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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. 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.

  4. 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

  5. Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Shoji, Emi; Woltjen, Knut; Sakurai, Hidetoshi

    2016-01-01

    Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening. PMID:25971915

  6. Preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages.

    PubMed

    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 (Ambystoma mexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream(1-4). PMID:24835774

  7. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy

    PubMed Central

    Roca, Isart; Requena, Jordi; Edel, Michael J.; Alvarez-Palomo, Ana Belén

    2015-01-01

    The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP) made from induced pluripotent stem cells (iPSCs) are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries. PMID:26239126

  8. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy.

    PubMed

    Roca, Isart; Requena, Jordi; Edel, Michael J; Alvarez-Palomo, Ana Belén

    2015-01-01

    The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP) made from induced pluripotent stem cells (iPSCs) are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries. PMID:26239126

  9. Antisense oligonucleotide induction of progerin in human myogenic cells.

    PubMed

    Luo, Yue-Bei; Mitrpant, Chalermchai; Adams, Abbie M; Johnsen, Russell D; Fletcher, Sue; Mastaglia, Frank L; Wilton, Steve D

    2014-01-01

    We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate

  10. Skeletal myogenic potential of human and mouse neural stem cells.

    PubMed

    Galli, R; Borello, U; Gritti, A; Minasi, M G; Bjornson, C; Coletta, M; Mora, M; De Angelis, M G; Fiocco, R; Cossu, G; Vescovi, A L

    2000-10-01

    Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues. PMID:11017170

  11. Lentivirus mediated HO-1 gene transfer enhances myogenic precursor cell survival after autologous transplantation in pig.

    PubMed

    Laumonier, Thomas; Yang, Sheng; Konig, Stephane; Chauveau, Christine; Anegon, Ignacio; Hoffmeyer, Pierre; Menetrey, Jacques

    2008-02-01

    Cell therapy for Duchenne muscular dystrophy and other muscle diseases is limited by a massive early cell death following injections. In this study, we explored the potential benefit of heme oxygenase-1 (HO-1) expression in the survival of porcine myogenic precursor cells (MPCs) transplanted in pig skeletal muscle. Increased HO-1 expression was assessed either by transient hyperthermia or by HO-1 lentiviral infection. One day after the thermic shock, we observed a fourfold and a threefold increase in HSP70/72 and HO-1 levels, respectively. This treatment protected 30% of cells from staurosporine-induced apoptosis in vitro. When porcine MPC were heat-shocked prior to grafting, we improved cell survival by threefold at 5 days after autologous transplantation (26.3 +/- 5.5% surviving cells). After HO-1 lentiviral transduction, almost 60% of cells expressed the transgene and kept their myogenic properties to proliferate and fuse in vitro. Apoptosis of HO-1 transduced cells was reduced by 50% in vitro after staurosporine induction. Finally, a fivefold enhancement in cell survival was observed after transplantation of HO-1-group (47.5 +/- 9.1% surviving cells) as compared to the nls-LacZ-group or control group. These results identify HO-1 as a protective gene against early MPC death post-transplantation. PMID:18026170

  12. Sparing of Extraocular Muscle in Aging and Muscular Dystrophies: A Myogenic Precursor Cell Hypothesis

    PubMed Central

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

    2011-01-01

    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. This 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. In addition, 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 were 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

  13. 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

  14. The role of myogenic mechanisms in human cerebrovascular regulation

    PubMed Central

    Tan, Can Ozan; Hamner, J W; Taylor, J Andrew

    2013-01-01

    Although myogenic mechanisms have been hypothesized to play a role in cerebrovascular regulation, previous data from both animals and humans have not provided an unequivocal answer. However, cerebral autoregulation is explicitly non-linear and most prior work relied on simple linear approaches for assessment, potentially missing important changes in autoregulatory characteristics. Therefore, we examined cerebral blood flow responses to augmented arterial pressure oscillations with and without calcium channel blockade (nicardipine) during blood pressure fluctuations (oscillatory lower body negative pressure, OLBNP) across a range of frequencies in 16 healthy subjects. Autoregulation was characterized via a robust non-linear method (projection pursuit regression, PPR). Blockade resulted in significant tachycardia, a modest but significant elevation in mean arterial pressure, and reductions in mean cerebral blood flow and end-tidal CO2 during OLBNP. The reductions in flow were directly related to the reductions in CO2 (r= 0.57). While linear cross-spectral analysis showed that the relationship between pressure–flow fluctuations was preserved after blockade, PPR showed that blockade significantly altered the non-linearity between pressure and flow, particularly at the slowest fluctuations. At 0.03 Hz, blockade reduced the range of pressure fluctuations that can be buffered (7.5 ± 1.0 vs. 3.7 ± 0.8 mmHg) while increasing the autoregulatory slope (0.10 ± 0.05 vs. 0.24 ± 0.08 cm s−1 mmHg−1). Furthermore, the same rate of change in pressure elicited a change in flow more than twice as large as at baseline. Thus, our results show that myogenic mechanisms play a significant role in cerebrovascular regulation but this may not be appreciated without adequately characterizing the non-linearities inherent in cerebrovascular regulation. PMID:23959681

  15. Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Yu, Haiyang; Lui, Yuan Siang; Xiong, Sijing; Leong, Wen Shing; Wen, Feng; Nurkahfianto, Himawan; Rana, Sravendra; Leong, David Tai; Ng, Kee Woei

    2013-01-01

    We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the β3 integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and α-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering. PMID:22765653

  16. 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. PMID:22396316

  17. Human ES- and iPS-Derived Myogenic Progenitors Restore Dystrophin and Improve Contractility upon Transplantation in Dystrophic Mice

    PubMed Central

    Darabi, Radbod; Arpke, Robert W.; Irion, Stefan; Dimos, John T.; Grskovic, Marica; Kyba, Michael; Perlingeiro, Rita C. R.

    2012-01-01

    SUMMARY A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of Pax7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived dystrophin-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment and engraftment is present over 11 months post-transplant. This study provides the proof-of-principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells, and highlights their potential for future therapeutic application in muscular dystrophies. PMID:22560081

  18. 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

  19. Dystrophin is a tumor suppressor in human cancers with myogenic programs.

    PubMed

    Wang, Yuexiang; Marino-Enriquez, Adrian; Bennett, Richard R; Zhu, Meijun; Shen, Yiping; Eilers, Grant; Lee, Jen-Chieh; Henze, Joern; Fletcher, Benjamin S; Gu, Zhizhan; Fox, Edward A; Antonescu, Cristina R; Fletcher, Christopher D M; Guo, Xiangqian; Raut, Chandrajit P; Demetri, George D; van de Rijn, Matt; Ordog, Tamas; Kunkel, Louis M; Fletcher, Jonathan A

    2014-06-01

    Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer. PMID:24793134

  20. Myogenic responses occur on a beat-to-beat basis in the resting human limb

    PubMed Central

    Fairfax, Seth T.; Padilla, Jaume; Vianna, Lauro C.; Holwerda, Seth W.; Davis, Michael J.

    2014-01-01

    Investigations of human myogenic responses typically use maneuvers that evoke robust changes in transmural pressure. Although this strategy has demonstrated peripheral myogenic responsiveness in the limbs, particularly in glabrous skin of the hand or foot, it has not considered the potential influence of the myogenic mechanism in beat-to-beat blood flow (BF) control during unprovoked rest. In the present study, we examined the interactions of spontaneous beat-to-beat mean arterial pressure (MAP; Finapres) with BF (Doppler ultrasound) supplying the forearm (brachial artery), lower leg (popliteal artery), and hand (ulnar artery) during 10 min of supine rest in healthy young men. Cross-correlation analyses revealed a negative association between MAP and BF, which was more prominent in the forearm than lower leg. The strongest correlation resulted when a −2-heart beat offset of MAP was applied (R = −0.53 ± 0.04 in the forearm and −0.23 ± 0.05 in the leg, P < 0.05), suggesting an ∼2-s delay from instances of high/low MAP to low/high BF. Negatively associated episodes (high MAP/low BF and low MAP/high BF) outnumbered positively associated data (P < 0.05). BF during low MAP values was greater than the steady-state average BF and vice versa. Wrist and ankle occlusion blunted the strength of correlations, homogenized the incidence of MAP and BF pairings, and reduced the magnitude of deviation from steady-state values. In contrast, these relationships were matched or accentuated for hand BF. Overall, these results suggest that myogenic responses are present and occur rapidly in human limbs during rest, overwhelm perfusion pressure gradient influences, and are primarily mediated by the distal limb circulation. PMID:25362138

  1. Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells.

    PubMed

    Bosurgi, L; Brunelli, S; Rigamonti, E; Monno, A; Manfredi, A A; Rovere-Querini, P

    2015-01-01

    Swift and regulated clearance of apoptotic cells prevents the accumulation of cell remnants in injured tissues and contributes to the shift of macrophages towards alternatively activated reparatory cells that sustain wound healing. Environmental signals, most of which are unknown, in turn control the efficiency of the clearance of apoptotic cells and as such determine whether tissues eventually heal. In this study we show that vessel-associated stem cells (mesoangioblasts) specifically modulate the expression of genes involved in the clearance of apoptotic cells and in macrophage alternative activation, including those of scavenger receptors and of molecules that bridge dying cells and phagocytes. Mesoangioblasts, but not immortalized myoblasts or neural precursor cells, enhance CD163 membrane expression in vitro as assessed by flow cytometry, indicating that the effect is specific. Mesoangioblasts transplanted in acutely or chronically injured skeletal muscles determine the expansion of the population of CD163(+) infiltrating macrophages and increase the extent of CD163 expression. Conversely, macrophages challenged with mesoangioblasts engulf significantly better apoptotic cells in vitro. Collectively, the data reveal a feed-forward loop between macrophages and vessel-associated stem cells, which has implications for the skeletal muscle homeostatic response to sterile injury and for diseases in which homeostasis is jeopardized, including muscle dystrophies and inflammatory myopathies. PMID:24749786

  2. Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors.

    PubMed

    Todaro, Marian; Quigley, Anita; Kita, Magdalena; Chin, Judy; Lowes, Kym; Kornberg, Andrew J; Cook, Mark J; Kapsa, Robert

    2007-08-01

    Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction. PMID:17394239

  3. Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

    PubMed Central

    Vauchez, Karine; Marolleau, Jean-Pierre; Schmid, Michel; Khattar, Patricia; Chapel, Alain; Catelain, Cyril; Lecourt, Séverine; Larghéro, Jérôme; Fiszman, Marc; Vilquin, Jean-Thomas

    2009-01-01

    Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH+ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDHbr) cells with low side scatter (SSClo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD+ (for skeletal muscle ALDH+) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD+/CD34− and SMALD+/CD34+ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD+/CD34+ cells developed in vitro as a heterogeneous population of CD56− cells able to differentiate in adipoblasts, the SMALD+/CD34− fraction developed in vitro as a highly enriched population of CD56+ myoblasts able to form myotubes. Moreover, only the SMALD+/CD34− population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. PMID:19738599

  4. 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. PMID:25637568

  5. 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.

  6. Assaying Human Myogenic Progenitor Cell Activity by Reconstitution of Muscle Fibers and Satellite Cells in Immunodeficient Mice.

    PubMed

    Parker, Maura H

    2016-01-01

    Comparing the functional myogenic potential of various human cell populations is an important step in the preclinical evaluation of cell transplantation as a means to treat human muscle disease and degeneration. Culture systems allow one to gage the potential of cell populations to proliferate and undergo myogenic differentiation under specific conditions. An in vivo assay evaluates the ability of cells to differentiate and generate muscle fibers within a natural environment, and importantly, evaluates the potential of donor cells to reconstitute the satellite cell niche. In this chapter, we describe a technique for isolating mononuclear cells from human muscle samples, and a method of xenotransplantation for assessing functional myogenic potential in vivo. Briefly, cell populations are injected into the pre-irradiated and regenerating muscle of immunodeficient mice. The injected muscle is frozen at specific time points after injection and cryosections analyzed by immunostaining. The number of human dystrophin-expressing fibers and the number of Pax7(+) human lamin A/C(+) nuclei are determined, which provides a quantitative method of comparing the in vivo functional potential of cell populations. PMID:27492175

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

    PubMed

    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

  8. 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

  9. Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

    PubMed Central

    Vella, Joseph B.; Thompson, Seth D.; Bucsek, Mark J.; Song, Minjung; Huard, Johnny

    2011-01-01

    Background Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity. Methodology/Principal Findings Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs. Conclusions The methodology of isolating myogenic cells on the basis of elevated ALDH activity

  10. 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

  11. 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

  12. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  13. 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. PMID:26486984

  14. Noninvasive examination of endothelial, sympathetic, and myogenic contributions to regional differences in the human cutaneous microcirculation.

    PubMed

    Hodges, Gary J; Del Pozzi, Andrew T

    2014-05-01

    The aim of this study was to examine whether there are regional differences in the cutaneous microvascular responses of the forearm and the leg. Utilizing a non-invasive measure (spectral analysis),we looked at the influence of the endothelial, sympathetic, and myogenic function between regions at thermoneutral conditions (33 °C) and in response to local skin warming (42 °C) using laser-Doppler flowmetry (LDF). We recruited 18 young, healthy participants, who visited the lab on 2 separate occasions. Participants were instrumented with LDF probes and local skin heater probe-holders, placed on the forearm or the leg. Blood pressure was recorded by oscillometry. At both 33 °C and during local skin warming to 42 °C, skin vasomotion for the forearm and leg were evaluated using spectral analysis of the LDF recordings. There were significant differences among all frequencies of interest between the forearm and the leg. At 33 °C the leg presented with higher (P=0.003) activity for endothelial (0.009-0.021 Hz), sympathetic (P=0.002) (0.021-0.052 Hz), and myogenic (P=0.004) (0.052-0.145 Hz) activity when compared to the forearm. In contrast, following 35 min of local skin warming, the forearm had greater endothelial (P=0.019), sympathetic (P=0.006), and myogenic (P=0.005) vasomotion than the leg. These outcomes indicate regional differences in the cutaneous microcirculation. The current results are similar to our previous work using invasive methods and pharmacological agents, indicating that non-invasive analyses may be useful in the diagnoses and understanding of the mechanisms that control the microvascular function of pathological conditions. PMID:24742702

  15. Neurally mediated propagating discrete clustered contractions superimposed on myogenic ripples in ex vivo segments of human ileum.

    PubMed

    Kuizenga, Merel H; Sia, Tiong C; Dodds, Kelsi N; Wiklendt, Lukasz; Arkwright, John W; Thomas, A; Brookes, Simon J; Spencer, Nick J; Wattchow, David A; Dinning, Phil G; Costa, Marcello

    2015-01-01

    Narrow muscle strips have been extensively used to study intestinal contractility. Larger specimens from laboratory animals have provided detailed understanding of mechanisms that underlie patterned intestinal motility. Despite progress in animal tissue, investigations of motor patterns in large, intact specimens of human gut ex vivo have been sparse. In this study, we tested whether neurally dependent motor patterns could be detected in isolated specimens of intact human ileum. Specimens (n = 14; 7-30 cm long) of terminal ileum were obtained with prior informed consent from patients undergoing colonic surgery for removal of carcinomas. Preparations were set up in an organ bath with an array of force transducers, a fiberoptic manometry catheter, and a video camera. Spontaneous and distension-evoked motor activity was recorded, and the effects of lidocaine, which inhibits neural activity, were studied. Myogenic contractions (ripples) occurred in all preparations (6.17 ± 0.36/min). They were of low amplitude and formed complex patterns by colliding and propagating in both directions along the specimen at anterograde velocities of 4.1 ± 0.3 mm/s and retrogradely at 4.9 ± 0.6 mm/s. In five specimens, larger amplitude clusters of contractions were seen (discrete clustered contractions), which propagated aborally at 1.05 ± 0.13 mm/s and orally at 1.07 ± 0.09 mm/s. These consisted of two to eight phasic contractions that aligned with ripples. These motor patterns were abolished by addition of lidocaine (0.3 mM). The ripples continued unchanged in the presence of this neural blocking agent. These results demonstrate that both myogenic and neurogenic motor patterns can be studied in isolated specimens of human small intestine. PMID:25394659

  16. 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

  17. Vascular Precursors in Developing Human Retina

    PubMed Central

    Hasegawa, Takuya; McLeod, D. Scott; Prow, Tarl; Merges, Carol; Grebe, Rhonda; Lutty, Gerard A.

    2016-01-01

    Purpose Prior investigation has demonstrated that angioblasts are present in the inner retinas of human embryos and fetuses and that they differentiate and organize to form the primordial retinal vasculature. The purpose of this study was to characterize these angioblasts further and examine ligands that might control their migration and differentiation. Methods Immunohistochemistry was used to localize stroma-derived factor-1 (SDF-1), its receptor CXCR4, stem cell factor (SCF), and its receptor c-Kit on sections obtained from human eyes at from 6 to 23 weeks’ gestation (WG). Coexpression of CD39 (marker for retinal angioblasts and endothelial cells) and CXCR4 or c-Kit was investigated by confocal microscopy. Results SDF-1 was prominent in inner retina with the greatest reaction product near the internal limiting membrane (ILM). SCF immunoreactivity was also confined to the inner retina and increased significantly between 7 and 12 WG. The level of both ligands declined by 22 WG. A layer of CXCR4+ and c-Kit+ precursors, some of which coexpressed CD39, existed in the inner retina from 7 to 12 WG. With migration, c-Kit was downregulated, whereas CD39+ cells continued to express CXCR4 as they formed cords. With canalization, CXCR4 expression was downregulated. Conclusions Embryonic human retina has a pool of precursors (CXCR4+ and c-Kit+) that enlarged centrifugally during fetal development. From this pool emerges angioblasts, which migrate anteriorly into the nerve fiber layer where SDF-1 and SCF levels are highest. c-Kit expression declines with apparent migration, and CXCR4 expression declines with canalization of new vessels. Both SCF and SDF-1 are associated with the differentiation of retinal precursors into angioblasts and their migration to sites of vessel assembly. PMID:18436851

  18. Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells.

