embryonic Stem Cells for DNA Methylomic Analysis.

    PubMed

    Roubal, Ivan; Park, Sun Joo; Kim, Yong

    2016-01-01

    Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application.

  3. Human Neural Precursor Cells Promote Neurologic Recovery in a Viral Model of Multiple Sclerosis

    PubMed Central

    Chen, Lu; Coleman, Ronald; Leang, Ronika; Tran, Ha; Kopf, Alexandra; Walsh, Craig M.; Sears-Kraxberger, Ilse; Steward, Oswald; Macklin, Wendy B.; Loring, Jeanne F.; Lane, Thomas E.

    2014-01-01

    Summary Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments. PMID:24936469

  4. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis.

  5. Tau isoforms imbalance impairs the axonal transport of the amyloid precursor protein in human neurons.

    PubMed

    Lacovich, Valentina; Espindola, Sonia L; Alloatti, Matías; Pozo Devoto, Victorio; Cromberg, Lucas; Čarná, Mária; Forte, Giancarlo; Gallo, Jean-Marc; Bruno, Luciana; Stokin, Gorazd B; Avale, M Elena; Falzone, Tomás L

    2016-11-11

    Tau, as a microtubule-associated protein, participates in key neuronal functions such as the regulation of microtubule dynamics, axonal transport and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) microtubule binding repeats. While tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated to the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here we addressed this question using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectories analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP-vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Contrarely, the imbalance towards the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoforms imbalances with APP abnormal metabolism in neurodegenerative processes.

  6. Tau Isoforms Imbalance Impairs the Axonal Transport of the Amyloid Precursor Protein in Human Neurons.

    PubMed

    Lacovich, Valentina; Espindola, Sonia L; Alloatti, Matías; Pozo Devoto, Victorio; Cromberg, Lucas E; Čarná, Mária E; Forte, Giancarlo; Gallo, Jean-Marc; Bruno, Luciana; Stokin, Gorazd B; Avale, M Elena; Falzone, Tomás L

    2017-01-04

    Tau, as a microtubule (MT)-associated protein, participates in key neuronal functions such as the regulation of MT dynamics, axonal transport, and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) MT binding repeats. Although tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated with the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here, we addressed this issue using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content, neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectory analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Conversely, the imbalance toward the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoform imbalances with APP abnormal metabolism in neurodegenerative processes.

  7. Effects of an oral glucose tolerance test on the myogenic response in healthy individuals.

    PubMed

    Lott, Mary E J; Hogeman, Cynthia; Herr, Michael; Gabbay, Robert; Sinoway, Lawrence I

    2007-01-01

    The myogenic response, the inherent ability of blood vessels to rapidly respond to changes in transmural pressure, is involved in local blood flow autoregulation. Animal studies suggest that both acute hyperglycemia and hyperinsulinemia may impair myogenic vasoconstriction. The purpose of this study was to examine the effects of an oral glucose load on brachial mean blood velocity (MBV) during increases in forearm transmural pressure in humans. Eight healthy men and women (38 +/- 5 yr) underwent an oral glucose tolerance test (OGTT). MBV (in cm/s; Doppler ultrasound) responses to a rise in forearm transmural pressure (arm tank suction, -50 mmHg) were studied before and every 30 min for 120 min during the OGTT. Before the start of the OGTT, MBV was lower than baseline values 30 and 60 s after the application of negative pressure. This suggests that myogenic constriction was present. During the OGTT, blood glucose rose from 88 +/- 2 to 120 +/- 6 mg/dl (P < 0.05) and insulin rose from 14 +/- 1 to 101 +/- 32 microU/ml (P < 0.05). Glucose loading attenuated the reduction in MBV with arm suction (Delta-0.73 +/- 0.14 vs. Delta-1.67 +/- 0.43 cm/s and Delta-1.07 +/- 0.14 vs. Delta-2.38 +/- 0.54 cm/s, respectively, during 30 and 60 s of suction postglucose compared with preglucose values; all P < 0.05). We observed no such time effect for myogenic responses during a sham OGTT. In an additional 5 subjects, glucose loading had no effect on brachial diameters with the application of negative pressure. Oral glucose loading leads to attenuated myogenic vasoconstriction in healthy individuals. The role that this diminished postglucose reactivity plays in mediating postprandial hypotension and/or orthostasis needs to be further explored.

  8. Myogenic Potential of Canine Craniofacial Satellite Cells

    PubMed Central

    La Rovere, Rita Maria Laura; Quattrocelli, Mattia; Pietrangelo, Tiziana; Di Filippo, Ester Sara; Maccatrozzo, Lisa; Cassano, Marco; Mascarello, Francesco; Barthélémy, Inès; Blot, Stephane; Sampaolesi, Maurilio; Fulle, Stefania

    2014-01-01

    The skeletal fibers have different embryological origin; the extraocular and jaw-closer muscles develop from prechordal mesoderm while the limb and trunk muscles from somites. These different origins characterize also the adult muscle stem cells, known as satellite cells (SCs) and responsible for the fiber growth and regeneration. The physiological properties of presomitic SCs and their epigenetics are poorly studied despite their peculiar characteristics to preserve muscle integrity during chronic muscle degeneration. Here, we isolated SCs from canine somitic [somite-derived muscle (SDM): vastus lateralis, rectus abdominis, gluteus superficialis, biceps femoris, psoas] and presomitic [pre-somite-derived muscle (PSDM): lateral rectus, temporalis, and retractor bulbi] muscles as myogenic progenitor cells from young and old animals. In addition, SDM and PSDM-SCs were obtained also from golden retrievers affected by muscular dystrophy (GRMD). We characterized the lifespan, the myogenic potential and functions, and oxidative stress of both somitic and presomitic SCs with the aim to reveal differences with aging and between healthy and dystrophic animals. The different proliferation rate was consistent with higher telomerase activity in PSDM-SCs compared to SDM-SCs, although restricted at early passages. SDM-SCs express early (Pax7, MyoD) and late (myosin heavy chain, myogenin) myogenic markers differently from PSDM-SCs resulting in a more efficient and faster cell differentiation. Taken together, our results showed that PSDM-SCs elicit a stronger stem cell phenotype compared to SDM ones. Finally, myomiR expression profile reveals a unique epigenetic signature in GRMD SCs and miR-206, highly expressed in dystrophic SCs, seems to play a critical role in muscle degeneration. Thus, miR-206 could represent a potential target for novel therapeutic approaches. PMID:24860499

  9. Myogenous temporomandibular disorders: diagnostic and management considerations.

    PubMed

    Fricton, James

    2007-01-01

    Myogenous temporomandibular disorders (or masticatory myalgia) are characterized by pain and dysfunction that arise from pathologic and functional processes in the masticatory muscles. There are several distinct muscle disorder subtypes in the masticatory system, including myofascial pain, myositis, muscle spasm, and muscle contracture. The major characteristics of masticatory myalgia include pain, muscle tenderness, limited range of motion, and other symptoms (eg, fatigability, stiffness, subjective weakness). Comorbid conditions and complicating factors also are common and are discussed. Management follows with stretching, posture, and relaxation exercises, physical therapy, reduction of contributing factors, and as necessary, muscle injections.

  10. ROCK2 and Its Alternatively Spliced Isoform ROCK2m Positively Control the Maturation of the Myogenic Program▿

    PubMed Central

    Pelosi, Michele; Marampon, Francesco; Zani, Bianca M.; Prudente, Sabrina; Perlas, Emerald; Caputo, Viviana; Cianetti, Luciano; Berno, Valeria; Narumiya, Shuh; Kang, Shin W.; Musarò, Antonio; Rosenthal, Nadia

    2007-01-01

    Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation. PMID:17606625

  11. Tripartite containing motif 32 modulates proliferation of human neural precursor cells in HIV-1 neurodegeneration

    PubMed Central

    Fatima, M; Kumari, R; Schwamborn, J C; Mahadevan, A; Shankar, S K; Raja, R; Seth, P

    2016-01-01

    In addition to glial cells, HIV-1 infection occurs in multipotent human neural precursor cells (hNPCs) and induces quiescence in NPCs. HIV-1 infection of the brain alters hNPC stemness, leading to perturbed endogenous neurorestoration of the CNS following brain damage by HIV-1, compounding the severity of dementia in adult neuroAIDS cases. In pediatric neuroAIDS cases, HIV-1 infection of neural stem cell can lead to delayed developmental milestones and impaired cognition. Using primary cultures of human fetal brain-derived hNPCs, we gained novel insights into the role of a neural stem cell determinant, tripartite containing motif 32 (TRIM32), in HIV-1 Tat-induced quiescence of NPCs. Acute HIV-1 Tat treatment of hNPCs resulted in proliferation arrest but did not induce differentiation. Cellular localization and levels of TRIM32 are critical regulators of stemness of NPCs. HIV-1 Tat exposure increased nuclear localization and levels of TRIM32 in hNPCs. The in vitro findings were validated by studying TRIM32 localization and levels in frontal cortex of HIV-1-seropositive adult patients collected at post mortem as well as by infection of hNPCs by HIV-1. We observed increased percentage of cells with nuclear localization of TRIM32 in the subventricular zone (SVZ) as compared with age-matched controls. Our quest for probing into the mechanisms revealed that TRIM32 is targeted by miR-155 as downregulation of miR-155 by HIV-1 Tat resulted in upregulation of TRIM32 levels. Furthermore, miR-155 or siRNA against TRIM32 rescued HIV-1 Tat-induced quiescence in NPCs. Our findings suggest a novel molecular cascade involving miR-155 and TRIM32 leading to HIV-1 Tat-induced attenuated proliferation of hNPCs. The study also uncovered an unidentified role for miR-155 in modulating human neural stem cell proliferation, helping in better understanding of hNPCs and diseased brain. PMID:26586575

  12. Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.

    PubMed

    Ciapetti, Gabriela; Di Pompo, Gemma; Avnet, Sofia; Martini, Desirée; Diez-Escudero, Anna; Montufar, Edgar B; Ginebra, Maria-Pau; Baldini, Nicola

    2017-03-01

    The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca(++)) and phosphorus (P(5+)) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca(++). Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.

  13. Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

    PubMed Central

    Swerdel, Mavis R.; Moore, Jennifer C.; Cohen, Rick I.; Wu, Hao; Sun, Yi E.; Hart, Ronald P.

    2009-01-01

    Background MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. Methodology/Principal Findings SOLiD ultra-deep sequencing identified >107 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. Conclusions/Significance Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation. PMID:19784364

  14. Treatment with Tyrosine a Neurotransmitter Precursor Reduces Environmental Stress in Humans

    DTIC Science & Technology

    1989-01-01

    brain norepinephrine and dopamine. catecholaminergic neurotransmitters. In animals, administration of tyrosine, a food constituent and precursor of the...Profile of Mood States. Stanford Sleepiness Scale) ENVIRONMENTAL STRESSORS that have been employed to evaluate a variety of psychoactive drugs foods ... tyramine . However. Plasma tyrosine levels were significantly elevated during behav- this amine is not detectable in the plasma of animals after they

  15. Narrow Band Ultraviolet B Treatment for Human Vitiligo Is Associated with Proliferation, Migration, and Differentiation of Melanocyte Precursors.

    PubMed

    Goldstein, Nathaniel B; Koster, Maranke I; Hoaglin, Laura G; Spoelstra, Nicole S; Kechris, Katerina J; Robinson, Steven E; Robinson, William A; Roop, Dennis R; Norris, David A; Birlea, Stanca A

    2015-08-01

    In vitiligo, the autoimmune destruction of epidermal melanocytes produces white spots that can be repigmented by melanocyte precursors from the hair follicles, following stimulation with UV light. We examined by immunofluorescence the distribution of melanocyte markers (C-KIT, DCT, PAX3, and TYR) coupled with markers of proliferation (KI-67) and migration (MCAM) in precursors and mature melanocytes from the hair follicle and the epidermis of untreated and narrow band UVB (NBUVB)-treated human vitiligo skin. NBUVB was associated with a significant increase in the number of melanocytes in the infundibulum and with restoration of the normal melanocyte population in the epidermis, which was lacking in the untreated vitiligo. We identified several precursor populations (melanocyte stem cells, melanoblasts, and other immature phenotypes), and progressively differentiating melanocytes, some with putative migratory and/or proliferative abilities. The primary melanocyte germ was present in the untreated and treated hair follicle bulge, whereas a possible secondary melanocyte germ composed of C-KIT+ melanocytes was found in the infundibulum and interfollicular epidermis of UV-treated vitiligo. This is an exceptional model for studying the mobilization of melanocyte stem cells in human skin. Improved understanding of this process is essential for designing better treatments for vitiligo, ultimately based on melanocyte stem cell activation and mobilization.

  16. Gastric secretion of platelet activating factor and precursors in healthy humans: effect of pentagastrin.

    PubMed Central

    Sobhani, I; Denizot, Y; Hochlaf, S; Rigaud, D; Vatier, J; Benveniste, J; Lewin, M J; Mignon, M

    1993-01-01

    The release of platelet activating factor (PAF-ACETHER or PAF) and its precursors in the gastric lumen was assessed in 13 normal subjects in basal condition and after stimulation by gastrin. Acid, pepsin, and sialic acid outputs were determined under the same conditions. Gastric juice was collected using a nasogastric tube after overnight fast in basal condition for 60 minutes, then under pentagastrin infusion (6 micrograms/kg/hr for 60 minutes). Platelet activating factor was detected at low concentration in 4/13 subjects under basal condition (mean (SEM) 1.2 (0.6) pg/hr) while high concentrations of lyso platelet activating factor (6.1 (1.8) microgram/hr) and of alkyl-acyl-glycerophosphocholine (AAGPC) (11.5 (3) micrograms/hr) were found in 13 and 11 subjects, respectively. Platelet activating factor was not detected during pentagastrin infusion, while lyso platelet activating factor and alkyl-acyl-glycerophosphocholine were detected in 13 and in 12 subjects, respectively. Compared with the basal condition these platelet activating factor precursors increased significantly (p < 0.001) going up to fivefold baseline (31.8 (6.8) micrograms/hr and 53 (9.3) micrograms/hr respectively) in response to pentagastrin. There was a positive correlation between platelet activating factor precursors and acid or pepsin output but not between platelet activating factor precursors and sialic acid. As sialic acid may be considered an index of mucus glycoprotein degradation, it seems that gastrin stimulation of gastric epithelial cells results in a concomittant secretion of platelet activating factor precursors, acid, and pepsin irrespective of mucus glycoprotein degradation. PMID:8174952

  17. Enhanced functional integration of human photoreceptor precursors into human and rodent retina in an ex vivo retinal explant model system.

    PubMed

    Yanai, Anat; Laver, Christopher R J; Gregory-Evans, Cheryl Y; Liu, Ran R; Gregory-Evans, Kevin

    2015-06-01

    Retinal disease is the major cause of irreversible blindness in developed countries. Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is a promising and widely applicable approach for the treatment of these blinding conditions. Previously, it has been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is low. The factors that inhibit PPC engraftment remain largely unknown, in part, because so many adverse factors could be at play during in vivo experiments. To advance our knowledge in overcoming potential adverse effects and optimize PPC transplantation, we have developed a novel ex vivo system. Harvested neural retina was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Explants cocultured with hESC-derived RPE maintained normal gross morphology and viability for up to 2 weeks, whereas the explants cultured on ARPE19 and RPE-J failed by 7 days. Furthermore, the proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when cocultured with hESC-derived RPE (20% and 10%, respectively), than when cocultured with ARPE19 (only 6% and 2%, respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs cocultured with hESC-derived RPE increased to ∼30% and 15%, respectively. These data demonstrate the utility of an ex vivo model system to define factors, such as TSP-1, which could influence integration efficiency in future in vivo experiments in models of retinal degeneration.

  18. Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

    PubMed Central

    Tenente, Inês M; Hayes, Madeline N; Ignatius, Myron S; McCarthy, Karin; Yohe, Marielle; Sindiri, Sivasish; Gryder, Berkley; Oliveira, Mariana L; Ramakrishnan, Ashwin; Tang, Qin; Chen, Eleanor Y; Petur Nielsen, G; Khan, Javed; Langenau, David M

    2017-01-01

    Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth. DOI: http://dx.doi.org/10.7554/eLife.19214.001 PMID:28080960

  19. Distribution of precursor amyloid-. beta. -protein messenger RNA in human cerebral cortex: relationship to neurofibrillary tangles and neuritic plaques

    SciTech Connect

    Lewis, D.A.; Higgins, G.A.; Young, W.G.; Goldgaber, D.; Gajdusek, D.C.; Wilson, M.C.; Morrison, J.H.

    1988-03-01

    Neurofibrillary tangles (NFT) and neuritic plaques (NP), two neuropathological markers of Alzheimer disease, may both contain peptide fragments derived from the human amyloid ..beta.. protein. However, the nature of the relationship between NFT and NP and the source of the amyloid ..beta.. proteins found in each have remained unclear. The authors used in situ hybridization techniques to map the anatomical distribution of precursor amyloid-..beta..-protein mRNA in the neocortex of brains from three subjects with no known neurologic disease and from five patients with Alzheimer disease. In brains from control subjects, positively hybridizing neurons were present in cortical regions and layers that contain a high density of neuropathological markers in Alzheimer disease, as well as in those loci that contain NP but few NFT. Quantitative analyses of in situ hybridization patterns within layers III and V of the superior frontal cortex revealed that the presence of high numbers of NFT in Alzheimer-diseased brains was associated with a decrease in the number of positively hybridizing neurons compared to controls and Alzheimer-diseased brains with few NFT. These findings suggest that the expression of precursor amyloid-..beta..-protein mRNA may be a necessary but is clearly not a sufficient prerequisite for NFT formation. In addition, these results may indicate that the amyloid ..beta.. protein, present in NP in a given region or layer of cortex, is not derived from the resident neuronal cell bodies that express the mRNA for the precursor protein.

  20. CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells

    PubMed Central

    Vacca, Paola; Vitale, Chiara; Montaldo, Elisa; Conte, Romana; Cantoni, Claudia; Fulcheri, Ezio; Darretta, Valeria; Moretta, Lorenzo; Mingari, Maria Cristina

    2011-01-01

    Natural killer (NK) cells are the main lymphoid population in the maternal decidua during the first trimester of pregnancy. Decidual NK (dNK) cells display a unique functional profile and play a key role in promoting tissue remodeling, neoangiogenesis, and immune modulation. However, little information exists on their origin and development. Here we discovered CD34+ hematopoietic precursors in human decidua (dCD34+). We show that dCD34+ cells differ from cord blood- or peripheral blood-derived CD34+ precursors. The expression of IL-15/IL-2 receptor common β-chain (CD122), IL-7 receptor α-chain (CD127), and mRNA for E4BP4 and ID2 transcription factors suggested that dCD34+ cells are committed to the NK cell lineage. Moreover, they could undergo in vitro differentiation into functional (i.e., IL-8– and IL-22–producing) CD56brightCD16−KIR+/− NK cells in the presence of growth factors or even upon coculture with decidual stromal cells. Their NK cell commitment was further supported by the failure to undergo myeloid differentiation in the presence of GM-CSF. Our findings strongly suggest that decidual NK cells may directly derive from CD34+ cell precursors present in the decidua upon specific cellular interactions with components of the decidual microenvironment. PMID:21248224

  1. miR-127 enhances myogenic cell differentiation by targeting S1PR3.

    PubMed

    Zhai, Lili; Wu, Rimao; Han, Wanhong; Zhang, Yong; Zhu, Dahai

    2017-03-30

    MicroRNAs (miRNAs) have recently been implicated in muscle stem cell function. miR-127 is known to be predominantly expressed in skeletal muscle, but its roles in myogenic differentiation and muscle regeneration are unknown. Here, we show that miR-127 is upregulated during C2C12 and satellite cell (SC) differentiation and, by establishing C2C12 cells stably expressing miR-127, demonstrate that overexpression of miR-127 in C2C12 cells enhances myogenic cell differentiation. To investigate the function of miR-127 during muscle development and regeneration in vivo, we generated miR-127 transgenic mice. These mice exhibited remarkably accelerated muscle regeneration compared with wild-type mice by promoting SC differentiation. Mechanistically, we demonstrated that the gene encoding sphingosine-1-phosphate receptor 3 (S1PR3), a G-protein-coupled receptor for sphingosine-1-phosphate, is a target of miR-127 required for its function in promoting myogenic cell differentiation. Importantly, overexpression of miR-127 in muscular dystrophy model mdx mice considerably ameliorated the disease phenotype. Thus, our findings suggest that miR-127 may serve as a potential therapeutic target for the treatment of skeletal muscle disease in humans.

  2. Human hepatitis B viral e antigen and its precursor P20 inhibit T lymphocyte proliferation

    SciTech Connect

    Purvina, Maija; Hoste, Astrid; Rossignol, Jean-Michel; Lagaudriere-Gesbert, Cecile

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer P20, precursor of the HBeAg, interacts with the cellular protein gC1qR. Black-Right-Pointing-Pointer HBeAg and P20 bind to T cell surface and inhibit mitogen-induced T cell division. Black-Right-Pointing-Pointer HBeAg and P20 inhibition of T cell proliferation is gC1qR and IL-1RAcP-independent. -- Abstract: The hepatitis B virus (HBV) Precore protein is processed through the secretory pathway directly as HBeAg or with the generation of an intermediate (P20). Precore gene has been shown to be implicated in viral persistence, but the functions of HBeAg and its precursors have not been fully elucidated. We show that the secreted proteins HBeAg and P20 interact with T cell surface and alter Kit-225 and primary T cells proliferation, a process which may facilitate the establishment of HBV persistence. Our data indicate that the N-terminal end of Precore is important for these inhibitory effects and exclude that they are dependent on the association of HBeAg and P20 with two characterized cell surface ligands, the Interleukin-1 Receptor Accessory Protein and gC1qR (present study).

  3. [Primary neurogenic and myogenic disorders of posture].

    PubMed

    Schranz, C; Meinck, H-M

    2004-05-01

    Disturbance of posture may occur in a variety of neurological disorders and occasionally is the presenting or even the only sign. In the majority of cases, the head or the trunk or both are bent forward (bent spine syndrome, dropped head syndrome). A feature of these primary neurogenic or myogenic postural disturbances that is in contrast to antalgic contraction or ankylosis is that they are not fixed, but the trunk or head are easily erected by the examiner and show a characteristic sagging. Neuromuscular disorders are a frequent cause. They may be confined to the paraspinal muscles. Axial computed tomography of the spine, electromyography of the involved muscles, and muscle biopsy help to make the diagnosis. However, also central movement disorders may lead to a sagging of the head or trunk or of both due to a lessened tone of the head and trunk extensors. This is frequently seen in the various parkinsonian syndromes which may, however, occur in association with a focal myopathy of the paraspinal muscles. Occasionally, sagging of the trunk is seen as a side effect of neuropharmacologic medication. Sagging of the trunk or head should be differentiated from a pathologically increased innervation of the ventral muscles in dystonic movement disorders such as antecollis or camptocormia. Pathologic reclination of the head or trunk or both is a rare disturbance of posture. It may occur in dystonia (retrocollis) or, occasionally, as a consequence of musculotendinous contractures secondary to certain neuromuscular disorders such as the rigid spine syndrome.

  4. Decorin expression in quiescent myogenic cells

    SciTech Connect

    Nishimura, Takanori Nozu, Kenjiro; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito

    2008-06-06

    Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.

  5. The amyloid precursor-like protein (APLP) gene maps to the long arm of human chromosome 19

    SciTech Connect

    Wasco, W.; Tanzi, R.E. ); Brook, J.D. )

    1993-01-01

    We have recently isolated a cDNA from a mouse brain library that encodes a protein whose predicted amino acid sequence is 42% identical and 64% similar to that of the amyloid [beta] protein precursor (APP; 16). This 653-amino-acid amyloid precursor-like protein (APLP) is similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are particularly strong in three distinct regions of the proteins where the identities are 47, 54, and 56% (16). All three of these regions are also conserved in the Drosophila APP-like gene, APPL (11). Notably, 12 cysteine residues and a N -glyco-sylation site are conserved in the extracellular portion of APLP and APP, and a clathrin-binding domain is conserved in the cytoplasmic domain. The cytoplasmic domain is also conserved in a partial CDNA reported to encode an APP-like gene in rat testes (17), These data suggest that APLP and APP are members of a highly conserved gene family. A panel of DNAs from 31 human-rodent somatic cell lines of known karyotype was digested with EcoR1. These DNAs were then probed with the human APLP cDNA clone and the hybridization pattern was consistent with the assignment of the APLP locus to chromosome 19. 17 refs., 1 fig.

  6. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    SciTech Connect

    Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

    2012-06-08

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

  7. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer's Disease.

    PubMed

    Brownjohn, Philip W; Smith, James; Portelius, Erik; Serneels, Lutgarde; Kvartsberg, Hlin; De Strooper, Bart; Blennow, Kaj; Zetterberg, Henrik; Livesey, Frederick J

    2017-03-06

    Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP) processing and the accumulation of APP-derived amyloid β (Aβ) peptides are key processes in Alzheimer's disease (AD). We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation.

  8. Most human non-GCIMP glioblastoma subtypes evolve from a common proneural-like precursor glioma

    PubMed Central

    Ozawa, Tatsuya; Riester, Markus; Cheng, Yu-Kang; Huse, Jason T; Squatrito, Massimo; Helmy, Karim; Charles, Nikki; Michor, Franziska; Holland, Eric C.

    2014-01-01

    SUMMARY To understand the relationships between the non-GCIMP glioblastoma (GBM) subgroups, we performed mathematical modeling to predict the temporal sequence of driver events during tumorigenesis. The most common order of evolutionary events is 1) chromosome (chr) 7 gain and chr10 loss, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes. We then developed a computational methodology to identify drivers of broad copy number changes, identifying PDGFA (chr7) and PTEN (chr10) as driving initial non-disjunction events. These predictions were validated using mouse modeling, showing that PDGFA is sufficient to induce proneural-like gliomas, and additional NF1 loss converts proneural to the mesenchymal subtype. Our findings suggest most non-GCIMP-mesenchymal GBMs arise as, and evolve from, a proneural-like precursor. PMID:25117714

  9. Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection

    PubMed Central

    Goldstein, Nathaniel B.; Koster, Maranke I.; Hoaglin, Laura G.; Wright, Michael J.; Robinson, Steven E.; Robinson, William A.; Roop, Dennis R.; Norris, David A.; Birlea, Stanca A.

    2017-01-01

    To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders. PMID:27193292

  10. Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide

    SciTech Connect

    Winton, E.F.; Colenda, K.W.

    1987-07-01

    The continued retrieval of progenitor cells (CFU-GEMM, BFU-E, CFU-E, CFU-GM) from human long-term marrow cultures (LTMC) is not uncommonly used as evidence that proliferation and differentiation are occurring in more primitive hematopoietic stem cells (HSC) in these cultures. Alternatively, the continued presence of progenitors in LTMC could be the result of survival and/or limited self-renewal of progenitor cells present when the culture was initiated, and such progenitors would have little relevance to the parent HSC. The following studies were designed to determine the relative contributions of precursors of progenitor cells to the total progenitor cells present in LTMC using a two-stage regeneration model. The adherent layer in LTMC was established over 3 weeks, irradiated (875 rad) to permanently eliminate resident hematopoietic cells, and recharged with autologous cryo-preserved marrow that was either treated or not treated (control) with 4-hydroperoxycyclophosphamide (4-HC, 100 micrograms/ml for 30 min). The 4-HC-treated marrow contained no progenitor cells, yet based on clinical autologous bone marrow transplant experience, has intact HSC. Within 1-3 weeks, progenitor cells reappeared in the irradiated LTMC recharged with 4-HC-treated marrow, and were preferentially located in the adherent layer. By 2-6 weeks, the number of progenitor cells in the adherent layer of LTMC recharged with 4-HC marrow was equivalent to control LTMC. The progenitors regenerating in the irradiated LTMC recharged with 4-HC-treated marrow appear to originate from precursors of progenitor cells, perhaps HSC. We propose this model may be useful in elucidating cellular and molecular correlates of progenitor cell regeneration from precursors.

  11. Immunohistochemical examination of gastrin, gastrin precursors, and gastrin/CCK-2 receptor in human esophageal squamous cell carcinomas.

    PubMed

    Yuan, Aping; Liu, Jinzhong; Liu, Yiqing; Bjørnsen, Tone; Varro, Andrea; Cui, Guanglin

    2008-12-01

    A promoting effect of gastrin on stimulating Barrett's oesophagus proliferation has been demonstrated, but whether it plays a regulating role for esophageal squamous cell carcinoma (ESCC) to date has not been fully investigated. The aim of this study is to examine the expressions of gastrin, gastrin precursors and gastrin/CCK-2 receptor in ESCC. Tissue specimen sections from 38 patients with ESSC obtained from a high incidence area of north China were assessed using immunohistochemistry for amidated gastrin, gastrin precursors (progastrin and glycine-extended gastrin) and gastrin/CCK-2 receptors. Their clinical histopathological significance was also analyzed. Of 38 ESCC, the immunoreactivities of gastrin, glycine-extended gastrin and progastrin were observed in 13.2% (5/38), 7.9% (3/38) and 23.68% (9/38) cases. The expression of progastrin was obviously higher than other gastrins, though not significantly (P > 0.05). In positive cases for gastrin or glycine-extended gastrin, the scores of positive tumor cell numbers were at a lower density (<10/abundant-distributed field). However, the scores of progastrin positive tumor cell density in five of nine positive cases were over 10/abundant-distributed field. The immunoreactivity of gastrin/CCK-2 receptor was also observed in 15.8% (6/38) ESCC cases. There was not significant correlation regarding immunohistochemical results with known histomorphological parameters i.e. gender, tumor location and TNM stages. Based on our current results, ESCC tumor cells could be a possible cellular source of gastrin precursors, which has been postulated to play a role in regulating the growth in some human tumor cells.

  12. Cytoglobin modulates myogenic progenitor cell viability and muscle regeneration.

    PubMed

    Singh, Sarvjeet; Canseco, Diana C; Manda, Shilpa M; Shelton, John M; Chirumamilla, Rajendra R; Goetsch, Sean C; Ye, Qiu; Gerard, Robert D; Schneider, Jay W; Richardson, James A; Rothermel, Beverly A; Mammen, Pradeep P A

    2014-01-07

    Mammalian skeletal muscle can remodel, repair, and regenerate itself by mobilizing satellite cells, a resident population of myogenic progenitor cells. Muscle injury and subsequent activation of myogenic progenitor cells is associated with oxidative stress. Cytoglobin is a hemoprotein expressed in response to oxidative stress in a variety of tissues, including striated muscle. In this study, we demonstrate that cytoglobin is up-regulated in activated myogenic progenitor cells, where it localizes to the nucleus and contributes to cell viability. siRNA-mediated depletion of cytoglobin from C2C12 myoblasts increased levels of reactive oxygen species and apoptotic cell death both at baseline and in response to stress stimuli. Conversely, overexpression of cytoglobin reduced reactive oxygen species levels, caspase activity, and cell death. Mice in which cytoglobin was knocked out specifically in skeletal muscle were generated to examine the role of cytoglobin in vivo. Myogenic progenitor cells isolated from these mice were severely deficient in their ability to form myotubes as compared with myogenic progenitor cells from wild-type littermates. Consistent with this finding, the capacity for muscle regeneration was severely impaired in mice deficient for skeletal-muscle cytoglobin. Collectively, these data demonstrate that cytoglobin serves an important role in muscle repair and regeneration.

  13. (-)-Epigallocatechin-3-gallate stimulates myogenic differentiation through TAZ activation.

    PubMed

    Kim, A Rum; Kim, Kyung Min; Byun, Mi Ran; Hwang, Jun-Ha; Park, Jung Il; Oh, Ho Taek; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2017-04-29

    Muscle loss is a typical process of aging. Green tea consumption is known to slow down the progress of aging. Their underlying mechanisms, however, remain largely unknown. In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, on myogenic differentiation and found that EGCG significantly increases myogenic differentiation. After EGCG treatment, the expression of myogenic marker genes, such as myosin heavy chain, are increased through activation of TAZ, a transcriptional coactivator with a PDZ-binding motif. TAZ-knockdown does not stimulate EGCG-induced myogenic differentiation. EGCG facilitates the interaction between TAZ and MyoD, which stimulates MyoD-mediated gene transcription. EGCG induces nuclear localization of TAZ through the dephosphorylation of TAZ at its Ser89 residue, which relieves 14-3-3 binding in the cytosol. Interestingly, inactivation of Lats kinase is observed after EGCG treatment, which is responsible for the production of dephosphorylated TAZ. Together, these results suggest that EGCG induces myogenic differentiation through TAZ, suggesting that TAZ plays an important role in EGCG induced muscle regeneration.

  14. Identification of mitochondrial proteins and some of their precursors in two-dimensional electrophoretic maps of human cells

    SciTech Connect

    Anderson, L.

    1981-04-01

    A set of at least 30 proteins disappears from the two-dimensional electrophoretic pattern of human lymphoid cells treated with various antimitochondrial agents. This set is similar to the set of proteins found in isolated mitochondria (except for the presence of actin in the latter group), indicating that the inhibitor effect stops production of a majority of mature mitochondrial proteins. Several proteins having the characteristics of precursors to the major cytoplasmically synthesized mitochondrial proteins can be observed in cells during fast-pulse experiments and in a reticulocyte lysate system fed with total lymphoid cell RNA. In the three major instances of mitochondrial precursor-product processing, the removal peptide is quite basic in each case, suggesting that a lysine- or arginine-rich terminal sequence may be necessary for initial recognition by the mitochondrial protein uptake apparatus. The inhibitor effect allows easy identification of a large set of mitochondrial proteins in two-dimensional maps of various cells, thereby specifying a particularly tractable and functionally distinctive subset of the cellular proteins. The nature and wide scope of the effect support the concept of energy-dependent vectorial processing and indicate that such a mechanism is generally applicable to the major class of cytoplasmically synthesized mitochondrial proteins in mammalian cells.

  15. Mitochondrial dysfunction in a transgenic mouse model expressing human amyloid precursor protein (APP) with the Arctic mutation.

    PubMed

    Rönnbäck, Annica; Pavlov, Pavel F; Mansory, Mansorah; Gonze, Prisca; Marlière, Nicolas; Winblad, Bengt; Graff, Caroline; Behbahani, Homira

    2016-02-01

    Accumulation of amyloid β-peptide (Aβ) in the brain is an important event in the pathogenesis of Alzheimer disease. We have used a transgenic mouse model expressing human amyloid precursor protein (APP) with the Arctic mutation to investigate whether Aβ deposition is correlated with mitochondrial functions in these animals. We found evidence of mitochondrial dysfunction (i.e., decreased mitochondrial membrane potential, increased production of reactive oxygen species and oxidative DNA damage) at 6 months of age, when the mice showed very mild Aβ deposition. More pronounced mitochondrial abnormalities were present in 24-month-old TgAPParc mice with more extensive Aβ pathology. This study demonstrates for the first time mitochondrial dysfunction in transgenic mice with a mutation within the Aβ peptide (the Arctic APP mutation), and confirms previous studies suggesting that mitochondrial dysfunction and oxidative stress is an early event in the pathogenesis of Alzheimer disease. This study demonstrates mitochondrial dysfunction in transgenic mice with a mutation within the amyloid beta (Aβ) peptide (the Arctic amyloid precursor protein (APP) mutation). We found evidence of mitochondrial dysfunction (i.e. decreased mitochondrial membrane potential (MMP), increased production of reactive oxygen species (ROS) and oxidative DNA damage) at 6 months of age, when very mild Aβ deposition is present in the mice. Also, the cytochrome c (COX) activity was significantly decreased in mitochondria from transgenic mice at 24 months of age.

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

    SciTech Connect

    Ohashi, Kazuya; Nagata, Yosuke; Wada, Eiji; Zammit, Peter S.; Shiozuka, Masataka; Matsuda, Ryoichi

    2015-05-01

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

  17. Functional plasticity of the N-methyl-d-aspartate receptor in differentiating human erythroid precursor cells

    PubMed Central

    Hänggi, Pascal; Telezhkin, Vsevolod; Kemp, Paul J.; Schmugge, Markus; Gassmann, Max; Goede, Jeroen S.; Speer, Oliver

    2015-01-01

    Calcium signaling is essential to support erythroid proliferation and differentiation. Precise control of the intracellular Ca2+ levels in erythroid precursor cells (EPCs) is afforded by coordinated expression and function of several cation channels, including the recently identified N-methyl-d-aspartate receptor (NMDAR). Here, we characterized the changes in Ca2+ uptake and electric currents mediated by the NMDARs occurring during EPC differentiation using flow cytometry and patch clamp. During erythropoietic maturation, subunit composition and properties of the receptor changed; in proerythroblasts and basophilic erythroblasts, fast deactivating currents with high amplitudes were mediated by the GluN2A subunit-dominated receptors, while at the polychromatic and orthochromatic erythroblast stages, the GluN2C subunit was getting more abundant, overriding the expression of GluN2A. At these stages, the currents mediated by the NMDARs carried the features characteristic of the GluN2C-containing receptors, such as prolonged decay time and lower conductance. Kinetics of this switch in NMDAR properties and abundance varied markedly from donor to donor. Despite this variability, NMDARs were essential for survival of EPCs in any subject tested. Our findings indicate that NMDARs have a dual role during erythropoiesis, supporting survival of polychromatic erythroblasts and contributing to the Ca2+ homeostasis from the orthochromatic erythroblast stage to circulating red blood cells. PMID:25788577

  18. Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.

    PubMed

    Turner, Paul R; Bourne, Katie; Garama, Daniel; Carne, Alan; Abraham, Wickliffe C; Tate, Warren P

    2007-08-15

    The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.

  19. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.

    PubMed Central

    Goldgaber, D; Harris, H W; Hla, T; Maciag, T; Donnelly, R J; Jacobsen, J S; Vitek, M P; Gajdusek, D C

    1989-01-01

    We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter. Images PMID:2508093

  20. Effects of ethanol on aggregation, serotonin release, and amyloid precursor protein processing in rat and human platelets.

    PubMed

    Ehrlich, Daniela; Humpel, Christian

    2014-01-01

    It is known that oxidative stress leads to amyloid precursor protein (APP) dysregulation in platelets. Ethanol (EtOH) is a vascular risk factor and induces oxidative stress. The aim of the present study was thus to investigate whether EtOH affects APP processing in rat and human platelets. Platelets were exposed to 50 mM EtOH with and without 2 mM calcium-chloride (CaCl₂) for 20 or 180 minutes at 37°C. Platelet aggregation, serotonin release and APP isoforms 130 and 106/110 kDa were analyzed. As a control, 100 mM H₂O₂ was tested in rat platelets. Our data show that EtOH alone did not affect any of the analyzed parameters, whereas CaCl₂ significantly increased aggregation of rat and human platelets. In addition, CaCl₂ alone enhanced serotonin release in rat platelets. EtOH counteracted CaCl₂-induced aggregation and serotonin release. In the presence of CaCl₂, EtOH reduced the 130 kDa APP isoform in rat and human platelets. In conclusion, this study shows that in the presence of CaCl₂, EtOH affects the platelet function and APP processing in rat and human platelets.

