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Sample records for mature mdx mice

  1. Upregulation of the creatine synthetic pathway in skeletal muscles of mature mdx mice

    PubMed Central

    McClure, Warrren C.; Rabon, Rick; Ogawa, Hirofumi; Tseng, Brian S.

    2009-01-01

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular human disease caused by dystrophin deficiency. The mdx mouse lacks dystrophin protein, yet does not exhibit the debilitating DMD phenotype. Investigating compensatory mechanisms in the mdx mouse is important. This study targets two metabolic genes, guanidinoacetate methyltransferase (GAMT) and arginine:glycine amidinotransferase (AGAT) which are required for creatine synthesis. We show that GAMT and AGAT mRNA are up-regulated 5.4 and 1.9-fold respectively in adult mdx muscle compared to C57. In addition, GAMT protein expression is up-regulated at least 2.5-fold in five different muscles of mdx vs. control. Furthermore, we find GAMT immunoreactivity in 80% of mature mdx muscle fibers in addition to small regenerating fibers and rare revertants; while GAMT immunoreactivity is equal to background levels in all muscle fibers of mature C57 mice. The up-regulation of the creatine synthetic pathway may help maintain muscle creatine levels and limit cellular energy failure in leaky mdx skeletal muscles. These results may help better understand the mild phenotype of the mdx mouse and may offer new treatment horizons for DMD. PMID:17588756

  2. Upregulation of the creatine synthetic pathway in skeletal muscles of mature mdx mice.

    PubMed

    McClure, Warren C; Rabon, Rick E; Ogawa, Hirofumi; Tseng, Brian S

    2007-08-01

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular human disease caused by dystrophin deficiency. The mdx mouse lacks dystrophin protein, yet does not exhibit the debilitating DMD phenotype. Investigating compensatory mechanisms in the mdx mouse may shed new insights into modifying DMD pathogenesis. This study targets two metabolic genes, guanidinoacetate methyltransferase (GAMT) and arginine:glycine amidinotransferase (AGAT) which are required for creatine synthesis. We show that GAMT and AGAT mRNA are up-regulated 5.4- and 1.9-fold respectively in adult mdx muscle compared to C57. In addition, GAMT protein expression is up-regulated at least 2.5-fold in five different muscles of mdx vs. control. Furthermore, we find GAMT immunoreactivity in up to 80% of mature mdx muscle fibers in addition to small regenerating fibers and rare revertants; while GAMT immunoreactivity is equal to background levels in all muscle fibers of mature C57 mice. The up-regulation of the creatine synthetic pathway may help maintain muscle creatine levels and limit cellular energy failure in leaky mdx skeletal muscles. These results may help better understand the mild phenotype of the mdx mouse and may offer new treatment horizons for DMD.

  3. Sequencing protocols to genotype mdx, mdx(4cv), and mdx(5cv) mice.

    PubMed

    Banks, Glen B; Combs, Ariana C; Chamberlain, Jeffrey S

    2010-08-01

    Currently available polymerase chain reaction (PCR) genotyping methods for point mutations in the mouse dystrophin gene can lead to false positives and result in wasted time and money due to breeding or treating the wrong mice. Here we describe a simple and accurate method for sequencing the point mutations in mdx, mdx(4cv), and mdx(5cv) mice. This method clearly distinguishes between wildtype, heterozygous, and mutant transcripts, and thereby time and money can be saved by avoiding false positives.

  4. Pharmacological activation of PPARbeta/delta stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice.

    PubMed

    Miura, Pedro; Chakkalakal, Joe V; Boudreault, Louise; Bélanger, Guy; Hébert, Richard L; Renaud, Jean-Marc; Jasmin, Bernard J

    2009-12-01

    A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.

  5. mdx(⁵cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice.

    PubMed

    Beastrom, Nicholas; Lu, Haiyan; Macke, Allison; Canan, Benjamin D; Johnson, Eric K; Penton, Christopher M; Kaspar, Brian K; Rodino-Klapac, Louise R; Zhou, Lan; Janssen, Paul M L; Montanaro, Federica

    2011-11-01

    Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle dysfunction leading to premature death by the third decade of life. The mdx mouse, the most widely used animal model of DMD, has been extremely useful to study disease mechanisms and to screen new therapeutics. However, unlike patients with DMD, mdx mice have a very mild motor function deficit, posing significant limitations for its use as a platform to assess the impact of treatments on motor function. It has been suggested that an mdx variant, the mdx(5cv) mouse, might be more severely affected. Here, we compared the motor activity, histopathology, and individual muscle force measurements of mdx and mdx(⁵cv) mice. Our study revealed that mdx(⁵cv) mice showed more severe exercise-induced fatigue, Rotarod performance deficits, and gait anomalies than mdx mice and that these deficits began at a younger age. Muscle force studies showed more severe strength deficits in the diaphragm of mdx(⁵cv) mice compared to mdx mice, but similar force generation in the extensor digitorum longus. Muscle histology was similar between the two strains. Differences in genetic background (genetic modifiers) probably account for these functional differences between mdx strains. Overall, our findings indicate that the mdx and mdx(⁵cv) mouse models of DMD are not interchangeable and identify the mdx(⁵cv) mouse as a valuable platform for preclinical studies that require assessment of muscle function in live animals.

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

    PubMed Central

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

    2014-01-01

    Background Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice. Results Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype. Conclusion We identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen. PMID:24626262

  7. Disease course in mdx:utrophin+/- mice: comparison of three mouse models of Duchenne muscular dystrophy.

    PubMed

    McDonald, Abby A; Hebert, Sadie L; Kunz, Matthew D; Ralles, Steven J; McLoon, Linda K

    2015-04-01

    The mdx mouse model of Duchenne muscular dystrophy (DMD) is used to study disease mechanisms and potential treatments, but its pathology is less severe than DMD patients. Other mouse models were developed to more closely mimic the human disease based on knowledge that upregulation of utrophin has a protective effect in mdx muscle. An mdx:utrophin(-/-) (dko) mouse was created, which had a severe disease phenotype and a shortened life span. An mdx:utrophin(+/-) mouse was also created, which had an intermediate disease phenotype compared to the mdx and dko mice. To determine the usefulness of mdx:utrophin(+/-) mice for long-term DMD studies, limb muscle pathology and function were assessed across the life span of wild-type, mdx, mdx:utrophin(+/-), and dko mice. Muscle function assessment, specifically grip duration and rotarod performance, demonstrated that mdx:utrophin(+/-) mice were weaker for a longer time than mdx mice. Mean myofiber area was smaller in mdx:utrophin(+/-) mice compared to mdx mice at 12 months. Mdx:utrophin(+/-) mice had a higher percentage of centrally nucleated myofibers compared to mdx mice at 6 and 12 months. Collagen I and IV density was significantly higher in mdx:utrophin(+/-) muscle compared to mdx at most ages examined. Generally, mdx:utrophin(+/-) mice showed an intermediate disease phenotype over a longer time course compared to the mdx and dko mice. While they do not genetically mirror human DMD, mdx:utrophin(+/-) mice may be a more useful animal model than mdx or dko mice for investigating long-term efficacy of potential treatments when fibrosis or muscle function is the focus.

  8. Intracellular calcium handling in ventricular myocytes from mdx mice.

    PubMed

    Williams, Iwan A; Allen, David G

    2007-02-01

    Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca(2+) into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca(2+) measurements using a fluorescent Ca(2+)-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca(2+) concentration ([Ca(2+)](i)). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca(2+) transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca(2+) transients showed that the Na(+)/Ca(2+) exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca(2+)](i) in old mdx mice, suggesting that SACs may be involved in the Ca(2+)-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca(2+) handling in young mdx mice.

  9. Bowman-Birk inhibitor attenuates dystrophic pathology in mdx mice.

    PubMed

    Morris, C A; Selsby, J T; Morris, L D; Pendrak, K; Sweeney, H L

    2010-11-01

    Bowman-Birk inhibitor concentrate (BBIC), a serine protease inhibitor, has been shown to diminish disuse atrophy of skeletal muscle. Duchenne muscular dystrophy (DMD) results from a loss of dystrophin protein and involves an ongoing inflammatory response, with matrix remodeling and activation of transforming growth factor (TGF)-β(1) leading to tissue fibrosis. Inflammatory-mediated increases in extracellular protease activity may drive much of this pathological tissue remodeling. Hence, we evaluated the ability of BBIC, an extracellular serine protease inhibitor, to impact pathology in the mouse model of DMD (mdx mouse). Mdx mice fed 1% BBIC in their diet had increased skeletal muscle mass and tetanic force and improved muscle integrity (less Evans blue dye uptake). Importantly, mdx mice treated with BBIC were less susceptible to contraction-induced injury. Changes consistent with decreased degeneration/regeneration, as well as reduced TGF-β(1) and fibrosis, were observed in the BBIC-treated mdx mice. While Akt signaling was unchanged, myostatin activitation and Smad signaling were reduced. Given that BBIC treatment increases mass and strength, while decreasing fibrosis in skeletal muscles of the mdx mouse, it should be evaluated as a possible therapeutic to slow the progression of disease in human DMD patients.

  10. N-Acetylcysteine ameliorates skeletal muscle pathophysiology in mdx mice

    PubMed Central

    Whitehead, Nicholas P; Pham, Catherine; Gervasio, Othon L; Allen, David G

    2008-01-01

    Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by a mutation in the gene encoding dystrophin, a protein linking the cytoskeleton to the extracellular matrix. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) provided protection against dystrophic muscle damage in the mdx mouse, an animal model of DMD. In isolated mdx muscles, NAC prevented the increased membrane permeability and reduced the force deficit associated with stretch-induced muscle damage. Three-week-old mdx mice were treated with NAC in the drinking water for 6 weeks. Dihydroethidium staining showed that NAC treatment reduced the concentration of reactive oxygen species (ROS) in mdx muscles. This was accompanied by a significant decrease in centrally nucleated fibres in muscles from NAC-treated mdx mice. Immunoblotting showed that NAC treatment decreased the nuclear protein expression of NF-κB, a transcription factor involved in pro-inflammatory cytokine expression. Finally, we show that NAC treatment reduced caveolin-3 protein levels and increased the sarcolemmal expression of β-dystroglycan and the dystrophin homologue, utrophin. Taken together, our findings suggest that ROS play an important role in the dystrophic pathogenesis, both in terms of activating damage pathways and in regulating the expression of some dystrophin-associated membrane proteins. These results offer the prospect that antioxidants such as NAC could have therapeutic potential for DMD patients. PMID:18258657

  11. N-Acetylcysteine ameliorates skeletal muscle pathophysiology in mdx mice.

    PubMed

    Whitehead, Nicholas P; Pham, Catherine; Gervasio, Othon L; Allen, David G

    2008-04-01

    Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by a mutation in the gene encoding dystrophin, a protein linking the cytoskeleton to the extracellular matrix. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) provided protection against dystrophic muscle damage in the mdx mouse, an animal model of DMD. In isolated mdx muscles, NAC prevented the increased membrane permeability and reduced the force deficit associated with stretch-induced muscle damage. Three-week-old mdx mice were treated with NAC in the drinking water for 6 weeks. Dihydroethidium staining showed that NAC treatment reduced the concentration of reactive oxygen species (ROS) in mdx muscles. This was accompanied by a significant decrease in centrally nucleated fibres in muscles from NAC-treated mdx mice. Immunoblotting showed that NAC treatment decreased the nuclear protein expression of NF-kappaB, a transcription factor involved in pro-inflammatory cytokine expression. Finally, we show that NAC treatment reduced caveolin-3 protein levels and increased the sarcolemmal expression of beta-dystroglycan and the dystrophin homologue, utrophin. Taken together, our findings suggest that ROS play an important role in the dystrophic pathogenesis, both in terms of activating damage pathways and in regulating the expression of some dystrophin-associated membrane proteins. These results offer the prospect that antioxidants such as NAC could have therapeutic potential for DMD patients.

  12. Disease course in mdx:utrophin+/− mice: comparison of three mouse models of Duchenne muscular dystrophy

    PubMed Central

    McDonald, Abby A; Hebert, Sadie L; Kunz, Matthew D; Ralles, Steven J; McLoon, Linda K

    2015-01-01

    The mdx mouse model of Duchenne muscular dystrophy (DMD) is used to study disease mechanisms and potential treatments, but its pathology is less severe than DMD patients. Other mouse models were developed to more closely mimic the human disease based on knowledge that upregulation of utrophin has a protective effect in mdx muscle. An mdx:utrophin−/− (dko) mouse was created, which had a severe disease phenotype and a shortened life span. An mdx:utrophin+/− mouse was also created, which had an intermediate disease phenotype compared to the mdx and dko mice. To determine the usefulness of mdx:utrophin+/− mice for long-term DMD studies, limb muscle pathology and function were assessed across the life span of wild-type, mdx, mdx:utrophin+/−, and dko mice. Muscle function assessment, specifically grip duration and rotarod performance, demonstrated that mdx:utrophin+/− mice were weaker for a longer time than mdx mice. Mean myofiber area was smaller in mdx:utrophin+/− mice compared to mdx mice at 12 months. Mdx:utrophin+/− mice had a higher percentage of centrally nucleated myofibers compared to mdx mice at 6 and 12 months. Collagen I and IV density was significantly higher in mdx:utrophin+/− muscle compared to mdx at most ages examined. Generally, mdx:utrophin+/− mice showed an intermediate disease phenotype over a longer time course compared to the mdx and dko mice. While they do not genetically mirror human DMD, mdx:utrophin+/− mice may be a more useful animal model than mdx or dko mice for investigating long-term efficacy of potential treatments when fibrosis or muscle function is the focus. PMID:25921779

  13. The role of reactive oxygen species in the hearts of dystrophin-deficient mdx mice.

    PubMed

    Williams, Iwan A; Allen, David G

    2007-09-01

    Duchenne muscular dystrophy (DMD) is caused by deficiency of the cytoskeletal protein dystrophin. Oxidative stress is thought to contribute to the skeletal muscle damage in DMD; however, little is known about the role of oxidative damage in the pathogenesis of the heart failure that occurs in DMD patients. The dystrophin-deficient (mdx) mouse is an animal model of DMD that also lacks dystrophin. The current study investigates the role of the antioxidant N-acetylcysteine (NAC) on mdx cardiomyocyte function, Ca(2+) handling, and the cardiac inflammatory response. Treated mice received 1% NAC in their drinking water for 6 wk. NAC had no effect on wild-type (WT) mice. Immunohistochemistry experiments revealed that mdx mice had increased dihydroethidine (DHE) staining, an indicator of superoxide production; NAC-treatment reduced DHE staining in mdx hearts. NAC treatment attenuated abnormalities in mdx cardiomyocyte Ca(2+) handling. Mdx cardiomyocytes had decreased fractional shortening and decreased Ca(2+) sensitivity; NAC treatment returned mdx fractional shortening to WT values but did not affect the Ca(2+) sensitivity. Immunohistochemistry experiments revealed that mdx hearts had increased levels of collagen type III and the macrophage-specific protein, CD68; NAC-treatment returned collagen type III and CD68 expression close to WT values. Finally, mdx hearts had increased NADPH oxidase activity, suggesting it could be a possible source of increased reactive oxygen species in mdx mice. This study is the first to demonstrate that oxidative damage may be involved in the pathogenesis of the heart failure that occurs in mdx mice. Therapies designed to reduce oxidative damage might be beneficial to DMD patients with heart failure.

  14. Talin, vinculin and nestin expression in orofacial muscles of dystrophin deficient mdx mice.

    PubMed

    Spassov, Alexander; Gredes, Tomasz; Pavlovic, Dragan; Gedrange, Tomasz; Lehmann, Christian; Lucke, Silke; Kunert-Keil, Christiane

    2012-04-01

    The activity of cytoskeletal proteins like talin, vinculin and nestin increases in muscle that regenerates. Little is known about their role or at least their expression in the process of regeneration in masticatory muscles of mdx mice, a model of Duchenne muscular dystrophy. To determine a potential role of cytoskeletal proteins in the regeneration process of mdx masticatory muscles, we examined the expression of talin 1, talin 2, vinculin and nestin in 100-day-old control and mdx mice using quantitative RT-PCR, Western blot analyses and histochemistry. The protein expression of talin 1, talin 2, nestin and vinculin in mdx muscles remained unchanged as compared with normal mice. However, in mdx masseter it was found a relative increase of nestin compared to controls. The protein expression of talin 1 and vinculin tended to be increased in mdx tongue and talin 2 to diminish in mdx masseter and temporal muscle. In mdx mice, we found significantly lower percentage of transcripts coding for nestin, talin 1, talin 2 and vinculin in masseter (p < 0.05) and temporal muscle (p < 0.001). In contrast, the mRNA expression of nestin was found to be increased in mdx tongue. Activated satellite cells, myoblasts and immature regenerated muscle fibres in mdx masseter and temporal revealed positive staining for nestin. The findings of the presented work suggest dystrophin-lack-associated changes in the expression of cytoskeletal proteins in mdx masticatory muscles could be compensatory for dystrophin absence. The expression of nestin may serve as an indicator for the regeneration in the orofacial muscles.

  15. Abnormalities in brain structure and biochemistry associated with mdx mice measured by in vivo MRI and high resolution localized (1)H MRS.

    PubMed

    Xu, Su; Shi, Da; Pratt, Stephen J P; Zhu, Wenjun; Marshall, Andrew; Lovering, Richard M

    2015-10-01

    Duchenne muscular dystrophy (DMD), an X-linked disorder caused by the lack of dystrophin, is characterized by the progressive wasting of skeletal muscles. To date, what is known about dystrophin function is derived from studies of dystrophin-deficient animals, with the most common model being the mdx mouse. Most studies on patients with DMD and in mdx mice have focused on skeletal muscle and the development of therapies to reverse, or at least slow, the severe muscle wasting and progressive degeneration. However, dystrophin is also expressed in the CNS. Both mdx mice and patients with DMD can have cognitive and behavioral changes, but studies in the dystrophic brain are limited. We examined the brain structure and metabolites of mature wild type (WT) and mdx mice using magnetic resonance imaging and spectroscopy (MRI/MRS). Both structural and metabolic alterations were observed in the mdx brain. Enlarged lateral ventricles were detected in mdx mice when compared to WT. Diffusion tensor imaging (DTI) revealed elevations in diffusion diffusivities in the prefrontal cortex and a reduction of fractional anisotropy in the hippocampus. Metabolic changes included elevations in phosphocholine and glutathione, and a reduction in γ-aminobutyric acid in the hippocampus. In addition, an elevation in taurine was observed in the prefrontal cortex. Such findings indicate a regional structural change, altered cellular antioxidant defenses, a dysfunction of GABAergic neurotransmission, and a perturbed osmoregulation in the brain lacking dystrophin.

  16. Prevention of muscle fibrosis and myonecrosis in mdx mice by suramin, a TGF-β1 blocker.

    PubMed

    Taniguti, Ana Paula Tiemi; Pertille, Adriana; Matsumura, Cíntia Yuri; Santo Neto, Humberto; Marques, Maria Julia

    2011-01-01

    Fibrosis is a pathological feature observed in patients with Duchenne muscular dystrophy (DMD) and in mdx mice, the experimental model of DMD. We evaluated the effect of suramin, a transforming growth factor-beta 1 (TGF-β1) blocker, on fibrosis in mdx mice. mdx mice (6 months old) received suramin for 7 weeks. Suramin- and saline-treated (control) mdx mice performed exercise on a treadmill to worsen disease progression. Immunoblotting showed an increase of TGF-β1 in mdx diaphragm, limb, and cardiac muscles. Suramin decreased creatine kinase in mdx mice and attenuated fibrosis in all muscles studied, except for cardiac muscle. Suramin protected limb muscles against damage and reduced the exercise-induced loss of strength over time. These findings support a role for TGF-β1 in fibrinogenesis and myonecrosis during the later stages of disease in mdx mice. Suramin might be a useful therapeutic alternative for the treatment of dystrophinopathies.

  17. Eicosapentaenoic acid decreases TNF-α and protects dystrophic muscles of mdx mice from degeneration.

    PubMed

    Machado, Rafael Ventura; Mauricio, Adriana Fogagnolo; Taniguti, Ana Paula Tiemi; Ferretti, Renato; Neto, Humberto Santo; Marques, Maria Julia

    2011-03-01

    In dystrophin-deficient fibers of mdx mice and in Duchenne muscular dystrophy, inflammation and increased production of tumor necrosis factor alpha (TNF-α) contribute to myonecrosis. We examined the effects of eicosapentaenoic acid (EPA) on dystrophic muscle degeneration. Mdx mice (14 days old) received EPA for 16 days. The sternomastoid, diaphragm and biceps brachii muscles were removed. Control mdx mice received vehicle. EPA decreased creatine kinase and myonecrosis and reduced the levels of TNF-α. These results suggest that EPA plays a protective role in dystrophic muscle degeneration, possibly by reducing TNF-α, and support further investigations of EPA as a potential therapy for dystrophinopathies.

  18. Increased catalase expression improves muscle function in mdx mice.

    PubMed

    Selsby, Joshua T

    2011-02-01

    It has been well established that oxidative stress contributes to pathology associated with Duchenne muscular dystrophy (DMD). I hypothesized that overexpression of the antioxidant enzyme catalase would improve muscle function in the mdx mouse, the mouse model of DMD. To test this hypothesis, neonatal mdx mice were injected with a recombinant adeno-associated virus driving the catalase transgene. Animals were killed 4 or 6 weeks or 6 months following injection. Muscle function was generally improved by catalase overexpression. Four weeks following injection, extensor digitorum longus specific tension was improved twofold, while soleus was similar between groups. Resistance to contraction-induced injury was similar between groups; however, resistance to fatigue was increased 25% in catalase-treated soleus compared with control muscle. Six weeks following injection, extensor digitorum longus specific tension was increased 15%, while soleus specific tension was similar between treated and untreated limbs. Catalase overexpression reduced contraction-induced injury by 30-45% and fatigue by 20% compared with control limbs. Six months following injection, diaphragm specific tension was similar between groups, but resistance to contraction-induced injury was improved by 35% and fatigue by 25%. Taken together, these data indicate that catalase can improve a subset of parameters of muscle function in dystrophin-deficient skeletal muscle.

  19. Recovery of damaged skeletal muscle in mdx mice through low-intensity endurance exercise.

    PubMed

    Frinchi, M; Macaluso, F; Licciardi, A; Perciavalle, V; Coco, M; Belluardo, N; Morici, G; Mudò, G

    2014-01-01

    The lack of dystrophin in mdx mice leads to cycles of muscle degeneration and regeneration processes. Various strategies have been proposed in order to reduce the muscle-wasting component of muscular dystrophy, including implementation of an exercise programme. The aim of this study was to examine how low-intensity endurance exercise affects the degeneration-regeneration process in dystrophic muscle of male mdx mice. Mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 days/week for 6 weeks. Histomorphological analysis showed a significant reduction of measured inflammatory-necrotic areas in both gastrocnemius and quadriceps muscle of exercised mdx mice as compared to matched sedentary mdx mice. The degenerative-regenerative process was also evaluated by examining the protein levels of connexin 39 (Cx39), a specific gene expressed in injured muscles. Cx39 was not detected in sedentary wild type mice, whereas it was found markedly increased in sedentary mdx mice, revealing active muscle degeneration-regeneration process. These Cx39 protein levels were significantly reduced in muscles of mdx mice exercised for 30 and 40 days, revealing together with histomorphological analysis a strong reduction of degeneration process in mice subjected to low-intensity endurance exercise. Muscles of exercised mdx mice did not show significant changes in force and fatigue resistance as compared to sedentary mdx mice. Overall in this study we found that specific low-intensity endurance exercise induces a beneficial effect probably by reducing the degeneration of dystrophic muscle.

  20. Arginine butyrate per os protects mdx mice against cardiomyopathy, kyphosis and changes in axonal excitability.

    PubMed

    Vianello, Sara; Bouyon, Sophie; Benoit, Evelyne; Sebrié, Catherine; Boerio, Delphine; Herbin, Marc; Roulot, Morgane; Fromes, Yves; de la Porte, Sabine

    2014-11-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by lack of dystrophin, a sub-sarcolemmal protein, which leads to dramatic muscle deterioration. We studied in mdx mice, the effects of oral administration of arginine butyrate (AB), a compound currently used for the treatment of sickle cell anemia in children, on cardiomyopathy, vertebral column deformation and electromyographic abnormalities. Monthly follow-up by echocardiography from the 8th month to the 14th month showed that AB treatment protected the mdx mice against drastic reduction (20-23%) of ejection fraction and fractional shortening, and also against the ≈20% ventricular dilatation and 25% cardiac hypertrophy observed in saline-treated mdx mice. The phenotypic improvement was corroborated by the decrease in serum CK level and by better fatigue resistance. Moreover, AB treatment protected against the progressive spinal deformity observed in mdx mice, another similarity with DMD patients. The value of the kyphosis index in AB-treated mice reached 94% of the value in C57BL/10 mice. Finally, axonal excitability parameters such as the membrane resting potential, the threshold and amplitude of the action potential, the absolute and relative refractory periods and the supernormal and subnormal periods, recorded from caudal and plantar muscles in response to excitability tests, that were modified in saline-treated mdx mice were not significantly changed, compared with wild-type animals, in AB-treated mdx mice. All of these results suggest that AB could be a potential treatment for DMD patients.

  1. Effect of genetic background on the dystrophic phenotype in mdx mice

    PubMed Central

    Coley, William D.; Bogdanik, Laurent; Vila, Maria Candida; Yu, Qing; Van Der Meulen, Jack H.; Rayavarapu, Sree; Novak, James S.; Nearing, Marie; Quinn, James L.; Saunders, Allison; Dolan, Connor; Andrews, Whitney; Lammert, Catherine; Austin, Andrew; Partridge, Terence A.; Cox, Gregory A.; Lutz, Cathleen; Nagaraju, Kanneboyina

    2016-01-01

    Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits. PMID:26566673

  2. Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice.

    PubMed

    Yajima, Hiroshi; Kawakami, Kiyoshi

    2016-08-01

    Muscle regeneration is an important process for skeletal muscle growth and recovery. Repair of muscle damage is exquisitely programmed by cellular mechanisms inherent in myogenic stem cells, also known as muscle satellite cells. We demonstrated previously the involvement of homeobox transcription factors, SIX1, SIX4 and SIX5, in the coordinated proliferation and differentiation of isolated satellite cells in vitro. However, their roles in adult muscle regeneration in vivo remain elusive. To investigate SIX4 and SIX5 functions during muscle regeneration, we introduced knockout alleles of Six4 and Six5 into an animal model of Duchenne Muscular Dystrophy (DMD), mdx (Dmd(mdx) /Y) mice, characterized by frequent degeneration-regeneration cycles in muscles. A lower number of small myofibers, higher number of thick ones and lower serum creatine kinase and lactate dehydrogenase activities were noted in 50-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice than Dmd(mdx) /Y mice, indicating improvement of dystrophic phenotypes of Dmd(mdx) /Y mice. Higher proportions of cells positive for MYOD1 and MYOG (markers of regenerating myonuclei) and SIX1 (a marker of regenerating myoblasts and newly regenerated myofibers) in 12-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice suggested enhanced regeneration, compared with Dmd(mdx) /Y mice. Although grip strength was comparable in Six4(+/-) 5(+/-) Dmd(mdx) /Y and Dmd(mdx) /Y mice, treadmill exercise did not induce muscle weakness in Six4(+/-) 5(+/-) Dmd(mdx) /Y mice, suggesting higher regeneration capacity. In addition, Six4(+/-) 5(+/-) Dmd(mdx) /Y mice showed 33.8% extension of life span. The results indicated that low Six4 and Six5 gene dosage improved dystrophic phenotypes of Dmd(mdx) /Y mice by enhancing muscle regeneration, and suggested that SIX4 and SIX5 are potentially useful de novo targets in therapeutic applications against muscle disorders, including DMD.

  3. Excitation-contraction coupling alterations in mdx and utrophin/dystrophin double knockout mice: a comparative study.

    PubMed

    Capote, Joana; DiFranco, Marino; Vergara, Julio L

    2010-05-01

    The double knockout mouse for utrophin and dystrophin (utr(-/-)/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and Ca(2+) transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and utr(-/-)/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and utr(-/-)/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in utr(-/-)/mdx (36%) than in mdx mice (27%), the Ca(2+) release elicited by single stimulation was found to be similarly depressed (32-38%) in utr(-/-)/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from utr(-/-)/mdx mice, group 1 fibers were able to sustain Ca(2+) release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from utr(-/-)/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model.