    PubMed

    Maffioletti, Sara M; Gerli, Mattia F M; Ragazzi, Martina; Dastidar, Sumitava; Benedetti, Sara; Loperfido, Mariana; VandenDriessche, Thierry; Chuah, Marinee K; Tedesco, Francesco Saverio

    2015-07-01

    Skeletal muscle is the most abundant human tissue; therefore, an unlimited availability of myogenic cells has applications in regenerative medicine and drug development. Here we detail a protocol to derive myogenic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, and we also provide evidence for its extension to human iPS cells cultured without feeder cells. The procedure, which does not require the generation of embryoid bodies or prospective cell isolation, entails four stages with different culture densities, media and surface coating. Pluripotent stem cells are disaggregated to single cells and then differentiated into expandable cells resembling human mesoangioblasts. Subsequently, transient Myod1 induction efficiently drives myogenic differentiation into multinucleated myotubes. Cells derived from patients with muscular dystrophy and differentiated using this protocol have been genetically corrected, and they were proven to have therapeutic potential in dystrophic mice. Thus, this platform has been demonstrated to be amenable to gene and cell therapy, and it could be extended to muscle tissue engineering and disease modeling. PMID:26042384

  19. Effects of droperidol, pentobarbital, and ketamine on myogenic transcranial magnetic motor-evoked responses in humans.

    PubMed

    Kalkman, C J; Drummond, J C; Patel, P M; Sano, T; Chesnut, R M

    1994-12-01

    Myogenic motor-evoked responses to transcranial magnetic stimulation of the motor cortex (tcmag-MERs) may become clinically useful for the noninvasive assessment of motor pathway conduction during surgery. However, application is hindered because most anesthetic regimens result in severe depression of tcmag-MER amplitudes. As part of our systematic attempts to identify anesthetic agents and supplements suitable for use during tcmag-MER recording, we studied the effect of bolus doses of pentobarbital (1.5 mg/kg), droperidol (0.07 mg/kg), or ketamine (1 mg/kg), administered intravenously, on compound muscle action potentials to transcranial magnetic stimulation in five healthy volunteers. The doses were chosen to be comparable with doses that might be suitable for supplementation of a nitrous oxide/opioid anesthetic technique. Droperidol administration resulted in sustained amplitude depression of both tibialis and adductor pollicis tc-MERs to 30 +/- 9% and 39 +/- 14% of baseline (P < 0.01). Tcmag-MER amplitude changes after pentobarbital were variable, ranging from no change to substantial amplitude depression (to 20% of baseline) in two subjects. In contrast, ketamine administration did not result in significant amplitude depression. In three subjects, tibialis anterior amplitude increased to 150 to 220% of control values in the first 10 minutes after ketamine. Onset latency was unchanged after any drug. These data indicate that tcmag-MERs are moderately depressed after droperidol and pentobarbital but well preserved after ketamine. Ketamine may be a more suitable supplement to opioid/nitrous oxide anesthesia than droperidol or pentobarbital. PMID:7885550

  20. Myogenic differentiation potential of human tonsil-derived mesenchymal stem cells and their potential for use to promote skeletal muscle regeneration

    PubMed Central

    PARK, SAEYOUNG; CHOI, YOONYOUNG; JUNG, NAMHEE; YU, YEONSIL; RYU, KYUNG-HA; KIM, HAN SU; JO, INHO; CHOI, BYUNG-OK; JUNG, SUNG-CHUL

    2016-01-01

    Stem cells are regarded as an important source of cells which may be used to promote the regeneration of skeletal muscle (SKM) which has been damaged due to defects in the organization of muscle tissue caused by congenital diseases, trauma or tumor removal. In particular, mesenchymal stem cells (MSCs), which require less invasive harvesting techniques, represent a valuable source of cells for stem cell therapy. In the present study, we demonstrated that human tonsil-derived MSCs (T-MSCs) may differentiate into myogenic cells in vitro and that the transplantation of myoblasts and myocytes generated from human T-MSCs mediates the recovery of muscle function in vivo. In order to induce myogenic differentiation, the T-MSC-derived spheres were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F-12) supplemented with 1 ng/ml transforming growth factor-β, non-essential amino acids and insulin-transferrin-selenium for 4 days followed by culture in myogenic induction medium [low-glucose DMEM containing 2% fetal bovine serum (FBS) and 10 ng/ml insulin-like growth factor 1 (IGF1)] for 14 days. The T-MSCs sequentially differentiated into myoblasts and skeletal myocytes, as evidenced by the increased expression of skeletal myogenesis-related markers [including α-actinin, troponin I type 1 (TNNI1) and myogenin] and the formation of myotubes in vitro. The in situ transplantation of T-MSCs into mice with a partial myectomy of the right gastrocnemius muscle enhanced muscle function, as demonstrated by gait assessment (footprint analysis), and restored the shape of SKM without forming teratomas. Thus, T-MSCs may differentiate into myogenic cells and effectively regenerate SKM following injury. These results demonstrate the therapeutic potential of T-MSCs to promote SKM regeneration following injury. PMID:27035161

  1. Myogenic differentiation potential of human tonsil-derived mesenchymal stem cells and their potential for use to promote skeletal muscle regeneration.

    PubMed

    Park, Saeyoung; Choi, Yoonyoung; Jung, Namhee; Yu, Yeonsil; Ryu, Kyung-Ha; Kim, Han Su; Jo, Inho; Choi, Byung-Ok; Jung, Sung-Chul

    2016-05-01

    Stem cells are regarded as an important source of cells which may be used to promote the regeneration of skeletal muscle (SKM) which has been damaged due to defects in the organization of muscle tissue caused by congenital diseases, trauma or tumor removal. In particular, mesenchymal stem cells (MSCs), which require less invasive harvesting techniques, represent a valuable source of cells for stem cell therapy. In the present study, we demonstrated that human tonsil-derived MSCs (T-MSCs) may differentiate into myogenic cells in vitro and that the transplantation of myoblasts and myocytes generated from human T-MSCs mediates the recovery of muscle function in vivo. In order to induce myogenic differentiation, the T-MSC-derived spheres were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F‑12) supplemented with 1 ng/ml transforming growth factor-β, non-essential amino acids and insulin‑transferrin-selenium for 4 days followed by culture in myogenic induction medium [low-glucose DMEM containing 2% fetal bovine serum (FBS) and 10 ng/ml insulin‑like growth factor 1 (IGF1)] for 14 days. The T-MSCs sequentially differentiated into myoblasts and skeletal myocytes, as evidenced by the increased expression of skeletal myogenesis-related markers [including α-actinin, troponin I type 1 (TNNI1) and myogenin] and the formation of myotubes in vitro. The in situ transplantation of T-MSCs into mice with a partial myectomy of the right gastrocnemius muscle enhanced muscle function, as demonstrated by gait assessment (footprint analysis), and restored the shape of SKM without forming teratomas. Thus, T-MSCs may differentiate into myogenic cells and effectively regenerate SKM following injury. These results demonstrate the therapeutic potential of T-MSCs to promote SKM regeneration following injury. PMID:27035161

  2. Effect of acute systemic hypoxia on human cutaneous microcirculation and endothelial, sympathetic and myogenic activity.

    PubMed

    Paparde, Artūrs; Plakane, Līga; Circenis, Kristaps; Aivars, Juris Imants

    2015-11-01

    The regulation of cutaneous vascular tone impacts vascular vasomotion and blood volume distribution as a challenge to hypoxia, but the regulatory mechanisms yet remain poorly understood. A skin has a very compliant circulation, an increase in skin blood flow results in large peripheral displacement of blood volume, which could be controlled by local and systemic regulatory factors. The aim of this study was to determine the acute systemic hypoxia influence on blood flow in skin, local regulatory mechanism fluctuations and changes of systemic hemodynamic parameters. Healthy subjects (n=11; 24.9±3.7years old) participated in this study and procedures were performed in siting position. After 20min of acclimatization 15min of basal resting period in normoxia (pO2=21%) was recorded, followed by 20min in acute systemic hypoxia (pO2=12%), and after 15min of recovery period in normoxia (pO2=21%). HRV was used to evaluate autonomic nervous system activity to heart from systemic hemodynamic parameters which continuously evaluated cardiac output, total peripheral resistance and mean arterial blood pressure. Regional blood flow was evaluated by venous occlusion plethysmography and skin blood flow by laser-Doppler flowmetry. To evaluate local factor influences to cutaneous circulation wavelet analysis was used; fluctuations in the frequency intervals of 0.0095-0.021, 0.021-0.052, and 0.052-0.145Hz correspondingly represent endothelial, sympathetic, and myogenic activities. Our results from HRV data suggest that acute systemic hypoxia causes statistically significant increase of sympathetic (LF/HF; N1=0.46±0.25 vs. H=0.67±0.36; P=0.027) and decrease of parasympathetic (RMSSD; 80.0±43.1 vs. H=69.9±40.4, ms; P=0.009) outflow to heart. Acute hypoxia causes statistically significant increase of heart rate (RR interval; N1=960.3±174.5 vs. H=864.7±134.6, ms; P=0.001) and cardiac output (CO; N1=5.4 (5.2; 7.9) vs. H=6.7±1.4, l/min; P=0.020). Regional blood flow and vascular

  3. 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

  4. Isolation of blood-vessel-derived multipotent precursors from human skeletal muscle.

    PubMed

    Chen, William C W; Saparov, Arman; Corselli, Mirko; Crisan, Mihaela; Zheng, Bo; Péault, Bruno; Huard, Johnny

    2014-01-01

    Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes. PMID:25177794

  5. 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

  6. DNA methylation analysis of human myoblasts during in vitro myogenic differentiation: de novo methylation of promoters of muscle-related genes and its involvement in transcriptional down-regulation

    PubMed Central

    Miyata, Kohei; Miyata, Tomoko; Nakabayashi, Kazuhiko; Okamura, Kohji; Naito, Masashi; Kawai, Tomoko; Takada, Shuji; Kato, Kiyoko; Miyamoto, Shingo; Hata, Kenichiro; Asahara, Hiroshi

    2015-01-01

    Although DNA methylation is considered to play an important role during myogenic differentiation, chronological alterations in DNA methylation and gene expression patterns in this process have been poorly understood. Using the Infinium HumanMethylation450 BeadChip array, we obtained a chronological profile of the genome-wide DNA methylation status in a human myoblast differentiation model, where myoblasts were cultured in low-serum medium to stimulate myogenic differentiation. As the differentiation of the myoblasts proceeded, their global DNA methylation level increased and their methylation patterns became more distinct from those of mesenchymal stem cells. Gene ontology analysis revealed that genes whose promoter region was hypermethylated upon myoblast differentiation were highly significantly enriched with muscle-related terms such as ‘muscle contraction’ and ‘muscle system process’. Sequence motif analysis identified 8-bp motifs somewhat similar to the binding motifs of ID4 and ZNF238 to be most significantly enriched in hypermethylated promoter regions. ID4 and ZNF238 have been shown to be critical transcriptional regulators of muscle-related genes during myogenic differentiation. An integrated analysis of DNA methylation and gene expression profiles revealed that de novo DNA methylation of non-CpG island (CGI) promoters was more often associated with transcriptional down-regulation than that of CGI promoters. These results strongly suggest the existence of an epigenetic mechanism in which DNA methylation modulates the functions of key transcriptional factors to coordinately regulate muscle-related genes during myogenic differentiation. PMID:25190712

  7. Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation.

    PubMed

    Tu, Chengjian; Bu, Yahao; Vujcic, Marija; Shen, Shichen; Li, Jun; Qu, Miao; Hangauer, David; Clements, James L; Qu, Jun

    2016-09-01

    Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic

  8. Isolation and in vitro differentiation of human erythroid precursor cells.

    PubMed

    Kim, H C; Marks, P A; Rifking, R A; Maniatis, G M; Bank, A

    1976-05-01

    There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia. PMID:1260133

  9. Intraspinal transplantation of mouse and human neural precursor cells

    PubMed Central

    Weinger, Jason G.; Chen, Lu; Coleman, Ronald; Leang, Ronika; Plaisted, Warren C.; Loring, Jeanne F.; Lane, Thomas E.

    2013-01-01

    This unit describes the preparation and transplantation of human neural precursor cells (hNPCs) and mouse neural precursor cells (mNPCs) into the thoracic region of the mouse spinal cord. The techniques in this unit also describe how to prepare the mouse for surgery by performing a laminectomy to expose the spinal cord for transplantation. Here we show NPCs genetically labeled with eGFP transplanted into the spinal cord of a mouse following viralmediated demyelination can efficiently be detected via eGFP expression. Transplantation of these cells into the spinal cord is an efficacious way to determine their effects in neurological disorders such as multiple sclerosis, Alzheimer's disease, and spinal cord injury. PMID:24510791

  10. Evidence for Vitamin D Receptor Expression and Direct Effects of 1α,25(OH)2D3 in Human Skeletal Muscle Precursor Cells.

    PubMed

    Olsson, Karl; Saini, Amarjit; Strömberg, Anna; Alam, Seher; Lilja, Mats; Rullman, Eric; Gustafsson, Thomas

    2016-01-01

    Presence of the vitamin D receptor and direct effects of vitamin D on the proliferation and differentiation of muscle precursor cells have been demonstrated in animal models. However, the effects and mechanisms of vitamin D actions in human skeletal muscle, and the presence of the vitamin D receptor in human adult skeletal muscle, remain to be established. Here, we investigated the role of vitamin D in human muscle cells at various stages of differentiation. We demonstrate that the components of the vitamin D-endocrine system are readily detected in human muscle precursor cells but are low to nondetectable in adult skeletal muscle and that human muscle cells lack the ability to convert the inactive vitamin D-metabolite 25-hydroxy-vitamin D3 to the active 1α,25-dihydroxy-vitamin D3 (1α,25(OH)2D3). In addition, we show that 1α,25(OH)2D3 inhibits myoblast proliferation and differentiation by altering the expression of cell cycle regulators and myogenic regulatory factors, with associated changes in forkhead box O3 and Notch signaling pathways. The present data add novel information regarding the direct effects of vitamin D in human skeletal muscle and provide functional and mechanistic insight to the regulation of myoblast cell fate decisions by 1α,25(OH)2D3. PMID:26469137

  11. CXCR7 Is Involved in Human Oligodendroglial Precursor Cell Maturation

    PubMed Central

    Göttle, Peter; Kuhlmann, Tanja; Hartung, Hans-Peter; Antel, Jack; Küry, Patrick

    2016-01-01

    Differentiation of oligodendroglial precursor cells (OPCs), a crucial prerequisite for central nervous system (CNS) remyelination in diseases such as Multiple Sclerosis (MS), is modulated by a multitude of extrinsic and intrinsic factors. In a previous study we revealed that the chemokine CXCL12 stimulates rodent OPC differentiation via activation of its receptor CXCR7. We could now demonstrate that CXCR7 is also expressed on NogoA- and Nkx2.2-positive oligodendroglial cells in human MS brains and that stimulation of cultured primary fetal human OPCs with CXCL12 promotes their differentiation as measured by surface marker expression and morphologic complexity. Pharmacological inhibition of CXCR7 effectively blocks these CXCL12-dependent effects. Our findings therefore suggest that a specific activation of CXCR7 could provide a means to promote oligodendroglial differentiation facilitating endogenous remyelination activities. PMID:26741980

  12. 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. PMID:26879731

  13. 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

  14. Future Human Precursor Mission Missions and Architectures to Achieve Humans to Mars

    NASA Astrophysics Data System (ADS)

    Beegle, L. W.; Kinnett, R.; Klien, E.

    2012-06-01

    We have studied a series of payload options that are in response to measurements identified by MEPAG as vital to human precursor activity. These payloads have the ability to make a significant dent in the needed measurements to send humans to Mars.