  1. Superoxide modulates myogenic contractions of mouse afferent arterioles.

    PubMed

    Lai, En Yin; Wellstein, Anton; Welch, William J; Wilcox, Christopher S

    2011-10-01

    Reactive oxygen species enhance or impair autoregulation. Because superoxide is a vasoconstrictor, we tested the hypothesis that stretch generates superoxide that mediates myogenic responses. Increasing perfusion pressure of mouse isolated perfused renal afferent arterioles from 40 to 80 mm Hg reduced their diameter by 13.3±1.8% (P<0.001) and increased reactive oxygen species (ethidium: dihydroethidium fluorescence) by 9.8±2.3% (P<0.05). Stretch-induced fluorescence was reduced significantly (P<0.05) by incubation with Tempol (3.7±0.8%), pegylated superoxide dismutase (3.2±1.0%), or apocynin (3.5±0.9%) but not by pegylated catalase, L-nitroarginine methylester, or Ca(2+)-free medium, relating it to Ca(2+)-independent vascular superoxide. Compared with vehicle, basal tone and myogenic contractions were reduced significantly (P<0.05) by pegylated superoxide dismutase (5.4±0.8), Tempol (4.1±1.0%), apocynin (1.0±1.3%), and diphenyleneiodinium (3.9±0.9%) but not by pegylated catalase (10.1±1.6%). L-Nitroarginine methylester enhanced basal tone, but neither it (15.8±3.3%) nor endothelial NO synthase knockout (10.2±1.8%) significantly changed myogenic contractions. Tempol had no further effect after superoxide dismutase but remained effective after catalase. H(2)O(2) >50 μmol/L caused contractions but at 25 μmol/L inhibited myogenic responses (7.4±0.8%; P<0.01). In conclusion, increasing the pressure within afferent arterioles led to Ca(2+)-independent increased vascular superoxide production from nicotinamide adenine dinucleotide phosphate oxidase, which enhanced myogenic contractions largely independent of NO, whereas H(2)O(2) impaired pressure-induced contractions but was not implicated in the normal myogenic response.

  2. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells.

    PubMed

    Studer, R; Baysang, G; Brack, C

    2001-01-01

    The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

  3. Detection of the single-chain precursor in the production and purification process of recombinant human insulin.

    PubMed

    Leng, Chunsheng; Li, Qingwei; Wu, Fenfang; Chen, Liyong; Su, Peng

    2013-08-01

    High quality recombinant insulin requires being free of single-chain precursor (proinsulin), a task that depends on the selectivity and sensitivity of the monitoring process for detecting proinsulin. In this study we developed an enzyme-linked immunosorbent assay (ELISA) system that was specifically tailored to detect recombinant proinsulin. The proinsulin consists of six components: an initiating methionine, 48 amino acids from human growth hormones (HGH, used as the protection peptide), first connecting Arg-residue, B-chain of insulin, and second connecting Arg-peptide and A-chain of insulin. This form of proinsulin is more stable and can be efficiently expressed by E. coli than insulin. Herein, we evaluated the specificity, precision, recovery, sensitivity, and detection range of the proinsulin ELISA kit. The results showed that the ELISA kit is a very useful tool for monitoring the proinsulin yield in early stages of insulin production as well as the residual proinsulin in the final product, insulin.

  4. Robosphere: Self Sustaining Robotic Ecologies as Precursors to Human Planetary Exploration

    NASA Technical Reports Server (NTRS)

    Colombano, Silvano P.

    2003-01-01

    The present sequential mission oriented approach to robotic planetary exploration, could be changed to an infrastructure building approach where a robotic presence is permanent, self sustaining and growing with each mission. We call this self-sustaining robotic ecology approach robosphere and discuss the technological issues that need to be addressed before this concept can be realized. One of the major advantages of this approach is that a robosphere would include much of the infrastructure required by human explorers and would thus lower the preparation and risk threshold inherent in the transition from robotic to human exploration. In this context we discuss some implications for space architecture.

  5. Bone Anabolic Effects of Soluble Si: In Vitro Studies with Human Mesenchymal Stem Cells and CD14+ Osteoclast Precursors

    PubMed Central

    Costa-Rodrigues, J.; Reis, S.; Castro, A.; Fernandes, M. H.

    2016-01-01

    Silicon (Si) is indispensable for many cellular processes including bone tissue metabolism. In this work, the effects of Si on human osteogenesis and osteoclastogenesis were characterized. Human mesenchymal stem cells (hMSC) and CD14+ stem cells, as osteoblast and osteoclast precursors, were treated with a wide range of Si concentrations, covering the physiological plasma levels. Si promoted a dose-dependent increase in hMSC proliferation, differentiation, and function, at levels similar to the normal basal plasma levels. Additionally, a decrease in the expression of the osteoclastogenic activators M-CSF and RANKL was observed. Also, Si elicited a decrease in osteoclastogenesis, which became significant at higher concentrations, as those observed after meals. Among the intracellular mechanisms studied, an upregulation of MEK and PKC signalling pathways was observed in both cell types. In conclusion, Si appears to have a direct positive effect on human osteogenesis, at basal plasma levels. On the other hand, it also seemed to be an inhibitor of osteoclastogenesis, but at higher concentrations, though yet in the physiological range. Further, an indirect effect of Si on osteoclastogenesis may also occur, through a downregulation of M-CSF and RANKL expression by osteoblasts. Thus, Si may be an important player in bone anabolic regenerative approaches. PMID:26798359

  6. Nanofiber Matrices Promote the Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors In Vitro

    PubMed Central

    Lim, Shawn H.; Christopherson, Gregory T.; Xu, Leyan; Nasonkin, Igor; Yu, Christopher; Mao, Hai-Quan; Koliatsos, Vassilis E.

    2011-01-01

    The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs. PMID:20973749

  7. Design of Photovoltaic Power System for a Precursor Mission for Human Exploration of Mars

    NASA Technical Reports Server (NTRS)

    Mcnatt, Jeremiah; Landis, Geoffrey; Fincannon, James

    2016-01-01

    This project analyzed the viability of a photovoltaic power source for technology demonstration mission to demonstrate Mars in-situ resource utilization (ISRU) to produce propellant for a future human mission, based on technology available within the next ten years. For this assessment, we performed a power-system design study for a scaled ISRU demonstrator lander on the Mars surface based on existing solar array technologies.

  8. Lunar precursor missions for human exploration of Mars--III: studies of system reliability and maintenance.

    PubMed

    Mendell, W W; Heydorn, R P

    2004-01-01

    Discussions of future human expeditions into the solar system generally focus on whether the next explorers ought to go to the Moon or to Mars. The only mission scenario developed in any detail within NASA is an expedition to Mars with a 500-day stay at the surface. The technological capabilities and the operational experience base required for such a mission do not now exist nor has any self-consistent program plan been proposed to acquire them. In particular, the lack of an Abort-to-Earth capability implies that critical mission systems must perform reliably for 3 years or must be maintainable and repairable by the crew. As has been previously argued, a well-planned program of human exploration of the Moon would provide a context within which to develop the appropriate technologies because a lunar expedition incorporates many of the operational elements of a Mars expedition. Initial lunar expeditions can be carried out at scales consistent with the current experience base but can be expanded in any or all operational phases to produce an experience base necessary to successfully and safely conduct human exploration of Mars.

  9. Lunar precursor missions for human exploration of Mars--III: studies of system reliability and maintenance

    NASA Technical Reports Server (NTRS)

    Mendell, W. W.; Heydorn, R. P.

    2004-01-01

    Discussions of future human expeditions into the solar system generally focus on whether the next explorers ought to go to the Moon or to Mars. The only mission scenario developed in any detail within NASA is an expedition to Mars with a 500-day stay at the surface. The technological capabilities and the operational experience base required for such a mission do not now exist nor has any self-consistent program plan been proposed to acquire them. In particular, the lack of an Abort-to-Earth capability implies that critical mission systems must perform reliably for 3 years or must be maintainable and repairable by the crew. As has been previously argued, a well-planned program of human exploration of the Moon would provide a context within which to develop the appropriate technologies because a lunar expedition incorporates many of the operational elements of a Mars expedition. Initial lunar expeditions can be carried out at scales consistent with the current experience base but can be expanded in any or all operational phases to produce an experience base necessary to successfully and safely conduct human exploration of Mars. Published by Elsevier Ltd.

  10. Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

    PubMed Central

    Salem, N; Wegher, B; Mena, P; Uauy, R

    1996-01-01

    It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants. PMID:8552667

  11. CD36 is required for myoblast fusion during myogenic differentiation

    SciTech Connect

    Park, Seung-Yoon; Yun, Youngeun; Kim, In-San

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  12. Electroconductive nanopatterned substrates for enhanced myogenic differentiation and maturation

    PubMed Central

    Yang, Hee Seok; Lee, Bora; Tsui, Jonathan H.; Macadangdang, Jesse; Jang, Seok-Young

    2016-01-01

    Electrically conductive materials provide a suitable platform for the in vitro study of excitable cells, such as skeletal muscle cells, due to their inherent conductivity and electro-activity. Here we demonstrate that bioinspired electroconductive nanopatterned substrates enhanced myogenic differentiation and maturation. The topographical cues from the highly-aligned collagen bundles that form the extracellular matrix (ECM) of skeletal muscle tissue were mimicked using nanopatterns created with capillary force lithography. Electron beam deposition was then utilized to conformally coat nanopatterned substrates with a thin layer of either gold or titanium to create electroconductive substrates with well-defined, large-area nanotopographical features. C2C12 cells, a myoblast cell line, were cultured for 7 days on substrates, and the effects of topography and electrical conductivity on cellular morphology and myogenic differentiation were assessed. We found that biomimetic nanotopography enhanced the formation of aligned myotubes, and the addition of an electroconductive coating promoted myogenic differentiation and maturation, as indicated by the upregulation of myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). These results suggest the suitability of electroconductive nanopatterned substrates as a biomimetic platform for the in vitro engineering of skeletal muscle tissue. PMID:25988569

  13. Clonal Human Fetal Ventral Mesencephalic Dopaminergic Neuron Precursors for Cell Therapy Research

    PubMed Central

    Ramos-Moreno, Tania; Lendínez, Javier G.; Pino-Barrio, María José; del Arco, Araceli; Martínez-Serrano, Alberto

    2012-01-01

    A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments. PMID:23300748

  14. Polydatin, a natural precursor of resveratrol, induces cell cycle arrest and differentiation of human colorectal Caco-2 cell

    PubMed Central

    2013-01-01

    Background Human colon adenocarcinoma cells are resistant to chemotherapeutic agents, such as anthracyclines, that induce death by increasing the reactive oxygen species. A number of studies have been focused on chemo-preventive use of resveratrol as antioxidant against cardiovascular diseases, aging and cancer. While resveratrol cytotoxic action was due to its pro-oxidant properties. In this study, we investigate whether the Resveratrol (trans-3,5,49-trihydroxystilbene) and its natural precursor Polydatin (resveratrol-3-O-b-mono- D-glucoside, the glycoside form of resveratrol) combination, might have a cooperative antitumor effect on either growing or differentiated human adenocarcinoma colon cancer cells. Methods The polydatin and resveratrol pharmacological interaction was evaluated in vitro on growing and differentiated Caco-2 cell lines by median drug effect analysis calculating a combination index with CalcuSyn software. We have selected a synergistic combination and we have evaluated its effect on the biological and molecular mechanisms of cell death. Results Simultaneous exposure to polydatin and resveratrol produced synergistic antiproliferative effects compared with single compound treatment. We demonstrated that polydatin alone or in combination with resveratrol at 3:1 molar ratio synergistically modulated oxidative stress, cell cycle, differentiation and apoptosis. Worthy of note treatment with polydatin induced a nuclear localization and decreased expression of heat shock protein 27, and vimentin redistributed within the cell. Conclusions From morphological, and biochemical outcome we obtained evidences that polydatin induced a transition from a proliferative morphology to cell-specific differentiated structures and caused human CaCo-2 cell death by induction of apoptosis. Our data suggest the potential use of polydatin in combination chemotherapy for human colon cancer. PMID:24138806

  15. Functional effects of cannabinoids during dopaminergic specification of human neural precursors derived from induced pluripotent stem cells.

    PubMed

    Stanslowsky, Nancy; Jahn, Kirsten; Venneri, Anna; Naujock, Maximilian; Haase, Alexandra; Martin, Ulrich; Frieling, Helge; Wegner, Florian

    2016-03-30

    Among adolescents cannabis is one of the most widely used illicit drugs. In adolescence brain development continues, characterized by neuronal maturation and synaptic plasticity. The endocannabinoid system plays an important role during brain development by modulating neuronal function and neurogenesis. Changes in endocannabinoid signaling by Δ(9) -tetrahydrocannabinol (THC), the psychoactive component of cannabis, might therefore lead to neurobiological changes influencing brain function and behavior. We investigated the functional maturation and dopaminergic specification of human cord blood-derived induced pluripotent stem cell (hCBiPSC)-derived small molecule neural precursor cells (smNPCs) after cultivation with the endogenous cannabinoid anandamide (AEA) and the exogenous THC, both potent agonists at the cannabinoid 1 receptor (CB1 R). Higher dosages of 10-μM AEA or THC significantly decreased functionality of neurons, indicated by reduced ion currents and synaptic activity. A lower concentration of 1-μM THC had no marked effect on neuronal and dopaminergic maturation, while 1-μM AEA significantly enhanced the frequency of synaptic activity. As there were no significant effects on DNA methylation in promotor regions of genes important for neuronal function, these cannabinoid actions seem to be mediated by another than this epigenetic mechanism. Our data suggest that there are concentration-dependent actions of cannabinoids on neuronal function in vitro indicating neurotoxic, dysfunctional effects of 10-μM AEA and THC during human neurogenesis.

  16. Protocol to Isolate a Large Amount of Functional Oligodendrocyte Precursor Cells from the Cerebral Cortex of Adult Mice and Humans

    PubMed Central

    Medina-Rodríguez, Eva María; Arenzana, Francisco Javier; Bribián, Ana; de Castro, Fernando

    2013-01-01

    During development, oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs), a cell type that is a significant proportion of the total cells (3-8%) in the adult central nervous system (CNS) of both rodents and humans. Adult OPCs are responsible for the spontaneous remyelination that occurs in demyelinating diseases like Multiple Sclerosis (MS) and they constitute an interesting source of cells for regenerative therapy in such conditions. However, there is little data regarding the neurobiology of adult OPCs isolated from mice since an efficient method to isolate them has yet to be established. We have designed a protocol to obtain viable adult OPCs from the cerebral cortex of different mouse strains and we have compared its efficiency with other well-known methods. In addition, we show that this protocol is also useful to isolate functional OPCs from human brain biopsies. Using this method we can isolate primary cortical OPCs in sufficient quantities so as to be able to study their survival, maturation and function, and to facilitate an evaluation of their utility in myelin repair. PMID:24303061

  17. Functional signature of human islet-derived precursor cells compared to bone marrow-derived mesenchymal stem cells.

    PubMed

    Limbert, Catarina; Ebert, Regina; Schilling, Tatjana; Path, Gunter; Benisch, Peggy; Klein-Hitpass, Ludger; Seufert, Jochen; Jakob, Franz

    2010-05-01

    Pancreatic islet beta-cell replenishment can be driven by epithelial cells from exocrine pancreas via epithelial-mesenchymal transition (EMT) and the reverse process MET, while specified pancreatic mesenchymal cells control islet cell development and maintenance. The role of human islet-derived precursor cells (hIPCs) in regeneration and support of endocrine islets is under investigation. Here, we analyzed hIPCs as to their immunophenotype, multilineage differentiation capacity, and gene profiling, in comparison to human bone marrow-derived mesenchymal stem cells (hBM-MSCs). hIPCs and hBM-MSCs display a common mesenchymal character and express lineage-specific marker genes upon induction toward pancreatic endocrine and mesenchymal pathways of differentiation. hIPCs can go further along endocrine pathways while lacking some core mesenchymal differentiation attributes. Significance analysis of microarray (SAM) from 5 hBM-MSC and 3 hIPC donors mirrored such differences. Candidate gene cluster analysis disclosed differential expression of key lineage regulators, indicated a HoxA gene-associated positional memory in hIPCs and hBM-MSCs, and showed as well a clear transition state from mesenchyme to epithelium or vice versa in hIPCs. Our findings raise new research platforms to further clarify the potential of hIPCs to undergo complete MET thus contributing to islet cell replenishment, maintenance, and function.

  18. apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein precursor.

    PubMed Central

    Daigle, I; Li, C

    1993-01-01

    The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. We have isolated several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans and have designated the corresponding gene as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP. Images Fig. 2 Fig. 3 PMID:8265668

  19. Ordered processing of the human immunodeficiency virus type 1 GagPol precursor is influenced by the context of the embedded viral protease.

    PubMed

    Pettit, Steven C; Clemente, Jose C; Jeung, Jennifer A; Dunn, Ben M; Kaplan, Andrew H

    2005-08-01

    Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.

  20. Cytotoxicity of gold nanoparticles in human neural precursor cells and rat cerebral cortex.

    PubMed

    Lee, Uhn; Yoo, Chan-Jong; Kim, Yong-Jung; Yoo, Young-Mi

    2016-03-01

    Nanoparticles are promising tools for the advancement of drug delivery, medical imaging, and as diagnostic sensor. Medical nanodevices should develop miniaturization, because it would be injected into a human body. Gold nanoparticles (GNPs) with different sizes and shapes have therapeutic potential as a result of their small size, robust nature, excellent biocompatibility and optical properties. However, the application of GNPs as medical nanodevices it is necessary to know the biodegradation, biocompatibility, and development of surface coating which avoid the accumulation of nanoparticles. In this study, we carry out an in vitro toxicity and in vivo gene expression study using two kinds of GNPs. We found that GNPs toxicity is dependent on the dose or size administrated after the injected GNPs into the brain, and small particle size GNPs appeared more nestin expression compared to large particle size at short term implantation. These findings of toxicity of GNPs may play an important role in development of in vivo tools for the safety of GNPs.

  1. Molten globule precursor states are conformationally correlated to amyloid fibrils of human beta-2-microglobulin.

    PubMed

    Skora, Lukasz; Becker, Stefan; Zweckstetter, Markus

    2010-07-14

    Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein beta-2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of beta-2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the beta-sheet-rich insoluble aggregates of beta-2-microglobulin.

  2. Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor

    SciTech Connect

    Jin-Qiu Chen; Hong-Tao Zhang; Mei-Hao Hu; Jian-Guo Tang

    1995-10-01

    The construction of a gene encoding Lys-human proinsulin, its direct expression in E.coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After a simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by tryspin and carboxypeptidase B treatment in later steps. After separation with DEAE Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L fermentation medium. 20 refs., 5 figs., 4 tabs.

  3. Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

    PubMed

    Kabadi, Ami M; Thakore, Pratiksha I; Vockley, Christopher M; Ousterout, David G; Gibson, Tyler M; Guilak, Farshid; Reddy, Timothy E; Gersbach, Charles A

    2015-06-19

    Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

  4. Enantioselective dehydroxylation of enterodiol and enterolactone precursors by human intestinal bacteria.

    PubMed

    Jin, Jong-Sik; Zhao, Yu-Feng; Nakamura, Norio; Akao, Teruaki; Kakiuchi, Nobuko; Min, Byung-Sun; Hattori, Masao

    2007-11-01

    During the course of experiments on the transformation of lignans to phytoestrogenic substances, such as enterodiol (END) and enterolactone (ENL), a previously isolated bacterium, Eubacterium (E.) sp. strain SDG-2, capable of phenolic p-dehydroxylation in the biotransformation of secoisolariciresinol diglucoside to END and ENL, was concluded to be Eggerthella (Eg.) lenta (Eg. sp. SDG-2) on the basis of 16S rRNA gene sequence analysis. The bacterium could transform (+)-dihydroxyenterodiol (DHEND, 3a) to (+)-END (1a), but not for (-)-DHEND (3b) to (-)-END (1b) under anaerobic conditions. By incubation of a mixture of (+)- and (-)-dihydroxyenterolactone (DHENL, 4a and 4b) with Eg. sp. SDG-2, only (-)-DHENL (4b) was converted to (-)-ENL (2b), selectively. On the other hand, we isolated a different bacterium, strain ARC-1, capable of dehydroxylating (-)-DHEND (3b) to (-)-END (1b) from human feces. Strain ARC-1 could transform not only (-)-DHEND (3b) to (-)-END (1b), but also (+)-DHENL (4a) to (+)-ENL (2b). However, the bacterium could not transform (+)-DHEND (3a) and (-)-DHENL (4b). Both bacterial strains demonstrated different enantioselective dehydroxylation.

  5. Myogenic progenitors and imaging single-cell flow analysis: a model to study commitment of adult muscle stem cells.

    PubMed

    Trapecar, Martin; Kelc, Robi; Gradisnik, Lidija; Vogrin, Matjaz; Rupnik, Marjan Slak

    2014-12-01

    Research on skeletal muscles suffers from a lack of appropriate human models to study muscle formation and regeneration on the regulatory level of single cells. This hampers both basic understanding and the development of new therapeutic approaches. The use of imaging multicolour flow cytometry and myogenic stem cells can help fill this void by allowing researchers to visualize and quantify the reaction of individual cultured cells to bioactives or other physiological impulses. As proof of concept, we subjected human CD56+ satellite cells to reference bioactives follistatin and Malva sylvestris extracts and then used imaging multicolor flow cytometry to visualize the stepwise activation of myogenic factors MyoD and myogenin in individual cells. This approach enabled us to evaluate the potency of these bioactives to stimulate muscle commitment. To validate this method, we used multi-photon confocal microscopy to confirm the potential of bioactives to stimulate muscle differentiation and expression of desmin. Imaging multicolor flow cytometry revealed statistically significant differences between treated and untreated groups of myogenic progenitors and we propose the utilization of this concept as an integral part of future muscle research strategies.

  6. Religious morality (and secular humanism) in Western civilization as precursors to medical ethics: A historic perspective

    PubMed Central

    Faria, Miguel A.

    2015-01-01

    In discussing bioethics and the formulation of neuroethics, the question has arisen as to whether secular humanism should be the sole philosophical guiding light, to the exclusion of any discussion (or even mention) of religious morality, in professional medical ethics. In addition, the question has arisen as to whether freedom or censorship should be part of medical (and neuroscience) journalism. Should independent medical journals abstain from discussing certain issues, or should only the major medical journals — i.e., the New England Journal of Medicine (NEJM), the Journal of the American Medical Association (JAMA) or Lancet — be heard, speaking with one “consensual,” authoritative voice? This issue is particularly important in controversial topics impacting medical politics — e.g., public health policy, socio-economics, bioethics, and the so-called redistributive justice in health care. Should all sides be heard when those controversial topics are discussed or only a consensual (monolithic) side? This historical review article discusses those issues and opts for freedom in medical and surgical practice as well as freedom in medical journalism, particularly in opinion pieces such as editorials, commentaries, or letters to the editor, as long as they relate to medicine and, in our special case, to neuroscience and neurosurgery. After answering those questions, and in response to a critical letter to the editor, this review article then expounds comprehensively on the historical and philosophical origins of ethics and religious morality. Necessarily, we discuss the Graeco-Roman legacy and the Judeo-Christian inheritance in the development of ethics and religious morality in Western civilization and their impact on moral conduct in general and on medical and neuroscience ethics in particular. PMID:26110085

  7. Feasibility of a Dragon-Derived Mars Lander for Scientific and Human-Precursor Missions

    NASA Technical Reports Server (NTRS)

    Karcz, John S.; Davis, Sanford S.; Allen, Gary A.; Glass, Brian J.; Gonzales, Andrew; Heldmann, Jennifer Lynne; Lemke, Lawrence G.; McKay, Chris; Stoker, Carol R.; Wooster, Paul Douglass; Zarchi, Kerry A.

    2013-01-01

    A minimally-modified SpaceX Dragon capsule launched on a Falcon Heavy rocket presents the possibility of a new low-cost, high-capacity Mars lander for robotic missions. We have been evaluating such a "Red Dragon" platform as an option for the Icebreaker Discovery Program mission concept. Dragon is currently in service ferrying cargo to and from the International Space Station, and a crew transport version is in development. The upcoming version, unlike other Earth-return vehicles, exhibits most of the capabilities necessary to land on Mars. In particular, it has a set of high-thrust, throttleable, storable bi-propellant "SuperDraco" engines integrated directly into the capsule that are intended for launch abort and powered landings on Earth. These thrusters provide the possibility of a parachute-free, fully-propulsive deceleration at Mars from supersonic speeds to the surface, a descent approach which would also scale well to larger future human landers. We will discuss the motivations for exploring a Red Dragon lander, the current results of our analysis of its feasibility and capabilities, and the implications of the platform for the Icebreaker mission concept. In particular, we will examine entry, descent, and landing (EDL) in detail. We will also describe the modifications to Dragon necessary for interplanetary cruise, EDL, and operations on the Martian surface. Our analysis to date indicates that a Red Dragon lander is feasible and that it would be capable of delivering more than 1000 kg of payload to sites at elevations three kilometers below the Mars Orbiter Laser Altimeter (MOLA) reference, which includes sites throughout most of the northern plains and Hellas.

  8. Restricted maternal nutrition alters myogenic regulatory factor expression in satellite cells of ovine offspring.

    PubMed

    Raja, J S; Hoffman, M L; Govoni, K E; Zinn, S A; Reed, S A

    2016-07-01

    Poor maternal nutrition inhibits muscle development and postnatal muscle growth. Satellite cells are myogenic precursor cells that contribute to postnatal muscle growth, and their activity can be evaluated by the expression of several transcription factors. Paired-box (Pax)7 is expressed in quiescent and active satellite cells. MyoD is expressed in activated and proliferating satellite cells and myogenin is expressed in terminally differentiating cells. Disruption in the expression pattern or timing of expression of myogenic regulatory factors negatively affects muscle development and growth. We hypothesized that poor maternal nutrition during gestation would alter the in vitro temporal expression of MyoD and myogenin in satellite cells from offspring at birth and 3 months of age. Ewes were fed 100% or 60% of NRC requirements from day 31±1.3 of gestation. Lambs from control-fed (CON) or restricted-fed (RES) ewes were euthanized within 24 h of birth (birth; n=5) or were fed a control diet until 3 months of age (n=5). Satellite cells isolated from the semitendinosus muscle were used for gene expression analysis or cultured for 24, 48 or 72 h and immunostained for Pax7, MyoD or myogenin. Fusion index was calculated from a subset of cells allowed to differentiate. Compared with CON, temporal expression of MyoD and myogenin was altered in cultured satellite cells isolated from RES lambs at birth. The percent of cells expressing MyoD was greater in RES than CON (P=0.03) after 24 h in culture. After 48 h of culture, there was a greater percent of cells expressing myogenin in RES compared with CON (P0.05). In satellite cells from RES lambs at 3 months of age, the percent of cells expressing MyoD and myogenin were greater than CON after 72 h in culture (P<0.05). Fusion index was reduced in RES lambs at 3 months of age compared with CON (P<0.001). Restricted nutrition during gestation alters the temporal expression of myogenic regulatory factors in satellite cells of the

  9. Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

    PubMed Central

    Huang, Xiaosong; Shah, Siddharth; Wang, Jing; Ye, Zhaohui; Dowey, Sarah N; Tsang, Kit Man; Mendelsohn, Laurel G; Kato, Gregory J; Kickler, Thomas S; Cheng, Linzhao

    2014-01-01

    There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 106–107-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~1068-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine. PMID:24002691

  10. APL-1, a Caenorhabditis elegans protein related to the human β-amyloid precursor protein, is essential for viability

    PubMed Central

    Hornsten, Angela; Lieberthal, Jason; Fadia, Shruti; Malins, Richard; Ha, Lawrence; Xu, Xiaomeng; Daigle, Isabelle; Markowitz, Mindy; O'Connor, Gregory; Plasterk, Ronald; Li, Chris

    2007-01-01

    Dominant mutations in the amyloid precursor protein (APP) gene are associated with rare cases of familial Alzheimer's disease; however, the normal functions of APP and related proteins remain unclear. The nematode Caenorhabditis elegans has a single APP-related gene, apl-1, that is expressed in multiple tissues. Loss of apl-1 disrupts several developmental processes, including molting and morphogenesis, and results in larval lethality. The apl-1 lethality can be rescued by neuronal expression of the extracellular domain of APL-1. These data highlight the importance of the extracellular domain of an APP family member and suggest that APL-1 acts noncell-autonomously during development. Overexpression of APL-1 also causes several defects, including a high level of larval lethality. Decreased activity of sel-12, a C. elegans homologue of the human γ-secretase component presenilin 1, partially rescues the lethality associated with APL-1 overexpression, suggesting that SEL-12 activity regulates APL-1 activity either directly or indirectly. PMID:17267616

  11. Expression of CD86 on human marrow CD34(+) cells identifies immunocompetent committed precursors of macrophages and dendritic cells.

    PubMed

    Ryncarz, R E; Anasetti, C

    1998-05-15

    Macrophages and dendritic cells derive from a hematopoietic stem cell and the existence of a common committed progenitor has been hypothesized. We have recently found in normal human marrow a subset of CD34(+) cells that constitutively expresses HLA-DR and low levels of CD86, a natural ligand for the T cell costimulation receptor CD28. This CD34(+) subset can elicit responses from allogeneic T cells. In this study, we show that CD34(+)/CD86(+) cells can also present tetanus toxoid antigen to memory CD4(+) T cells. CD86 is expressed at low levels in macrophages and high levels in dendritic cells. Therefore, we have tested the hypothesis that CD34(+)/CD86(+) cells are the common precursors of both macrophages and dendritic cells. CD34(+)/CD86(+) marrow cells cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated macrophages. In contrast, CD34(+)/CD86(-) cells cultured in GM-CSF generated a predominant population of granulocytes. CD34(+)/CD86(+) cells cultured in GM-CSF plus tumor necrosis factor-alpha (TNF-alpha) generated almost exclusively CD1a+/CD83(+) dendritic cells. In contrast, CD34(+)/CD86(-) cells cultured in GM-CSF plus TNF-alpha generated a variety of cell types, including a small population of dendritic cells. In addition, CD34(+)/CD86(+) cells cultured in granulocyte colony-stimulating factor failed to generate CD15(+) granulocytes. Therefore, CD34(+)/CD86(+) cells are committed precursors of both macrophages and dendritic cells. The ontogeny of dendritic cells was recapitulated by stimulation of CD34(+)/CD86(-) cells with TNF-alpha that induced expression of CD86. Subsequent costimulation of CD86(+) cells with GM-CSF plus TNF-alpha lead to expression of CD83 and produced terminal dendritic cell differentiation. Thus, expression of CD86 on hematopoietic progenitor cells is regulated by TNF-alpha and denotes differentiation towards the macrophage or dendritic cell lineages.

  12. Myogenic transcription factors regulate pro-metastatic miR-182.

    PubMed

    Dodd, R D; Sachdeva, M; Mito, J K; Eward, W C; Brigman, B E; Ma, Y; Dodd, L; Kim, Y; Lev, D; Kirsch, D G

    2016-04-07

    Approximately 30% of patients with soft-tissue sarcoma die from pulmonary metastases. The mechanisms that drive sarcoma metastasis are not well understood. Recently, we identified miR-182 as a driver of sarcoma metastasis in a primary mouse model of soft-tissue sarcoma. We also observed elevated miR-182 in a subset of primary human sarcomas that metastasized to the lungs. Here, we show that myogenic differentiation factors regulate miR-182 levels to contribute to metastasis in mouse models. We find that MyoD directly binds the miR-182 promoter to increase miR-182 expression. Furthermore, mechanistic studies revealed that Pax7 can promote sarcoma metastasis in vivo through MyoD-dependent regulation of pro-metastatic miR-182. Taken together, these results suggest that sarcoma metastasis can be partially controlled through Pax7/MyoD-dependent activation of miR-182 and provide insight into the role that myogenic transcription factors have in sarcoma progression.

  13. Myogenic transcription factors regulate pro-metastatic miR-182

    PubMed Central

    Dodd, Rebecca D.; Sachdeva, Mohit; Mito, Jeffrey K.; Eward, William C.; Brigman, Brian E.; Ma, Yan; Dodd, Leslie; Kim, Youngbaek; Lev, Dina; Kirsch, David G.

    2015-01-01

    Approximately thirty percent of patients with soft-tissue sarcoma die from pulmonary metastases. The mechanisms that drive sarcoma metastasis are not well understood. Recently, we identified miR-182 as a driver of sarcoma metastasis in a primary mouse model of soft-tissue sarcoma. We also observed elevated miR-182 in a subset of primary human sarcomas that metastasized to the lungs. Here, we show that myogenic differentiation factors regulate miR-182 levels to contribute to metastasis in mouse models. We find that MyoD directly binds the miR-182 promoter to increase miR-182 expression. Furthermore, mechanistic studies revealed that Pax7 can promote sarcoma metastasis in vivo through MyoD-dependent regulation of pro-metastatic miR-182. Taken together, these results suggest that sarcoma metastasis can be partially controlled through Pax7/MyoD-dependent activation of miR-182 and provide insight into the role that myogenic transcription factors play in sarcoma progression. PMID:26234681

  14. Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction.

    PubMed

    Parsons, Xuejun H; Teng, Yang D; Parsons, James F; Snyder, Evan Y; Smotrich, David B; Moore, Dennis A

    2011-11-03

    To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture

  15. Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: Alternative exon usage predicts two different precursor proteins

    SciTech Connect

    Rorsman, F.; Bywater, M.; Knott, T.J.; Scott, J.; Betsholtz, C.

    1988-02-01

    The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

  16. Transcriptome analysis of post-hatch breast muscle in legacy and modern broiler chickens reveals enrichment of several regulators of myogenic growth.

    PubMed

    Davis, Richard V N; Lamont, Susan J; Rothschild, Max F; Persia, Michael E; Ashwell, Chris M; Schmidt, Carl J

    2015-01-01

    Agriculture provides excellent model systems for understanding how selective pressure, as applied by humans, can affect the genomes of plants and animals. One such system is modern poultry breeding in which intensive genetic selection has been applied for meat production in the domesticated chicken. As a result, modern meat-type chickens (broilers) exhibit enhanced growth, especially of the skeletal muscle, relative to their legacy counterparts. Comparative studies of modern and legacy broiler chickens provide an opportunity to identify genes and pathways affected by this human-directed evolution. This study used RNA-seq to compare the transcriptomes of a modern and a legacy broiler line to identify differentially enriched genes in the breast muscle at days 6 and 21 post-hatch. Among the 15,945 genes analyzed, 10,841 were expressed at greater than 0.1 RPKM. At day 6 post-hatch 189 genes, including several regulators of myogenic growth and development, were differentially enriched between the two lines. The transcriptional profiles between lines at day 21 post-hatch identify 193 genes differentially enriched and still include genes associated with myogenic growth. This study identified differentially enriched genes that regulate myogenic growth and differentiation between the modern and legacy broiler lines. Specifically, differences in the ratios of several positive (IGF1, IGF1R, WFIKKN2) and negative (MSTN, ACE) myogenic growth regulators may help explain the differences underlying the enhanced growth characteristics of the modern broilers.

  17. Transcriptome Analysis of Post-Hatch Breast Muscle in Legacy and Modern Broiler Chickens Reveals Enrichment of Several Regulators of Myogenic Growth

    PubMed Central

    Davis, Richard V. N.; Lamont, Susan J.; Rothschild, Max F.; Persia, Michael E.; Ashwell, Chris M.; Schmidt, Carl J.

    2015-01-01

    Agriculture provides excellent model systems for understanding how selective pressure, as applied by humans, can affect the genomes of plants and animals. One such system is modern poultry breeding in which intensive genetic selection has been applied for meat production in the domesticated chicken. As a result, modern meat-type chickens (broilers) exhibit enhanced growth, especially of the skeletal muscle, relative to their legacy counterparts. Comparative studies of modern and legacy broiler chickens provide an opportunity to identify genes and pathways affected by this human-directed evolution. This study used RNA-seq to compare the transcriptomes of a modern and a legacy broiler line to identify differentially enriched genes in the breast muscle at days 6 and 21 post-hatch. Among the 15,945 genes analyzed, 10,841 were expressed at greater than 0.1 RPKM. At day 6 post-hatch 189 genes, including several regulators of myogenic growth and development, were differentially enriched between the two lines. The transcriptional profiles between lines at day 21 post-hatch identify 193 genes differentially enriched and still include genes associated with myogenic growth. This study identified differentially enriched genes that regulate myogenic growth and differentiation between the modern and legacy broiler lines. Specifically, differences in the ratios of several positive (IGF1, IGF1R, WFIKKN2) and negative (MSTN, ACE) myogenic growth regulators may help explain the differences underlying the enhanced growth characteristics of the modern broilers. PMID:25821972

  18. Non-myogenic tumors display altered expression of dystrophin (DMD) and a high frequency of genetic alterations

    PubMed Central

    Luce, Leonela N.; Abbate, Mercedes

    2017-01-01

    DMD gene mutations have been associated with the development of Dystrophinopathies. Interestingly, it has been recently reported that DMD is involved in the development and progression of myogenic tumors, assigning DMD a tumor suppressor activity in these types of cancer. However, there are only few reports that analyze DMD in non-myogenic tumors. Our study was designed to examine DMD expression and genetic alterations in non-myogenic tumors using public repositories. We also evaluated the overall survival of patients with and without DMD mutations. We studied 59 gene expression microarrays (GEO database) and RNAseq (cBioPortal) datasets that included 9817 human samples. We found reduced DMD expression in 15/27 (56%) pairwise comparisons performed (Fold-Change (FC) ≤ 0.70; p-value range = 0.04-1.5×10−20). The analysis of RNAseq studies revealed a median frequency of DMD genetic alterations of 3.4%, higher or similar to other well-known tumor suppressor genes. In addition, we observed significant poorer overall survival for patients with DMD mutations. The analyses of paired tumor/normal tissues showed that the majority of tumor specimens had lower DMD expression compared to their normal adjacent counterpart. Interestingly, statistical significant over-expression of DMD was found in 6/27 studies (FC ≥ 1.4; p-value range = 0.03-3.4×10−15). These results support that DMD expression and genetic alterations are frequent and relevant in non-myogenic tumors. The study and validation of DMD as a new player in tumor development and as a new prognostic factor for tumor progression and survival are warranted. PMID:27391342

  19. Myogenic potential of chick limb bud mesenchyme in micromass culture.

    PubMed

    Archer, C W; Langille, R M; Teran, M A; Solursh, M

    1992-01-01

    The myogenic potential of chick limb mesenchyme from stages 18-25 was assessed by micromass culture under conditions conductive to myogenesis, and was measured as the proportion of differentiated (muscle myosin-positive) mononucleated cells detected. It was found that similar myogenic potentials existed in mesenchyme from whole limbs between stages 18 and 19, but this potential was halved by stage 20. At stage 21, proximal mesenchyme showed significantly more myogenesis than distal mesenchyme, but this difference was abolished by stage 22. Thereafter, myogenesis was increasingly restricted from the distal mesenchyme, whilst the potential in more proximal regions did not significantly increase after stage 23. When the ratio between total limb myoblasts which differentiated on days 1 and 4 of culture was analysed, it was found that two distinct peaks existed at stages 20 and 23. The significance of these ratio peaks is unclear, but may be related to different proliferative potentials of the pre-myoblasts at these stages.

  20. Myogenic progenitors contribute to open but not closed fracture repair

    PubMed Central

    2011-01-01

    Background Bone repair is dependent on the presence of osteocompetent progenitors that are able to differentiate and generate new bone. Muscle is found in close association with orthopaedic injury, however its capacity to make a cellular contribution to bone repair remains ambiguous. We hypothesized that myogenic cells of the MyoD-lineage are able to contribute to bone repair. Methods We employed a MyoD-Cre+:Z/AP+ conditional reporter mouse in which all cells of the MyoD-lineage are permanently labeled with a human alkaline phosphatase (hAP) reporter. We tracked the contribution of MyoD-lineage cells in mouse models of tibial bone healing. Results In the absence of musculoskeletal trauma, MyoD-expressing cells are limited to skeletal muscle and the presence of reporter-positive cells in non-muscle tissues is negligible. In a closed tibial fracture model, there was no significant contribution of hAP+ cells to the healing callus. In contrast, open tibial fractures featuring periosteal stripping and muscle fenestration had up to 50% of hAP+ cells detected in the open fracture callus. At early stages of repair, many hAP+ cells exhibited a chondrocyte morphology, with lesser numbers of osteoblast-like hAP+ cells present at the later stages. Serial sections stained for hAP and type II and type I collagen showed that MyoD-lineage cells were surrounded by cartilaginous or bony matrix, suggestive of a functional role in the repair process. To exclude the prospect that osteoprogenitors spontaneously express MyoD during bone repair, we created a metaphyseal drill hole defect in the tibia. No hAP+ staining was observed in this model suggesting that the expression of MyoD is not a normal event for endogenous osteoprogenitors. Conclusions These data document for the first time that muscle cells can play a significant secondary role in bone repair and this knowledge may lead to important translational applications in orthopaedic surgery. Please see related article: http

  1. Intracellular Aβ pathology and early cognitive impairments in a transgenic rat overexpressing human amyloid precursor protein: a multidimensional study.