  4. Excitation-contraction coupling alterations in mdx and utrophin/dystrophin double knockout mice: a comparative study

    PubMed Central

    Capote, Joana; DiFranco, Marino

    2010-01-01

    The double knockout mouse for utrophin and dystrophin (utr−/−/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and Ca2+ transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and utr−/−/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and utr−/−/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in utr−/−/mdx (36%) than in mdx mice (27%), the Ca2+ release elicited by single stimulation was found to be similarly depressed (32–38%) in utr−/−/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from utr−/−/mdx mice, group 1 fibers were able to sustain Ca2+ release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from utr−/−/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model. PMID:20130206

  5. Endogenous mesenchymal stromal cells in bone marrow are required to preserve muscle function in mdx mice.

    PubMed

    Fujita, Ryo; Tamai, Katsuto; Aikawa, Eriko; Nimura, Keisuke; Ishino, Saki; Kikuchi, Yasushi; Kaneda, Yasufumi

    2015-03-01

    The physiological role of "endogenous" bone marrow (BM) mesenchymal stromal cells (MSCs) in tissue regeneration is poorly understood. Here, we show the significant contribution of unique endogenous BM-MSC populations to muscle regeneration in Duchenne muscular dystrophy (DMD) mice (mdx). Transplantation of BM cells (BMCs) from 10-week-old mdx into 3-4-week-old mdx mice increased inflammation and fibrosis and reduced muscle function compared with mdx mice that received BMCs from 10-week-old wild-type mice, suggesting that the alteration of BMC populations in mdx mice affects the progression of muscle pathology. Two distinct MSC populations in BM, that is, hematopoietic lineage (Lin)(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) cells, were significantly reduced in 10-week-old mdx mice in disease progression. The results of a whole-transcriptome analysis indicated that these two MSC populations have distinct gene expression profiles, indicating that the Lin(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) MSC populations are proliferative- and dormant-state populations in BM, respectively. BM-derived Lin(-) /CD106(+) /CD44(+) MSCs abundantly migrated to damaged muscles and highly expressed tumor necrosis factor-alpha-stimulated gene/protein-6 (TSG-6), an anti-inflammatory protein, in damaged muscles. We also demonstrated that TSG-6 stimulated myoblast proliferation. The injection of Lin(-) /ckit(-) /CD106(+) /CD44(+) MSCs into the muscle of mdx mice successfully ameliorated muscle dysfunction by decreasing inflammation and enhancing muscle regeneration through TSG-6-mediated activities. Thus, we propose a novel function of the unique endogenous BM-MSC population, which countered muscle pathology progression in a DMD model.

  6. Caveolin-1, caveolin-3 and VEGF expression in the masticatory muscles of mdx mice.

    PubMed

    Kunert-Keil, Christiane; Gredes, Tomasz; Lucke, Silke; Morgenstern, Sven; Mielczarek, Agnieszka; Sporniak-Tutak, Katarzyna; Gedrange, Tomasz; Spassov, Alexander

    2011-01-01

    Duchenne muscular dystrophy (DMD) and murine X-linked muscular dystrophy (mdx), its murine model, are characterized by muscle damage and muscle weakness associated with inflammation and new vessel formation. Caveolins, dystrophin-associated proteins, are involved in the pathogenesis of DMD, because increased numbers of caveolae are found in DMD and mdx hindlimb muscles. Caveolae influence angiogenesis due to their content of vascular endothelial growth factor (VEGF) receptors. Orofacial muscles in mdx mice undergo muscle necrosis followed by muscle regeneration. To ascertain the role of caveolins and VEGF in the pathogenesis of dystrophic masticatory muscles, we examined the expression of caveolin-1 (cav-1), caveolin-3 (cav-3) and VEGF in control and mdx mice. In mdx masticatory muscles, no changes in transcript and protein levels of VEGF were found, whereas cav-1 and cav-3 expression was increased. Using immunohistochemistry, a strong sarcolemmal staining of caveolin-3 in regenerated muscle fibers was found. Furthermore, immunohistochemistry with the caveolin-1 antibody showed an increase in the amount of blood vessels in areas with regenerating muscle fibers. Dystrophic masticatory muscles showed changes comparable to those of hindlimb muscles in the expression of cav-1 and cav-3. The angiogenesis seems to be unaffected in the jaw muscles of mdx mice. We speculate that the increased caveolin expression could cause extensive and efficient muscle regeneration.

  7. Telomere shortening in diaphragm and tibialis anterior muscles of aged mdx mice.

    PubMed

    Lund, Troy C; Grange, Robert W; Lowe, Dawn A

    2007-09-01

    The progression of Duchenne muscular dystrophy (DMD) is, in part, due to satellite cell senescence driven by high replicative pressure as these muscle stem cells repeatedly divide and fuse to damaged muscle fibers. We hypothesize that telomere shortening in satellite cells underlies their senescence. To test this hypothesis, we evaluated the diaphragm and a leg muscle from dystrophic mice of various ages for telomere dynamics. We found 30% telomere shortening in tibialis anterior muscles from 600-day-old mdx mice relative to age-matched wildtype mice. We also found a more severe shortening of telomere length in diaphragm muscles of old mdx mice. In those muscles, telomeres were shortened by approximately 15% and 40% in 100- and 600-day-old mdx mice, respectively. These findings indicate that satellite cells undergo telomere erosion, which may contribute to the inability of these cells to perpetually repair DMD muscle.

  8. Microdystrophin delivery in dystrophin-deficient (mdx) mice by genetically-corrected syngeneic MSCs transplantation.

    PubMed

    Xiong, F; Xu, Y; Zheng, H; Lu, X; Feng, S; Shang, Y; Li, Y; Zhang, Y; Jin, S; Zhang, C

    2010-09-01

    Cell transplantation and gene therapy are two promising therapeutical approaches for the treatment on Duchenne Muscular Dystrophy (DMD). However, both strategies have met many hurdles, mainly because of the absence of an efficient systemic delivery system on gene therapy and immune reactionns on cell transplantation. In this project, we investigated the strategy based on combination of these two basic ones, ie, transplantation of transgene-corrected mdx mesenchymal stem cells (MSCs) into mdx mice to cure DMD. The MSCs isolated from male mdx mice were transduced with recombinant adenovirus including human microdystrophin gene and labeled with BrdU were transplanted into female mdx mice, the Chimerism with the sex-determinant Y chromosome and human microdystrophin expression were detected. Simultaneously, the plasma creatine kinase (CK) activity, the improvement with the muscles' pathology and contractile propertie were evaluated. The results clearly demonstrated that some human dystrophin and BrdU expression collectively were detected in some muscles of transplanted mdx mice. Moreover, the CK activity and percentage of centrally nucleated fiber (CNF) decreased slightly after transplanation. Regrettably, the protective effect on contraction-induced injury in TA and diaphragm muscles wasn't significantly improvement after transplantation. Our results suggested, if enhancement on the efficiency with cell transplantation, that the transplantation of autologous MSCs corrected by dystrophin may be a form to treat DMD patients in future.

  9. Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

    PubMed Central

    Williams, D A; Head, S I; Lynch, G S; Stephenson, D G

    1993-01-01

    1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which

  10. Long term dietary quercetin enrichment as a cardioprotective countermeasure in mdx mice.

    PubMed

    Ballmann, Christopher; Denney, Thomas; Beyers, Ronald J; Quindry, Tiffany; Romero, Matthew; Selsby, Joshua T; Quindry, John C

    2017-02-13

    Duchenne Muscular Dystrophy (DMD) causes declines in cardiac health resulting in premature mortality. As a potential countermeasure, quercetin is a polyphenol possessing inherent anti-inflammatory and antioxidant effects that activate proliferator-activated γ coactivator 1α (PGC-1α) increasing mitochondrial biogenesis protein abundance. We investigated the extent to which lifelong 0.2% dietary quercetin enrichment attenuates dystrophic cardiopathology in mdx mice. Dystrophic animals were fed quercetin or control diet for 12 months while control C57 mice were fed a control diet. Cardiac function was assessed via 7T MRI at 2, 10, and 14 months. At 14 months hearts were collected for histology and western blotting. Results indicate mdx strain dependent declines in cardiac performance at 14 months and that dietary quercetin enrichment did not attenuate functional losses. In contrast, histological analyses provide evidence that quercetin feeding was associated with decreased fibronectin, and indirect damage indices (Hematoxylin & Eosin) as compared to mdx. Dietary quercetin enrichment increased cardiac protein abundance of PGC-1α, cytochrome-c, ETC complexes I-V, citrate synthase, SOD2, and GPX versus untreated mdx. Protein abundance of inflammatory markers NFκB, P-NFκB, and P-IKBα were decreased by quercetin compared to untreated mdx, while preserving IKBα compared to mdx. Furthermore, quercetin decreased TGF-β1, COX2, and F4/80 versus untreated mdx mice. Data suggest that long term quercetin enrichment does not impact physiologic parameters of cardiac function but improves indices of mitochondrial biogenesis and antioxidant enzymes, facilitated DGC assembly, and decreased inflammation in dystrophic hearts. This article is protected by copyright. All rights reserved.

  11. Muscle injury induced by different types of contractions in dystrophic mdx mice.

    PubMed

    Lou, Jianwei; Bi, Wenbo; Li, Wei; Zhao, Yuying; Liu, Shuping; Zheng, Jinfan; Yan, Chuanzhu

    2012-03-01

    Studies on comparing the effect of lengthening, isometric and shortening contractions on dystrophin-deficient muscles are unavailable. We hypothesized that different types of contractions lead to different extents to which dystrophin-deficient muscles are injured. For this purpose, we developed protocols for different types of contraction-induced injury to mdx muscles in vitro. Force deficits and percentages of procion orange dye positive fibers were employed to assess the extent of injury to each muscle. Our results revealed that both the lengthening and isometric contractions resulted in significantly greater injury to extensor digitorum longus (EDL) muscles of mdx mice than to that of control (C57BL/6) mice. In contrast, the shortening contractions induced very mild and identical injury to EDL muscles of mdx and C57BL/6 mice. Then another protocol was carried out in vivo to ascertain the effect of shortening contractions on mdx muscles by achillotenotomy. Histological assessment revealed that the triceps surae muscles with excised Achilles tendon (EAT) displayed little and significantly milder injury than the normal ones did. In conclusions, the unloaded shortening contractions induce little injury to mdx muscles. The in vitro protocol for different types of contraction-induced injury is sensitive and reliable.

  12. Differential expression of genes involved in the calcium homeostasis in masticatory muscles of MDX mice.

    PubMed

    Kunert-Keil, C H; Gredes, T; Lucke, S; Botzenhart, U; Dominiak, M; Gedrange, T

    2014-04-01

    Duchenne Muscular Dystrophy (DMD) and its murine model, mdx, are characterized by Ca(2+) induced muscle damage and muscle weakness followed by distorted dentofacial morphology. In both, DMD patients and in mdx mice, could be proven so far that only the extraocular muscles (EOM) are not affected by muscular dystrophy. The EOMs are protected against calcium overload by enhanced expression of genes involved in the Ca(2+) homeostasis. We could recently demonstrate that masticatory muscles of mdx mice are differentially affected by muscle dystrophy. The dystrophic masseter and temporalis shows muscle histology comparable to all other skeletal muscles in this animal model, whereas dystrophic tongue muscles seem to develop a milder phenotype. Due to this fact it is to hypothesize that an altered Ca(2+) homeostasis seems to underlie the mdx masticatory muscle pathology. Aim of this study was to examine the mRNA and protein levels of the sarcoplasmic reticulum Ca(2+) ATPases SERCA1 and SERCA2, the plasma membrane Ca(2+) ATPases Atp2b1 and Atp2b4, the sodium/calcium exchanger NCX1, the ryanodine receptor 1, parvalbumin, sarcolipin, phospholamban and the L-type Ca(2+) channel alpha-1 subunit (Cacna1s) in Musculus masseter, temporalis, and tongue of 100 day old control and mdx mice. In mdx masseter muscle significant increased mRNA levels of NCX1 and Cacna1s were found compared to control mice. In contrast, the mRNA amount of RYR1 was significant reduced in mdx temporalis muscle, whereas ATP2b4 was significant increased. In mdx tongue a down-regulation of the ATP2b1, sarcolipin and parvalbumin mRNA expression was found, whereas the phospholamban mRNA level was significantly increased compared to controls. These data were verified by western blot analyses. Our findings revealed that mdx masticatory muscles showed an unequally altered expression of genes involved in the Ca(2+) homeostasis that can support the differences in masticatory muscles response to dystrophin deficiency.

  13. Sparing of the Extraocular Muscles in mdx Mice with Absent or Reduced Utrophin Expression: A Life Span Analysis

    PubMed Central

    McDonald, Abby A.; Hebert, Sadie L.; McLoon, Linda K.

    2015-01-01

    Sparing of the extraocular muscles in muscular dystrophy is controversial. To address the potential role of utrophin in this sparing, mdx:utrophin+/− and mdx:utrophin−/− mice were examined for changes in myofiber size, central nucleation, and Pax7-positive and MyoD-positive cell density at intervals over their life span. Known to be spared in the mdx mouse, and contrary to previous reports, the extraocular muscles from both the mdx:utrophin+/− and mdx:utrophin−/− mice were also morphologically spared. In the mdx:utrophin+/− mice, which have a normal life span compared to the mdx:utrophin−/− mice, the myofibers were larger at 3 and 12 months than the wild type age-matched eye muscles. While there was a significant increase in central nucleation in the extraocular muscles from all mdx:utrophin+/− mice, the levels were still very low compared to age-matched limb skeletal muscles. Pax7- and MyoD-positive myogenic precursor cell populations were retained and similar to age-matched wild type controls. These results support the hypothesis that utrophin is not involved in extraocular muscle sparing in these genotypes. In addition, it appears these muscles retain the myogenic precursors that would allow them to maintain their regenerative capacity and normal morphology over a lifetime even in these more severe models of muscular dystrophy. PMID:26429098

  14. Sparing of the extraocular muscles in mdx mice with absent or reduced utrophin expression: A life span analysis.

    PubMed

    McDonald, Abby A; Hebert, Sadie L; McLoon, Linda K

    2015-11-01

    Sparing of the extraocular muscles in muscular dystrophy is controversial. To address the potential role of utrophin in this sparing, mdx:utrophin(+/-) and mdx:utrophin(-/-) mice were examined for changes in myofiber size, central nucleation, and Pax7-positive and MyoD-positive cell density at intervals over their life span. Known to be spared in the mdx mouse, and contrary to previous reports, the extraocular muscles from both the mdx:utrophin(+/-) and mdx:utrophin(-/-) mice were also morphologically spared. In the mdx:utrophin(+/)(-) mice, which have a normal life span compared to the mdx:utrophin(-/-) mice, the myofibers were larger at 3 and 12 months than the wild type age-matched eye muscles. While there was a significant increase in central nucleation in the extraocular muscles from all mdx:utrophin(+/)(-) mice, the levels were still very low compared to age-matched limb skeletal muscles. Pax7- and MyoD-positive myogenic precursor cell populations were retained and were similar to age-matched wild type controls. These results support the hypothesis that utrophin is not involved in extraocular muscle sparing in these genotypes. In addition, it appears that these muscles retain the myogenic precursors that would allow them to maintain their regenerative capacity and normal morphology over a lifetime even in these more severe models of muscular dystrophy.

  15. Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice

    PubMed Central

    Huynh, Tony; Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Heier, Christopher R; Van Der Meulen, Jack H; Yu, Qing; Harris, Mark; Nolan, Christopher J; Haegeman, Guy; Grounds, Miranda D; Nagaraju, Kanneboyina

    2014-01-01

    The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2Kb-tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective

  16. Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice.

    PubMed

    Huynh, Tony; Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Heier, Christopher R; Van Der Meulen, Jack H; Yu, Qing; Harris, Mark; Nolan, Christopher J; Haegeman, Guy; Grounds, Miranda D; Nagaraju, Kanneboyina

    2013-10-01

    The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective

  17. Fetal muscle-derived cells can repair dystrophic muscles in mdx mice.

    PubMed

    Auda-Boucher, Gwenola; Rouaud, Thierry; Lafoux, Aude; Levitsky, Dmitri; Huchet-Cadiou, Corinne; Feron, Marie; Guevel, Laetitia; Talon, Sophie; Fontaine-Pérus, Josiane; Gardahaut, Marie-France

    2007-03-10

    We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

  18. Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

    PubMed

    Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie M R; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi

    2012-08-21

    Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.

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

    PubMed

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

    2010-03-01

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

  20. Fiber type composition of the sternomastoid and diaphragm muscles of dystrophin-deficient mdx mice.

    PubMed

    Guido, Anderson Neri; Campos, Gerson Eduardo Rocha; Neto, Humberto Santo; Marques, Maria Julia; Minatel, Elaine

    2010-10-01

    The muscle fiber phenotype is mainly determined by motoneuron innervation and changes in neuromuscular interaction alter the muscle fiber type. In dystrophin-deficient mdx mice, changes in the molecular assembly of the neuromuscular junction and in nerve terminal sprouting occur in the sternomastoid (STN) muscle during early stages of the disease. In this study, we were interested to see whether early changes in neuromuscular assembly are correlated with alterations in fiber type in dystrophic STN at 2 months of age. A predominance of hybrid fast myofibers (about 52% type IIDB) was observed in control (C57Bl/10) STN. In mdx muscle, the lack of dystrophin did not change this profile (about 54% hybrid type IIDB). Pure fast type IID fibers predominated in normal and dystrophic diaphragm (DIA; about 39% in control and 30% in mdx muscle) and a population of slow Type I fibers was also present (about 10% in control and 13% in mdx muscle). In conclusion, early changes in neuromuscular assembly do not affect the fiber type composition of dystrophic STN. In contrast to the pure fast fibers of the more affected DIA, the hybrid phenotype of the STN may permit dynamic adaptations during progression of the disease.

  1. Rapid depletion of muscle progenitor cells in dystrophic mdx/utrophin-/- mice.

    PubMed

    Lu, Aiping; Poddar, Minakshi; Tang, Ying; Proto, Jonathan D; Sohn, Jihee; Mu, Xiaodong; Oyster, Nicholas; Wang, Bing; Huard, Johnny

    2014-09-15

    Duchenne muscular dystrophy (DMD) patients lack dystrophin from birth; however, muscle weakness becomes apparent only at 3-5 years of age, which happens to coincide with the depletion of the muscle progenitor cell (MPC) pools. Indeed, MPCs isolated from older DMD patients demonstrate impairments in myogenic potential. To determine whether the progression of muscular dystrophy is a consequence of the decline in functional MPCs, we investigated two animal models of DMD: (i) dystrophin-deficient mdx mice, the most commonly utilized model of DMD, which has a relatively mild dystrophic phenotype and (ii) dystrophin/utrophin double knock-out (dKO) mice, which display a similar histopathologic phenotype to DMD patients. In contrast to age-matched mdx mice, we observed that both the number and regeneration potential of dKO MPCs rapidly declines during disease progression. This occurred in MPCs at both early and late stages of myogenic commitment. In fact, early MPCs isolated from 6-week-old dKO mice have reductions in proliferation, resistance to oxidative stress and multilineage differentiation capacities compared with age-matched mdx MPCs. This effect may potentially be mediated by fibroblast growth factor overexpression and/or a reduction in telomerase activity. Our results demonstrate that the rapid disease progression in the dKO model is associated, at least in part, with MPC depletion. Therefore, alleviating MPC depletion could represent an approach to delay the onset of the histopathologies associated with DMD patients.

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

    PubMed

    Jørgensen, Louise Helskov; Jensen, Charlotte Harken; Wewer, Ulla M; Schrøder, Henrik Daa

    2007-11-01

    Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.

  3. Comparison of Experimental Protocols of Physical Exercise for mdx Mice and Duchenne Muscular Dystrophy Patients

    PubMed Central

    Hyzewicz, Janek; Ruegg, Urs T.; Takeda, Shin’ichi

    2015-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene coding for dystrophin and leads to muscle degeneration, wheelchair dependence and death by cardiac or respiratory failure. Physical exercise has been proposed as a palliative therapy for DMD to maintain muscle strength and prevent contractures for as long as possible. However, its practice remains controversial because the benefits of training may be counteracted by muscle overuse and damage. The effects of physical exercise have been investigated in muscles of dystrophin-deficient mdx mice and in patients with DMD. However, a lack of uniformity among protocols limits comparability between studies and translatability of results from animals to humans. In the present review, we summarize and discuss published protocols used to investigate the effects of physical exercise on mdx mice and DMD patients, with the objectives of improving comparability between studies and identifying future research directions. PMID:27858750

  4. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice

    PubMed Central

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J. A.; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose–fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  5. Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2.

    PubMed

    Sun, Chengcao; Yang, Cuili; Xue, Ruilin; Li, Shujun; Zhang, Ting; Pan, Lei; Ma, Xuejiao; Wang, Liang; Li, Dejia

    2015-01-15

    Sulforaphane (SFN), one of the most important isothiocyanates in the human diet, is known to have chemo-preventive and antioxidant activities in different tissues via activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. However, its effects on muscular dystrophy remain unknown. This work was undertaken to evaluate the effects of SFN on Duchenne muscular dystrophy. Four-week-old mdx mice were treated with SFN by gavage (2 mg·kg body wt(-1)·day(-1) for 8 wk), and our results demonstrated that SFN treatment increased the expression and activity of muscle phase II enzymes NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1 with a Nrf2-dependent manner. SFN significantly increased skeletal muscle mass, muscle force (∼30%), running distance (∼20%), and GSH-to-GSSG ratio (∼3.2-fold) of mdx mice and decreased the activities of plasma creatine phosphokinase (∼45%) and lactate dehydrogenase (∼40%), gastrocnemius hypertrophy (∼25%), myocardial hypertrophy (∼20%), and malondialdehyde levels (∼60%). Furthermore, SFN treatment also reduced the central nucleation (∼40%), fiber size variability, and inflammation and improved the sarcolemmal integrity of mdx mice. Collectively, these results show that SFN can improve muscle function and pathology and protect dystrophic muscle from oxidative damage in mdx mice associated with Nrf2 signaling pathway, which indicate Nrf2 may have clinical implications for the treatment of patients with muscular dystrophy.

  6. Truncated dystrophins reduce muscle stiffness in the extensor digitorum longus muscle of mdx mice

    PubMed Central

    Hakim, Chady H.

    2013-01-01

    Muscle stiffness is a major clinical feature in Duchenne muscular dystrophy (DMD). DMD is the most common lethal inherited muscle-wasting disease in boys, and it is caused by the lack of the dystrophin protein. We recently showed that the extensor digitorum longus (EDL) muscle of mdx mice (a DMD mouse model) exhibits disease-associated muscle stiffness. Truncated micro- and mini-dystrophins are the leading candidates for DMD gene therapy. Unfortunately, it has never been clear whether these truncated genes can mitigate muscle stiffness. To address this question, we examined the passive properties of the EDL muscle in transgenic mdx mice that expressed a representative mini- or micro-gene (ΔH2-R15, ΔR2-15/ΔR18-23/ΔC, or ΔR4-23/ΔC). The passive properties were measured at the ages of 6 and 20 mo and compared with those of age-matched wild-type and mdx mice. Despite significant truncation of the gene, surprisingly, the elastic and viscous properties were completely restored to the wild-type level in every transgenic strain we examined. Our results demonstrated for the first time that truncated dystrophin genes may effectively treat muscle stiffness in DMD. PMID:23221959

  7. The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (mdx) mice.

    PubMed

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-06-01

    The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechano-growth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.

  8. Mechanical and/or neural activity-dependent regulation of soleus muscle fibers of mdx mice

    NASA Astrophysics Data System (ADS)

    Terada, Masahiro; Kawano, Fuminori; Lan, Yong Bo; Matsuoka, Yoshikazu; Wang, Xiao Dong; Ohira, Yoshinobu

    2005-08-01

    Roles of mechanical and/or neural activity in the necrosis -regeneration cycle in the soleus muscle fibers of mdxmicewerestudied. Five-weeks-oldmalemdxand wild type (WT) mice were separated into tenotomy (T), denervation (D), and T+D groups. The distal tendons of the left plantarflexors (soleus, plantaris, and gastrocnemius) were ablated in the T group. The left sciatic nerve was transected at the gluteal region in the D group. The right limb was kept intact as the normal control. Ambulation was allowed after the surgery. Soleus muscle was sampled 14 days after the surgery and analyses were performed in cross-section of whole muscle and in single fibers removed longitudinally. The total fiber number of the untreated muscle was 913±19 (Mean±SEM) and 872±45 in WT and mdx mice, respectively. The fiber number in mdx mice was decreased 48% by T and 31-35% by D and T+D, which induced fiber atrophy, may be due to either inhibited regeneration or stimulated necrosis. Although fibers with central nuclei or necrosis were not observed in WT muscle, 25-40% of fibers (vs. 40% in the contralateral control side) in treated muscles of mdx mice, analyzed cross-sectionally, were central-nucleated. However, fibers with only central nuclei were not detected in the longitudinally isolated fibers of treated groups, may be due to the phenomenon that the fibers with necrosis were lost in the relaxing solution. But % fibers with both central and peripheral nuclei were decreased and those with peripheral nuclei alone were increased by T. In both cross-sectional and longitudinal analyses, the % distribution of the central-nucleated relative to total fiber number was not affected by D, but decreased by T in mdx mice (p>0.05). Myonuclear number per mm of fiber length was identical generally, although the number was increased by T. Furthermore, DNA fragmentation was noted in the mdx fibers with necrosis. These data suggested that the localization of myonuclei, as well as either necrosis or

  9. Absence of Cardiac Benefit with Early Combination ACE Inhibitor and Beta Blocker Treatment in mdx Mice.

    PubMed

    Blain, Alison; Greally, Elizabeth; Laval, Steven H; Blamire, Andrew M; MacGowan, Guy A; Straub, Volker W

    2015-04-01

    Most patients with Duchenne muscular dystrophy (DMD) will develop cardiomyopathy; however, the evidence for prophylactic treatment of children with cardiac medications is limited. We have used the mdx mouse model of DMD to assess if early combination treatment with beta blocker (BB) and ACE inhibitor (AI) is superior to single treatment with either one of these drugs. Mice were assessed with cardiac MRI (ventricular structure and function, in vivo calcium influx (manganese-enhanced MRI)), pressure-volume loops, and histopathology. Combination treatment did not show benefits over treatment with AI or BB alone. Indeed, some beneficial aspects of BB and AI were lost when used in combination. None of the treatments impacted RV function. Combination treatment had no significant effect on sarcolemmal damage or histopathology. The study suggests that combined BB and AI may not confer an advantage at an early stage in DMD cardiomyopathy. However, limitations of the mdx model should be considered.

  10. Green tea extract decreases muscle pathology and NF-κB immunostaining in regenerating muscle fibers of mdx mice

    PubMed Central

    Evans, Nicholas P.; Call, Jarrod A.; Bassaganya-Riera, Josep; Robertson, John L.; Grange, Robert W.

    2009-01-01

    BACKGROUND & AIMS Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in afflicted boys. The primary cause of this disease is mutations in the dystrophin gene resulting in massive muscle degeneration and inflammation. The purpose of this study was to determine if dystrophic muscle pathology and inflammation were decreased by pre-natal and early dietary intervention with green tea extract. METHODS Mdx breeder mice and pups were fed diets containing 0.25% or 0.5% green tea extract and compared to untreated mdx and C57BL/6J mice. Serum creatine kinase was assessed as a systemic indicator of muscle damage. Quantitative histopathological and immunohistochemical techniques were used to determine muscle pathology, macrophage infiltration, and NF-κB localization. RESULTS Early treatment of mdx mice with green tea extract significantly decreased serum creatine kinase by ~85% at age 42 days (P≤0.05). In these mice, the area of normal fiber morphology was increased by as much as ~32% (P≤0.05). The primary histopathological change was a ~21% decrease in the area of regenerating fibers (P≤0.05). NF-κB staining in regenerating muscle fibers was also significantly decreased in green tea extract-treated mdx mice when compared to untreated mdx mice. CONCLUSION Early treatment with green tea extract decreases dystrophic muscle pathology potentially by regulating NF-κB activity in regenerating muscle fibers. PMID:19897286

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

    PubMed

    Altamirano, Francisco; Valladares, Denisse; Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R; Allen, Paul D; Jaimovich, Enrique

    2013-01-01

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

  12. Nifedipine Treatment Reduces Resting Calcium Concentration, Oxidative and Apoptotic Gene Expression, and Improves Muscle Function in Dystrophic mdx Mice

    PubMed Central

    Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R.; Allen, Paul D.; Jaimovich, Enrique

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca2+]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca2+]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca2+]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91phox/p47phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca2+]r in mdx skeletal muscle cells. The results in this work open new perspectives towards

  13. Dystrophic mdx mice develop severe cardiac and respiratory dysfunction following genetic ablation of the anti-inflammatory cytokine IL-10.

    PubMed

    Nitahara-Kasahara, Yuko; Hayashita-Kinoh, Hiromi; Chiyo, Tomoko; Nishiyama, Akiyo; Okada, Hironori; Takeda, Shin'ichi; Okada, Takashi

    2014-08-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory and cardiac failure. Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, but its role and regulation in the disease time course has not been sufficiently examined. In the present study, we used IL-10(-/-)/mdx mice lacking both dystrophin and the anti-inflammatory cytokine, interleukin-10 (IL-10), to investigate whether a predisposition to inflammation affects the severity of DMD with advancing age. The IL-10 deficiency caused a profound DMD phenotype in the dystrophic heart such as muscle degeneration and extensive myofiber loss, but the limb muscle and diaphragm morphology of IL-10(-/) (-)/mdx mice was similar to that of mdx mice. Extensive infiltrates of pro-inflammatory M1 macrophages in regeneration of cardiotoxin-injured muscle, altered M1/M2 macrophage phenotype and increased pro-inflammatory cytokines/chemokines production were observed in the diaphragm and heart of IL-10(-/-)/mdx mice. We characterized the IL-10(-/-)/mdx mice as a dystrophic model with chronic inflammation and severe cardiorespiratory dysfunction, as evidenced by decreased percent fractional shortening (%FS) and ejection fraction percent (EF%) on echocardiography, reduced lower tidal volume on whole-body plethysmography. This study suggests that a predisposition to inflammation is an important indicator of DMD disease progression. Therefore, the development of anti-inflammatory strategies may help in slowing down the cardiorespiratory dysfunction on DMD.

  14. Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice.