  15. An ESA precursor mission to human exploration of the Moon

    NASA Astrophysics Data System (ADS)

    Carpenter, James; Fisackerly, Richard; Houdou, Berengere; Pradier, Alain; de Rossa, Diego; Vanoutryve, Benjamin; Jojaghaian, Aliac; Espinasse, Sylvie; Gardini, Bruno

    2010-05-01

    The coming decades will once again see humans on the surface of the Moon. Unlike the Apollo missions of the 1960s this new lunar exploration will be an international effort, with long duration missions and a goal to pave the way for further human expansion into the solar system. Ensuring the success and sustainability of this exploration poses significant challenges for all involved. ESA is currently preparing its first contribution to this international lunar exploration effort; a lunar lander mission, which will be a precursor to a future, Ariane V launched, ESA cargo and logistics capability to the Moon. The precursor mission will demonstrate soft precision landing with hazard avoidance capabilities, which will be required by a future cargo lander. In addition the mission can be applied as a preparation for future human exploration activities and help to ensure the sustainability of future exploration efforts. Activities have included Phase A and B1 mission design studies and technology development activities (both reported in another paper) and the definition of mission objectives and a model payload. The mission objectives have been derived by the Lunar Exploration Definition Team, a group derived of European specialists in various areas of exploration related science and technology, supported by ESA. Major inputs to the definition process were the 195 responses received to a request for information for potential payload contributions to the mission. The group was tasked with establishing how such a mission could best prepare for future human exploration. It was determined that the mission's goal should be to enable sustainable exploration and objectives were identified within a number of themes: health, habitation, resources, mobility and scientific preparations for future human activities. Investigations seek to characterise the lunar environment (e.g. radiation, dust etc.) and its effects and the properties of a landing site (potential resources, geological

  16. An ESA precursor mission to human exploration of the Moon

    NASA Astrophysics Data System (ADS)

    Carpenter, James; Fisackerly, Richard; Houdou, Berengere; Pradier, Alain; de Rossa, Diego; Vanoutryve, Benjamine; Jojaghaian, Aliac; Espinasse, Sylvie; Gardini, Bruno

    The coming decades will once again see humans on the surface of the Moon. Unlike the Apollo missions of the 1960s this new lunar exploration will be an international effort, with long duration missions and a goal to pave the way for further human expansion into the solar system. Ensuring the success and sustainability of this exploration poses significant challenges for all involved. ESA is currently preparing its first contribution to this international lunar exploration effort; a lunar lander mission, which will be a precursor to a future, Ariane V launched, ESA cargo and logistics capability to the Moon. The precursor mission will demonstrate soft precision landing with hazard avoidance capabilities, which will be required by a future cargo lander. In addition the mission can be applied as a preparation for future human exploration activities and help to ensure the sustainability of future exploration efforts. Activities have included Phase A and B1 mission design studies and technology development activities (both reported in another paper) and the definition of mission objectives and a model payload. The mission objectives have been derived by the Lunar Exploration Definition Team, a group derived of European specialists in various areas of exploration related science and technology, supported by ESA. Major inputs to the definition process were the 195 responses received to a request for information for potential payload contributions to the mission. The group was tasked with establishing how such a mission could best prepare for future human exploration. It was determined that the mission's goal should be to enable sustainable exploration and objectives were identified within a number of themes: health, habitation, resources, mobility and scientific preparations for future human activities. Investigations seek to characterise the lunar environment (e.g. radiation, dust etc.) and its effects and the properties of a landing site (potential resources, geological

  17. 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. PMID:26294751

  18. Expression of CD34 on human B cell precursors.

    PubMed Central

    Schmitt, C; Eaves, C J; Lansdorp, P M

    1991-01-01

    CD34 is a 110-kD glycoprotein previously shown by a variety of monoclonal antibodies (MoAbs) to be expressed selectively on immature hematopoietic cells. However, more detailed characterization of CD34+ cells has been hampered by lack of anti-CD34 MoAbs that can be labelled directly with fluorochromes to facilitate subpopulation analysis by multi-parameter flow cytometry. We have recently isolated a murine anti-CD34 MoAb, designated as 8G12, that can be directly labelled with fluorochromes such as FITC. In this study, we have exploited this property of 8G12 to compare the reactivity of 8G12 and My10 with normal and leukaemic human marrow cells and to characterize normal early human B cell precursors by two- and three-colour immunofluorescence analysis. Comparison of three-colour staining profiles of normal bone marrow cells incubated with both 8G12 and MY10, and either anti-CD10 or anti-CD19 MoAb revealed the reactivity patterns of 8G12 and MY10 to be indistinguishable. This conclusion was confirmed by a similar comparative analysis of 8G12 and MY10 staining of blood and bone marrow cells from 4 patients with B lineage acute lymphoblastic leukaemia (ALL). Of interest, both 8G12 and MY10 detected a CD34+CD10+CD19- population in normal adult bone marrow. To determine whether a CD34+CD10+CD19- precursor population previously reported by others to exist in fetal liver could also be identified, CD10+CD16- marrow cells were first isolated by FACS and the sorted cells then re-analysed for expression of CD19 and CD34. These studies showed that all of the sorted CD10+ cells that expressed CD34 appeared to coexpress CD19. No CD34+CD10+CD19- cells were detected (at a sensitivity of less than or equal to 0.1%). Further studies will be required to determine whether a very minor population of CD34+CD10+CD19- cells may still be generated in the normal development of B cells in adult human marrow. PMID:1712682

  19. Hedgehog-driven myogenic tumors recapitulate skeletal muscle cellular heterogeneity.

    PubMed

    Hettmer, Simone; Lin, Michael M; Tchessalova, Daria; Tortorici, Sara J; Castiglioni, Alessandra; Desai, Tushar; Mao, Junhao; McMahon, Andrew P; Wagers, Amy J

    2016-01-01

    Hedgehog (Hh) pathway activation in R26-SmoM2;CAGGS-CreER mice, which carry a tamoxifen-inducible activated Smoothened allele (SmoM2), results in numerous microscopic tumor foci in mouse skeletal muscle. These tumors exhibit a highly differentiated myogenic phenotype and resemble human fetal rhabdomyomas. This study sought to apply previously established strategies to isolate lineally distinct populations of normal mouse myofiber-associated cells in order to examine cellular heterogeneity in SmoM2 tumors. We demonstrate that established SmoM2 tumors are composed of cells expressing myogenic, adipocytic and hematopoietic lineage markers and differentiation capacity. SmoM2 tumors thus recapitulate the phenotypic and functional hetereogeneity observed in normal mouse skeletal muscle. SmoM2 tumors also contain an expanded population of PAX7+ and MyoD+ satellite-like cells with extremely low clonogenic activity. Selective activation of Hh signaling in freshly isolated muscle satellite cells enhanced terminal myogenic differentiation without stimulating proliferation. Our findings support the conclusion that SmoM2 tumors represent an aberrant skeletal muscle state and demonstrate that, similar to normal muscle, myogenic tumors contain functionally distinct cell subsets, including cells lacking myogenic differentiation potential. PMID:26460176

  20. Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for myogenic differentiation

    PubMed Central

    De Palma, C; Falcone, S; Pisoni, S; Cipolat, S; Panzeri, C; Pambianco, S; Pisconti, A; Allevi, R; Bassi, MT; Cossu, G; Pozzan, T; Moncada, S; Scorrano, L; Brunelli, S; Clementi, E

    2011-01-01

    During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. The functional relevance and the molecular mechanisms driving the formation of this mitochondrial network are unknown. We now show that mitochondrial elongation is required for myogenesis to occur and that this event depends on the cellular generation of nitric oxide (NO). Inhibition of NO synthesis in myogenic precursor cells leads to inhibition of mitochondrial elongation and of myogenic differentiation. This is due to the enhanced activity, translocation and docking of the pro-fission GTPase dynamin-related protein-1 (Drp1) to mitochondria, leading also to a latent mitochondrial dysfunction that increased sensitivity to apoptotic stimuli. These effects of NO inhibition were not observed in myogenic precursor cells containing a dominant-negative form of Drp1. Both NO-dependent repression of Drp1 action and maintenance of mitochondrial integrity and function were mediated through the soluble guanylate cyclase. These data uncover a novel level of regulation of differentiation linking mitochondrial morphology and function to myogenic differentiation. PMID:20467441

  1. 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

  2. Two myogenic lineages within the developing somite.

    PubMed

    Ordahl, C P; Le Douarin, N M

    1992-02-01

    It is well known that the muscles of the vertebrate body are derived from the somite. Precursor cells within the somite proper form the back or axial muscles while other precursor cells migrate away from the somite to populate the muscle of the limbs and ventral body wall. Although both types of muscle are generally thought of as arising from a common progenitor population, the myotome, recent evidence points to developmental differences in these two groups of muscles which may reflect different developmental lineages. To test the lineage hypothesis, we used microsurgery and the chick-quail nucleolar marker system to follow the developmental fate of the lateral and medial halves of somites at the wing level. The results showed that the structures of the mature somite (myotome and sclerotome) are derived virtually exclusively from cells residing in the medial half of the newly formed somite. On the other hand, virtually all of the cells residing in the lateral half of the newly formed somite are destined to leave the somite proper and populate the limb muscle and, probably, other somite-derived mesenchymal structures in the limb and ventral body wall. Switch-graft experiments show that the two halves of newly formed somites are largely interchangeable demonstrating that their ultimate developmental fate is position-dependent and that it becomes fixed as a result of extrinsic influences which act during later stages of somitogenesis. We conclude that at least two distinct myogenic lineages exist in the somite; one giving rise to the muscles of the back and the other giving rise to the limb musculature. PMID:1591996

  3. 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. PMID:18094147

  4. 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. PMID:11708369

  5. Crystal Structure of the Human Laminin Receptor Precursor

    SciTech Connect

    Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.

    2008-01-01

    The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

  6. Determination of Dideoxyosone Precursors of AGEs in Human Lens Proteins

    PubMed Central

    Linetsky, Mikhail; Johar, Kaid; Meltretter, Jasmin; Padmanabha, Smitha; Parmar, Trilok; Vasavada, Abhay R.; Pischetsrieder, Monika; Nagaraj, Ram H.

    2011-01-01

    Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation end products (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-{[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl) benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 AU/mg protein vs. 31.7±19.5 AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates. PMID:21820400

  7. Determination of dideoxyosone precursors of AGEs in human lens proteins.

    PubMed

    Linetsky, Mikhail; Kaid Johar, S R; Meltretter, Jasmin; Padmanabha, Smitha; Parmar, Trilok; Vasavada, Abhay R; Pischetsrieder, Monika; Nagaraj, Ram H

    2011-10-01

    Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates. PMID:21820400

  8. 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.

  9. Complete nucleotide sequence of the human corticotropin-beta-lipotropin precursor gene.

    PubMed Central

    Takahashi, H; Hakamata, Y; Watanabe, Y; Kikuno, R; Miyata, T; Numa, S

    1983-01-01

    The nucleotide sequence of an 8658-base-pair human genomic DNA segment containing the entire corticotropin-beta-lipotropin precursor gene has been determined, and some sequence features of the gene and its flanking regions have been analysed. The gene is composed of 7665 base pairs including two introns of 3708 and 2886 base pairs. Comparison of the 5'-flanking sequences of the human, bovine and mouse corticotropin-beta-lipotropin precursor genes reveals the presence of a highly conserved region, which contains sequences of 14-15 base pairs homologous with sequences located upstream of the mRNA start site of other glucocorticoid-regulated genes. PMID:6314261

  10. 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.

  11. 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.

  12. Human embryonic stem cell differentiation toward regional specific neural precursors.

    PubMed

    Erceg, Slaven; Ronaghi, Mohammad; Stojković, Miodrag

    2009-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration. PMID:18845761

  13. Human Embryonic Stem Cell Differentiation Toward Regional Specific Neural Precursors

    PubMed Central

    Erceg, Slaven; Ronaghi, Mohammad; Stojković, Miodrag

    2009-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that have the capacity to differentiate into a wide variety of cell types. This potentiality represents a promising source to overcome many human diseases by providing an unlimited supply of all cell types, including cells with neural characteristics. Therefore, this review summarizes early neural development and the potential of hESCs to differentiate under in vitro conditions, examining at the same time the potential use of differentiated hESCs for therapeutic applications for neural tissue and cell regeneration. PMID:18845761

  14. 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 ...

  15. 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...

  16. 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.

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

    PubMed

    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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.…

  3. 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. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23868863

  4. Circulating precursors of human CD1c+ and CD141+ dendritic cells

    PubMed Central

    Breton, Gaëlle; Lee, Jaeyop; Zhou, Yu Jerry; Schreiber, Joseph J.; Keler, Tibor; Puhr, Sarah; Anandasabapathy, Niroshana; Schlesinger, Sarah; Caskey, Marina

    2015-01-01

    Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c+ and CD141+ Clec9a+ cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L. PMID:25687281

  5. 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

    2015-11-01

    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. PMID:26385918

  6. 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

  7. 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

  8. Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

    PubMed Central

    Suram, Anitha; Kaplunov, Jessica; Patel, Priyanka L; Ruan, Haihe; Cerutti, Aurora; Boccardi, Virginia; Fumagalli, Marzia; Di Micco, Raffaella; Mirani, Neena; Gurung, Resham Lal; Hande, Manoor Prakash; d'Adda di Fagagna, Fabrizio; Herbig, Utz

    2012-01-01

    In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans. PMID:22569128

  9. 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

  10. 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

  11. 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…

  12. Ability of human oral microbiota to produce wine odorant aglycones from odourless grape glycosidic aroma precursors.

    PubMed

    Muñoz-González, Carolina; Cueva, Carolina; Ángeles Pozo-Bayón, M; Victoria Moreno-Arribas, M

    2015-11-15

    Grape aroma precursors are odourless glycosides that represent a natural reservoir of potential active odorant molecules in wines. Since the first step of wine consumption starts in the oral cavity, the processing of these compounds in the mouth could be an important factor in influencing aroma perception. Therefore, the objective of this work has been to evaluate the ability of human oral microbiota to produce wine odorant aglycones from odourless grape glycosidic aroma precursors previously isolated from white grapes. To do so, two methodological approaches involving the use of typical oral bacteria or the whole oral microbiota isolated from human saliva were followed. Odorant aglycones released in the culture mediums were isolated and analysed by HS-SPME-GC/MS. Results showed the ability of oral bacteria to hydrolyse grape aroma precursors, releasing different types of odorant molecules (terpenes, benzenic compounds and lipid derivatives). The hydrolytic activity seemed to be bacteria-dependent and was subject to large inter-individual variability. PMID:25977005

  13. 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. PMID:18429699

  14. 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.

  15. Antiadipogenic properties of retinol in primary cultured differentiating human adipocyte precursor cells.

    PubMed

    Garcia, E; Lacasa, D; Agli, B; Giudicelli, Y; Castelli, D

    2000-04-01

    The aim of this study was to investigate the effect of retinol on the human adipose conversion process using primary cultured human adipocyte precursor cells. When these cells were seeded in a medium containing retinol (concentrations ranging from 3.5 nM to 3.5 muM), cell proliferation was slightly inhibited by high concentrations of retinol, as demonstrated by cell counting and [(3)H]-thymidine incorporation. Moreover, the differentiation capacities of these cells were markedly and dose-dependently inhibited by retinol, as shown by the reduced expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase and by microscopic morphological analysis. These results strongly suggest that retinol, by inhibiting the ability of human preadipocytes to convert into mature adipocytes, could be of potential interest in the prevention of human adipose tissue development in general and of cellulitis in particular. PMID:18503465

  16. Adenovirus-mediated expression of myogenic differentiation factor 1 (MyoD) in equine and human dermal fibroblasts enables their conversion to caffeine-sensitive myotubes.

    PubMed

    Fernandez-Fuente, Marta; Martin-Duque, Pilar; Vassaux, Georges; Brown, Susan C; Muntoni, Francesco; Terracciano, Cesare M; Piercy, Richard J

    2014-03-01

    Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy. PMID:24342283

  17. Cloning and characterization of human liver cDNA encoding a protein S precursor

    SciTech Connect

    Hoskins, J.; Norman, D.K.; Beckmann, R.J.; Long, G.L.

    1987-01-01

    Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approx. = 0.01%. Blot hybridization of electrophoretically fractionated poly(A)/sup +/ RNA revealed a major mRNA approx. = 4 kilobases long and two minor forms of approx. = 3.1 and approx. = 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid leader peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal ..gamma..-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approx. = 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function.

  18. Human Embryonic Stem Cell-Derived Neural Precursors Develop Into Neurons and Integrate Into the Host Brain

    PubMed Central

    Guillaume, Daniel J.; Johnson, M. Austin; Li, Xue-Jun; Zhang, Su-Chun

    2009-01-01

    Whether and how in-vitro-produced human neural precursors mature and integrate into the brain are crucial to the utility of human embryonic stem (hES) cells in treating neurological disorders. After transplantation into the ventricles of neonatal immune-deficient mice, hES-cell-derived neural precursors stopped expressing the cell division marker Ki67, except in neurogenic areas, and differentiated into neurons and then glia in a temporal course intrinsic to that of human cells regardless of location. The human cells located in the gray matter became neurons in the olfactory bulb and striatum, whereas those in the white matter produced exclusively glia. Importantly, the grafted human cells formed synapses. Thus, the in-vitro-produced human neural precursors follow their intrinsic temporal program to produce neurons and glia and, in response to environmental signals, generate cells appropriate to their target regions and integrate into the brain. PMID:16941479

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

    PubMed Central

    Uckun, Fatih M.; Goodman, Patricia; Ma, Hong; Dibirdik, Ilker; Qazi, Sanjive

    2010-01-01

    Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients’ primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia. PMID:20841423

  5. Relative Contributions of Sympathetic, Cholinergic, and Myogenic Mechanisms to Cerebral Autoregulation

    PubMed Central

    Hamner, J.W.; Tan, Can Ozan

    2014-01-01

    Background and Purpose Prior work aimed at improving our understanding of human cerebral autoregulation has explored individual physiologic mechanisms of autoregulation in isolation, but none has attempted to consolidate the individual roles of these mechanisms into a comprehensive model of the overall cerebral pressure–flow relation. Methods We retrospectively analyzed this relation before and after pharmacologic blockade of alpha-adrenergic, muscarinic, and calcium channel-mediated mechanisms in 43 healthy volunteers to determine the relative contributions of the sympathetic, cholinergic, and myogenic controllers to cerebral autoregulation. Projection pursuit regression was used to assess the effect of pharmacologic blockade on the cerebral pressure–flow relation. Subsequently, analysis of covariance decomposition was used to determine the cumulative effect of these three mechanisms on cerebral autoregulation and whether they can fully explain it. Results Sympathetic, cholinergic, and myogenic mechanisms together accounted for 62% of the cerebral pressure–flow relation (p < 0.05), with significant and distinct contributions from each of the three effectors. ANCOVA decomposition demonstrated that myogenic effectors were the largest determinant of the cerebral pressure–flow relation but their effect was outside of the autoregulatory region where neurogenic control appeared prepotent. Conclusions Our results suggest that myogenic effects occur outside the active region of autoregulation, whereas neurogenic influences are largely responsible for cerebral blood flow control within it. However, our model of cerebral autoregulation left 38% of the cerebral pressure–flow relation unexplained, suggesting that there are other physiologic mechanisms that contribute to cerebral autoregulation. PMID:24723314

  6. Different Blood-Borne Human Osteoclast Precursors Respond in Distinct Ways to IL-17A.

    PubMed

    Sprangers, Sara; Schoenmaker, Ton; Cao, Yixuan; Everts, Vincent; de Vries, Teun J

    2016-06-01

    Osteoclasts are bone-degrading cells that are formed through fusion of their monocytic precursors. Three distinct subsets of monocytes have been identified in human peripheral blood: classical, intermediate, and non-classical monocytes. They are known to play different roles in physiology and pathology, but their capacity to differentiate into osteoclasts and whether inflammatory cytokines influence this differentiation is unknown. We hypothesized that classical, intermediate, and non-classical monocytes generate functionally different osteoclasts and that they respond in different ways to the inflammatory cytokine interleukin-17A (IL-17A). To investigate this, the different monocyte subsets were isolated from human peripheral blood and osteoclastogenesis was induced with the cytokines M-CSF and RANKL, with or without IL-17A. We found that all subsets are able to differentiate into osteoclasts in vitro, and that both osteoclastogenesis and subsequent bone resorption was distinctly affected by IL-17A. Osteoclastogenesis and bone resorption by osteoclasts derived from classical monocytes remained unaffected by IL-17A, while osteoclast formation from intermediate monocytes was inhibited by the cytokine. Surprisingly, bone resorption by osteoclasts derived from intermediate monocytes remained at similar levels as control cultures, indicating an increased bone resorbing activity by these osteoclasts. Limited numbers of osteoclasts were formed from non-classical monocytes on bone and no bone resorption was detected, which suggest that these cells belong to a cell lineage different from the osteoclast. By providing more insight into osteoclast formation from human blood monocytes, this study contributes to the possible targeting of specific osteoclast precursors as a therapeutic approach for diseases associated with inflammatory bone loss. J. Cell. Physiol. 231: 1249-1260, 2016. © 2015 Wiley Periodicals, Inc. PMID:26491867

  7. Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

    PubMed Central

    Salomonis, Nathan; Nelson, Brandon; Vranizan, Karen; Pico, Alexander R.; Hanspers, Kristina; Kuchinsky, Allan; Ta, Linda; Mercola, Mark; Conklin, Bruce R.