    PubMed

    Iulita, M Florencia; Allard, Simon; Richter, Luise; Munter, Lisa-Marie; Ducatenzeiler, Adriana; Weise, Christoph; Do Carmo, Sonia; Klein, William L; Multhaup, Gerhard; Cuello, A Claudio

    2014-06-05

    Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-β (Aβ) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular Aβ remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the Aβ pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal Aβ in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble Aβ42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. Aβ38, Aβ39, Aβ40 and Aβ42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular Aβ may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (Aβ, APP and CTFs) of which a considerable component is Aβ; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.

  2. ICV-transplanted human glial precursor cells are short-lived yet exert immunomodulatory effects in mice with EAE.

    PubMed

    Kim, Heechul; Walczak, Piotr; Muja, Naser; Campanelli, James T; Bulte, Jeff W M

    2012-07-01

    Human glial precursor cells (hGPs) have potential for remyelinating lesions and are an attractive cell source for cell therapy of multiple sclerosis (MS). To investigate whether transplanted hGPs can affect the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we evaluated the therapeutic effects of transplanted hGPs together with the in vivo fate of these cells using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). At 14 days post-EAE induction, mice (n = 19) were intracerebroventricularly (ICV) injected with 5 × 10(5) hGPs that were magnetically labeled with superparamagnetic iron oxide (SPIO) particles as MR contrast agent and transduced with firefly luciferase for BLI of cell survival. Control mice (n = 18) received phosphate buffered saline (PBS) vehicle only. The severity of EAE clinical disability in the hGP-transplanted group was significantly suppressed (P < 0.05) with concomitant inhibition of ConA and MOG-specific T cell proliferation in the spleen. Astrogliosis was reduced and a lower activity of macrophages and/or microglia was observed in the spinal cord (P < 0.05). On MRI, SPIO signal was detected within the lateral ventricle from 1 day post-transplantation and remained there for up to 34 days. BLI indicated that most cells did not survive beyond 5-10 days, consistent with the lack of detectable migration into the brain parenchyma and the histological presence of an abundance of apoptotic cells. Transplanted hGPs could not be detected in the spleen. We conclude that ICV transplantation of short-lived hGPs can have a remote therapeutic effect through immunomodulation from within the ventricle, without cells directly participating in remyelination.

  3. Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors.

    PubMed

    Surrao, Denver C; Boon, Kathryn; Borys, Breanna; Sinha, Sarthak; Kumar, Ranjan; Biernaskie, Jeff; Kallos, Michael S

    2016-12-01

    Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an

  4. In-situ resource utilization for the human exploration of Mars : a Bayesian approach to valuation of precursor missions

    NASA Technical Reports Server (NTRS)

    Smith, Jeffrey H.

    2006-01-01

    The need for sufficient quantities of oxygen, water, and fuel resources to support a crew on the surface of Mars presents a critical logistical issue of whether to transport such resources from Earth or manufacture them on Mars. An approach based on the classical Wildcat Drilling Problem of Bayesian decision theory was applied to the problem of finding water in order to compute the expected value of precursor mission sample information. An implicit (required) probability of finding water on Mars was derived from the value of sample information using the expected mass savings of alternative precursor missions.

  5. The Krüppel-like Factor 15 as a Molecular Link between Myogenic Factors and a Chromosome 4q Transcriptional Enhancer Implicated in Facioscapulohumeral Dystrophy*

    PubMed Central

    Dmitriev, Petr; Petrov, Andrei; Ansseau, Eugenie; Stankevicins, Luiza; Charron, Sébastien; Kim, Elena; Bos, Tomas Jan; Robert, Thomas; Turki, Ahmed; Coppée, Frédérique; Belayew, Alexandra; Lazar, Vladimir; Carnac, Gilles; Laoudj, Dalila; Lipinski, Marc; Vassetzky, Yegor S.

    2011-01-01

    Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We report here that the enhancer within the D4Z4 repeat binds the Krüppel-like factor KLF15. KLF15 was found to be up-regulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts, the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15-binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes, and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and it thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD. PMID:21937448

  6. The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts

    PubMed Central

    Khor, Shy Cian; Razak, Azraul Mumtazah; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Abdul Karim, Norwahidah; Makpol, Suzana

    2016-01-01

    Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts. PMID:26885980

  7. Clinical application of vestibular evoked myogenic potential (VEMP).

    PubMed

    Murofushi, Toshihisa

    2016-08-01

    The author reviewed clinical aspects of vestibular evoked myogenic potentials (VEMPs). Now two types of VEMPs are available. The first one is cervical VEMP, which is recorded in the sternocleidomastoid muscle and predominantly reflects sacculo-collic reflex. The other is ocular VEMP, which is usually recorded below the lower eye lid and predominantly reflects utriculo-ocular reflex. VEMPs play important roles not only for assessment of common vestibular diseases but also for establishment of new clinical entities. Clinical application in Meniere's disease, vestibular neuritis, benign paroxysmal positional vertigo, vestibular migraine, idiopathic otolithic vertigo, and central vertigo/dizziness was reviewed.

  8. Polysialic acid modification of the synaptic cell adhesion molecule SynCAM 1 in human embryonic stem cell-derived oligodendrocyte precursor cells.

    PubMed

    Werneburg, Sebastian; Buettner, Falk F R; Mühlenhoff, Martina; Hildebrandt, Herbert

    2015-05-01

    Oligodendrocyte precursor cells (OPCs) are the progenitors of myelinating oligodendrocytes in brain development and repair. Successful myelination depends on the control of adhesiveness during OPC migration and axon contact formation. The decoration of cell surface proteins with the glycan polysialic acid (polySia) is a key regulatory element of OPC interactions during development and under pathological conditions. By far the major protein carrier of polySia is the neural cell adhesion molecule NCAM, but recently, polysialylation of the synaptic cell adhesion molecule SynCAM 1 has been detected in the developing mouse brain. In mice, polySia-SynCAM 1 is associated with cells expressing NG2, a marker of a heterogeneous precursor cell population, which is the primary source for oligodendrocytes in development and myelin repair but can also give rise to astrocytes and possibly neurons. It is not yet clear if polySia-SynCAM 1 is expressed by OPCs and its occurrence in humans is elusive. By generating uniform human embryonic stem cell-derived OPC cultures, we demonstrate that polySia is present on human OPCs but down-regulated during differentiation into myelin basic protein-positive oligodendrocytes. PolySia on NCAM resides on the isoforms NCAM-180 and NCAM-140, and SynCAM 1 is identified as a novel polySia acceptor in human OPCs.

  9. Expression of the myogenic gene MRF4 during Xenopus development.

    PubMed

    Jennings, C G

    1992-05-01

    In a search for myogenic genes in Xenopus, I have cloned homologs of the mammalian myogenic genes MRF4 and myogenin. The myogenin clone is a genomic fragment encoding an amino acid sequence with 62% identity to the N-terminal region of rat myogenin. No myogenin transcript has been detected and no cDNA has been isolated, suggesting that Xenopus myogenin, if it is expressed at all, is likely to be expressed at low levels or transiently during development. A Xenopus MRF4 cDNA has been isolated and encodes an amino acid sequence with 72% identity to rat MRF4. In adult frogs, MRF4 RNA is detectable only in skeletal muscle (whereas MyoD, unexpectedly, is also expressed at low levels in the heart). During embryonic development, MRF4 RNA appears later than MyoD, at a time when the embryonic musculature already shows many differentiated features. This implies that MRF4 is not involved in the commitment or early differentiation of muscle cells. The accumulation of Xenopus MRF4 RNA overlaps with the formation of neuromuscular connections, suggesting that it may be induced by innervation. Consistent with this possibility, the level of Xenopus MRF4, but not MyoD, RNA is reduced in response to denervation of adult frog muscle.

  10. Vestibular evoked myogenic potentials: past, present and future.

    PubMed

    Rosengren, S M; Welgampola, M S; Colebatch, J G

    2010-05-01

    Since the first description of sound-evoked short-latency myogenic reflexes recorded from neck muscles, vestibular evoked myogenic potentials (VEMPs) have become an important part of the neuro-otological test battery. VEMPs provide a means of assessing otolith function: stimulation of the vestibular system with air-conducted sound activates predominantly saccular afferents, while bone-conducted vibration activates a combination of saccular and utricular afferents. The conventional method for recording the VEMP involves measuring electromyographic (EMG) activity from surface electrodes placed over the tonically-activated sternocleidomastoid (SCM) muscles. The "cervical VEMP" (cVEMP) is thus a manifestation of the vestibulo-collic reflex. However, recent research has shown that VEMPs can also be recorded from the extraocular muscles using surface electrodes placed near the eyes. These "ocular VEMPs" (oVEMPs) are a manifestation of the vestibulo-ocular reflex. Here we describe the historical development and neurophysiological properties of the cVEMP and oVEMP and provide recommendations for recording both reflexes. While the cVEMP has documented diagnostic utility in many disorders affecting vestibular function, relatively little is known as yet about the clinical value of the oVEMP. We therefore outline the known cVEMP and oVEMP characteristics in common central and peripheral disorders encountered in neuro-otology clinics.

  11. Expression of Transthyretin during bovine myogenic satellite cell differentiation.

    PubMed

    Pokharel, Smritee; Kamli, Majid Rasool; Mir, Bilal Ahmad; Malik, Adeel; Lee, Eun Ju; Choi, Inho

    2014-09-01

    Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T4) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T4 decreased significantly following TTR knockdown, indicating the critical role of TTR in T4 transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T4 and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.

  12. Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes.

    PubMed

    Tavi, Pasi; Korhonen, Topi; Hänninen, Sandra L; Bruton, Joseph D; Lööf, Sara; Simon, Andras; Westerblad, Håkan

    2010-05-01

    Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F-actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax-7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination.

  13. Arterial Myogenic Activation through Smooth Muscle Filamin A.

    PubMed

    Retailleau, Kevin; Arhatte, Malika; Demolombe, Sophie; Peyronnet, Rémi; Baudrie, Véronique; Jodar, Martine; Bourreau, Jennifer; Henrion, Daniel; Offermanns, Stefan; Nakamura, Fumihiko; Feng, Yuanyi; Patel, Amanda; Duprat, Fabrice; Honoré, Eric

    2016-03-08

    Mutations in the filamin A (FlnA) gene are frequently associated with severe arterial abnormalities, although the physiological role for this cytoskeletal element remains poorly understood in vascular cells. We used a conditional mouse model to selectively delete FlnA in smooth muscle (sm) cells at the adult stage, thus avoiding the developmental effects of the knockout. Basal blood pressure was significantly reduced in conscious smFlnA knockout mice. Remarkably, pressure-dependent tone of the resistance caudal artery was lost, whereas reactivity to vasoconstrictors was preserved. Impairment of the myogenic behavior was correlated with a lack of calcium influx in arterial myocytes upon an increase in intraluminal pressure. Notably, the stretch activation of CaV1.2 was blunted in the absence of smFlnA. In conclusion, FlnA is a critical upstream element of the signaling cascade underlying the myogenic tone. These findings allow a better understanding of the molecular basis of arterial autoregulation and associated disease states.

  14. Vestibular evoked myogenic potentials in patients with rheumatoid arthritis

    PubMed Central

    Heydari, Nahid; Hajiabolhassani, Fahimeh; Fatahi, Jamileh; Movaseghi, Shafieh; Jalaie, Shohreh

    2015-01-01

    Background: Rheumatoid arthritis (RA) is an autoimmune systemic disease. Most common autoimmune diseases are multisystem disorders that may also present with otological manifestations, and autoimmune inner ear disease accompanied by vestibular dysfunction. This study aimed to compare the vestibular function between RA patients and normal subjects using cervical vestibular evoked myogenic potentials (cVEMPs). Methods: In this cross- sectional study, 25patients with RA (19 female and 6 male: mean (±SD) age, 40.00 (±7.92) years) and 20 healthy subjects (15 female and 5 male: mean (±SD) age, 35.35 (±10.48) years) underwent cVEMPs, using 500 Hz-tone bursts at 95 dB nHL intensity level. Data were analyzed using independent sample t-test through SPSS software v. 16. Results: The mean peak latency of p13 was significantly higher in RA patients (p<0.001). The mean peak latency of n23 was significantly higher in patients in the left ear (p=0.03). Vestibular evoked myogenic potential (VEMP) responses were present in all (100%) of the participants. There were no significant differences in mean peak to peak amplitude and amplitude ratio between the two groups. Conclusion: According to the prolonged latency of VEMP responses in RA patients, lesions in the retrolabyrinthine, especially in the vestibulospinal tract are suspected. PMID:26478874

  15. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage.

    PubMed

    Quek, Lynn; Otto, Georg W; Garnett, Catherine; Lhermitte, Ludovic; Karamitros, Dimitris; Stoilova, Bilyana; Lau, I-Jun; Doondeea, Jessica; Usukhbayar, Batchimeg; Kennedy, Alison; Metzner, Marlen; Goardon, Nicolas; Ivey, Adam; Allen, Christopher; Gale, Rosemary; Davies, Benjamin; Sternberg, Alexander; Killick, Sally; Hunter, Hannah; Cahalin, Paul; Price, Andrew; Carr, Andrew; Griffiths, Mike; Virgo, Paul; Mackinnon, Stephen; Grimwade, David; Freeman, Sylvie; Russell, Nigel; Craddock, Charles; Mead, Adam; Peniket, Andrew; Porcher, Catherine; Vyas, Paresh

    2016-07-25

    Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34(-), there are multiple, nonhierarchically arranged CD34(+) and CD34(-) LSC populations. Within CD34(-) and CD34(+) LSC-containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34(-) LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34(-) mature granulocyte-macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis.

  16. Genetically distinct leukemic stem cells in human CD34− acute myeloid leukemia are arrested at a hemopoietic precursor-like stage

    PubMed Central

    Quek, Lynn; Garnett, Catherine; Karamitros, Dimitris; Stoilova, Bilyana; Doondeea, Jessica; Kennedy, Alison; Metzner, Marlen; Ivey, Adam; Sternberg, Alexander; Hunter, Hannah; Price, Andrew; Virgo, Paul; Grimwade, David; Freeman, Sylvie; Russell, Nigel; Mead, Adam

    2016-01-01

    Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34−, there are multiple, nonhierarchically arranged CD34+ and CD34− LSC populations. Within CD34− and CD34+ LSC–containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34− LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34− mature granulocyte–macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis. PMID:27377587

  17. Grafted Human iPS Cell-Derived Oligodendrocyte Precursor Cells Contribute to Robust Remyelination of Demyelinated Axons after Spinal Cord Injury

    PubMed Central

    Kawabata, Soya; Takano, Morito; Numasawa-Kuroiwa, Yuko; Itakura, Go; Kobayashi, Yoshiomi; Nishiyama, Yuichiro; Sugai, Keiko; Nishimura, Soraya; Iwai, Hiroki; Isoda, Miho; Shibata, Shinsuke; Kohyama, Jun; Iwanami, Akio; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

    2015-01-01

    Summary Murine- and human-induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NS/PCs) promote functional recovery following transplantation into the injured spinal cord in rodents and primates. Although remyelination of spared demyelinated axons is a critical mechanism in the regeneration of the injured spinal cord, human iPSC-NS/PCs predominantly differentiate into neurons both in vitro and in vivo. We therefore took advantage of our recently developed protocol to obtain human-induced pluripotent stem cell-derived oligodendrocyte precursor cell-enriched neural stem/progenitor cells and report the benefits of transplanting these cells in a spinal cord injury (SCI) model. We describe how this approach contributes to the robust remyelination of demyelinated axons and facilitates functional recovery after SCI. PMID:26724902

  18. Influence of Connexin40 on the renal myogenic response in murine afferent arterioles

    PubMed Central

    Jacobsen, Jens Christian B; Sorensen, Charlotte M

    2015-01-01

    Renal autoregulation consists of two main mechanisms; the myogenic response and the tubuloglomerular feedback mechanism (TGF). Increases in renal perfusion pressure activate both mechanisms causing a reduction in diameter of the afferent arteriole (AA) resulting in stabilization of the glomerular pressure. It has previously been shown that connexin-40 (Cx40) is essential in the renal autoregulation and mediates the TGF mechanism. The aim of this study was to characterize the myogenic properties of the AA in wild-type and connexin-40 knockout (Cx40KO) mice using both in situ diameter measurements and modeling. We hypothesized that absence of Cx40 would not per se affect myogenic properties as Cx40 is expressed primarily in the endothelium and as the myogenic response is known to be present also in isolated, endothelium-denuded vessels. Methods used were the isolated perfused juxtamedullary nephron preparation to allow diameter measurements of the AA. A simple mathematical model of the myogenic response based on experimental parameters was implemented. Our findings show that the myogenic response is completely preserved in the AA of the Cx40KO and if anything, the stress sensitivity of the smooth muscle cell in the vascular wall is increased rather than reduced as compared to the WT. These findings are compatible with the view of the myogenic response being primarily a local response to the local transmural pressure. PMID:26009638

  19. The effect of hyperammonemia on myostatin and myogenic regulatory factor gene expression in broiler embryos

    PubMed Central

    Stern, R.A.; Ashwell, C.M.; Dasarathy, S.; Mozdziak, P.E.

    2015-01-01

    Myogenesis is facilitated by four myogenic regulatory factors and is significantly inhibited by myostatin. The objective of the current study was to examine embryonic gene regulation of myostatin/myogenic regulatory factors, and subsequent manipulations of protein synthesis, in broiler embryos under induced hyperammonemia. Broiler eggs were injected with ammonium acetate solution four times over 48 hours beginning on either embryonic day (ED) 15 or 17. Serum ammonia concentration was significantly higher (P < 0.05) in ammonium acetate injected embryos for both ED17 and ED19 collected samples when compared to sham-injected controls. Expression of mRNA, extracted from pectoralis major of experimental and control embryos, was measured using real-time quantitative PCR for myostatin, myogenic regulatory factors myogenic factor 5, myogenic determination factor 1, myogenin, myogenic regulatory factor 4, and paired box 7. A significantly lower (P < 0.01) myostatin expression was accompanied by a higher serum ammonia concentration in both ED17 and ED19 collected samples. Myogenic factor 5 expression was higher (P < 0.05) in ED17 collected samples administered ammonium acetate. In both ED17 and ED19 collected samples, myogenic regulatory factor 4 was lower (P ≤ 0.05) in ammonium acetate injected embryos. No significant difference was seen in myogenic determination factor 1, myogenin, or paired box 7 expression between treatment groups for either age of sample collection. Additionally, there was no significant difference in BrdU staining of histological samples taken from treated and control embryos. Myostatin protein levels were evaluated by Western blot analysis, and also showed lower myostatin expression (P < 0.05). Overall, it appears possible to inhibit myostatin expression through hyperammonemia, which is expected to have a positive effect on embryonic myogenesis and postnatal muscle growth. PMID:25689990

  20. Caspase-3, myogenic transcription factors and cell cycle inhibitors are regulated by leukemia inhibitory factor to mediate inhibition of myogenic differentiation

    PubMed Central

    2011-01-01

    Background Leukemia inhibitory factor (LIF) is known to inhibit myogenic differentiation as well as to inhibit apoptosis and caspase-3 activation in non-differentiating myoblasts. In addition caspase-3 activity is required for myogenic differentiation. Therefore the aim of this study was to further investigate mechanisms of the differentiation suppressing effect of LIF in particular the possibility of a caspase-3 mediated inhibition of differentiation. Results LIF dependent inhibition of differentiation appeared to involve several mechanisms. Differentiating myoblasts that were exposed to LIF displayed increased transcripts for c-fos. Transcripts for the cell cycle inhibitor p21 as well as muscle regulatory factors myoD and myogenin were decreased with LIF exposure. However, LIF did not directly induce a proliferative effect under differentiation conditions, but did prevent the proportion of myoblasts that were proliferating from decreasing as differentiation proceeded. LIF stimulation decreased the percentage of cells positive for active caspase-3 occurring during differentiation. Both the effect of LIF inhibiting caspase-3 activation and differentiation appeared dependent on mitogen activated protein kinase and extracellular signal regulated kinase kinase (MEK) signalling. The role of LIF in myogenic differentiation was further refined to demonstrate that myoblasts are unlikely to secrete LIF endogenously. Conclusions Altogether this study provides a more comprehensive view of the role of LIF in myogenic differentiation including LIF and receptor regulation in myoblasts and myotubes, mechanisms of inhibition of differentiation and the link between caspase-3 activation, apoptosis and myogenic differentiation. PMID:21798094

  1. Effect of regional precursor emission controls on long-range ozone transport - Part 2: Steady-state changes in ozone air quality and impacts on human mortality

    NASA Astrophysics Data System (ADS)

    West, J. J.; Naik, V.; Horowitz, L. W.; Fiore, A. M.

    2009-08-01

    Large-scale changes in ozone precursor emissions affect ozone directly in the short term, and also affect methane, which in turn causes long-term changes in ozone that affect surface ozone air quality. Here we assess the effects of changes in ozone precursor emissions on the long-term change in surface ozone via methane, as a function of the emission region, by modeling 10% reductions in anthropogenic nitrogen oxide (NOx) emissions from each of nine world regions. Reductions in NOx emissions from all world regions increase methane and long-term surface ozone. While this long-term increase is small compared to the intra-regional short-term ozone decrease, it is comparable to or larger than the short-term inter-continental ozone decrease for some source-receptor pairs. The increase in methane and long-term surface ozone per ton of NOx reduced is greatest in tropical and Southern Hemisphere regions, exceeding that from temperate Northern Hemisphere regions by roughly a factor of ten. We also assess changes in premature ozone-related human mortality associated with regional precursor reductions and long-range transport, showing that for 10% regional NOx reductions, the strongest inter-regional influence is for emissions from Europe affecting mortalities in Africa. Reductions of NOx in North America, Europe, the Former Soviet Union, and Australia are shown to reduce more mortalities outside of the source regions than within. Among world regions, NOx reductions in India cause the greatest number of avoided mortalities per ton, mainly in India itself. Finally, by increasing global methane, NOx reductions in one hemisphere tend to cause long-term increases in ozone concentration and mortalities in the opposite hemisphere. Reducing emissions of methane, and to a lesser extent carbon monoxide and non-methane volatile organic compounds, alongside NOx reductions would avoid this disbenefit.

  2. Effect of regional precursor emission controls on long-range ozone transport - Part 2: steady-state changes in ozone air quality and impacts on human mortality

    NASA Astrophysics Data System (ADS)

    West, J. J.; Naik, V.; Horowitz, L. W.; Fiore, A. M.

    2009-03-01

    Large-scale changes in ozone precursor emissions affect ozone directly in the short term, and also affect methane, which in turn causes long-term changes in ozone that affect surface ozone air quality. Here we assess the effects of changes in ozone precursor emissions on the long-term change in surface ozone via methane, as a function of the emission region, by modeling 10% reductions in anthropogenic nitrogen oxide (NOx) emissions from each of nine world regions. Reductions in NOx emissions from all world regions increase methane and long-term surface ozone. While this long-term increase is small compared to the intra-regional short-term ozone decrease, it is comparable to or larger than the short-term inter-continental ozone decrease for some source-receptor pairs. The increase in methane and long-term surface ozone per ton of NOx reduced is greatest in tropical and Southern Hemisphere regions, exceeding that from temperate Northern Hemisphere regions by roughly a factor of ten. We also assess changes in premature ozone-related human mortality associated with regional precursor reductions and long-range transport, showing that for 10% regional NOx reductions, the strongest inter-regional influence is for emissions from Europe affecting mortalities in Africa. Reductions of NOx in North America, Europe, the Former Soviet Union, and Australia are shown to reduce more mortalities outside of the source regions than within. Among world regions, NOx reductions in India cause the greatest number of avoided mortalities per ton, mainly in India itself. Finally, by increasing global methane, NOx reductions in one hemisphere tend to cause long-term increases in ozone concentration and mortalities in the opposite hemisphere. Reducing emissions of methane, and to a lesser extent carbon monoxide and non-methane volatile organic compounds, alongside NOx reductions would avoid this disbenefit.

  3. Proinflammatory Macrophages Enhance the Regenerative Capacity of Human Myoblasts by Modifying Their Kinetics of Proliferation and Differentiation

    PubMed Central

    Bencze, Maximilien; Negroni, Elisa; Vallese, Denis; Yacoub–Youssef, Houda; Chaouch, Soraya; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Chazaud, Bénédicte; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent; Riederer, Ingo

    2012-01-01

    Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2−/− γC−/− immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy. PMID:23070116

  4. Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.

    PubMed

    Bencze, Maximilien; Negroni, Elisa; Vallese, Denis; Yacoub-Youssef, Houda; Chaouch, Soraya; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Chazaud, Bénédicte; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent; Riederer, Ingo

    2012-11-01

    Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

  5. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  6. Temporal gene expression profile of human precursor B leukemia cells induced by adhesion receptor: identification of pathways regulating B-cell survival.

    PubMed

    Astier, Anne Laurence; Xu, Ronghui; Svoboda, Marek; Hinds, Esther; Munoz, Olivier; de Beaumont, Rosalie; Crean, Colin Daniel; Gabig, Theodore; Freedman, Arnold Stephen

    2003-02-01

    The physical interactions between B cells and stromal cells from the lymphoid tissue microenvironment are critical to the survival of normal and malignant B cells. They are principally mediated by integrins expressed on B cells and counterreceptors on stromal cells. Specifically, alpha4beta1 integrin engagement rescues B cells from physiological or drug-induced apoptosis. Therefore, in order to understand the mechanisms by which integrins prevent apoptosis in leukemia B cells, we compared the temporal gene expression profiles induced by beta1-integrin ligation with fibronectin (Fn) or adhesion by poly-L-Lysine in serum-starved precursor B leukemia cells. Among the 38 selected differentially expressed genes, 6 genes involved in adhesion (VAV2, EPB41L1, CORO1A), proliferation (FRAP1, CCT4), and intercellular communication (GJB3) were validated by real-time quantitative polymerase chain reaction (RT-Q-PCR). Gene expression modulation could also be validated at the protein level for 5 other genes. We show that integrin stimulation up-regulated FBI-1 expression but inhibited CD79a, Requiem, c-Fos, and caspase 7 induction when the cells underwent apoptosis. We further demonstrate that Fn stimulation also inhibits caspase 3 activation but increases XIAP and survivin expression. Moreover, integrin stimulation also prevents caspase activation induced by doxorubicin. Therefore, we identified genes modulated by adhesion of human precursor B leukemia cells that regulate proliferation and apoptosis, highlighting new pathways that might provide insights into future therapy aiming at targeting apoptosis of leukemia cells.

  7. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  8. Myogenic determination and differentiation of the mouse palatal muscle in relation to the developing mandibular nerve.

    PubMed

    Zhang, L; Yoshimura, Y; Hatta, T; Otani, H

    1999-08-01

    The vertebrate palatal muscles are derived from the cranial paraxial mesoderm and start myogenesis by the expression of myogenic regulatory factors (MRFs). Predetermined myogenic cells migrate from the cranial paraxial mesoderm into the branchial arches, followed by myogenic differentiation. The objective of this study was to elucidate whether the determination, migration, and differentiation of myogenic cells during the myogenesis of the palatal muscles, particularly the tensor veli palatini (TVP), are related to the extending mandibular nerve in mouse embryos. By immunohistochemical staining at embryonic day (E) 9.5, MyoD1 and myogenin have been expressed in the mandibular arch, into which the mandibular nerve had not yet extended. At E11.5, these myogenic cells encircled the extending mandibular nerve and were distributed from the distal and lateral to the trigeminal ganglion and into the mandibular arch to form the muscle plate, a girdle-like structure. By E12.5, these myogenic cells lost their girdle-like pattern, vacated the trunk area of the mandibular nerve, and were separated into several incompletely divided masses encircling the collateral branches of the mandibular nerve. The TVP started differentiation at E13.5 with the appearance of myofilaments and acetylcholinesterase (AchE), whereas the other palatal muscles began differentiation at E14.5. We defined the differentiation process of mouse palatal muscles into five stages based on the present findings. These results suggest that the determination and initial migration of the palatal myogenic cells into the mandibular arch occur before the mandibular nerve extends out of the trigeminal ganglion, whereas the myogenic cells migrating into the final sites of differentiation intimately relate to the extending nerve.

  9. Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

    1985-12-01

    A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

  10. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury.

    PubMed Central

    Mestril, R; Chi, S H; Sayen, M R; O'Reilly, K; Dillmann, W H

    1994-01-01

    Myocardial ischemia markedly increases the expression of several members of the stress/heat shock protein (HSP) family, especially the inducible HSP70 isoforms. Increased expression of HSP70 has been shown to exert a protective effect against a lethal heat shock. We have examined the possibility of using this resistance to a lethal heat shock as a protective effect against an ischemic-like stress in vitro using a rat embryonic heart-derived cell line H9c2 (2-1). Myogenic cells in which the heat shock proteins have been induced by a previous heat shock are found to become resistant to a subsequent simulated ischemic stress. In addition, to address the question of how much does the presence of the HSP70 contribute to this protective effect, we have generated stably transfected cell lines overexpressing the human-inducible HSP70. Embryonal rat heart-derived H9c2(2-1) cells were used for this purpose. This stably transfected cell line was found to be significantly more resistant to an ischemic-like stress than control myogenic cells only expressing the selectable marker (neomycin) or the parental cell line H9c2(2-1). This finding implicates the inducible HSP70 protein as playing a major role in protecting cardiac cells against ischemic injury. Images PMID:8113409

  11. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma.

    PubMed

    Xiong, Ye; Liu, Liyun; Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-02-12

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion.

  12. In vitro myogenic and adipogenic differentiation model of genetically engineered bovine embryonic fibroblast cell lines.

    PubMed

    Yin, Jinlong; Jin, Xun; Beck, Samuel; Kang, Dong Ho; Hong, Zhongshan; Li, Zhehu; Jin, Yongcheng; Zhang, Qiankun; Choi, Yun-Jaie; Kim, Sung-Chan; Kim, Hyunggee

    2010-02-01

    Our current understanding of muscle and adipose tissue development has been largely restricted to the study of murine myogenic and adipogenic cell lines, since attempts to establish these cell lines from other species have met with only limited success. Here we report that a spontaneously immortalized bovine embryonic fibroblast cell line (BEFS) undergoes differentiation into adipogenic or myogenic lineages when ectopically transduced with PPARgamma2 (an adipogenic lineage determinant) or MyoD (a myogenic lineage determinant) and grown in adipogenic and myogenic differentiation culture media (ADCM and MDCM, respectively). We also found that PPARgamma2-overexpressing BEFS cells (BEFS-PPARgamma2) grown in ADCM with or without the PPARgamma2 ligand, troglitazone, preferentially differentiate into adipogenic cells in the presence of ectopic MyoD expression. Ectopic expression of PPARgamma2 in the inducible MyoD-overepxressing BEFS cells (BEFS-TetOn-MyoD) completely suppresses myogenic differentiation and leads to a significant increase in adipogenic differentiation, suggesting that the adipogenic differentiation program might be dominant. Therefore, BEFS, BEFS-PPARgamma2, and BEFS-TetOn-MyoD would be a valuable biological model for understanding a fundamental principle underlying myogenic and adipogenic development, and for isolating various genetic and chemical factors that enable muscle and adipocyte differentiation.

  13. A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

    PubMed Central

    Venuti, J M; Goldberg, L; Chakraborty, T; Olson, E N; Klein, W H

    1991-01-01

    Using the basic helix-loop-helix domain of the myogenic factor myogenin as a probe, we identified a clone from a sea urchin cDNA library with considerable sequence similarity to the vertebrate myogenic factors. This cDNA, sea urchin myogenic factor 1 (SUM-1), transactivated a muscle creatine kinase-chloramphenicol acetyltransferase reporter gene in 10T1/2 fibroblasts to a level comparable to that of the vertebrate myogenic factors. In addition, bacterially expressed beta-galactosidase-SUM-1 fusion protein interacted directly with the kappa E-2 site in the muscle creatine kinase enhancer core as assayed by electrophoretic mobility shift assays. Stably transfected SUM-1 activated the muscle differentiation program and converted 10T1/2 cells from fibroblasts to myotubes. In sea urchin embryos, SUM-1 RNA was not detected before gastrulation. It accumulated to its highest levels during the prism stage when myoblasts were first detected by myosin immunostaining and then diminished as myocytes differentiated. SUM-1 protein was localized in secondary mesenchyme cells when they could first be identified as muscle cells by myosin immunostaining. These results implicate SUM-1 as a regulatory factor involved in the early decision of a pluripotent secondary mesenchyme cell to convert to a myogenic fate. SUM-1 is an example of an invertebrate myogenic factor that is capable of functioning in mammalian cells. Images PMID:2068103

  14. Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

    PubMed

    Matlawska-Wasowska, K; Ward, E; Stevens, S; Wang, Y; Herbst, R; Winter, S S; Wilson, B S

    2013-06-01

    This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

  15. DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation.

    PubMed

    Megiorni, Francesca; Camero, Simona; Ceccarelli, Simona; McDowell, Heather P; Mannarino, Olga; Marampon, Francesco; Pizer, Barry; Shukla, Rajeev; Pizzuti, Antonio; Marchese, Cinzia; Clerico, Anna; Dominici, Carlo

    2016-11-29

    Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS.

  16. DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation

    PubMed Central

    Megiorni, Francesca; Camero, Simona; Ceccarelli, Simona; McDowell, Heather P.; Mannarino, Olga; Marampon, Francesco; Pizer, Barry; Shukla, Rajeev; Pizzuti, Antonio; Marchese, Cinzia; Clerico, Anna; Dominici, Carlo

    2016-01-01

    Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway. Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS. PMID:27764816

  17. Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture.

    PubMed

    Gao, Liyang; Zhao, Mingyan; Li, Peng; Kong, Junchao; Liu, Zhijun; Chen, Yonghua; Huang, Rui; Chu, Jiaqi; Quan, Juanhua; Zeng, Rong

    2017-01-01

    The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/β-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.

  18. A Vision for the Exploration of Mars: Robotic Precursors Followed by Humans to Mars Orbit in 2033

    NASA Technical Reports Server (NTRS)

    Sellers, Piers J.; Garvin, James B.; Kinney, Anne L.; Amato, Michael J.; White, Nicholas E.

    2012-01-01

    The reformulation of the Mars program gives NASA a rare opportunity to deliver a credible vision in which humans, robots, and advancements in information technology combine to open the deep space frontier to Mars. There is a broad challenge in the reformulation of the Mars exploration program that truly sets the stage for: 'a strategic collaboration between the Science Mission Directorate (SMD), the Human Exploration and Operations Mission Directorate (HEOMD) and the Office of the Chief Technologist, for the next several decades of exploring Mars'.Any strategy that links all three challenge areas listed into a true long term strategic program necessitates discussion. NASA's SMD and HEOMD should accept the President's challenge and vision by developing an integrated program that will enable a human expedition to Mars orbit in 2033 with the goal of returning samples suitable for addressing the question of whether life exists or ever existed on Mars

  19. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    SciTech Connect

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-12-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2/sup +/, CD3/sup +/, CD4/sup +/ or CD2/sup +/, CD3/sup +/, CD8/sup +/) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by /sup 51/Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.

  20. Isolation and myogenic differentiation of mesenchymal stem cells for urologic tissue engineering.

    PubMed

    Wu, Rongpei; Liu, Guihua; Bharadwaj, Shantaram; Zhang, Yuanyuan

    2013-01-01

    the bladders repopulate and reorganize the tissue rapidly, thus reducing fibrosis and restoring appropriate neural functionality.In this chapter, we describe the methods we use for the isolation of human bone marrow mesenchymal stem cells (BMSC), and demonstrate evidence of their myogenic differentiation capacity for potential use in urologic tissue engineering.

  1. Time course and side-by-side analysis of mesodermal, pre-myogenic, myogenic and differentiated cell markers in the chicken model for skeletal muscle formation

    PubMed Central

    Berti, Federica; Nogueira, Júlia Meireles; Wöhrle, Svenja; Sobreira, Débora Rodrigues; Hawrot, Katarzyna; Dietrich, Susanne

    2015-01-01

    The chicken is a well-established model for amniote (including human) skeletal muscle formation because the developmental anatomy of chicken skeletal muscle matches that of mammals. The accessibility of the chicken in the egg as well as the sequencing of its genome and novel molecular techniques have raised the profile of this model. Over the years, a number of regulatory and marker genes have been identified that are suited to monitor the progress of skeletal myogenesis both in wildtype and in experimental embryos. However, in the various studies, differing markers at different stages of development have been used. Moreover, contradictory results on the hierarchy of regulatory factors are now emerging, and clearly, factors need to be able to cooperate. Thus, a reference paper describing in detail and side-by-side the time course of marker gene expression during avian myogenesis is needed. We comparatively analysed onset and expression patterns of the key markers for the chicken immature paraxial mesoderm, for muscle-competent cells, for cells committed to myogenesis and for cells entering terminal differentiation. We performed this analysis from stages when the first paraxial mesoderm is being laid down to the stage when mesoderm formation comes to a conclusion. Our data show that, although the sequence of marker gene expression is the same at the various stages of development, the timing of the expression onset is quite different. Moreover, marker gene expression in myogenic cells being deployed from the dorsomedial and ventrolateral lips of the dermomyotome is different from those being deployed from the rostrocaudal lips, suggesting different molecular programs. Furthermore, expression of Myosin Heavy Chain genes is overlapping but different along the length of a myotube. Finally, Mef2c is the most likely partner of Mrf proteins, and, in contrast to the mouse and more alike frog and zebrafish fish, chicken Mrf4 is co-expressed with MyoG as cells enter terminal

  2. Regeneration and myogenic cell proliferation correlate with taurine levels in dystrophin- and MyoD-deficient muscles.

    PubMed

    McIntosh, L M; Garrett, K L; Megeney, L; Rudnicki, M A; Anderson, J E

    1998-10-01

    This study coupled proton magnetic resonance spectroscopy (1H-NMR) and in situ hybridization plus autoradiography in a novel examination of different phenotypes of adult myogenesis that arise from genetic disruptions in mice. Study of muscle extracts from normal and dystrophin-deficient mdx limb and diaphragm muscles confirmed our previous findings linking taurine and muscle regeneration at the peak of damage and repair. 1H-NMR distinguished biochemical differences in regenerating muscles that were consistent with the extent of repair in three strains: mdx dystrophic mice; MyoD(-/-) mice that lack expression of the early myogenic regulatory gene MyoD; and a double-mutant mdx:MyoD(-/-) strain lacking expression of both MyoD and dystrophin. We tested the hypothesis that differences in spectra according to genotype and the regeneration phenotype are related specifically to proliferation by committed myogenic precursor cells. 1H-NMR distinguished the three mutant strains: Taurine was highest in mdx muscles, with the phenotype of most effective regeneration; lowest in MyoD(-/-) muscles, with the least effective formation of new muscle in repair, as reported previously; and intermediate in double-mutant muscles, now reported to show an intermediate repair phenotype. The early and late muscle precursors (mpcs) expressing myf5 and myogenin were examined for proliferation. Eighteen percent of mdx myf5-positive mpcs were proliferative, whereas myf5-positive mpcs did not proliferate in regenerating muscles that lacked MyoD expression. By contrast, whereas 30% of myogenin-positive mpcs were proliferative in mdx muscles, almost none were proliferative in MyoD(-/-) muscles, and 12% were proliferative in double-mutant muscles. Therefore, the extent of accumulated structural regeneration, taurine levels, and proliferation of late mpc (expressing myogenin) were congruent across genotypes. Proliferation by early mpc (expressing myf5) was inhibited by the lack of MyoD expression

  3. Vestibular evoked myogenic potentials in patients with BPPV

    PubMed Central

    Korres, Stavros; Gkoritsa, Eleni; Giannakakou-Razelou, Dimitra; Yiotakis, Ioannis; Riga, Maria; Nikolpoulos, Thomas P.