    PubMed

    Mozzetta, Chiara; Consalvi, Silvia; Saccone, Valentina; Tierney, Matthew; Diamantini, Adamo; Mitchell, Kathryn J; Marazzi, Giovanna; Borsellino, Giovanna; Battistini, Luca; Sassoon, David; Sacco, Alessandra; Puri, Pier Lorenzo

    2013-04-01

    HDAC inhibitors (HDACi) exert beneficial effects in mdx mice, by promoting endogenous regeneration; however, the cellular determinants of HDACi activity on dystrophic muscles have not been determined. We show that fibroadipogenic progenitors (FAP) influence the regeneration potential of satellite cells during disease progression in mdx mice and mediate HDACi ability to selectively promote regeneration at early stages of disease. FAPs from young mdx mice promote, while FAPs from old mdx mice repress, satellite cell-mediated formation of myotubes. In young mdx mice HDACi inhibited FAP adipogenic potential, while enhancing their ability to promote differentiation of adjacent satellite cells, through upregulation of the soluble factor follistatin. By contrast, FAPs from old mdx mice were resistant to HDACi-mediated inhibition of adipogenesis and constitutively repressed satellite cell-mediated formation of myotubes. We show that transplantation of FAPs from regenerating young muscles restored HDACi ability to increase myofibre size in old mdx mice. These results reveal that FAPs are key cellular determinants of disease progression in mdx mice and mediate a previously unappreciated stage-specific beneficial effect of HDACi in dystrophic muscles.

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

    PubMed

    Hindi, Sajedah M; Shin, Jonghyun; Ogura, Yuji; Li, Hong; Kumar, Ashok

    2013-01-01

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

  16. Motor performance of young dystrophic mdx mice treated with long-circulating prednisolone liposomes.

    PubMed

    Weller, Charlotte; Zschüntzsch, Jana; Makosch, Gregor; Metselaar, Josbert M; Klinker, Florian; Klinge, Lars; Liebetanz, David; Schmidt, Jens

    2012-05-01

    For Duchenne muscular dystrophy (DMD), a common myopathy that leads to severe disability, no causal therapy is available. Glucocorticosteroids improve patients' muscle strength, but their long-term use is limited by negative side effects. Thus, pharmacological modifications of glucocorticosteroids are required to increase the efficacy by drug targeting. Liposomal encapsulation augments systemic half-life and local tissue concentrations of glucocorticosteroids and, at the same time, reduces systemic side effects. In this study, the efficacy of novel, long-circulating, polyethylene-glycol-coated liposomes encapsulating prednisolone was compared with free prednisolone in the treatment of mdx mice, a well-established animal model for DMD. Using an objective and sensitive computerized 24-hr detection system of voluntary wheel-running in single cages, we demonstrate a significant impairment of the running performance in mdx compared with black/10 control mice aged 3-6 weeks. Treatment with liposomal or free prednisolone did not improve running performance compared with saline control or empty liposomes. Histopathological parameters, including the rate of internalized nuclei and fiber size variation, and mRNA and protein expression levels of transforming growth factor (TGF)-β and monocytes chemotactic protein (MCP)-1 also remained unchanged. Bioactivity in skeletal muscle of liposomal and free prednisolone was demonstrated by elevated mRNA expression of muscle ring finger protein 1 (MuRF1), a mediator of muscle atrophy, and its forkhead box transcription factors (Foxo1/3). Our data support the assessment of voluntary running to be a robust and reproducible outcome measure of skeletal muscle performance during the early disease course of mdx mice and suggest that liposomal encapsulation is not superior in treatment efficacy compared with conventional prednisolone. Our study helps to improve the future design of experimental treatment in animal models of neuromuscular

  17. Myogenic Akt signaling attenuates muscular degeneration, promotes myofiber regeneration and improves muscle function in dystrophin-deficient mdx mice

    PubMed Central

    Kim, Michelle H.; Kay, Danielle I.; Rudra, Renuka T.; Chen, Bo Ming; Hsu, Nigel; Izumiya, Yasuhiro; Martinez, Leonel; Spencer, Melissa J.; Walsh, Kenneth; Grinnell, Alan D.; Crosbie, Rachelle H.

    2011-01-01

    Duchenne muscular dystrophy, the most common form of childhood muscular dystrophy, is caused by X-linked inherited mutations in the dystrophin gene. Dystrophin deficiencies result in the loss of the dystrophin–glycoprotein complex at the plasma membrane, which leads to structural instability and muscle degeneration. Previously, we induced muscle-specific overexpression of Akt, a regulator of cellular metabolism and survival, in mdx mice at pre-necrotic (<3.5 weeks) ages and demonstrated upregulation of the utrophin–glycoprotein complex and protection against contractile-induced stress. Here, we found that delaying exogenous Akt treatment of mdx mice after the onset of peak pathology (>6 weeks) similarly increased the abundance of compensatory adhesion complexes at the extrasynaptic sarcolemma. Akt introduction after onset of pathology reverses the mdx histopathological measures, including decreases in blood serum albumin infiltration. Akt also improves muscle function in mdx mice as demonstrated through in vivo grip strength tests and in vitro contraction measurements of the extensor digitorum longus muscle. To further explore the significance of Akt in myofiber regeneration, we injured wild-type muscle with cardiotoxin and found that Akt induced a faster regenerative response relative to controls at equivalent time points. We demonstrate that Akt signaling pathways counteract mdx pathogenesis by enhancing endogenous compensatory mechanisms. These findings provide a rationale for investigating the therapeutic activation of the Akt pathway to counteract muscle wasting. PMID:21245083

  18. Prevention of exercised induced cardiomyopathy following Pip-PMO treatment in dystrophic mdx mice.

    PubMed

    Betts, Corinne A; Saleh, Amer F; Carr, Carolyn A; Hammond, Suzan M; Coenen-Stass, Anna M L; Godfrey, Caroline; McClorey, Graham; Varela, Miguel A; Roberts, Thomas C; Clarke, Kieran; Gait, Michael J; Wood, Matthew J A

    2015-03-11

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder caused by mutations in the Dmd gene. In addition to skeletal muscle wasting, DMD patients develop cardiomyopathy, which significantly contributes to mortality. Antisense oligonucleotides (AOs) are a promising DMD therapy, restoring functional dystrophin protein by exon skipping. However, a major limitation with current AOs is the absence of dystrophin correction in heart. Pip peptide-AOs demonstrate high activity in cardiac muscle. To determine their therapeutic value, dystrophic mdx mice were subject to forced exercise to model the DMD cardiac phenotype. Repeated peptide-AO treatments resulted in high levels of cardiac dystrophin protein, which prevented the exercised induced progression of cardiomyopathy, normalising heart size as well as stabilising other cardiac parameters. Treated mice also exhibited significantly reduced cardiac fibrosis and improved sarcolemmal integrity. This work demonstrates that high levels of cardiac dystrophin restored by Pip peptide-AOs prevents further deterioration of cardiomyopathy and pathology following exercise in dystrophic DMD mice.

  19. Voluntary physical activity protects from susceptibility to skeletal muscle contraction-induced injury but worsens heart function in mdx mice.

    PubMed

    Hourdé, Christophe; Joanne, Pierre; Medja, Fadia; Mougenot, Nathalie; Jacquet, Adeline; Mouisel, Etienne; Pannerec, Alice; Hatem, Stéphane; Butler-Browne, Gillian; Agbulut, Onnik; Ferry, Arnaud

    2013-05-01

    It is well known that inactivity/activity influences skeletal muscle physiological characteristics. However, the effects of inactivity/activity on muscle weakness and increased susceptibility to muscle contraction-induced injury have not been extensively studied in mdx mice, a murine model of Duchenne muscular dystrophy with dystrophin deficiency. In the present study, we demonstrate that inactivity (ie, leg immobilization) worsened the muscle weakness and the susceptibility to contraction-induced injury in mdx mice. Inactivity also mimicked these two dystrophic features in wild-type mice. In contrast, we demonstrate that these parameters can be improved by activity (ie, voluntary wheel running) in mdx mice. Biochemical analyses indicate that the changes induced by inactivity/activity were not related to fiber-type transition but were associated with altered expression of different genes involved in fiber growth (GDF8), structure (Actg1), and calcium homeostasis (Stim1 and Jph1). However, activity reduced left ventricular function (ie, ejection and shortening fractions) in mdx, but not C57, mice. Altogether, our study suggests that muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle could be attributable, at least in part, to inactivity. It also suggests that activity exerts a beneficial effect on dystrophic skeletal muscle but not on the heart.

  20. CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.

    PubMed

    Xu, Li; Park, Ki Ho; Zhao, Lixia; Xu, Jing; El Refaey, Mona; Gao, Yandi; Zhu, Hua; Ma, Jianjie; Han, Renzhi

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD.

  1. Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice.

    PubMed

    Lee, Eun-Joo; Kim, Ah-Young; Lee, Eun-Mi; Lee, Myeong-Mi; Min, Chang-Woo; Kang, Kyung-Ku; Park, Jin-Kyu; Hwang, Meeyul; Kwon, Soon-Hak; Tremblay, Jacques P; Jeong, Kyu-Shik

    2014-08-01

    Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

  2. Ventilation during air breathing and in response to hypercapnia in 5 and 16 month-old mdx and C57 mice

    PubMed Central

    Gayraud, Jérome; Matécki, Stefan; Hnia, Karim; Mornet, Dominique; Préfaut, Christian; Mercier, Jacques; Michel, Alain; Ramonatxo, Michèle

    2007-01-01

    Previous studies have shown a blunted ventilatory response to hypercapnia in mdx mice older than 7 months. We test the hypothesis that in the mdx mice ventilatory response changes with age, concomitantly with the increased functional impairment of the respiratory muscles. We thus studied the ventilatory response to CO2 in 5 and 16 month-old mdx and C57BL10 mice (n = 8 for each group). Respiratory rate (RR), tidal volume (VT), and minute ventilation (VE) were measured, using whole-body plethysmography, during air breathing and in response to hypercapnia (3, 5 and 8% CO2). The ventilatory protocol was completed by histological analysis of the diaphragm and intercostals muscles. During air breathing, the 16 month-old mdx mice showed higher RR and, during hypercapnia (at 8% CO2 breathing), significantly lower RR (226 ± 26 vs. 270 ± 21 breaths/min) and VE (1.81 ± 0.35 vs. 3.96 ± 0.59 ml min−1 g−1)(P < 0.001) in comparison to C57BL10 controls. On the other hand, 5 month-old C57BL10 and mdx mice did not present any difference in their ventilatory response to air breathing and to hypercapnia. In conclusion, this study shows similar ventilation during air breathing and in response to hypercapnia in the 5 month-old mdx and control mice, in spite of significant pathological structural changes in the respiratory muscles of the mdx mice. However in the 16 month-old mdx mice we observed altered ventilation under air and blunted ventilation response to hypercapnia compared to age-matched control mice. Ventilatory response to hypercapnia thus changes with age in mdx mice, in line with the increased histological damage of their respiratory muscles. PMID:17431804

  3. Effects of Mechanical Overloading on the Properties of Soleus Muscle Fibers, with or without Damage in MDX and Wild Type Mice

    NASA Astrophysics Data System (ADS)

    Terada, Masahiro; Kawano, Fuminori; Ohira, Takashi; Oke, Yoshihiko; Nakai, Naoya; Ohira, Yoshinobu

    2008-06-01

    Effects of mechanical overloading on the characteristics of regenerating or not-regenerating soleus muscle fibers were studied. The muscle fibers of mdx mice were characterized by the localization of myonuclei. Muscle damage was also induced in wild type (WT) mice by injection of cardiotoxin (CTX) into soleus muscle. Overloading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mice by removing the distal tendons of plantaris and gastrocnemius muscles. The contralateral muscle served as the normal control. These animals were then allowed ambulation recovery in the cage. Central myonuclei were noted in many fibers of mdx and CTX-injected mice with or without overloading. In general, the fibers with central nuclei were considered as regenerating fibers. The fibers with more central nuclei were increased in mdx mice, but the fibers with more peripheral nuclei were increased in CTX-injected WT mice by overloading. The muscle satellite cells, neuromuscular junctions (NMJ), and myonuclei were stained. Most of the properties, such as number of myonuclei and satellite cells, size of NMJ, and fiber length, were not influenced by mechanical overloading in all mice. Approximately 0.6% branched fibers were seen in the intact soleus of mdx mice, although these fibers were not detected in WT mice. However, the percentage of these fibers was increased by overloading especially in mdx mice (~50% vs. ~2.5% in WT). In CTX-injected WT mice, these fibers were ~15% with or without overloading. The fiber cross sectional area in normal WT, but not in mdx and CTX-injected WT mice, was increased by overloading (p<0.05). These results suggested that the functional overload induced muscle damage in mdx mice, but promoted the regeneration in CTX-injected WT mice.

  4. NGF-dependent axon growth and regeneration are altered in sympathetic neurons of dystrophic mdx mice.

    PubMed

    Lombardi, Loredana; Persiconi, Irene; Gallo, Alessandra; Hoogenraad, Casper C; De Stefano, Maria Egle

    2017-02-02

    Duchenne muscular dystrophy (DMD) is a lethal disease, determined by lack of dystrophin (Dp427), a muscular cytoskeletal protein also expressed by selected neuronal populations. Consequently, besides muscular wasting, both human patients and DMD animal models suffer several neural disorders. In previous studies on the superior cervical ganglion (SCG) of wild type and dystrophic mdx mice (Lombardi et al. 2008), we hypothesized that Dp427 could play some role in NGF-dependent axonal growth, both during development and adulthood. To address this issue, we first analyzed axon regeneration potentials of SCG neurons of both genotypes after axotomy in vivo. While noradrenergic innervation of mdx mouse submandibular gland, main source of nerve growth factor (NGF), recovered similarly to wild type, iris innervation (muscular target) never did. We, therefore, evaluated whether dystrophic SCG neurons were poorly responsive to NGF, especially at low concentration. Following in vitro axotomy in the presence of either 10 or 50ng/ml NGF, the number of regenerated axons in mdx mouse neuron cultures was indeed reduced, compared to wild type, at the lower concentration. Neurite growth parameters (i.e. number, length), growth cone dynamics and NGF/TrkA receptor signaling in differentiating neurons (not injured) were also significantly reduced when cultured with 10ng/ml NGF, but also with higher NGF concentrations. In conclusion, we propose a role for Dp427 in NGF-dependent cytoskeletal dynamics associated to growth cone advancement, possibly through indirect stabilization of TrkA receptors. Considering NGF activity in nervous system development/remodeling, this aspect could concur in some of the described DMD-associated neural dysfunctions.

  5. The Proton Pump Inhibitor Lansoprazole Improves the Skeletal Phenotype in Dystrophin Deficient mdx Mice

    PubMed Central

    Sali, Arpana; Many, Gina M.; Gordish-Dressman, Heather; van der Meulen, Jack H.; Phadke, Aditi; Spurney, Christopher F.; Cnaan, Avital; Hoffman, Eric P.; Nagaraju, Kanneboyina

    2013-01-01

    Background In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology. Methodology/Principal Findings We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions. Conclusions/Significance Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings warrant

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

    2014-01-01

    Background Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy. Methods mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice. Results mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells. Conclusions These results suggest that andrographolide could be used to improve quality of life in individuals with DMD. PMID:24655808

  8. Selective activation of α7 nicotinic acetylcholine receptor (nAChRα7) inhibits muscular degeneration in mdx dystrophic mice.

    PubMed

    Leite, Paulo Emílio Correa; Gandía, Luís; de Pascual, Ricardo; Nanclares, Carmen; Colmena, Inés; Santos, Wilson C; Lagrota-Candido, Jussara; Quirico-Santos, Thereza

    2014-07-21

    Amount evidence indicates that α7 nicotinic acetylcholine receptor (nAChRα7) activation reduces production of inflammatory mediators. This work aimed to verify the influence of endogenous nAChRα7 activation on the regulation of full-blown muscular inflammation in mdx mouse with Duchenne muscular dystrophy. We used mdx mice with 3 weeks-old at the height myonecrosis, and C57 nAChRα7(+/+) wild-type and nAChRα7(-/-) knockout mice with muscular injury induced with 60µL 0.5% bupivacaine (bp) in the gastrocnemius muscle. Pharmacological treatment included selective nAChRα7 agonist PNU282987 (0.3mg/kg and 1.0mg/kg) and the antagonist methyllycaconitine (MLA at 1.0mg/kg) injected intraperitoneally for 7 days. Selective nAChRα7 activation of mdx mice with PNU282987 reduced circulating levels of lactate dehydrogenase (LDH, a marker of cell death by necrosis) and the area of perivascular inflammatory infiltrate, and production of inflammatory mediators TNFα and metalloprotease MMP-9 activity. Conversely, PNU282987 treatment increased MMP-2 activity, an indication of muscular tissue remodeling associated with regeneration, in both mdx mice and WTα7 mice with bp-induced muscular lesion. Treatment with PNU282987 had no effect on α7KO, and MLA abolished the nAChRα7 agonist-induced anti-inflammatory effect in both mdx and WT. In conclusion, nAChRα7 activation inhibits muscular inflammation and activates tissue remodeling by increasing muscular regeneration. These effects were not accompanied with fibrosis and/or deposition of non-functional collagen. The nAChRα7 activation may be considered as a potential target for pharmacological strategies to reduce inflammation and activate mechanisms of muscular regeneration.

  9. The Dietary Supplement Protandim® Decreases Plasma Osteopontin and Improves Markers of Oxidative Stress in Muscular Dystrophy Mdx Mice

    PubMed Central

    Qureshi, Muhammad Muddasir; McClure, Warren C.; Arevalo, Nicole L.; Rabon, Rick E.; Mohr, Benjamin; Bose, Swapan K.; McCord, Joe M.; Tseng, Brian S.

    2010-01-01

    Therapeutic options for Duchenne muscular dystrophy (DMD), the most common and lethal neuromuscular disorder in children, remain elusive. Oxidative damage is implicated as a pertinent factor involved in its pathogenesis. Protandim® is an over-the-counter supplement with the ability to induce antioxidant enzymes. In this study we investigated whether Protandim® provided benefit using surrogate markers and functional measures in the dystrophin-deficient (mdx)mouse model of DMD. Male 3-week-old mdx mice were randomized into two treatment groups: control (receiving standard rodent chow) and Protandim®-supplemented standard rodent chow. The diets were continued for 6-week and 6-month studies. The endpoints included the oxidative stress marker thiobarbituric acid-reactive substances (TBARS), plasma osteopontin (OPN), plasma paraoxonase (PON1) activity, H&E histology, gadolinium-enhanced magnetic resonance imaging (MRI) of leg muscle and motor functional measurements. The Protandim® chow diet in mdx mice for 6 months was safe and well tolerated. After 6 months of Protandim®, a 48% average decrease in plasma TBARS was seen; 0.92 nmol/mg protein in controls versus 0.48 nmol/mg protein in the Protandim® group (p = .006). At 6 months, plasma OPN was decreased by 57% (p = .001) in the Protandim®-treated mice. Protandim® increased the plasma antioxidant enzyme PON1 activity by 35% (p = .018). After 6 months, the mdx mice with Protandim® showed 38% less MRI signal abnormality (p = .07) than mice on control diet. In this 6-month mdx mouse study, Protandim® did not significantly alter motor function nor histological criteria. PMID:20740052

  10. The Dietary Supplement Protandim Decreases Plasma Osteopontin and Improves Markers of Oxidative Stress in Muscular Dystrophy Mdx Mice.

    PubMed

    Qureshi, Muhammad Muddasir; McClure, Warren C; Arevalo, Nicole L; Rabon, Rick E; Mohr, Benjamin; Bose, Swapan K; McCord, Joe M; Tseng, Brian S

    2010-06-01

    Therapeutic options for Duchenne muscular dystrophy (DMD), the most common and lethal neuromuscular disorder in children, remain elusive. Oxidative damage is implicated as a pertinent factor involved in its pathogenesis. Protandim((R)) is an over-the-counter supplement with the ability to induce antioxidant enzymes. In this study we investigated whether Protandim((R)) provided benefit using surrogate markers and functional measures in the dystrophin-deficient (mdx)mouse model of DMD. Male 3-week-old mdx mice were randomized into two treatment groups: control (receiving standard rodent chow) and Protandim((R))-supplemented standard rodent chow. The diets were continued for 6-week and 6-month studies. The endpoints included the oxidative stress marker thiobarbituric acid-reactive substances (TBARS), plasma osteopontin (OPN), plasma paraoxonase (PON1) activity, H&E histology, gadolinium-enhanced magnetic resonance imaging (MRI) of leg muscle and motor functional measurements. The Protandim((R)) chow diet in mdx mice for 6 months was safe and well tolerated. After 6 months of Protandim((R)), a 48% average decrease in plasma TBARS was seen; 0.92 nmol/mg protein in controls versus 0.48 nmol/mg protein in the Protandim((R)) group (p = .006). At 6 months, plasma OPN was decreased by 57% (p = .001) in the Protandim((R))-treated mice. Protandim((R)) increased the plasma antioxidant enzyme PON1 activity by 35% (p = .018). After 6 months, the mdx mice with Protandim((R)) showed 38% less MRI signal abnormality (p = .07) than mice on control diet. In this 6-month mdx mouse study, Protandim((R)) did not significantly alter motor function nor histological criteria.

  11. Molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a truncated dystrophin.

    PubMed

    Banks, Glen B; Combs, Ariana C; Chamberlain, Joel R; Chamberlain, Jeffrey S

    2008-12-15

    Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophin(DeltaR4-R23)). We found that muscles with myotendinous strain injury had an increased expression of utrophin and alpha7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophin(DeltaR4-R23)/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSA(LR) (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease.

  12. Increased taurine in pre-weaned juvenile mdx mice greatly reduces the acute onset of myofibre necrosis and dystropathology and prevents inflammation

    PubMed Central

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

    2016-01-01

    Background: The mdx mouse model for the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD) shows a very mild pathology once growth has ceased, with low levels of myofibre necrosis in adults. However, from about 3 weeks of post-natal age, muscles of juvenile mdx mice undergo an acute bout of severe necrosis and inflammation: this subsequently decreases and stabilises to lower adult levels by about 6 weeks of age. Prior to the onset of this severe dystropathology, we have shown that mdx mice are deficient in the amino acid taurine (potentially due to weaning), and we propose that this exacerbates myofibre necrosis and inflammation in juvenile mdx mice. Objectives: The purpose of this study was to increase taurine availability to pre-weaned juvenile mdx mice (from 14 days of age), to evaluate the impact on levels of myofibre necrosis and inflammation (at 22 days) during the acute period of severe dystropathology. Results: Untreated 22 day old mdx muscle was not deficient in taurine, with similar levels to normal C57 control muscle. However taurine treatment, which increased the taurine content of young dystrophic muscle (by 40%), greatly reduced myofibre necrosis (by 75%) and prevented significant increases in 3 markers of inflammation. Conclusion: Taurine was very effective at preventing the acute phase of muscle damage that normally results in myofibre necrosis and inflammation in juvenile mdx mice, supporting continued research into the use of taurine as a therapeutic intervention for protecting growing muscles of young DMD boys PMID:27679740

  13. Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

    PubMed Central

    2012-01-01

    Background The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive

  14. A Multidisciplinary Evaluation of the Effectiveness of Cyclosporine A in Dystrophic Mdx Mice

    PubMed Central

    De Luca, Annamaria; Nico, Beatrice; Liantonio, Antonella; Paola Didonna, Maria; Fraysse, Bodvael; Pierno, Sabata; Burdi, Rosa; Mangieri, Domenica; Rolland, Jean-François; Camerino, Claudia; Zallone, Alberta; Confalonieri, Paolo; Andreetta, Francesca; Arnoldi, Elisa; Courdier-Fruh, Isabelle; Magyar, Josef P.; Frigeri, Antonio; Pisoni, Michela; Svelto, Maria; Conte-Camerino, Diana

    2005-01-01

    Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy. PMID:15681831

  15. Comparison of the myoplasmic calcium transient elicited by an action potential in intact fibres of mdx and normal mice.

    PubMed

    Hollingworth, Stephen; Zeiger, Ulrike; Baylor, Stephen M

    2008-11-01

    The myoplasmic free [Ca2+] transient elicited by an action potential (Delta[Ca2+]) was compared in fast-twitch fibres of mdx (dystrophin null) and normal mice. Methods were used that maximized the likelihood that any detected differences apply in vivo. Small bundles of fibres were manually dissected from extensor digitorum longus muscles of 7- to 14-week-old mice. One fibre within a bundle was microinjected with furaptra, a low-affinity rapidly responding fluorescent calcium indicator. A fibre was accepted for study if it gave a stable, all-or-nothing fluorescence response to an external shock. In 18 normal fibres, the peak amplitude and the full-duration at half-maximum (FDHM) of Delta[Ca2+] were 18.4 +/- 0.5 microm and 4.9 +/- 0.2 ms, respectively (mean +/- s.e.m.; 16 degrees C). In 13 mdx fibres, the corresponding values were 14.5 +/- 0.6 microm and 4.7 +/- 0.2 ms. The difference in amplitude is statistically highly significant (P = 0.0001; two-tailed t test), whereas the difference in FDHM is not (P = 0.3). A multi-compartment computer model was used to estimate the amplitude and time course of the sarcoplasmic reticulum (SR) calcium release flux underlying Delta[Ca2+]. Estimates were made based on several differing assumptions: (i) that the resting myoplasmic free Ca2+ concentration ([Ca2+]R) and the total concentration of parvalbumin ([Parv(T)]) are the same in mdx and normal fibres, (ii) that [Ca2+](R) is larger in mdx fibres, (iii) that [Parv(T)] is smaller in mdx fibres, and (iv) that [Ca2+]R is larger and [Parv(T)] is smaller in mdx fibres. According to the simulations, the 21% smaller amplitude of Delta[Ca2+] in mdx fibres in combination with the unchanged FDHM of Delta[Ca2+] is consistent with mdx fibres having a approximately 25% smaller flux amplitude, a 6-23% larger FDHM of the flux, and a 9-20% smaller total amount of released Ca2+ than normal fibres. The changes in flux are probably due to a change in the gating of the SR Ca2+-release channels and

  16. Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy.

    PubMed

    Pertille, Adriana; de Carvalho, Candida Luiza Tonizza; Matsumura, Cintia Yuri; Neto, Humberto Santo; Marques, Maria Julia

    2010-02-01

    Duchenne muscular dystrophy is one of the most common hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more susceptible to necrosis and a rise in intracellular calcium is an important initiating event in dystrophic muscle pathogenesis. In the mdx mice, muscles are affected with different intensities and some muscles are spared. We investigated the levels of the calcium-binding proteins calsequestrin and calmodulin in the non-spared axial (sternomastoid and diaphragm), limb (tibialis anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of control and mdx mice. Immunoblotting analysis showed a significant increase of the proteins in the spared mdx EOM and a significant decrease in the most affected diaphragm. Both proteins were comparable to the cardiac muscle controls. In limb and sternomastoid muscles, calmodulin and calsequestrin were affected differently. These results suggest that differential levels of the calcium-handling proteins may be involved in the pathogenesis of myonecrosis in mdx muscles. Understanding the signaling mechanisms involving Ca(2+)-calmodulin activation and calsequestrin expression may be a valuable way to develop new therapeutic approaches to the dystrophinopaties.

  17. Effect of the IL-1 Receptor Antagonist Kineret® on Disease Phenotype in mdx Mice

    PubMed Central

    Benny Klimek, Margaret E.; Sali, Arpana; Rayavarapu, Sree; Van der Meulen, Jack H.; Nagaraju, Kanneboyina

    2016-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by mutations in the dystrophin gene. The pathology of DMD manifests in patients with progressive muscle weakness, loss of ambulation and ultimately death. One of the characteristics of DMD is muscle inflammation, and dystrophin-deficient skeletal muscles produce higher levels of the pro-inflammatory cytokine interleukin 1β (IL-1β) in response to toll like receptor (TLR) stimulation compared to controls; therefore, blocking the IL-1β pathway could improve the disease phenotype in mdx mice, a mouse model of DMD. Kineret® or IL-1Ra is a recombinant IL-1 receptor antagonist approved by the FDA for treating rheumatoid arthritis. To determine the efficacy of IL-1Ra in a DMD model, we administered subcutaneous injections of saline control or IL-1Ra (25 mg/kg/day) to mdx mice daily for 45 days beginning at 5 weeks of age. Functional and histological parameters were measured at the conclusion of the study. IL-1Ra only partially inhibited this signaling pathway in this study; however, there were still interesting observations to be noted. For example, although not significantly changed, splenocytes from the IL-1Ra-treated group secreted less IL-1β after LPS stimulation compared to control mice indicating a blunted response and incomplete inhibition of the pathway (37% decrease). In addition, normalized forelimb grip strength was significantly increased in IL-1Ra-treated mice. There were no changes in EDL muscle-specific force measurements, histological parameters, or motor coordination assessments in the dystrophic mice after IL-1Ra treatment. There was a significant 27% decrease in the movement time and total distance traveled by the IL-1Ra treated mice, correlating with previous studies examining effects of IL-1 on behavior. Our studies indicate partial blocking of IL-1β with IL-1Ra significantly altered only a few behavioral and strength related disease parameters; however, treatment with

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

    PubMed

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

    2012-05-01

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

  19. The effect of taurine and β-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice.

    PubMed

    Horvath, Deanna M; Murphy, Robyn M; Mollica, Janelle P; Hayes, Alan; Goodman, Craig A

    2016-11-01

    This study investigated the effect of taurine and β-alanine supplementation on muscle function and muscle taurine transporter (TauT) protein expression in mdx mice. Wild-type (WT) and mdx mice (5 months) were supplemented with taurine or β-alanine for 4 weeks, after which in vitro contractile properties, fatigue resistance and force recovery, and the expression of the TauT protein and proteins involved in excitation-contraction (E-C) coupling were examined in fast-twitch muscle. There was no difference in basal TauT protein expression or basal taurine content between mdx than WT muscle. Supplementation with taurine and β-alanine increased and reduced taurine content, respectively, in muscle from WT and mdx mice but had no effect of TauT protein. Taurine supplementation reduced body and muscle mass, and enhanced fatigue resistance and force recovery in mdx muscle. β-Alanine supplementation enhanced fatigue resistance in WT and mdx muscle. There was no difference in the basal expression of key E-C coupling proteins [ryanodine receptor 1 (RyR1), dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca(2+)-ATPase 1 (SERCA1) or calsequestrin 1 (CSQ1)] between WT and mdx mice, and the expression of these proteins was not altered by taurine or β-alanine supplementation. These findings suggest that TauT protein expression is relatively insensitive to changes in muscle taurine content in WT and mdx mice, and that taurine and β-alanine supplementation may be viable therapeutic strategies to improve fatigue resistance of dystrophic skeletal muscle.