    2009-01-01

    The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation. PMID:19893621

  8. Increased cellular turnover in response to fluoxetine in neuronal precursors derived from human embryonic stem cells.

    PubMed

    Chang, Eun-Ah; Beyhan, Zeki; Yoo, Myung-Sik; Siripattarapravat, Kannika; Ko, Tak; Lookingland, Keith J; Madhukar, Burra V; Cibelli, Jose B

    2010-01-01

    Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (p<.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics. PMID:19598107

  9. Development of methods for characterizing fetal and adult somatic mutations detected in human erythroid precursor

    SciTech Connect

    Langlois, R.G.; Manchester, D.K.

    1994-12-31

    The glycophorin A (GPA) assay was developed to quantify somatic mutations in humans by measuring the frequency of peripheral erythrocytes with mutant phenotypes that are presumed to be progeny of mutated erythroid precursor cells. This assay has been used to identify GPA variant cells in unexposed individuals at a frequency of {approximately}10 per million erythrocytes, and to demonstrate significant increases in variant frequency after mutagenic exposures. Characterization of the mutations responsible for these variant cells requires that the assay be modified to allow flow analysis and sorting of variant erythroid precursor cells that contain nucleic acids. Cord blood samples contain low levels of both reticulocytes and nucleated erythrocytes. We have developed enrichment methods using centrifugation that yield samples containing up to 30% nucleated erythrocytes, and immunomagnetic separation methods that yield samples containing up to 90% reticulocytes. Enrichment methods for these two cell types are also being developed for adult bone marrow samples. We have confirmed that enrichment and labeling with a nucleic acid-specific dye are compatible with GPA analysis of erythrocytes, reticulocytes, and nucleated erythrocytes. Enriched samples have been successfully used for flow cytometric detection of GPA variant reticulocytes in cord blood. PCR-based analysis methods are being developed for molecular characterization of sorted variant cells at the mRNA level.

  10. Semaphorin 3F and neuropilin-2 control the migration of human T-cell precursors.

    PubMed

    Mendes-da-Cruz, Daniella Arêas; Brignier, Anne Colette; Asnafi, Vahid; Baleydier, Frederic; Messias, Carolina Valença; Lepelletier, Yves; Bedjaoui, Nawel; Renand, Amedée; Smaniotto, Salete; Canioni, Danielle; Milpied, Pierre; Balabanian, Karl; Bousso, Philippe; Leprêtre, Stéphane; Bertrand, Yves; Dombret, Hervé; Ifrah, Norbert; Dardenne, Mireille; Macintyre, Elizabeth; Savino, Wilson; Hermine, Olivier

    2014-01-01

    Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions. PMID:25068647

  11. 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.

  12. Acute effect of protein or carbohydrate breakfasts on human cerebrospinal fluid monoamine precursor and metabolite levels.

    PubMed

    Teff, K L; Young, S N; Marchand, L; Botez, M I

    1989-01-01

    Patients with normal pressure hydrocephalus who had three lumbar punctures during 1 week ingested either water, a protein breakfast, or a carbohydrate breakfast 2.5 h before each of the lumbar punctures. The CSF was analyzed for biogenic amine precursors and metabolites. The protein meal raised CSF tyrosine levels, a finding consistent with animal data, but did not alter those of tryptophan or any of the biogenic amine metabolites. The carbohydrate meal increased CSF 3-methoxy-4-hydroxyphenylethylene glycol, an unexplained finding. The carbohydrate meal did not affect CSF tryptophan, tyrosine, 5-hydroxyindoleacetic acid, or homovanillic acid. Our results support the idea that in humans protein or carbohydrate meals do not alter plasma amino acid levels sufficiently to cause appreciable changes in CNS tryptophan levels or 5-hydroxytryptamine synthesis. PMID:2462018

  13. Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells

    PubMed Central

    Kozhich, O; Hamilton, RS; Mallon, BS

    2012-01-01

    Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAMpos and Tra-1–60neg by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments. PMID:22388559

  14. Efficient Production of Photoreceptor Precursor Cells from Human Embryonic Stem Cells.

    PubMed

    Yanai, Anat; Laver, Christopher; Joe, Aaron W; Gregory-Evans, Kevin

    2016-01-01

    Transplantation of photoreceptor precursor cells (PPCs) differentiated from human embryonic stem cells (hESCs) is a promising approach to treat common blinding diseases such as age-related macular degeneration and retinitis pigmentosa. However, existing PPC generation methods are inefficient. To enhance differentiation protocols for rapid and high-yield production of PPCs, we focused on optimizing the handling of the cells by including feeder-independent growth of hESCs, using size-controlled embryoid bodies (EBs), and addition of triiodothyronine (T3) and taurine to the differentiation medium, with subsequent removal of undifferentiated cells via negative cell-selection. Our novel protocol produces higher yields of PPCs than previously reported while reducing the time required for differentiation, which will help understand retinal diseases and facilitate large-scale preclinical trials. PMID:24301073

  15. 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.

  16. 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

  17. Gene amplification during myogenic differentiation

    PubMed Central

    Fischer, Ulrike; Ludwig, Nicole; Raslan, Abdulrahman; Meier, Carola; Meese, Eckart

    2016-01-01

    Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians. PMID:26760505

  18. An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors

    PubMed Central

    Mihardja, Shirley S.; Liszewski, Walter; Erle, David J.; Lee, Randall J.; Bernstein, Harold S.

    2011-01-01

    Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease. PMID:21245908

  19. Human HMGA2 protein overexpressed in mice induces precursor T-cell lymphoblastic leukemia

    PubMed Central

    Efanov, A; Zanesi, N; Coppola, V; Nuovo, G; Bolon, B; Wernicle-Jameson, D; Lagana, A; Hansjuerg, A; Pichiorri, F; Croce, C M

    2014-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a neoplasia of thymocytes characterized by the rapid accumulation of the precursors of T lymphocytes. HMGA2 (high-mobility group AT-hook 2) gene expression is extremely low in normal adult tissues, but it is overexpressed in many tumors. To identify the biological function of HMGA2, we generated transgenic mice carrying the human HMGA2 gene under control of the VH promoter/Eμ enhancer. Approximately 90% of Eμ-HMGA2 transgenic mice became visibly sick between 4 and 8 months due to the onset and progression of a T-ALL-like disease. Characteristic features included severe alopecia (30% of mice); enlarged lymph nodes and spleen; and profound immunological abnormalities (altered cytokine levels, hypoimmunoglobulinemia) leading to reduced immune responsiveness. Immunophenotyping showed accumulation of CD5+CD4+, CD5+CD8+ or CD5+CD8+CD4+ T-cell populations in the spleens and bone marrow of sick animals. These findings show that HMGA2-driven leukemia in mice closely resembles spontaneous human T-ALL, indicating that HMGA2 transgenic mice should serve as an important model for investigating basic mechanisms and potential new therapies of relevance to human T-ALL. PMID:25014774

  20. p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps

    PubMed Central

    Wu, Zhenguo; Woodring, Pamela J.; Bhakta, Kunjan S.; Tamura, Kumiko; Wen, Fang; Feramisco, James R.; Karin, Michael; Wang, Jean Y. J.; Puri, Pier Lorenzo

    2000-01-01

    The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun–N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38α−/− embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes. PMID

  1. 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

  2. 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. PMID:17185060

  3. Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes.

    PubMed

    Kvedar, J C; Manabe, M; Phillips, S B; Ross, B S; Baden, H P

    1992-04-01

    The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin

  4. Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression.

    PubMed

    Schnorr, J J; Xanthakos, S; Keikavoussi, P; Kämpgen, E; ter Meulen, V; Schneider-Schaulies, S

    1997-05-13

    As well as inducing a protective immune response against reinfection, acute measles is associated with a marked suppression of immune functions against superinfecting agents and recall antigens, and this association is the major cause of the current high morbidity and mortality rate associated with measles virus (MV) infections. Dendritic cells (DCs) are antigen-presenting cells crucially involved in the initiation of primary and secondary immune responses, so we set out to define the interaction of MV with these cells. We found that both mature and precursor human DCs generated from peripheral blood monocytic cells express the major MV protein receptor CD46 and are highly susceptible to infection with both MV vaccine (ED) and wild-type (WTF) strains, albeit with different kinetics. Except for the down-regulation of CD46, the expression pattern of functionally important surface antigens on mature DCs was not markedly altered after MV infection. However, precursor DCs up-regulated HLA-DR, CD83, and CD86 within 24 h of WTF infection and 72 h after ED infection, indicating their functional maturation. In addition, interleukin 12 synthesis was markedly enhanced after both ED and WTF infection in DCs. On the other hand, MV-infected DCs strongly interfered with mitogen-dependent proliferation of freshly isolated peripheral blood lymphocytes in vitro. These data indicate that the differentiation of effector functions of DCs is not impaired but rather is stimulated by MV infection. Yet, mature, activated DCs expressing MV surface antigens do give a negative signal to inhibit lymphocyte proliferation and thus contribute to MV-induced immunosuppression. PMID:9144236

  5. Human BCAS3 Expression in Embryonic Stem Cells and Vascular Precursors Suggests a Role in Human Embryogenesis and Tumor Angiogenesis

    PubMed Central

    Siva, Kavitha; Venu, Parvathy; Mahadevan, Anita; S. K., Shankar; Inamdar, Maneesha S.

    2007-01-01

    Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3) is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES) cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker. PMID:18030336

  6. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence.

    PubMed

    Wang, Zhe; Wen, Yan; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-03-15

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 10(6) cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  7. Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor.

    PubMed Central

    Lebacq-Verheyden, A M; Kasprzyk, P G; Raum, M G; Van Wyke Coelingh, K; Lebacq, J A; Battey, J F

    1988-01-01

    The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands. Images PMID:3211139

  8. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence

    PubMed Central

    Wang, Zhe; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-01-01

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 106 cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  9. 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.

  10. 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.

  11. Identification of a human splenic marginal zone B cell precursor with NOTCH2-dependent differentiation properties

    PubMed Central

    Descatoire, Marc; Weller, Sandra; Irtan, Sabine; Sarnacki, Sabine; Feuillard, Jean; Storck, Sébastien; Guiochon-Mantel, Anne; Bouligand, Jérôme; Morali, Alain; Cohen, Joseph; Jacquemin, Emmanuel; Iascone, Maria; Bole-Feysot, Christine; Cagnard, Nicolas

    2014-01-01

    Mouse splenic marginal zone precursors (MZPs) differentiate into marginal zone B (MZB) cells under a signaling pathway involving Notch2 and its ligand, delta-like 1 ligand (Dll1). We report the identification of an MZP subset in the spleen of young children. These MZPs differentiate into MZ-like B cells in vitro in the presence of OP9 cells expressing human DLL1, as demonstrated by the up-regulation of classical MZB cell markers. A set of diagnostic genes discriminating IgM+IgD+CD27+ blood and splenic MZB cells from switched B cells was identified (up-regulation of SOX7, down-regulation of TOX, COCH, and HOPX), and their expression during the induction assay mirrored the one of MZB cells. Moreover, Alagille patients with a NOTCH2 haploinsufficiency display a marked reduction of IgM+IgD+CD27+ B cells in blood, whereas their switched memory B cells are not affected. Altogether, these results argue in favor of the existence of a rodent-like MZB cell lineage in humans. PMID:24733829

  12. Interleukin 4 inhibits in vitro proliferation of leukemic and normal human B cell precursors.

    PubMed Central

    Pandrau, D; Saeland, S; Duvert, V; Durand, I; Manel, A M; Zabot, M T; Philippe, N; Banchereau, J

    1992-01-01

    In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells. Images PMID:1385474

  13. The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

    PubMed

    Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

    2011-12-01

    We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. PMID:21889203

  14. Genetically engineered mouse models of human B-cell precursor leukemias

    PubMed Central

    Hauer, Julia; Borkhardt, Arndt; Sánchez-García, Isidro; Cobaleda, César

    2014-01-01

    B-cell precursor acute lymphoblastic leukemias (pB-ALLs) are the most frequent type of malignancies of the childhood, and also affect an important proportion of adult patients. In spite of their apparent homogeneity, pB-ALL comprises a group of diseases very different both clinically and pathologically, and with very diverse outcomes as a consequence of their biology, and underlying molecular alterations. Their understanding (as a prerequisite for their cure) will require a sustained multidisciplinary effort from professionals coming from many different fields. Among all the available tools for pB-ALL research, the use of animal models stands, as of today, as the most powerful approach, not only for the understanding of the origin and evolution of the disease, but also for the development of new therapies. In this review we go over the most relevant (historically, technically or biologically) genetically engineered mouse models (GEMMs) of human pB-ALLs that have been generated over the last 20 years. Our final aim is to outline the most relevant guidelines that should be followed to generate an “ideal” animal model that could become a standard for the study of human pB-ALL leukemia, and which could be shared among research groups and drug development companies in order to unify criteria for studies like drug testing, analysis of the influence of environmental risk factors, or studying the role of both low-penetrance mutations and cancer susceptibility alterations. PMID:25486471

  15. 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

    2007-12-01

    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. PMID:17947800

  16. Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells.

    PubMed

    Xi, G; Kamanga-Sollo, E; Hathaway, M R; Dayton, W R; White, M E

    2006-07-01

    We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells. PMID:16233971

  17. Evidence of an evolutionary precursor to human language affixation in a non-human primate.

    PubMed

    Endress, Ansgar D; Cahill, Donal; Block, Stefanie; Watumull, Jeffrey; Hauser, Marc D

    2009-12-23

    Human language, and grammatical competence in particular, relies on a set of computational operations that, in its entirety, is not observed in other animals. Such uniqueness leaves open the possibility that components of our linguistic competence are shared with other animals, having evolved for non-linguistic functions. Here, we explore this problem from a comparative perspective, asking whether cotton-top tamarin monkeys (Saguinus oedipus) can spontaneously (no training) acquire an affixation rule that shares important properties with our inflectional morphology (e.g. the rule that adds -ed to create the past tense, as in the transformation of walk into walk-ed). Using playback experiments, we show that tamarins discriminate between bisyllabic items that start with a specific 'prefix' syllable and those that end with the same syllable as a 'suffix'. These results suggest that some of the computational mechanisms subserving affixation in a diversity of languages are shared with other animals, relying on basic perceptual or memory primitives that evolved for non-linguistic functions. PMID:19586963

  18. CEACAM1 in Cervical Cancer and Precursor Lesions: Association With Human Papillomavirus Infection

    PubMed Central

    Albarran-Somoza, Benibelks; Franco-Topete, Ramon; Delgado-Rizo, Vidal; Cerda-Camacho, Felipe; Acosta-Jimenez, Lourdes; Lopez-Botet, Miguel; Daneri-Navarro, Adrian

    2006-01-01

    Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an adhesion molecule expressed in a wide variety of tissues including epithelial cells, leukocytes, and tumors that may establish both homotypic and heterotypic interactions. The aim of this work was to study the protein expression pattern of CEACAM1 in cervical cancer and precursor lesions in the context of human papillomavirus (HPV) infection. We used immunohistochemistry to analyze CEACAM1 expression in formalin-fixed, paraffin-embedded cervical tissues from 15 healthy women, 15 patients with low-grade squamous intraepithelial lesions (SIL), 15 patients with high-grade SIL, and 15 patients with squamous carcinomas. HPV types were identified by PCR. CEACAM1 was either undetectable (13/15) or low (2/15) in normal cervical tissues. By contrast, CEACAM1 expression was increased in high-grade SIL (10 samples staining intermediate/high and 4 samples staining low) as compared with low-grade SIL with undetectable (n=3) or low (n= 12) expression. CEACAM1 expression was undetectable or low in cervical carcinoma. Our results suggest that CEACAM1 may be an interesting progression marker in SIL and cervical cancer, in particular due to reported immunoregulatory properties. PMID:16924126

  19. Human neural precursor cells promote neurologic recovery in a viral model of multiple sclerosis.

    PubMed

    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

    2014-06-01

    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

  20. Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

    PubMed Central

    Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J.; Alani, Sara; Bocchetta, Maurizio

    2015-01-01

    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. PMID:25283437

  1. Intravenous Administration of Human ES-derived Neural Precursor Cells Attenuates Cuprizone-induced CNS Demyelination

    PubMed Central

    Crocker, Stephen J.; Bajpai, Ruchi; Moore, Craig S.; Frausto, Ricardo F.; Brown, Graham D.; Pagarigan, Roberto R.; Whitton, J. Lindsay; Terskikh, Alexey V.