    2011-01-01

    Summary Background The probable cause of Benign Paroxysmal Positional Vertigo (BPPV) is a degeneration of the oto lithic organs (utricle and saccule). The aim of the study is to find possible alterations in Vestibular Evoked Myogenic Potentials (VEMP) recordings in BPPV patients, because the saccule is part of the VEMP pathway. Material/Methods 27 BPPV patients (24 unilateral and 3 bilateral) aged 20 to 70 years and 30 healthy age matched controls. BPPV was diagnosed by the upbeating geotropic nystagmus found in the supine position with the head overextended towards one side. The subjects were investigated with pure tone audiometry, bi-thermal caloric test with electronystagmographic (ENG) recording, and VEMP recording. Results P1 latency and N1 latency did not present any statistical difference between control ears and affected ears of the BPPV population. The percentage of abnormal VEMP in the BPPV population was statistically higher than in the control ears (p<0.005). No significant relationship could be shown between the occurrence of Canal Paresis and abnormal VEMP. No relationship was found between the side (right or left ear) where BPPV appeared clinically and the side where abnormal VEMP was registered. Conclusions BPPV is a clinical entity associated with increased occurrence of abnormal VEMP recordings, possibly due to degeneration of the saccular macula, which is part of the neural VEMP pathway. PMID:21169909

  4. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  5. PFOS or PreFOS? Are perfluorooctane sulfonate precursors (PreFOS) important determinants of human and environmental perfluorooctane sulfonate (PFOS) exposure?

    PubMed

    Martin, Jonathan W; Asher, Brian J; Beesoon, Sanjay; Benskin, Jonathan P; Ross, Matthew S

    2010-11-01

    The extent to which perfluorooctanesulfonate precursors (PreFOS) play a role in human or environmental exposure to perfluorooctanesulfonate (PFOS) is not well characterized. The diversity of manufactured PreFOS and its degradation products (e.g. C(8)F(17)SO(2)R and C(8)F(17)SO(2)NR'R'', where R is H or F, and R' and R'' are various) has made it difficult to track their fate. Temporal trends of PFOS in both humans and wildlife are discrepant, thus it is difficult to predict future exposure, and hypotheses about the role of PreFOS have been raised. Although abiotic degradation of commercially important PreFOS materials requires further research, current data suggest that the yield of PFOS is negligible or minor. On the other hand, in vivo biotransformation of PreFOS yields PFOS as the major metabolite, and >32% yields have been observed. In Canadians, exposure to PreFOS was equivalent or greater than direct PFOS exposure prior to 2002. In most ocean water, PFOS is dominant to PreFOS, but in the oceans east of Greenland there may be more PreFOS than PFOS, consistent with the fact that whales and humans in this region also show evidence of substantial PreFOS exposure. Quantitative assessments of PFOS body-burdens coming from PreFOS are complicated by the fact that PreFOS partitions to the cellular fraction of blood, thus biomonitoring in serum under predicts PreFOS relative to PFOS. Many unknowns exist that prevent accurate modelling, thus analytical methods that can distinguish directly manufactured PFOS, from PFOS that has been biotransformed from PreFOS, should be applied in future human and environmental monitoring. Two new source tracking principles are presented and applied to human serum.

  6. Methods to uncover an antibody epitope in the KPI domain of Alzheimer's amyloid precursor protein for immunohistochemistry in human brain.

    PubMed

    Campbell, E; Pearson, R C; Parkinson, D

    1999-11-15

    A novel polyclonal antibody (Ab993), specific for a KPI domain epitope of APP, was characterised for use in immunoprecipitation, Western blotting and immunohistochemistry. Conditioned medium from NTera2/D1 cells was used for immunoprecipitation and Western blots. Paraffin-embedded human brain sections were used for immunohistochemistry. The antibody recognised KPI-containing APP on Western blots after standard solubilisation but immunoprecipitation of soluble APP required reduction with 2-mercaptoethanol followed by alkylation of reduced sulphydryl bonds with sodium iodoacetate. Immunohistochemical staining of human brain sections was significantly enhanced by this pre-treatment. Microwaving of sections also increased immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation in 80% formic acid did not confer any enhancement of immunoreactivity. Ab993, applied with the methods reported here, is expected to be valuable in investigations of the pathogenesis of Alzheimer's disease to determine the source of the beta-amyloid peptide.

  7. American Cancer Society Guideline for human papillomavirus (HPV) vaccine use to prevent cervical cancer and its precursors.

    PubMed

    Saslow, Debbie; Castle, Philip E; Cox, J Thomas; Davey, Diane D; Einstein, Mark H; Ferris, Daron G; Goldie, Sue J; Harper, Diane M; Kinney, Walter; Moscicki, Anna-Barbara; Noller, Kenneth L; Wheeler, Cosette M; Ades, Terri; Andrews, Kimberly S; Doroshenk, Mary K; Kahn, Kelly Green; Schmidt, Christy; Shafey, Omar; Smith, Robert A; Partridge, Edward E; Garcia, Francisco

    2007-01-01

    The American Cancer Society (ACS) has developed guidelines for the use of the prophylactic human papillomavirus (HPV) vaccine for the prevention of cervical intraepithelial neoplasia and cervical cancer. These recommendations are based on a formal review of the available evidence. They address the use of prophylactic HPV vaccines, including who should be vaccinated and at what age, as well as a summary of policy and implementation issues. Implications for screening are also discussed.

  8. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

    PubMed Central

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  9. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    PubMed

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.

  10. Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

    SciTech Connect

    Hagiwara, Hiroki; Saito, Fumiaki; Masaki, Toshihiro; Ikeda, Miki; Nakamura-Ohkuma, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of TSA, one of most potent HDACIs, on myogenesis using the C2C12 skeletal muscle cell line. Black-Right-Pointing-Pointer TSA enhances the expression of myosin heavy chain without affecting DAPC expression. Black-Right-Pointing-Pointer TSA enhances the expression of the early MRFs, Myf5 and MEF2, and suppresses the late MRF, myogenin, after 24 h treatment. Black-Right-Pointing-Pointer TSA enhances the expression of the myogenic repressors, Ids, which inhibit myogenic differentiation. Black-Right-Pointing-Pointer TSA promotes myogenesis by coordinating the expression of MRFs and myogenic repressors. -- Abstract: Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.

  11. Adipogenic and myogenic gene expression in rotator cuff muscle of the sheep after tendon tear.

    PubMed

    Frey, Eric; Regenfelder, Felix; Sussmann, Patrick; Zumstein, Matthias; Gerber, Christian; Born, Walter; Fuchs, Bruno

    2009-04-01

    Chronic rotator cuff tendon tears lead to fatty infiltration and muscle atrophy with impaired physiological functions of the affected muscles. However, the cellular and molecular mechanisms of corresponding pathophysiological processes remain unknown. The purpose of this study was to characterize the expression pattern of adipogenic (PPARgamma, C/EBPbeta) and myogenic (myostatin, myogenin, Myf-5) transcription factors in infraspinatus muscle of sheep after tenotomy, implantation of a tension device, refixation of the tendon, and rehabilitation, reflecting a model of chronic rotator cuff tears. In contrast to human patients, the presented sheep model allows a temporal evaluation of the expression of a given marker in the same individual over time. Semiquantitative RT/PCR analysis of PPARgammaã, myostatin, myogenin, Myf-5, and C/EBPbeta transcript levels was carried out with sheep muscle biopsy-derived total RNA. We found a significantly increased expression of Myf-5 and PPARgamma after tenotomy and a significant change for Myf-5 and C/EBPbeta after continuous traction and refixation. This experimental sheep model allows the molecular analysis of pathomechanisms of muscular changes after rotator cuff tear. The results point to a crucial role of the transcription factors PPARgamma, C/EBPbeta, and Myf-5 in impairment and regeneration of rotator cuff muscles after tendon tears in sheep.

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

    PubMed

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

    2006-06-06

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

  13. The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors.

    PubMed

    Mervis, R J; Ahmad, N; Lillehoj, E P; Raum, M G; Salazar, F H; Chan, H W; Venkatesan, S

    1988-11-01

    Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.

  14. Optimization of heme precursors for the expression of human cytochrome P450 2A13 and its co-expression with oxidoreductase in baculovirus/sf9 system.

    PubMed

    Lu, Hui-Yuan; Qiu, Liang-Lin; Yang, Xue-Jiao; Zhang, Xiao-Ming; Zhang, Zhan; Wang, Shou-Lin

    2013-06-01

    Human cytochrome P450 2A13 (CYP2A13), mainly expressed in respiratory tract, is active towards numerous toxicants. To establish the metabolism in vitro, we expressed CYP2A13 and NADPH-CYP450 oxidoreductase (POR) in a baculovirus/sf9 system. Due to the deficiency of sf9 cells in heme incorporation, we investigated the effects of different heme precursors on the expression of CYP2A13, POR and their co-expression. The present results showed that both CYP2A13 and POR were presented the highest expression levels or activity with 0.2 mM δ-aminolaevulinic acid (5-ALA), 0.02 mM Fe(3+) and 0.5-1.0 μg/ml hemin. The combination of 0.2 mM 5-ALA and 0.02 mM Fe(3+) significantly improved CYP2A13 expression and content compared with heme precursors alone, so was POR activity. A multiplicity of infection (MOI) value of 5 pfu/cell for CYP2A13 baculovirus particles induced very high CYP2A13 expression. When co-infected with different POR MOI values, a viral ratio of 5 : 2 was associated with the highest CYP2A13 activity, whereas POR activity dose dependently increased with POR MOI. Furthermore, the expressed CYP2A13 in the optimized conduction could eliminate its substrate aflatoxin B1 at a significantly higher than those in other condition (P < 0.01). Our results provide an efficient approach for expressing functionally characterized, highly active and homogeneous CYP2A13 proteins.

  15. Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells.

    PubMed Central

    Murakami, Y; Mizuno, S; Uehara, Y

    1994-01-01

    The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8037692

  16. Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis

    PubMed Central

    Plaisted, Warren C.; Zavala, Angel; Hingco, Edna; Tran, Ha; Coleman, Ronald; Lane, Thomas E.; Loring, Jeanne F.; Walsh, Craig M.

    2016-01-01

    We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement. PMID:27310015

  17. Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro.

    PubMed

    Hyysalo, Anu; Ristola, Mervi; Joki, Tiina; Honkanen, Mari; Vippola, Minnamari; Narkilahti, Susanna

    2017-03-15

    Stem cell transplantations for spinal cord injury (SCI) have been studied extensively for the past decade in order to replace the damaged tissue with human pluripotent stem cell (hPSC)-derived neural cells. Transplanted cells may, however, benefit from supporting and guiding structures or scaffolds in order to remain viable and integrate into the host tissue. Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. All cell types are shown to efficiently spread over the nanofiber platform and orient according to the fiber alignment. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment. In this work, clinically relevant materials and substrates for nanofibers, fiber coatings, and hydrogel scaffolds are used and combined with cells suitable for developing functional cell grafts for SCI repair.

  18. Targeted analysis of sphingoid precursors in human biofluids by solid-phase extraction with in situ derivatization prior to μ-LC-LIF determination.

    PubMed

    Sánchez, B Alvarez; Capote, F Priego; Luque de Castro, M D

    2011-05-01

    A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2 ng mL(-1) for serum and 0.56-1.36 ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

  19. Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

    PubMed

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E; Notarangelo, Luigi D; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-11-17

    Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

  20. IL-12 involvement in myogenic differentiation of C2C12 in vitro.

    PubMed

    Romanazzo, Sara; Forte, Giancarlo; Morishima, Keisuke; Taniguchi, Akiyoshi

    2015-03-01

    Recently, the extracellular microenvironment has been shown to be critical for the correct differentiation of stem cells to specific tissues. Many factors, including physical (e.g. biomaterial stiffness and topography) and biological (as growth factors, cytokines and chemokines) components, cooperate to create an ideal microenvironment for muscle stem cells, with many of these factors having been widely investigated. We previously demonstrated that the use of non-proliferating muscle-specific and unrelated cells as feeder layers for skeletal muscle progenitor cell differentiation resulted in significant differences in the ability to form myotubes, suggesting the importance of biological factors in myogenic differentiation. In this study, we investigated the biological factors involved in this process, analyzing the expression profile of 84 genes coding for cytokines and chemokines. We successfully identified a novel role for the cytokine IL-12 in the myogenic differentiation of C2C12 mouse skeletal muscle cells. Experiments involving the overexpression or silencing of the IL-12 gene in C2C12 showed that IL-12 enhanced the myogenic differentiation process. Moreover, when IL-12 was overexpressed in non-biologically related feeder cells, the new co-culture system was able to improve myogenic differentiation of C2C12 seeded on top. Although IL-12 is known to be a cytokine involved in inflammatory responses, it also appears to be involved in the myogenic differentiation process, acting as a positive regulator of this mechanism. This fact is expected to prove to be important for the development of functional biomaterials.

  1. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    SciTech Connect

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  2. Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells.

    PubMed

    Li, Xuehui; McFarland, Douglas C; Velleman, Sandra G

    2008-10-01

    Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. Decorin, a small proteoglycan in the extracellular matrix, binds to TGF-beta1 and modulates the activity of TGF-beta1 during muscle cell growth and development. However, its interaction with TGF-beta1 and involvement in myogenesis is not well characterized. In the present study, chicken myogenic satellite cells, myogenic precursors for muscle growth and repair, were isolated from the pectoralis major muscle and used to investigate the biological function of TGF-beta1 and decorin during myogenesis. The over-expression of decorin in satellite cells significantly increased cell proliferation, compared to the control cells. Consistent with this result, reducing decorin expression decreased cell proliferation, which suggests a decorin-mediated mechanism is involved in the regulation of myogenic satellite cell proliferation. Satellite cells over-expressing decorin were less sensitive to TGF-beta1 during proliferation, which indicates that decorin may sequester TGF-beta1 leading to increased proliferation. During satellite cell differentiation, the over-expression of decorin induced differentiation by increasing the muscle specific creatine kinase concentration. However, the addition of TGF-beta1 diminished decorin-mediated cell responsiveness to TGF-beta1 during differentiation. Taken together, these results suggest that decorin induces myogenic satellite cell proliferation and differentiation by regulating cellular responsiveness to TGF-beta1. An alternative TGF-beta1-independent pathway may be involved in the regulation of satellite cells by decorin.

  3. Community effect triggers terminal differentiation of myogenic cells derived from muscle satellite cells by quenching Smad signaling

    SciTech Connect

    Yanagisawa, Michiko; Mukai, Atsushi; Shiomi, Kosuke; Song, Si-Yong; Hashimoto, Naohiro

    2011-01-15

    A high concentration of bone morphogenetic proteins (BMPs) stimulates myogenic progenitor cells to undergo heterotopic osteogenic differentiation. However, the physiological role of the Smad signaling pathway during terminal muscle differentiation has not been resolved. We report here that Smad1/5/8 was phosphorylated and activated in undifferentiated growing mouse myogenic progenitor Ric10 cells without exposure to any exogenous BMPs. The amount of phosphorylated Smad1/5/8 was severely reduced during precocious myogenic differentiation under the high cell density culture condition even in growth medium supplemented with a high concentration of serum. Inhibition of the Smad signaling pathway by dorsomorphin, an inhibitor of Smad activation, or noggin, a specific antagonist of BMP, induced precocious terminal differentiation of myogenic progenitor cells in a cell density-dependent fashion even in growth medium. In addition, Smad1/5/8 was transiently activated in proliferating myogenic progenitor cells during muscle regeneration in rats. The present results indicate that the Smad signaling pathway is involved in a critical switch between growth and differentiation of myogenic progenitor cells both in vitro and in vivo. Furthermore, precocious cell density-dependent myogenic differentiation suggests that a community effect triggers the terminal muscle differentiation of myogenic cells by quenching the Smad signaling.

  4. Calcium dynamics underlying the myogenic response of the renal afferent arteriole

    PubMed Central

    Edwards, Aurélie

    2013-01-01

    The renal afferent arteriole reacts to an elevation in blood pressure with an increase in muscle tone and a decrease in luminal diameter. This effect, known as the myogenic response, is believed to stabilize glomerular filtration and to protect the glomerulus from systolic blood pressure increases, especially in hypertension. To study the mechanisms underlying the myogenic response, we developed a mathematical model of intracellular Ca2+ signaling in an afferent arteriole smooth muscle cell. The model represents detailed transmembrane ionic transport, intracellular Ca2+ dynamics, the kinetics of myosin light chain phosphorylation, and the mechanical behavior of the cell. It assumes that the myogenic response is initiated by pressure-induced changes in the activity of nonselective cation channels. Our model predicts spontaneous vasomotion at physiological luminal pressures and KCl- and diltiazem-induced diameter changes comparable to experimental findings. The time-periodic oscillations stem from the dynamic exchange of Ca2+ between the cytosol and the sarcoplasmic reticulum, coupled to the stimulation of Ca2+-activated potassium (KCa) and chloride (ClCa) channels, and the modulation of voltage-activated L-type channels; blocking sarco/endoplasmic reticulum Ca2+ pumps, ryanodine receptors (RyR), KCa, ClCa, or L-type channels abolishes these oscillations. Our results indicate that the profile of the myogenic response is also strongly dependent on the conductance of ClCa and L-type channels, as well as the activity of plasmalemmal Ca2+ pumps. Furthermore, inhibition of KCa is not necessary to induce myogenic contraction. Lastly, our model suggests that the kinetic behavior of L-type channels results in myogenic kinetics that are substantially faster during constriction than during dilation, consistent with in vitro observations (Loutzenhiser R, Bidani A, Chilton L. Circ. Res. 90: 1316–1324, 2002). PMID:24173354

  5. Structural Characterization of the E2 Domain of APL-1, a C. Elegans Homolog of Human Amyloid Precursor Protein, and its Heparin Binding Site

    SciTech Connect

    Hoopes, J.; Liu, X; Xu, X; Demeler, B; Folta-Stogniew, E; Li, C; Ha, Y

    2010-01-01

    The amyloid {beta}-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.

  6. Effects of Intermittent Administration of Parathyroid Hormone (1-34) on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

    PubMed Central

    Wang, Xiaoxiao; Wang, Yanlan; Dai, Xubin; Chen, Tianyu; Yang, Fanqiao; Dai, Shuangye; Ou, Qianmin; Wang, Yan; Lin, Xuefeng

    2016-01-01

    Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs) may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1) has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH) has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+) and STRO-1(−) hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+) hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+) hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R) than STRO-1(−) hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+) hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+) hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+) hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis. PMID:27069479

  7. Structural Characterization of the E2 Domain of APL-1, a Caenorhabditis elegans Homolog of Human Amyloid Precursor Protein, and Its Heparin Binding Site*

    PubMed Central

    Hoopes, James T.; Liu, Xuying; Xu, Xiaomeng; Demeler, Borries; Folta-Stogniew, Ewa; Li, Chris; Ha, Ya

    2010-01-01

    The amyloid β-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1. PMID:19906646

  8. [Functional organization of myogenic pacemaker of the stomach in conditions of hunger and satiation].

    PubMed

    Kromin, A A; Kuznetsov, A M

    2002-06-01

    In chronic experiments, we have studied electrical activity of muscles of the gastro-esophageal sphincter, small curvature, corpus and antrum of the stomach in conditions of hunger, food intake behaviour and satiation of the rabbits. The aim of this study involved particularities of the electrical activity of myogenic pacemaker zone of the stomach. It has been shown that function of myogenic pacemaker of the rabbit stomach is performed by smooth muscles of the small curvature of the stomach. Pacemaker properties of muscles of the small curvature of the stomach are performed in conditions of food intake behaviour and satiation.

  9. Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype

    PubMed Central

    Baybutt, Herbert; Diack, Abigail B.; Kellett, Katherine A. B.; Piccardo, Pedro; Manson, Jean C.

    2016-01-01

    The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer’s disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production. PMID:27447728

  10. OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells.

    PubMed

    Kaur, Ravinder; Aiken, Christopher; Morrison, Ludivine Coudière; Rao, Radhika; Del Bigio, Marc R; Rampalli, Shravanti; Werbowetski-Ogilvie, Tamra

    2015-10-01

    Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression.

  11. Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events.

    PubMed

    Jackers, P; Clausse, N; Fernandez, M; Berti, A; Princen, F; Wewer, U; Sobel, M E; Castronovo, V

    1996-02-07

    A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution.

  12. Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells.

    PubMed

    Iwasa, Masaki; Miura, Yasuo; Fujishiro, Aya; Fujii, Sumie; Sugino, Noriko; Yoshioka, Satoshi; Yokota, Asumi; Hishita, Terutoshi; Hirai, Hideyo; Andoh, Akira; Ichinohe, Tatsuo; Maekawa, Taira

    2017-01-02

    The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

  13. Band limited chirp stimulation in vestibular evoked myogenic potentials.

    PubMed

    Walther, Leif Erik; Cebulla, Mario

    2016-10-01

    Air conducted vestibular evoked myogenic potentials (VEMP) can be elicited by various low frequency and intense sound stimuli, mainly clicks or short tone bursts (STB). Chirp stimuli are increasingly used in diagnostic audiological evaluations as an effective means to obtain acoustically evoked responses in narrowed or extended frequency ranges. We hypothesized in this study that band limited chirp stimulation, which covers the main sensitivity range of sound sensitive otolithic afferents (around 500 Hz), might be useful for application in cervical and ocular VEMP to air conduction. For this purpose we designed a chirp stimulus ranging 250-1000 Hz (up chirp). The chirp stimulus was delivered with a stimulus intensity of 100 dB nHL in normal subjects (n = 10) and patients with otolith involvement (vestibular neuritis) (n = 6). Amplitudes of the designed chirp ("CW-VEMP-chirp, 250-1000 Hz") were compared with amplitudes of VEMPs evoked by click stimuli (0.1 ms) and a short tone burst (STB, 1-2-1, 8 ms, 500 Hz). CVEMPs and oVEMPs were detectable in 9 of 10 normal individuals. Statistical evaluation in healthy patients revealed significantly larger cVEMP and oVEMP amplitudes for CW-VEMP-chirp (250-1000 Hz) stimuli. CVEMP amplitudes evoked by CW-VEMP-chirp (250-1000 Hz) showed a high stability in comparison with click and STB stimulation. CW-VEMP-chirp (250-1000 Hz) showed abnormal cVEMP and oVEMP amplitudes in patients with vestibular neuritis, with the same properties as click and STB stimulated VEMPs. We conclude that the designed CW-VEMP-chirp (250-1000 Hz) is an effective stimulus which can be further used in VEMP diagnostic. Since a chirp stimulus can be easily varied in its properties, in particular with regard to frequency, this might be a promising tool for further investigations.

  14. CD19-antigen specific nanoscale liposomal formulation of a SYK P-site inhibitor causes apoptotic destruction of human B-precursor leukemia cells

    PubMed Central

    Myers, Dorothea E.; Yiv, Seang; Qazi, Sanjive; Ma, Hong; Cely, Ingrid; Shahidzadeh, Anoush; Arellano, Martha; Finestone, Erin; Gaynon, Paul; Termuhlen, Amanda; Cheng, Jianjun; Uckun, Fatih

    2014-01-01

    We report the anti-leukemic potency of a unique biotargeted nanoscale liposomal nanoparticle (LNP) formulation of the spleen tyrosine kinase (SYK) P-site inhibitor C61. C61-loaded LNP were decorated with a murine CD19-specific monoclonal antibody directed against radiation-resistant CD19-receptor positive aggressive B-precursor acute lymphoblastic leukemia (ALL) cells. The biotargeted C61-LNP were more potent than untargeted C61-LNP and consistently caused apoptosis in B-precursor ALL cells. The CD19-directed C61-LNP also destroyed B-precursor ALL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. This unique nanostructural therapeutic modality targeting the SYK-dependent anti-apoptotic blast cell survival machinery shows promise for overcoming the clinical radiochemotherapy resistance of B-precursor ALL cells. PMID:24910947

  15. Synthetic carbon precursor materials

    SciTech Connect

    Frame, B.J.

    1986-03-01

    Synthetic carbon precursor systems offer advantages over natural petroleum and coal-tar pitch precursors in that they can reproducibly provide a material with a known and uniform composition. They also permit controlled modifications of the derived carbon's properties through variations in the precursor's properties and processing conditions. Extensive research efforts at Oak Ridge have been directed toward the production and characterization of synthetic carbon precursors and the correlations that exist between carbon precursor properties and the properties of the ultimate carbon. This report describes how synthetic carbon precursors can be used to tailor and develop reproducible carbon structures for advanced materials applications. The potential and capability for performing carbon material development at Oak Ridge is also described.

  16. Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone

    PubMed Central

    Kauffenstein, Gilles; Tamareille, Sophie; Prunier, Fabrice; Roy, Charlotte; Ayer, Audrey; Toutain, Bertrand; Billaud, Marie; Isakson, Brant E.; Grimaud, Linda; Loufrani, Laurent; Rousseau, Pascal; Abraham, Pierre; Procaccio, Vincent; Monyer, Hannah; de Wit, Cor; Boeynaems, Jean-Marie; Robaye, Bernard; Kwak, Brenda R.; Henrion, Daniel

    2017-01-01

    Objective Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. Approach and Results We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6−/− arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6−/− mice were protected against MT elevation in myocardial infarction–induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA–GTP binding, myosin light chain, P42–P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43+/− and P2rx7−/− mesenteric resistance arteries. Conclusions Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases. PMID:27255725

  17. Pregnancy increases myometrial artery myogenic tone via NOS- or COX-independent mechanisms.

    PubMed

    Eckman, Delrae M; Gupta, Ridhima; Rosenfeld, Charles R; Morgan, Timothy M; Charles, Shelton M; Mertz, Heather; Moore, Lorna G

    2012-08-15

    Myogenic tone (MT) is a primary modulator of blood flow in the resistance vasculature of the brain, kidney, skeletal muscle, and perhaps in other high-flow organs such as the pregnant uterus. MT is known to be regulated by endothelium-derived factors, including products of the nitric oxide synthase (NOS) and/or the cyclooxygenase (COX) pathways. We asked whether pregnancy influenced MT in myometrial arteries (MA), and if so, whether such an effect could be attributed to alterations in NOS and/or COX. MA (200-300 μm internal diameter, 2-3 mm length) were isolated from 10 nonpregnant and 12 pregnant women undergoing elective hysterectomy or cesarean section, respectively. In the absence of NOS and/or COX inhibition, pregnancy was associated with increased MT in endothelium-intact MA compared with MA from nonpregnant women (P < 0.01). The increase in MT was not due to increased Ca(2+) entry via voltage-dependent channels since both groups of MA exhibited similar levels of constriction when exposed to 50 mM KCl. NOS inhibition (N(ω)-nitro-L-arginine methyl ester, L-NAME) or combined NOS/COX inhibition (L-NAME/indomethacin) increased MT in MA from pregnant women (P = 0.001 and P = 0.042, respectively) but was without effect in arteries from nonpregnant women. Indomethacin alone was without effect on MT in MA from either nonpregnant or pregnant women. We concluded that MT increases in MA during human pregnancy and that this effect was partially opposed by enhanced NOS activity.

  18. Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene.

    PubMed Central

    Saitoh, O; Fujisawa-Sehara, A; Nabeshima, Y; Periasamy, M

    1993-01-01

    In this study, we have isolated and characterized the chicken Myf5 gene, and cDNA clones encoding chicken MyoD1 and myogenin. The chicken Myf5 and MRF4 genes are tandemly located on a single genomic DNA fragment, and the chicken Myf5 gene is organized into at least three exons. Using genomic and cDNA probes, we further analyzed the mRNA levels of four myogenic factors during chicken breast muscle development. This analysis revealed that myogenin expression is restricted to in ovo stages in breast muscle, and is not detectable in neonatal and adult stages. On the other hand, Myf5 expression is detectable until day 7 post-hatching, and is not found in adult muscle, whereas high levels of MyoD1 and MRF4 are detectable at all stages. To further understand the roles of innervation on muscle maturation, we analyzed the expression of the four myogenic factors in denervated adult breast muscle. We found that MyoD1, myogenin, and MRF4 are induced at high levels in denervated muscle, whereas no change occurs in the level of Myf5. These studies suggest that innervation controls the relative abundance and type of myogenic factors that are expressed in adult muscle, and that when nerve control is removed, the muscle reverts to a neonatal phenotype, with the enhanced expression of three myogenic factors (MyoD1, myogenin, and MRF4). Images PMID:8389445

  19. Neurogenic and myogenic motor patterns of rabbit proximal, mid, and distal colon.

    PubMed

    Dinning, P G; Costa, M; Brookes, S J; Spencer, N J

    2012-07-01

    The rabbit colon consists of four distinct regions. The motility of each region is controlled by myogenic and neurogenic mechanisms. Associating these mechanisms with specific motor patterns throughout all regions of the colon has not previously been achieved. Three sections of the colon (the proximal, mid, and distal colon) were removed from euthanized rabbits. The proximal colon consists of a triply teniated region and a single tenia region. Spatio-temporal maps were constructed from video recordings of colonic wall diameter, with associated intraluminal pressure recorded from the aboral end. Hexamethonium (100 μM) and tetrodotoxin (TTX; 0.6 μM) were used to inhibit neural activity. Four distinct patterns of motility were detected: 1 myogenic and 3 neurogenic. The myogenic activity consisted of circular muscle (CM) contractions (ripples) that occurred throughout the colon and propagated in both antegrade (anal) and retrograde (oral) directions. The neural activity of the proximal colon consisted of slowly (0.1 mm/s) propagating colonic migrating motor complexes, which were abolished by hexamethonium. These complexes were observed in the region of the proximal colon with a single band of tenia. In the distal colon, tetrodotoxin-sensitive, thus neurally mediated, but hexamethonium-resistant, peristaltic (anal) and antiperistaltic (oral) contractions were identified. The distinct patterns of neurogenic and myogenic motor activity recorded from isolated rabbit colon are specific to each anatomically distinct region. The regional specificity motor pattern is likely to facilitate orderly transit of colonic content from semi-liquid to solid composition of feces.

  20. Invited Review: Extrinsic regulation of domestic animal-derived myogenic satellite cells II

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The existence of myogenic satellite cells was reported some forty-seven years ago, and, since that time, satellite cell research has flourished. So much new information is generated (daily) on these cells that it can be difficult for individuals to keep abreast of important issues related to the act...

  1. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.

  2. MicroRNA regulation of myogenic satellite cell proliferation and differentiation.

    PubMed

    Harding, Rachel L; Velleman, Sandra G

    2016-01-01

    Myogenic satellite cells are stem cells responsible for muscle growth and regeneration. MicroRNAs (miRNAs) play significant roles in regulating numerous cellular processes. Two genes essential to satellite cell function are syndecan-4 and glypican-1. To determine if miRNAs influence myogenic satellite cell function, one miRNA predicted to bind syndecan-4 (miR-128) and two predicted to bind glypican-1 (miR-24 and miR-16) were inhibited in vitro by transfection of inhibitors targeting each miRNA. Inhibition of these miRNAs differentially affected the expression of syndecan-4, glypican-1, and myogenic regulatory factors myoD and myogenin. Inhibition of miR-16 reduced proliferation of satellite cells at 72 h. Inhibition of miR-128 and miR-24 did not affect proliferation. Inhibition of miRNAs reduced differentiation of satellite cells into myotubes at 48 and 72 h except for miR-16, which only affected differentiation at 72 h. Inhibition of all three miRNAs decreased myotube width at 24 h of differentiation and increased myotube width at 48 h of differentiation. Inhibiting these miRNAs also increased the number of nuclei per myotube at 72 h of differentiation. These data demonstrate individual miRNAs regulate genes essential for myogenic satellite cell proliferation and differentiation.

  3. ROK and Arteriolar Myogenic Tone Generation: Molecular Evidence in Health and Disease

    PubMed Central

    El-Yazbi, Ahmed F.; Abd-Elrahman, Khaled S.

    2017-01-01

    The myogenic response is an inherent property of resistance arteries that warrants a relatively constant blood flow in response to changes in perfusion pressure and protect delicate organs from vascular insufficiencies and excessive blood flow. This fundamental phenomenon has been extensively studied aiming to elucidate the underlying mechanisms triggering smooth muscle contraction in response to intraluminal pressure elevation, particularly, Rho-associated kinase (ROK)-mediated Ca2+-independent mechanisms. The size of the resistance arteries limits the capacity to examine changes in protein phosphorylation/expression levels associated with ROK signaling. A highly sensitive biochemical detection approach was beneficial in examining the role of ROK in different force generation mechanisms along the course of myogenic constriction. In this mini review, we summarize recent results showing direct evidence for the contribution of ROK in development of myogenic response at the level of mechanotransduction, myosin light chain phosphatase inhibition and dynamic actin cytoskeleton reorganization. We will also present evidence that alterations in ROK signaling could underlie the progressive loss in myogenic response in a rat model of type 2 diabetes. PMID:28280468

  4. 5α-Androst-16-en-3α-ol β-D-glucuronide, precursor of 5α-androst-16-en-3α-ol in human sweat.

    PubMed

    Starkenmann, Christian; Mayenzet, Fabienne; Brauchli, Robert; Troccaz, Myriam

    2013-12-01

    5α-Androst-16-en-3α-ol (α-androstenol) is an important contributor to human axilla sweat odor. It is assumed that α-andostenol is excreted from the apocrine glands via a H2 O-soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H2 O-soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α-androstenol, β-androstenol sulfates, 5α-androsta-5,16-dien-3β-ol (β-androstadienol) sulfate, α-androstenol β-glucuronide, α-androstenol α-glucuronide, β-androstadienol β-glucuronide, and α-androstenol β-glucuronide furanose. The occurrence of α-androstenol β-glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative-ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α-androstenol was observed after incubation of the sterile human sweat or α-androstenol β-glucuronide with a commercial glucuronidase enzyme, the urine-isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β-glucuronidase activities. We demonstrated that if α- and β-androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H2 O-soluble precursor of α-androstenol in apocrine secretion should be a β-glucuronide.

  5. A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell.

    PubMed

    Siegel, Ashley L; Gurevich, David B; Currie, Peter D

    2013-09-01

    The cellular basis for mammalian muscle regeneration has been an area of intense investigation over recent decades. The consensus is that a specialized self-renewing stem cell, termed the satellite cell, plays a major role during the process of regeneration in amniotes. How broadly this mechanism is deployed within the vertebrate phylogeny remains an open question. A lack of information on the role of cells analogous to the satellite cell in other vertebrate systems is even more unexpected given the fact that satellite cells were first designated in frogs. An intriguing aspect of this debate is that a number of amphibia and many fish species exhibit epimorphic regenerative processes in specific tissues, whereby regeneration occurs by the dedifferentiation of the damaged tissue, without deploying specialized stem cell populations analogous to satellite cells. Hence, it is feasible that a cellular process completely distinct from that deployed during mammalian muscle regeneration could operate in species capable of epimorphic regeneration. In this minireview, we examine the evidence for the broad phylogenetic distribution of satellite cells. We conclude that, in the vertebrates examined so far, epimorphosis does not appear to be deployed during muscle regeneration, and that analogous cells expressing similar marker genes to satellite cells appear to be deployed during the regenerative process. However, the functional definition of these cells as self-renewing muscle stem cells remains a final hurdle to the definition of the satellite cell as a generic vertebrate cell type.

  6. Variation of erythroid and myeloid precursors in the marrow and peripheral blood of volunteer subjects infected with human parvovirus (B19).

    PubMed Central

    Potter, C G; Potter, A C; Hatton, C S; Chapel, H M; Anderson, M J; Pattison, J R; Tyrrell, D A; Higgins, P G; Willman, J S; Parry, H F

    1987-01-01

    Infection of normal individuals with human parvovirus (B19) results in a mild disease (erythema infectiosum) but gives rise to aplastic crises in patients with chronic hemolytic anemias. The effects of this disease on hemopoiesis were investigated following intranasal inoculation of the virus into three volunteers. A typical disease ensued with a viremia peaking at 9 d. Marrow morphology 6 d after inoculation appeared normal but at 10 d there was a severe loss of erythroid precursors followed by a 1-2-g drop in hemoglobin, and an increase in serum immunoreactive erythropoietin. Erythroid burst-forming units (BFU-E) from the peripheral blood were considerably reduced, starting at the time of viremia and persisting for 4-8 d depending on the individual. Granulocyte-macrophage colony-forming units (CFU-GM) were also affected but the loss started 2 d later. Both CFU-GM and BFU-E showed a sharp overshoot at recovery. In the marrow, BFU-E and CFU-E were reduced at 6 and 10 d in the individual having the longest period of peripheral progenitor loss. In contrast, there was an increase in BFU-E and CFU-E in the subject with least change in peripheral progenitors. In the third subject, with an intermediate picture, there was a loss at 6 d but an increase at 10 d of erythroid progenitors. It is suggested that the architecture of the marrow might partially isolate progenitors from high titers of virus in the serum and individual variation in this respect might give the results observed. PMID:3033026

  7. Early in vivo Effects of the Human Mutant Amyloid-β Protein Precursor (hAβPPSwInd) on the Mouse Olfactory Bulb.

    PubMed

    Rusznák, Zoltán; Kim, Woojin Scott; Hsiao, Jen-Hsiang T; Halliday, Glenda M; Paxinos, George; Fu, YuHong

    2016-01-01

    The amyloid-β protein precursor (AβPP) has long been linked to Alzheimer's disease (AD). Using J20 mice, which express human AβPP with Swedish and Indiana mutations, we studied early pathological changes in the olfactory bulb. The presence of AβPP/amyloid-β (Aβ) was examined in mice aged 3 months (before the onset of hippocampal Aβ deposition) and over 5 months (when hippocampal Aβ deposits are present). The number of neurons, non-neurons, and proliferating cells was assessed using the isotropic fractionator method. Our results demonstrate that although AβPP is overexpressed in some of the mitral cells, widespread Aβ deposition and microglia aggregates are not prevalent in the olfactory bulb. The olfactory bulbs of the younger J20 group harbored significantly fewer neurons than those of the age-matched wild-type mice (5.57±0.13 million versus 6.59±0.36 million neurons; p = 0.011). In contrast, the number of proliferating cells was higher in the young J20 than in the wild-type group (i.e., 6617±425 versus 4455±623 cells; p = 0.011). A significant increase in neurogenic activity was also observed in the younger J20 olfactory bulb. In conclusion, our results indicate that (1) neurons participating in the mouse olfactory function overexpress AβPP; (2) the cellular composition of the young J20 olfactory bulb is different from that of wild-type littermates; (3) these differences may reflect altered neurogenic activity and/or delayed development of the J20 olfactory system; and (4) AβPP/Aβ-associated pathological changes that take place in the J20 hippocampus and olfactory bulb are not identical.

  8. Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid

    PubMed Central

    Halim, Adnan; Brinkmalm, Gunnar; Rüetschi, Ulla; Westman-Brinkmalm, Ann; Portelius, Erik; Zetterberg, Henrik; Blennow, Kaj; Larson, Göran; Nilsson, Jonas

    2011-01-01

    The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid β (Aβ)-peptides, e.g., Aβ1-42, is considered a critical step in the pathogenesis of Alzheimer’s disease (AD). Although APP is a well-known membrane glycoprotein carrying both N- and O-glycans, nothing is known about the occurrence of released APP/Aβ glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified Aβ peptides and glycopeptides from CSF samples and then liquid chromatography—tandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/Aβ peptides, we identified 37 APP/Aβ glycopeptides with sialylated core 1 like O-glycans attached to Thr(−39, −21, −20, and −13), in a series of APP/AβX-15 glycopeptides, where X was −63, −57, −52, and −45, in relation to Asp1 of the Aβ sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the Aβ1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)1-2Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5Acα2,8Neu5Ac linkage. We could not detect any glycosylation of the Aβ1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated Aβ peptides in CSF in six AD patients compared to seven non-AD patients. APP/Aβ sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD. PMID:21712440

  9. Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells

    PubMed Central

    Peschansky, Veronica J.; Pastori, Chiara; Zeier, Zane; Motti, Dario; Wentzel, Katya; Velmeshev, Dmitry; Magistri, Marco; Bixby, John L.; Lemmon, Vance P.; Silva, José P.; Wahlestedt, Claes

    2015-01-01

    CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. “Full”-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions. PMID:26322075

  10. Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

    PubMed Central

    2010-01-01

    Background The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries. Results A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth. Conclusions A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture. PMID:20462406

  11. Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor.