  20. Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1.

    PubMed

    De Luca, Annamaria; Pierno, Sabata; Liantonio, Antonella; Cetrone, Michela; Camerino, Claudia; Fraysse, Bodvael; Mirabella, Massimo; Servidei, Serenella; Rüegg, Urs T; Conte Camerino, Diana

    2003-01-01

    A preclinical screening for prompt-to-use drugs that are safer than steroids and beneficial in Duchenne muscular dystrophy was performed. Compounds able to reduce calcium-induced degeneration (taurine or creatine 10% in chow) or to stimulate regeneration [insulin-like growth factor-1 (IGF-1); 50 or 500 microg/kg s.c.] were administered for 4 to 8 weeks to mdx mice undergoing chronic exercise on a treadmill, a protocol to worsen dystrophy progression. alpha-Methyl-prednisolone (PDN; 1 mg/kg) was used as positive control. The effects were evaluated in vivo on forelimb strength and in vitro electrophysiologically on the macroscopic chloride conductance (gCl), an index of degeneration-regeneration events in mdx muscles, and on the mechanical threshold, a calcium-sensitive index of excitation-contraction coupling. The exercise produced a significant weakness and an impairment of gCl, by further decreasing the already low value of degenerating diaphragm (DIA) and fully hampering the increase of gCl typical of regenerating extensor digitorum longus (EDL) mdx muscle. The already negative voltage threshold for contraction of mdx EDL was also slightly worsened. Taurine > creatine > IGF-1 counteracted the exercise-induced weakness. The amelioration of gCl was drug- and muscle-specific: taurine was effective in EDL, but not in DIA muscle; IGF-1 and PDN were fully restorative in both muscles, whereas creatine was ineffective. An acute effect of IGF-1 on gCl was observed in vitro in untreated, but not in IGF-1-treated exercised mdx muscles. Taurine > PDN > IGF-1, but not creatine, significantly ameliorated the negative threshold voltage values of the EDL fibers. The results predict a potential benefit of taurine and IGF-1 for treating human dystrophy.

  1. Pregnancy-Induced Amelioration of Muscular Dystrophy Phenotype in mdx Mice via Muscle Membrane Stabilization Effect of Glucocorticoid

    PubMed Central

    Shimizu-Motohashi, Yuko; Asakura, Yoko; Motohashi, Norio; Belur, Nandkishore R.; Baumrucker, Michael G.; Asakura, Atsushi

    2015-01-01

    Duchenne muscular dystrophy (DMD), the most common and severe type of dystrophinopathy, is an X-linked recessive genetic disease caused by the absence of dystrophin, which leads to fragility and vulnerability of the sarcolemma to mechanical stretching with increased membrane permeability. Currently, glucocorticoids such as prednisolone are the only medication available for DMD. However, molecular pathways responsible for this effect are still unclear. In addition, it remains unclear whether sex-related factors, including pregnancy and the postpartum period, affect the phenotype of dystrophinopathy. Here, we report the amelioration of muscle membrane permeability in the diaphragm muscle of pregnant and postpartum, but not in nulliparous, mdx mice, an animal model for DMD, during the physiological surge of corticosterone, the most abundant glucocorticoid in rodents. Cultures of single muscle fibers and myotubes isolated from mdx mouse diaphragm demonstrate resistance to hypo-osmotic shock when treated with corticosterone but not with estradiol or progesterone. This corticosterone-mediated resistance was diminished by an antagonist of corticosterone, indicating that the glucocorticoid-glucocorticoid receptor axis plays a role in this membrane stabilization effect on muscle. Moreover, subcutaneous injection of corticosterone into mdx mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in mdx mice due to the membrane stabilization effect of corticosterone. We also propose that this membrane stabilization effect is exerted through annexin A1 up-regulation as the molecular mechanisms of glucocorticoid effects on DMD muscle. Furthermore, single muscle fiber culture studies provide a sensitive chemical screening platform for muscular dystrophies. PMID:25775477

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

    PubMed

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

    2014-04-01

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

  3. Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice.

    PubMed

    Nakatani, Masashi; Takehara, Yuka; Sugino, Hiromu; Matsumoto, Mitsuru; Hashimoto, Osamu; Hasegawa, Yoshihisa; Murakami, Tatsuya; Uezumi, Akiyoshi; Takeda, Shin'ichi; Noji, Sumihare; Sunada, Yoshihide; Tsuchida, Kunihiro

    2008-02-01

    Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin-related proteins, and myostatin antibodies. Although follistatin shows potent myostatin-inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (K(d)) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (K(d)) of a follistatin-derived myostatin inhibitor, designated FS I-I, to activin and myostatin are 64.3 microM and 46.8 nM, respectively. Transgenic mice expressing FS I-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I-I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I-I mice. These results indicate that myostatin blockade by FS I-I has a therapeutic potential for muscular dystrophy.

  4. Myocardial fibrosis is unaltered by long-term administration of L-arginine in dystrophin deficient mdx mice: a histomorphometric analysis.

    PubMed

    Marques, Maria Julia; Barbin, Isabel Cristina Chagas; Taniguti, Ana Paula Tiemi; Oggian, Daniela Silva; Ferretti, R; Santo Neto, H

    2010-06-01

    Cardiac failure secondary to myocardial fibrosis (MF) significantly contributes to death in Duchenne muscular dystrophy (DMD), a fatal form of muscle disease. In aging, the mdx mice, an animal model of DMD, MF is similar to that observed in humans. Nitric oxide-based therapy has been proposed to retard MF in DMD and a candidate is L-arginine (L-arg). In this study we evaluated the effects of long-term therapy with L-arg in the MF of mdx mice. mdx mice (6 months old) were treated with L-arg in drinking water. Control mdx mice received water only. After 15 months of treatment, hearts were stained with Masson's trichrome for analysis of MF and with hematoxilyn and eosin for analysis of inflammation and cardiomyocyte damage. We observed that MF was not affected (29.5 +/- 2.5% of MF area for control vs 31.4 +/- 2% for L-arginine-treated animals; P > 0.05). The density of inflammatory cells was reduced (169 +/- 12 cells/mm 2 in control vs 102 +/- 9 cells/mm 2 in L-arg-treated; P < 0.05). The present study shows that long-term administration of L-arg is not effective in retarding MF in mdx dystrophinopathy.

  5. A muscle resident cell population promotes fibrosis in hindlimb skeletal muscles of mdx mice through the Wnt canonical pathway.

    PubMed

    Trensz, Frédéric; Haroun, Sonia; Cloutier, Alexandre; Richter, Martin V; Grenier, Guillaume

    2010-11-01

    Previous work has pointed to a role for the Wnt canonical pathway in fibrosis formation in aged skeletal muscles. In the present study, we studied the dystrophic mdx mouse, which displays skeletal muscle fibrosis. Our results indicated that the muscle resident stromal cell (mrSC) population in the muscles of dystrophic mice is higher than in the muscles of age-matched wild-type mice. Wnt3a promoted the proliferation of and collagen expression by cultured mrSCs but arrested the growth of and collagen expression by cultured myoblasts. Injections of Wnt3A in the tibialis anterior muscles of adult wild-type mice significantly enhanced the mrSC population and collagen deposition compared with the contralateral muscles. Conversely, an injection of the Wnt antagonist Dickkof protein (DKK1) into the skeletal muscles of mdx mice significantly reduced collagen deposition. These results suggested that the Wnt canonical pathway expands the population of mrSCs and stimulates their production of collagen as observed during aging and in various myopathies.

  6. Therapeutic effects of mouse adipose-derived stem cells and losartan in the skeletal muscle of injured mdx mice.

    PubMed

    Lee, Eun-Mi; Kim, Ah-Young; Lee, Eun-Joo; Park, Jin-Kyu; Lee, Myeong-Mi; Hwang, Meeyul; Kim, Choong-Yong; Kim, Shin-Yoon; Jeong, Kyu-Shik

    2015-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder caused by mutations in the dystrophin gene. Adipose-derived stem cells (ASCs) are an attractive source of cells for stem cell therapy. Losartan has been reported to improve ASC transplantation in injured mouse muscles. In the present study, we investigated whether the combined treatment of losartan and ASCs in the injured muscles of mdx mice improves regeneration. The combined treatment of ASCs and losartan remarkably improved muscle regeneration and induced muscle hypertrophy. In addition, ASCs and losartan treatment downregulated transforming growth factor-β and inhibited muscle fibrosis. We observed cells coexpressing green fluorescent protein (GFP) and dystrophin in the muscle samples of mice transplanted with GFP-positive ASCs. In the coculture in vitro experiment, we also observed that the GFP ASCs differentiated into dystrophin-expressing myotubes. The present study shows that the combination of transplanted ASCs and treatment with losartan ameliorated muscle fibrosis and improved muscle regeneration in injured mdx mice. Thus, we suggest that combined treatment with losartan and ASCs could help to improve muscle regeneration in the muscles of injured patients, including DMD patients.

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

    PubMed

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

    2015-01-15

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

  8. Combination of Myostatin Pathway Interference and Dystrophin Rescue Enhances Tetanic and Specific Force in Dystrophic mdx Mice

    PubMed Central

    Dumonceaux, Julie; Marie, Solenne; Beley, Cyriaque; Trollet, Capucine; Vignaud, Alban; Ferry, Arnaud; Butler-Browne, Gillian; Garcia, Luis

    2010-01-01

    Duchenne muscular dystrophy is characterized by muscular atrophy, fibrosis, and fat accumulation. Several groups have demonstrated that in the mdx mouse, the exon-skipping strategy can restore a quasi-dystrophin in almost 100% of the muscle fibers. On the other hand, inhibition of the myostatin pathway in adult mice has been described to enhance muscle growth and improve muscle force. Our aim was to combine these two strategies to evaluate a possible additive effect. We have chosen to inhibit the myostatin pathway using the technique of RNA interference directed against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). The restoration of a quasi-dystrophin was mediated by the vectorized U7 exon-skipping technique (U7-DYS). Adeno-associated vectors carrying either the sh-AcvrIIb construct alone, the U7-DYS construct alone, or a combination of both constructs were injected in the tibialis anterior (TA) muscle of dystrophic mdx mice. We show that even if each separate approach has some effects on muscle physiology, the combination of the dystrophin rescue and the downregulation of the myostatin receptor is required to massively improve both the tetanic force and the specific force. This study provides a novel pharmacogenetic strategy for treatment of certain neuromuscular diseases associated with muscle wasting. PMID:20104211

  9. Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice.

    PubMed

    Matsumura, Cíntia Yuri; Taniguti, Ana Paula Tiemi; Pertille, Adriana; Santo Neto, Humberto; Marques, Maria Julia

    2011-12-01

    In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.

  10. Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice.

    PubMed

    Martin, Paul T; Xu, Rui; Rodino-Klapac, Louise R; Oglesbay, Elaine; Camboni, Marybeth; Montgomery, Chrystal L; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Sahenk, Zarife; Mendell, Jerry R; Janssen, Paul M L

    2009-03-01

    The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:beta1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcbeta1,4[NeuAc(orGc)alpha2, 3]Galbeta1,4GlcNAcbeta-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications.

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

    PubMed Central

    Todorov, Teodor

    2016-01-01

    The most widespread animal model to investigate Duchenne muscular dystrophy is the mdx-mouse. In contrast to humans, phases of muscle degeneration are replaced by regeneration processes; hence there is only a restricted time slot for research. The aim of the study was to investigate if an intramuscular injection of BTX-A is able to break down muscle regeneration and has direct implications on the gene expression of myosin heavy chains in the corresponding treated and untreated muscles. Therefore, paralysis of the right masseter muscle was induced in adult healthy and dystrophic mice by a specific intramuscular injection of BTX-A. After 21 days the mRNA expression and protein content of MyHC isoforms of the right and left masseter, temporal, and the tongue muscle were determined using quantitative RT-PCR and Western blot technique. MyHC-IIa and MyHC-I-mRNA expression significantly increased in the paralyzed masseter muscle of control-mice, whereas MyHC-IIb and MyHC-IIx/d-mRNA were decreased. In dystrophic muscles no effect of BTX-A could be detected at the level of MyHC. This study suggests that BTX-A injection is a suitable method to simulate DMD-pathogenesis in healthy mice but further investigations are necessary to fully analyse the BTX-A effect and to generate sustained muscular atrophy in mdx-mice. PMID:27689088

  12. Compared with that of MUFA, a high dietary intake of n-3 PUFA does not reduce the degree of pathology in mdx mice.

    PubMed

    Henderson, Gregory C; Evans, Nicholas P; Grange, Robert W; Tuazon, Marc A

    2014-05-28

    Duchenne muscular dystrophy (DMD) is a severe muscle disease that affects afflicted males from a young age, and the mdx mouse is an animal model of this disease. Although new drugs are in development, it is also essential to assess potential dietary therapies that could assist in the management of DMD. In the present study, we compared two diets, high-MUFA diet v. high-PUFA diet, in mdx mice. To generate the high-PUFA diet, a portion of dietary MUFA (oleic acid) was replaced with the dietary essential n-3 PUFA α-linolenic acid (ALA). We sought to determine whether ALA, compared with oleic acid, was beneficial in mdx mice. Consumption of the high-PUFA diet resulted in significantly higher n-3 PUFA content and reduced arachidonic acid content in skeletal muscle phospholipids (PL), while the high-MUFA diet led to higher oleate content in PL. Mdx mice on the high-MUFA diet exhibited 2-fold lower serum creatine kinase activity than those on the high-PUFA diet (P< 0·05) as well as a lower body fat percentage (P< 0·05), but no significant difference in skeletal muscle histopathology results. There was no significant difference between the dietary groups with regard to phosphorylated p65 (an inflammatory marker) in skeletal muscle. In conclusion, alteration of PL fatty acid (FA) composition by the high-PUFA diet made mdx muscle more susceptible to sarcolemmal leakiness, while the high-MUFA diet exhibited a more favourable impact. These results may be important for designing dietary treatments for DMD patients, and future work on dietary FA profiles, such as comparing other FA classes and dose effects, is needed.

  13. Granulocyte colony-stimulating factor (G-CSF) positive effects on muscle fiber degeneration and gait recovery after nerve lesion in MDX mice

    PubMed Central

    Simões, Gustavo F; Benitez, Suzana U; Oliveira, Alexandre L R

    2014-01-01

    Background G-CSF has been shown to decrease inflammatory processes and to act positively on the process of peripheral nerve regeneration during the course of muscular dystrophy. Aims The aims of this study were to investigate the effects of treatment of G-CSF during sciatic nerve regeneration and histological analysis in the soleus muscle in MDX mice. Methods Six-week-old male MDX mice underwent left sciatic nerve crush and were G-CSF treated at 7 days prior to and 21 days after crush. Ten and twenty-one days after surgery, the mice were euthanized, and the sciatic nerves were processed for immunohistochemistry (anti-p75NTR and anti-neurofilament) and transmission electron microscopy. The soleus muscles were dissected out and processed for H&E staining and subsequent morphologic analysis. Motor function analyses were performed at 7 days prior to and 21 days after sciatic crush using the CatWalk system and the sciatic nerve index. Results Both groups treated with G-CSF showed increased p75NTR and neurofilament expression after sciatic crush. G-CSF treatment decreased the number of degenerated and regenerated muscle fibers, thereby increasing the number of normal muscle fibers. Conclusions The reduction in p75NTR and neurofilament indicates a decreased regenerative capacity in MDX mice following a lesion to a peripheral nerve. The reduction in motor function in the crushed group compared with the control groups may reflect the cycles of muscle degeneration/regeneration that occur postnatally. Thus, G-CSF treatment increases motor function in MDX mice. Nevertheless, the decrease in baseline motor function in these mice is not reversed completely by G-CSF. PMID:25328849

  14. Unloaded speed of shortening in voltage-clamped intact skeletal muscle fibers from wt, mdx, and transgenic minidystrophin mice using a novel high-speed acquisition system.

    PubMed

    Friedrich, O; Weber, C; von Wegner, F; Chamberlain, J S; Fink, R H A

    2008-06-01

    Skeletal muscle unloaded shortening has been indirectly determined in the past. Here, we present a novel high-speed optical tracking technique that allows recording of unloaded shortening in single intact, voltage-clamped mammalian skeletal muscle fibers with 2-ms time resolution. L-type Ca(2+) currents were simultaneously recorded. The time course of shortening was biexponential: a fast initial phase, tau(1), and a slower successive phase, tau(2,) with activation energies of 59 kJ/mol and 47 kJ/mol. Maximum unloaded shortening speed, v(u,max), was faster than that derived using other techniques, e.g., approximately 14.0 L(0) s(-1) at 30 degrees C. Our technique also allowed direct determination of shortening acceleration. We applied our technique to single fibers from C57 wild-type, dystrophic mdx, and minidystrophin-expressing mice to test whether unloaded shortening was affected in the pathophysiological mechanism of Duchenne muscular dystrophy. v(u,max) and a(u,max) values were not significantly different in the three strains, whereas tau(1) and tau(2) were increased in mdx fibers. The results were complemented by myosin heavy and light chain (MLC) determinations that showed the same myosin heavy chain IIA profiles in the interossei muscles from the different strains. In mdx muscle, MLC-1f was significantly increased and MLC-2f and MLC-3f somewhat reduced. Fast initial active shortening seems almost unaffected in mdx muscle.

  15. Lack of dystrophin leads to the selective loss of superior cervical ganglion neurons projecting to muscular targets in genetically dystrophic mdx mice.

    PubMed

    De Stefano, M Egle; Leone, Lucia; Lombardi, Loredana; Paggi, Paola

    2005-12-01

    Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.

  16. Proteasome inhibitor (MG132) rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx (5cv) mice.

    PubMed

    Rougier, Jean-Sébastien; Gavillet, Bruno; Abriel, Hugues

    2013-01-01

    The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (l Na) were both decreased in cardiomyocytes of dystrophin-deficient mdx (5cv) mice. In this study, wild-type and mdx (5cv) mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and l Na but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

  17. Age-related changes in dystrophin-glycoprotein complex and in utrophin are not correlated with intrinsic laryngeal muscles protection in mdx mice.

    PubMed

    Ferretti, Renato; Pertille, Adriana; Santo Neto, Humberto; Marques, Maria Julia

    2011-12-01

    In this study we investigate whether dystrophic intrinsic laryngeal muscles (ILM) from aged mdx mice show alterations in dystrophin-glycoprotein complex (DGC) components.Immunofluorescence and immunoblotting analyses of beta-sarcoglycan, beta-dystroglycan, and utrophin showed that aged ILM had a similar pattern of changes in aged affected muscles (diaphragm and limb), suggesting that aging leads to changes in utrophin and DGC proteins in dystrophic ILM that cannot be correlated with their protection from dystrophic change.

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

    Matsuzaka, Yasunari; Tanihata, Jun; Komaki, Hirofumi; Ishiyama, Akihiko; Oya, Yasushi; Rüegg, Urs; Takeda, Shin-ichi; Hashido, Kazuo

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs), is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration. PMID:27977725

  1. Enhanced sensitivity of hippocampal pyramidal neurons from mdx mice to hypoxia-induced loss of synaptic transmission.

    PubMed Central

    Mehler, M F; Haas, K Z; Kessler, J A; Stanton, P K

    1992-01-01

    The gene at the Duchenne/Becker muscular dystrophy locus encodes dystrophin, a member of a protein superfamily that links the actin cytoskeleton to transmembrane plasmalemmal proteins. In mature skeletal myocytes, the absence of dystrophin is associated with decreased membrane stability, altered kinetics of several calcium channels, and increased intracellular calcium concentration. In the central nervous system, dystrophin is restricted to specific neuronal populations that show heightened susceptibility to excitotoxic damage and is localized in proximal dendrites and the neuronal somata. We report that CA1 pyramidal neurons in a hippocampal slice preparation from a dystrophin-deficient mouse genetic model of Duchenne muscular dystrophy (the mdx mouse) exhibit significant increased susceptibility to hypoxia-induced damage to synaptic transmission. This selective vulnerability was substantially ameliorated by pretreatment with diphenylhydantoin, an anticonvulsant that blocks both sodium-dependent action potentials and low-threshold transient calcium conductances. These findings suggest that dystrophin deficiency could predispose susceptible neuronal populations to cumulative hypoxic insults that may contribute to the development of cognitive deficits in Duchenne/Becker muscular dystrophy patients and that the effects of such periods of hypoxia may be pharmacologically remediable. PMID:1549609

  2. D-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity.

    PubMed

    Reay, Daniel P; Bastacky, Sheldon I; Wack, Kathryn E; Stolz, Donna B; Robbins, Paul D; Clemens, Paula R

    2015-05-22

    In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using L-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring L-isoform peptide due to the longer persistence of the D-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of L- and D-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both L- or D-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the D-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.

  3. Human adipose tissue derived pericytes increase life span in Utrn (tm1Ked) Dmd (mdx) /J mice.

    PubMed

    Valadares, M C; Gomes, J P; Castello, G; Assoni, A; Pellati, M; Bueno, C; Corselli, M; Silva, H; Bartolini, P; Vainzof, M; Margarido, P F; Baracat, E; Péault, B; Zatz, M

    2014-12-01

    Duchenne muscular dystrophy (DMD) is still an untreatable lethal X-linked disorder, which affects 1 in 3500 male births. It is caused by the absence of muscle dystrophin due to mutations in the dystrophin gene. The potential regenerative capacity as well as immune privileged properties of mesenchymal Stem Cells (MSC) has been under investigation for many years in an attempt to treat DMD. One of the questions to be addressed is whether stem cells from distinct sources have comparable clinical effects when injected in murine or canine muscular dystrophy animal models. Many studies comparing different stem cells from various sources were reported but these cells were obtained from different donors and thus with different genetic backgrounds. Here we investigated whether human pericytes obtained from 4 different tissues (muscle, adipose tissue, fallopian tube and endometrium) from the same donor have a similar clinical impact when injected in double mutant Utrn (tm1Ked) Dmd (mdx) /J mice, a clinically relevant model for DMD. After a weekly regimen of intraperitoneal injections of 10(6) cells per 8 weeks we evaluated the motor ability as well as the life span of the treated mice as compared to controls. Our experiment showed that only adipose tissue derived pericytes are able to increase significantly (39 days on average) the life span of affected mice. Microarray analysis showed an inhibition of the interferon pathway by adipose derived pericytes. Our results suggest that the clinical benefit associated with intraperitoneal injections of these adult stem cells is related to immune modulation rather than tissue regeneration.

  4. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

    PubMed Central

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-01-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  5. Dystrophic changes in extraocular muscles after gamma irradiation in mdx:utrophin(+/-) mice.

    PubMed

    McDonald, Abby A; Kunz, Matthew D; McLoon, Linda K

    2014-01-01

    Extraocular muscles (EOM) have a strikingly different disease profile than limb skeletal muscles. It has long been known that they are spared in Duchenne (DMD) and other forms of muscular dystrophy. Despite many studies, the cause for this sparing is not understood. We have proposed that differences in myogenic precursor cell properties in EOM maintain normal morphology over the lifetime of individuals with DMD due to either greater proliferative potential or greater resistance to injury. This hypothesis was tested by exposing wild type and mdx:utrophin(+/-) (het) mouse EOM and limb skeletal muscles to 18 Gy gamma irradiation, a dose known to inhibit satellite cell proliferation in limb muscles. As expected, over time het limb skeletal muscles displayed reduced central nucleation mirrored by a reduction in Pax7-positive cells, demonstrating a significant loss in regenerative potential. In contrast, in the first month post-irradiation in the het EOM, myofiber cross-sectional areas first decreased, then increased, but ultimately returned to normal compared to non-irradiated het EOM. Central nucleation significantly increased in the first post-irradiation month, resembling the dystrophic limb phenotype. This correlated with decreased EECD34 stem cells and a concomitant increase and subsequent return to normalcy of both Pax7 and Pitx2-positive cell density. By two months, normal het EOM morphology returned. It appears that irradiation disrupts the normal method of EOM remodeling, which react paradoxically to produce increased numbers of myogenic precursor cells. This suggests that the EOM contain myogenic precursor cell types resistant to 18 Gy gamma irradiation, allowing return to normal morphology 2 months post-irradiation. This supports our hypothesis that ongoing proliferation of specialized regenerative populations in the het EOM actively maintains normal EOM morphology in DMD. Ongoing studies are working to define the differences in the myogenic precursor cells

  6. Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the αMβ2 leukocyte integrin receptor.

    PubMed

    Vidal, Berta; Ardite, Esther; Suelves, Mònica; Ruiz-Bonilla, Vanessa; Janué, Anna; Flick, Matthew J; Degen, Jay L; Serrano, Antonio L; Muñoz-Cánoves, Pura

    2012-05-01

    In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)β(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)β(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)β(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)β(2) interactions may provide a novel strategy for DMD treatment.

  7. Comparative study of inorganic elements determined in whole blood from Dmd(mdx)/J mice strain by EDXRF and NAA analytical techniques.

    PubMed

    Redígolo, M M; Sato, I M; Metairon, S; Zamboni, C B

    2016-04-01

    Several diseases can be diagnosed observing the variation of specific elements concentration in body fluids. In this study the concentration of inorganic elements in blood samples of dystrophic (Dmd(mdx)/J) and C57BL/6J (control group) mice strain were determined. The results obtained from Energy Dispersive X-ray Fluorescence (EDXRF) were compared with Neutron Activation Analysis (NAA) technique. Both analytical techniques showed to be appropriate and complementary offering a new contribution for veterinary medicine as well as detailed knowledge of this pathology.

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

    PubMed

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

    2013-09-01

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

  9. Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

    PubMed

    De Luca, Annamaria; Nico, Beatrice; Rolland, Jean-François; Cozzoli, Anna; Burdi, Rosa; Mangieri, Domenica; Giannuzzi, Viviana; Liantonio, Antonella; Cippone, Valentina; De Bellis, Michela; Nicchia, Grazia Paola; Camerino, Giulia Maria; Frigeri, Antonio; Svelto, Maria; Camerino, Diana Conte

    2008-11-01

    Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.

  10. Evaluation of Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked polyethylenimine as antisense morpholino oligomer delivery vehicle in cell culture and dystrophic mdx mice.

    PubMed

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

    2014-05-01

    Hyperbranched poly(ester amine)s (PEAs) based on tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) cross-linked low-molecular-weight polyethylenimine (Mw: 0.8k/1.2k/2.0k) have been evaluated for delivering antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in vivo in the dystrophic mdx mouse. The results show that the PEAs constructed with polyethylenimine (PEI) 2.0k (C series) improved PMO delivery more efficiently than those constructed with PEI 0.8k (A series) or 1.2k (B series) in a GFP reporter-based C2C12 mouse myoblast culture system. The highest efficiency of exon-skipping in vitro with the PMO oligonucleotide targeting human dystrophin exon 50 was obtained when the PEA C12 [TAEI-PEI 2.0k (1:2)] was used. Nearly all of the PEAs improved dystrophin expression in mdx mice by local injection with a 2-4-fold increase when compared with PMO alone. Improved transfection efficiency and lower toxicity indicate the potential of the biodegradable PEA polymers as safe and efficient PMO delivery vectors for in vivo applications.

  11. Isobaric Tagging-Based Quantification for Proteomic Analysis: A Comparative Study of Spared and Affected Muscles from mdx Mice at the Early Phase of Dystrophy

    PubMed Central

    Durbeej, Madeleine; Marques, Maria Julia

    2013-01-01

    Duchenne muscular dystrophy (DMD) is the most common childhood myopathy, characterized by muscle loss and cardiorespiratory failure. While the genetic basis of DMD is well established, secondary mechanisms associated with dystrophic pathophysiology are not fully clarified yet. In order to obtain new insights into the molecular mechanisms of muscle dystrophy during earlier stages of the disease, we performed a comparative proteomic profile of the spared extraocular muscles (EOM) vs. affected diaphragm from the mdx mice, using a label based shotgun proteomic approach. Out of the 857 identified proteins, 42 to 62 proteins had differential abundance of peptide ions. The calcium-handling proteins sarcalumenin and calsequestrin-1 were increased in control EOM compared with control DIA, reinforcing the view that constitutional properties of EOM are important for their protection against myonecrosis. The finding that galectin-1 (muscle regeneration), annexin A1 (anti-inflammatory) and HSP 47 (fibrosis) were increased in dystrophic diaphragm provides novel insights into the mechanisms through which mdx affected muscles are able to counteract dystrophy, during the early stage of the disease. Overall, the shotgun technique proved to be suitable to perform quantitative comparisons between distinct dystrophic muscles and allowed the suggestion of new potential biomarkers and drug targets for dystrophinopaties. PMID:23823696

  12. Muscle weakness and atrophy are associated with decreased regenerative capacity and changes in mTOR signaling in skeletal muscles of venerable (18-24-month-old) dystrophic mdx mice.