    2011-01-01

    Aims Previous studies have demonstrated the therapeutic potential for human embryonic stem cell-derived neural precursor cells (hES-NPCs) in autoimmune and genetic animal models of demyelinating diseases. Herein, we tested whether intravenous (i.v) administration of hES-NPCs would impact central nervous system (CNS) demyelination in a cuprizone model of demyelination. Methods C57Bl/6 mice were fed cuprizone (0.2%) for two weeks and then separated into two groups that either received an i.v. injection of hES-NPCs or i.v. administration of media without these cells. After an additional two weeks of dietary cuprizone treatment, CNS tissues were analyzed for detection of transplanted cells and differences in myelination in the region of the corpus callosum (CC). Results Cuprizone-induced demyelination in the CC was significantly reduced in mice treated with hES-NPCs compared with cuprizone-treated controls that did not receive stem cells. hES-NPCs were identified within the brain tissues of treated mice and revealed migration of transplanted cells into the CNS. A limited number of human cells were found to express the mature oligodendrocyte marker, O1, or the astrocyte marker, GFAP. Reduced apoptosis and attenuated microglial and astrocytic responses were also observed in the CC of hES-NPC-treated mice. Conclusions These findings indicated that systemically-administered hES-NPCs migrated from circulation into a demyelinated lesion within the CNS and effectively reduced demyelination. Observed reductions in astrocyte and microglial responses, and (c) the benefit of hES-NPC treatment in this model of myelin injury was not obviously accountable to tissue replacement by exogenously administered cells. PMID:21276029

  2. Introduction of yeast artificial chromosomes containing mutant human amyloid precursor protein genes into transgenic mice

    SciTech Connect

    Call, L.M.; Lamb, B.T.; Boese, K.F.

    1994-09-01

    Several hypothetical mechanisms have been proposed for the generation and deposition of the amyloid beta (A{beta}) peptide in Alzheimer`s disease (AD). These include overexpression of the amyloid precursor protein (APP) gene, as suggested by Down Syndrome (DS, trisomy 21), and mutation of APP, as suggested by mutations associated with the presence of disease/amyloid deposition in some cases of familial AD (FAD). Although numerous in vitro studies have lead to certain insights into the molecular basis for amyloid deposition, the mechanisms(s) of amyloidogenesis in vivo remains poorly defined. To examine the effect of FAD mutations on amyloidogenesis in an animal model, we have focused on producing APP YAC transgenic mice containing the human APP gene with FAD mutations. These APP YAC transgenics are being produced by introduction of a 650 kb APP YAC through lipid-mediated transfection of ES cells. This strategy has two principal advantages: the APP genomic sequences contain transcriptional regulatory elements required for proper spatial and temporal expression and contain appropriate splice donor and acceptor sites needed to generate the entire spectrum of alternatively spliced APP transcripts. As a first step, we cloned the genomic regions surrounding APP exons 16 and 17 from an APP YAC sublibrary. Both the Swedish and the 717 mutations were then introduced into exons 16 and 17, respectively, by PCR mutagenesis, and subsequently transferred into the 650 kb APP YAC by a two step gene replacement in yeast. The mutant YACs have been introduced into ES cells, and we have determined that these cells are expressing human mutant APP mRNA and protein. These cells are being used to generate transgenic mice. This paradigm should provide the appropriate test of whether a mutant APP gene is capable of producing AD-like pathology in a mouse model.

  3. 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. PMID:25283437

  4. Human umbilical cord Wharton's jelly-derived oligodendrocyte precursor-like cells for axon and myelin sheath regeneration★

    PubMed Central

    Chen, Hong; Zhang, Yan; Yang, Zhijun; Zhang, Hongtian

    2013-01-01

    Human umbilical mesenchymal stem cells from Wharton's jelly of the umbilical cord were induced to differentiate into oligodendrocyte precursor-like cells in vitro. Oligodendrocyte precursor cells were transplanted into contused rat spinal cords. Immunofluorescence double staining indicated that transplanted cells survived in injured spinal cord, and differentiated into mature and immature oligodendrocyte precursor cells. Biotinylated dextran amine tracing results showed that cell transplantation promoted a higher density of the corticospinal tract in the central and caudal parts of the injured spinal cord. Luxol fast blue and toluidine blue staining showed that the volume of residual myelin was significantly increased at 1 and 2 mm rostral and caudal to the lesion epicenter after cell transplantation. Furthermore, immunofluorescence staining verified that the newly regenerated myelin sheath was derived from the central nervous system. Basso, Beattie and Bresnahan testing showed an evident behavioral recovery. These results suggest that human umbilical mesenchymal stem cell-derived oligodendrocyte precursor cells promote the regeneration of spinal axons and myelin sheaths. PMID:25206380

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

    PubMed

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

    2016-05-01

    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

  6. 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

  7. In-Situ Cryogenic Propellant Liquefaction and Storage for a Precursor to a Human Mars Mission

    NASA Astrophysics Data System (ADS)

    Mueller, Paul; Durrant, Tom

    The current mission plan for the first human mission to Mars is based on an in-situ propellant production (ISPP) approach to reduce the amount of propellants needed to be taken to Mars and ultimately to reduce mission cost. Recent restructuring of the Mars Robotic Exploration Program has removed ISPP from the early sample return missions. A need still exists to demonstrate ISPP technologies on one or more robotic missions prior to the first human mission. This paper outlines a concept for an ISPP-based precursor mission as a technology demonstration prior to the first human mission. It will also return Martian soil samples to Earth for scientific analysis. The mission will primarily demonstrate cryogenic oxygen and fuel production, liquefaction, and storage for use as propellants for the return trip. Hydrogen will be brought from Earth as a feedstock to produce the hydrocarbon fuel (most likely methane). The analysis used to develop the mission concept includes several different thermal control and liquefaction options for the cryogens. Active cooling and liquefaction devices include Stirling, pulse tube, and Brayton-cycle cryocoolers. Insulation options include multilayer insulation, evacuated microspheres, aerogel blankets, and foam insulation. The cooling capacity and amount of insulation are traded off against each other for a minimum-mass system. In the case of hydrogen feedstock, the amount of hydrogen boiloff allowed during the trip to Mars is also included in the tradeoff. The spacecraft concept includes a Lander (including the propellant production plant) with a Mars Ascent Vehicle (MAV) mounted atop it. An option is explored where the engines on the MAV are also used for descent and landing on the Martian surface at the beginning of the mission. So the MAV propellant tanks would contain oxygen and methane during the trip from Earth. This propellant would be consumed in descent to the Martian surface, resulting in nearly-empty MAV tanks to be filled by the

  8. Histone methyltransferase KMT1A restrains entry of alveolar rhabdomyosarcoma cells into a myogenic differentiated state.

    PubMed

    Lee, Min-Hyung; Jothi, Mathivanan; Gudkov, Andrei V; Mal, Asoke K

    2011-06-01

    Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric muscle cancer, which arrested during the process of skeletal muscle differentiation. In muscle myoblast cells, ectopic expression of the histone H3 lysine 9 (H3K9) methytransferase KMT1A blocks differentiation by repressing a myogenic gene expression program. In this study, we tested the hypothesis that activation of a KMT1A-mediated program of transcriptional repression prevents ARMS cells from differentiating. We investigated whether KMT1A represses the expression of differentiation-associated genes in ARMS cells, thereby blocking muscle differentiation. Our results show that expression of KMT1A is induced in human ARMS cancer cell lines when cultured under differentiation-permissible conditions. shRNA-mediated knockdown of KMT1A decreased anchorage dependent and independent cell proliferation and tumor xenograft growth, increased expression of differentiation-associated genes, and promoted the appearance of a terminally differentiated-like phenotype. Finally, shRNA-directed KMT1A knockdown restored the impaired transcriptional activity of the myogenic regulator MyoD. Together, our results suggested that high levels of KMT1A in ARMS cells under differentiation conditions impairs MyoD function, thereby arresting myogenic differentiation in these tumor cells. Thus, targeting KMT1A may be a novel strategy for the treatment of this disease. PMID:21493592

  9. Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates

    PubMed Central

    Quattrocelli, Mattia; Giacomazzi, Giorgia; Broeckx, Sarah Y.; Ceelen, Liesbeth; Bolca, Selin; Spaas, Jan H.; Sampaolesi, Maurilio

    2016-01-01

    Summary Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage. PMID:26771353

  10. Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates.

    PubMed

    Quattrocelli, Mattia; Giacomazzi, Giorgia; Broeckx, Sarah Y; Ceelen, Liesbeth; Bolca, Selin; Spaas, Jan H; Sampaolesi, Maurilio

    2016-01-12

    Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage. PMID:26771353

  11. Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

    PubMed Central

    Padma Priya, P.; Grover, Manish; Tatu, Utpal S.; Natarajan, Vasant

    2015-01-01

    Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes. PMID:26317863

  12. Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

    PubMed

    Dilsen, S; Paul, W; Sandgathe, A; Tippe, D; Freudl, R; Thömmes, J; Kula, M R; Takors, R; Wandrey, C; Weuster-Botz, D

    2000-09-01

    A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract). PMID:11030573

  13. Loquat leaf extract enhances myogenic differentiation, improves muscle function and attenuates muscle loss in aged rats.

    PubMed

    Sung, Bokyung; Hwang, Seong Yeon; Kim, Min Jo; Kim, Minjung; Jeong, Ji Won; Kim, Cheol Min; Chung, Hae Young; Kim, Nam Deuk

    2015-09-01

    A main characteristic of aging is the debilitating, progressive and generalized impairment of biological functions, resulting in an increased vulnerability to disease and death. Skeletal muscle comprises approximately 40% of the human body; thus, it is the most abundant tissue. At the age of 30 onwards, 0.5‑1% of human muscle mass is lost each year, with a marked acceleration in the rate of decline after the age of 65. Thus, novel strategies that effectively attenuate skeletal muscle loss and enhance muscle function are required to improve the quality of life of older subjects. The aim of the present study was to determine whether loquat (Eriobotrya japonica) leaf extract (LE) can prevent the loss of skeletal muscle function in aged rats. Young (5-month-old) and aged (18‑19-month-old) rats were fed LE (50 mg/kg/day) for 35 days and the changes in muscle mass and strength were evaluated. The age‑associated loss of grip strength was attenuated, and muscle mass and muscle creatine kinase (CK) activity were enhanced following the administration of LE. Histochemical analysis also revealed that LE abrogated the age‑associated decrease in cross‑sectional area (CSA) and decreased the amount of connective tissue in the muscle of aged rats. To investigate the mode of action of LE, C2C12 murine myoblasts were used to evaluate the myogenic potential of LE. The expression levels of myogenic proteins (MyoD and myogenin) and functional myosin heavy chain (MyHC) were measured by western blot analysis. LE enhanced MyoD, myogenin and MyHC expression. The changes in the expression of myogenic genes corresponded with an increase in the activity of CK, a myogenic differentiation marker. Finally, LE activated the Akt/mammalian target of rapamycin (mTOR) signaling pathway, which is involved in muscle protein synthesis during myogenesis. These findings suggest that LE attenuates sarcopenia by promoting myogenic differentiation and subsequently promoting muscle protein synthesis

  14. 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. PMID:25822579

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:25822579

  16. Deconvolution of the vestibular evoked myogenic potential.

    PubMed

    Lütkenhöner, Bernd; Basel, Türker

    2012-02-01

    The vestibular evoked myogenic potential (VEMP) and the associated variance modulation can be understood by a convolution model. Two functions of time are incorporated into the model: the motor unit action potential (MUAP) of an average motor unit, and the temporal modulation of the MUAP rate of all contributing motor units, briefly called rate modulation. The latter is the function of interest, whereas the MUAP acts as a filter that distorts the information contained in the measured data. Here, it is shown how to recover the rate modulation by undoing the filtering using a deconvolution approach. The key aspects of our deconvolution algorithm are as follows: (1) the rate modulation is described in terms of just a few parameters; (2) the MUAP is calculated by Wiener deconvolution of the VEMP with the rate modulation; (3) the model parameters are optimized using a figure-of-merit function where the most important term quantifies the difference between measured and model-predicted variance modulation. The effectiveness of the algorithm is demonstrated with simulated data. An analysis of real data confirms the view that there are basically two components, which roughly correspond to the waves p13-n23 and n34-p44 of the VEMP. The rate modulation corresponding to the first, inhibitory component is much stronger than that corresponding to the second, excitatory component. But the latter is more extended so that the two modulations have almost the same equivalent rectangular duration. PMID:22079097

  17. 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.

  18. 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.

  19. Myomir dysregulation and reactive oxygen species in aged human satellite cells.

    PubMed

    Di Filippo, Ester Sara; Mancinelli, Rosa; Pietrangelo, Tiziana; La Rovere, Rita Maria Laura; Quattrocelli, Mattia; Sampaolesi, Maurilio; Fulle, Stefania

    2016-04-29

    Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells. PMID:26975470

  20. The TPR-MET oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions.

    PubMed Central

    Soman, N R; Correa, P; Ruiz, B A; Wogan, G N

    1991-01-01

    The TPR-MET oncogenic rearrangement was originally observed in an in vitro transformed human osteosarcoma cell line. Recently, we detected the expression of this rearrangement at very low levels in several cell lines derived from human tumors of nonhematopoietic origin using a highly sensitive method based on polymerase chain reaction amplification of the transcript. We report here the results of analysis of TPR-MET expression in cell lines derived from human gastric tumors and 22 biopsy samples of human gastric mucosa showing cancer or precursor lesions. The rearranged RNA was expressed in all four cell lines as well as in biopsy samples from 12 of the 22 patients. Overexpression of TPR-MET RNA in superficial gastritis lesions with hyperplasia of glandular neck cells suggests the possible involvement of this oncogene at an early stage of gastric tumorigenesis. Analysis of gastric biopsy samples for RAS gene mutations showed base substitutions occurring in the codon 12 region of Ki- and Ha-RAS genes in four cases, including two precursor lesions. Images PMID:2052572

  1. Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation

    PubMed Central

    Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

    2016-01-01

    Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation. PMID:26954143

  2. Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

    PubMed

    Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

    2016-07-01

    Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation. PMID:26954143

  3. Myogenic potential of adipose-tissue-derived cells.

    PubMed

    Di Rocco, Giuliana; Iachininoto, Maria Grazia; Tritarelli, Alessandra; Straino, Stefania; Zacheo, Antonella; Germani, Antonia; Crea, Filippo; Capogrossi, Maurizio C

    2006-07-15

    Adipose-tissue-derived mesenchymal stem cells can be directed towards a myogenic phenotype in vitro by the addition of specific inductive media. However, the ability of these or other adipose-tissue-associated cells to respond to ;natural' myogenic cues such as a myogenic environment has never been investigated in detail. Here, we provide evidence that a restricted subpopulation of freshly harvested adipose-tissue-derived cells possesses an intrinsic myogenic potential and can spontaneously differentiate into skeletal muscle. Conversion of adipose-tissue-derived cells to a myogenic phenotype is enhanced by co-culture with primary myoblasts in the absence of cell contact and is maximal when the two cell types are co-cultured in the same plate. Conversely, in vitro expanded adipose-tissue-derived mesenchymal stem cells require direct contact with muscle cells to generate skeletal myotubes. Finally, we show that uncultured adipose-tissue-associated cells have a high regenerative capacity in vivo since they can be incorporated into muscle fibers following ischemia and can restore significantly dystrophin expression in mdx mice. PMID:16825428

  4. 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.

  5. CID-miRNA: A web server for prediction of novel miRNA precursors in human genome

    SciTech Connect

    Tyagi, Sonika; Vaz, Candida; Gupta, Vipin; Bhatia, Rohit; Maheshwari, Sachin; Srinivasan, Ashwin; Bhattacharya, Alok

    2008-08-08

    microRNAs (miRNA) are a class of non-protein coding functional RNAs that are thought to regulate expression of target genes by direct interaction with mRNAs. miRNAs have been identified through both experimental and computational methods in a variety of eukaryotic organisms. Though these approaches have been partially successful, there is a need to develop more tools for detection of these RNAs as they are also thought to be present in abundance in many genomes. In this report we describe a tool and a web server, named CID-miRNA, for identification of miRNA precursors in a given DNA sequence, utilising secondary structure-based filtering systems and an algorithm based on stochastic context free grammar trained on human miRNAs. CID-miRNA analyses a given sequence using a web interface, for presence of putative miRNA precursors and the generated output lists all the potential regions that can form miRNA-like structures. It can also scan large genomic sequences for the presence of potential miRNA precursors in its stand-alone form. The web server can be accessed at (http://mirna.jnu.ac.in/cidmirna/)

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

    PubMed

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

    2011-02-01

    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) CD56(bright)CD16(-)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

  7. 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.

  8. Adenoviral-Mediated Endothelial Precursor Cell Delivery of Soluble CD115 Suppresses Human Prostate Cancer Xenograft Growth in Mice

    PubMed Central

    Lucas, Trevor; Abraham, Dietmar; Untergasser, Gerold; Zins, Karin; Hofer, Erhard; Gunsilius, Eberhard; Aharinejad, Seyedhossein

    2009-01-01

    Prostate cancer tumor growth and neovascularization is promoted by an interplay between migratory tumor stromal cells such as specialized tumor-associated macrophages (TAMs) and circulating endothelial precursor cells (CEPs). As vehicles for tumor therapy, human CEPs are relatively easy to isolate from peripheral blood, are able to proliferate long-term in vitro, are amenable to viral manipulation, and preferentially home to regions of ischemia found in growing tumors. We show here that human peripheral blood CEPs expanded ex vivo migrate to prostate cancer cells in vitro and efficiently home to human prostate tumor xenografts in vivo. Infection of precursors ex vivo with an adenovirus constructed to secrete a soluble form of the colony-stimulating factor-1 receptor CD115 that inhibits macrophage viability and migration in vitro significantly decreases the number of TAMs in xenografts (p < .05), reduces proliferation (p < .01) and vascular density (p < .03), and suppresses the growth of xenografts (p < .03). These data show for the first time that targeting stromal cell processes with cellular therapy has the potential to retard prostate tumor growth. PMID:19522014

  9. Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage marked differentiated muscle to myogenic progenitor cells

    PubMed Central

    Paliwal, Preeti; Conboy, Irina M

    2011-01-01

    Summary Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. Here, we used the Cre-Lox method to specifically label post-mitotic primary multinucleated myotubes and then utilized small molecule inhibitors of tyrosine phosphatases and apoptosis to de-differentiate these myotubes into proliferating myogenic cells, without gene over expression. The reprogrammed, fusion competent, muscle precursor cells contributed to muscle regeneration in vitro and in vivo and were unequivocally distinguished from reactivated reserve cells due to the lineage marking method. The small molecule inhibitors down-regulated cell cycle inhibitors and chromatin remodeling factors known to promote and maintain the cell fate of myotubes, facilitating cell fate reversal. Our findings enhance understanding of cell-fate determination and create novel therapeutic approaches for improved muscle repair. PMID:21944754

  10. Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells.