    PubMed

    Poser, Ina; Rahman, Qamar; Lohani, Mohtashim; Yadav, Santosh; Becker, Hans-Henner; Weiss, Dieter G; Schiffmann, Dietmar; Dopp, Elke

    2004-04-11

    The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.

  12. Endoplasmic reticulum stress induces myostatin precursor protein and NF-kappaB in cultured human muscle fibers: relevance to inclusion body myositis.

    PubMed

    Nogalska, Anna; Wojcik, Slawomir; Engel, W King; McFerrin, Janis; Askanas, Valerie

    2007-04-01

    Sporadic-inclusion body myositis (s-IBM) is the most common progressive muscle disease of older persons. It leads to pronounced muscle fiber atrophy and weakness, and there is no successful treatment. We have previously shown that myostatin precursor protein (MstnPP) and myostatin (Mstn) dimer are increased in biopsied s-IBM muscle fibers, and proposed that MstnPP/Mstn increase may contribute to muscle fiber atrophy and weakness in s-IBM patients. Mstn is known to be a negative regulator of muscle fiber mass. It is synthesized as MstnPP, which undergoes posttranslational processing in the muscle fiber to produce mature, active Mstn. To explore possible mechanisms involved in Mstn abnormalities in s-IBM, in the present study we utilized primary cultures of normal human muscle fibers and experimentally modified the intracellular micro-environment to induce endoplasmic-reticulum (ER)-stress, thereby mimicking an important aspect of the s-IBM muscle fiber milieu. ER stress was induced by treating well-differentiated cultured muscle fibers with either tunicamycin or thapsigargin, both well-established ER stress inducers. Our results indicate for the first time that the ER stress significantly increased MstnPP mRNA and protein. The results also suggest that in our system ER stress activates NF-kappaB, and we suggest that MstnPP increase occurred through the ER-stress-activated NF-kappaB. We therefore propose a novel mechanism leading to the Mstn increase in s-IBM. Accordingly, interfering with pathways inducing ER stress, NF-kappaB activation or its action on the MstnPP gene promoter might prevent Mstn increase and provide a new therapeutic approach for s-IBM and, possibly, for muscle atrophy in other neuromuscular diseases.

  13. Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages.

    PubMed

    Dupas, Tanaelle; Rouaud, Thierry; Rouger, Karl; Lieubeau, Blandine; Cario-Toumaniantz, Chrystelle; Fontaine-Pérus, Josiane; Gardahaut, Marie-France; Auda-Boucher, Gwenola

    2011-11-01

    We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.

  14. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

    PubMed

    Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-06-15

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

  15. Ocular Vestibular Evoked Myogenic Potentials Using Head Striker Stimulation

    NASA Technical Reports Server (NTRS)

    De Dios, Y. E.; Gadd, N. E.; Kofman, I. S.; Peters, B. T.; Reschke, M.; Bloomberg, J. J.; Wood, S. J.; Noohibezanjani, F.; Kinnaird, C.; Seidler, R. D.; Mulavara, A. P.

    2016-01-01

    Introduction: Over the last two decades, several studies have been published on the impact of long-duration (i.e., 22 days or longer) spaceflight on the central nervous system (CNS). In consideration of the health and performance of crewmembers in flight and post-flight, we are conducting a controlled prospective longitudinal study to investigate the effects of spaceflight on the extent, longevity and neural bases of sensorimotor, cognitive, and neural changes. Multiple studies have demonstrated the effects of spaceflight on the vestibular system. One of the supporting tests conducted in this protocol is the Vestibular Evoked Myogenic Potential (VEMP) test that provides a unilateral measure of otolith (saccule and utricle) function. A different approach was taken for ocular VEMP (oVEMP) testing using a head striker system (Wackym et al. 2012). The oVEMP is generally considered to be a measure of utricle function. The the otolithic input to the inferior oblique muscle is predominately from the utricular macula. Thus, quantitatively, oVEMP tests utricular function. Another practical extension of these relationships is that the oVEMP reflects the superior vestibular nerve function. Methods: Ground testing was administered on 16 control subjects and for 8 subjects over four repeated sessions spanning 70 days. The oVEMP was elicitied via a hand held striker by a vibrotactile pulse presented at the rate of 1 Hz for 24 seconds on the side of the head as subjects lay supine on a gurney. Subjects were directed to gaze approximately 25 degrees above straight ahead in semi-darkness. For the oVEMP electromyograms will be recorded with active bipolar electrodes (Delsys Inc., Boston, MA) on the infra-orbital ridge 1 cm below the eyelid with a reference electrode on the below the knee cap. The EMG potentials were amplified; band-pass filtered using a BagnoliTM Desktop EMG System (Delsys Inc., Boston, MA, USA). This EMG signal is sampled at 10 kHz and the data stimulus onset to

  16. Validation of ICA-Based Myogenic Artifact Correction for Scalp and Source-Localized EEG

    PubMed Central

    McMenamin, Brenton W.; Shackman, Alexander J.; Maxwell, Jeffrey S.; Bachhuber, David R. W.; Koppenhaver, Adam M.; Greischar, Lawrence L.; Davidson, Richard J.

    2009-01-01

    Muscle electrical activity, or “electromyogenic” (EMG) artifact, poses a serious threat to the validity of electroencephalography (EEG) investigations in the frequency domain. EMG is sensitive to a variety of psychological processes and can mask genuine effects or masquerade as legitimate neurogenic effects across the scalp in frequencies at least as low as the alpha band (8–13Hz). Although several techniques for correcting myogenic activity have been described, most are subjected to only limited validation attempts. Attempts to gauge the impact of EMG correction on intracerebral source models (source “localization” analyses) are rarer still. Accordingly, we assessed the sensitivity and specificity of one prominent correction tool, independent component analysis (ICA), on the scalp and in the source-space using high-resolution EEG. Data were collected from 17 participants while neurogenic and myogenic activity was independently varied. Several protocols for classifying and discarding components classified as myogenic and non-myogenic artifact (e.g., ocular) were systematically assessed, leading to the exclusion of one-third to as much as three-quarters of the variance in the EEG. Some, but not all, of these protocols showed adequate performance on the scalp. Indeed, performance was superior to previously validated regression-based techniques. Nevertheless, ICA-based EMG correction exhibited low validity in the intracerebral source-space, likely owing to incomplete separation of neurogenic from myogenic sources. Taken with prior work, this indicates that EMG artifact can substantially distort estimates of intracerebral spectral activity. Neither regression- nor ICA-based EMG correction techniques provide complete safeguards against such distortions. In light of these results, several practical suggestions and recommendations are made for intelligently using ICA to minimize EMG and other common artifacts. PMID:19833218

  17. Common Phenolic Metabolites of Flavonoids, but Not Their Unmetabolized Precursors, Reduce the Secretion of Vascular Cellular Adhesion Molecules by Human Endothelial Cells123

    PubMed Central

    Warner, Emily F; Zhang, Qingzhi; Raheem, K Saki; O’Hagan, David; O’Connell, Maria A; Kay, Colin D

    2016-01-01

    Background: Flavonoids have been implicated in the prevention of cardiovascular disease; however, their mechanisms of action have yet to be elucidated, possibly because most previous in vitro studies have used supraphysiological concentrations of unmetabolized flavonoids, overlooking their more bioavailable phenolic metabolites. Objective: We aimed to explore the effects of phenolic metabolites and their precursor flavonoids at physiologically achievable concentrations, in isolation and combination, on soluble vascular cellular adhesion molecule-1 (sVCAM-1). Method: Fourteen phenolic acid metabolites and 6 flavonoids were screened at 1 μM for their relative effects on sVCAM-1 secretion by human umbilical vein endothelial cells stimulated with tumor necrosis factor alpha (TNF-α). The active metabolites were further studied for their response at different concentrations (0.01 μM–100 μM), structure-activity relationships, and effect on vascular cellular adhesion molecule (VCAM)-1 mRNA expression. In addition, the additive activity of the metabolites and flavonoids was investigated by screening 25 unique mixtures at cumulative equimolar concentrations of 1 μM. Results: Of the 20 compounds screened at 1 μM, inhibition of sVCAM-1 secretion was elicited by 4 phenolic metabolites, of which protocatechuic acid (PCA) was the most active (−17.2%, P = 0.05). Investigations into their responses at different concentrations showed that PCA significantly reduced sVCAM-1 15.2–36.5% between 1 and 100 μM, protocatechuic acid-3-sulfate and isovanillic acid reduced sVCAM-1 levels 12.2–54.7% between 10 and 100 μM, and protocatechuic acid-4-sulfate and isovanillic acid-3-glucuronide reduced sVCAM-1 secretion 27.6% and 42.8%, respectively, only at 100 μM. PCA demonstrated the strongest protein response and was therefore explored for its effect on VCAM-1 mRNA, where 78.4% inhibition was observed only after treatment with 100 μM PCA. Mixtures of the metabolites showed no

  18. Polyimide Precursor Solid Residuum

    NASA Technical Reports Server (NTRS)

    Weiser, Erik S. (Inventor); St.Clair, Terry L. (Inventor); Echigo, Yoshiaki (Inventor); Kaneshiro, Hisayasu (Inventor)

    2001-01-01

    A polyimide precursor solid residuum is an admixture of an aromatic dianhydride or derivative thereof and an aromatic diamine or derivative thereof plus a complexing agent, which is complexed with the admixture by hydrogen bonding. The polyimide precursor solid residuum is effectively employed in the preparation of polyimide foam and the fabrication of polyimide foam structures.

  19. Viability, differentiation capacity, and detectability of super-paramagnetic iron oxide-labeled muscle precursor cells for magnetic-resonance imaging.

    PubMed

    Azzabi, Fahd; Rottmar, Markus; Jovaisaite, Virginija; Rudin, Markus; Sulser, Tullio; Boss, Andreas; Eberli, Daniel

    2015-02-01

    Cell therapies are a promising approach for the treatment of a variety of human conditions including stress urinary incontinence, but their success greatly depends on the biodistribution, migration, survival, and differentiation of the transplanted cells. Noninvasive in vivo cell tracking therefore presents an important aspect for translation of such a procedure into the clinics. Upon labeling with superparamagnetic iron oxide (SPIO) nanoparticles, cells can be tracked by magnetic resonance imaging (MRI), but possible adverse effect of the labeling have to be considered when labeling stem cells with SPIOs. In this study, human muscle precursor cells (hMPC) were labeled with increasing concentrations of SPIO nanoparticles (100-1600 μg/mL) and cell viability and differentiation capacity upon labeling was assessed in vitro. While a linear dependence between cell viability and nanoparticle concentration could be observed, differentiation capacity was not affected by the presence of SPIOs. Using a nude mouse model, a concentration (400 μg/mL) could be defined that allows reliable detection of hMPCs by MRI but does not influence myogenic in vivo differentiation to mature and functional muscle tissue. This suggests that such an approach can be safely used in a clinical setting to track muscle regeneration in patients undergoing cell therapy without negative effects on the functionality of the bioengineered muscle.

  20. Viability, Differentiation Capacity, and Detectability of Super-Paramagnetic Iron Oxide-Labeled Muscle Precursor Cells for Magnetic-Resonance Imaging

    PubMed Central

    Azzabi, Fahd; Rottmar, Markus; Jovaisaite, Virginija; Rudin, Markus; Sulser, Tullio; Boss, Andreas

    2015-01-01

    Cell therapies are a promising approach for the treatment of a variety of human conditions including stress urinary incontinence, but their success greatly depends on the biodistribution, migration, survival, and differentiation of the transplanted cells. Noninvasive in vivo cell tracking therefore presents an important aspect for translation of such a procedure into the clinics. Upon labeling with superparamagnetic iron oxide (SPIO) nanoparticles, cells can be tracked by magnetic resonance imaging (MRI), but possible adverse effect of the labeling have to be considered when labeling stem cells with SPIOs. In this study, human muscle precursor cells (hMPC) were labeled with increasing concentrations of SPIO nanoparticles (100–1600 μg/mL) and cell viability and differentiation capacity upon labeling was assessed in vitro. While a linear dependence between cell viability and nanoparticle concentration could be observed, differentiation capacity was not affected by the presence of SPIOs. Using a nude mouse model, a concentration (400 μg/mL) could be defined that allows reliable detection of hMPCs by MRI but does not influence myogenic in vivo differentiation to mature and functional muscle tissue. This suggests that such an approach can be safely used in a clinical setting to track muscle regeneration in patients undergoing cell therapy without negative effects on the functionality of the bioengineered muscle. PMID:24988198

  1. Effects of ROCK inhibitor Y27632 and EGFR inhibitor PD168393 on human neural precursors co-cultured with rat auditory brainstem explant.

    PubMed

    Novozhilova, E; Englund-Johansson, U; Kale, A; Jiao, Y; Olivius, P

    2015-02-26

    Hearing function lost by degeneration of inner ear spiral ganglion neurons (SGNs) in the auditory nervous system could potentially be compensated by cellular replacement using suitable donor cells. Donor cell-derived neuronal development with functional synaptic formation with auditory neurons of the cochlear nucleus (CN) in the brainstem is a prerequisite for a successful transplantation. Here a rat auditory brainstem explant culture system was used as a screening platform for donor cells. The explants were co-cultured with human neural precursor cells (HNPCs) to determine HNPCs developmental potential in the presence of environmental cues characteristic for the auditory brainstem region in vitro. We explored effects of pharmacological inhibition of GTPase Rho with its effector Rho-associated kinase (ROCK) and epidermal growth factor receptor (EGFR) signaling on the co-cultures. Pharmacological agents ROCK inhibitor Y27632 and EGFR blocker PD168393 were tested. Effect of the treatment on explant penetration by green fluorescent protein (GFP)-labeled HNPCs was evaluated based on the following criteria: number of GFP-HNPCs located within the explant; distance migrated by the GFP-HNPCs deep into the explant; length of the GFP+/neuronal class III β-tubulin (TUJ1)+ processes developed and phenotypes displayed. In a short 2-week co-culture both inhibitors had growth-promoting effects on HNPCs, prominent in neurite extension elongation. Significant enhancement of migration and in-growth of HNPCs into the brain slice tissue was only observed in Y27632-treated co-cultures. Difference between Y27632- and PD168393-treated HNPCs acquiring neuronal fate was significant, though not different from the fates acquired in control co-culture. Our data suggest the presence of inhibitory mechanisms in the graft-host environment of the auditory brainstem slice co-culture system with neurite growth arresting properties which can be modulated by administration of signaling pathways

  2. Precursors to Lymphoproliferative Malignancies

    PubMed Central

    Goldin, Lynn R.; McMaster, Mary L.; Caporaso, Neil E.

    2013-01-01

    We review monoclonal B-cell lymphocytosis (MBL) as a precursor to chronic lymphocytic leukemia and monoclonal gammopathy of undetermined significance (MGUS) as a precursor to plasma cell disorders. These conditions are present in the general population and increase with age. These precursors aggregate with lymphoproliferative malignancies in families suggesting shared inheritance. MBL and MGUS may share some of the same risk factors as their related malignancies but data are limited. While these conditions are characterized by enhanced risk for the associated malignancy, the majority of individuals with these conditions do not progress to malignancy. A key focus for current work is to identify markers that predict progression to malignancy. PMID:23549397

  3. Murine Muscle Engineered from Dermal Precursors: An In Vitro Model for Skeletal Muscle Generation, Degeneration, and Fatty Infiltration

    PubMed Central

    García-Parra, Patricia; Naldaiz-Gastesi, Neia; Maroto, Marcos; Padín, Juan Fernando; Goicoechea, María; Aiastui, Ana; Fernández-Morales, José Carlos; García-Belda, Paula; Lacalle, Jaione; Álava, Jose Iñaki; García-Verdugo, José Manuel; García, Antonio G.

    2014-01-01

    Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5–7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na+- and Ca2+-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca2+ transients; fiber contractions coupled to these Ca2+ transients suggest that Ca2+ entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca2+ transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration. PMID:23631552

  4. Differentiated evolutionary relationships among chordates from comparative alignments of multiple sequences of MyoD and MyoG myogenic regulatory factors.

    PubMed

    Oliani, L C; Lidani, K C F; Gabriel, J E

    2015-10-16

    MyoD and MyoG are transcription factors that have essential roles in myogenic lineage determination and muscle differentiation. The purpose of this study was to compare multiple amino acid sequences of myogenic regulatory proteins to infer evolutionary relationships among chordates. Protein sequences from Mus musculus (P10085 and P12979), human Homo sapiens (P15172 and P15173), bovine Bos taurus (Q7YS82 and Q7YS81), wild pig Sus scrofa (P49811 and P49812), quail Coturnix coturnix (P21572 and P34060), chicken Gallus gallus (P16075 and P17920), rat Rattus norvegicus (Q02346 and P20428), domestic water buffalo Bubalus bubalis (D2SP11 and A7L034), and sheep Ovis aries (Q90477 and D3YKV7) were searched from a non-redundant protein sequence database UniProtKB/Swiss-Prot, and subsequently analyzed using the Mega6.0 software. MyoD evolutionary analyses revealed the presence of three main clusters with all mammals branched in one cluster, members of the order Rodentia (mouse and rat) in a second branch linked to the first, and birds of the order Galliformes (chicken and quail) remaining isolated in a third. MyoG evolutionary analyses aligned sequences in two main clusters, all mammalian specimens grouped in different sub-branches, and birds clustered in a second branch. These analyses suggest that the evolution of MyoD and MyoG was driven by different pathways.

  5. Earthquakes: hydrogeochemical precursors

    USGS Publications Warehouse

    Ingebritsen, Steven E.; Manga, Michael

    2014-01-01

    Earthquake prediction is a long-sought goal. Changes in groundwater chemistry before earthquakes in Iceland highlight a potential hydrogeochemical precursor, but such signals must be evaluated in the context of long-term, multiparametric data sets.

  6. Selective androgen receptor modulator, YK11, regulates myogenic differentiation of C2C12 myoblasts by follistatin expression.

    PubMed

    Kanno, Yuichiro; Ota, Rumi; Someya, Kousuke; Kusakabe, Taichi; Kato, Keisuke; Inouye, Yoshio

    2013-01-01

    The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11.

  7. Pax3/Pax7 mark a novel population of primitive myogenic cells during development

    PubMed Central

    Kassar-Duchossoy, Lina; Giacone, Ellen; Gayraud-Morel, Barbara; Jory, Aurélie; Gomès, Danielle; Tajbakhsh, Shahragim

    2005-01-01

    Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies. PMID:15964993

  8. Pax3/Pax7 mark a novel population of primitive myogenic cells during development.

    PubMed

    Kassar-Duchossoy, Lina; Giacone, Ellen; Gayraud-Morel, Barbara; Jory, Aurélie; Gomès, Danielle; Tajbakhsh, Shahragim

    2005-06-15

    Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.

  9. Myogenic program induction in mature fat tissue (with MyoD expression)

    SciTech Connect

    Kocaefe, Y.C. . E-mail: kocaefe@hacettepe.edu.tr; Israeli, D.; Ozguc, M.; Danos, O.; Garcia, L. . E-mail: garcia@genethon.fr

    2005-08-15

    MyoD exerts a master transcriptional control over the myogenic differentiation cascade. Here, we study different approaches to induce myogenic transdifferentiation in mature adipocytes utilizing MyoD gene transfer. Organotypic cultures of fat tissue and a long-term culture of in vitro differentiated adipocytes deduced that MyoD provoked morphological changes in mature adipocytes that can be summarized as loss of fat content, acquisition of a fusiform shape and eventual fusion with committed neighbor cells. In vivo, MyoD gene transfer into rat interscapular and inguinal fat pads demonstrated that while structural proteins of muscle lineage were expressed, they co-existed with specific adipocyte proteins. Expression of these proteins diminished over time likewise the fat content. The transdifferentiation process initiated by MyoD did not require cell cycle progression and was well tolerated by the fully differentiated and mature adipocytes.

  10. Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration

    PubMed Central

    Park, Seung-Yoon; Yun, Youngeun; Lim, Jung-Suk; Kim, Mi-Jin; Kim, Sang-Yeob; Kim, Jung-Eun; Kim, In-San

    2016-01-01

    Myoblast fusion is essential for the formation of skeletal muscle myofibres. Studies have shown that phosphatidylserine is necessary for myoblast fusion, but the underlying mechanism is not known. Here we show that the phosphatidylserine receptor stabilin-2 acts as a membrane protein for myoblast fusion during myogenic differentiation and muscle regeneration. Stabilin-2 expression is induced during myogenic differentiation, and is regulated by calcineurin/NFAT signalling in myoblasts. Forced expression of stabilin-2 in myoblasts is associated with increased myotube formation, whereas deficiency of stabilin-2 results in the formation of small, thin myotubes. Stab2-deficient mice have myofibres with small cross-sectional area and few myonuclei and impaired muscle regeneration after injury. Importantly, myoblasts lacking stabilin-2 have reduced phosphatidylserine-dependent fusion. Collectively, our results show that stabilin-2 contributes to phosphatidylserine-dependent myoblast fusion and provide new insights into the molecular mechanism by which phosphatidylserine mediates myoblast fusion during muscle growth and regeneration. PMID:26972991

  11. Nitric oxide drives embryonic myogenesis in chicken through the upregulation of myogenic differentiation factors.

    PubMed

    Cazzato, Denise; Assi, Emma; Moscheni, Claudia; Brunelli, Silvia; De Palma, Clara; Cervia, Davide; Perrotta, Cristiana; Clementi, Emilio

    2014-01-15

    The muscle-specific variant of neuronal nitric oxide (NO) synthase (NOS-I), is developmentally regulated in mouse suggesting a role of NO during myogenesis. In chick embryo, a good model of development, we found that the expression of NOS-I is up-regulated, but only in the early phase of development. Through a pharmacological intervention in ovo we found that NO signalling plays a relevant role during embryonic development. The inhibition of NOS-I decreased the growth of embryo, in particular of muscle tissue, while the restoring of physiological NO levels, via administration of a NO donor, reversed this effect. We found a selective action of NO, produced by NOS-I, on regulatory factors involved in myogenic differentiation in the early phase of chick embryo development: inhibition of NO generation leads to a decreased expression of the Myocyte enhancer factor 2a (Mef2a), Mef2c, Myogenin and Myosin, which was reversed by the administration of a NO donor. NO had no effects on Myf5 and MyoD, the myogenic regulatory factors necessary for myogenic determination. The action of NO on the myogenic regulatory factors was mediated via generation of cyclic GMP (cGMP) and activation of the cGMP-dependent protein kinase G (PKG). Finally we found in myoblasts in vitro that the activation of Mef2c was the key event mediating the NO-induced modulation of myogenesis. Our results identify NO produced by NOS-I as a key messenger in the early phase of embryonic development of chicken, acting as a critical determinant of myogenesis through its physiological cGMP/PKG pathway.

  12. Functional studies of the Ciona intestinalis myogenic regulatory factor reveal conserved features of chordate myogenesis.

    PubMed

    Izzi, Stephanie A; Colantuono, Bonnie J; Sullivan, Kelly; Khare, Parul; Meedel, Thomas H

    2013-04-15

    Ci-MRF is the sole myogenic regulatory factor (MRF) of the ascidian Ciona intestinalis, an invertebrate chordate. In order to investigate its properties we developed a simple in vivo assay based on misexpressing Ci-MRF in the notochord of Ciona embryos. We used this assay to examine the roles of three structural motifs that are conserved among MRFs: an alanine-threonine (Ala-Thr) dipeptide of the basic domain that is known in vertebrates as the myogenic code, a cysteine/histidine-rich (C/H) domain found just N-terminal to the basic domain, and a carboxy-terminal amphipathic α-helix referred to as Helix III. We show that the Ala-Thr dipeptide is necessary for normal Ci-MRF function, and that while eliminating the C/H domain or Helix III individually has no demonstrable effect on Ci-MRF, simultaneous loss of both motifs significantly reduces its activity. Our studies also indicate that direct interaction between CiMRF and an essential E-box of Ciona Troponin I is required for the expression of this muscle-specific gene and that multiple classes of MRF-regulated genes exist in Ciona. These findings are consistent with substantial conservation of MRF-directed myogenesis in chordates and demonstrate for the first time that the Ala/Thr dipeptide of the basic domain of an invertebrate MRF behaves as a myogenic code.

  13. Correcting myogenic ptosis accompanying extraocular muscle weakness: The "Bobby Pin" procedure.

    PubMed

    Vemuri, Swapna; Christianson, Murray D; Demirci, Hakan

    2016-10-01

    This article evaluates the "Bobby Pin" procedure in the correction of myogenic ptosis accompanying extraocular muscle weakness. We retrospectively reviewed 26 eyelids of 13 patients who underwent "Bobby Pin" procedure for myogenic ptosis accompanying extraocular muscle weakness. We evaluated the patients' clinical features such as age, etiology of ptosis, symptoms, standard ptosis measurements, associated systemic diseases, additional ophthalmic conditions, complications, and recurrence. Etiology of myogenic ptosis and extraocular muscle weakness was oculopharyngeal dystrophy in 4 (31%) patients, chronic progressive external ophthalmoplegia in 4 (31%) patients, myotonic dystrophy in 2 (23%) patients, and idiopathic in 3 (15%) patients. The mean levator function was approximately 5 mm pre- and post-operatively (range 1 to 12 mm). The mean margin-to-reflex distance 1 increased from -1.1 mm (below the light reflex) pre-operatively to +0.4 mm (above the light reflex) post-operatively. After a mean follow-up of 40 months, only 1 (8%) patient experienced ptosis recurrence. Upper eyelids were symmetric in both contour and height in all patients. Mild superficial keratopathy involving less than 10% of cornea was observed in 4 (31%) patients. The "Bobby Pin" procedure is an effective and long-lasting treatment option for correcting acquired ptosis accompanying extraocular muscle weakness. The procedure is safe, simple, easily learned, time- and cost-effective, and does not require any expensive equipment.

  14. Middle Ear Myoclonus: Two Informative Cases and a Systematic Discussion of Myogenic Tinnitus

    PubMed Central

    Ellenstein, Aviva; Yusuf, Nadia; Hallett, Mark

    2013-01-01

    Background The term middle ear myoclonus (MEM) has been invoked to explain symptoms of tinnitus presumably caused by the dysfunctional movement of either of the two muscles that insert in the middle ear: tensor tympani and stapedius. MEM has been characterized through heterogeneous case reports in the otolaryngology literature, where clinical presentation is variable, phenomenology is scarcely described, the pathogenic muscle is usually not specified, natural history is unknown, and the presumptive definitive treatment, tensor tympani or stapedius tendon lysis, is inconsistently effective. It is not surprising that no unique acoustogenic mechanism or pathophysiologic process has been identified to explain MEM, one of several descriptive diagnoses associated with the complicated disorders of myogenic tinnitus. Methods Here, we explore MEM from the neurologist’s perspective. Following the detailed descriptions of two informative cases from our clinic, we systematically evaluate the different mechanisms and movement disorder phenomena that could lead to a diagnosis of MEM. Results From a functional neuroanatomic perspective, we explain how tensor tympani MEM is best explained as a form of peritubal myogenic tinnitus, similar to the related disorder of essential palatal tremor. From a pathogenic perspective, we discuss how MEM symptomatology may reflect different mechanical and neurologic processes. We emphasize the diagnostic imperative to recognize when myogenic tinnitus is consistent with a psychogenic origin. Discussion Both individual patient care and further elucidation of MEM will rely on more detailed clinical characterization as well as multidisciplinary input from neurology, otolaryngology, and dentistry. PMID:23610741

  15. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

    PubMed

    Sin, Jon; Andres, Allen M; Taylor, David J R; Weston, Thomas; Hiraumi, Yoshimi; Stotland, Aleksandr; Kim, Brandon J; Huang, Chengqun; Doran, Kelly S; Gottlieb, Roberta A

    2016-01-01

    Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

  16. FGFR4 and its novel splice form in myogenic cells: Interplay of glycosylation and tyrosine phosphorylation.

    PubMed

    Kwiatkowski, Boguslaw A; Kirillova, Irina; Richard, Robert E; Israeli, David; Yablonka-Reuveni, Zipora

    2008-06-01

    The family of fibroblast growth factor receptors (FGFRs) is encoded by four distinct genes. FGFR1 and FGFR4 are both expressed during myogenesis, but whereas the function of FGFR1 in myoblast proliferation has been documented, the role of FGFR4 remains unknown. Here, we report on a new splice form of FGFR4 cloned from primary cultures of mouse satellite cells. This form, named FGFR4(-16), lacks the entire exon 16, resulting in a deletion within the FGFR kinase domain. Expression of FGFR4(-16) coincided with that of wild-type FGFR4 in all FGFR4-expressing tissues examined. Moreover, expression of both FGFR4 forms correlated with the onset of myogenic differentiation, as determined in mouse C2C12 cells and in the inducible myogenic system of 10T(1/2)-MyoD-ER cell line. Both endogenous and overexpressed forms of FGFR4 exhibited N-glycosylation. In contrast to FGFR1, induced homodimerization of FGFR4 proteins did not result in receptor tyrosine phosphorylation. Surprisingly, coexpression of FGFR4 forms and a chimeric FGFR1 protein resulted in FGFR4 tyrosine phosphorylation, raising the possibility that FGFR4 phosphorylation might be enabled by a heterologous tyrosine kinase activity. Collectively, the present study reveals novel characteristics of mouse FGFR4 gene products and delineates their expression pattern during myogenesis. Our findings suggest that FGFR4 functions in a distinctly different manner than the prototype FGFR during myogenic differentiation.

  17. IPMK and β-catenin mediate PLC-β1-dependent signaling in myogenic differentiation.

    PubMed

    Ramazzotti, Giulia; Billi, Anna Maria; Manzoli, Lucia; Mazzetti, Cristina; Ruggeri, Alessandra; Erneux, Christophe; Kim, Seyun; Suh, Pann-Ghill; Cocco, Lucio; Faenza, Irene

    2016-12-20

    In previous studies, we have reported that phospholipase C (PLC)-β1 plays a crucial role in myogenic differentiation and we determined the importance of its catalytic activity for the initiation of this process. Here we define the effectors that take part to its signaling pathway. We show that the Inositol Polyphosphate Multikinase (IPMK) is able to promote myogenic differentiation since its overexpression determines the up-regulation of several myogenic markers. Moreover, we demonstrate that IPMK activates the same cyclin D3 promoter region targeted by PLC-β1 and that IPMK-induced promoter activation relies upon c-jun binding to the promoter, as we have shown previously for PLC-β1. Furthermore, our data shows that IPMK overexpression causes an increase in β-catenin translocation and accumulation to the nuclei of differentiating myoblasts resulting in higher MyoD activation. Finally, we describe that PLC-β1 overexpression determines too an increase in β-catenin translocation and that PLC-β1, IPMK and β-catenin are mediators of the same signaling pathway since their overexpression results in cyclin D3 and myosin heavy chain (MYH) induction.

  18. Limit-cycle-based control of the myogenic wingbeat rhythm in the fruit fly Drosophila

    PubMed Central

    Bartussek, Jan; Mutlu, A. Kadir; Zapotocky, Martin; Fry, Steven N.

    2013-01-01

    In many animals, rhythmic motor activity is governed by neural limit cycle oscillations under the control of sensory feedback. In the fruit fly Drosophila melanogaster, the wingbeat rhythm is generated myogenically by stretch-activated muscles and hence independently from direct neural input. In this study, we explored if generation and cycle-by-cycle control of Drosophila's wingbeat are functionally separated, or if the steering muscles instead couple into the myogenic rhythm as a weak forcing of a limit cycle oscillator. We behaviourally tested tethered flying flies for characteristic properties of limit cycle oscillators. To this end, we mechanically stimulated the fly's ‘gyroscopic’ organs, the halteres, and determined the phase relationship between the wing motion and stimulus. The flies synchronized with the stimulus for specific ranges of stimulus amplitude and frequency, revealing the characteristic Arnol'd tongues of a forced limit cycle oscillator. Rapid periodic modulation of the wingbeat frequency prior to locking demonstrates the involvement of the fast steering muscles in the observed control of the wingbeat frequency. We propose that the mechanical forcing of a myogenic limit cycle oscillator permits flies to avoid the comparatively slow control based on a neural central pattern generator. PMID:23282849

  19. Myogenic capacity of muscle progenitor cells from head and limb muscles.

    PubMed

    Grefte, Sander; Kuijpers, Mette A R; Kuijpers-Jagtman, Anne M; Torensma, Ruurd; Von den Hoff, Johannes W

    2012-02-01

    The restoration of muscles in the soft palate of patients with cleft lip and/or palate is accompanied by fibrosis, which leads to speech and feeding problems. Treatment strategies that improve muscle regeneration have only been tested in limb muscles. Therefore, in the present study the myogenic potential of muscle progenitor cells (MPCs) isolated from head muscles was compared with that of limb muscles. Muscle progenitor cells were isolated from the head muscles and limb muscles of rats and cultured. The proliferation of MPCs was analysed by DNA quantification. The differentiation capacity was analysed by quantifying the numbers of fused cells, and by measuring the mRNA levels of differentiation markers. Muscle progenitor cells were stained to quantify the expression of paired box protein Pax 7 (Pax-7), myoblast determination protein 1 (MyoD), and myogenin. Proliferation was similar in the head MPCs and the limb MPCs. Differentiating head and limb MPCs showed a comparable number of fused cells and mRNA expression levels of myosin-1 (Myh1), myosin-3 (Myh3), and myosin-4 (Myh4). During proliferation and differentiation, the number of Pax-7(+), MyoD(+), and myogenin(+) cells in head and limb MPCs was equal. It was concluded that head and limb MPCs show similar myogenic capacities in vitro. Therefore, in vivo myogenic differences between those muscles might rely on the local microenvironment. Thus, regenerative strategies for limb muscles might also be used for head muscles.

  20. BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model.

    PubMed

    Shi, SongTing; Hoogaars, Willem M H; de Gorter, David J J; van Heiningen, Sandra H; Lin, Herbert Y; Hong, Charles C; Kemaladewi, Dwi U; Aartsma-Rus, Annemieke; ten Dijke, Peter; 't Hoen, Peter A C

    2011-02-01

    Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.

  1. Conditional Cripto overexpression in satellite cells promotes myogenic commitment and enhances early regeneration

    PubMed Central

    Prezioso, Carolina; Iaconis, Salvatore; Andolfi, Gennaro; Zentilin, Lorena; Iavarone, Francescopaolo; Guardiola, Ombretta; Minchiotti, Gabriella

    2015-01-01

    Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRedloxP/loxPCripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury. PMID:26052513

  2. Conditional Cripto overexpression in satellite cells promotes myogenic commitment and enhances early regeneration.

    PubMed

    Prezioso, Carolina; Iaconis, Salvatore; Andolfi, Gennaro; Zentilin, Lorena; Iavarone, Francescopaolo; Guardiola, Ombretta; Minchiotti, Gabriella

    2015-01-01

    Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRed (loxP/loxP) Cripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury.

  3. Calcium Homeostasis in Myogenic Differentiation Factor 1 (MyoD)-Transformed, Virally-Transduced, Skin-Derived Equine Myotubes

    PubMed Central

    Fernandez-Fuente, Marta; Terracciano, Cesare M.; Martin-Duque, Pilar; Brown, Susan C.; Vassaux, Georges; Piercy, Richard J.

    2014-01-01

    Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells’ calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans. PMID:25148524

  4. Biomechanical signals upregulate myogenic gene induction in the presence or absence of inflammation

    PubMed Central

    Chandran, Ravi; Knobloch, Thomas J.; Anghelina, Mirela; Agarwal, Sudha

    2016-01-01

    Inflammation of the muscle invariably leads to muscle cell damage and impaired regeneration. Biomechanical signals play a vital role in the regulation of myogenesis in healthy and inflamed muscle. We hypothesized that biomechanical signals counteract the actions of proinflammatory mediators and upregulate the basic helix-loop-helix and MADS box transcription enhancer factor 2 (MEF2) families of transcription factors, leading to increased myogenesis in inflamed muscle cells. For this purpose, C2C12 cells plated on collagenized silastic membranes were subjected to equibiaxial cyclic tensile strain (CTS) in the presence or absence of TNF-α, and the myogenic gene induction was examined over a period of 72 h. Exposure of cells to CTS resulted in a significant upregulation of mRNA expressions and synthesis of myogenic regulatory factors, MYOD1, myogenin (MYOG), MEF2A, and cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) as well as muscle structural proteins like myosin heavy chain (MYHC) isoforms (MYH1, MYH2, and MYH4) and α-tropomyosin (TPM1), eventually leading to an increase in myotube formation. Contrarily, TNF-α suppressed the expression of all of the above differentiation-inducing factors in C2C12 cells. Further results revealed that simultaneous exposure of C2C12 cells to CTS and TNF-α abrogated the TNF-α-mediated downregulation of myogenic differentiation. In fact, the mRNA expression and protein synthesis of all myogenic factors (Myod1, Myog, Mef2a, Cdkn1a, Myh1, Myh2, Myh4, and Tpm1) were increased in stretched C2C12 cells despite the sustained presence of TNF-α. These results demonstrate that mechanotransduction regulates multiple signaling molecules involved in C2C12 cell differentiation. On one hand, these signals are potent transducers of myotube phenotype in myoblasts; on the other, these signals counteract catabolic actions of proinflammatory cytokines like TNF-α and allow the expression of myogenic genes to upregulate muscle cell differentiation

  5. Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors

    PubMed Central

    2013-01-01

    Background In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. Results Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast

  6. Myogenic-induced mesenchymal stem cells are capable of modulating the immune response by regulatory T cells

    PubMed Central

    Joo, Sunyoung; Lim, Hyun Ju; Jackson, John D; Atala, Anthony

    2014-01-01

    Cell therapy for patients who have intractable muscle disorders may require highly regenerative cells from young, healthy allogeneic donors. Mesenchymal stem cells are currently under clinical investigation because they are known to induce muscle regeneration and believed to be immune privileged, thus making them suitable for allogeneic applications. However, it is unclear whether allogeneic and myogenic-induced mesenchymal stem cells retain their immunomodulatory characteristics. Therefore, our aim was to evaluate the effects of mesenchymal stem cell differentiation on the immune characteristics of cells in vitro. We investigated the immunologic properties of mesenchymal stem cells after myogenic induction. Mesenchymal stem cells were obtained from C57BL/6 mice and the C3H/10T1/2 murine mesenchymal stem cell line. Two different 5-aza-2′-deoxycytidine doses (0.5 and 3 µM) were evaluated for their effects on mesenchymal stem cell skeletal myogenic differentiation potential, immune antigen expression, and mixed lymphocytic reactions. Using a mixed lymphocytic reaction, we determined the optimal splenocyte proliferation inhibition dose. The induction of regulatory T cells was markedly increased by the addition of 3 µM 5-aza-2′-deoxycytidine–treated mesenchymal stem cells. Myogenic-induced mesenchymal stem cells do not elicit alloreactive lymphocyte proliferative responses and are able to modulate immune responses. These findings support the hypothesis that myogenic-induced mesenchymal stem cells may be transplantable across allogeneic barriers. PMID:24555015

  7. The effect of nutritional status and myogenic satellite cell age on turkey satellite cell proliferation, differentiation, and expression of myogenic transcriptional regulatory factors and heparan sulfate proteoglycans syndecan-4 and glypican-1.