    PubMed

    Mouisel, E; Vignaud, A; Hourdé, C; Butler-Browne, G; Ferry, A

    2010-06-01

    The muscles of mdx mice progressively deteriorate with age. We wanted to know whether this is associated with a decrease in regenerative capacity and/or changes in the mammalian target of rapamycin complex (mTOR) signaling pathway. Muscles of mdx mice aged 5 weeks, 5, 12, and 18-24 months were studied. Maximal force and muscle weight of the older mice were decreased as compared to younger adult mice. Activation of the mTOR signaling pathway, i.e., phosphorylation of Akt (also known as protein kinase B) and ribosomal protein S6 was also reduced in the older mice. Moreover, 14 days after cardiotoxin injury the degree of recovery of maximal force and muscle weight were less in the older mice. In contrast to younger mice, there was also activation of the mTOR pathway during regeneration in the older mice. Progressive muscle weakness and atrophy in mdx mouse muscle is associated with a decline in regenerative potential and changes in activation of the mTOR signaling pathway.

  13. AAV micro-dystrophin gene therapy alleviates stress-induced cardiac death but not myocardial fibrosis in >21-m-old mdx mice, an end-stage model of Duchenne muscular dystrophy cardiomyopathy.

    PubMed

    Bostick, Brian; Shin, Jin-Hong; Yue, Yongping; Wasala, Nalinda B; Lai, Yi; Duan, Dongsheng

    2012-08-01

    Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by the absence of the sarcolemmal protein dystrophin. Dilated cardiomyopathy leading to heart failure is a significant source of morbidity and mortality in DMD. We recently demonstrated amelioration of DMD heart disease in 16 to 20-m-old dystrophin-null mdx mice using adeno-associated virus (AAV) mediated micro-dystrophin gene therapy. DMD patients show severe heart disease near the end of their life expectancy. Similarly, mdx mice exhibit profoundly worsening heart disease when they reach beyond 21 months of age. To more rigorously test micro-dystrophin therapy, we treated mdx mice that were between 21.2 and 22.7-m-old (average, 22.1 ± 0.2 months; N=8). The ∆R4-23/∆C micro-dystrophin gene was packaged in the cardiotropic AAV-9 virus. 5×10(12) viral genome particles/mouse were delivered to mdx mice via the tail vein. AAV transduction, myocardial fibrosis and heart function were examined 1.7 ± 0.2 months after gene therapy. Efficient micro-dystrophin expression was observed in the myocardium of treated mice. Despite the robust dystrophin expression, myocardial fibrosis was not mitigated. Most hemodynamic parameters were not improved either. However, ECG abnormalities were partially corrected. Importantly, treated mice became more resistant to dobutamine-induced cardiac death. In summary, we have revealed for the first time the potential benefits and limitations of AAV micro-dystrophin therapy in end-stage Duchenne dilated cardiomyopathy. Our findings have important implications for the use of AAV gene therapy in dilated cardiomyopathy and heart failure.

  14. Electrical Stimuli Are Anti-Apoptotic in Skeletal Muscle via Extracellular ATP. Alteration of This Signal in Mdx Mice Is a Likely Cause of Dystrophy

    PubMed Central

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. PMID:24282497

  15. Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy.

    PubMed

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies.

  16. Differential expression of utrophin-A and -B promoters in the central nervous system (CNS) of normal and dystrophic mdx mice.

    PubMed

    Baby, Santhosh M; Bogdanovich, Sasha; Willmann, Gabriel; Basu, Utpal; Lozynska, Olga; Khurana, Tejvir S

    2010-03-01

    Utrophin (Utrn) is the autosomal homolog of dystrophin, the Duchene Muscular Dystrophy (DMD) locus product and of therapeutic interest, as its overexpression can compensate dystrophin's absence. Utrn is transcribed by Utrn-A and -B promoters with mRNAs differing at their 5' ends. However, previous central nervous system (CNS) studies used C-terminal antibodies recognizing both isoforms. As this distinction may impact upregulation strategies, we generated Utrn-A and -B promoter-specific antibodies, Taqman Polymerase chain reaction (PCR)-based absolute copy number assays, and luciferase-reporter constructs to study CNS of normal and dystrophic mdx mice. Differential expression of Utrn-A and -B was noted in microdissected and capillary-enriched fractions. At the protein level, Utrn-B was predominantly expressed in vasculature and ependymal lining, whereas Utrn-A was expressed in neurons, astrocytes, choroid plexus and pia mater. mRNA quantification demonstrated matching patterns of differential expression; however, transcription-translation mismatch was noted for Utrn-B in caudal brain regions. Utrn-A and Utrn-B proteins were significantly upregulated in olfactory bulb and cerebellum of mdx brain. Differential promoter activity, mRNA and protein expressions were studied in cultured C2C12, bEnd3, neurons and astrocytes. Promoter activity ranking for Utrn-A and -B was neurons > astrocytes > C2C12 > bEnd3 and bEnd3 > astrocytes > neurons > C2C12, respectively. Our results identify promoter usage patterns for therapeutic targeting and define promoter-specific differential distribution of Utrn isoforms in normal and dystrophic CNS.

  17. Polytrauma Increases Susceptibility to Pseudomonas Pneumonia in Mature Mice.

    PubMed

    Turnbull, Isaiah R; Ghosh, Sarbani; Fuchs, Anja; Hilliard, Julia; Davis, Christopher G; Bochicchio, Grant V; Southard, Robert E

    2016-05-01

    Pneumonia is the most common complication observed in patients with severe injuries. Although the average age of injured patients is 47 years, existing studies of the effect of injury on the susceptibility to infectious complications have focused on young animals, equivalent to a late adolescent human. We hypothesized that mature adult animals are more susceptible to infection after injury than younger counterparts. To test this hypothesis, we challenged 6 to 8-month-old mature mice to a polytrauma injury followed by Pseudomonas aeruginosa pneumonia and compared them to young (8-10-week-old) animals. We demonstrate that polytrauma injury increases mortality from pneumonia in mature animals (sham-pneumonia 21% vs. polytrauma-pneumonia 62%) but not younger counterparts. After polytrauma, pneumonia in mature mice is associated with higher bacterial burden in lung, increased incidence of bacteremia, and elevated levels of bacteria in the blood, demonstrating that injury decreases the ability to control the infectious challenge. We further find that polytrauma did not induce elevations in circulating cytokine levels (TNF-alpha, IL-6, KC, and IL-10) 24  h after injury. However, mature mice subjected to polytrauma demonstrated an exaggerated circulating inflammatory cytokine response to subsequent Pseudomonas pneumonia. Additionally, whereas prior injury increases LPS-stimulated IL-6 production by peripheral blood leukocytes from young (8-10-week-old) mice, injury does not prime IL-6 production by cell from mature adult mice. We conclude that in mature mice polytrauma results in increased susceptibility to Pseudomonas pneumonia while priming an exaggerated but ineffective inflammatory response.

  18. Multivariate Data EXplorer (MDX)

    SciTech Connect

    Steed, Chad Allen

    2012-08-01

    The MDX toolkit facilitates exploratory data analysis and visualization of multivariate datasets. MDX provides and interactive graphical user interface to load, explore, and modify multivariate datasets stored in tabular forms. MDX uses an extended version of the parallel coordinates plot and scatterplots to represent the data. The user can perform rapid visual queries using mouse gestures in the visualization panels to select rows or columns of interest. The visualization panel provides coordinated multiple views whereby selections made in one plot are propagated to the other plots. Users can also export selected data or reconfigure the visualization panel to explore relationships between columns and rows in the data.

  19. Pluronic-PEI copolymers enhance exon-skipping of 2'-O-methyl phosphorothioate oligonucleotide in cell culture and dystrophic mdx mice.

    PubMed

    Wang, M; Wu, B; Lu, P; Tucker, J D; Milazi, S; Shah, S N; Lu, Q L

    2014-01-01

    A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2'-O-methyl phosphorothioate RNA (2'-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100-300 nm nanoparticles. Exon-skipping effect of 2'-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2'-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k-5kDa) and intermediate hydrophilic-lipophilic balance (7-23) of pluronics, enhanced exon-skipping of 2'-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases.

  20. Impaired response of mature adipocytes of diabetic mice to hypoxia

    SciTech Connect

    Hong, Seok Jong Jin, Da P.; Buck, Donald W.; Galiano, Robert D.; Mustoe, Thomas A.

    2011-10-01

    Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.

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

    PubMed

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

    2015-09-01

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

  2. Dietary phosphorus overload aggravates the phenotype of the dystrophin-deficient mdx mouse.

    PubMed

    Wada, Eiji; Yoshida, Mizuko; Kojima, Yoriko; Nonaka, Ikuya; Ohashi, Kazuya; Nagata, Yosuke; Shiozuka, Masataka; Date, Munehiro; Higashi, Tetsuo; Nishino, Ichizo; Matsuda, Ryoichi

    2014-11-01

    Duchenne muscular dystrophy is a lethal X-linked disease with no effective treatment. Progressive muscle degeneration, increased macrophage infiltration, and ectopic calcification are characteristic features of the mdx mouse, a murine model of Duchenne muscular dystrophy. Because dietary phosphorus/phosphate consumption is increasing and adverse effects of phosphate overloading have been reported in several disease conditions, we examined the effects of dietary phosphorus intake in mdx mice phenotypes. On weaning, control and mdx mice were fed diets containing 0.7, 1.0, or 2.0 g phosphorus per 100 g until they were 90 days old. Dystrophic phenotypes were evaluated in cryosections of quadriceps and tibialis anterior muscles, and maximal forces and voluntary activity were measured. Ectopic calcification was analyzed by electron microscopy to determine the cells initially responsible for calcium deposition in skeletal muscle. Dietary phosphorus overload dramatically exacerbated the dystrophic phenotypes of mdx mice by increasing inflammation associated with infiltration of M1 macrophages. In contrast, minimal muscle necrosis and inflammation were observed in exercised mdx mice fed a low-phosphorus diet, suggesting potential beneficial therapeutic effects of lowering dietary phosphorus intake on disease progression. To our knowledge, this is the first report showing that dietary phosphorus intake directly affects muscle pathological characteristics of mdx mice. Dietary phosphorus overloading promoted dystrophic disease progression in mdx mice, whereas restricting dietary phosphorus intake improved muscle pathological characteristics and function.

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

    PubMed

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

    2015-01-01

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

  4. Maturation of "neocortex isole" in vivo in mice.

    PubMed

    Zhou, Libing; Gall, David; Qu, Yibo; Prigogine, Cynthia; Cheron, Guy; Tissir, Fadel; Schiffmann, Serge N; Goffinet, Andre M

    2010-06-09

    How much neocortical development depends on connections remains elusive. Here, we show that Celsr3|Dlx mutant mice have no extrinsic neocortical connections yet survive to postnatal day 20, acquire a basic behavioral repertoire, and display spontaneous hyperactivity, with abnormal light/dark activity cycling. Except for hallmarks related to thalamic input, such as barrels in somatosensory cortex, cortical arealization and laminar maturation proceeded normally. However, the tangential extension of the mature cortex was diminished, with radial thickness less severely affected. Deep layer neurons were reduced in number, and their apical and basal dendritic arbors were blunted, with reduced synapse density. Interneurons reached the cortex, and their density was comparable with wild type. The excitability of mutant pyramidal neurons, measured in vitro in patch-clamp experiments in acute slices, was decreased. However, their firing activity in vivo was quite similar to the wild type, except for the presence of rapid firing exhaustion in some mutant neurons. Local field potential and electrocorticogram showed similar range of oscillations, albeit with higher frequency peaks and reduced left-right synchrony in the mutant. Thus, "protomap" formation, namely cortical lamination and arealization, proceed normally in absence of extrinsic connections, but survival of projection neurons and acquisition of mature morphological and some electrophysiological features depend on the establishment of normal cortical-subcortical relationships.

  5. Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy.

    PubMed

    Morales, Maria Gabriela; Gutierrez, Jaime; Cabello-Verrugio, Claudio; Cabrera, Daniel; Lipson, Kenneth E; Goldschmeding, Roel; Brandan, Enrique

    2013-12-15

    In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-β (TGF-β), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.

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

    SciTech Connect

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

    1991-01-01

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

  7. Changes in calsequestrin, TNF-α, TGF-β and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps, diaphragm and intrinsic laryngeal muscles.

    PubMed

    Barros Maranhão, Juliana; de Oliveira Moreira, Drielen; Maurício, Adriana Fogagnolo; de Carvalho, Samara Camaçari; Ferretti, Renato; Pereira, Juliano Alves; Santo Neto, Humberto; Marques, Maria Julia

    2015-10-01

    In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium-related protein), tumour necrosis factor (TNF-α; pro-inflammatory cytokine), tumour growth factor (TGF-β; pro-fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF-α, TGF-β and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF-α and TGF-β serve as markers of dystrophy primarily for the diaphragm.

  8. Ventilatory Chemosensory Drive Is Blunted in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)

    PubMed Central

    Mosqueira, Matias; Baby, Santhosh M.; Khurana, Tejvir S.

    2013-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure. PMID:23922741

  9. Fibrosis and inflammation are greater in muscles of beta-sarcoglycan-null mouse than mdx mouse.

    PubMed

    Gibertini, Sara; Zanotti, Simona; Savadori, Paolo; Curcio, Maurizio; Saredi, Simona; Salerno, Franco; Andreetta, Francesca; Bernasconi, Pia; Mantegazza, Renato; Mora, Marina

    2014-05-01

    The Sgcb-null mouse, with knocked-down β-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-β1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-β1 was present in areas of degeneration-regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis

  10. Difference between mammary epithelial cells from mature virgin and primiparous mice.

    PubMed

    Vonderhaar, B K; Smith, G H; Pauley, R J; Rosen, J M; Topper, Y J

    1978-11-01

    Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

  11. Impaired regenerative capacity and lower revertant fibre expansion in dystrophin-deficient mdx muscles on DBA/2 background

    PubMed Central

    Rodrigues, Merryl; Echigoya, Yusuke; Maruyama, Rika; Lim, Kenji Rowel Q.; Fukada, So-ichiro; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy, one of the most common lethal genetic disorders, is caused by mutations in the DMD gene and a lack of dystrophin protein. In most DMD patients and animal models, sporadic dystrophin-positive muscle fibres, called revertant fibres (RFs), are observed in otherwise dystrophin-negative backgrounds. RFs are thought to arise from skeletal muscle precursor cells and clonally expand with age due to the frequent regeneration of necrotic fibres. Here we examined the effects of genetic background on muscle regeneration and RF expansion by comparing dystrophin-deficient mdx mice on the C57BL/6 background (mdx-B6) with those on the DBA/2 background (mdx-DBA), which have a more severe phenotype. Interestingly, mdx-DBA muscles had significantly lower RF expansion than mdx-B6 in all age groups, including 2, 6, 12, and 18 months. The percentage of centrally nucleated fibres was also significantly lower in mdx-DBA mice compared to mdx-B6, indicating that less muscle regeneration occurs in mdx-DBA. Our study aligns with the model that RF expansion reflects the activity of precursor cells in skeletal muscles, and it serves as an index of muscle regeneration capacity. PMID:27924830

  12. Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy.

    PubMed

    Au, Carol G; Butler, Tanya L; Sherwood, Megan C; Egan, Jonathan R; North, Kathryn N; Winlaw, David S

    2011-02-01

    Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP-1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMP-2, MMP-9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

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

    PubMed Central

    2012-01-01

    Background IL-6 is a pleiotropic cytokine that modulates inflammatory responses and plays critical roles in muscle maintenance and remodeling. In the mouse model (mdx) of Duchenne Muscular Dystrophy, IL-6 and muscle inflammation are elevated, which is believed to contribute to the chronic inflammation and failure of muscle regeneration in DMD. The purpose of the current study was to examine the effect of blocking IL-6 signaling on the muscle phenotype including muscle weakness and pathology in the mdx mouse. Methods A monoclonal antibody against the IL-6 receptor (IL-6r mAb) that blocks local and systemic IL-6 signaling was administered to mdx and BL-10 mice for 5 weeks and muscle function, histology, and inflammation were examined. Results IL-6r mAb treatment increased mdx muscle inflammation including total inflammation score and ICAM-1 positive lumens in muscles. There was no significant improvement in muscle strength nor muscle pathology due to IL-6r mAb treatment in mdx mice. Conclusions These results showed that instead of reducing inflammation, IL-6 signaling blockade for 5 weeks caused an increase in muscle inflammation, with no significant change in indices related to muscle regeneration and muscle function. The results suggest a potential anti-inflammatory instead of the original hypothesized pro-inflammatory role of IL-6 signaling in the mdx mice. PMID:22716658

  14. Diaphragm degeneration and cardiac structure in mdx mouse: potential clinical implications for Duchenne muscular dystrophy.

    PubMed

    Barbin, Isabel Cristina Chagas; Pereira, Juliano Alves; Bersan Rovere, Matheus; de Oliveira Moreira, Drielen; Marques, Maria Julia; Santo Neto, Humberto

    2016-05-01

    We examined the effects of exercise on diaphragm degeneration and cardiomyopathy in dystrophin-deficient mdx mice. Mdx mice (11 months of age) were exercised (swimming) for 2 months to worsen diaphragm degeneration. Control mdx mice were kept sedentary. Morphological evaluation demonstrated increased fibrosis in the diaphragm of exercised mdx mice (33.3 ± 6.0% area of fibrosis) compared with control mdx mice (20.9 ± 1.7% area of fibrosis). Increased (26%) activity of MMP-2, a marker of fibrosis, was detected in the diaphragms from exercised mdx mice. Morphological evaluation of the heart demonstrated a 45% increase in fibrosis in the right ventricle (8.3 ± 0.6% in sedentary vs. 12.0 ± 0.6% of fibrosis in exercised) and in the left ventricle (35% increase) in the exercised mdx mice. The density of inflammatory cells-degenerating cardiomyocytes increased 95% in the right ventricle (2.3 ± 0.6 in sedentary vs. 4.5 ± 0.8 in exercised) and 71% in the left ventricle (1.4 ± 0.6 sedentary vs. 2.4 ± 0.5 exercised). The levels of both active MMP-2 and the pro-fibrotic factor transforming growth factor beta were elevated in the hearts of exercised compared with sedentary mdx mice. The wall thickness to lumen diameter ratio of the pulmonary trunk was significantly increased in the exercised mdx mice (0.11 ± 0.04 in sedentary vs. 0.28 ± 0.12 in exercised), as was the thickness of the right ventricle wall, which suggests the occurrence of pulmonary hypertension in those animals. It is suggested that diaphragm degeneration is a main contributor to right ventricle dystrophic pathology. These findings may be relevant for future interventional studies for Duchenne muscular dystrophy-associated cardiomyopathy.

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

    SciTech Connect

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

    1991-07-01

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

  16. Myocarditis induced by coxsackie B3 virus in mature mice.

    PubMed

    Jaśkiewicz, K; Mrozińska, B

    1975-01-01

    Forty female mice during breast-feeding were infected intraperitoneally with coxackie B3 virus. Gross and microscopic examination of the hearts of the mice 7, 20, 44 and 120 days after infection revealed myocarditis typical of the acute stage of the disease, not reported previously, and gradually increasing intensity of immunologic changes in the chronic stage.

  17. Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype.

    PubMed

    Villalta, S Armando; Rinaldi, Chiara; Deng, Bo; Liu, Grace; Fedor, Brian; Tidball, James G

    2011-02-15

    M1 macrophages play a major role in worsening muscle injury in the mdx mouse model of Duchenne muscular dystrophy. However, mdx muscle also contains M2c macrophages that can promote tissue repair, indicating that factors regulating the balance between M1 and M2c phenotypes could influence the severity of the disease. Because interleukin-10 (IL-10) modulates macrophage activation in vitro and its expression is elevated in mdx muscles, we tested whether IL-10 influenced the macrophage phenotype in mdx muscle and whether changes in IL-10 expression affected the pathology of muscular dystrophy. Ablation of IL-10 expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treating mdx muscle macrophages with IL-10 reduced activation of the M1 phenotype, assessed by iNOS expression, and macrophages from IL-10 null mutant mice were more cytolytic than macrophages isolated from wild-type mice. Our data also showed that muscle cells in mdx muscle expressed the IL-10 receptor, suggesting that IL-10 could have direct effects on muscle cells. We assayed whether ablation of IL-10 in mdx mice affected satellite cell numbers, using Pax7 expression as an index, but found no effect. However, IL-10 mutation significantly increased myogenin expression in vivo during the acute and the regenerative phase of mdx pathology. Together, the results show that IL-10 plays a significant regulatory role in muscular dystrophy that may be caused by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation and modulating muscle differentiation.

  18. Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer.

    PubMed

    Foster, Helen; Sharp, Paul S; Athanasopoulos, Takis; Trollet, Capucine; Graham, Ian R; Foster, Keith; Wells, Dominic J; Dickson, George

    2008-11-01

    Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (DeltaAB/R3-R18/DeltaCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (DeltaAB/R3-R18/DeltaCT and DeltaR4-R23/DeltaCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of DeltaAB/R3-R18/DeltaCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized DeltaAB/R3-R18/DeltaCT. However, codon-optimized microdystrophin DeltaR4-R23/DeltaCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.

  19. Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis.

    PubMed

    Cea, Luis A; Puebla, Carlos; Cisterna, Bruno A; Escamilla, Rosalba; Vargas, Aníbal A; Frank, Marina; Martínez-Montero, Paloma; Prior, Carmen; Molano, Jesús; Esteban-Rodríguez, Isabel; Pascual, Ignacio; Gallano, Pía; Lorenzo, Gustavo; Pian, Héctor; Barrio, Luis C; Willecke, Klaus; Sáez, Juan C

    2016-07-01

    Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca(2+) levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice was significantly better than that of control mdx Cx43(fl/fl)Cx45(fl/fl) mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

  20. Evidence for a mature B cell subpopulation in Peyer's patches of young adult xid mice

    PubMed Central

    1983-01-01

    Peyer's patch (PP) and mesenteric lymph node (MLN) cell cultures from young adult X-linked immunodeficient (xid) CBA/N and (CBA/N X DBA/2) F1 male mice support primary anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) responses, which suggests that gut-associated lymphoreticular tissue (GALT) contains a normal B lymphocyte subpopulation. Further support for this was provided by the observation that PP cells from xid mice gave responses to both TI-1 and TI-2 antigens that were similar to the responses of PP cell cultures from normal mice. Spleen cell cultures from xid mice were unresponsive to SRBC and TI-2 antigens. Proof that GALT of xid mice contain mature B lymphocytes was provided by the demonstration of PP B cells that bear a low density of surface immunoglobulin M. When these cells were separated by flow cytometry and immunized with trinitrophenyl (TNP)- Ficoll in vitro, good anti-TNP PFC responses were observed. These results suggest that GALT of young adult xid mice contain mature B cells and may represent the origin for the mature B cell responses seen in aged xid mice. PMID:6600493

  1. Comparative transcriptome analysis of muscular dystrophy models Large(myd), Dmd(mdx)/Large(myd) and Dmd(mdx): what makes them different?

    PubMed

    Almeida, Camila F; Martins, Poliana Cm; Vainzof, Mariz

    2016-08-01

    Muscular dystrophies (MD) are a clinically and genetically heterogeneous group of Mendelian diseases. The underlying pathophysiology and phenotypic variability in each form are much more complex, suggesting the involvement of many other genes. Thus, here we studied the whole genome expression profile in muscles from three mice models for MD, at different time points: Dmd(mdx) (mutation in dystrophin gene), Large(myd-/-) (mutation in Large) and Dmd(mdx)/Large(myd-/-) (both mutations). The identification of altered biological functions can contribute to understand diseases and to find prognostic biomarkers and points for therapeutic intervention. We identified a substantial number of differentially expressed genes (DEGs) in each model, reflecting diseases' complexity. The main biological process affected in the three strains was immune system, accounting for the majority of enriched functional categories, followed by degeneration/regeneration and extracellular matrix remodeling processes. The most notable differences were in 21-day-old Dmd(mdx), with a high proportion of DEGs related to its regenerative capacity. A higher number of positive embryonic myosin heavy chain (eMyHC) fibers confirmed this. The new Dmd(mdx)/Large(myd-/-) model did not show a highly different transcriptome from the parental lineages, with a profile closer to Large(myd-/-), but not bearing the same regenerative potential as Dmd(mdx). This is the first report about transcriptome profile of a mouse model for congenital MD and Dmd(mdx)/Large(myd). By comparing the studied profiles, we conclude that alterations in biological functions due to the dystrophic process are very similar, and that the intense regeneration in Dmd(mdx) involves a large number of activated genes, not differentially expressed in the other two strains.

  2. Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

    PubMed Central

    Koo, Taeyoung; Malerba, Alberto; Athanasopoulos, Takis; Trollet, Capucine; Boldrin, Luisa; Ferry, Arnaud; Popplewell, Linda; Foster, Helen; Foster, Keith

    2011-01-01

    Abstract Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy. PMID:21453126

  3. The effect of respiratory muscle training with CO2 breathing on cellular adaptation of mdx mouse diaphragm

    PubMed Central

    Matécki, Stefan; Rivier, François; Hugon, Gerald; Koechlin, Christelle; Michel, Alain; Préfaut, Christian; Mornet, Dominique; Ramonatxo, Michèle

    2005-01-01

    The aim of our study was to investigate the cellular mechanisms induced by hypercapnic stimulation of ventilation, during 6 weeks/30 min per day, in 10 mdx and 8 C57BL10 mice (10G0.2 months old). Ten mdx and eight C57BL10 mice served as control group. This respiratory training increases in vitro maximal tetanic tension of the diaphragm only in mdx mice. Western blot analysis of diaphragm showed: (1) an over-expression of a-dystrobrevin in mdx and C57BL10 training group compared to control group (8100G710 versus 6100G520 and 2800G400 versus 2200G250 arbitrary units); (2) a decrease in utrophin expression only in mdx training group compared to control group (2100G320 versus 3100G125 arbitrary units). Daily respiratory muscle training in mdx mice, induces a beneficial effect on diaphragm strength, with an over-expression of a-dystrobrevin. Further studies are needed to determine if, in absence of dystrophin, the over-expression of a-dystrobrevin could be interpreted as a possible pathway to improve function of dystrophic muscle. PMID:15907290

  4. Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of duchenne muscular dystrophy.

    PubMed

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

    2014-01-01

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

  5. Moderate beer consumption does not change early or mature atherosclerosis in mice

    PubMed Central

    Escolà-Gil, Joan Carles; Calpe-Berdiel, Laura; Ribas, Vicent; Blanco-Vaca, Francisco

    2004-01-01

    Background Although the consumption of wine in particular has been associated with a lower risk of atherothrombotic cardiovascular disease, systematic reviews differ as to the relative protective effect of beer, wine and spirits. Two previous studies showed that red wine reduces fatty streak formation (early atherosclerosis) but not mature atherosclerosis in apolipoprotein (apo) E-deficient (apoE-/-) mice. Aim of the study To determine whether a moderate beer intake would affect early and mature atherosclerotic lesion formation using control C57BL/6 and apoE-/- mice, respectively, as models. Methods Control C57BL/6 and apoE-/- mice were randomized to receive either water, ethanol, mild beer, dark beer or ethanol-free beer. The level of beer was designed to approximate the alcohol intake currently believed to be beneficial in reducing human vascular risk. Control C57BL/6 mice were fed a Western diet for 24 weeks, and apoE-/- mice a chow diet for 12 weeks. At the end of the trial period, mice were euthanized and atherosclerotic lesions quantified. Plasma lipid concentrations were also measured. Results The amount of atherosclerosis and average number of lesions in the proximal aortic region did not differ among groups in control C57BL/6 mice (p = 0.32 and p = 0.29, respectively) and apoE-/- mice (p = 0.19 and p = 0.59, respectively). No consistent differences were observed in plasma lipid and lipoprotein concentrations among water, ethanol and beer groups. Conclusions Moderate beer consumption does not change the development of early or mature atherosclerosis in mice. Our findings do not support the hypothesis of an anti-atherogenic effect of beer. Other potential protective actions of moderate beer consumption such as plaque stabilization, a reduction in plaque intrinsic thrombogenicity, or a reduction in the systemic propensity to thrombosis, remain to be studied. PMID:14725716

  6. Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies

    PubMed Central

    Kobayashi, Yvonne M.; Rader, Erik P.; Crawford, Robert W.; Campbell, Kevin P.