    PubMed

    Paliwal, Preeti; Conboy, Irina M

    2011-09-23

    Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. Here, we used the Cre-Lox method to specifically label postmitotic primary multinucleated myotubes and then utilized small molecule inhibitors of tyrosine phosphatases and apoptosis to dedifferentiate these myotubes into proliferating myogenic cells, without gene overexpression. The reprogrammed, fusion competent, muscle precursor cells contributed to muscle regeneration in vitro and in vivo and were unequivocally distinguished from reactivated reserve cells because of the lineage marking method. The small molecule inhibitors downregulated cell cycle inhibitors and chromatin remodeling factors known to promote and maintain the cell fate of myotubes, facilitating cell fate reversal. Our findings enhance understanding of cell-fate determination and create novel therapeutic approaches for improved muscle repair. PMID:21944754

  11. Vestibular evoked myogenic potentials in Bell's palsy.

    PubMed

    Krbot Skoric, Magdalena; Adamec, Ivan; Habek, Mario

    2014-10-01

    The aim of the present study was to evaluate vestibular nerve involvement in patients with Bell's palsy with ocular and cervical vestibular evoked myogenic potentials (oVEMP and cVEMP). Ten patients who were diagnosed with Bell's palsy and ten healthy controls were included. All patients underwent VEMP recordings within 6 days after their initial presentation. Patients with Bell's palsy had greater oVEMP asymmetry ratio comparing to healthy controls (-38.4 ± 28.7 % vs -1.3 ± 19.3 %, p = 0.005). As well N10 latencies of the oVEMP response were prolonged comparing to healthy controls (11.575 vs 9.72 ms). There was no difference in cVEMP asymmetry ratio or latencies between groups. We found no correlation between House-Brackmann grading scale and oVEMP asymmetry ratio (r = 0.003, p = 0.994). There are three possible explanations for increased oVEMP amplitudes on the affected side: (1) oVEMP response on the ipsilateral eye could be contaminated by facial nerve activity (blink reflex); (2) the amplitude of N10-P33 could be affected through the stapedial reflex; and (3) increased oVEMP amplitude could be the consequence of the vestibular nerve dysfunction itself, with prolonged latencies of the N10 oVEMP further supporting this explanation. The results of this study indicate possible involvement of the superior branch of the vestibular nerve in patients with Bell's palsy. PMID:24916836

  12. 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).

  13. Long-term, stable differentiation of human embryonic stem cell-derived neural precursors grafted into the adult mammalian neostriatum.

    PubMed

    Nasonkin, Igor; Mahairaki, Vasiliki; Xu, Leyan; Hatfield, Glen; Cummings, Brian J; Eberhart, Charles; Ryugo, David K; Maric, Dragan; Bar, Eli; Koliatsos, Vassilis E

    2009-10-01

    Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic AMP-regulated phosphoprotein. Graft-derived neurons formed mature synapses with host postsynaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin(+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches. PMID:19609935

  14. Long-Term, Stable Differentiation Of Human Embryonic Stem Cell-Derived Neural Precursors Grafted Into The Adult Mammalian Neostriatum

    PubMed Central

    Nasonkin, I.; Mahairaki, V.; Xu, L.; Hatfield, G.; Cummings, B.J.; Eberhart, C.; Ryugo, D.; Maric, D.; Bar, E.; Koliatsos, V.E.

    2010-01-01

    Stem-cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker Dopamine- and cyclic AMP-Regulated Phosphoprotein. Graft-derived neurons formed mature synapses with host post-synaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency towards glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin (+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular CNS niches. PMID:19609935

  15. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.

    PubMed

    Kadowaki, N; Ho, S; Antonenko, S; Malefyt, R W; Kastelein, R A; Bazan, F; Liu, Y J

    2001-09-17

    Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens. PMID:11561001

  16. 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.

  17. Subsets of Human Dendritic Cell Precursors Express Different Toll-like Receptors and Respond to Different Microbial Antigens

    PubMed Central

    Kadowaki, Norimitsu; Ho, Stephen; Antonenko, Svetlana; de Waal Malefyt, Rene; Kastelein, Robert A.; Bazan, Fernando; Liu, Yong-Jun

    2001-01-01

    Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c+ immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-α. CD11c+ imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-α, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-α and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens. PMID:11561001

  18. Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.

    PubMed Central

    Ryan, D H; Nuccie, B L; Abboud, C N; Winslow, J M

    1991-01-01

    Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment. Images PMID:1715889

  19. Prophylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors

    PubMed Central

    Arbyn, Marc; Bryant, Andrew; Beutels, Philippe; Martin-Hirsch, Pierre PL; Paraskevaidis, Evangelos; Van Hoof, Elke; Steben, Marc; Qiao, Youlin; Zhao, Fang-Hui; Schneider, Achim; Kaufmann, Andreas; Dillner, Joakim; Markowitz, Lauri; Hildesheim, Allan

    2014-01-01

    This is the protocol for a review and there is no abstract. The objectives are as follows: To evaluate the immunogenicity, clinical efficacy, and safety of prophylactic HPV vaccines in females. The assessment of clinical efficacy will address protection against HPV infection (for homologous and heterologous HPV types), against re-infection, against cervical cancer and its precursors (high-grade CIN (grade 2 or grade 3), adenocarcinoma in situ) in women previously not exposed to HPV infection (negative at enrolment for both HPV DNA and antibodies against the vaccine HPV types). We will assess clinical effectiveness by evaluating outcomes in all women, irrespective of the HPV DNA or serology status at enrolment. Evaluation by fine age and time since sexual debut categories is also planned. PMID:25267916

  20. p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma.

    PubMed

    Moskaluk, C A; Hruban, R H; Kern, S E

    1997-06-01

    Pancreatic adenocarcinoma is thought to arise from a noninvasive neoplastic precursor, the pancreatic intraductal lesion (PIL). Mutations of the K-ras gene are known to occur in PILs, but their high prevalence among PILs within the general population probably limit the use of K-ras as a marker of eventual clinical risk. In search of genetic constellations that might indicate the progression of some PILs toward an invasive phenotype, mutations at both the K-ras and p16 genes were sought within PILs of 10 pancreata resected for adenocarcinoma. K-ras mutations were present in most PILs and in nearly all PILs having nuclear atypia. In half of the patients, two or more unique K-ras mutations were identified among distinct PILs, which is evidence for the separate clonal evolution of multiple pancreatic neoplasms within individual patients. p16 alterations (one homozygous deletion and three point mutations) were found in 4 of the 10 carcinomas; these four pancreata harbored p16 alterations in three of nine PILs, of which one was a "histologically early" lesion. Two patients had p16 alterations in PILs matching those of the associated carcinomas. p16 mutations were not found in PILs of pancreata having wild-type p16 in the carcinoma, nor were they found in ducts having normal histology. It is suggested that alterations of the p16 gene affect a subset of PILs that contain mutations of the K-ras gene and that these mutations might identify high-risk precursors of the invasive malignancy. PMID:9187111

  1. 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.

  2. 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.

  3. Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells.

    PubMed

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

    2015-04-01

    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 increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-μ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency. PMID:25467851

  4. Quantitative variation in obesity-related traits and insulin precursors linked to the OB gene region on human chromosome 7

    SciTech Connect

    Duggirala, R.; Stern, M.P.; Reinhart, L.J.

    1996-09-01

    Despite the evidence that human obesity has strong genetic determinants, efforts at identifying specific genes that influence human obesity have largely been unsuccessful. Using the sibship data obtained from 32 low-income Mexican American pedigrees ascertained on a type II diabetic proband and a multipoint variance-components method, we tested for linkage between various obesity-related traits plus associated metabolic traits and 15 markers on human chromosome 7. We found evidence for linkage between markers in the OB gene region and various traits, as follows: D7S514 and extremity skinfolds (LOD = 3.1), human carboxypeptidase A1 (HCPA1) and 32,33-split proinsulin level (LOD = 4.2), and HCPA1 and proinsulin level (LOD = 3.2). A putative susceptibility locus linked to the marker D7S514 explained 56% of the total phenotypic variation in extremity skinfolds. Variation at the HCPA1 locus explained 64% of phenotypic variation in proinsulin level and {approximately}73% of phenotypic variation in split proinsulin concentration, respectively. Weaker evidence for linkage to several other obesity-related traits (e.g., waist circumference, body-mass index, fat mass by bioimpedance, etc.) was observed for a genetic location, which is {approximately}15 cM telomeric to OB. In conclusion, our study reveals that the OB region plays a significant role in determining the phenotypic variation of both insulin precursors and obesity-related traits, at least in Mexican Americans. 66 refs., 3 figs., 4 tabs.

  5. 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

  6. Regulation of proteolytic cleavage of brain-derived neurotrophic factor precursor by antidepressants in human neuroblastoma cells

    PubMed Central

    Lin, Pao-Yen

    2015-01-01

    Evidence has supported the role of brain-derived neurotrophic factor (BDNF) in antidepressant effect. The precursor of BDNF (proBDNF) often exerts opposing biological effects on mature BDNF (mBDNF). Hence, the balance between proBDNF and mBDNF might be critical in total neurotrophic effects, leading to susceptibility to or recovery from depression. In the current study, we measured the protein expression levels of proBDNF, and its proteolytic products, truncated BDNF, and mBDNF, in human SH-SY5Y cells treated with different antidepressants. We found that the treatment significantly increased the production of mBDNF, but decreased the production of truncated BDNF and proBDNF. These results support that antidepressants can promote proBDNF cleavage. Further studies are needed to clarify whether proBDNF cleavage plays a role in antidepressant mechanisms. PMID:26491331

  7. Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

    PubMed

    Höfling, Corinna; Morawski, Markus; Zeitschel, Ulrike; Zanier, Elisa R; Moschke, Katrin; Serdaroglu, Alperen; Canneva, Fabio; von Hörsten, Stephan; De Simoni, Maria-Grazia; Forloni, Gianluigi; Jäger, Carsten; Kremmer, Elisabeth; Roßner, Steffen; Lichtenthaler, Stefan F; Kuhn, Peer-Hendrik

    2016-10-01

    Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals. PMID:27470171

  8. Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

    PubMed Central

    Killian, Rhiannon L.; Flippin, Jessica D.; Herrera, Cheryl M.; Almenar-Queralt, Angels; Goldstein, Lawrence S. B.

    2012-01-01

    The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases. PMID:22272245

  9. 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

  10. 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.

  11. 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

  12. PROBLEMS IN THE ESTIMATION OF HUMAN EXPOSURE TO COMPONENTS OF ACID PRECIPITATION PRECURSORS

    EPA Science Inventory

    Problems associated with estimation of human exposures to ambient air pollution are discussed. Ideally the authors would prefer to have some indication of actual dose. For most pollutants this is not presently feasible. Specific problems discussed are adequacy of outdoor monitors...

  13. Exercise training reverses aging-induced impairment of myogenic constriction in skeletal muscle arterioles

    PubMed Central

    Ghosh, Payal; Mora Solis, Fredy R.; Dominguez, James M.; Spier, Scott A.; Donato, Anthony J.; Delp, Michael D.

    2015-01-01

    To investigate whether exercise training can reverse age-related impairment of myogenic vasoconstriction in skeletal muscle arterioles, young (4 mo) and old (22 mo) male Fischer 344 rats were randomly assigned to either sedentary or exercise-trained groups. The roles of the endothelium and Kv1 channels in age- and exercise training-induced adaptations of myogenic responses were assessed through evaluation of pressure-induced constriction in endothelium-intact and denuded soleus muscle arterioles in the presence and absence of the Kv1 channel blocker, correolide. Exercise training enhanced myogenic constriction in arterioles from both old and young rats. In arterioles from old rats, exercise training restored myogenic constriction to a level similar to that of arterioles from young sedentary rats. Removal of the endothelium did not alter myogenic constriction of arterioles from young sedentary rats, but reduced myogenic constriction in arterioles from young exercise-trained rats. In contrast, endothelial removal had no effect on myogenic constriction of arterioles from old exercise-trained rats, but increased myogenic vasoconstriction in old sedentary rats. The effect of Kv1 channel blockade was also dependent on age and training status. In arterioles from young sedentary rats, Kv1 blockade had little effect on myogenic constriction, whereas in old sedentary rats Kv1 blockade increased myogenic constriction. After exercise training, Kv1 channel blockade increased myogenic constriction in arterioles from both young and old rats. Thus exercise training restores myogenic constriction of arterioles from old rats and enhances myogenic constriction from young rats through adaptations of the endothelium and smooth muscle Kv1 channels. PMID:25634999

  14. 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

  15. Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro.

    PubMed

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

    2011-03-01

    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

  16. Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength.

    PubMed

    Bentzinger, C Florian; von Maltzahn, Julia; Dumont, Nicolas A; Stark, Danny A; Wang, Yu Xin; Nhan, Kevin; Frenette, Jérôme; Cornelison, D D W; Rudnicki, Michael A

    2014-04-14

    Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle. PMID:24711502

  17. Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

    PubMed Central

    Bentzinger, C. Florian; von Maltzahn, Julia; Dumont, Nicolas A.; Stark, Danny A.; Wang, Yu Xin; Nhan, Kevin; Frenette, Jérôme; Cornelison, DDW

    2014-01-01

    Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle. PMID:24711502

  18. 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

  19. Lentivirus Live Cell Array for Quantitative Assessment of Gene and Pathway Activation during Myogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Tian, Jun; Gaile, Daniel P.; Andreadis, Stelios T.

    2015-01-01

    Stem cell differentiation involves multiple cascades of transcriptional regulation that govern the cell fate. To study the real-time dynamics of this complex process, quantitative and high throughput live cell assays are required. Herein, we developed a lentiviral library of promoters and transcription factor binding sites to quantitatively capture the gene expression dynamics over a period of several days during myogenic differentiation of human mesenchymal stem cells (MSCs) harvested from two different anatomic locations, bone marrow and hair follicle. Our results enabled us to monitor the sequential activation of signaling pathways and myogenic gene promoters at various stages of differentiation. In conjunction with chemical inhibitors, the lentiviral array (LVA) results also revealed the relative contribution of key signaling pathways that regulate the myogenic differentiation. Our study demonstrates the potential of LVA to monitor the dynamics of gene and pathway activation during MSC differentiation as well as serve as a platform for discovery of novel molecules, genes and pathways that promote or inhibit complex biological processes. PMID:26505747

  20. 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.

  1. 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. PMID:15806749

  2. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  3. Myogenic Progenitors from Mouse Pluripotent Stem Cells for Muscle Regeneration.

    PubMed

    Magli, Alessandro; Incitti, Tania; Perlingeiro, Rita C R

    2016-01-01

    Muscle homeostasis is maintained by resident stem cells which, in both pathologic and non-pathologic conditions, are able to repair or generate new muscle fibers. Although muscle stem cells have tremendous regenerative potential, their application in cell therapy protocols is prevented by several restrictions, including the limited ability to grow ex vivo. Since pluripotent stem cells have the unique potential to both self-renew and expand almost indefinitely, they have become an attractive source of progenitors for regenerative medicine studies. Our lab has demonstrated that embryonic stem cell (ES)-derived myogenic progenitors retain the ability to repair existing muscle fibers and contribute to the pool of resident stem cells. Because of their relevance in both cell therapy and disease modeling, in this chapter we describe the protocol to derive myogenic progenitors from murine ES cells followed by their intramuscular delivery in a murine muscular dystrophy model. PMID:27492174

  4. Determination, diversification and multipotency of mammalian myogenic cells.

    PubMed

    Cossu, G; De Angelis, L; Borello, U; Berarducci, B; Buffa, V; Sonnino, C; Coletta, M; Vivarelli, E; Bouche, M; Lattanzi, L; Tosoni, D; Di Donna, S; Berghella, L; Salvatori, G; Murphy, P; Cusella-De Angelis, M G; Molinaro, M

    2000-01-01

    In amniotes, myogenic commitment appears to be dependent upon signaling from neural tube and dorsal ectoderm, that can be replaced by members of the Wnt family and by Sonic hedgehog. Once committed, myoblasts undergo different fates, in that they can differentiate immediately to form the myotome, or later to give rise to primary and secondary muscle fibers. With fiber maturation, satellite cells are first detected; these cells contribute to fiber growth and regeneration during post-natal life. We will describe recent data, mainly from our laboratory, that suggest a different origin for some of the cells that are incorporated into the muscle fibers during late development. We propose the possibility that these myogenic cells are derived from the vasculature, are multi-potent and become committed to myogenesis by local signaling, when ingressing a differentiating muscle tissue. The implications for fetal and perinatal development of the whole mesoderm will also be discussed. PMID:11061434

  5. An essential role of phosphatidylinositol 3-kinase in myogenic differentiation

    PubMed Central

    Jiang, Bing-Hua; Zheng, Jenny Z.; Vogt, Peter K.