    PubMed

    Harthan, Laura B; McFarland, Douglas C; Velleman, Sandra G

    2014-01-01

    Posthatch satellite cell mitotic activity is a critical component of muscle development and growth. Satellite cells are myogenic stem cells that can be induced by nutrition to follow other cellular developmental pathways, and whose mitotic activity declines with age. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation, expression of myogenic transcriptional regulatory factors myogenic determination factor 1, myogenin, and myogenic regulatory factor 4, and expression of the heparan sulfate proteoglycans syndecan-4 and glypican-1 in satellite cells isolated from 1-d-, 7-wk-, and 16-wk-old turkey pectoralis major muscle (1 d, 7 wk, and 16 wk cells, respectively) by using variable concentrations of Met and Cys. Four Met concentrations-30 (control), 7.5, 3, or 0 mg/L with 3.2 mg/L of Cys per 1 mg/L of Met-were used for culture of satellite cells to determine the effect of nutrition and age on satellite cell behavior during proliferation and differentiation. Proliferation was reduced by lower Met and Cys concentrations in all ages at 96 h of proliferation. Differentiation was increased in the 1 d Met-restricted cells, whereas the 7 wk cells treated with 3 mg/L of Met had decreased differentiation. Reduced Met and Cys levels from the control did not significantly affect the 16 wk cells at 72 h of differentiation. However, medium with no Met or Cys suppressed differentiation at all ages. The expression of myogenic determination factor 1, myogenin, myogenic regulatory factor 4, syndecan-4, and glypican-1 was differentially affected by age and Met or Cys treatment. These data demonstrate the age-specific manner in which turkey pectoralis major muscle satellite cells respond to nutritional availability and the importance of defining optimal nutrition to maximize satellite cell proliferation and differentiation for subsequent muscle mass accretion.

  8. Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from Fetal Bovine Bone Marrow.

    PubMed

    Okamura, Lucas Hidenori; Cordero, Paloma; Palomino, Jaime; Parraguez, Victor Hugo; Torres, Cristian Gabriel; Peralta, Oscar Alejandro

    2017-03-07

    The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.

  9. Myogenic differentiation induces taurine transporter in association with taurine-mediated cytoprotection in skeletal muscles.

    PubMed

    Uozumi, Yoriko; Ito, Takashi; Hoshino, Yuki; Mohri, Tomomi; Maeda, Makiko; Takahashi, Kyoko; Fujio, Yasushi; Azuma, Junichi

    2006-03-15

    Skeletal muscle homoeostasis is maintained by a variety of cytoprotective mechanisms. Since ablation of the TauT (taurine transporter) gene results in susceptibility to exercise-induced muscle weakness in vivo, it has been suggested that TauT is essential for skeletal muscle function. However, the regulatory mechanisms of TauT expression remain to be elucidated. In the present study, we demonstrated that TauT was up-regulated during myogenesis in C2C12 cells. Treatment with bFGF (basic fibroblast growth factor), which inhibited muscle differentiation, abrogated myogenic induction of TauT. The promoter activities of TauT were up-regulated during muscle differentiation in C2C12 cells. Database analyses identified an MEF2 (myocyte enhancer binding factor 2) consensus sequence at -844 in the rat TauT gene. Truncation of the promoter region containing the MEF2 site significantly reduced the promoter activity, demonstrating the functional importance of the MEF2 site. Electrophoretic mobility-shift assays confirmed that MEF2 bound to the MEF2 consensus sequence and that DNA-protein complex levels were increased during differentiation. Promoter analyses using mutated promoter-reporter plasmids demonstrated that this site was functional. Importantly, transfection with a MyoD expression vector markedly enhanced TauT promoter activity in the (non-myogenic) 10T1/2 cells. Moreover, co-transfection with an MEF2 expression vector augmented MyoD-induced TauT promoter activity, suggesting that MEF2 is required for full activation of TauT expression. Finally, we examined the effects of taurine on myotube atrophy to clarify the biological significance of the up-regulation of TauT, and demonstrated that taurine attenuated muscle atrophy induced by dexamethasone. TauT expression is regulated under the control of the myogenic programme, and we propose that this is the mechanism for taurine-mediated resistance to muscle atrophy.

  10. Effect of coculturing on the myogenic and adipogenic marker gene expression.

    PubMed

    Muthuraman, Pandurangan

    2014-05-01

    The present experiment was carried out to evaluate the effect of coculturing on myogenic and adipogenic marker gene expressions with the use of C2C12 and 3 T3-L1 preadipocyte cells under the coculture system. C2C12 and 3 T3-L1 cells were cocultured using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3 T3-L1 cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3 T3-L1 cells were transferred to C2C12 plates, and inserts containing C2C12 cells were transferred to 3 T3-L1 plates. After coculture of the C2C12 and 3 T3-L1 cells for 48 and 72 h, the cells in the lower well were harvested for analysis, and this process was carried out for both cells. Myogenic markers such as myogenin, MyoD, Myf5, PAX3, and PAX7 mRNA expressions were analyzed in the cocultured C2C12 cells. Adipogenic markers such as fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activating receptor (PPARγ), CCAAT/enhancer-binding protein (CEBPA), adiponectin, lipoprotein lipase, and fatty acid synthase mRNA expressions were analyzed in the cocultured 3 T3-L1 cells. Myogenic and adipogenic marker gene mRNA expressions were significantly altered in the cocultured C2C12 and 3 T3-L1 cells when compared with the monocultured C2C12 and 3 T3-L1 cells.

  11. Vitamin D induces myogenic differentiation in skeletal muscle derived stem cells.

    PubMed

    Braga, Melissa; Simmons, Zena; Norris, Keith C; Ferrini, Monica G; Artaza, Jorge N

    2017-04-01

    Skeletal muscle wasting is a serious disorder associated with health conditions such as aging, chronic kidney disease and AIDS. Vitamin D is most widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance. Recently, vitamin D supplementation has been shown to improve muscle performance and reduce the risk of falls in vitamin D deficient older adults. However, little is known of the underlying molecular mechanism(s) or the role it plays in myogenic differentiation. We examined the effect of 1,25-D3 on myogenic cell differentiation in skeletal muscle derived stem cells. Primary cultures of skeletal muscle satellite cells were isolated from the tibialis anterior, soleus and gastrocnemius muscles of 8-week-old C57/BL6 male mice and then treated with 1,25-D3 The efficiency of satellite cells isolation determined by PAX7+ cells was 81%, and they expressed VDR. Incubation of satellite cells with 1,25-D3 induces increased expression of: (i) MYOD, (ii) MYOG, (iii) MYC2, (iv) skeletal muscle fast troponin I and T, (v) MYH1, (vi) IGF1 and 2, (vii) FGF1 and 2, (viii) BMP4, (ix) MMP9 and (x) FST. It also promotes myotube formation and decreases the expression of MSTN. In conclusion, 1,25-D3 promoted a robust myogenic effect on satellite cells responsible for the regeneration of muscle after injury or muscle waste. This study provides a mechanistic justification for vitamin D supplementation in conditions characterized by loss of muscle mass and also in vitamin D deficient older adults with reduced muscle mass and strength, and increased risk of falls.

  12. Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity

    PubMed Central

    Chen, Xiaona; He, Liangqiang; Zhao, Yu; Li, Yuying; Zhang, Suyang; Sun, Kun; So, Karl; Chen, Fengyuan; Zhou, Liang; Lu, Leina; Wang, Lijun; Zhu, Xihua; Bao, Xichen; Esteban, Miguel A; Nakagawa, Shinichi; Prasanth, Kannanganattu V; Wu, Zhenguo; Sun, Hao; Wang, Huating

    2017-01-01

    Malat1 is one of the most abundant long non-coding RNAs in various cell types; its exact cellular function is still a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both in vitro and in vivo assays, we demonstrate that Malat1 has a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knockdown of Malat1 accelerates the myogenic differentiation in cultured cells. Consistently, Malat1 knockout mice display enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic mdx mice also improves the muscle regeneration. Mechanistically, in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD-binding loci, causing trimethylation of histone 3 lysine 9 (H3K9me3), which suppresses the target gene expression. Upon differentiation, the pro-myogenic miR-181a is increased and targets the nuclear Malat1 transcripts for degradation through Ago2-dependent nuclear RNA-induced silencing complex machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1β/HDAC1-repressive complex and displacement by a Set7-containing activating complex, which allows MyoD trans-activation to occur. Together, our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation. PMID:28326190

  13. Ammonia elicits a different myogenic response in avian and murine myotubes.

    PubMed

    Stern, Rachel A; Dasarathy, Srinivasan; Mozdziak, Paul E

    2017-02-01

    Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10 mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10 mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48 h (P < 0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P < 0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2', 7'- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24 h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P < 0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment.

  14. Thermographic characterization of masticatory muscle regions in volunteers with and without myogenous temporomandibular disorder: preliminary results

    PubMed Central

    Brioschi, M L; Vardasca, R; Weber, M; Crosato, E M; Arita, E S

    2014-01-01

    Objectives: This study aims to conduct a non-invasive measurement of the cutaneous temperature of selected masticatory muscle regions of volunteers with and without myogenous temporomandibular disorder (TMD), using infrared thermography. Methods: 23 females (10 myogenous TMD volunteers and 13 controls) were recruited and studied. The temperature at the surface of the facial area over the anterior temporalis and masseter muscles was assessed by medical thermography, using regional lateral views and clinical examination. Results: The temperature levels measured at the masseter and anterior temporalis muscle regions in myogenous TMD volunteers (32.85 ± 0.85 and 34.37 ± 0.64 ºC, respectively) were significantly lower (p < 0.05) than those measured in controls (33.49 ± 0.92 and 34.78 ± 0.44 ºC, respectively). Medical infrared imaging indicated a mean difference of 1.4 ºC between the masseter and anterior temporalis regions. Analysis of the comparison between the absolute and normalized mean temperatures was performed using the pairwise comparison of receiver operating characteristic curves, and no statistically significant difference was observed (p > 0.05). The sensitivity and specificity of the thermographic assessment for the masseter region was of 70% and 73%, respectively and for the anterior temporalis region was of 80% and 62%, respectively. Conclusions: This method of evaluating masticatory muscle regions of this preliminary study seems to indicate that it can be used as an aid in complimentary diagnosing of TMDs. PMID:25144605

  15. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    SciTech Connect

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  16. Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

    PubMed

    Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

    2014-01-01

    Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

  17. Histone H3 Methyltransferase Suv39h1 Prevents Myogenic Terminal Differentiation by Repressing MEF2 Activity in Muscle Cells

    PubMed Central

    Jin, Wei; Shang, Yangyang; Peng, Jian; Jiang, Siwen

    2016-01-01

    The myogenic regulatory factors (MRFs) and myocyte enhancer factor 2 (MEF2) transcription factors have been extensively studied as key transcription factors that regulate myogenic gene expression. However, few reports on the molecular mechanism that modulates chromatin remodeling during skeletal muscle differentiation are available. We reported here that the expression of the H3-K9 methyltransferase Suv39h1 was decreased during myoblast differentiation. Ectopic expression of Suv39h1 could inhibit myoblast differentiation, increasing H3-K9 methylation levels, whereas knockdown of Suv39h1 stimulated myoblast differentiation. Furthermore, Suv39h1 interacted with MEF2C directly and inhibited MEF2 transcription activity in a dose-dependent manner. Together, our studies revealed a molecular mechanism wherein Suv39h1 modulated myogenic gene expression and activation during skeletal muscle differentiation. PMID:27916793

  18. Prenatal cocaine exposure causes sex-dependent impairment in the myogenic reactivity of coronary arteries in adult offspring

    PubMed Central

    Xiao, DaLiao; Yang, Shumei; Zhang, Lubo

    2009-01-01

    Cocaine abuse is a significant problem among pregnant women. The present study tested the hypothesis that prenatal cocaine exposure impairs myogenic reactivity of coronary arteries in adult offspring. Pregnant rats received cocaine (30 mg kg−1 day−1) or saline from days 15 to 21 of gestational age and experiments were conducted in 3-month-old offspring. In pressurized coronary septal arteries, the diameter and vessel wall intracellular Ca2+ concentrations were measured simultaneously in the same tissue as a function of intraluminal pressure. Cocaine did not affect KCl-induced contractions of coronary arteries in either males or females, but decreased the distensibility in male vessels. In male offspring, cocaine treatment resulted in a significant decease in pressure-dependent myogenic contractions. Inhibition of eNOS with NG-nitro-L-arginine did not alter the myogenic response in either saline control or cocaine-treated animals. In females, cocaine caused a significant increase in pressure-dependent myogenic contractions. NG-nitro-L-arginine did not affect the myogenic response in the control animals, but blocked the cocaine-mediated effect. In both males and females, the presure-induced increases in vessel wall Ca2+ concentrations were not significantly different between cocaine and saline groups. The ratio of changes in the diameter to Ca2+ concentrations in the presurized arteries was significantly less in male but greater in female offspring after cocaine treatment. The results suggest that prenatal cocaine exposure causes reprogramming of coronary myogenic tone via changes in the Ca2+ sensitivity in a sex-dependent manner, leading to an increased risk of dysfunction of coronary autoregulation in adult offspring. PMID:19704103

  19. Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling.

    PubMed

    Liu, Chang; Chen, Lin; Zeng, Jing; Cui, Jian; Ning, Jiao-Nin; Wang, Guan-Song; Belguise, Karine; Wang, Xiaobo; Qian, Gui-Sheng; Lu, Kai-Zhi; Yi, Bin

    2015-08-01

    Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling.

  20. Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

    SciTech Connect

    Liu, Chang; Chen, Lin; Zeng, Jing; Cui, Jian; Ning, Jiao-nin; Wang, Guan-song; Belguise, Karine; Wang, Xiaobo; Qian, Gui-sheng; Lu, Kai-zhi; Yi, Bin

    2015-08-01

    Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic

  1. The EM Earthquake Precursor

    NASA Astrophysics Data System (ADS)

    Jones, K. B., II; Saxton, P. T.

    2013-12-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After the 1989 Loma Prieta Earthquake, American earthquake investigators predetermined magnetometer use and a minimum earthquake magnitude necessary for EM detection. This action was set in motion, due to the extensive damage incurred and public outrage concerning earthquake forecasting; however, the magnetometers employed, grounded or buried, are completely subject to static and electric fields and have yet to correlate to an identifiable precursor. Secondly, there is neither a networked array for finding any epicentral locations, nor have there been any attempts to find even one. This methodology needs dismissal, because it is overly complicated, subject to continuous change, and provides no response time. As for the minimum magnitude threshold, which was set at M5, this is simply higher than what modern technological advances have gained. Detection can now be achieved at approximately M1, which greatly improves forecasting chances. A propagating precursor has now been detected in both the field and laboratory. Field antenna testing conducted outside the NE Texas town of Timpson in February, 2013, detected three strong EM sources along with numerous weaker signals. The antenna had mobility, and observations were noted for recurrence, duration, and frequency response. Next, two

  2. A case of small round cell tumor of the thoracopulmonary region with myogenic and neurogenic elements.

    PubMed

    Goji, J; Sano, K; Murakami, R; Nakamura, H; Ninomiya, M; Ito, H

    1992-02-01

    We here report a unique case of a young boy with an intrathoracic tumor which consisted of neurogenic and myogenic elements. The initial pathological diagnosis was alveolar rhabdomyosarcoma. The tumor tissue from surgical resection was composed of three parts, each showing a different histological appearance, i.e. a monotonous small cell area, an alveolar area, and an area consisting of pleomorphic rhabdomyoblasts. The small round cells in the monotonous area were immunoreactive with the antibodies for Leu7, neuron-specific enolase (NSE), neurofilament proteins (NFP), and beta 2 microglobulin, but not with the antibody for desmin. These cells also had dense core granules. The tumor cells in the alveolar area were immunoreactive with the antibodies for Leu7 and desmin, but not with the antibody for NFP. Pleomorphic rhabdomyoblasts were immunoreactive with the antibody for desmin, but not with the antibodies for Leu7 and NFP. These findings imply that this tumor consisted of neurogenic and myogenic elements and is considered to be a special type of rhabdomyosarcoma.

  3. Derivation of a continuous myogenic cell culture from an embryo of common killifish, Fundulus heteroclitus.

    PubMed

    Gignac, Sarah J; Vo, Nguyen T K; Mikhaeil, Michael S; Alexander, J Andrew N; MacLatchy, Deborah L; Schulte, Patricia M; Lee, Lucy E J

    2014-09-01

    The common killifish or mummichog (Fundulus heteroclitus) is an estuarine teleost increasingly used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions and ease of maintenance has made them popular aquatic research organisms. Scientific advances with most popular model organisms have been assisted with the availability of continuous cell lines; however, cell lines from F. heteroclitus appear to be unavailable. The development of a killifish cell line, KFE-5, derived from the mid trunk region of a late stage embryo is described here. KFE-5 grows well in Leibovitz's L-15 media with 10% fetal bovine serum (FBS). This cell line has been passaged over 60 times in a span of three years, and cells at various passages have been successfully cryopreserved and thawed. The cells are mostly fibroblastic but contain myogenic cells that differentiate into mono-, bi- and multi-nucleated striated myocytes. Immunofluorescence detection of muscle specific antigens such as α-actinin, desmin, and myosin confirms KFE-5 as a myogenic cell line. KFE-5 has a temperature preference for 26-28°C and has been shown to withstand temperatures up to 37°C. The cell line responds to chemical signals including growth factors, hormones and extracellular matrix components. KFE-5 could thus be useful not only for mummichog's thermobiology but also for studies in fish muscle physiology and development.

  4. Comparison of Cervical and Ocular Vestibular Evoked Myogenic Potentials in Dancers and Non-Dancers

    PubMed Central

    Sinha, Sujeet Kumar; Bohra, Vaishnavi; Sanju, Himanshu Kumar

    2013-01-01

    The objective of the study was to assess the sacculocollic and otolith ocular pathway function using cervical vestibular evoked myogenic potentials (cVEMP) and ocular vestibular myogenic potentials (oVEMP) in dancers and non dancers. Total 16 subjects participated in the study. Out of 16 participants, 8 were trained in Indian classical form of dance (dancers) and other 8 participants who were not trained in any dance form (non dancers). cVEMP and oVEMP responses were recorded for all the subjects. Non Parametric Mann-Whitney U test revealed no significant difference between dancers and non dancers for the latency and amplitude parameter for cVEMP and oVEMP, i.e. P13, N23 latency and P13-N23 complex amplitude and N10, P14 latency, N10-P14 complex amplitude respectively. The vestibular system comprises of several structures. It is possible that the dance style practiced by the dancer’s group assessed in this study does not contribute towards improving the plasticity of the sacculocollic and otolith-ocular pathways. It can be concluded that not all forms of dance training brings about a change in the plasticity of the sacculocollic and otolithocular pathways. PMID:26557344

  5. Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats

    NASA Technical Reports Server (NTRS)

    Marsh, D. R.; Criswell, D. S.; Carson, J. A.; Booth, F. W.

    1997-01-01

    Myogenic factor mRNA expression was examined during muscle regeneration after bupivacaine injection in Fischer 344/Brown Norway F1 rats aged 3, 18, and 31 mo of age (young, adult, and old, respectively). Mass of the tibialis anterior muscle in the young rats had recovered to control values by 21 days postbupivacaine injection but in adult and old rats remained 40% less than that of contralateral controls at 21 and 28 days of recovery. During muscle regeneration, myogenin mRNA was significantly increased in muscles of young, adult, and old rats 5 days after bupivacaine injection. Subsequently, myogenin mRNA levels in young rat muscle decreased to postinjection control values by day 21 but did not return to control values in 28-day regenerating muscles of adult and old rats. The expression of MyoD mRNA was also increased in muscles at day 5 of regeneration in young, adult, and old rats, decreased to control levels by day 14 in young and adult rats, and remained elevated in the old rats for 28 days. In summary, either a diminished ability to downregulate myogenin and MyoD mRNAs in regenerating muscle occurs in old rat muscles, or the continuing myogenic effort includes elevated expression of these mRNAs.

  6. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

    PubMed

    Lee, Eun Ju; Jan, Arif Tasleem; Baig, Mohammad Hassan; Ashraf, Jalaluddin Mohammad; Nahm, Sang-Soep; Kim, Yong-Woon; Park, So-Young; Choi, Inho

    2016-08-01

    Differentiation of muscle satellite cells (MSCs) involves interaction of the proteins present in the extracellular matrix (ECM) with MSCs to regulate their activity, and therefore phenotype. Herein, we report fibromodulin (FMOD), a member of the proteoglycan family participating in the assembly of ECM, as a novel regulator of myostatin (MSTN) during myoblast differentiation. In addition to having a pronounced effect on the expression of myogenic marker genes [myogenin (MYOG) and myosin light chain 2 (MYL2)], FMOD was found to maintain the transcriptional activity of MSTN Moreover, coimmunoprecipitation and in silico studies performed to investigate the interaction of FMOD helped confirm that it antagonizes MSTN function by distorting its folding and preventing its binding to activin receptor type IIB. Furthermore, in vivo studies revealed that FMOD plays an active role in healing by increasing satellite cell recruitment to sites of injury. Together, these findings disclose a hitherto unrecognized regulatory role for FMOD in MSCs and highlight new mechanisms whereby FMOD circumvents the inhibitory effects of MSTN and triggers myoblast differentiation. These findings offer a basis for the design of novel MSTN inhibitors that promote muscle regeneration after injury or for the development of pharmaceutical agents for the treatment of different muscle atrophies.-Lee, E. J., Jan, A. T., Baig, M. H., Ashraf, J. M., Nahm, S.-S., Kim, Y.-W., Park, S.-Y., Choi, I. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

  7. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy.

    PubMed

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C R

    2009-11-01

    Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.

  8. Overexpression of facioscapulohumeral muscular dystrophy region gene 1 causes primary defects in myogenic stem cells.

    PubMed

    Xynos, Alexandros; Neguembor, Maria Victoria; Caccia, Roberta; Licastro, Danilo; Nonis, Alessandro; Di Serio, Clelia; Stupka, Elia; Gabellini, Davide

    2013-05-15

    Overexpression of facioscapulohumeral muscular dystrophy region gene 1 (FRG1) in mice, frogs and worms leads to muscular and vascular abnormalities. Nevertheless, the mechanism that follows FRG1 overexpression and finally leads to muscular defects is currently unknown. Here, we show that the earliest phenotype displayed by mice overexpressing FRG1 is a postnatal muscle-growth defect. Long before the development of muscular dystrophy, FRG1 mice also exhibit a muscle regeneration impairment. Ex vivo and in vivo experiments revealed that FRG1 overexpression causes myogenic stem cell activation and proliferative, clonogenic and differentiation defects. A comparative gene expression profiling of muscles from young pre-dystrophic wild-type and FRG1 mice identified differentially expressed genes in several gene categories and networks that could explain the emerging tissue and myogenic stem cell defects. Overall, our study provides new insights into the pathways regulated by FRG1 and suggests that muscle stem cell defects could contribute to the pathology of FRG1 mice.

  9. Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene

    PubMed Central

    Himeda, Charis L.; Debarnot, Céline; Homma, Sachiko; Beermann, Mary Lou; Miller, Jeffrey B.

    2014-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl. PMID:24636994

  10. Transthyretin: A Transporter Protein Essential for Proliferation of Myoblast in the Myogenic Program

    PubMed Central

    Lee, Eun Ju; Pokharel, Smritee; Jan, Arif Tasleem; Huh, Soyeon; Galope, Richelle; Lim, Jeong Ho; Lee, Dong-Mok; Choi, Sung Wook; Nahm, Sang-Soep; Kim, Yong-Woon; Park, So-Young; Choi, Inho

    2017-01-01

    Irregularities in the cellular uptake of thyroid hormones significantly affect muscle development and regeneration. Herein, we report indispensable role of transthyretin (TTR) in maintaining cellular thyroxine level. TTR was found to enhance recruitment of muscle satellite cells to the site of injury, thereby regulating muscle regeneration. Fluorescence-activated cell sorting (FACS) and immunofluorescence analysis of TTRwt (TTR wild type) and TTRkd (TTR knock-down) cells revealed that TTR controlled cell cycle progression by affecting the expression of Cyclin A2. Deiodinase 2 (D2) mediated increases in triiodothyronine levels were found to regulate the expression of myogenic marker, myogenin (MYOG). Moreover, use of a coumarin derivative (CD) revealed a significant reduction in cellular thyroxine, thereby indicating that TTR play a role in the transport of thyroxine. Taken together, these findings suggest that TTR mediated transport of thyroxine represents a survival mechanism necessary for the myogenic program. The results of this study will be highly useful to the strategic development of novel therapeutics to combat muscular dystrophies. PMID:28075349

  11. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy

    PubMed Central

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C.R.

    2009-01-01

    Muscular dystrophies (MD) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon, however to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell- derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with Facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD. PMID:19682990

  12. The migration of myogenic cells from the somites at the wing level in avian embryos.

    PubMed

    Solursh, M; Drake, C; Meier, S

    1987-06-01

    This study is concerned with establishing a morphological basis for the initiation of migration of putative myogenic cells from the somites into the presumptive wing bud in avian embryos. At the 22 somite stage (stage 14) vasculogenesis is a prevalent activity. By use of a quail specific monoclonal antibody to vascular endothelial cells, vascular cells are recognized in the lateral plate, on the intermediate mesoderm, and on somite surfaces. Cells that are found between the lateral plate mesoderm and somites are shown to be vascular endothelial cells. The lateral body folds progressively bring the lateral plate mesoderm close to the lateral margin of the somites and vascular elements disappear from surface view. It is not until the 24 somite stage (stage 15) that some cells in the ventral lateral margin of somites at the wing level can be seen in scanning electron micrographs to extend basal cell processes toward adjacent vascular tubes. These results provide a morphological basis for the early migratory behavior of myogenic cells and demonstrate their close proximity to the prepatterned vascular network.

  13. Amyloid precursor protein expression is enhanced in human platelets from subjects with Alzheimer's disease and Frontotemporal lobar degeneration: A Real-time PCR study.

    PubMed

    Vignini, Arianna; Morganti, Stefano; Salvolini, Eleonora; Sartini, Davide; Luzzi, Simona; Fiorini, Rosamaria; Provinciali, Leandro; Di Primio, Roberto; Mazzanti, Laura; Emanuelli, Monica

    2013-10-26

    Frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) represent the most frequent causes of early-onset and late-onset degenerative dementia, respectively. A correct diagnosis entails the choice of appropriate therapies. In this view the present study aimed to identify biomarkers that could improve the differential diagnosis. We recently found an overexpression of platelet amyloid precursor protein (APP) in AD; furthermore, recent studies have suggested the presence of changes in APP processing in FTLD. In this context, we analyzed the mRNA expression level of Total APP (TOT) and APP containing a Kunitz-type serine protease inhibitor domain (KPI) in platelets obtained from AD patients, subjects with FTLD, and healthy subjects. In addition, we evaluated the correlation between platelet APP mRNA expression levels and cognitive impairment. Differential gene expression measurements revealed a significant up-regulation of APP TOT and APP KPI in both AD and FTLD patients compared to the controls (being AD/Controls: 1.67 for APP TOT and 1.47 for APP KPI; FTLD/Controls: 1.62 for APP TOT and 1.51 for APP KPI; p<0.05) , although it is interesting to note that in FTLD patients this expression did not correlate with the severity of cognitive impairment. This could be related to a reduced beta-amyloid (Aβ) formation, caused by an alteration of secretase enzymatic activity, even though a post-transcriptional regulation of APP mRNAs in FTLD cannot be excluded.

  14. Amyloid precursor protein expression is enhanced in human platelets from subjects with Alzheimer's disease and frontotemporal lobar degeneration: a real-time PCR study.

    PubMed

    Vignini, Arianna; Morganti, Stefano; Salvolini, Eleonora; Sartini, Davide; Luzzi, Simona; Fiorini, Rosamaria; Provinciali, Leandro; Di Primio, Roberto; Mazzanti, Laura; Emanuelli, Monica

    2013-12-01

    Frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) represent the most frequent causes of early-onset and late-onset degenerative dementia, respectively. A correct diagnosis entails the choice of appropriate therapies. In this view the present study aimed to identify biomarkers that could improve the differential diagnosis. We recently found an overexpression of platelet amyloid precursor protein (APP) in AD; furthermore, recent studies have suggested the presence of changes in APP processing in FTLD. In this context, we analyzed the mRNA expression level of Total APP (TOT) and APP containing a Kunitz-type serine protease inhibitor domain (KPI) in platelets obtained from AD patients, subjects with FTLD, and healthy subjects. In addition, we evaluated the correlation between platelet APP mRNA expression levels and cognitive impairment.Differential gene expression measurements revealed a significant up-regulation of APP TOT and APP KPI in both AD and FTLD patients compared to the controls (being AD/Controls: 1.67 for APP TOT and 1.47 for APP KPI; FTLD/Controls: 1.62 for APP TOT and 1.51 for APP KPI; p < 0.05), although it is interesting to note that in FTLD patients this expression did not correlate with the severity of cognitive impairment.This could be related to a reduced beta-amyloid (Aβ) formation, caused by an alteration of secretase enzymatic activity, even though a post-transcriptional regulation of APP mRNAs in FTLD cannot be excluded.

  15. GZD824 suppresses the growth of human B cell precursor acute lymphoblastic leukemia cells by inhibiting the SRC kinase and PI3K/AKT pathways.

    PubMed

    Ye, Wei; Jiang, Zhiwu; Lu, Xiaoyun; Ren, Xiaomei; Deng, Manman; Lin, Shouheng; Xiao, Yiren; Lin, Simiao; Wang, Suna; Li, Baiheng; Zheng, Yi; Lai, Peilong; Weng, Jianyu; Wu, Donghai; Ma, Yuguo; Chen, Xudong; Wen, Zhesheng; Chen, Yaoyu; Feng, Xiaoyan; Li, Yangqiu; Liu, Pentao; Du, Xin; Pei, Duanqing; Yao, Yao; Xu, Bing; Ding, Ke; Li, Peng

    2016-07-28

    Available therapeutic options for advanced B cell precursor acute lymphoblastic leukemia (pre-B ALL) are limited. Many lead to neutropenia, leaving patients at risk of life-threatening infections and result in bad outcomes. New treatment options are needed to improve overall survival. We previously showed that GZD824, a novel BCR-ABL tyrosine kinase inhibitor, has anti-tumor activity in Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia cells and tumor models. Here, we show that GZD824 decreases cell viability, induces cell-cycle arrest, and causes apoptosis in pre-B ALL cells. Furthermore, Ph- pre-B ALL cells were more sensitive to GZD824 than Ph+ pre-B ALL cells. GZD824 consistently reduced tumor loads in Ph- pre-B ALL xenografts but failed to suppress Ph+ pre-B ALL xenografts. GZD824 decreased phosphorylation of SRC kinase, STAT3, RB and C-myc. It also downregulated the expression of BCL-XL, CCND1 and CDK4 and upregulated expression of CCKN1A. Expression of IRS1 was decreased in GZD824-treated pre-B ALL cells, blocking the PI3K/AKT pathway. These data demonstrate that GZD824 suppresses pre-B ALL cells through inhibition of the SRC kinase and PI3K/AKT pathways and may be a potential therapeutic agent for the management of pre-B ALL.

  16. Myogenic Potential of Whole Bone Marrow Mesenchymal Stem Cells In Vitro and In Vivo for Usage in Urinary Incontinence

    PubMed Central

    Giammò, Alessandro; Boido, Marina; Rustichelli, Deborah; Mareschi, Katia; Errichiello, Edoardo; Parola, Maurizio; Ferrero, Ivana; Fagioli, Franca; Vercelli, Alessandro; Carone, Roberto

    2012-01-01

    Urinary incontinence, defined as the complaint of any involuntary loss of urine, is a pathological condition, which affects 30% females and 15% males over 60, often following a progressive decrease of rhabdosphincter cells due to increasing age or secondary to damage to the pelvic floor musculature, connective tissue and/or nerves. Recently, stem cell therapy has been proposed as a source for cell replacement and for trophic support to the sphincter. To develop new therapeutic strategies for urinary incontinence, we studied the interaction between mesenchymal stem cells (MSCs) and muscle cells in vitro; thereafter, aiming at a clinical usage, we analyzed the supporting role of MSCs for muscle cells in vitro and in in vivo xenotransplantation. MSCs can express markers of the myogenic cell lineages and give rise, under specific cell culture conditions, to myotube-like structures. Nevertheless, we failed to obtain mixed myotubes both in vitro and in vivo. For in vivo transplantation, we tested a new protocol to collect human MSCs from whole bone marrow, to get larger numbers of cells. MSCs, when transplanted into the pelvic muscles close to the external urethral sphincter, survived for a long time in absence of immunosuppression, and migrated into the muscle among fibers, and towards neuromuscular endplates. Moreover, they showed low levels of cycling cells, and did not infiltrate blood vessels. We never observed formation of cell masses suggestive of tumorigenesis. Those which remained close to the injection site showed an immature phenotype, whereas those in the muscle had more elongated morphologies. Therefore, MSCs are safe and can be easily transplanted without risk of side effects in the pelvic muscles. Further studies are needed to elucidate their integration into muscle fibers, and to promote their muscular transdifferentiation either before or after transplantation. PMID:23029081

  17. SH2B1 modulates chromatin state and MyoD occupancy to enhance expressions of myogenic genes.

    PubMed

    Chen, Kuan-Wei; Chang, Yu-Jung; Yeh, Chia-Ming; Lian, Yen-Ling; Chan, Michael W Y; Kao, Cheng-Fu; Chen, Linyi

    2017-02-01

    As mesoderm-derived cell lineage commits to myogenesis, a spectrum of signaling molecules, including insulin growth factor (IGF), activate signaling pathways and ultimately instruct chromatin remodeling and the transcription of myogenic genes. MyoD is a key transcription factor during myogenesis. In this study, we have identified and characterized a novel myogenic regulator, SH2B1. Knocking down SH2B1 delays global chromatin condensation and decreases the formation of myotubes. SH2B1 interacts with histone H1 and is required for the removal of histone H1 from active transcription sites, allowing for the expressions of myogenic genes, IGF2 and MYOG. Chromatin immunoprecipitation assays suggest the requirement of SH2B1 for the induction of histone H3 lysine 4 trimethylation as well as the reduction of histone H3 lysine 9 trimethylation at the promoters and/or enhancers of IGF2 and MYOG genes during myogenesis. Furthermore, SH2B1 is required for the transcriptional activity of MyoD and MyoD occupancy at the enhancer/promoter regions of IGF2 and MYOG during myogenesis. Together, this study demonstrates that SH2B1 fine-tunes global-local chromatin states, expressions of myogenic genes and ultimately promotes myogenesis.

  18. Effects of matrix metalloproteinase-1 on the myogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro

    SciTech Connect

    Zheng, Zhenyang; Leng, Yan; Zhou, Chen; Ma, Zhenyu; Zhong, Zhigang; Shi, Xing-Ming; Zhang, Weixi

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer MMP-1 is a member of the zinc-dependent endopeptidase family. Black-Right-Pointing-Pointer MMP-1 has no cytotoxic effects on BMSCs. Black-Right-Pointing-Pointer MMP-1 can promote the myogenic differentiation of BMSCs. Black-Right-Pointing-Pointer MyoD and desmin were chosen as myogenic markers in this study. -- Abstract: Matrix metalloproteinase-1 (MMP-1) is a member of the family of zinc-dependent endopeptidases that are capable of degrading extracellular matrix (ECM) and certain non-matrix proteins. It has been shown that MMP-1 can enhance muscle regeneration by improving the differentiation and migration of myoblasts. However, it is still not known whether MMP-1 can promote the myogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). To address this question, we isolated BMSCs from C57BL/6J mice and investigated the effects of MMP-1 on their proliferation and myogenic differentiation. Our results showed that MMP-1 treatment, which had no cytotoxic effects on BMSCs, increased the mRNA and protein levels of MyoD and desmin in a dose-dependent manner, indicating that MMP-1 promoted myogenic differentiation of BMSCs in vitro. These results suggest that BMSCs may have a therapeutic potential for treating muscular disorders.

  19. Effect of anthocyanidins on myogenic differentiation in induced and non-induced primary myoblasts from rainbow trout (Oncorhynchus mykiss).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to test whether an anthocyanidin mixture (peonidin, cyanidin and pelargonidin chloride) modulates myogenesis in both induced and non-induced myogenic cells from juvenile rainbow trout (Oncorhynchus mykiss). We evaluated three different anthocyanidin concentrations (1X, 2.5X and...

  20. Long-term engraftment of myogenic progenitors from adipose-derived stem cells and muscle regeneration in dystrophic mice.

    PubMed

    Zhang, Yu; Zhu, Yuling; Li, Yaqin; Cao, Jiqing; Zhang, Huili; Chen, Menglong; Wang, Liang; Zhang, Cheng

    2015-11-01

    Stem cell therapy is a promising approach for treating Duchenne muscular dystrophy (DMD); however, its application is hindered by poor cell engraftment. There have been no reports to date describing the efficient generation of myogenic progenitors from adipose-derived stem cells (ADSCs) that can contribute to muscle regeneration. In this study, we examined the in vivo myogenic potential of progenitors differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3β inhibitor 6-bromoindirubin-3'-oxime as well as the supernatant of ADSC cultures. The results indicate that a proliferative population of myogenic progenitors can be derived from ADSCs that have characteristics similar to muscle satellite cells and are capable of terminal differentiation into multinucleated myotubes. When transplanted into DMD model mdx mice either by intramuscular injection or systemic delivery, progenitors were successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression and contributed to the satellite cell compartment. These findings highlight the potential application of myogenic progenitors derived from ADSCs to the treatment of muscular dystrophy.

  1. Arsenic affects expression and processing of amyloid precursor protein (APP) in primary neuronal cells overexpressing the Swedish mutation of human APP.

    PubMed

    Zarazúa, Sergio; Bürger, Susanne; Delgado, Juan M; Jiménez-Capdeville, Maria E; Schliebs, Reinhard

    2011-06-01

    Arsenic poisoning due to contaminated water and soil, mining waste, glass manufacture, select agrochemicals, as well as sea food, affects millions of people world wide. Recently, an involvement of arsenic in Alzheimer's disease (AD) has been hypothesized (Gong and O'Bryant, 2010). The present study stresses the hypothesis whether sodium arsenite, and its main metabolite, dimethylarsinic acid (DMA), may affect expression and processing of the amyloid precursor protein (APP), using the cholinergic cell line SN56.B5.G4 and primary neuronal cells overexpressing the Swedish mutation of APP, as experimental approaches. Exposure of cholinergic SN56.B5.G4 cells with either sodium arsenite or DMA decreased cell viability in a concentration- and exposure-time dependent manner, and affected the activities of the cholinergic enzymes acetylcholinesterase and choline acetyltransferase. Both sodium arsenite and DMA exposure of SN56.B5.G4 cells resulted in enhanced level of APP, and sAPP in the membrane and cytosolic fractions, respectively. To reveal any effect of arsenic on APP processing, the amounts of APP cleavage products, sAPPβ, and β-amyloid (Aβ) peptides, released into the culture medium of primary neuronal cells derived from transgenic Tg2576 mice, were assessed by ELISA. Following exposure of neuronal cells by sodium arsenite for 12h, the membrane-bound APP level was enhanced, the amount of sAPPβ released into the culture medium was slightly higher, while the levels of Aβ peptides in the culture medium were considerably lower as compared to that assayed in the absence of any drug. The sodium arsenite-induced reduction of Aβ formation suggests an inhibition of the APP γ-cleavage step by arsenite. In contrast, DMA exposure of neuronal cells considerably increased formation of Aβ and sAPPβ, accompanied by enhanced membrane APP level. The DMA-induced changes in APP processing may be the result of the enhanced APP expression. Alternatively, increased Aβ production

  2. Statistically and clinically important change of pain scores in patients with myogenous temporomandibular disorders.

    PubMed

    van Grootel, Robert J; van der Glas, Hilbert W

    2009-05-01

    A within-patient change in pain score after treatment is statistically 'reliable' when it exceeds the smallest detectable difference (SDD). The aims of the present study were (i) to determine SDD for scoring pain behavior on a 0-5 point adjectival scale, and (ii) to explore the relationship between SDD, clinically important difference (CID) and effect size (ES) following treatment of known efficacy, and to compare these parameters of pain behavior with those of VAS-scores of pain intensity [van Grootel RJ, van der Bilt A, van der Glas HW. Long-term reliable change of pain scores in individual myogenous TMD patients. Eur J Pain 2007;11:635-43]. SDD was determined using duplicate scores on pain behavior from a pre-treatment diary that was completed by 118 patients with myogenous temporomandibular disorders (TMD). CID was determined as the mean change in score following treatment, and Cohen's ES as the ratio between mean change and SD of baseline values. The SDDs were 2-3 units (40-60% of the scale range) for test-retest intervals of 1-13 days. CID was 1.13 units (22.6%) and ES was 1.38. The normalized SDD and CID values and ES were similar for VAS-scores of pain intensity, i.e., 38-49% (SDD), 24.2% (CID) and 1.09 (ES). Because reliable change (change>SDD) exceeds CID, the responsiveness of scoring of pain variables is low for detecting CID. The finding of ES values that are larger than 0.5 (ES for patients with chronic degenerative diseases [Norman GR, Sloan JA, Wyrwich KW. Interpretation of changes in health-related quality of life. The remarkable universality of half a standard deviation. Med Care 2003;41:582-92]) suggests that for myogenous TMD (chronic pain not caused by somatic disease and with a large chance on recovery following treatment), there are higher expectations of what constitutes important change.