    2011-01-01

    Loss of mobility influences the quality of life for patients with neuromuscular diseases. Common measures of mobility and chronic muscle damage are the six-minute walk test and serum creatine kinase. Despite extensive pre-clinical studies of therapeutic approaches, characterization of these measures is incomplete. To address this, a six-minute ambulation assay, serum creatine kinase, and myoglobinuria were investigated for the mdx mouse, a dystrophinopathy mouse model commonly used in pre-clinical studies. Mdx mice ambulated shorter distances than normal controls, a disparity accentuated after mild exercise. An asymmetric pathophysiology in mdx mice was unmasked with exercise, and peak measurements of serum creatine kinase and myoglobinuria were identified. Our data highlights the necessity to consider asymmetric pathology and timing of biomarkers when testing potential therapies for muscular dystrophy. PMID:22154712

  7. Biotin deficiency blocks thymocyte maturation, accelerates thymus involution, and decreases nose-rump length in mice.

    PubMed

    Báez-Saldaña, Armida; Ortega, Enrique

    2004-08-01

    Biotin deficiency in experimental animals causes low body weight as well as several phenomena suggestive of an altered immune system. We reported previously that chronic biotin deficiency in mice decreases body weight and alters the number and proportion of lymphocyte subpopulations in the spleen. To further characterize the effects of biotin deficiency, we studied in detail the maturation of thymocytes and the status of biotin in the thymus, as well as the body length of biotin-deficient mice. Male Balb/cAnN mice were fed for up to 20 wk either standard control diet, a biotin-deficient diet, or a biotin-sufficient diet. At different times, nose-rump length, weight of the thymus, spleen and liver, total number of cells in the spleen and thymus, pyruvate carboxylase (PC) and propionyl CoA carboxylase (PCC) activity in thymus cells, and the proportion of distinct thymocyte subsets were determined. These variables did not differ between mice fed the control and biotin-sufficient diets. In contrast, biotin-deficient mice differed from biotin-sufficient mice in all of the analyzed variables. PC and PCC specific activities of thymocytes of mice fed the biotin-depleting diet decreased during the first 4 wk by 84.5%. The maturation of thymocytes in biotin-deficient mice was arrested at the double-negative stage. Our results suggest that biotin deficiency in mice causes an accelerated involution of the thymus and decreases nose-rump length, but these effects do not correlate in magnitude or in temporality with the sharp decrease in the activity of the biotin-dependent carboxylases. As such, the possibility that the aforementioned effects are not related directly to the prosthetic function of biotin should be considered.

  8. Impaired mast cell maturation and degranulation and attenuated allergic responses in Ndrg1-deficient mice.

    PubMed

    Taketomi, Yoshitaka; Sunaga, Kohei; Tanaka, Satoshi; Nakamura, Masanori; Arata, Satoru; Okuda, Tomohiko; Moon, Tae-Chul; Chang, Hyeun-Wook; Sugimoto, Yukihiko; Kokame, Koichi; Miyata, Toshiyuki; Murakami, Makoto; Kudo, Ichiro

    2007-06-01

    We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcepsilonRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.

  9. Effect of Friend Leukemia Virus and Rowson-Parr Virus on Immunological Maturation of Mice

    PubMed Central

    Bendinelli, M.

    1971-01-01

    The effect of neonatal infection with Friend virus (FV) and Rowson-Parr virus (RPV) on the maturation of the capacity to respond to sheep red cells, as measured by the numbers of hemolytic plaque-forming cells in the spleen, was investigated in BALB/c mice. Both viruses affected immunological maturation but there were significant differences between their effects. The development with age of the ability to produce plaque-forming cells in response to antigen was virtually abolished by FV and only slightly impaired by RPV. Furthermore, FV also suppressed the development of background plaque-forming cells, whereas RPV did not. PMID:4343401

  10. Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity.

    PubMed Central

    Spencer, M J; Walsh, C M; Dorshkind, K A; Rodriguez, E M; Tidball, J G

    1997-01-01

    Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis. PMID:9169505

  11. Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

    PubMed

    Ren, Yue; Dashtsoodol, Nyambayar; Watarai, Hiroshi; Koseki, Haruhiko; Quan, Chengshi; Taniguchi, Masaru

    2014-10-01

    NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αβT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells.

  12. Estrogen Receptor beta mediates decreased occlusal loading induced inhibition of chondrocyte maturation in female mice

    PubMed Central

    Polur, Ilona; Kamiya, Yosuke; Xu, Manshan; Cabri, Bianca S.; Alshabeeb, Marwa; Wadhwa, Sunil; Chen, Jing

    2015-01-01

    Objective Temporomandibular joint (TMJ) disorders predominantly afflict women, suggesting that estrogen may play a role in the disease process. Defects in mechanical loading-induced TMJ remodeling are believed to be a major etiological factor in TMJ degenerative disease. Previously, we found that, decreased occlusal loading caused a significant decrease in early chondrocyte maturation markers (Sox9 and Col 2) in female, but not male, C57BL/6 wild type mice (1). The goal of this study was to examine the role of Estrogen Receptor (ER) beta in mediating these effects. Design 21-day-old male (n=24) and female (n=25) ER beta KO mice were exposed to decreased occlusal loading (soft diet administration and incisor trimming) for 4 weeks. At 49 days of age the mice were sacrificed. Proliferation, gene expression, Col 2 immunohistochemistry and micro-CT analysis were performed on the mandibular condyles. Results Decreased occlusal loading triggered similar effects in male and female ER beta KO mice; specifically, significant decreases in Col 10 expression, subchondral total volume, bone volume, and trabecular number. Conclusion Decreased occlusal loading induced inhibition of chondrocyte maturation markers (Sox9 and Col 2) did not occur in female ER beta deficient mice. PMID:25791327

  13. Exopolysaccharide Produced by Lactobacillus Plantarum Induces Maturation of Dendritic Cells in BALB/c Mice.

    PubMed

    Tang, Yanjun; Dong, Wei; Wan, Keyu; Zhang, Ligang; Li, Chun; Zhang, Lili; Liu, Ning

    2015-01-01

    Lactobacillus plantarum (L. plantarum) exopolysaccharide (EPS) is an important bioactive component in fermented functional foods. However, there is a lack of data concerning the effects of L. plantarum EPS on maturation of mouse dendritic cells (DCs). In this study, we purified L. plantarum EPS and examined its effects on cytokines production by dendritic cells in serum and intestinal fluid of BALB/c mice, then investigated its effects on phenotypic and functional maturation of mouse bone marrow-derived dendritic cells (BMDCs). Cytokines (nitric oxide, IL-12p70, IL-10 and RANTES) in serum and intestinal fluid were analyzed by enzyme linked immunosorbent assay (ELISA) after the mice received EPS for 2, 5 and 7 days, respectively. DCs derived from bone marrow of BALB/c mouse were treated with EPS, then the phenotypic maturation of BMDCs was analyzed using flow cytometer and the functional maturation of BMDCs was analyzed by ELISA, and, lastly, mixed lymphocyte proliferation was performed. We found the molecular weight of purified EPS was approximately 2.4×106 Da and it was composed of ribose, rhamnose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2:1:1:10:4:205:215. We observed that L. plantarum EPS enriched production of nitric oxide, IL-12p70 and RANTES, and decreased the secretion of IL-10 in the serum or intestinal fluid as well as in the supernatant of DCs treated with the EPS. The EPS also up-regulated the expression of MHC II and CD86 on DCs surface and promoted T cells to proliferate in vitro. Our data provide direct evidence to suggest that L. plantarum EPS can effectively induce maturation of DCs in mice.

  14. Behavioral responses to and brain distribution of morphine in mature adult and aged mice

    SciTech Connect

    Burton, C.K.; Ho, I.K.; Hoskins, B.

    1986-03-01

    Mature adult (3-6 mo old) and aged (2 yr old) male ICR mice were injected with 10 to 100 mg/kg morphine, s.c. The ED50 values for running behavior (as measured using Stoelting activity monitors and having each mouse serve as its own control) representing 5 times control activity was approximately 7.5 mg/kg for aged mice and approximately 17.5 mg/kg for the mature adults. The ED50 values for analgesia 1 hr after morphine administration using the tail-flick method (max. response time = 8 sec) were approx. 70 mg/kg for the aged mice and 15 mg/kg for the mature adults. One hour after injecting /sup 3/H-morphine at doses of 30 and 100 mg/kg, 0.13 and 0.14% of the doses appeared in brains of aged and mature adult mice, respectively. Regional distribution of the morphine was the same for both age groups. Expressed as percent of total brain morphine, it was as follows: cortex, 30%; midbrain, 18%; cerebellum, 17%; medulla, 12%; pons, 9%; striatum, 8% and periaqueductal gray, 6%. Expressed as g morphine/g tissue for the 2 doses, the distribution was; periaqueductal gray, 30 and 80; striatum, 9 and 34; medulla, 6 and 20 pons; 5 and 19; cerebellum, 4 and 13; midbrain 2.5 and 8.5 and cortex, 2 and 8. These results suggest that the differences in response to morphine by the two age groups were due to age-related differences in opioid receptor populations and/or affinities.

  15. Effect of Holothuria leucospilota extracted saponin on maturation of mice oocyte and granulosa cells

    PubMed Central

    Moghadam, Fereshteh Delghandi; Baharara, Javad; Balanezhad, Saeedeh Zafar; Jalali, Mohsen; Amini, Elaheh

    2016-01-01

    Sea cucumbers saponins are triterpenoid glycosides which exert beneficial biomedical effects. This study was performed to assess the effect of saponin extracted from sea cucumber Holothuria leucospilota (H. leucospilota) on maturation of mice oocytes and granulosa cells. The germinal vesicles oocytes were collected from 6–8 weeks old Naval Medical Research Institute (NMRI) mice ovaries, randomly divided into untreated and four experimental groups and cultured In vitro. Maturation medium was supplemented with 0, 1, 2, 4 and 8 μg/ml saponin for 12 days. The rates of maturation were recorded through morphological observation by measurement of follicle diameter during treatment. After 4 days, the effects of saponin on granulosa cells were investigated by reactive oxygen species (ROS) measurement, super oxide dismutase (SOD) activity, caspase assay and tumor necrosis factor-alpha (TNF-α) expression. The oocyte maturation rate was significantly higher in treated groups (1 μg/ml). The ROS and SOD assays demonstrated the antioxidant potential of saponin. The caspase assay exhibited that optimum concentrations of saponin (1, 2 μg/ml) reduced caspase activity in granulosa cells. Flow cytometry showed that optimum concentration of saponin promoted oocyte maturation via down regulation of TNF-α as follicular degenerative factor in nursing cells. These results proposed that maturation rate were obtained after the incorporation of 1 μg/ml sea cucumber saponin. Moreover, the extracted saponin at concentrations of 1, 2 μg/ml enhanced follicle growth which is accompanied by attenuating ROS formation, elevating SOD activity and reducing TNF-α expression in granulosa cells. But, further examinations are required to understand precise mechanisms of saponin action on oocyte and granulosa cells. PMID:27168752

  16. Altered nuclear dynamics in MDX myofibers.

    PubMed

    Iyer, Shama R; Shah, Sameer B; Valencia, Ana P; Schneider, Martin F; Hernández-Ochoa, Erick O; Stains, Joseph P; Blemker, Silvia S; Lovering, Richard M

    2017-03-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to progressive muscle degeneration and weakness. Although the genetic basis is known, the pathophysiology of dystrophic skeletal muscle remains unclear. We examined nuclear movement in wild-type (WT) and muscular dystrophy mouse model for DMD (MDX) (dystrophin-null) mouse myofibers. We also examined expression of proteins in the linkers of nucleoskeleton and cytoskeleton (LINC) complex, as well as nuclear transcriptional activity via histone H3 acetylation and polyadenylate-binding nuclear protein-1. Because movement of nuclei is not only LINC dependent but also microtubule dependent, we analyzed microtubule density and organization in WT and MDX myofibers, including the application of a unique 3D tool to assess microtubule core structure. Nuclei in MDX myofibers were more mobile than in WT myofibers for both distance traveled and velocity. MDX muscle shows reduced expression and labeling intensity of nesprin-1, a LINC protein that attaches the nucleus to the microtubule and actin cytoskeleton. MDX nuclei also showed altered transcriptional activity. Previous studies established that microtubule structure at the cortex is disrupted in MDX myofibers; our analyses extend these findings by showing that microtubule structure in the core is also disrupted. In addition, we studied malformed MDX myofibers to better understand the role of altered myofiber morphology vs. microtubule architecture in the underlying susceptibility to injury seen in dystrophic muscles. We incorporated morphological and microtubule architectural concepts into a simplified finite element mathematical model of myofiber mechanics, which suggests a greater contribution of myofiber morphology than microtubule structure to muscle biomechanical performance.NEW & NOTEWORTHY Microtubules provide the means for nuclear movement but show altered organization in the muscular dystrophy mouse model (MDX

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

    PubMed

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

    2014-11-01

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

  18. Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice.

    PubMed

    Pfannenstiel, Lukas W; Lam, Samuel S K; Emens, Leisha A; Jaffee, Elizabeth M; Armstrong, Todd D

    2010-01-01

    Subclinical doses of Paclitaxel (PTX) given 1day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu(+) tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8(+) T cells with enhanced lytic activity against neu(+) tumors. PTX treatment also enhances maturation marker expression on CD11c(+) DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8(+) T cells with greater lytic capability than DC's from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-gamma-secreting CD8(+) T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.

  19. Survival of mature T cells in the periphery is intrinsically dependent on GIMAP1 in mice

    PubMed Central

    Datta, Preeta; Webb, Louise M.C.; Avdo, Inxhina; Pascall, John

    2016-01-01

    An effective immune system depends upon the survival of mature T cells in the periphery. Members of the GIMAP family of GTPases have been proposed to regulate this homeostasis, supported by the paucity of peripheral T cells in rodents deficient for either GIMAP1 or GIMAP5. It is unclear whether this lack of T cells is a consequence of an ontological defect, causing the thymus to generate and export T cells incapable of surviving in the periphery, or whether (alternatively or additionally) mature T cells intrinsically require GIMAP1 for survival. Using the ERT2Cre+ transgene, we conditionally deleted Gimap1 in C57BL/6 mice and demonstrate that GIMAP1 is intrinsically required for the survival of mature T cells in the periphery. We show that, in contrast to GIMAP5, this requirement is independent of the T‐cells' activation status. We investigated the nature of the survival defect in GIMAP1‐deficient CD4+ T cells and show that the death occurring after GIMAP1 ablation is accompanied by mitochondrial depolarization and activation of the extrinsic apoptotic pathway. This study shows that GIMAP1 is critical for maintaining the peripheral T‐cell pool in mice and offers a potent target for the treatment of T‐cell‐mediated diseases. PMID:27792288

  20. Acute food restriction increases collagen breakdown and phagocytosis by mature decidual cells of mice.

    PubMed

    Spadacci-Morena, D D; Katz, S G

    2001-06-01

    An ultrastructural study was undertaken on antimesometrial mature decidual tissue of fed and food-restricted mice, on day 9 of pregnancy. The mean ad libitum food intake was established on mice from the 8th till the 9th day of pregnancy. Fed mice were used as controls. Experimental animals were divided into two groups: one was allowed to feed 25% of normal diet and the other 50%. Extracellular collagen fibrils were scarce in fed animals and conspicuous in food restriction. Granular electron-dense deposits and filamentous aggregates of disintegrating collagen fibrils were observed in all food-deprived mice but were rarely noted in fed animals. Intracellular vacuolar structures exhibited other typical cross-banded collagen immersed in finely granular electron-translucent material (clear vacuole) or electron-dense material containing collagen fibrils with a faint periodicity (dark vacuole). The clear and dark vacuoles were scarce in fed animals and evident in food-restricted mice, mainly in those 25% food restricted. Although collagen breakdown may be part of the normal process of decidual tissue remodelling our results suggest that it is enhanced in food-restricted animals. Thus it seems that collagen breakdown is a normal mechanism that may be regulated by the food intake of the pregnant animal.

  1. Novel B-cell maturation factor from spontaneously autoimmune viable motheaten mice.

    PubMed

    Sidman, C L; Marshall, J D; Masiello, N C; Roths, J B; Shultz, L D

    1984-11-01

    Both in vivo and in vitro, mice homozygous for the viable motheaten mutation show severe immunodeficiency, polyclonal B-cell activation and Ig secretion, and spontaneous production of a lymphokine [B-cell maturation factor (BMF)] that directly drives the maturation of normal or tumor B cells to the state of active Ig secretion. BMF from motheaten mice is distinct from previously identified forms in its cells of origin (B cells) and biochemical characteristics (apparent Mr 15,000 by gel filtration and NaDodSO4/PAGE; pI 4.3 by chromatofocusing). Among the known murine single-gene models of autoimmunity, only motheaten mice show high levels of spontaneous BMF production, which therefore may be an important component in the development of this form of autoimmunity/immunodeficiency disease. The coincidence of spontaneous BMF production and uncontrolled Ig secretion within the same mutant mouse constitutes the strongest evidence to date for a significant physiological (in vivo) role for BMFs.

  2. Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice.

    PubMed

    Watanabe, Shigeru; Yamamori, Saori; Otsuka, Shintaro; Saito, Masanori; Suzuki, Eiji; Kataoka, Masakazu; Miyaoka, Hitoshi; Takahashi, Masami

    2015-09-01

    Snap25(S187A/S187A) mouse is a knock-in mouse with a single amino acid substitution at a protein kinase C-dependent phosphorylation site of the synaptosomal-associated protein of 25 kDa (SNAP-25), which is a target-soluble NSF attachment protein receptor (t-SNARE) protein essential for neurotransmitter release. Snap25(S187A/S187A) mice exhibit several distinct phenotypes, including reductions in dopamine and serotonin release in the brain, anxiety-like behavior, and cognitive dysfunctions. Homozygous mice show spontaneous epileptic convulsions, and about 15% of the mice die around three weeks after birth. The remaining mice survive for almost two years and exhibit spontaneous recurrent seizures throughout their lifetime. Here, we conducted long-term continuous video electroencephalogram recording of the mice and analyzed the process of epileptogenesis and epileptic maturation in detail. Spikes and slow-wave discharges (SWDs) were observed in the cerebral cortex and thalamus before epileptic convulsions began. SWDs showed several properties similar to those observed in absence seizures including (1) lack of in the hippocampus, (2) movement arrest during SWDs, and (3) inhibition by ethosuximide. Multiple generalized seizures occurred in all homozygous mice around three weeks after birth. However, seizure generation stopped within several days, and a seizure-free latent period began. Following a spike-free quiet period, the number of spikes increased gradually, and epileptic seizures reappeared. Subsequently, spontaneous seizures occurred cyclically throughout the life of the mice, and several progressive changes in seizure frequency, seizure duration, seizure cycle interval, seizure waveform, and the number and waveform of epileptic discharges during slow-wave sleep occurred with different time courses over 10 weeks. Anxiety-related behaviors appeared suddenly within three days after epileptic seizures began and were delayed markedly by oral administration of

  3. Scrapie Affects the Maturation Cycle and Immune Complex Trapping by Follicular Dendritic Cells in Mice

    PubMed Central

    McGovern, Gillian; Mabbott, Neil; Jeffrey, Martin

    2009-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurological disorders of man and animals, characterised by abnormal disease-associated prion protein (PrPd) accumulations in the brain and lymphoreticular system (LRS). Prior to neuroinvasion, TSE agents often accumulate to high levels within the LRS, apparently without affecting immune function. However, our analysis of scrapie-affected sheep shows that PrPd accumulations within the LRS are associated with morphological changes to follicular dendritic cells (FDCs) and tingible body macrophages (TBMs). Here we examined FDCs and TBMs in the mesenteric lymph nodes (MLNs) of scrapie-affected mice by light and electron microscopy. In MLNs from uninfected mice, FDCs could be morphologically categorised into immature, mature and regressing forms. However, in scrapie-affected MLNs this maturation cycle was adversely affected. FDCs characteristically trap and retain immune complexes on their surfaces, which they display to B-lymphocytes. In scrapie-affected MLNs, some FDCs were found where areas of normal and abnormal immune complex retention occurred side by side. The latter co-localised with PrPd plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrPd accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrPd. Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrPd accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function. PMID:19997557

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

    PubMed Central

    2012-01-01

    Background Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists) increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice. Methods Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR), separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s)' effects. Results We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage. Conclusions The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice. PMID:22908954

  5. Parvalbumin-positive GABAergic interneurons are increased in the dorsal hippocampus of the dystrophic mdx mouse.

    PubMed

    Del Tongo, Claudia; Carretta, Donatella; Fulgenzi, Gianluca; Catini, Claudio; Minciacchi, Diego

    2009-12-01

    Duchenne muscular dystrophy (DMD) is characterized by variable alterations of the dystrophin gene and by muscle weakness and cognitive impairment. We postulated an association between cognitive impairment and architectural changes of the hippocampal GABAergic system. We investigated a major subpopulation of GABAergic neurons, the parvalbumin-immunopositive (PV-I) cells, in the dorsal hippocampus of the mdx mouse, an acknowledged model of DMD. PV-I neurons were quantified and their distribution was compared in CA1, CA2, CA3, and dentate gyrus in wild-type and mdx mice. The cell morphology and topography of PV-I neurons were maintained. Conversely, the number of PV-I neurons was significantly increased in the mdx mouse. The percent increase of PV-I neurons was from 45% for CA2, up to 125% for the dentate gyrus. In addition, the increased parvalbumin content in the mdx hippocampus was confirmed by Western blot. A change in the hippocampus processing abilities is the expected functional counterpart of the modification displayed by PV-I GABAergic neurons. Altered hippocampal functionality can be responsible for part of the cognitive impairment in DMD.

  6. Injection of vessel-derived stem cells prevents dilated cardiomyopathy and promotes angiogenesis and endogenous cardiac stem cell proliferation in mdx/utrn-/- but not aged mdx mouse models for duchenne muscular dystrophy.

    PubMed

    Chun, Ju Lan; O'Brien, Robert; Song, Min Ho; Wondrasch, Blake F; Berry, Suzanne E

    2013-01-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. DMD patients lack dystrophin protein and develop skeletal muscle pathology and dilated cardiomyopathy (DCM). Approximately 20% succumb to cardiac involvement. We hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent DCM in animal models of DMD. ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and α-actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn(-/-) mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin(+) cardiac stem cells were detected in ADM-injected heart. Nestin(+) striated cells were also detected in four of five mdx/utrn(-/-) hearts injected with ADMs. In contrast, when ADMs were injected into the heart of aged mdx mice with advanced fibrosis, no functional improvement was detected by echocardiography. Instead, ADMs exacerbated some features of DCM. No dystrophin protein, increase in CD31 expression, or increase in Nestin(+) cell proliferation was detected following ADM injection in aged mdx heart. Dystrophin was observed following transplantation of ADMs into the hearts of young mdx mice, however, suggesting that pathology in aged mdx heart may alter the fate of donor stem cells. In summary, ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.

  7. Utrophin A is essential in mediating the functional adaptations of mdx mouse muscle following chronic AMPK activation.

    PubMed

    Al-Rewashdy, Hasanen; Ljubicic, Vladimir; Lin, Wei; Renaud, Jean-Marc; Jasmin, Bernard J

    2015-03-01

    Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin along muscle fibers. An attractive therapeutic avenue for DMD consists in the upregulation of utrophin A, a protein with high sequence identity and functional redundancy with dystrophin. Recent work has shown that pharmacological interventions that induce a muscle fiber shift toward a slower, more oxidative phenotype with increased expression of utrophin A confer morphological and functional improvements in mdx mice. Whether such improvements result from the increased expression of utrophin A per se or are linked to other beneficial adaptations associated with the slow, oxidative phenotype remain to be established. To address this central issue, we capitalized on the use of double knockout (dKO) mice, which are mdx mice also deficient in utrophin. We first compared expression of signaling molecules and markers of the slow, oxidative phenotype in muscles of mdx versus dKO mice and found that both strains exhibit similar phenotypes. Chronic activation of 5' adenosine monophosphate-activated protein kinase with 5-amino-4-imidazolecarboxamide riboside (AICAR) resulted in expression of a slower, more oxidative phenotype in both mdx and dKO mice. In mdx mice, this fiber type shift was accompanied by clear functional improvements that included reductions in central nucleation, IgM sarcoplasmic penetration and sarcolemmal damage resulting from eccentric contractions, as well as in increased grip strength. These important morphological and functional adaptations were not seen in AICAR-treated dKO mice. Our findings show the central role of utrophin A in mediating the functional benefits associated with expression of a slower, more oxidative phenotype in dystrophic animals.

  8. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse.

    PubMed

    Rayavarapu, Sree; Coley, William; Cakir, Erdinc; Jahnke, Vanessa; Takeda, Shin'ichi; Aoki, Yoshitsugu; Grodish-Dressman, Heather; Jaiswal, Jyoti K; Hoffman, Eric P; Brown, Kristy J; Hathout, Yetrib; Nagaraju, Kanneboyina

    2013-05-01

    Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with (13)C6-lysine or stable isotope labeling in mammals (SILAM) with (15)N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of

  9. Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy.

    PubMed

    Adamo, Candace M; Dai, Dao-Fu; Percival, Justin M; Minami, Elina; Willis, Monte S; Patrucco, Enrico; Froehner, Stanley C; Beavo, Joseph A

    2010-11-02

    Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack expression of the protein dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies as well as cardiomyopathy, which manifests as congestive heart failure and arrhythmias. Like humans, dystrophin-deficient mice (mdx mice) show cardiac dysfunction as evidenced by a decrease in diastolic function followed by systolic dysfunction later in life. We have investigated whether sildenafil citrate (Viagra), a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction present in the mdx mice. By using echocardiography, we show that chronic sildenafil treatment reduces functional deficits in the cardiac performance of aged mdx mice, with no effect on normal cardiac function in WT controls. More importantly, when sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. It is recognized that PDE5 inhibitors can have cardioprotective effects in other models of cardiac damage, but the present study reports a prevention and reversal of pathological cardiac dysfunction as measured by functional analysis in a mouse model of DMD. Overall, the data suggest that PDE5 inhibitors may be a useful treatment for the cardiomyopathy affecting patients with DMD at early and late stages of the disease.

  10. Quantitative evaluation of the beneficial effects in the mdx mouse of epigallocatechin gallate, an antioxidant polyphenol from green tea.

    PubMed

    Nakae, Yoshiko; Dorchies, Olivier M; Stoward, Peter J; Zimmermann, Benno F; Ritter, Christina; Ruegg, Urs T

    2012-06-01

    In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (-)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients.

  11. Quantitative T2 combined with texture analysis of nuclear magnetic resonance images identify different degrees of muscle involvement in three mouse models of muscle dystrophy: mdx, Largemyd and mdx/Largemyd.

    PubMed

    Martins-Bach, Aurea B; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C M; Almeida, Camila F; Caldeira, Waldir; Ribeiro, Alberto F; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G; Vainzof, Mariz

    2015-01-01

    Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant-T2-measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.

  12. Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx, Largemyd and mdx/Largemyd

    PubMed Central

    Martins-Bach, Aurea B.; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C. M.; Almeida, Camila F.; Caldeira, Waldir; Ribeiro, Alberto F.; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G.; Vainzof, Mariz

    2015-01-01

    Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research

  13. Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice.

    PubMed

    Kueakhai, Pornanan; Changklungmoa, Narin; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Itagaki, Tadashi; Sobhon, Prasert

    2015-10-01

    In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immuno-blotting to react with the native FgCatL1 in the extract of newly excysted juveniles (NEJ), 4-week-old juveniles and the ES products of 4 week-old juveniles. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune response, respectively, it was found that both Th1 and Th2 responses were significantly increased in rproFgCatL1- and rmatFgCatL1-immunized groups compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rmatFgCatL1-immunized group showed a significant decrease when compared to rproFgCatL1-immunized group, indicating that rmatFgCatL1-vaccinated mice had reduced liver parenchyma damage. The pathological lesions of liver in vaccinated groups were significantly decreased when compared with control groups. This study indicates that r

  14. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice

    PubMed Central

    Fu, Chun; Begum, Khurshida; Jordan, Philip W.; He, Yan; Overbeek, Paul A.

    2016-01-01

    After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance) protein. Fanconi anemia (FA) proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2–3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line. PMID:27486799

  15. Deletion of Type 3 Adenylyl Cyclase Perturbs the Postnatal Maturation of Olfactory Sensory Neurons and Olfactory Cilium Ultrastructure in Mice

    PubMed Central

    Zhang, Zhe; Yang, Dong; Zhang, Mengdi; Zhu, Ning; Zhou, Yanfen; Storm, Daniel R.; Wang, Zhenshan

    2017-01-01

    Type 3 adenylyl cyclase (Adcy3) is localized to the cilia of olfactory sensory neurons (OSNs) and is an essential component of the olfactory cyclic adenosine monophosphate (cAMP) signaling pathway. Although the role of this enzyme in odor detection and axonal projection in OSNs was previously characterized, researchers will still have to determine its function in the maturation of postnatal OSNs and olfactory cilium ultrastructure. Previous studies on newborns showed that the anatomic structure of the main olfactory epithelium (MOE) of Adcy3 knockout mice (Adcy3-/-) is indistinguishable from that of their wild-type littermates (Adcy3+/+), whereas the architecture and associated composition of MOE are relatively underdeveloped at this early age. The full effects of sensory deprivation on OSNs may not also be exhibited in such age. In the present study, following a comparison of postnatal OSNs in seven-, 30-, and 90-day-old Adcy3-/- mice and wild-type controls (Adcy3+/+), we observed that the absence of Adcy3 leads to cumulative defects in the maturation of OSNs. Upon aging, Adcy3-/- OSNs exhibited increase in immature cells and reduction in mature cells along with elevated apoptosis levels. The density and ultrastructure of Adcy3-/- cilia were also disrupted in mice upon aging. Collectively, our results reveal an indispensable role of Adcy3 in postnatal maturation of OSNs and maintenance of olfactory cilium ultrastructure in mice through adulthood. PMID:28154525

  16. MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy.

    PubMed

    Yuasa, Katsutoshi; Hagiwara, Yasuko; Ando, Masanori; Nakamura, Akinori; Takeda, Shin'ichi; Hijikata, Takao

    2008-01-01

    miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.

  17. A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.

    PubMed

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

    2013-08-01

    Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice, we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx(4Cv) mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx(4Cv) mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx(4Cv) recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function and the utility of the NSG-mdx(4Cv) model for studies on muscle regeneration and Duchenne muscular dystrophy therapy.

  18. Long-term treatment with naproxcinod significantly improves skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy.