    1998-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis. PMID:9826674

  6. MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors.

    PubMed

    Masterson, Allan J; Sombroek, Claudia C; De Gruijl, Tanja D; Graus, Yvo M F; van der Vliet, Hans J J; Lougheed, Sinéad M; van den Eertwegh, Alfons J M; Pinedo, Herbert M; Scheper, Rik J

    2002-07-15

    Many human myeloid leukemia-derived cell lines possess the ability to acquire a dendritic cell (DC) phenotype. However, cytokine responsiveness is generally poor, requiring direct manipulation of intracellular signaling mechanisms for differentiation. In contrast, the CD34+ human acute myeloid leukemia cell line MUTZ-3 responds to granulocyte macrophage- colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNFalpha), cytokines known to be pivotal both in vivo and in vitro for DC generation from monocytes and CD34+ stem cells. In all respects, MUTZ-3 cells behave as the immortalized equivalent of CD34+ DC precursors. Upon stimulation with specific cytokine cocktails, they acquire a phenotype consistent with either interstitial- or Langerhans-like DCs and upon maturation (mDC), express CD83. MUTZ-3 DC display the full range of functional antigen processing and presentation pathways. These findings demonstrate the unique suitability of MUTZ-3 cells as an unlimited source of CD34+ DC progenitors for the study of cytokine-induced DC differentiation. PMID:12091369

  7. 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

  8. Hydroxyapatite surface roughness: complex modulation of the osteoclastogenesis of human precursor cells.

    PubMed

    Costa-Rodrigues, João; Fernandes, Anabela; Lopes, Maria A; Fernandes, Maria H

    2012-03-01

    It is recognized that the surface roughness affects osteoblastic differentiation, but little information is available regarding its effect on osteoclastogenesis. With this work, the osteoclastogenic behaviour of human peripheral blood mononuclear cells (PBMCs), cultured isolated (1.5×10(6)cellscm(-2)) or co-cultured with human bone marrow cells (hBMCs; 10(3)cellscm(-2)), was assessed on surface-abraded hydroxyapatite disks with three different surface roughnesses (R(a) 0.0437-0.582 μm). Monocultures and co-cultures were performed for 21 days in the absence or presence of recombinant M-CSF and RANKL. Results showed that PBMCs supplemented with M-CSF and RANKL or co-cultured with hBMCs displayed typical osteoclastic features, i.e. multinucleated cells with actin rings, vitronectin and calcitonin receptors, gene expression of TRAP, cathepsin K, carbonic anhydrase 2, c-myc and c-src, TRAP activity and resorbing activity. The osteoclastogenic response increased with surface roughness in PBMCs cultured with M-CSF and RANKL but decreased in PBMCs co-cultured with hBMCs. However, co-cultures supplemented with the osteoclastogenic inducers displayed high and similar levels of osteoclast differentiation in the three tested surfaces. In conclusion, modulation of osteoclast differentiation by surface roughness seemed to be dependent on the mechanisms subjacent to the osteoclastogenic stimulus, i.e. the presence of soluble factors or direct cell-to-cell contacts between osteoblastic and osteoclastic cells. PMID:22178652

  9. Occurring of In Vitro Functional Vasculogenic Pericytes from Human Circulating Early Endothelial Precursor Cell Culture

    PubMed Central

    Bianchi, Francesca; Galletti, Margherita; Galiè, Nazzareno; Ventura, Carlo

    2015-01-01

    Pericytes are periendothelial cells of the microcirculation which contribute to tissue homeostasis and hemostasis by regulating microvascular morphogenesis and stability. Because of their multipotential ex vivo differentiation capabilities, pericytes are becoming very interesting in regenerative medicine field. Several studies address this issue by attempting to isolate pericyte/mesenchymal-like cells from peripheral blood; however the origin of these cells and their culture conditions are still debated. Here we showed that early Endothelial Progenitor Cells (EPCs) expressing CD45+/CD146+/CD31+ can be a source of cells with pericyte/mesenchymal phenotype and function, identified as human Progenitor Perivascular Cells (hPPCs). We provided evidence that hPPCs have an immunophenotype consistent with Mesenchymal Stem Cells (MSCs) from human adipose tissue (hASCs) and fetal membranes of term placenta (FM-hMSCs). In addition, hPPCs can be subcultured and exhibit expression of pluripotent genes (OCT-4, KLF-4, and NANOG) as well as a remarkable vasculogenic potential. Our findings could be helpful to develop innovative cell-based therapies for future clinical applications with distinct therapeutic purposes. PMID:26064139

  10. 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

  11. Ocular vestibular evoked myogenic potentials in patients with acoustic neuroma.

    PubMed

    Piras, Gianluca; Brandolini, Cristina; Castellucci, Andrea; Modugno, Giovanni Carlo

    2013-02-01

    To assess the usefulness of vestibular testing in patients with acoustic neuroma, considering two main aspects: to compare diagnostic sensitivity of the current vestibular tests, especially considering ocular vestibular evoked myogenic potentials (OVEMPs) and to identify pre-operative localization of the tumor (inferior vestibular nerve vs. superior vestibular nerve) only with the help of vestibular electrophysiological data. Twenty-six patients with unilateral acoustic neuroma (mainly intracanalicular type) were studied with a full audio-vestibular test battery (pure tone and speech audiometry, caloric bithermal test, vibration-induced nystagmus test (VIN), cervical and OVEMPs). 18 patients (69 %) showed abnormal caloric responses. 12 patients (46.2 %) showed a pattern of VIN test suggestive of vestibular asymmetry. 16 patients (61.5 %) showed abnormal OVEMPs (12 only to AC, 4 both to AC and BC). 10 patients (38.5 %) showed abnormal cervical vestibular evoked myogenic potentials (5 both to AC and BC, 5 only to AC). In one case, results of vestibular evoked potentials and caloric test were confirmed by intra-operative and post-operative findings. Results of electrophysiological tests in AN patients could be helpful for planning the proper surgical approach, considering that sensitivity of every exam is quite low in intracanalicular lesion; clinical data allow a better interpretation of vestibular evoked myogenic potentials. PMID:22526579

  12. Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors

    SciTech Connect

    Zanocco-Marani, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio . E-mail: sergio@unimo.it

    2006-12-10

    The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

  13. 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

  14. 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.

  15. Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.

    PubMed

    Hübner, Juwita; Hoseini, Shahabuddin S; Suerth, Julia D; Hoffmann, Dirk; Maluski, Marcel; Herbst, Jessica; Maul, Holger; Ghosh, Arnab; Eiz-Vesper, Britta; Yuan, Qinggong; Ott, Michael; Heuser, Michael; Schambach, Axel; Sauer, Martin G

    2016-08-01

    Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer. PMID:27138041

  16. Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

    PubMed Central

    Askanas, V; McFerrin, J; Baqué, S; Alvarez, R B; Sarkozi, E; Engel, W K

    1996-01-01

    As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8577761

  17. Lipid supplemented medium induces lamellar bodies and precursors of barrier lipids in cultured analogues of human skin.

    PubMed

    Boyce, S T; Williams, M L

    1993-08-01

    Barrier function of cultured skin substitutes (CSS) is required for their effective use in clinical treatment of skin wounds, and for percutaneous absorption in vitro. Arachidonic, palmitic, oleic, and linoleic free fatty acids, in conjunction with the antioxidant alpha-tocopherol acetate (lipid supplements, "LS"), were added to nutrient media of CSS to provide precursors of epidermal barrier lipids. CSS were composed of human keratinocytes (HK), fibroblasts (HF), and collagen-glycosaminoglycan substrates, and were incubated for 14 d submerged or lifted to the air-liquid interface in media based on MCDB 153 +/- LS. Duplicate samples (30 cm2) were harvested and the epidermal analogue was analyzed for total protein, total DNA, total lipid, lipid fractions including acylglucosylceramide (AGC), and presence of lamellar bodies. Significant increases (p < 0.05) were detected between CSS incubated in +LS medium for total lipid, total DNA, ceramide, glucosylceramide, triglycerides, and diglycerides. AGC and lamellar bodies were detected only in epithelia of CSS incubated in +LS medium. These data show that free fatty acids, vitamin E, and lifting of CSS promote increased epithelial morphogenesis compared to CSS cultured submerged without lipid supplements. Presence of lamellar bodies and AGC suggests enhanced production in vitro of barrier-associated epidermal lipids. PMID:8345218

  18. Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum.

    PubMed Central

    Crise, B; Rose, J K

    1992-01-01

    The cell surface glycoprotein, CD4, is the receptor for human immunodeficiency virus (HIV) in T lymphocytes. Following HIV infection, there is reduced expression of CD4 on the cell surface, and this downregulation probably results, at least in part, from the formation of complexes containing the HIV type 1 (HIV-1) glycoprotein precursor (gp160) and CD4 that are not transported from the endoplasmic reticulum (ER). At the plasma membrane of T cells, CD4 is tightly associated with a cytoplasmic tyrosine kinase (p56lck) that is involved in T-cell activation. Using a transient expression system with HeLa cells, we show by pulse-labeling and immunoprecipitation that newly synthesized CD4 can associate with p56lck before CD4 is transported from the ER. In the presence of HIV-1 gp160, a ternary complex of gp160-CD4 and p56lck forms in the ER. Using confocal immunofluorescence microscopy, we observed complete retention of p56lck in the ER. Such mislocation of a tyrosine kinase to the cytoplasmic face of the ER could play a role in lymphocyte killing caused by HIV infection or expression of gp160 alone. Images PMID:1548763

  19. Structural Analysis using SHALiPE to Reveal RNA G-Quadruplex Formation in Human Precursor MicroRNA.

    PubMed

    Kwok, Chun Kit; Sahakyan, Aleksandr B; Balasubramanian, Shankar

    2016-07-25

    RNA G-quadruplex (rG4) structures are of fundamental importance to biology. A novel approach is introduced to detect and structurally map rG4s at single-nucleotide resolution in RNAs. The approach, denoted SHALiPE, couples selective 2'-hydroxyl acylation with lithium ion-based primer extension, and identifies characteristic structural fingerprints for rG4 mapping. We apply SHALiPE to interrogate the human precursor microRNA 149, and reveal the formation of an rG4 structure in this non-coding RNA. Additional analyses support the SHALiPE results and uncover that this rG4 has a parallel topology, is thermally stable, and is conserved in mammals. An in vitro Dicer assay shows that this rG4 inhibits Dicer processing, supporting the potential role of rG4 structures in microRNA maturation and post-transcriptional regulation of mRNAs. PMID:27355429

  20. Assembly-defective point mutants of the human immunodeficiency virus type 1 Gag precursor phenotypically expressed in recombinant baculovirus-infected cells.

    PubMed Central

    Hong, S S; Boulanger, P

    1993-01-01

    Two substitution mutants of the human immunodeficiency virus type 1 gag gene product were isolated after nitrous acid mutagenesis of a recombinant baculovirus expressing a non-N-myristylated, p6-deleted Gag precursor (Pr49). Both mutants failed to assemble intracellular Gag virus-like particles, as does the parental recombinant, and therefore expressed a self-assembly defective (Sad) phenotype in insect cells. The mutations consisted of nonconservative changes involving highly conserved hydrophobic residues in the p24 domain, Leu to Pro at position 268 (L268P) and Leu to Ser at amino acid 322 (L322S). Experimental data suggested that the two mutated residues belonged to functionally different regions of the Gag precursor. (i) A partial complementation effect between the two mutants for Gag precursor assembly was observed in coinfection experiments. (ii) The two mutations showed different phenotypes when placed in the N-myristylated context, of which only the L268P mutation abolished extracellular budding and release of Gag particles at the plasma membrane. Both L268P and L322S mutants had a trans-dominant negative effect on the intracellular assembly of a non-N-myristylated, full-length (Pr55) Gag precursor expressed by a coinfecting recombinant. None of the mutants, however, showed any detectable effect in trans on membrane targeting and budding of the coexpressed N-myristylated wild-type Gag precursor. Images PMID:8474175

  1. Effect of recombinant porcine IGFBP-3 on IGF-I and long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells.

    PubMed

    Xi, G; Kamanga-Sollo, E; Pampusch, M S; White, M E; Hathaway, M R; Dayton, William R

    2004-09-01

    Insulin-like growth factor (IGF)-I stimulates both proliferation and differentiation of myogenic precursor cells. In vivo, IGFs are bound to one of the members of a family of six high-affinity IGF binding proteins (IGFBP 1-6) that regulate their biological activity. One of these binding proteins, IGFBP-3, affects cell proliferation via both IGF-dependent and IGF-independent mechanisms and it has generally been shown to suppress proliferation of cultured cells; however, it also may stimulate proliferation depending upon the cell type and the assay conditions. Cultured porcine embryonic myogenic cells (PEMCs) produce IGFBP-3 and its level drops significantly immediately prior to differentiation. Additionally, IGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of embryonic porcine myogenic cells. In this study, we have examined the effects of recombinant porcine IGFBP-3 (rpIGFBP-3) on IGF-I- and Long-R3-IGF-I-stimulated proliferation and differentiation of the L6 myogenic cell line. L6 cells potentially provide a good model for studying the actions of IGFBP-3 on muscle because they contain no non-muscle cells and they do not produce detectable levels of IGFBP-3. RpIGFBP-3 suppresses both IGF-I and Long-R3-IGF-I-stimulated proliferation of L6 cells, indicating that it suppresses proliferation via both IGF-dependent and IGF-independent mechanisms. Our data also show that rpIGFBP-3 causes IGF-independent suppression of proliferation without increasing the level of phosphosmad-2 in L6 cultures. Additionally, rpIGFBP-3 suppresses IGF-I-stimulated differentiation of L6 cells. In contrast, however, rpIGFBP-3 does not suppress Long-R3-IGF-I-stimulated differentiation. This suggests that rpIGFBP-3 does not have IGF-independent effects on L6 cell differentiation. PMID:15254966

  2. 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

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

    PubMed

    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

  4. 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

  5. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.

    PubMed

    Balasubramanian, Anuradha; Kawahara, Genri; Gupta, Vandana A; Rozkalne, Anete; Beauvais, Ariane; Kunkel, Louis M; Gussoni, Emanuela

    2014-07-01

    Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in differentiating human muscle cells and regenerating muscle tissue. In vitro, human muscle cells were treated with histone-deacetylase (HDAC) inhibitors, and expression of Fam65b and interacting proteins was studied. Nontreated cells were used as controls. In vivo, expression of Fam65b was down-regulated in developing zebrafish to determine the effects on muscle development. Fam65b binds to HDAC6 and dysferlin, the protein mutated in limb girdle muscular dystrophy 2B. The tricomplex Fam65b-HDAC6-dysferlin is transient, and Fam65b expression is necessary for the complex to form. Treatment of myogenic cells with pan-HDAC or HDAC6-specific inhibitors alters Fam65b expression, while dysferlin expression does not change. Inhibition of Fam65b expression in developing zebrafish results in abnormal muscle, with low birefringence, tears at the myosepta, and increased embryo lethality. Fam65b is an essential component of the HDAC6-dysferlin complex. Down-regulation of Fam65b in developing muscle causes changes consistent with muscle disease.-Balasubramanian, A., Kawahara, G., Gupta, V. A., Rozkalne, A., Beauvais, A., Kunkel, L. M., Gussoni, E. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation. PMID:24687993

  6. 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. PMID:26791591

  7. 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

    2014-01-01

    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. PMID:24903713

  8. Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (βAPP).

    PubMed

    Jęśko, Henryk; Wilkaniec, Anna; Cieślik, Magdalena; Hilgier, Wojciech; Gąssowska, Magdalena; Lukiw, Walter J; Adamczyk, Agata

    2016-01-01

    Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer's disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing humanprecursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD. PMID:26971935

  9. Human papillomavirus infection is rare in nonmalignant tonsil tissue in the UK: implications for tonsil cancer precursor lesions.

    PubMed

    Palmer, Elizabeth; Newcombe, Robert G; Green, Adele C; Kelly, Carole; Noel Gill, O; Hall, Gillian; Fiander, Alison N; Pirotte, Evelyne; Hibbitts, Sam J; Homer, Jarrod; Powell, Ned G

    2014-11-15

    The incidence of human papillomavirus (HPV)-associated tonsil cancer is increasing but the prevalence of HPV, and of premalignant precursors, in tonsil tissue is unknown. We aimed to assess prevalence of HPV infection in nonmalignant tonsillar crypt epithelia and to histopathologically characterise positive samples. Formalin-fixed paraffin-embedded (FFPE) tonsil tissue specimens were obtained from an age- and sex-stratified random sample of patients aged 0-69 years whose paired tonsils were archived following elective tonsillectomy at hospitals throughout England and Southern Scotland from 2004 to 2008. Homogenised fresh-frozen tonsil tissue was also obtained from archive for two random subsets of males aged 25-34 and over 44. HPV status was assessed in all samples for 20 mucosal HPV types by GP5+/6+ polymerase chain reaction (PCR) enzyme immunoassay and by HPV16 type-specific PCR targeting the E6 gene. In the homogenised material, HPV status was also assessed for 44 HPV types by SPF10-PCR enzyme immunoassay. Of 4,095 randomly sampled FFPE specimens, amplifiable DNA was extracted from 3,377 (82.5%) and from 511 of 524 (97.5%) homogenised tonsils. HPV DNA was identified in 0 of 3,377 (0%, 95% CI 0-0.089%) fixed samples and 0 of 511 (0%, 95% CI 0-0.58%) homogenised samples. This suggests HPV infection may be rare in tonsil reticulated crypt epithelia. Furthermore, we found no evidence of HPV-associated premalignant neoplasia. These data suggest that if HPV-associated premalignant lesions do occur, they are likely to be rare and may have a high risk of progression to carcinoma. PMID:24723209

  10. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors.