  3. Evaluation of the effect of static magnetic fields combined with human hepatocyte growth factor on human satellite cell cultures.

    PubMed

    Birk, Richard; Sommer, Ulrich; Faber, Anne; Aderhold, Christoph; Schulz, Johannes D; Hörmann, Karl; Goessler, Ulrich Reinhart; Stern-Straeter, Jens

    2014-06-01

    Tissue engineering is a promising research field, which aims to create new functional muscle tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal muscle tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.

  4. MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells.

    PubMed

    Collins, Carol M; Ellis, Joseph A; Holaska, James M

    2017-04-01

    Mutations in the gene encoding emerin cause Emery-Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo.

  5. Pregnancy upregulates large-conductance Ca(2+)-activated K(+) channel activity and attenuates myogenic tone in uterine arteries.

    PubMed

    Hu, Xiang-Qun; Xiao, Daliao; Zhu, Ronghui; Huang, Xiaohui; Yang, Shumei; Wilson, Sean; Zhang, Lubo

    2011-12-01

    Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K(+) current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K(+) currents to the same extent in uterine arterial myocytes. The BK(Ca) channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BK(Ca) channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17β-estradiol and progesterone caused a significant increase in the BK(Ca) channel current density. Western blot analyses demonstrated a significant increase of the β1, but not α, subunit of BK(Ca) channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the β1, but not α, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the β1 subunit and BK(Ca) channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.

  6. The effect of nutritional status on myogenic gene expression of satellite cells derived from different muscle types.

    PubMed

    Powell, D J; McFarland, D C; Cowieson, A J; Muir, W I; Velleman, S G

    2014-09-01

    Satellite cells (SC) are a multipotential stem cell population responsible for facilitating posthatch muscle fiber hypertrophy. The proliferation and differentiation of SC is sensitive to nutritional regimen, and the SC response to nutrition varies depending upon their muscle type of origin. The objective of the current study was to determine the effect of altering protein synthesis on the expression of several key genes regulating SC activity and the effect of muscle type. Satellite cells isolated from the fast glycolytic pectoralis major and the fast oxidative and glycolytic biceps femoris were studied. These genes included the myogenic regulatory factors myogenic determination factor 1 (MyoD) and myogenin, the cell-membrane associated proteoglycans syndecan-4 and glypican-1, the extracellular matrix proteoglycan decorin, and the transcription factor paired box 7. Protein synthesis potential varied by the concentration of the sulfur amino acids Met and Cys during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3.0/9.6, 1.0/3.2, or 0/0 mg/L. A consistent pattern of gene expression emerged following Met/Cys manipulation as increasing reductions in mRNA expression for all genes were observed as Met/Cys concentration decreased, whereas increased Met/Cys concentration caused either no change or had a small negative effect on mRNA expression. Reduced paired box 7 expression would limit myogenic specification of SC, whereas decreased myogenic regulatory factor expression would affect subsequent myogenic development of the SC. Decreased levels of decorin affect SC response to growth factors like myostatin and transforming growth factor β, and extracellular matrix organization. These data highlight the importance of nutrition on the expression of genes critical for satellite cell activation, proliferation and differentiation, and growth factor signal transduction.

  7. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus

    PubMed Central

    1990-01-01

    We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0- 2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human

  8. Identified EM Earthquake Precursors

    NASA Astrophysics Data System (ADS)

    Jones, Kenneth, II; Saxton, Patrick

    2014-05-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After a number of custom rock experiments, two hypotheses were formed which could answer the EM wave model. The first hypothesis concerned a sufficient and continuous electron movement either by surface or penetrative flow, and the second regarded a novel approach to radio transmission. Electron flow along fracture surfaces was determined to be inadequate in creating strong EM fields, because rock has a very high electrical resistance making it a high quality insulator. Penetrative flow could not be corroborated as well, because it was discovered that rock was absorbing and confining electrons to a very thin skin depth. Radio wave transmission and detection worked with every single test administered. This hypothesis was reviewed for propagating, long-wave generation with sufficient amplitude, and the capability of penetrating solid rock. Additionally, fracture spaces, either air or ion-filled, can facilitate this concept from great depths and allow for surficial detection. A few propagating precursor signals have been detected in the field occurring with associated phases using custom-built loop antennae. Field testing was conducted in Southern California from 2006-2011, and outside the NE Texas town of Timpson in February, 2013. The antennae have mobility and observations were noted for

  9. An interstellar precursor mission

    NASA Technical Reports Server (NTRS)

    Jaffe, L. D.; Ivie, C.; Lewis, J. C.; Lipes, R. G.; Norton, H. N.; Stearns, J. W.; Stimpson, L.; Weissman, P.

    1977-01-01

    A mission out of the planetary system, with launch about the year 2000, could provide valuable scientific data as well as test some of the technology for a later mission to another star. Primary scientific objectives for the precursor mission concern characteristics of the heliopause, the interstellar medium, stellar distances (by parallax measurements), low energy cosmic rays, interplanetary gas distribution, and mass of the solar system. Secondary objectives include investigation of Pluto. Candidate science instruments are suggested. Individual spacecraft systems for the mission were considered, technology requirements and problem areas noted, and a number of recommendations made for technology study and advanced development. The most critical technology needs include attainment of 50-yr spacecraft lifetime and development of a long-life NEP system.

  10. Development of Bipotent Cardiac/Skeletal Myogenic Progenitors from MESP1+ Mesoderm.

    PubMed

    Chan, Sunny Sun-Kin; Hagen, Hannah R; Swanson, Scott A; Stewart, Ron; Boll, Karly A; Aho, Joy; Thomson, James A; Kyba, Michael

    2016-01-12

    The branchiomeric skeletal muscles co-evolved with new chambers of the heart to enable predatory feeding in chordates. These co-evolved tissues develop from a common population in anterior splanchnic mesoderm, referred to as cardiopharyngeal mesoderm (CPM). The regulation and development of CPM are poorly understood. We describe an embryonic stem cell-based system in which MESP1 drives a PDGFRA+ population with dual cardiac and skeletal muscle differentiation potential, and gene expression resembling CPM. Using this system, we investigate the regulation of these bipotent progenitors, and find that cardiac specification is governed by an antagonistic TGFβ-BMP axis, while skeletal muscle specification is enhanced by Rho kinase inhibition. We define transcriptional signatures of the first committed CPM-derived cardiac and skeletal myogenic progenitors, and discover surface markers to distinguish cardiac (PODXL+) from the skeletal muscle (CDH4+) CPM derivatives. These tools open an accessible window on this developmentally and evolutionarily important population.

  11. Clinical utility of ocular vestibular-evoked myogenic potentials (oVEMPs).

    PubMed

    Weber, Konrad P; Rosengren, Sally M

    2015-05-01

    Over the last years, vestibular-evoked myogenic potentials (VEMPs) have been established as clinical tests of otolith function. Complementary to the cervical VEMPs, which assess mainly saccular function, ocular VEMPs (oVEMPs) test predominantly utricular otolith function. oVEMPs are elicited either with air-conducted (AC) sound or bone-conducted (BC) skull vibration and are recorded from beneath the eyes during up-gaze. They assess the vestibulo-ocular reflex and are a crossed excitatory response originating from the inferior oblique eye muscle. Enlarged oVEMPs have proven to be sensitive for screening of superior canal dehiscence, while absent oVEMPs indicate a loss of superior vestibular nerve otolith function, often seen in vestibular neuritis (VN) or vestibular Schwannoma.

  12. Ageing Effect on Air-Conducted Ocular Vestibular Evoked Myogenic Potential.

    PubMed

    Kumar, Kaushlendra; Bhat, Jayashree S; Sequeira, Nimalka Maria; Bhojwani, Kiran M

    2015-06-11

    One of the recent diagnostic tests to assess the function of otolithic organs is through vestibular evoked myogenic potential (VEMP) testing. There are equivocal findings on effect of aging on ocular VEMP (oVEMP) parameters with reference to latencies. Hence this study was taken up to investigate the age related changes in oVEMP parameters. This present study considered 30 participants in each age group i.e., young adults, middle-aged adults and older adults. oVEMP were recorded using insert earphone at 100dBnHL at 500hZ short duration tone burst. The results showed in older adult significant difference in response rate, latencies and amplitude as compared to young and middle adult. Hence age should be taken into consideration when interpreting oVEMP results.

  13. Dropped head syndrome due to myogenic atrophy - a case report of surgical treatment.

    PubMed

    Zenmyo, Michihisa; Abematsu, Masahiko; Yamamoto, Takuya; Ishidou, Yasuhiro; Komiya, Setsuro; Ijiri, Kosei

    2011-01-19

    We report a case of a 69-year-old man with dropped head syndrome associated with isolated neck extensor myopathy (INEM). Over a period of 2 years, he exhibited progressive inability to lift his chin off his chest, resulting in the dropped head position that impaired his activities of daily living. He had a disturbed gait with severe imbalance of spinal alignment. Computed tomography revealed osseous contracture of cervical vertebral bodies in flexed position. Anterior combined posterior reconstruction surgery yielded a successful outcome in his activities of daily living, including his walking balance of spinal alignment. Pathologic study confirmed myogenic atrophy in the cervical extensor muscles. We suggest that consideration for surgical management should be given to dropped head syndrome especially due to INEM.

  14. Comparison of chromosome centromere topology in differentiating cells with myogenic potential.

    PubMed

    Mikołajczak, Bartosz; Wiland, Ewa; Rozwadowska, Natalia; Rucinski, Marek; Mietkiewski, Tomasz; Kurpisz, Maciej

    2009-01-01

    Chromosome territories (CT's) constitute the critical element of the intranuclear architecture. Position of these compartmentalized structures plays an important role in functioning of entire genome. Present study was to examine whether the centromeres position of chromosomes 4, X and Y can be changed during differentiation from myoblasts to myotubes. Topological analysis of these centromeres was based on two-dimensional fluorescent hybridization in situ (2D-FISH). During differentiation process the majority of X chromosome centromeres analyzed shifted to the peripheral part of a nucleus and similar phenomenon was observed with one of the chromosome 4 centromeres. Completely different tendency was noticed when investigating the location of the chromosome Y centromeres. Centromeres of this chromosome migrated to the centre of a nucleus. The results obtained demonstrated visible changes in chromosome topology along the myogenic stem cells differentiation.

  15. Imaging Approaches in Functional Assessment of Implantable Myogenic Biomaterials and Engineered Muscle Tissue

    PubMed Central

    Gargiulo, Paolo

    2015-01-01

    The fields of tissue engineering and regenerative medicine utilize implantable biomaterials and engineered tissues to regenerate damaged cells or replace lost tissues. There are distinct challenges in all facets of this research, but functional assessments and monitoring of such complex environments as muscle tissues present the current strategic priority. Many extant methods for addressing these questions result in the destruction or alteration of tissues or cell populations under investigation. Modern advances in non-invasive imaging modalities present opportunities to rethink some of the anachronistic methods, however, their standard employment may not be optimal when considering advancements in myology. New image analysis protocols and/or combinations of established modalities need to be addressed. This review focuses on efficacies and limitations of available imaging modalities to the functional assessment of implantable myogenic biomaterials and engineered muscle tissues. PMID:26913149

  16. Asymmetric vestibular evoked myogenic potentials in unilateral Menière patients.

    PubMed

    Kingma, C M; Wit, H P

    2011-01-01

    Vestibular evoked myogenic potentials (VEMPs) were measured in 22 unilateral Menière patients with monaural and binaural stimulation with 250 and 500 Hz tone bursts. For all measurement situations significantly lower VEMP amplitudes were on average measured at the affected side compared to the unaffected side. Unilateral Menière patients have, in contrast to normal subjects, asymmetric VEMPs, indicating a permanently affected vestibular (most likely otolith) system at the side of hearing loss. The diagnostic value of VEMP amplitude asymmetry measurement in individual patients is low, because of the large overlap of the VEMP amplitude asymmetry range for unilateral Menière patients with that for normal subjects.

  17. Generation of Nonlinear Vortex Precursors

    NASA Astrophysics Data System (ADS)

    Chen, Yue-Yue; Feng, Xun-Li; Liu, Chengpu

    2016-07-01

    We numerically study the propagation of a few-cycle pulse carrying orbital angular momentum (OAM) through a dense atomic system. Nonlinear precursors consisting of high-order vortex harmonics are generated in the transmitted field due to carrier effects associated with ultrafast Bloch oscillation. The nonlinear precursors survive to propagation effects and are well separated with the main pulse, which provides a straightforward way to measure precursors. By virtue of carrying high-order OAM, the obtained vortex precursors as information carriers have potential applications in optical information and communication fields where controllable loss, large information-carrying capacity, and high speed communication are required.

  18. Perinatal Nicotine Exposure Induces Myogenic Differentiation, But Not Epithelial–Mesenchymal Transition in Rat Offspring Lung

    PubMed Central

    Sakurai, Reiko; Liu, Jie; Gong, Ming; Bo, Ji; Rehan, Virender K.

    2016-01-01

    Summary Objective Perinatal nicotine exposure alters offspring lung structure and function; however, the underlying mechanisms remain incompletely understood. Whether epithelial– mesenchymal transition (EMT), a known contributor to pulmonary pathology, occurs following moderate perinatal nicotine exposure is not known. Methods Pregnant, pair-fed Sprague Dawley rat dams received either placebo (diluent) or nicotine [1 mg/kg, subcutaneously] once daily from embryonic day (e) 6 to postnatal day (PND) 21. Generation 1 (F1) and 3 (F3) offspring lungs were isolated at PND 21, and using Western analysis, q-RT-PCR and immunohistochemistry examined for evidence of EMT. To gain further supportive evidence for nicotine-induced EMT, embryonic day 19 primary rat lung alveolar type II (ATII) cells were cultured and treated with nicotine for 24 hr. Results Protein levels of α-smooth muscle actin, fibronectin, and calponin (myogenic differentiation markers) increased significantly. However, surfactant proteins B and C, and cholinephosphate cytidylyltransferase- α (epithelial cell markers), as well as the typical markers of EMT, E-cadherin, N-cadherin, and fibroblast specific protein (FSP)-1, in both F1 and F3 generation lungs, showed no significant change between the nicotine exposed and control dams. Immunostaining of lung sections and data from in vitro treated ATII cells strongly supported the Western data. Conclusions Enhanced myogenic molecular profile, without evidence of EMT, as evidenced by the absence of the loss of E-cadherin or gains in N-cadherin and FSP-1, suggest that perinatal nicotine exposure does not result in EMT, but it leads to myogenesis, which predominantly accounts for the lung phenotype seen in perinatally nicotine exposed rat offspring. PMID:27183179

  19. Genetic basis of the impaired renal myogenic response in FHH rats

    PubMed Central

    Burke, Marilyn; Pabbidi, Malikarjuna; Fan, Fan; Ge, Ying; Liu, Ruisheng; Williams, Jan Michael; Sarkis, Allison; Lazar, Jozef; Jacob, Howard J.

    2013-01-01

    This study examined the effect of substitution of a 2.4-megabase pair (Mbp) region of Brown Norway (BN) rat chromosome 1 (RNO1) between 258.8 and 261.2 Mbp onto the genetic background of fawn-hooded hypertensive (FHH) rats on autoregulation of renal blood flow (RBF), myogenic response of renal afferent arterioles (AF-art), K+ channel activity in renal vascular smooth muscle cells (VSMCs), and development of proteinuria and renal injury. FHH rats exhibited poor autoregulation of RBF, while FHH.1BN congenic strains with the 2.4-Mbp BN region exhibited nearly perfect autoregulation of RBF. The diameter of AF-art from FHH rats increased in response to pressure but decreased in congenic strains containing the 2.4-Mbp BN region. Protein excretion and glomerular and interstitial damage were significantly higher in FHH rats than in congenic strains containing the 2.4-Mbp BN region. K+ channel current was fivefold greater in VSMCs from renal arterioles of FHH rats than cells obtained from congenic strains containing the 2.4-Mbp region. Sequence analysis of the known and predicted genes in the 2.4-Mbp region of FHH rats revealed amino acid-altering variants in the exons of three genes: Add3, Rbm20, and Soc-2. Quantitative PCR studies indicated that Mxi1 and Rbm20 were differentially expressed in the renal vasculature of FHH and FHH.1BN congenic strain F. These data indicate that transfer of this 2.4-Mbp region from BN to FHH rats restores the myogenic response of AF-art and autoregulation of RBF, decreases K+ current, and slows the progression of proteinuria and renal injury. PMID:23220727

  20. Grp94 acts as a mediator of curcumin-induced antioxidant defence in myogenic cells.

    PubMed

    Pizzo, Paola; Scapin, Cristina; Vitadello, Maurizio; Florean, Cristina; Gorza, Luisa

    2010-04-01

    Curcumin is a non-toxic polyphenol with pleiotropic activities and limited bioavailability. We investigated whether a brief exposure to low doses of curcumin would induce in the myogenic C2C12 cell line an endoplasmic reticulum (ER) stress response and protect against oxidative stress. A 3-hr curcumin administration (5-10 microM) increased protein levels of the ER chaperone Grp94, without affecting those of Grp78, calreticulin and haeme-oxygenase-1 (HO-1). Exposure of cells to hydrogen peroxide 24 hrs after the curcumin treatment decreased caspase-12 activation, total protein oxidation and translocation of NF-kappaB to the nucleus, compared with untreated cells. Grp94 overexpression, achieved by means of either stable or transient trasfection, induced comparable cytoprotective effects to hydrogen peroxide. The delayed cytoprotection induced by curcumin acted through Grp94, because the curcumin-induced increase in Grp94 expression was hampered by either stable or transient transfection with antisense cDNA; in these latter cells, the extent of total protein oxidation, as well as the translocation of NF-kappaB to the nucleus, and the percentage of apoptotic cells were comparable to those observed in both curcumin-untreated wild-type and empty vector transfected cells. Defining the mechanism(s) by which Grp94 exerts its antioxidant defence, the determination of cytosolic calcium levels in C2C12 cells by fura-2 showed a significantly reduced amount of releasable calcium from intracellular stores, both in conditions of Grp94 overexpression and after curcumin pre-treatment. Therefore, a brief exposure to curcumin induces a delayed cytoprotection against oxidative stress in myogenic cells by increasing Grp94 protein level, which acts as a regulator of calcium homeostasis.

  1. Long-term reliable change of pain scores in individual myogenous TMD patients.

    PubMed

    van Grootel, Robert J; van der Bilt, Andries; van der Glas, Hilbert W

    2007-08-01

    A within-patient change in pain score after treatment is statistically 'reliable' when it exceeds the smallest detectable difference (SDD). The aims of the present study were to: (i) determine SDDs for VAS-scores of pain intensity, for sufficiently long test-retest intervals to include most biological fluctuations, (ii) examine whether SDD is invariant to baseline score, and (iii) discuss the value of reliable change (RC) for detecting clinically important difference (CID) or as a possible indicator of successful treatment. SDDs were determined using duplicate data from 118 patients with myogenous Temporomandibular disorders: (1) VAS-scores of pain intensity from the masticatory system in a pre-treatment diary, and (2) VAS-scores of pain intensity from the hand (cold-pressor test). RC was determined in VAS-scores from a pre- and post-treatment questionnaire. The long-term SDD was 49mm. A regression analysis on duplicate VAS-scores showed that SDD was largely invariant to the baseline level. Because RC (change>SDD) exceeded CID, it might serve as an indicator of successful treatment. However, only 17% of the patients showed RC after treatment, mainly because the baseline was smaller than SDD in 67% of the patients thus making detection of any treatment effect impossible. For patients with possible detection (33%), the frequency of RC was 51%. If the detection threshold would be avoided by provoking pain in patients with a low baseline, a long-term RC in VAS-scores might occur in about half of all myogenous TMD patients and might then serve as an indicator of cases of treatment success.

  2. Using Zinc Finger Nuclease Technology to Generate CRX‐Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation

    PubMed Central

    Collin, Joseph; Mellough, Carla B; Dorgau, Birthe; Przyborski, Stefan; Moreno‐Gimeno, Inmaculada

    2015-01-01

    Abstract The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone‐Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX‐GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX‐GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation. Stem Cells 2016;34:311–321 PMID:26608863

  3. Dengue virus-specific human CD4+ T-lymphocyte responses in a recipient of an experimental live-attenuated dengue virus type 1 vaccine: bulk culture proliferation, clonal analysis, and precursor frequency determination.

    PubMed Central

    Green, S; Kurane, I; Edelman, R; Tacket, C O; Eckels, K H; Vaughn, D W; Hoke, C H; Ennis, F A

    1993-01-01

    We analyzed the CD4+ T-lymphocyte responses to dengue, West Nile, and yellow fever viruses 4 months after immunization of a volunteer with an experimental live-attenuated dengue virus type 1 vaccine (DEN-1 45AZ5). We examined bulk culture proliferation to noninfectious antigens, determined the precursor frequency of specific CD4+ T cells by limiting dilution, and established and analyzed CD4+ T-cell clones. Bulk culture proliferation was predominantly dengue virus type 1 specific with a lesser degree of cross-reactive responses to other dengue virus serotypes, West Nile virus, and yellow fever virus. Precursor frequency determination by limiting dilution in the presence of noninfectious dengue virus antigens revealed a frequency of antigen-reactive cells of 1 in 1,686 peripheral blood mononuclear cells (PBMC) for dengue virus type 1, 1 in 9,870 PBMC for dengue virus type 3, 1 in 14,053 PBMC for dengue virus type 2, and 1 in 17,690 PBMC for dengue virus type 4. Seventeen CD4+ T-cell clones were then established by using infectious dengue virus type 1 as antigen. Two patterns of dengue virus specificity were found in these clones. Thirteen clones were dengue virus type 1 specific, and four clones recognized both dengue virus types 1 and 3. Analysis of human leukocyte antigen (HLA) restriction revealed that five clones are HLA-DRw52 restricted, one clone is HLA-DP3 restricted, and one clone is HLA-DP4 restricted. These results indicate that in this individual, the CD4+ T-lymphocyte responses to immunization with live-attenuated dengue virus type 1 vaccine are predominantly serotype specific and suggest that a multivalent vaccine may be necessary to elicit strong serotype-cross-reactive CD4+ T-lymphocyte responses in such individuals. PMID:8371350

  4. Human skeletal muscle xenograft as a new preclinical model for muscle disorders

    PubMed Central

    Zhang, Yuanfan; King, Oliver D.; Rahimov, Fedik; Jones, Takako I.; Ward, Christopher W.; Kerr, Jaclyn P.; Liu, Naili; Emerson, Charles P.; Kunkel, Louis M.; Partridge, Terence A.; Wagner, Kathryn R.

    2014-01-01

    Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1null IL2rγnull immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics. PMID:24452336

  5. Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines

    PubMed Central

    Eaton, Mary J.; Berrocal, Yerko; Wolfe, Stacey Q.

    2012-01-01

    Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain. PMID:22619713

  6. Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

    PubMed Central

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-01-01

    SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

  7. Albumin Redhill (-1 Arg, 320 Ala yields Thr): A glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site

    SciTech Connect

    Brennan, S.O.; Myles, T.; Peach, R.J.; George, P.M. ); Donaldson, D. )

    1990-01-01

    Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind {sup 63}Ni{sup 2+} and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni{sup 2+} was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala {yields} Thr. This introduces as Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg {yields} Cys. The authors propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

  8. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.

    PubMed

    Amer, Luke D; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J; Bryant, Stephanie J

    2015-08-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15)μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced under both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (∼ 2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1(+)/Nkx6.1(+) cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.

  9. Restricted spontaneous in vitro differentiation and region-specific migration of long-term expanded fetal human neural precursor cells after transplantation into the adult rat brain.

    PubMed

    Maciaczyk, Jaroslaw; Singec, Ilyas; Maciaczyk, Donata; Klein, Alexander; Nikkhah, Guido

    2009-09-01

    Human fetal neural stem/progenitor cells (hNSCs) are investigated for their potential as a cell source for cell-based therapies in neurodegenerative diseases. However, the limited availability of fetal tissue and insufficient understanding of the lineage-dependent pattern of survival, migration, and differentiation following engraftment are still unresolved issues. In the current study hNSCs derived from different brain regions were long-term expanded in vitro to yield proliferating neurospheres giving rise to neurons, astro-, and oligodendroglial cells and assessed for their potential for migration, differentiation, and anatomical integration following intracerebral grafting into rats. hNSCs isolated from neocortex, striatum, midbrain, and spinal cord (SC) proliferated following in vitro differentiation, and showed a significant decrease of newly formed neurons along the rostrocaudal axis of the developing central nervous system (CNS). Most of the mature neurons were positive for the neurotransmitter GABA. In vivo all cell types survived up to 9 weeks posttransplantation. Intrastriatally grafted hNSCs migrated extensively along white matter tracts reaching both rostral (forceps minor) and caudal (midbrain, cerebral peduncle) brain regions. The majority of migratory cells expressed the stem cell marker, nestin. A fraction of grafted cells acquired a neuronal phenotype expressing doublecortin, beta-III-tubulin, or GABA. These data demonstrate efficient in vitro propagation, region-specific long-term survival, long-distance migration, and neuronal differentiation of hNSCs after transplantation into the adult rat brain. The availability of a large pool of in vitro expanded nestin-positive cells offers the possibility for further ex vivo manipulations and the recruitment of different neuronal phenotypes for cell replacement strategies for CNS disorders.

  10. [Presentation of the Lunar Precursor Robotics Program

    NASA Technical Reports Server (NTRS)

    Lavoie, Anthony R.

    2008-01-01

    The Lunar Precursor Robotics Program (LPRP) is the host program for the Exploration Systems Mission Directorate's (ESMD) lunar robotic precursor missions to the Moon. The program includes two missions, the Lunar Reconnaissance Orbiter (LRO), and the Lunar CRater Observation and Sensing Satellite (LCROSS). Both missions will provide the required lunar information to support development and operations of those systems required for Human lunar return. LPRP is developing a lunar mapping plan, Called the Lunar Mapping and Modeling Project, to create the capability to archive and present all data from LRO, LCROSS, historical lunar missions, and international lunar missions for future mission planning and operations. LPRP is also developing its educational and public outreach activities for the Vision for Space Exploration's first missions. LPRP is working closely with the Science Mission Directorate as their lunar activities come into focus.

  11. Evidence for a common mechanism for spontaneous rhythmic contraction and myogenic contraction induced by quick stretch in detrusor smooth muscle

    PubMed Central

    Komari, S Omid; Headley, Patrick C; Klausner, Adam P; Ratz, Paul H; Speich, John E

    2013-01-01

    Detrusor smooth muscle exhibits myogenic contraction in response to a quick stretch (QS) as well as spontaneous rhythmic contraction (SRC); however, whether the same population of actomyosin crossbridges with a common regulatory mechanism is responsible for these two types of contraction has not been determined. Detrusor strips from New Zealand white rabbit bladders were allowed to develop SRC at a reference muscle length (Lref), or rhythmic contraction (RC) was induced with tetraethylammonium (TEA). Multiple 10-msec stretches of 15% Lref were then imposed at Lref randomly during the rhythm cycle, and the nadir-to-peak (NTP) tension amplitude of the resulting myogenic contraction was measured. The amplitude and period of the rhythm cycle were measured prior to each QS. NTP was larger when a QS was imposed during a portion the cycle when tension was smaller (n = 3 each SRC and TEA-induced RC). These data suggest that when the rhythmic mechanism was mostly inactive and tension was near a minimum, a larger portion of a shared population of crossbridges was available to produce a myogenic response to a QS. Rho kinase, cyclooxygenase-1, and cyclooxygenase-2 inhibitors (H-1152, SC-560, and NS-398) affected SRC amplitude and NTP amplitude following a QS to the same degree (n = 3 each drug), providing additional evidence to support the hypothesis that a common mechanism is responsible for SRC and myogenic contraction due to QS. If a common mechanism exists, then QS is a potential mechanical probe to study SRC regulation and its alteration in overactive bladder. PMID:24400167

  12. N-terminal 5-mer peptide analog P165 of amyloid precursor protein inhibits UVA-induced MMP-1 expression by suppressing the MAPK pathway in human dermal fibroblasts.

    PubMed

    Wang, Ying; Chen, Hui; Wang, Wen; Wang, Rong; Liu, Zi-Lian; Zhu, Wei; Lian, Shi

    2014-07-05

    Exposure to ultraviolet (UV) radiation leads to a progressive increase in dermal damage through the degradation of collagen, which is mediated by matrix metalloproteinases (MMPs). UV radiation alters the intracellular signaling events that regulate the elaboration of MMPs. Our previous study showed that P165, the N-terminal 5-mer peptide analog of amyloid precursor protein, exerts a protective effect on ultraviolet A (UVA)-induced loss of collagen type I in human dermal fibroblasts (HDFs) by inhibiting the generation of intracellular reactive oxygen species and MMP-1. In this study, we focused on specific signal transduction pathways to elucidate the possible photoprotective mechanisms of P165 in controlling MMP-1 inhibition. Results from western blot analyses indicated that pretreatment with P165 dose-dependently inhibited UVA-induced phosphorylation of extracellular regulated protein kinases (ERK), c-Jun N-terminal kniase (JNK), p38 mitogen-activated protein kinases (MAPKs), and the phosphorylation of their downstream targets c-Jun and c-Fos. The photoprotective effects of P165 were further demonstrated in collagen type I secretion and cellular senescence induced by UVA irradiation. These findings suggest that P165 exerts photoprotective activity in UVA-treated HDFs by regulating MMP-1 generation. This activity may be mediated by inhibiting the MAPK signaling pathways. Thus, P165 is a potential agent for the prevention of skin photoaging.

  13. Suppression of interleukin 2-dependent human T cell growth in vitro by prostaglandin E (PGE) and their precursor fatty acids. Evidence for a PGE-independent mechanism of inhibition by the fatty acids.

    PubMed Central

    Santoli, D; Phillips, P D; Colt, T L; Zurier, R B

    1990-01-01

    PGE represent oxygenation products of polyunsaturated essential fatty acids and are important regulators of cell-mediated immune responses. Because oils enriched in such fatty acids reduce inflammation and tissue injury in vivo, we examined the effects of these PGE precursors on IL-2-driven growth of human T lymphocytes. Dihomogamma linoleic acid (DGLA), AA, and their metabolites (PGE1 and PGE2, respectively) strongly inhibited short- and long-term growth of IL-2-dependent T cell cultures; EPA was much less inhibitory and its product, PGE3, failed to suppress IL-2 responses. Short-term pretreatment of the cells with DGLA or AA and removal of the fatty acids before the proliferation assay resulted in a smaller reduction in [3H]TdR incorporation. PGE and fatty acids did not alter the number of high affinity IL-2 binding sites on the T cell cultures but reduced the percentage of cells expressing CD25 and HLA class II molecules. No PGE was detected in supernatants from the fatty acid-treated cultures. Moreover, indomethacin, a cyclooxygenase inhibitor, did not reverse the antiproliferative effects of the fatty acids. Together, these findings indicate that fatty acids can inhibit IL-2-driven T cell growth via a PGE-independent mechanism and might be relevant to inflammatory diseases associated with persistent T cell activation. Images PMID:2298918

  14. Long non-coding RNA Linc-RAM enhances myogenic differentiation by interacting with MyoD.

    PubMed

    Yu, Xiaohua; Zhang, Yong; Li, Tingting; Ma, Zhao; Jia, Haixue; Chen, Qian; Zhao, Yixia; Zhai, Lili; Zhong, Ran; Li, Changyin; Zou, Xiaoting; Meng, Jiao; Chen, Antony K; Puri, Pier Lorenzo; Chen, Meihong; Zhu, Dahai

    2017-01-16

    Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes. Here we report on functional identification and characterization of a novel long intergenic non-coding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to the differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD-Baf60c-Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodelling and gene expression.

  15. Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

    PubMed

    Zhong, Xia; Wang, Qian-Qian; Li, Jian-Wei; Zhang, Yu-Mei; An, Xiao-Rong; Hou, Jian

    2017-03-08

    Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression.

  16. Roles of adherent myogenic cells and dynamic culture in engineered muscle function and maintenance of satellite cells.

    PubMed

    Juhas, Mark; Bursac, Nenad

    2014-11-01

    Highly functional engineered skeletal muscle constructs could serve as physiological models of muscle function and regeneration and have utility in therapeutic replacement of damaged or diseased muscle tissue. In this study, we examined the roles of different myogenic cell fractions and culturing conditions in the generation of highly functional engineered muscle. Fibrin-based muscle bundles were fabricated using either freshly-isolated myogenic cells or their adherent fraction pre-cultured for 36 h. Muscle bundles made of these cells were cultured in both static and dynamic conditions and systematically characterized with respect to early myogenic events and contractile function. Following 2 weeks of culture, we observed both individual and synergistic benefits of using the adherent cell fraction and dynamic culture on muscle formation and function. In particular, optimal culture conditions resulted in significant increase in the total cross-sectional muscle area (- 3-fold), myofiber size (- 1.6-fold), myonuclei density (- 1.2-fold), and force generation (- 9-fold) compared to traditional use of freshly-isolated cells and static culture. Curiously, we observed that only a simultaneous use of the adherent cell fraction and dynamic culture resulted in accelerated formation of differentiated myofibers which were critical for providing a niche-like environment for maintenance of a satellite cell pool early during culture. Our study identifies key parameters for engineering large-size, highly functional skeletal muscle tissues with improved ability for retention of functional satellite cells.

  17. Distinct Neural Circuits Control Rhythm Inhibition and Spitting by the Myogenic Pharynx of C. elegans.

    PubMed

    Bhatla, Nikhil; Droste, Rita; Sando, Steven R; Huang, Anne; Horvitz, H Robert

    2015-08-17

    Neural circuits have long been known to modulate myogenic muscles such as the heart, yet a mechanistic understanding at the cellular and molecular levels remains limited. We studied how light inhibits pumping of the Caenorhabditis elegans pharynx, a myogenic muscular pump for feeding, and found three neural circuits that alter pumping. First, light inhibits pumping via the I2 neuron monosynaptic circuit. Our electron microscopic reconstruction of the anterior pharynx revealed evidence for synapses from I2 onto muscle that were missing from the published connectome, and we show that these "missed synapses" are likely functional. Second, light inhibits pumping through the RIP-I1-MC neuron polysynaptic circuit, in which an inhibitory signal is likely transmitted from outside the pharynx into the pharynx in a manner analogous to how the mammalian autonomic nervous system controls the heart. Third, light causes a novel pharyngeal behavior, reversal of flow or "spitting," which is induced by the M1 neuron. These three neural circuits show that neurons can control a myogenic muscle organ not only by changing the contraction rate but also by altering the functional consequences of the contraction itself, transforming swallowing into spitting. Our observations also illustrate why connectome builders and users should be cognizant that functional synaptic connections might exist despite the absence of a declared synapse in the connectome.

  18. Skeletal muscle hypertrophy and regeneration: interplay between the myogenic regulatory factors (MRFs) and insulin-like growth factors (IGFs) pathways.

    PubMed

    Zanou, Nadège; Gailly, Philippe

    2013-11-01

    Adult skeletal muscle can regenerate in response to muscle damage. This ability is conferred by the presence of myogenic stem cells called satellite cells. In response to stimuli such as injury or exercise, these cells become activated and express myogenic regulatory factors (MRFs), i.e., transcription factors of the myogenic lineage including Myf5, MyoD, myogenin, and Mrf4 to proliferate and differentiate into myofibers. The MRF family of proteins controls the transcription of important muscle-specific proteins such as myosin heavy chain and muscle creatine kinase. Different growth factors are secreted during muscle repair among which insulin-like growth factors (IGFs) are the only ones that promote both muscle cell proliferation and differentiation and that play a key role in muscle regeneration and hypertrophy. Different isoforms of IGFs are expressed during muscle repair: IGF-IEa, IGF-IEb, or IGF-IEc (also known as mechano growth factor, MGF) and IGF-II. MGF is expressed first and is observed in satellite cells and in proliferating myoblasts whereas IGF-Ia and IGF-II expression occurs at the state of muscle fiber formation. Interestingly, several studies report the induction of MRFs in response to IGFs stimulation. Inversely, IGFs expression may also be regulated by MRFs. Various mechanisms are proposed to support these interactions. In this review, we describe the general process of muscle hypertrophy and regeneration and decipher the interactions between the two groups of factors involved in the process.

  19. Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

    PubMed Central

    Zhong, Xia; Wang, Qian-Qian; Li, Jian-Wei; Zhang, Yu-Mei; An, Xiao-Rong; Hou, Jian

    2017-01-01

    Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression. PMID:28272491

  20. Pitx genes are redeployed in adult myogenesis where they can act to promote myogenic differentiation in muscle satellite cells.

    PubMed

    Knopp, Paul; Figeac, Nicolas; Fortier, Mathieu; Moyle, Louise; Zammit, Peter S

    2013-05-01

    Skeletal muscle retains a resident stem cell population called satellite cells. Although mitotically quiescent in mature muscle, satellite cells can be activated to produce myoblast progeny to generate myonuclei for skeletal muscle homoeostasis, hypertrophy and repair. Regulation of satellite cell function in adult requires redeployment of many of the regulatory networks fundamental to developmental myogenesis. Involved in such control of muscle stem cell fate in embryos are members of the Pitx gene family of bicoid-class homeodomain proteins. Here, we investigated the expression and function of all three Pitx genes in muscle satellite cells of adult mice. Endogenous Pitx1 was undetectable, whilst Pitx2a, Pitx2b and Pitx2c were at low levels in proliferating satellite cells, but increased during the early stages of myogenic differentiation. By contrast, proliferating satellite cells expressed robust amounts of Pitx3, with levels then decreasing as cells differentiated, although Pitx3 remained expressed in unfused myoblasts. To examine the role of Pitx genes in satellite cell function, retroviral-mediated expression of Pitx1, all Pitx2 isoforms or Pitx3, was used. Constitutive expression of any Pitx isoform suppressed satellite cell proliferation, with the cells undergoing enhanced myogenic differentiation. Conversely, myogenic differentiation into multinucleated myotubes was decreased when Pitx2 or Pitx3 levels were reduced using siRNA. Together, our results show that Pitx genes play a role in regulating satellite cell function during myogenesis in adult.

  1. Long non-coding RNA Linc-RAM enhances myogenic differentiation by interacting with MyoD

    PubMed Central

    Yu, Xiaohua; Zhang, Yong; Li, Tingting; Ma, Zhao; Jia, Haixue; Chen, Qian; Zhao, Yixia; Zhai, Lili; Zhong, Ran; Li, Changyin; Zou, Xiaoting; Meng, Jiao; Chen, Antony K.; Puri, Pier Lorenzo; Chen, Meihong; Zhu, Dahai

    2017-01-01

    Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes. Here we report on functional identification and characterization of a novel long intergenic non-coding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to the differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD–Baf60c–Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodelling and gene expression. PMID:28091529

  2. Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains.

    PubMed Central

    Wood, L J; Baxter, M K; Plafker, S M; Gibson, W

    1997-01-01

    We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus. PMID:8985337

  3. Preparation of superconductor precursor powders

    DOEpatents

    Bhattacharya, Raghunath

    1998-01-01

    A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.

  4. Identification of Retinoic Acid in a High Content Screen for Agents that Overcome the Anti-Myogenic Effect of TGF-Beta-1

    PubMed Central

    Krueger, Chateen; Hoffmann, F. Michael

    2010-01-01

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

  5. miR-206 represses hypertrophy of myogenic cells but not muscle fibers via inhibition of HDAC4.