    PubMed

    Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Van Der Meulen, Jack H; Yu, Qing; Phadke, Aditi; Miller, Brittany K; Gordish-Dressman, Heather; Ongini, Ennio; Miglietta, Daniela; Nagaraju, Kanneboyina

    2014-06-15

    In Duchenne muscular dystrophy (DMD) patients and the mouse model of DMD, mdx, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to functional impairments. Previous studies have shown that nitric oxide (NO) donation associated with anti-inflammatory action has beneficial effects in dystrophic mouse models. In this study, we have systematically investigated the effects of naproxcinod, an NO-donating naproxen derivative, on the skeletal and cardiac disease phenotype in mdx mice. Four-week-old mdx and C57BL/10 mice were treated with four different concentrations (0, 10, 21 and 41 mg/kg) of naproxcinod and 0.9 mg/kg of prednisolone in their food for 9 months. All mice were subjected to twice-weekly treadmill sessions, and functional and behavioral parameters were measured at 3, 6 and 9 months of treatment. In addition, we evaluated in vitro force contraction, optical imaging of inflammation, echocardiography and blood pressure (BP) at the 9-month endpoint prior to sacrifice. We found that naproxcinod treatment at 21 mg/kg resulted in significant improvement in hindlimb grip strength and a 30% decrease in inflammation in the fore- and hindlimbs of mdx mice. Furthermore, we found significant improvement in heart function, as evidenced by improved fraction shortening, ejection fraction and systolic BP. In addition, the long-term detrimental effects of prednisolone typically seen in mdx skeletal and heart function were not observed at the effective dose of naproxcinod. In conclusion, our results indicate that naproxcinod has significant potential as a safe therapeutic option for the treatment of muscular dystrophies.

  19. Distinct roles of TRAF6 at early and late stages of muscle pathology in the mdx model of Duchenne muscular dystrophy.

    PubMed

    Hindi, Sajedah M; Sato, Shuichi; Choi, Yongwon; Kumar, Ashok

    2014-03-15

    Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by loss of functional dystrophin protein. Accumulating evidence suggests that the deficiency of dystrophin leads to aberrant activation of many signaling pathways which contribute to disease progression. However, the proximal signaling events leading to the activation of various pathological cascades in dystrophic muscle remain less clear. TNF receptor-associated factor 6 (TRAF6) is an adaptor protein which acts as a signaling intermediate for several receptor-mediated signaling events leading to the context-dependent activation of a number of signaling pathways. TRAF6 is also an E3 ubiquitin ligase and an important regulator of autophagy. However, the role of TRAF6 in pathogenesis of DMD remains unknown. Here, we demonstrate that the levels and activity of TRAF6 are increased in skeletal muscle of mdx (a mouse model of DMD) mice. Targeted deletion of TRAF6 improves muscle strength and reduces fiber necrosis, infiltration of macrophages and the activation of proinflammatory transcription factor nuclear factor-kappa B (NF-κB) in 7-week-old mdx mice. Ablation of TRAF6 also increases satellite cells proliferation and myofiber regeneration in young mdx mice. Intriguingly, ablation of TRAF6 exacerbates muscle injury and increases fibrosis in 9-month-old mdx mice. TRAF6 inhibition reduces the markers of autophagy and Akt signaling in dystrophic muscle of mdx mice. Collectively, our study suggests that while the inhibition of TRAF6 improves muscle structure and function in young mdx mice, its continued inhibition causes more severe myopathy at later stages of disease progression potentially through repressing autophagy.

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

    PubMed

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

    2013-11-15

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

  1. Variable maturation and oviposition by female Schistosoma japonicum in mice: the effects of irradiation of the host prior to infection

    SciTech Connect

    Cheever, A.W.; Duvall, R.H.

    1987-11-01

    The maturation of female Schistosoma japonicum was found to vary greatly within each of two Philippine strains of this parasite and some females did not contain uterine eggs 7 to 15 weeks after infection while others contained numerous eggs before the fifth week of infection. It was found that female worms containing less than 20 uterine eggs contributed little to the accumulation of eggs in the tissues of infected mice. Such worms also generally appeared to be immature. The variable rate of maturation of worms is likely to have profound effects on the immune reactions of mice as well as on the pathologic response to infection. Systematic delay in oviposition was serendipitously found in worms from mice which had been irradiated for other purposes prior to exposure to S. japonicum, and from the fourth to the sixth week after infection egg production by worms in irradiated mice lagged well behind that in intact mice. Seven to 10 weeks after infection these worms were laying normal numbers of eggs, as judged by egg passage per worm pair in the feces and the accumulation of eggs in the tissues. S. mansoni developed normally in irradiated mice.

  2. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue.

    PubMed

    Nobusue, Hiroyuki; Endo, Tsuyoshi; Kano, Koichiro

    2008-06-01

    We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro.

  3. The p35 human invariant chain in transgenic mice restores mature B cells in the absence of endogenous CD74.

    PubMed

    Genève, Laetitia; Ménard, Catherine; Labrecque, Nathalie; Thibodeau, Jacques

    2012-10-01

    The invariant chain (Ii; CD74) has pleiotropic functions and Ii-deficient mice show defects in MHC class II (MHC II) transport and B cell maturation. In humans, but not in mice, a minor Iip35 isoform of unknown function includes an endoplasmic reticulum-retention motif that is masked upon binding of MHC II molecules. To gain further insight into the roles of Ii in B cell homeostasis, we generated Iip35 transgenic mice (Tgp35) and bred these with mice deficient for Ii (Tgp35/mIiKO). Iip35 was shown to compete with mIi for the binding to I-A(b) . In addition, classical endosomal degradation products (p20/p10) and the class II-associated invariant chain peptide (CLIP) fragment were detected. Moreover, Iip35 favored the formation of compact peptide-MHC II complexes in the Tgp35/mIiKO mice. I-A(b) levels were restored at the plasma membrane of mature B cells but Iip35 affected the fine conformation of MHC II molecules as judged by the increased reactivity of the AF6-120.1 antibody in permeabilized cells. However, the human Iip35 cannot fully replace the endogenous Ii. Indeed, most immature B cells in the bone marrow and spleen of transgenic mice had reduced surface expression of MHC II molecules, demonstrating a dominant-negative effect of Iip35 in Tgp35 mice. Interestingly, while maturation to follicular B cells was normal, Iip35 expression appeared to reduce the proportions of marginal zone B cells. These results emphasize the importance of Ii in B cell homeostasis and suggest that Iip35 could have regulatory functions.

  4. In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse.

    PubMed

    Aoki, Yoshitsugu; Nakamura, Akinori; Yokota, Toshifumi; Saito, Takashi; Okazawa, Hitoshi; Nagata, Tetsuya; Takeda, Shin'ichi

    2010-11-01

    A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

  5. Lack of mature lymphocytes results in obese but metabolically healthy mice when fed a high-fat diet

    PubMed Central

    Liu, X; Huh, JY; Gong, H; Chamberland, JP; Brinkoetter, MT; Hamnvik, O-PR; Mantzoros, CS

    2017-01-01

    BACKGROUND/OBJECTIVES Obesity is characterized by chronic inflammation and immune dysregulation, as well as insulin resistance, but the link between obesity and adaptive immunity remains to be fully studied. METHODS To elucidate the role of adaptive immunity on body composition, glucose homeostasis and inflammation, recombination-activating gene 1 knockout (Rag1 − / −) mice, without mature T-lymphocytes or B-lymphocytes, were maintained on a low- or high-fat diet (LFD and HFD, respectively) for 11 weeks. RESULTS Rag1 − / − mice fed HFD gained significantly more weight and had increased body fat compared with wild type. Downregulation of energy expenditure as well as brown fat uncoupling protein UCP-1 and UCP-3 gene expression were noticed in HFD-fed Rag1 − / − mice compared with LFD. HFD mice had significantly decreased energy intake compared with LFD mice, consistent with decreased agouti-related protein and increased pro-opiomelanocortin gene expression levels in the hypothalamus. Moreover, compared with wild type, Rag1 − / − mice had lower interleukin (IL)-4 levels, a cytokine recently found to induce browning in white adipocytes, and higher IL-12 levels in HFD-fed Rag1 − / − mice. Despite that HFD Rag1 − / − mice were more obese, they had similar glucose, insulin and adiponectin levels, while leptin was marginally increased. CONCLUSIONS Mice with deficiency in adaptive immunity are obese, partly owing to decreased energy expenditure, but are metabolically normal, suggesting that mature lymphocytes have necessary roles in the development of obesity-related metabolic dysregulation. PMID:25994806

  6. Bile acid-FXRα pathways regulate male sexual maturation in mice

    PubMed Central

    Vega, Aurélie; Sédes, Lauriane; Rouaisnel, Betty; de Haze, Angélique; Baron, Silvère; Schoonjans, Kristina; Caira, Françoise; Volle, David H.

    2016-01-01

    The bile acid receptor Farnesol-X-Receptor alpha (FRXα) is a member of the nuclear receptor superfamily. FRXα is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. In adult, short term treatment (12 hours) with FRXα agonist inhibits the expression of steroidogenic genes via the induction of the Small heterodimer partner (SHP). However the consequences of FRXα activation on testicular pathophysiology have never been evaluated. We demonstrate here that mice fed a diet supplemented with bile acid during pubertal age show increased incidence of infertility. This is associated with altered differentiation and increase apoptosis of germ cells due to lower testosterone levels. At the molecular level, next to the repression of basal steroidogenesis via the induction expression of Shp and Dax-1, two repressors of steroidogenesis, the main action of the BA-FRXα signaling is through lowering the Leydig cell sensitivity to the hypothalamo-pituitary axis, the main regulator of testicular endocrine function. In conclusion, BA-FRXα signaling is a critical actor during sexual maturation. PMID:26848619

  7. Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients

    PubMed Central

    Hathout, Yetrib; Marathi, Ramya L.; Rayavarapu, Sree; Zhang, Aiping; Brown, Kristy J.; Seol, Haeri; Gordish-Dressman, Heather; Cirak, Sebahattin; Bello, Luca; Nagaraju, Kanneboyina; Partridge, Terry; Hoffman, Eric P.; Takeda, Shin'ichi; Mah, Jean K.; Henricson, Erik; McDonald, Craig

    2014-01-01

    It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials. PMID:25027324

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

    PubMed

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

    2011-05-01

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

  9. Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients.

    PubMed

    Hathout, Yetrib; Marathi, Ramya L; Rayavarapu, Sree; Zhang, Aiping; Brown, Kristy J; Seol, Haeri; Gordish-Dressman, Heather; Cirak, Sebahattin; Bello, Luca; Nagaraju, Kanneboyina; Partridge, Terry; Hoffman, Eric P; Takeda, Shin'ichi; Mah, Jean K; Henricson, Erik; McDonald, Craig

    2014-12-15

    It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials.

  10. Suppression of Dendritic Cell Maturation and T Cell Proliferation by Synovial Fluid Myeloid Cells from Mice with Autoimmune Arthritis

    PubMed Central

    Egelston, Colt; Kurkó, Júlia; Besenyei, Timea; Tryniszewska, Beata; Rauch, Tibor A.; Glant, Tibor T.; Mikecz, Katalin

    2012-01-01

    Objective To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells, harvested from the joints of mice with proteoglycan (PG)-induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen-specific T-cell proliferation. Methods We monitored DC maturation (MHC class II and CD86 expression) by flow cytometry upon co-culture of DCs with SF or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T-cell proliferation were studied using T cells purified from PG-specific T cell receptor transgenic (PG-TCR-Tg) mice. Phenotypic analysis of myeloid cells was performed employing immunostaining, RT-PCR, Western blot, and biochemical assays. Results Inflammatory SF cells significantly suppressed the maturation of DCs upon co-culture. PG-TCR-Tg T cells cultured with antigen-loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28-stimulated proliferation of the same T cells, suggesting a DC-dependent mechanism. SF cells exhibited all of the characteristics of myeloid-derived suppressor cells (MDSCs), and exerted suppression primarily through production of nitric oxide and reactive oxygen species by granulocyte-like cells. Conclusion SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T-cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation. PMID:22492217

  11. Structural and functional maturation of distal femoral cartilage and bone during postnatal development and growth in humans and mice.

    PubMed

    Chan, Elaine F; Harjanto, Ricky; Asahara, Hiroshi; Inoue, Nozomu; Masuda, Koichi; Bugbee, William D; Firestein, Gary S; Hosalkar, Harish S; Lotz, Martin K; Sah, Robert L

    2012-04-01

    The size and shape of joints markedly affect their biomechanical properties, but the macroscopic 3-dimensional (3-D) mechanism and extent of cartilage and joint maturation during normal growth are largely unknown. This study qualitatively illustrates the development of the bone-cartilage interface in the knee during postnatal growth in humans and C57BL/6 wild-type mice, quantitatively defines the 3-D shape using statistical shape modeling, and assesses growth strain rates in the mouse distal femur. Accurate quantification of the cartilage-bone interface geometry is imperative for furthering the understanding of the macroscopic mechanisms of cartilage maturation and overall joint development.

  12. Effect of insulin supplementation on in vitro maturation of pre-antral follicles from adult and pre-pubertal mice.

    PubMed

    Nath, Amar; Hakim, Bilal Ahmad; Rajender, Singh; Singh, Kavita; Sachdev, Monika; Konwar, Rituraj

    2016-05-01

    This study was aimed to determine the impact of insulin concentrations on in vitro pre-antral follicle growth, survival, antrum formation rate, and retrieval of mature oocytes in mice. Mice pre-antral follicle growth were recorded on days 2, 4, 6, 8, 10, and 12 in α-modified essential media (α-MEM) supplemented with insulin concentrations of 6, 8, and 10 μg/ml along with 10% FBS, 100 mIU/ml follicle stimulating hormone, 10 mIU/ml luteinizing hormone, 100 μg/ml penicillin, and 50 μg/ml streptomycin. After 12 d of growth in vitro, follicles were allowed to mature for 16-18 h in α-MEM supplemented with 1.5 IU/ml human chorionic gonadotrophin (hCG) and 5 ng/ml epidermal growth factor (EGF). The initial diameter (54.86 ± 2.5 μm) of mice oocyte progressively increased in all the three insulin concentration groups and attained a maximum size on day 12 (71.90 ± 2.8 μm). Supplementation with higher concentrations of insulin (both 8 and 10 μg/ml) significantly enhanced antrum formation without effecting the oocyte diameter and percent retrieval of mature oocyte in all the three concentration groups. Both in vitro cultured as well as in vivo collected follicles and oocytes showed similar localization and expression of oocyte maturation markers SAS1B and GDF9. Insulin concentration of 8 μg/ml was found to be optimal for in vitro follicle culture of adult mice (42-49 d). Optimized follicle culture conditions were also assessed successfully with pre-pubertal mice (12-14 d); however, adult mice showed higher follicle survival, antrum formation, and more mature oocytes production in comparison to pre-pubertal mice.

  13. Mature adult dystrophic mouse muscle environment does not impede efficient engrafted satellite cell regeneration and self-renewal.

    PubMed

    Boldrin, Luisa; Zammit, Peter Steven; Muntoni, Francesco; Morgan, Jennifer Elizabeth

    2009-10-01

    Changes that occur in the skeletal muscle environment with the progress of muscular dystrophies may affect stem cell function and result in impaired muscle regeneration. It has previously been suggested that the success of stem cell transplantation could therefore be dependent both on the properties of the cell itself and on the host muscle environment. Here we engrafted young and mature adult mdx-nude mice, which are the genetic homolog of Duchenne muscular dystrophy, with a small number of satellite cells freshly isolated from young, normal donor mice. We found that the donor satellite cells contributed to muscle regeneration and self-renewal as efficiently within mature adult, as in young, dystrophic host muscle. Donor-derived satellite cells also contributed to robust regeneration after further injury, showing that they were functional despite the more advanced dystrophic muscle environment. These findings provide evidence that muscle tissue in a later stage of dystrophy may be effectively treated by stem cells.

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

    PubMed Central

    CARBERRY, STEVEN; BRINKMEIER, HEINRICH; ZHANG, YAXIN; WINKLER, CLAUDIA K.; OHLENDIECK, KAY

    2013-01-01

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

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

    PubMed

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

    2013-09-01

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

  16. Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice

    PubMed Central

    Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Aydiner, Fulya; Sasson, Isaac; Ilbay, Orkan; Sakkas, Denny; Lowther, Katie M.; Mehlmann, Lisa M.; Seli, Emre

    2014-01-01

    Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab-knockout mice. Although Epab−/− males and Epab+/− of both sexes were fertile, Epab−/− female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab−/− oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Ccnb1 (cyclin B1) and Dazl (deleted in azoospermia-like) mRNAs. Microinjection of Epab mRNA into Epab−/− germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab−/− mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding the EGF (epidermal growth factor)-like growth factors Areg (amphiregulin), Ereg (epiregulin) and Btc (betacellulin), and their downstream regulators, Ptgs2 (prostaglandin synthase 2), Has2 (hyaluronan synthase 2) and Tnfaip6 (tumour necrosis factor α-induced protein 6). The findings from the present study indicate that EPAB is necessary for oogenesis, folliculogenesis and female fertility in mice. PMID:22621333

  17. Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy.

    PubMed

    Rooney, Jachinta E; Gurpur, Praveen B; Burkin, Dean J

    2009-05-12

    Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The alpha(7)beta(1)-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the alpha(7)-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate alpha(7)-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for alpha(7)-integrin expression. We show that laminin-111 (alpha(1), beta(1), gamma(1)), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased alpha(7)-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of alpha(7)-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.

  18. Propagation in the transverse tubular system and voltage dependence of calcium release in normal and mdx mouse muscle fibres

    PubMed Central

    Woods, Christopher E; Novo, David; DiFranco, Marino; Capote, Joana; Vergara, Julio L

    2005-01-01

    Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation–contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca2+ release by measuring Ca2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca2+ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca2+ may be altered in the mdx mouse. PMID:16123111

  19. Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

    PubMed

    Keck, Zhen-Yong; Wang, Yong; Lau, Patrick; Lund, Garry; Rangarajan, Sneha; Fauvelle, Catherine; Liao, Grant C; Holtsberg, Frederick W; Warfield, Kelly L; Aman, M Javad; Pierce, Brian G; Fuerst, Thomas R; Bailey, Justin R; Baumert, Thomas F; Mariuzza, Roy A; Kneteman, Norman M; Foung, Steven K H

    2016-12-01

    Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation.

  20. Preclinical studies in the mdx mouse model of duchenne muscular dystrophy with the histone deacetylase inhibitor givinostat.

    PubMed

    Consalvi, Silvia; Mozzetta, Chiara; Bettica, Paolo; Germani, Massimiliano; Fiorentini, Francesco; Del Bene, Francesca; Rocchetti, Maurizio; Leoni, Flavio; Monzani, Valmen; Mascagni, Paolo; Puri, Pier Lorenzo; Saccone, Valentina

    2013-05-20

    Previous work has established the existence of dystrophin-nitric oxide (NO) signaling to histone deacetylases (HDACs) that is deregulated in dystrophic muscles. As such, pharmacological interventions that target HDACs (that is, HDAC inhibitors) are of potential therapeutic interest for the treatment of muscular dystrophies. In this study, we explored the effectiveness of long-term treatment with different doses of the HDAC inhibitor givinostat in mdx mice--the mouse model of Duchenne muscular dystrophy (DMD). This study identified an efficacy for recovering functional and histological parameters within a window between 5 and 10 mg/kg/d of givinostat, with evident reduction of the beneficial effects with 1 mg/kg/d dosage. The long-term (3.5 months) exposure of 1.5-month-old mdx mice to optimal concentrations of givinostat promoted the formation of muscles with increased cross-sectional area and reduced fibrotic scars and fatty infiltration, leading to an overall improvement of endurance performance in treadmill tests and increased membrane stability. Interestingly, a reduced inflammatory infiltrate was observed in muscles of mdx mice exposed to 5 and 10 mg/kg/d of givinostat. A parallel pharmacokinetic/pharmacodynamic analysis confirmed the relationship between the effective doses of givinostat and the drug distribution in muscles and blood of treated mice. These findings provide the preclinical basis for an immediate translation of givinostat into clinical studies with DMD patients.

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

    PubMed

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

    2012-10-10

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

  2. Changes in surface antigens of Hymenolepis nana during differentiation and maturation in mice.

    PubMed

    Ito, A; Onitake, K

    1987-06-01

    The surface antigens of oncosphere, cysticercoid, adult scolex and adult strobila (other than scolex) of Hymenolepis nana differ critically from one another. When the oncosphere of H. nana undergoes differentiation and development into the mature tapeworm, the infected mouse first produces anti-oncosphere antibody, followed by anti-cysticercoid, anti-adult scolex and finally anti-strobila (other than scolex region) antibodies of IgG, IgM and IgA isotypes as detected by indirect immunofluorescent antibody test. The parasite changed its surface antigens throughout its differentiation and maturation, and all developmental stages were recognized by the infected mouse host. However, there appeared no further changes in surface antigens during aging after maturation. The antibody responses were always delayed compared with the differentiation and maturation of the parasite.

  3. Loss of function of KIF1B impairs oocyte meiotic maturation and early embryonic development in mice.

    PubMed

    Kong, Xiang-Wei; Wang, Dong-Hui; Zhou, Cheng-Jie; Zhou, Hong-Xia; Liang, Cheng-Guang

    2016-11-01

    Kinesin family member 1B (KIF1B) is an important microtubule-dependent monomeric motor in mammals, although little is known about its role in meiosis. We profiled KIF1B expression and localization during oocyte maturation and early embryonic development in mice, revealing a dynamic pattern throughout meiotic progression. Depletion or inhibition of KIF1B leads to abnormal polar body extrusion, disordered spindle dynamics, defects in chromosome congression, increased aneuploidy, and impaired embryonic development. Further, KIF1B depletion affects the distribution of mitochondria and abundance of ATP. Taken together, our study demonstrates that mouse KIF1B is important for spindle assembly, chromosome congression, and mitochondrial distribution during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 83: 1027-1040, 2016 © 2016 Wiley Periodicals, Inc.

  4. Early life stress accelerates behavioral and neural maturation of the hippocampus in male mice

    PubMed Central

    Bath, K.G.; Manzano-Nieves, G.; Goodwill, H.

    2017-01-01

    Early life stress (ELS) increases the risk for later cognitive and emotional dysfunction. ELS is known to truncate neural development through effects on suppressing cell birth, increasing cell death, and altering neuronal morphology, effects that have been associated with behavioral profiles indicative of precocious maturation. However, how earlier silencing of growth drives accelerated behavioral maturation has remained puzzling. Here, we test the novel hypothesis that, ELS drives a switch from growth to maturation to accelerate neural and behavioral development. To test this, we used a mouse model of ELS, fragmented maternal care, and a cross-sectional dense sampling approach focusing on hippocampus and measured effects of ELS on the ontogeny of behavioral development and biomarkers of neural maturation. Consistent with previous work, ELS was associated with an earlier developmental decline in expression of markers of cell proliferation (Ki-67) and differentiation (doublecortin). However, ELS also led to a precocious arrival of Parvalbumin-positive cells, led to an earlier switch in NMDA receptor subunit expression (marker of synaptic maturity), and was associated with an earlier rise in myelin basic protein expression (key component of the myelin sheath). In addition, in a contextual fear-conditioning task, ELS accelerated the timed developmental suppression of contextual fear. Together, these data provide support for the hypothesis that ELS serves to switch neurodevelopment from processes of growth to maturation and promotes accelerated development of some forms of emotional learning. PMID:27155103

  5. Early life stress accelerates behavioral and neural maturation of the hippocampus in male mice.

    PubMed

    Bath, K; Manzano-Nieves, G; Goodwill, H

    2016-06-01

    Early life stress (ELS) increases the risk for later cognitive and emotional dysfunction. ELS is known to truncate neural development through effects on suppressing cell birth, increasing cell death, and altering neuronal morphology, effects that have been associated with behavioral profiles indicative of precocious maturation. However, how earlier silencing of growth drives accelerated behavioral maturation has remained puzzling. Here, we test the novel hypothesis that, ELS drives a switch from growth to maturation to accelerate neural and behavioral development. To test this, we used a mouse model of ELS, fragmented maternal care, and a cross-sectional dense sampling approach focusing on hippocampus and measured effects of ELS on the ontogeny of behavioral development and biomarkers of neural maturation. Consistent with previous work, ELS was associated with an earlier developmental decline in expression of markers of cell proliferation (Ki-67) and differentiation (doublecortin). However, ELS also led to a precocious arrival of Parvalbumin-positive cells, led to an earlier switch in NMDA receptor subunit expression (marker of synaptic maturity), and was associated with an earlier rise in myelin basic protein expression (key component of the myelin sheath). In addition, in a contextual fear-conditioning task, ELS accelerated the timed developmental suppression of contextual fear. Together, these data provide support for the hypothesis that ELS serves to switch neurodevelopment from processes of growth to maturation and promotes accelerated development of some forms of emotional learning.

  6. Galanin synaptic input to gonadotropin-releasing hormone perikarya in juvenile and adult female mice: implications for sexual maturity.

    PubMed

    Rajendren, G; Li, X

    2001-11-26

    Changes in connectivity of the gonadotropin-releasing hormone (GnRH) neuronal system are believed to occur during the transition from juvenile to adulthood in females. Experiments were designed to investigate whether there is any difference in the number of galanin inputs to GnRH cells located in the organum vasculosum of lamina terminalis-rostral preoptic area (OVLT-rPOA) between juvenile (2 weeks old) and adult (10 weeks old) female mice. Triple label immunofluorescence staining of brain sections for galanin, GnRH and the presynaptic vesicle marker synaptophysin coupled with confocal microscopy was employed to identify galanin synapses to GnRH perikarya. The number of galanin synapses to GnRH cells and the proportion of GnRH cells with galanin input were significantly higher in adults than in juvenile mice. In adult mice, the proportions of GnRH cells with 0, 1, 2, 3, 4, 5 or 6 galanin synapses/cell were comparable to each other whereas in the juveniles the vast majority of them received no galanin synaptic input. A greater number of galanin synapses in adult as compared with juvenile female mice suggests a functional role for galanin in the maturation of the GnRH system.

  7. Reduced Connexin26 in the Mature Cochlea Increases Susceptibility to Noise-Induced Hearing Loss in Mice

    PubMed Central

    Zhou, Xing-Xing; Chen, Sen; Xie, Le; Ji, Yu-Zi; Wu, Xia; Wang, Wen-Wen; Yang, Qi; Yu, Jin-Tao; Sun, Yu; Lin, Xi; Kong, Wei-Jia

    2016-01-01

    Connexin26 (Cx26, encoded by GJB2) mutations are the most common cause of non-syndromic deafness. GJB2 is thought to be involved in noise-induced hearing loss (NIHL). However, the role of Cx26 in NIHL is still obscure. To explore the association between Cx26 and NIHL, we established a Cx26 knockdown (KD) mouse model by conditional knockdown of Cx26 at postnatal day 18 (P18), and then we observed the auditory threshold and morphologic changes in these mice with or without noise exposure. The Cx26 KD mice did not exhibit substantial hearing loss and hair cell degeneration, while the Cx26 KD mice with acoustic trauma experienced higher hearing loss than simple noise exposure siblings and nearly had no recovery. Additionally, extensive outer hair cell loss and more severe destruction of the basal organ of Corti were observed in Cx26 KD mice after noise exposure. These data indicate that reduced Cx26 expression in the mature mouse cochlea may increase susceptibility to noise-induced hearing loss and facilitate the cell degeneration in the organ of Corti. PMID:26927086

  8. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    SciTech Connect

    Ryu, Min Sook; Woo, Min-Yeong; Kwon, Daeho; Hong, Allen E.; Song, Kye Yong; Park, Sun; Lim, In Kyoung

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.

  9. Histological study of masseter muscle in a mouse muscular dystrophy model (mdx mouse).

    PubMed

    Abe, S; Kasahara, N; Amano, M; Yoshii, M; Watanabe, H; Ide, Y

    2000-08-01

    Histological changes in the masseter muscle were observed over time in mdx mice, a muscular dystrophy model. It was found that marked necrosis occurs about the time of weaning at around 4 weeks of age; then the tissue actively regenerates at 8 weeks and stabilizes as regenerated muscle with centronuclei at 15 weeks old. This study examined the centronucleus in regenerated muscle. The process from necrosis to regeneration in muscle fibers occurs a little later in the masseter muscle than in other limbic muscles. Regenerated muscles observed around 15 weeks after birth showed a moth-eaten appearance. Transmission Electron Microscope (TEM) observation of transverse sections of muscle fibers revealed that myofibrils surrounded lost regions in the area showing a moth-eaten appearance. Thus, some defensive mechanism may affect the ability of muscle fibers to maintain a function close to normal in mdx mice even though the muscle fibers develop muscular dystrophy. The function of the masseter muscle drastically changes from sucking to mastication behavior at around 4 weeks, and this was considered to influence the morphological changes in the muscle tissue. The moth-eaten appearance seen at 15 weeks may represent an appropriate myofibril reconstruction preventing invasion of the lost regions.