    PubMed

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels H H; Borregaard, Niels

    2013-10-01

    Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes. PMID:23650620

  11. Cerebral Myogenic Reactivity and Blood Flow in Type 2 Diabetic Rats: Role of Peroxynitrite in Hypoxia-Mediated Loss of Myogenic Tone

    PubMed Central

    Kelly-Cobbs, Aisha I.; Prakash, Roshini; Coucha, Maha; Knight, Robert A.; Li, Weiguo; Ogbi, Safia N.; Johnson, Maribeth

    2012-01-01

    Dysregulation of cerebral vascular function and, ultimately, cerebral blood flow (CBF) may contribute to complications such as stroke and cognitive decline in diabetes. We hypothesized that 1) diabetes-mediated neurovascular and myogenic dysfunction impairs CBF and 2) under hypoxic conditions, cerebral vessels from diabetic rats lose myogenic properties because of peroxynitrite (ONOO−)-mediated nitration of vascular smooth muscle (VSM) actin. Functional hyperemia, the ability of blood vessels to dilate upon neuronal stimulation, and myogenic tone of isolated middle cerebral arteries (MCAs) were assessed as indices of neurovascular and myogenic function, respectively, in 10- to 12-week control and type 2 diabetic Goto-Kakizaki rats. In addition, myogenic behavior of MCAs, nitrotyrosine (NY) levels, and VSM actin content were measured under normoxic and hypoxic [oxygen glucose deprivation (OGD)] conditions with and without the ONOO− decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl) prophyrinato iron (III), chloride (FeTPPs). The percentage of myogenic tone was higher in diabetes, and forced dilation occurred at higher pressures. Functional hyperemia was impaired. Consistent with these findings, baseline CBF was lower in diabetes. OGD reduced the percentage of myogenic tone in both groups, and FeTPPs restored it only in diabetes. OGD increased VSM NY in both groups, and although FeTPPs restored basal levels, it did not correct the reduced filamentous/globular (F/G) actin ratio. Acute alterations in VSM ONOO− levels may contribute to hypoxic myogenic dysfunction, but this cannot be solely explained by the decreased F/G actin ratio due to actin nitration, and mechanisms may differ between control and diabetic animals. Our findings also demonstrate that diabetes alters the ability of cerebral vessels to regulate CBF under basal and hypoxic conditions. PMID:22570365

  12. Skeletal Myogenic Progenitors Originating from Embryonic Dorsal Aorta Coexpress Endothelial and Myogenic Markers and Contribute to Postnatal Muscle Growth and Regeneration

    PubMed Central

    De Angelis, Luciana; Berghella, Libera; Coletta, Marcello; Lattanzi, Laura; Zanchi, Malvina; Gabriella, M.; Ponzetto, Carola; Cossu, Giulio

    1999-01-01

    Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met−/− embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells. The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system. PMID:10562287

  13. An experimental method to identify neurogenic and myogenic active mechanical states of intestinal motility

    PubMed Central

    Costa, Marcello; Wiklendt, Lukasz; Arkwright, John W.; Spencer, Nicholas J.; Omari, Taher; Brookes, Simon J. H.; Dinning, Phil G.

    2013-01-01

    Excitatory and inhibitory enteric neural input to intestinal muscle acting on ongoing myogenic activity determines the rich repertoire of motor patterns involved in digestive function. The enteric neural activity cannot yet be established during movement of intact intestine in vivo or in vitro. We propose the hypothesis that is possible to deduce indirectly, but reliably, the state of activation of the enteric neural input to the muscle from measurements of the mechanical state of the intestinal muscle. The fundamental biomechanical model on which our hypothesis is based is the “three-element model” proposed by Hill. Our strategy is based on simultaneous video recording of changes in diameters and intraluminal pressure with a fiber-optic manometry in isolated segments of rabbit colon. We created a composite spatiotemporal map (DPMap) from diameter (DMap) and pressure changes (PMaps). In this composite map rhythmic myogenic motor patterns can readily be distinguished from the distension induced neural peristaltic contractions. Plotting the diameter changes against corresponding pressure changes at each location of the segment, generates “orbits” that represent the state of the muscle according to its ability to contract or relax actively or undergoing passive changes. With a software developed in MatLab, we identified twelve possible discrete mechanical states and plotted them showing where the intestine actively contracted and relaxed isometrically, auxotonically or isotonically, as well as where passive changes occurred or was quiescent. Clustering all discrete active contractions and relaxations states generated for the first time a spatio-temporal map of where enteric excitatory and inhibitory neural input to the muscle occurs during physiological movements. Recording internal diameter by an impedance probe proved equivalent to measuring external diameter, making possible to further develop similar strategy in vivo and humans. PMID:23596400

  14. Guidance of myogenic cell migration by oriented deposits of fibronectin.

    PubMed

    Turner, D C; Lawton, J; Dollenmeier, P; Ehrismann, R; Chiquet, M

    1983-02-01

    Fibronectin mediates myoblast-substratum attachment; one region of the molecule binds directly to the cell surface, while others bind to collagen, glycosaminoglycans, and other fibronectin molecules. There is evidence to suggest that fibronectin-containing extracellular matrices guide cell migration in vivo. We describe a method for producing regular deposits of fibronectin in vitro that can serve as a model system for studying cell-substrate interactions, cell orientation, and contact guidance. The novel culture substrate is prepared by allowing an aqueous solution of fibronectin and urea to dry in a culture dish and then washing away the urea crystals. Myogenic cells in vitro adhere to, align with, and migrate along, parallel streaks of fibronectin. This leads to the formation of myotubes that are long and thin, with little branching. Myogenic clones are highly elongated in the direction of the deposits, in contrast with the roughly circular clones seen in conventional cultures. Fibroblasts and limb bud mesenchymal cells align with fibronectin deposits, assuming a bipolar shape. PMID:6825944

  15. Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

    PubMed Central

    y Schnitzler, Michael Mederos; Storch, Ursula; Meibers, Simone; Nurwakagari, Pascal; Breit, Andreas; Essin, Kirill; Gollasch, Maik; Gudermann, Thomas

    2008-01-01

    Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist-independent activation of Gq/11-coupled receptors, which subsequently signal to TRPC channels in a G protein- and phospholipase C-dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of β-arrestin. Agonist-independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT1 angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT1 receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G-protein signalling-2. These findings suggest that Gq/11-coupled receptors function as sensors of membrane stretch in VSM cells. PMID:18987636

  16. Vestibular evoked myogenic potentials (VEMPs) in central neurological disorders.

    PubMed

    Venhovens, J; Meulstee, J; Verhagen, W I M

    2016-01-01

    Several types of acoustic stimulation (i.e. tone bursts or clicks), bone-conducted vibration, forehead taps, and galvanic stimulation elicit myogenic potentials. These can be recorded in cervical and ocular muscles, the so called vestibular evoked myogenic potentials (VEMPs). The cervical VEMP (cVEMP) resembles the vestibulo-collic reflex and the responses can be recorded from the ipsilateral sternocleidomastoid muscle. The ocular VEMP resembles the vestibulo-ocular reflex and can be recorded from extra-ocular muscles by a surface electrode beneath the contralateral infraorbital margin. Initially, the literature concerning VEMPs was limited to peripheral vestibular disorders, however, the field of VEMP testing is rapidly expanding, with an increasing focus on central neurological disorders. The current literature concerning VEMP abnormalities in central neurological disorders is critically reviewed, especially regarding the methodological aspects in relation to quality as well as the clinical interpretation of the VEMP results. Suggestions for further research are proposed as well as some clinically useful indications. PMID:25649969

  17. 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

    2016-03-01

    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. PMID:26923587

  18. 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.

  19. Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells

    PubMed Central

    2010-01-01

    Introduction This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation. Methods Human MPCs were maintained in monolayer, pellet or micromass cultures (MMC) for up to 10 days with PPS at concentrations of 0 to 20 μg/ml. MPC viability and proliferation was assessed using the WST-1 assay and 3H-thymidine incorporation into DNA, while apoptosis was monitored by flow cytometry. Proteoglycan (PG) biosynthesis was determined by 35SO42- incorporation and staining with Alcian blue. Proteoglycan and collagen type I and collagen type II deposition in pellet cultures was also examined by Toluidine blue and immunohistochemical staining, respectively. The production of hyaluronan (HA) by MPCs in MMC was assessed by ELISA. The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC. Gene expression of MPCs in 7-day and 10-day MMC was examined using real-time PCR. MPC differentiation was investigated by co-culturing with PPS in osteogenic or adipogenic inductive culture media for 28 days. Results Significant MPC proliferation was evident by day 3 at PPS concentrations of 1 to 5 μg/ml (P < 0.01). In the presence of 1 to 10 μg/ml PPS, a 38% reduction in IL-4/IFNγ-induced MPC apoptosis was observed. In 5-day MMC, 130% stimulation of PG synthesis occurred at 2.5 μg/ml PPS (P < 0.0001), while 5.0 μg/ml PPS achieved maximal stimulation in the 7-day and 10-day cultures (P < 0.05). HA and DS at ≥ 5 μg/ml inhibited PG synthesis (P < 0.05) in 5-day MMC. Collagen type II deposition by MMC was significant at ≥ 0.5 μg/ml PPS (P < 0.001 to 0.05). In MPC-PPS pellet cultures, more PG, collagen type II but less collagen type I was deposited than in controls. Real-time PCR results were consistent with the protein data. At 5 and 10 μg/ml PPS, MPC osteogenic differentiation was suppressed (P < 0.01). Conclusions This is

  20. Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line

    SciTech Connect

    Bryant, M.L.; Ratner, L.; Duronio, R.J.; Gordon, J.I. ); Kishore, N.S. ); Devadas, B.; Adams, S.P. )

    1991-03-15

    Covalent linkage of myristate (tetradecanoate; 14:0) to the NH{sub 2}-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55{sup gag}) is necessary for its proteolytic processing and viral assembly. The authors have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase despite their reduced hydrophobicity. To examine the mechanism of their antiviral effects, they performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. ({sup 3}H)Myristate and ({sup 3}H)13-OxaMyr were incorporated into Pr55{sup gag} with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55{sup gag}.

  1. Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

    PubMed

    Maslova, E V; Andreeva, E R; Andrianova, I V; Bobyleva, P I; Romanov, Yu A; Kabaeva, N V; Balashova, E E; Ryaskina, S S; Dugina, T N; Buravkova, L B

    2014-02-01

    We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine. PMID:24771453

  2. MiR-124 inhibits myogenic differentiation of mesenchymal stem cells via targeting Dlx5.

    PubMed

    Qadir, Abdul S; Woo, Kyung Mi; Ryoo, Hyun-Mo; Yi, TacGhee; Song, Sun U; Baek, Jeong-Hwa

    2014-09-01

    MicroRNAs (miRNAs), including miR-1, miR-133, and miR-206, play a crucial role in muscle development by regulating muscle cell proliferation and differentiation. The aim of the present study was to define the effect of miR-124 on myogenic differentiation of mesenchymal stem cells (MSCs). The expression level of miR-124 in skeletal muscles was much lower than those in primary cultured bone marrow-derived MSCs and the bone, fat and brain tissues obtained from C57BL/6 mice. Myogenic stimuli significantly decreased the expression levels of miR-124 in mouse bone marrow-derived MSCs and C2C12 cells. Forced expression of miR-124 suppressed the expression of myogenic marker genes such as Myf5, Myod1, myogenin and myosin heavy chain and multinucleated myotube formation. Blockade of endogenous miR-124 with a hairpin inhibitor enhanced myogenic marker gene expression and myotube formation. During myogenic differentiation of MSCs and C2C12 cells, the levels of Dlx5, a known target of miR-124, were inversely regulated with those of miR-124. Furthermore, overexpression of Dlx5 increased myogenic differentiation, whereas knockdown of Dlx5 using siRNA inhibited myogenesis in C2C12 cells. These results suggest that miR-124 is a negative regulator of myogenic differentiation of MSCs and that upregulation of Dlx5 accompanied with downregulation of miR-124 by myogenic stimuli is necessary for the proper progression of myogenic differentiation. PMID:24733577

  3. 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

  4. Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms.

    PubMed Central

    Brown, N F; Sen, A; Soltis, D A; Jones, B; Foster, D W; McGarry, J D

    1993-01-01

    cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5' end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3' to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8363589

  5. Vestibular Evoked Myogenic Potentials in a Female Population with Migraine.

    PubMed

    Yetiser, Sertac; Gok, Meltem Hale; Kutukcu, Yasar; Ince, Dilay

    2016-06-01

    The objective is to analyze the vestibular system by vestibular evoked myogenic potential (VEMP) in 30 female patients with migraine and balance problem in a controlled study. Thirty female patients with migraine and vestibular problems were enrolled in the study (2009-2012). Fifteen age-matched healthy subjects were selected as the controls. Air conduction cervical VEMP was used. Tone-burst sound stimuli of 95 dB nHL with rarefaction polarity, 5 Hz stimulus repetition rate, 1 ms rise/fall time and 2 ms plateau time were delivered at 500 Hz. 200 sweeps were averaged. Myogenic responses were amplified and band-pass filtered (800-10 Hz). The latency and the amplitude of p1 and n1 waves and interpeak amplitude and latency differences were measured. Results were given as mean and SDs. Interaural p1 and n1 amplitude greater than 30 % asymmetry was accepted as abnormal. VEMP results were compared with controls. The One-way ANOVA test was used. Statistical significance was set at P < 0.05. VEMP responses were elicited in all controls and the patients. Comparative analysis of p1 amplitude between the patients and the controls was statistically significant (P = 0.010). P1n1 interaural amplitude difference was greater than 30 % in 4 patients (13.4 %). No statistically significant difference was found when comparing latency of all wave forms between the patients and healthy controls (P > 0.05). VEMP is an useful tool to test the vestibular system in patients with migraine and balance problem at the very early period. Clinicians should always consider migraine in patients with vertigo. PMID:27340638

  6. Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM

    PubMed Central

    Wentzensen, Nicolas; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren; Lorey, Thomas S.; Sahasrabuddhe, Vikrant V.; Lamere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Castle, Philip E.

    2014-01-01

    Objective Anal cancer incidence is high in HIV-infected MSM. Screening for anal intraepithelial lesions and cancers is performed at specialized clinics and relies on high-resolution anoscopy (HRA) and anal cytology. Both approaches have limited reproducibility and sensitivity for detecting anal cancer precursors. We evaluated biomarkers for human papillomavirus (HPV)-related disease in a population of HIV-infected MSM. Methods A cross-sectional screening study with passive follow-up included 363 MSM followed at a HIV/AIDS clinic. All men had anal cytology samples taken and were evaluated using HRA and anal biopsies. Using a composite endpoint of biopsy results and cytology, we compared the performance of HPV16/18 genotyping, HPVE6/E7 mRNA expression, and p16/Ki-67 cytology to detect high-grade anal intraepithelial neoplasias (AINs). Results For all biomarkers analyzed, there was a significant trend of increasing percentage of men testing positive with increasing severity of disease (P< 0.001). HPV DNA testing had the highest sensitivity for anal intraepithelial neoplasia grade 2 and anal intraepithelial neoplasia grade 3 (AIN3), followed by p16/Ki-67, HPVE6/E7 mRNA testing, and HPV16/18 genotyping. The highest Youden's index was observed for HPVE6/E7 mRNA testing, followed by HPV16/18 genotyping, p16/Ki-67 cytology, and HPV DNA testing. Increasing the threshold for positivity of p16/Ki-67 to five or more positive cells led to significantly higher specificity, but unchanged sensitivity for detecting AIN3. Conclusion Molecular features of anal disease categories are similar to those of corresponding cervical lesions. Biomarkers evaluated for cervical cancer screening may be used for primary anal cancer screening or to decide who should require immediate treatment vs. expectant management. PMID:23018436

  7. Folic acid promotes the myogenic differentiation of C2C12 murine myoblasts through the Akt signaling pathway.

    PubMed

    Hwang, Seong Yeon; Kang, Yong Jung; Sung, Bokyung; Kim, Minjung; Kim, Dong Hwan; Lee, Yujin; Yoo, Mi-Ae; Kim, Cheol Min; Chung, Hae Young; Kim, Nam Deuk

    2015-10-01

    Folic acid is a water-soluble vitamin in the B-complex group, and an exogenous intake is required for health, growth and development. As a precursor to co-factors, folic acid is required for one-carbon donors in the synthesis of DNA bases and other essential biomolecules. A lack of dietary folic acid can lead to folic acid deficiency and can therefore result in several health problems, including macrocytic anemia, elevated plasma homocysteine levels, cardiovascular disease, birth defects, carcinogenesis, muscle weakness and difficulty in walking. Previous studies have indicated that folic acid exerts a positive effect on skeletal muscle functions. However, the precise role of folic acid in skeletal muscle cell differentiation remains poorly understood. Thus, in the present study, we examined the effects of folic acid on neo-myotube maturation and differentiation using C2C12 murine myoblasts. We found that folic acid promoted the formation of multinucleated myotubes, and increased the fusion index and creatine kinase (CK) activity in a concentration-dependent manner. In addition, western blot analysis revealed that the expression levels of the muscle-specific marker, myosin heavy chain (MyHC), as well as those of the myogenic regulatory factors (MRFs), MyoD and myogenin, were increased in the folic acid-treated myotubes during myogenic differentiation. Folic acid also promoted the activation of the Akt pathway, and this effect was inhibited by treatment of the C2C12 cells with LY294002 (Akt inhibitor). Blocking of the Akt pathway with a specific inhibitor revealed that it was necessary for mediating the stimulatory effects of folic acid on muscle cell differentiation and fusion. Taken together, our data suggest that folic acid promotes the differentiation of C2C12 cells through the activation of the Akt pathway. PMID:26310574

  8. Potential Role of Omega-3 Fatty Acids on the Myogenic Program of Satellite Cells

    PubMed Central

    Bhullar, Amritpal S.; Putman, Charles T.; Mazurak, Vera C.

    2016-01-01

    Skeletal muscle loss is associated with aging as well as pathological conditions. Satellite cells (SCs) play an important role in muscle regeneration. Omega-3 fatty acids are widely studied in a variety of muscle wasting diseases; however, little is known about their impact on skeletal muscle regeneration. The aim of this review is to evaluate studies examining the effect of omega-3 fatty acids, α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid on the regulation of SC proliferation and differentiation. This review highlights mechanisms by which omega-3 fatty acids may modulate the myogenic program of the stem cell population within skeletal muscles and identifies considerations for future studies. It is proposed that minimally three myogenic transcriptional regulatory factors, paired box 7 (Pax7), myogenic differentiation 1 protein, and myogenin, should be measured to confirm the stage of SCs within the myogenic program affected by omega-3 fatty acids. PMID:26884682

  9. Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation

    PubMed Central

    McKinnon, Timothy; Venier, Rosemarie; Dickson, Brendan C.; Kabaroff, Leah; Alkema, Manon; Chen, Li; Shern, Jack F.; Yohe, Marielle E.; Khan, Javed; Gladdy, Rebecca A.

    2015-01-01

    While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53−/− myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53−/− myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum. PMID:25992772

  10. 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.