    PubMed

    Winbanks, Catherine E; Beyer, Claudia; Hagg, Adam; Qian, Hongwei; Sepulveda, Patricio V; Gregorevic, Paul

    2013-01-01

    microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass in vivo, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 "sponge," featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype

  6. Activation of non-myogenic mesenchymal stem cells during the disease progression in dystrophic dystrophin/utrophin knockout mice.

    PubMed

    Sohn, Jihee; Lu, Aiping; Tang, Ying; Wang, Bing; Huard, Johnny

    2015-07-01

    Ectopic calcification as well as fatty and fibrotic tissue accumulation occurs in skeletal muscle during the disease progression of Duchenne muscular dystrophy (DMD), a degenerative muscle disorder caused by mutations in the dystrophin gene. The cellular origin and the environmental cues responsible for this ectopic calcification, fatty and fibrotic infiltration during the disease progression, however, remain unknown. Based on a previously published preplate technique, we isolated two distinct populations of muscle-derived cells from skeletal muscle: (i) a rapidly adhering cell population, which is non-myogenic, Pax7(-) and express the mesenchymal stem cell (MSC) marker platelet-derived growth factor receptor alpha; hence, we termed this population of cells non-myogenic MSCs (nmMSCs); and (ii) a slowly adhering cell population which is Pax7(+) and highly myogenic, termed muscle progenitor cells (MPCs). Previously, we demonstrated that the rapid progression of skeletal muscle histopathologies in dystrophin/utrophin knockout (dys(-/-) utro(-/-) dKO) mice is closely associated with a rapid depletion of the MPC population pool. In the current study, we showed that in contrast to the MPCs, the nmMSCs become activated during the disease progression in dKO mice, displaying increased proliferation and differentiation potentials (adipogenesis, osteogenesis and fibrogenesis). We also found that after co-culturing the dKO-nmMSCs with dKO-MPCs, the myogenic differentiation potential of the dKO-MPCs was reduced. This effect was found to be potentially mediated by the secretion of secreted frizzled-related protein 1 by the dKO-nmMSCs. We therefore posit that the rapid occurrence of fibrosis, ectopic calcification and fat accumulation, in dKO mice, is not only attributable to the rapid depletion of the MPC pool, but is also the consequence of nmMSC activation. Results from this study suggest that approaches to alleviate muscle weakness and wasting in DMD patients should not only

  7. Ocular vestibular evoked myogenic potentials to air conduction (AC oVEMP): useful in clinical practice?

    PubMed

    Walther, L E; Rogowski, M; Hörmann, K; Schaaf, H; Löhler, J

    2011-01-01

    Cervical vestibular-evoked myogenic potential (cVEMP) and ocular VEMP (oVEMP) stimuli can be used to measure otolith function using air (AC) and bone conducted (BC) stimuli. Cervical VEMPs reflect saccular function and can be recorded using air conduction (AC), whereas oVEMPs reflect probably predominantly utricular function. Air- and bone-conducted vibration can be used, because AC oVEMP methodology seems to be fast and simple in clinical practice to measure otolith function. In this study we discuss the advantages and problems of AC oVEMP stimulation. AC oVEMP can be easily and quickly obtained within a few seconds. N10 (first negative peak) and p15 (first positive peak) latencies may be used as parameters for clinical interpretation but amplitude fluctuations are relatively large. For daily clinical use of VEMP visualization in a normogram seems feasible. Especially the AC oVEMP methodology (100 dB nHL, tone burst 500 Hz) is fast and efficient in clinical practice to measure otolith function, predominantly utricular function.

  8. Expression of myogenes in longissimus dorsi muscle during prenatal development in commercial and local Piau pigs

    PubMed Central

    dos Reis, Evelyze Pinheiro; Paixão, Débora Martins; Brustolini, Otávio José Bernardes; Silva, Fabyano Fonseca e; Silva, Walmir; de Araújo, Flávio Marcos Gomes; Salim, Anna Christina de Matos; Oliveira, Guilherme; Guimarães, Simone Eliza Facioni

    2016-01-01

    Abstract This study used qRT-PCR to examine variation in the expression of 13 myogenes during muscle development in four prenatal periods (21, 40, 70 and 90 days post-insemination) in commercial (the three-way Duroc, Landrace and Large-White cross) and local Piau pig breeds that differ in muscle mass. There was no variation in the expression of the CHD8, EID2B, HIF1AN, IKBKB, RSPO3, SOX7 and SUFU genes at the various prenatal ages or between breeds. The MAP2K1 and RBM24 genes showed similar expression between commercial and Piau pigs but greater expression (p < 0.05) in at least one prenatal period. Pair-wise comparisons of prenatal periods in each breed showed that only the CSRP3, LEF1, MRAS and MYOG genes had higher expression (p < 0.05) in at least one prenatal period in commercial and Piau pigs. Overall, these results identified the LEF1 gene as a primary candidate to account for differences in muscle mass between the pig breeds since activation of this gene may lead to greater myoblast fusion in the commercial breed compared to Piau pigs. Such fusion could explain the different muscularity between breeds in the postnatal periods. PMID:27801482

  9. Saccular function in otosclerosis patients: bone conducted-vestibular evoked myogenic potential analysis.

    PubMed

    Amali, Amin; Mahdi, Parvane; Karimi Yazdi, Alireza; Khorsandi Ashtiyani, Mohammad Taghi; Yazdani, Nasrin; Vakili, Varasteh; Pourbakht, Akram

    2014-01-01

    Vestibular involvements have long been observed in otosclerotic patients. Among vestibular structures saccule has the closest anatomical proximity to the sclerotic foci, so it is the most prone vestibular structure to be affected during the otosclerosis process. The aim of this study was to investigate the saccular function in patients suffering from otosclerosis, by means of Vestibular Evoked Myogenic Potential (VEMP). The material consisted of 30 otosclerosis patients and 20 control subjects. All participants underwent audiometric and VEMP testing. Analysis of tests results revealed that the mean values of Air-Conducted Pure Tone Average (AC-PTA) and Bone-Conducted Pure Tone Average (BC-PTA) in patients were 45.28 ± 15.57 and 19.68 ± 10.91, respectively and calculated 4 frequencies Air Bone Gap (ABG) was 25.64 ± 9.95. The VEMP response was absent in 14 (28.57%) otosclerotic ears. A statistically significant increase in latency of the p13 was found in the affected ears (P=0.004), differences in n23 latency did not reach a statistically significant level (P=0.112). Disparities in amplitude of p13-n23 in between two study groups was statistically meaningful (P=0.009), indicating that the patients with otosclerosis had lower amplitudes. This study tends to suggest that due to the direct biotoxic effect of the materials released from the otosclerosis foci on saccular receptors, there might be a possibility of vestibular dysfunction in otosclerotic patients.

  10. A Dual Mode Pulsed Electro-Magnetic Cell Stimulator Produces Acceleration of Myogenic Differentiation

    PubMed Central

    Leon-Salas, Walter D.; Rizk, Hatem; Mo, Chenglin; Weisleder, Noah; Brotto, Leticia; Abreu, Eduardo; Brotto, Marco

    2013-01-01

    This paper presents the design and test of a dual-mode electric and magnetic biological stimulator (EM-Stim). The stimulator generates pulsing electric and magnetic fields at programmable rates and intensities. While electric and magnetic stimulators have been reported before, this is the first device that combines both modalities. The ability of the dual stimulation to target bone and muscle tissue simultaneously has the potential to improve the therapeutic treatment of osteoporosis and sarcopenia. The device is fully programmable, portable and easy to use, and can run from a battery or a power supply. The device can generate magnetic fields of up to 1.6 mT and output voltages of +/−40 V. The EM-Stim accelerated myogenic differentiation of myoblasts into myotubes as evidenced by morphometric, gene expression, and protein content analyses. Currently, there are many patents concerned with the application of single electrical or magnetic stimulation, but none that combine both simultaneously. However, we applied for and obtained a provisional patent for new device to fully explore its therapeutic potential in pre-clinical models. PMID:23445453

  11. Phospholipase D Is Involved in Myogenic Differentiation through Remodeling of Actin Cytoskeleton

    PubMed Central

    Komati, Hiba; Naro, Fabio; Mebarek, Saida; De Arcangelis, Vania; Adamo, Sergio; Lagarde, Michel; Prigent, Annie-France; Némoz, Georges

    2005-01-01

    We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation. PMID:15616193

  12. Non-myogenic Contribution to Muscle Development and Homeostasis: The Role of Connective Tissues

    PubMed Central

    Nassari, Sonya; Duprez, Delphine; Fournier-Thibault, Claire

    2017-01-01

    Skeletal muscles belong to the musculoskeletal system, which is composed of bone, tendon, ligament and irregular connective tissue, and closely associated with motor nerves and blood vessels. The intrinsic molecular signals regulating myogenesis have been extensively investigated. However, muscle development, homeostasis and regeneration require interactions with surrounding tissues and the cellular and molecular aspects of this dialogue have not been completely elucidated. During development and adult life, myogenic cells are closely associated with the different types of connective tissue. Connective tissues are defined as specialized (bone and cartilage), dense regular (tendon and ligament) and dense irregular connective tissue. The role of connective tissue in muscle morphogenesis has been investigated, thanks to the identification of transcription factors that characterize the different types of connective tissues. Here, we review the development of the various connective tissues in the context of the musculoskeletal system and highlight their important role in delivering information necessary for correct muscle morphogenesis, from the early step of myoblast differentiation to the late stage of muscle maturation. Interactions between muscle and connective tissue are also critical in the adult during muscle regeneration, as impairment of the regenerative potential after injury or in neuromuscular diseases results in the progressive replacement of the muscle mass by fibrotic tissue. We conclude that bi-directional communication between muscle and connective tissue is critical for a correct assembly of the musculoskeletal system during development as well as to maintain its homeostasis in the adult. PMID:28386539

  13. Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton.

    PubMed

    Komati, Hiba; Naro, Fabio; Mebarek, Saida; De Arcangelis, Vania; Adamo, Sergio; Lagarde, Michel; Prigent, Annie-France; Némoz, Georges

    2005-03-01

    We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.

  14. Non-myogenic Contribution to Muscle Development and Homeostasis: The Role of Connective Tissues.

    PubMed

    Nassari, Sonya; Duprez, Delphine; Fournier-Thibault, Claire

    2017-01-01

    Skeletal muscles belong to the musculoskeletal system, which is composed of bone, tendon, ligament and irregular connective tissue, and closely associated with motor nerves and blood vessels. The intrinsic molecular signals regulating myogenesis have been extensively investigated. However, muscle development, homeostasis and regeneration require interactions with surrounding tissues and the cellular and molecular aspects of this dialogue have not been completely elucidated. During development and adult life, myogenic cells are closely associated with the different types of connective tissue. Connective tissues are defined as specialized (bone and cartilage), dense regular (tendon and ligament) and dense irregular connective tissue. The role of connective tissue in muscle morphogenesis has been investigated, thanks to the identification of transcription factors that characterize the different types of connective tissues. Here, we review the development of the various connective tissues in the context of the musculoskeletal system and highlight their important role in delivering information necessary for correct muscle morphogenesis, from the early step of myoblast differentiation to the late stage of muscle maturation. Interactions between muscle and connective tissue are also critical in the adult during muscle regeneration, as impairment of the regenerative potential after injury or in neuromuscular diseases results in the progressive replacement of the muscle mass by fibrotic tissue. We conclude that bi-directional communication between muscle and connective tissue is critical for a correct assembly of the musculoskeletal system during development as well as to maintain its homeostasis in the adult.

  15. Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

    SciTech Connect

    Capkovic, Katie L.; Stevenson, Severin; Johnson, Marc C.; Thelen, Jay J.; Cornelison, D.D.W.

    2008-04-15

    Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography-tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.

  16. Comparison of chirp versus click and tone pip stimulation for cervical vestibular evoked myogenic potentials.

    PubMed

    Wang, Bo-Chen; Liang, Yong; Liu, Xiao-Long; Zhao, Jing; Liu, You-Li; Li, Yan-Fei; Zhang, Wei; Li, Qi

    2014-12-01

    The current study explored differences among cervical vestibular evoked myogenic potentials (cVEMP) that were evoked by CE-chirp and click and tone pip in healthy controls, and tried to explain the differences of cVEMP between the three of them. Thirty normal volunteers were used as subjects for CE-chirp and click and tone-pip (Blackman pip) stimuli. The latency of P1, N1, peak-to-peak P1-N1 amplitude, and cVEMP interaural difference were obtained and analyzed. The response rates of cVEMP were 93 % for click and 100 % for both Blackman pip and CE-chirp, respectively. The P1 and N1 latencies of cVEMP evoked by CE-chirp were the shortest, followed by click, with Blackman pip the longest (F = 6,686.852, P < 0.001). All indices of cVEMP evoked by the three stimuli showed no significant difference between the left and right ears or between genders. cVEMP responses were significantly different between the three stimuli. Compared with the currently used stimulus, CE-chirp can evoke cVEMP with shorter latencies and demonstrates increased speed and reliability.

  17. Correlation between acceleration magnitude and ocular vestibular-evoked myogenic potential.

    PubMed

    Wang, Shou-Jen; Jaw, Fu-Shan; Young, Yi-Ho

    2012-05-10

    This study combined bone-conducted vibration (BCV) stimulation with triaxial accelerometry to correlate the acceleration magnitudes of BCV stimuli with ocular vestibular-evoked myogenic potential (oVEMP) test results. Fourteen healthy volunteers underwent oVEMP test using BCV stimuli with simultaneous monitoring the triaxial acceleration. All (100%) subjects exhibited clear oVEMPs in response to BCV stimuli from a vibrator. The lowest acceleration magnitudes for eliciting oVEMPs along the x-, y- and z-axes were 0.05±0.01 g, 0.16±0.08 g, and 0.04±0.01 g, respectively, exhibiting significantly higher acceleration magnitude along the y-axis than those along the x- and z-axes. In addition, significantly positive correlations were noted between the acceleration magnitude along each axis and the oVEMP amplitude. In conclusion, measuring the acceleration magnitude throughout oVEMP testing revealed a significant correlation between linear acceleration and oVEMP responses. Restated, increasing acceleration magnitude may have more synchronization of firing of vestibular afferents, resulting in more synchronized evoked potentials and greater oVEMP amplitude.

  18. A FINE-STRUCTURAL ANALYSIS OF THE FUSION OF MYOGENIC CELLS

    PubMed Central

    Lipton, Bruce H.; Konigsberg, Irwin R.

    1972-01-01

    The fusion of myogenic cells has been examined on the fine-structural level in muscle cell cultures of embryonic Japanese Coturnix quail. Cells, selected by light microscopy, were serially sectioned normal to their long axis. In this plane, oblique sections of cell membranes are rare and plasmalemmal profiles are more easily traced between adjacent cells. In seven cases, pairs of cells, apparently fixed in the process of fusion, are joined by a single cytoplasmic bridge. Since obliquely sectioned membranes often suggest cytoplasmic confluence, tilting stage analysis was employed to resolve cell membranes in suspect cases. In contrast to such artifacts of superposition, however, the observed intercommunicating pores are contained within a pair of culs-de-sac formed by the fused membranes of both cells. These blind pouches can be traced back between the cells to the external space. The confluent regions are clearly demarcated and they are not simply areas between vesicular profiles. The results of this analysis suggest that (a) at no time is there any loss of integrity of the cellular envelope, and (b) fusion is most probably initiated at single sites between pairs of cells, the pore enlarging, leaving first vestiges and eventually no trace of the original intervening membranes. PMID:4554365

  19. Stem Cell Differentiation Toward the Myogenic Lineage for Muscle Tissue Regeneration: A Focus on Muscular Dystrophy.

    PubMed

    Ostrovidov, Serge; Shi, Xuetao; Sadeghian, Ramin Banan; Salehi, Sahar; Fujie, Toshinori; Bae, Hojae; Ramalingam, Murugan; Khademhosseini, Ali

    2015-12-01

    Skeletal muscle tissue engineering is one of the important ways for regenerating functionally defective muscles. Among the myopathies, the Duchenne muscular dystrophy (DMD) is a progressive disease due to mutations of the dystrophin gene leading to progressive myofiber degeneration with severe symptoms. Although current therapies in muscular dystrophy are still very challenging, important progress has been made in materials science and in cellular technologies with the use of stem cells. It is therefore useful to review these advances and the results obtained in a clinical point of view. This article focuses on the differentiation of stem cells into myoblasts, and their application in muscular dystrophy. After an overview of the different stem cells that can be induced to differentiate into the myogenic lineage, we introduce scaffolding materials used for muscular tissue engineering. We then described some widely used methods to differentiate different types of stem cell into myoblasts. We highlight recent insights obtained in therapies for muscular dystrophy. Finally, we conclude with a discussion on stem cell technology. We discussed in parallel the benefits brought by the evolution of the materials and by the expansion of cell sources which can differentiate into myoblasts. We also discussed on future challenges for clinical applications and how to accelerate the translation from the research to the clinic in the frame of DMD.

  20. Nitric Oxide Donor Molsidomine Positively Modulates Myogenic Differentiation of Embryonic Endothelial Progenitors

    PubMed Central

    Tirone, Mario; Conti, Valentina; Manenti, Fabio; Nicolosi, Pier Andrea; D’Orlando, Cristina; Azzoni, Emanuele

    2016-01-01

    Embryonic VE-Cadherin-expressing progenitors (eVE-Cad+), including hemogenic endothelium, have been shown to generate hematopoietic stem cells and a variety of other progenitors, including mesoangioblasts, or MABs. MABs are vessel-associated progenitors with multilineage mesodermal differentiation potential that can physiologically contribute to skeletal muscle development and regeneration, and have been used in an ex vivo cell therapy setting for the treatment of muscular dystrophy. There is currently a therapeutic need for molecules that could improve the efficacy of cell therapy protocols; one such good candidate is nitric oxide. Several studies in animal models of muscle dystrophy have demonstrated that nitric oxide donors provide several beneficial effects, including modulation of the activity of endogenous cell populations involved in muscle repair and the delay of muscle degeneration. Here we used a genetic lineage tracing approach to investigate whether the therapeutic effect of nitric oxide in muscle repair could derive from an improvement in the myogenic differentiation of eVE-Cad+ progenitors during embryogenesis. We show that early in vivo treatment with the nitric oxide donor molsidomine enhances eVE-Cad+ contribution to embryonic and fetal myogenesis, and that this effect could originate from a modulation of the properties of yolk sac hemogenic endothelium. PMID:27760216

  1. Diagnostic Value of Vestibular Evoked Myogenic Potentials in Endolymphatic Hydrops: A Meta-Analysis

    PubMed Central

    Zhang, Sulin; Leng, Yangming; Liu, Bo; Shi, Hao; Lu, Meixia; Kong, Weijia

    2015-01-01

    In this study, we evaluated the clinical diagnostic value of vestibular evoked myogenic potentials (VEMPs) for endolymphatic hydrops (EH) by systematic review and Meta-analysis. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio and area under summary receiver operating characteristic curves (AUC) were calculated. Subgroup analysis and publication bias assessment were also conducted. The pooled sensitivity and the specificity were 49% (95% CI: 46% to 51%) and 95% (95% CI: 94% to 96%), respectively. The pooled positive likelihood ratio was 18.01 (95% CI: 9.45 to 34.29) and the pooled negative likelihood ratio was 0.54 (95% CI: 0.47 to 0.61). AUC was 0.78 and the pooled diagnostic odds ratio of VEMPs was 39.89 (95% CI: 20.13 to 79.03). In conclusion, our present meta-analysis has demonstrated that VEMPs test alone is not sufficient for Meniere’s disease or delayed endolymphatic hydrops diagnosis, but that it might be an important component of a test battery for diagnosing Meniere’s disease or delayed endolymphatic hydrops. Moreover, VEMPs, due to its high specificity and non-invasive nature, might be used as a screening tool for EH. PMID:26455332

  2. Ocular vestibular evoked myogenic potential frequency tuning in certain Menière's disease.

    PubMed

    Jerin, Claudia; Berman, Albert; Krause, Eike; Ertl-Wagner, Birgit; Gürkov, Robert

    2014-04-01

    Ocular vestibular evoked myogenic potentials (oVEMP) represent extraocular muscle activity in response to vestibular stimulation. To specify the value of oVEMP in the diagnostics of Menière's disease, the amplitude ratio between 500 and 1000 Hz stimuli was investigated. Thirty-nine patients with certain Menière's disease, i.e. definite Menière's disease with visualization of endolymphatic hydrops by magnetic resonance imaging and 19 age-matched healthy controls were enrolled in this study. oVEMP were recorded using 500 and 1000 Hz air-conducted tone bursts. For Menière's ears, the 500/1000 Hz amplitude ratio (mean ratio = 1.20) was significantly smaller when compared to unaffected ears of Menière's patients (mean ratio = 1.80; p = 0.008) or healthy controls (mean ratio = 1.81; p = 0.011). The amplitude ratio was neither correlated with the degree of endolymphatic hydrops nor with the duration of disease. While an older age was associated with a diminished amplitude ratio in healthy controls, there was no correlation between the amplitude ratio and age in Menière's ears. Hence, the calculation of the oVEMP 500/1000 Hz amplitude ratio may be a valuable diagnostic tool for Menière's disease.

  3. Analysis of Saccular Function With Vestibular Evoked Myogenic Potential Test in Meniere's Disease.

    PubMed

    Dabiri, Sasan; Yazdani, Nasrin; Esfahani, Mahdis; Tari, Niloufar; Adil, Susan; Mahvi, Zahra; Rezazadeh, Nima

    2017-02-01

    Meniere's disease is the disorder of inner ear characterized by vertigo, tinnitus and sensorineural hearing loss. The vestibular evoked myogenic potential (VEMP) test could be useful in the analysis of saccular function, and diagnosis of Meniere's disease. In this study, we've analyzed the saccular function, using VEMP test in different groups of Meniere's disease. Patients were categorized as possible, probable or definite Meniere's disease groups according to the guideline of American Academy of Otolaryngology-Head and Neck Surgery. The exclusion criteria were neuromuscular system diseases, diseases of central nervous system, inner ear disorders, conductive hearing loss, a history of ototoxic drug consumption, being a drug abuser and a positive history of inner ear surgery or manipulations. The VEMP test is the recording of positive and negative waves from sternocleidomastoid muscle that is made by an auditory click to the ear. From the total of 100 patients, the waves of VEMP test was seen in 59 patients which 19 patients had abnormal amplitude, and latency and 40 patients were with normally recorded waves. There was a significant relationship between the severity of hearing loss and a VEMP test without any recorded waves. Most of the cases with 'no wave recorded' VEMP test, were patients with severe hearing loss. However, there wasn't any relation between the pattern of hearing loss and 'no wave recorded' VEMP test. VEMP test could be a valuable diagnostic clue especially in patients with definite Meniere's disease.

  4. Validation of Regression-Based Myogenic Correction Techniques for Scalp and Source-Localized EEG

    PubMed Central

    McMenamin, Brenton W.; Shackman, Alexander J.; Maxwell, Jeffrey S.; Greischar, Lawrence L.; Davidson, Richard J.

    2008-01-01

    EEG and EEG source-estimation are susceptible to electromyographic artifacts (EMG) generated by the cranial muscles. EMG can mask genuine effects or masquerade as a legitimate effect - even in low frequencies, such as alpha (8–13Hz). Although regression-based correction has been used previously, only cursory attempts at validation exist and the utility for source-localized data is unknown. To address this, EEG was recorded from 17 participants while neurogenic and myogenic activity were factorially varied. We assessed the sensitivity and specificity of four regression-based techniques: between-subjects, between-subjects using difference-scores, within-subjects condition-wise, and within-subject epoch-wise on the scalp and in data modeled using the LORETA algorithm. Although within-subject epoch-wise showed superior performance on the scalp, no technique succeeded in the source-space. Aside from validating the novel epoch-wise methods on the scalp, we highlight methods requiring further development. PMID:19298626

  5. The histone demethylase KDM4B interacts with MyoD to regulate myogenic differentiation in C2C12 myoblast cells.

    PubMed

    Choi, Jang Hyun; Song, Young Joon; Lee, Hansol

    2015-01-24

    Enzymes that mediate posttranslational modifications of histone and nonhistone proteins have been implicated in regulation of skeletal muscle differentiation. However, functions of histone demethylases that could counter the actions of H3-K9 specific histone methyltransferases remain still obscure. Here we present evidences that KDM4B histone demethylase regulates expression of myogenic regulators such as MyoD and thereby controls myogenic differentiation of C2C12 myoblast cells. We demonstrate that expression of KDM4B gradually increases during myogenic differentiation and depletion of KDM4B using shRNA results in inhibition of differentiation in C2C12 myoblast cells, which is correlated with decreased expression of MyoD and myogenin. In addition, we find that KDM4B shRNA represses expression of MyoD promoter-driven luciferase reporter and exogenous expression of MyoD rescues myogenic potential in KDM4B-depleted myoblast cells. We further show that KDM4B interacts with MyoD, binds to MyoD and myogenin promoters in vivo, and finally, is involved in demethylation of tri-methylated H3-K9 on promoters of MyoD and myogenin. Taken together, our data suggest that KDM4B plays key roles in myogenic differentiation of C2C12 cells, presumably by its function as a H3-K9 specific histone demethylase.

  6. Ebf2 is a selective marker of brown and beige adipogenic precursor cells.

    PubMed

    Wang, Wenshan; Kissig, Megan; Rajakumari, Sona; Huang, Li; Lim, Hee-Woong; Won, Kyoung-Jae; Seale, Patrick

    2014-10-07

    Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α(+), myogenic factor 5(Cre)-lineage-marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2(GFP) embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.

  7. The Clinical Utility of Vestibular Evoked Myogenic Potentials in Patients of Benign Paroxysmal Positional Vertigo

    PubMed Central

    Sreenivasan, Anuprasad; Parida, Pradiptata Kumar; Alexander, Arun; Saxena, Sunil Kumar; Suria, Gopalakrishnan

    2015-01-01

    Context Vestibular Evoked Myogenic Potentials (VEMP) is an emerging tool to diagnose Benign Paroxysmal Positional Vertigo (BPPV). The clinical utility of VEMP has been reported only in small accord in Indian literature. Aim To study the latency and amplitude of VEMP in patients with BPPV and compare it with that of normal subjects. Settings and Design The study included two groups. Group one (control group) were 18 normal subjects. Group two (test group) were 15 subjects with unilateral BPPV. Materials and Methods Those subjects who fulfilled the selection criteria based on case history and audiological assessment were taken for the VEMP recording. The VEMP response consist of positive and negative successive waves (pI-nI), with latency values in adults about 13 and 23 milliseconds respectively. Statistical Analysis Data was analysed using Statistical Package for Social Sciences (SPSS) version 12 (Chicago, IL, USA). Unpaired t-test was employed to measure the statistical difference between control group and test group. Results The difference in n23 and the peak to peak amplitude between the ipsilateral and contralateral ears of the test group were statistically significant, whereas such a difference in the p13 latency turned out to be statistically insignificant. It should be noted that, out of 15 patients in the test group, five patients showed only artifact tracer recordings in both the ears which is considered as no response. The heterogeneity of the results extended from absence of VEMP to prolongation of both p13, n23; prolongation of p13 alone; and even side to side variations. Conclusion Absent response from the ipsilateral ear, prolonged latency of n23 and decreased peak to peak amplitude (p13, n23), indicates the disease pathology. However, large sample size is required to draw further conclusions and to consolidate the usage of VEMP in the diagnosis of BPPV. PMID:26266140

  8. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

    PubMed

    Alway, Stephen E; Degens, Hans; Lowe, Dawn A; Krishnamurthy, Gururaj

    2002-02-01

    The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

  9. Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

    PubMed Central

    Gard, D L; Lazarides, E

    1982-01-01

    The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation. Images PMID:6294504

  10. Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

    SciTech Connect

    Retamales, A.; Zuloaga, R.; Valenzuela, C.A.; Gallardo-Escarate, C.; Molina, A.; Valdés, J.A.

    2015-08-21

    Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletal myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.

  11. Myogenic temporomandibular disorders: Clinical systemic comorbidities in a female population sample

    PubMed Central

    Mesa-Jiménez, Juan; Fernández-de-las-Peñas, César; de-la-Hoz-Aizpurua, José-Luis

    2016-01-01

    Background Myogenic temporomandibular disorders (MTMD) frequently coexist with other clinical conditions in the same individual. In the last decades, several authors have analyzed these comorbidities looking for the origin of this overlapping. Objetives The aim of this study was to perform a comparative anaylisis between a group of patients with MTMD and a control group of dental patients without dysfunctional pathology to assess whether there are significant differences in the presence of systemic medical comorbidities between the two groups. Material and Methods Restrospective epidemiological analysis, based on medical questionnaires in a group of 31 patients, women, aged from 24 to 58 (average 39.96 years), diagnosed with MTMD (Masticatory Myofascial Pain), with a control group with the same number of individuals, gender and age range to evaluate if there is a significant statistical difference in the presence of medical comorbidities in this group of patients with MTMD and if they are in a higher risk of suffering different pathological conditions. Results It was found that the group affected by MTMD presented many more associated medical conditions than the control group: health changes during the last year, medical evaluations and treatments, presence of pain, sinus disease, tinnitus, headache, joint pain, ocular disorders, fatigue, dizziness, genitourinary disorders and xerostomia among others; and they were also in a higher risk to suffer other pathological entities as headaches and articular pain. Conclusions These results reinforce our hypothesis that MTMD belong to a group of medical conditions triggered by a loss of equilibrium of the individual’s Psycho-Neuro-Endocrine-Immune (PNEI) Axis that produces alterations in the response against external stimuli in some genetically predisposed individuals. It is, therefore, necessary to change the way of diagnosing and managing these individual’s medical conditions, being mandatory to look from a more

  12. MicroRNA-146b promotes myogenic differentiation and modulates multiple gene targets in muscle cells.

    PubMed

    Khanna, Nidhi; Ge, Yejing; Chen, Jie

    2014-01-01

    MicroRNAs are established as crucial modulators of skeletal myogenesis, but our knowledge about their identity and targets remains limited. In this study, we have identified microRNA-146b (miR-146b) as a novel regulator of skeletal myoblast differentiation. Following up on a previous microRNA profiling study, we establish that the expression of miR-146b is up-regulated during myoblast differentiation in vitro and muscle regeneration in vivo. Inhibition of miR-146b led to reduced myoblast differentiation, whereas overexpression of miR-146b enhanced differentiation. Computational prediction combined with gene expression information has revealed candidates for miR-146b targets in muscles. Among them, the expression of Smad4, Notch1, and Hmga2 are significantly suppressed by miR-146b overexpression in myocytes. In addition, expression levels of Smad4, Notch1 and Hmga2 are decreased during myoblast differentiation and muscle regeneration, inversely correlating to the levels of miR-146b. Importantly, inhibition of endogenous miR-146b prevents the down-regulation of Smad4, Notch1 and Hmga2 during differentiation. Furthermore, miR-146b directly targets the microRNA response elements (MREs) in the 3'UTR of those genes as assessed by reporter assays. Reporters with the seed regions of MREs mutated are insensitive to miR-146b, further confirming the specificity of targeting. In conclusion, miR-146b is a positive regulator of myogenic differentiation, possibly acting through multiple targets.

  13. Splenius capitis is a reliable target for measuring cervical vestibular evoked myogenic potentials in adults.

    PubMed

    Camp, Aaron J; Gu, Chao; Cushing, Sharon L; Gordon, Karen A; Corneil, Brian D

    2017-02-08

    The cervical vestibular evoked myogenic potential (cVEMP) is a common and simple test of vestibulospinal reflex patency. In the clinic, cVEMPs are measured in response to loud sounds from the sternocleidomastoid (SCM) on the ventral neck, as subjects maintain an uncomfortable head posture needed to recruit SCM. Here we characterize the cVEMP in a dorsal neck turner (splenius capitis; SPL), and compare it with the SCM cVEMP. cVEMPs were recorded simultaneously via surface electromyography from SCM and SPL from 17 healthy subjects in a variety of postures, including head-turned postures adopted while either seated or standing, and the clinical posture. Like the SCM cVEMP recorded ipsilateral to the side of sound stimulation, the cVEMP on the contralateral SPL (synergistic with ipsilateral SCM) was characterized by a biphasic wave of muscle activity that began at ~ 13 ms. cVEMP reliability was higher on SPL vs. SCM in standing postures (chi-squared; P < 0.05), and equivalent results were obtained from SPL in a standing or seated posture. In 9 of the 17 subjects, we also obtained bilateral intramuscular (IM) recordings from SPL at the same time as the surface recordings. In these subjects, the initial surface response in SPL was associated with a consistent decrease in multi-unit IM SPL activity. Overall, these results demonstrate that SPL recordings offer a complimentary target for cVEMP assessments. The expression of SPL cVEMPs in simple head-turned postures may also improve the utility of cVEMP testing for vestibular assessment in children, the elderly, or non-compliant.

  14. Precursor decay in several aluminas

    NASA Astrophysics Data System (ADS)

    Murray, N. H.; Bourne, N. K.; Rosenberg, Z.

    1996-05-01

    Plate impact experiments were performed on three ceramics with alumina content varying from 88 to 99.9% using a 50 mm single stage gas gun. Tiles of ceramic with thicknesses varying from 2 to 12 mm were impacted above their Hugoniot Elastic Limits (HELs) and the rate dependent strength was investigated by monitoring the variation in amplitude of the elastic precursor with propagation distance. Stress levels in the target were recorded using manganin stress transducers and a 1 GS s-1 storage oscilloscope. All grades of alumina were found to exhibit some elastic precursor decay indicating strain rate sensitivity.

  15. Myogenic origin of the hypotension induced by rapid changes in posture in awake dogs following autonomic blockade

    PubMed Central

    Wong, Brett J.; Sheriff, Don D.

    2008-01-01

    The “push-pull” effect denotes the reduced tolerance to +Gz (hypergravity) when +Gz stress is preceded by exposure to hypogravity, i.e., fractional, zero, or negative Gz. The purpose of this study was to test the hypothesis that an exaggerated, myogenically mediated rise in leg vascular conductance contributes to the push-pull effect, using heart level arterial blood pressure as a measure of G tolerance. The approach was to impose control (30 s of 30° head-up tilt) and push-pull (30 s of 30° head-up tilt immediately preceded by 10 s of −15° head-down tilt) gravitational stress after administration of hexamethonium (5 mg/kg) to inhibit autonomic ganglionic neurotransmission in seven dogs. Cardiac output or thigh level arterial pressure (myogenic stimulus) was maintained constant by computer-controlled ventricular pacing. The animals were sedated with acepromazine and lightly restrained in lateral recumbency on a tilt table. Following the onset of head-up tilt, the magnitude of the fall in heart level arterial pressure from baseline was −11.6 ± 2.9 and −17.1 ± 2.2 mmHg for the control and push-pull trials, respectively (P < 0.05), when cardiac output was maintained constant. Over 40% of the exaggerated fall in heart level arterial pressure was attributable to an exaggerated rise in hindlimb vascular conductance (P < 0.05). Maintaining thigh level arterial pressure constant abolished the exaggerated rise in hindlimb blood flow. Thus a push-pull effect largely attributable to a myogenically induced rise in leg vascular conductance occurs when autonomic function is inhibited. PMID:18927267

  16. Identification of L- and T-type Ca2+ channels in rat cerebral arteries: role in myogenic tone development

    PubMed Central

    El-Rahman, Rasha R. Abd; Harraz, Osama F.; Brett, Suzanne E.; Anfinogenova, Yana; Mufti, Rania E.; Goldman, Daniel

    2013-01-01

    L-type Ca2+ channels are broadly expressed in arterial smooth muscle cells, and their voltage-dependent properties are important in tone development. Recent studies have noted that these Ca2+ channels are not singularly expressed in vascular tissue and that other subtypes are likely present. In this study, we ascertained which voltage-gated Ca2+ channels are expressed in rat cerebral arterial smooth muscle and determined their contribution to the myogenic response. mRNA analysis revealed that the α1-subunit of L-type (Cav1.2) and T-type (Cav3.1 and Cav3.2) Ca2+ channels are present in isolated smooth muscle cells. Western blot analysis subsequently confirmed protein expression in whole arteries. With the use of patch clamp electrophysiology, nifedipine-sensitive and -insensitive Ba2+ currents were isolated and each were shown to retain electrical characteristics consistent with L- and T-type Ca2+ channels. The nifedipine-insensitive Ba2+ current was blocked by mibefradil, kurtoxin, and efonidpine, T-type Ca2+ channel inhibitors. Pressure myography revealed that L-type Ca2+ channel inhibition reduced tone at 20 and 80 mmHg, with the greatest effect at high pressure when the vessel is depolarized. In comparison, the effect of T-type Ca2+ channel blockade on myogenic tone was more limited, with their greatest effect at low pressure where vessels are hyperpolarized. Blood flow modeling revealed that the vasomotor responses induced by T-type Ca2+ blockade could alter arterial flow by ∼20–50%. Overall, our findings indicate that L- and T-type Ca2+ channels are expressed in cerebral arterial smooth muscle and can be electrically isolated from one another. Both conductances contribute to myogenic tone, although their overall contribution is unequal. PMID:23103495

  17. Regulation of turkey myogenic satellite cell migration by MicroRNAs miR-128 and miR-24.

    PubMed

    Velleman, S G; Harding, R L

    2016-12-05

    Myogenic satellite cells are an adult stem cell responsible for all post-hatch muscle growth in poultry. As a stem cell population, satellite cells are highly heterogeneous, but the origin of this heterogeneity remains unclear. Heterogeneity is, in part, regulated by gene expression. One method of endogenous gene regulation that may contribute to heterogeneity is microRNAs (miRNAs). Two miRNAs previously shown to regulate poultry myogenic satellite cell proliferation and differentiation, miR-128 and miR-24, were studied to determine if they also affected satellite cell migration. Satellite cell migration is an essential step for both proliferation and differentiation. During proliferation, satellite cells will migrate and align to form new myofibers or donate their nuclei to existing myofibers leading to muscle fiber hypertrophy or regeneration. Transient transfection of miRNA specific mimics to each miRNA reduced migration of satellite cells following a cell culture scratch at 72 h of proliferation when the cultures were 90 to 100% confluent. However, only the migration in cells transfected with miR-24 mimics at 24 and 30 h following the scratch was significantly reduced (P ≤ 0.05) to around 70% of the distance migrated by controls. Alternately, transfection with inhibitors specific to miR-128 or miR-24 significantly (P ≤ 0.05) increased migration between 147 and 252% compared to their controls between 24 and 48 h following the scratch. These data demonstrate that miR-128 and miR-24 play a role in myogenic satellite cell migration, which will impact muscle development and growth.

  18. Effect of pregnancy and nitric oxide on the myogenic vasodilation of posterior cerebral arteries and the lower limit of cerebral blood flow autoregulation.

    PubMed

    Chapman, Abbie C; Cipolla, Marilyn J; Chan, Siu-Lung

    2013-09-01

    Hemorrhage during parturition can lower blood pressure beyond the lower limit of cerebral blood flow (CBF) autoregulation that can cause ischemic brain injury. However, the impact of pregnancy on the lower limit of CBF autoregulation is unknown. We measured myogenic vasodilation, a major contributor of CBF autoregulation, in isolated posterior cerebral arteries (PCAs) from nonpregnant and late-pregnant rats (n = 10/group) while the effect of pregnancy on the lower limit of CBF autoregulation was studied in the posterior cerebral cortex during controlled hemorrhage (n = 8). Pregnancy enhanced myogenic vasodilation in PCA and shifted the lower limit of CBF autoregulation to lower pressures. Inhibition of nitric oxide synthase (NOS) prevented the enhanced myogenic vasodilation during pregnancy but did not affect the lower limit of CBF autoregulation. The shift in the autoregulatory curve to lower pressures during pregnancy is likely protective of ischemic injury during hemorrhage and appears to be independent of NOS.

  19. Preparation of superconductor precursor powders

    DOEpatents

    Bhattacharya, R.

    1998-08-04

    A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products. 7 figs.

  20. Preparation of superconductor precursor powders

    DOEpatents

    Bhattacharya, Raghunath; Blaugher, Richard D.

    1995-01-01

    A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals, such as nitrate salts of thallium, barium, calcium, and copper, which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of thallium in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.