  10. Influenza Infection in Mice Induces Accumulation of Lung Mast Cells through the Recruitment and Maturation of Mast Cell Progenitors

    PubMed Central

    Zarnegar, Behdad; Mendez-Enriquez, Erika; Westin, Annika; Söderberg, Cecilia; Dahlin, Joakim S.; Grönvik, Kjell-Olov; Hallgren, Jenny

    2017-01-01

    Mast cells (MCs) are powerful immune cells that mature in the peripheral tissues from bone marrow (BM)-derived mast cell progenitors (MCp). Accumulation of MCs in lung compartments where they are normally absent is thought to enhance symptoms in asthma. The enrichment of lung MCs is also observed in mice subjected to models of allergic airway inflammation. However, whether other types of lung inflammation trigger increased number of MCp, which give rise to MCs, is unknown. Here, mouse-adapted H1N1 influenza A was used as a model of respiratory virus infection. Intranasal administration of the virus induced expression of VCAM-1 on the lung vascular endothelium and an extensive increase in integrin β7hi lung MCp. Experiments were performed to distinguish whether the influenza-induced increase in the number of lung MCp was triggered mainly by recruitment or in situ cell proliferation. A similar proportion of lung MCp from influenza-infected and PBS control mice were found to be in a proliferative state. Furthermore, BM chimeric mice were used in which the possibility of influenza-induced in situ cell proliferation of host MCp was prevented. Influenza infection in the chimeric mice induced a similar number of lung MCp as in normal mice. These experiments demonstrated that recruitment of MCp to the lung is the major mechanism behind the influenza-induced increase in lung MCp. Fifteen days post-infection, the influenza infection had elicited an immature MC population expressing intermediate levels of integrin β7, which was absent in controls. At the same time point, an increased number of toluidine blue+ MCs was detected in the upper central airways. When the inflammation was resolved, the MCs that accumulated in the lung upon influenza infection were gradually lost. In summary, our study reveals that influenza infection induces a transient accumulation of lung MCs through the recruitment and maturation of MCp. We speculate that temporary augmented numbers of lung MCs

  11. Protective Effect of Boiled and Freeze-dried Mature Silkworm Larval Powder Against Diethylnitrosamine-induced Hepatotoxicity in Mice

    PubMed Central

    Cho, Jae-Min; Kim, Kee-Young; Ji, Sang-Deok; Kim, Eun-Hee

    2016-01-01

    Background Hepatocellular carcinoma (HCC) is a representative inflammation-associated cancer and known to be the most frequent tumors. HCC may also induce important pro- and anti-tumor immune reactions. However, the underlying mechanisms are unsatisfactorily identified. We investigated the protective effect of boiled and freeze-dried mature silkworm larval powder (BMSP) on diethylnitrosamine (DEN)-induced hepatotoxicity in mice. Methods Mice were fed with diet containing BMSP (0.1, 1, and 10 g/kg) for two weeks and DEN (100 mg/kg, intraperitoneally) was injected 18 hours before the end of this experiment. Liver toxicity was determined in serum and histopathological examination was assessed in the liver tissues. Infiltration of immune cells and expressions of inflammatory cytokines and chemokines were also examined. Results Pretreatment with BMSP reduced necrotic and histopathological changes induced by DEN in the liver. Measurement of serum biochemical indicators, the levels of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase, showed that pretreatment with BMSP also decreased DEN-induced hepatotoxicity. In addition, BMSP inhibited the macrophage and CD31 infiltration in a dose-dependent manner. The expressions of interleukin-1β, IFN-γ and chemokines for T cell activation were decreased in BMSP pretreatment groups. Conclusions BMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice. PMID:27722143

  12. Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine

    PubMed Central

    De Luca, Annamaria; Pierno, Sabata; Liantonio, Antonella; Cetrone, Michela; Camerino, Claudia; Simonetti, Simonetta; Papadia, Francesco; Camerino, Diana Conte

    2001-01-01

    No clear data is available about functional alterations in the calcium-dependent excitation-contraction (e-c) coupling mechanism of dystrophin-deficient muscle of mdx mice. By means of the intracellular microelectrode ‘point' voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8–12 weeks, during the active regeneration of hind limb muscles, and 6–8 months, when regeneration is complete. The EDL muscle fibres of 8–12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction- and relaxation-related calcium movements) with respect to control mice of 6–8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without effect on 6–8-month-old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse. HPLC determination of taurine content at 6–8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid. The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid. PMID:11226135

  13. Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine.

    PubMed

    De Luca, A; Pierno, S; Liantonio, A; Cetrone, M; Camerino, C; Simonetti, S; Papadia, F; Camerino, D C

    2001-03-01

    No clear data is available about functional alterations in the calcium-dependent excitation-contraction (e-c) coupling mechanism of dystrophin-deficient muscle of mdx mice. By means of the intracellular microelectrode "point" voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8 - 12 weeks, during the active regeneration of hind limb muscles, and 6 - 8 months, when regeneration is complete. The EDL muscle fibres of 8 - 12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction- and relaxation-related calcium movements) with respect to control mice of 6 - 8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without effect on 6 - 8-month-old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse. HPLC determination of taurine content at 6 - 8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid. The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid.

  14. Ovariectomy in mature mice does not increase food intake, but increases adiposity and adipose tissue inflammation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Menopause, characterized by reduced estrogen (E2), is associated with increased adiposity and metabolic pathology. Molecular mechanisms underlying this association between low E2 status and metabolic disease are not fully elucidated. When mice are fed a high fat diet (HFD) to induce obesity and diab...

  15. Essential Role of Cyclin-G–associated Kinase (Auxilin-2) in Developing and Mature Mice

    PubMed Central

    Lee, Dong-won; Zhao, Xiaohong; Yim, Yang-In; Eisenberg, Evan

    2008-01-01

    Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis. PMID:18434600

  16. Developmental programming of long non-coding RNAs during postnatal liver maturation in mice.

    PubMed

    Peng, Lai; Paulson, Ariel; Li, Hua; Piekos, Stephanie; He, Xi; Li, Linheng; Zhong, Xiao-Bo

    2014-01-01

    The liver is a vital organ with critical functions in metabolism, protein synthesis, and immune defense. Most of the liver functions are not mature at birth and many changes happen during postnatal liver development. However, it is unclear what changes occur in liver after birth, at what developmental stages they occur, and how the developmental processes are regulated. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation. Here, we analyzed the transcriptome of lncRNAs in mouse liver from perinatal (day -2) to adult (day 60) by RNA-Sequencing, with an attempt to understand the role of lncRNAs in liver maturation. We found around 15,000 genes expressed, including about 2,000 lncRNAs. Most lncRNAs were expressed at a lower level than coding RNAs. Both coding RNAs and lncRNAs displayed three major ontogenic patterns: enriched at neonatal, adolescent, or adult stages. Neighboring coding and non-coding RNAs showed the trend to exhibit highly correlated ontogenic expression patterns. Gene ontology (GO) analysis revealed that some lncRNAs enriched at neonatal ages have their neighbor protein coding genes also enriched at neonatal ages and associated with cell proliferation, immune activation related processes, tissue organization pathways, and hematopoiesis; other lncRNAs enriched at adolescent ages have their neighbor protein coding genes associated with different metabolic processes. These data reveal significant functional transition during postnatal liver development and imply the potential importance of lncRNAs in liver maturation.

  17. Long-term wheel running compromises diaphragm function but improves cardiac and plantarflexor function in the mdx mouse.

    PubMed

    Selsby, Joshua T; Acosta, Pedro; Sleeper, Meg M; Barton, Elisabeth R; Sweeney, H Lee

    2013-09-01

    Dystrophin-deficient muscles suffer from free radical injury, mitochondrial dysfunction, apoptosis, and inflammation, among other pathologies that contribute to muscle fiber injury and loss, leading to wheelchair confinement and death in the patient. For some time, it has been appreciated that endurance training has the potential to counter many of these contributing factors. Correspondingly, numerous investigations have shown improvements in limb muscle function following endurance training in mdx mice. However, the effect of long-term volitional wheel running on diaphragm and cardiac function is largely unknown. Our purpose was to determine the extent to which long-term endurance exercise affected dystrophic limb, diaphragm, and cardiac function. Diaphragm specific tension was reduced by 60% (P < 0.05) in mice that performed 1 yr of volitional wheel running compared with sedentary mdx mice. Dorsiflexor mass (extensor digitorum longus and tibialis anterior) and function (extensor digitorum longus) were not altered by endurance training. In mice that performed 1 yr of volitional wheel running, plantarflexor mass (soleus and gastrocnemius) was increased and soleus tetanic force was increased 36%, while specific tension was similar in wheel-running and sedentary groups. Cardiac mass was increased 15%, left ventricle chamber size was increased 20% (diastole) and 18% (systole), and stroke volume was increased twofold in wheel-running compared with sedentary mdx mice. These data suggest that the dystrophic heart may undergo positive exercise-induced remodeling and that limb muscle function is largely unaffected. Most importantly, however, as the diaphragm most closely recapitulates the human disease, these data raise the possibility of exercise-mediated injury in dystrophic skeletal muscle.

  18. Androgen receptor action in osteoblasts in male mice is dependent on their stage of maturation.

    PubMed

    Russell, Patricia K; Clarke, Michele V; Cheong, Karey; Anderson, Paul H; Morris, Howard A; Wiren, Kristine M; Zajac, Jeffrey D; Davey, Rachel A

    2015-05-01

    Androgen action via the androgen receptor (AR) is essential for normal skeletal growth and bone maintenance post-puberty in males; however, the molecular and cellular mechanisms by which androgens exert their actions in osteoblasts remains relatively unexplored in vivo. To identify autonomous AR actions in osteoblasts independent of AR signaling in other tissues, we compared the extent to which the bone phenotype of the Global-ARKO mouse was restored by replacing the AR in osteoblasts commencing at either the (1) proliferative or (2) mineralization stage of their maturation. In trabecular bone, androgens stimulated trabecular bone accrual during growth via the AR in proliferating osteoblasts and maintained trabecular bone post-puberty via the AR in mineralizing osteoblasts, with its predominant action being to inhibit bone resorption by decreasing the ratio of receptor activator of NF-κB ligand (RANKL) to osteoprotegerin (OPG) gene expression. During growth, replacement of the AR in proliferating but not mineralizing osteoblasts of Global-ARKOs was able to partially restore periosteal circumference, supporting the concept that androgen action in cortical bone to increase bone size during growth is mediated via the AR in proliferating osteoblasts. This study provides further significant insight into the mechanism of androgen action via the AR in osteoblasts, demonstrating that it is dependent on the stage of osteoblast maturation.

  19. GluRδ2 Expression in the Mature Cerebellum of Hotfoot Mice Promotes Parallel Fiber Synaptogenesis and Axonal Competition

    PubMed Central

    Cesa, Roberta; Premoselli, Federica; Cesare, Paolo; Strata, Piergiorgio

    2009-01-01

    Glutamate receptor delta 2 (GluRδ2) is selectively expressed in the cerebellum, exclusively in the spines of the Purkinje cells (PCs) that are in contact with parallel fibers (PFs). Although its structure is similar to ionotropic glutamate receptors, it has no channel function and its ligand is unknown. The GluRδ2-null mice, such as knockout and hotfoot have profoundly altered cerebellar circuitry, which causes ataxia and impaired motor learning. Notably, GluRδ2 in PC-PF synapses regulates their maturation and strengthening and induces long term depression (LTD). In addition, GluRδ2 participates in the highly territorial competition between the two excitatory inputs to the PC; the climbing fiber (CF), which innervates the proximal dendritic compartment, and the PF, which is connected to spiny distal branchlets. Recently, studies have suggested that GluRδ2 acts as an adhesion molecule in PF synaptogenesis. Here, we provide in vivo and in vitro evidence that supports this hypothesis. Through lentiviral rescue in hotfoot mice, we noted a recovery of PC-PF contacts in the distal dendritic domain. In the proximal domain, we observed the formation of new spines that were innervated by PFs and a reduction in contact with the CF; ie, the pattern of innervation in the PC shifted to favor the PF input. Moreover, ectopic expression of GluRδ2 in HEK293 cells that were cocultured with granule cells or in cerebellar Golgi cells in the mature brain induced the formation of new PF contacts. Collectively, our observations show that GluRδ2 is an adhesion molecule that induces the formation of PF contacts independently of its cellular localization and promotes heterosynaptic competition in the PC proximal dendritic domain. PMID:19370152

  20. Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice

    PubMed Central

    Foltz, Steven J.; Modi, Jill N.; Melick, Garrett A.; Abousaud, Marin I.; Luan, Junna; Fortunato, Marisa J.; Beedle, Aaron M.

    2016-01-01

    Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury. PMID:26751696

  1. [Comparison observation on the mature alveolar of Echinococcus sibiricensis and Echinococcus multilocularis in the experimentally infected white mice].

    PubMed

    Tang, C T; Chen, J A; Tang, L; Cui, G W; Qian, Y C; Kang, Y M; Lu, H C

    2001-12-01

    The alveolar echinococcus is one of the most dangerous worm parasites in man. Rausch and Schiller reported a new species, Echinococcus sibiricensis n. sp. from arctic fox, Alpex logopus, on St. Lawrence Island of Alaska, USA. According to the view of Vogel, the sibiricensis form is only a geographical race or subspecies of Europe Echinococcus multilocularis. So far, the two names, Echinococcus multiocularis multilocularis and Echinococcus multilocularis sibiricensis, existed in many references and text books. We have found the adults of Echinococcus sibiricensis and Echinococcus multilocularis from sand foxes, Vulpes corsac and their larval stages (alveolar echinococcus) from field voles, Microtus brandti in the Hulunbeier Pasture of Inner Mongolia, northeastern China in 1985 and 1998-1999. Two types of metacestodes with quite different styles of early development of E. sibiricensis and E. multilocularis were found from field voles and laboratory experimental white mice. As one characteristic of alveolar E. multilocularis, the capsules are produced by the exogenous budding of germinal cell layer together with cyst wall. The protoscoleces grow from germinal cells on germinal cell layer. The peduncles of early protoscoleces attached to the germinal cell layer on the inner surface of capsule wall(Plate I, Figs. 1-2). Some protoscoleces in reticular structure were linked with the inner surface of capsule wall (Plate I, Fig. 3) in livers of mice in 9.5th month postinfection. In 14th month old alveolar multilocularis, large number of mature protoscoleces in reticular structure were still linked to the inner surface of capsule wall (Plate I, Figs. 4-8). The cavities of some capsules were filled with protoscoleces in meshes of reticular structure which were also linked around with the inner surface of capsule wall (Plate I, Fig. 9). The superficial surface of livers of positive field voles and experimental mice never showed any hyperemic phenomenon. The superficial

  2. Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKα1.

    PubMed

    Noppe, Gauthier; Dufeys, Cécile; Buchlin, Patricia; Marquet, Nicolas; Castanares-Zapatero, Diego; Balteau, Magali; Hermida, Nerea; Bouzin, Caroline; Esfahani, Hrag; Viollet, Benoit; Bertrand, Luc; Balligand, Jean-Luc; Vanoverschelde, Jean-Louis; Beauloye, Christophe; Horman, Sandrine

    2014-09-01

    Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodeling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodeling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase (TGF-β1/p38 MAPK) pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodeling, as indicated by increased LV diastolic dimension 30days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.

  3. P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment.

    PubMed

    Young, Christopher N J; Brutkowski, Wojciech; Lien, Chun-Fu; Arkle, Stephen; Lochmüller, Hanns; Zabłocki, Krzysztof; Górecki, Dariusz C

    2012-05-01

    Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca(2+) homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca(2+) and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca(2+) influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration-regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.

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

    PubMed

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

    2016-09-01

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

  5. In vivo reference point indentation measurement variability in skeletally mature inbred mice.

    PubMed

    Srisuwananukorn, Andrew; Allen, Matthew R; Brown, Drew M; Wallace, Joseph M; Organ, Jason M

    2015-01-01

    Reference point indentation (RPI) was developed to measure material-level mechanical properties of bone in vivo. Studies using RPI in vivo have discriminated between human subjects with previous skeletal fractures and those without and among dogs given different anti-remodeling drugs. Recently, this technology was extended to rats, providing the first in vivo data for rodents. The goal of the present study was to perform in vivo RPI measurements in mice, the most common animal model used to study bone. Twelve 16-week-old female C57BL/6 mice were subjected to RPI (three tests) on the anterior tibia, followed by a repeat test session on the contralateral limb 28 days later. A custom MATLAB program was used to derive several outcome parameters from RPI force-displacement curves: first cycle indentation distance (ID-1st), ID increase (IDI), total ID (TID), first cycle unloading slope (US-1st) and first cycle energy dissipation (ED-1st). Data within an individual were averaged across the three tests for each time point. Within-animal variation of all RPI parameters on day 1 ranged from 12.8 to 33.4% and from 14.1 to 22.4% on day 28. Between-animal variation on day 1 ranged from 11.4% to 22.8% and from 7.5% to 24.7% on day 28. At both time points, within- and between-animals, US-1st was the least variable parameter and IDI was most variable. All parameters were nonsignificantly lower at day 28 compared with day 1. These data are important to demonstrate the feasibility of collecting bone material property data longitudinally in mice and will inform the design of future studies in terms of statistical power and appropriate sample size considerations.

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

    PubMed Central

    Zhang, Aiping; Uaesoontrachoon, Kitipong; Shaughnessy, Conner; Das, Jharna R.; Rayavarapu, Sree; Brown, Kristy J; Ray, Patricio E.; Nagaraju, Kanneboyina; van den Anker, John N.; Hoffman, Eric P; Hathout, Yetrib

    2015-01-01

    Phosphorodiamidate morpholino oligonucleotides (PMO) are used as a promising exon-skipping gene therapy for Duchenne Muscular Dystrophy (DMD). One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM) strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23). We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10) mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg). Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI). The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at day 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO. Chemical compounds studied in this article: Phosphorodiamidate morpholino (PubChem CID: 22140692); isoflurane (PubChem CID: 3763); formic acid (PubChem CID: 284); acetonitrile (PubChem CID: 6342); acetone (PubChem CID: 180); methanol (PubChem CID: 887) PMID:26213685

  7. The Molecular Basis of Muscular Dystrophy in the mdx Mouse: A Point Mutation

    NASA Astrophysics Data System (ADS)

    Sicinski, Piotr; Geng, Yan; Ryder-Cook, Allan S.; Barnard, Eric A.; Darlison, Mark G.; Barnard, Pene J.

    1989-06-01

    The mdx mouse is an X-linked myopathic mutant, an animal model for human Duchenne muscular dystrophy. In both mouse and man the mutations lie within the dystrophin gene, but the phenotypic differences of the disease in the two species confer much interest on the molecular basis of the mdx mutation. The complementary DNA for mouse dystrophin has been cloned, and the sequence has been used in the polymerase chain reaction to amplify normal and mdx dystrophin transcripts in the area of the mdx mutation. Sequence analysis of the amplification products showed that the mdx mouse has a single base substitution within an exon, which causes premature termination of the polypeptide chain.

  8. Andrographolide reduces cognitive impairment in young and mature AβPPswe/PS-1 mice.

    PubMed

    Serrano, Felipe G; Tapia-Rojas, Cheril; Carvajal, Francisco J; Hancke, Juan; Cerpa, Waldo; Inestrosa, Nibaldo C

    2014-12-18

    Alzheimer's disease (AD) is a neurodegenerative disorder in which the amyloid-β (Aβ) oligomers are a key factor in synaptic impairment and in spatial memory decline associated with neuronal dysfunction. This impairment includes synaptic failure associated with the loss of synaptic proteins that contribute to AD progression. Interestingly, the use of natural compounds is an emergent conceptual strategy in the search for drugs with therapeutic potentials for treating neurodegenerative disorders. In the present study, we report that andrographolide (ANDRO), which is a labdane diterpene extracted from Andrographis paniculata, increases slope of field excitatory postsynaptic potentials (fEPSP) in the CA1 region of hippocampal slices and inhibits long-term depression (LTD), protecting the long-term potentiation (LTP) against the damage induced by Aβ oligomers in vitro, most likely by inhibiting glycogen synthase kinase-3β (GSK-3β). Additionally, ANDRO prevents changes in neuropathology in two different age groups (7- and 12-month-old mice) of an AβPPswe/PS-1 Alzheimer's model. ANDRO reduces the Aβ levels, changing the ontogeny of amyloid plaques in hippocampi and cortices in 7-month-old mice, and reduces tau phosphorylation around the Aβ oligomeric species in both age groups. Additionally, we observed that ANDRO recovers spatial memory functions that correlate with protecting synaptic plasticity and synaptic proteins in two different age groups. Our results suggest that ANDRO could be used in a potential preventive therapy during AD progression.

  9. Overview of MDX-A System for Medical Diagnosis

    PubMed Central

    Mittal, S.; Chandrasekaran, B.; Smith, J.

    1979-01-01

    We describe the design and performance of MDX, an experimental medical diagnosis system, which currently diagnoses in the syndrome called Cholestasis. The needed medical knowledge is represented in a scheme called conceptual structures, which can be viewed as a collection of conceptual experts interacting according to certain well-defined principles. MDX has three components: the diagnostic system, a patient data base and a radiology consultant. We describe these components, the inter-expert communication system and the query language used by these components. The system is illustrated by means of its performance on a real case.

  10. Maturation of intestinal defenses against peroral infection with group B coxsackievirus in mice.

    PubMed Central

    Loria, R M; Shadoff, N; Kibrick, S; Broitman, S

    1976-01-01

    The intestinal tract of adult mice provides effective protection against peroral infection with group B coxsackievirus. This protective function consists of at least two separate components. One is a barrier effect that prevents virus from passing through the mucosal side of the gut into the circulation. It becomes clearly evident at 18 days of life and is present thereafter. The other is a clearance mechanism that acts to eliminate virus from the enteric tract after infection has occurred. This is first demonstrable at about 14 to 18 days and also persists. The appearance of these protective functions coincides with the known development of enzymatic and morphological changes in the gut associated with the transition from suckling to weanling. PMID:1270146

  11. Maturation of intestinal defenses against peroral infection with group B coxsackievirus in mice.

    PubMed

    Loria, R M; Shadoff, N; Kibrick, S; Broitman, S

    1976-05-01

    The intestinal tract of adult mice provides effective protection against peroral infection with group B coxsackievirus. This protective function consists of at least two separate components. One is a barrier effect that prevents virus from passing through the mucosal side of the gut into the circulation. It becomes clearly evident at 18 days of life and is present thereafter. The other is a clearance mechanism that acts to eliminate virus from the enteric tract after infection has occurred. This is first demonstrable at about 14 to 18 days and also persists. The appearance of these protective functions coincides with the known development of enzymatic and morphological changes in the gut associated with the transition from suckling to weanling.

  12. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B−/− mice

    PubMed Central

    Peixoto, Rui T.; Wang, Wengang; Croney, Donyell M.; Kozorovitskiy, Yevgenia; Sabatini, Bernardo L.

    2016-01-01

    Some autistic individuals exhibit abnormal development of the caudate nucleus and associative cortical areas, suggesting potential dysfunction of cortico-basal ganglia (BG) circuits. Using optogenetic and electrophysiological approaches in mice we identified a narrow postnatal period characterized by extensive glutamatergic synaptogenesis in striatal spiny projection neurons (SPNs) and a concomitant increase in corticostriatal circuit activity. SPNs during early development have high intrinsic excitability and respond strongly to cortical afferents despite sparse excitatory inputs. As a result, striatum and corticostriatal connectivity are highly sensitive to acute and chronic changes in cortical activity, suggesting that early imbalances in cortical function alter BG development. Indeed, a mouse model of autism with deletions in SHANK3 (Shank3B−/−) has early cortical hyperactivity, which triggers increased SPN excitatory synapse and corticostriatal hyper-connectivity. These results show a tight functional coupling between cortex and striatum during early postnatal development and suggest a potential common circuit dysfunction caused by cortical hyperactivity. PMID:26928064

  13. Activated satellite cells are present in uninjured extraocular muscles of mature mice.

    PubMed Central

    McLoon, Linda K; Wirtschafter, Jonathan

    2002-01-01

    PURPOSE: We recently demonstrated that there is a continuous process of myonuclear addition into normal, uninjured adult myofibers in rabbit extraocular muscles (EOM). This phenomenon is not seen in skeletal muscles from normal, adult limbs. These features may explain the selective involvement of the EOM in progressive external ophthalmoplegia and oculopharyngeal muscular dystrophy due to an accumulation of damaged DNA in mitochondria and nuclei within the EOM as a result of repeated cycling of the muscle satellite cells. Many testable hypotheses flow from these observations. We investigated whether continuous myonuclear addition is present in normal mouse EOM so that mouse models of genetic disorders can be used to study the pathogenic mechanisms and to test potential therapies for human muscle disorders. METHODS: Bromodeoxyuridine (brdU) was injected intraperitoneally into C57 adult mice every 2 hours for 12 hours. Twenty-four hours later the animals were sacrificed, and the globes with the muscles attached were prepared for immunohistochemical localization of brdU-positive nuclei within the EOM. All cross sections were immunostained for both brdU and either dystrophin or laminin. RESULTS: All the rectus muscles from the mouse EOM examined contained both satellite cells and myonuclei that were positive for brdU. This demonstrates the division of satellite cells and the fusion of their daughter cells with existing adult EOM myofibers in mice. CONCLUSIONS: These data indicate that the process of continuous myonuclear addition is also active in mouse EOM. These findings will allow various mutant mouse models to be used to study the pathogenesis and treatment of various muscle disorders. The existence of continuous myonuclear addition in adult, uninjured EOM fundamentally changes the accepted notion that EOM myofibers are postmitotic. PMID:12545684

  14. Accelerated Maturation of Human Stem Cell-Derived Pancreatic Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice

    PubMed Central

    Bruin, Jennifer E.; Asadi, Ali; Fox, Jessica K.; Erener, Suheda; Rezania, Alireza; Kieffer, Timothy J.

    2015-01-01

    Summary Pluripotent human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19–21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature β cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation. PMID:26677767

  15. Schistosoma mansoni: interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

    SciTech Connect

    James, E.R.; Dobinson, A.R.

    1984-06-01

    Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of 60Co irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CI). Development into adult worms was prevented with 4 krad for IC schistosomula, but for CI schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed.

  16. Skeletal maturation substantially affects elastic tissue properties in the endosteal and periosteal regions of loaded mice tibiae.

    PubMed

    Checa, Sara; Hesse, Bernhard; Roschger, Paul; Aido, Marta; Duda, Georg N; Raum, Kay; Willie, Bettina M

    2015-07-01

    Although it is well known that the bone adapts to changes in the mechanical environment by forming and resorbing the bone matrix, little is known about the influence of mechanical loading on tissue material properties of the pre-existing and newly formed bone. In this study, we analyzed the newly formed and pre-existing tissue after two weeks of controlled in vivo axial compressive loading in tibia of young (10 week-old) and adult (26 week-old) female mice and compared to the control contralateral limb, by means of scanning acoustic microscopy. Additionally, we used quantitative backscattered electron imaging to determine the bone mineral density distribution within the newly formed and pre-existing bone of young mice. No significant differences were found in tissue stiffness or mineral density in the pre-existing bone tissue as a result of external loading. In the endosteal region, 10 and 26 week loaded animals showed a 9% reduction in bone tissue stiffness compared to control animals. An increase of 200% in the mineral apposition rate in this region was observed in both age groups. In the periosteal region, the reduction in bone tissue stiffness and the increase in bone mineral apposition rate as a result of loading were two times higher in the 10 compared to the 26 week old animals. These data suggest that, during growth and skeletal maturation, the response of bone to mechanical loading is a deposition of new bone matrix, where the tissue amount but not its mineral or elastic properties are influenced by animal age.

  17. Plasmid Vector-Linked Maturation of Natural Killer (NK) Cells Is Coupled to Antigen-Dependent NK Cell Activation during DNA-Based Immunization in Mice

    PubMed Central

    Zhu, Ren; Mancini-Bourgine, Maryline; Zhang, Xiao Ming; Bayard, Florence; Deng, Qiang; Michel, Marie-Louise

    2011-01-01

    Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1+ CD27− NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1+ CD27+ NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity. PMID:21775455

  18. Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

    PubMed

    Seki, Takao; Yamamoto, Mami; Taguchi, Yuu; Miyauchi, Maki; Akiyama, Nobuko; Yamaguchi, Noritaka; Gohda, Jin; Akiyama, Taishin; Inoue, Jun-ichiro

    2015-12-01

    RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.

  19. Prednisolone attenuates improvement of cardiac and skeletal contractile function and histopathology by lisinopril and spironolactone in the mdx mouse model of Duchenne muscular dystrophy.

    PubMed

    Janssen, Paul M L; Murray, Jason D; Schill, Kevin E; Rastogi, Neha; Schultz, Eric J; Tran, Tam; Raman, Subha V; Rafael-Fortney, Jill A

    2014-01-01

    Duchenne muscular dystrophy (DMD) is an inherited disease that causes striated muscle weakness. Recently, we showed therapeutic effects of the combination of lisinopril (L), an angiotensin converting enzyme (ACE) inhibitor, and spironolactone (S), an aldosterone antagonist, in mice lacking dystrophin and haploinsufficient for utrophin (utrn(+/-);mdx, het mice); both cardiac and skeletal muscle function and histology were improved when these mice were treated early with LS. It was unknown to what extent LS treatment is effective in the most commonly used DMD murine model, the mdx mouse. In addition, current standard-of-care treatment for DMD is limited to corticosteroids. Therefore, potentially useful alternative or additive drugs need to be both compared directly to corticosteroids and tested in presence of corticosteroids. We evaluated the effectiveness of this LS combination in the mdx mouse model both compared with corticosteroid treatment (prednisolone, P) or in combination (LSP). We tested the additional combinatorial treatment containing the angiotensin II receptor blocker losartan (T), which is widely used to halt and treat the developing cardiac dysfunction in DMD patients as an alternative to an ACE inhibitor. Peak myocardial strain rate, assessed by magnetic resonance imaging, showed a negative impact of P, whereas in both diaphragm and extensor digitorum longus (EDL) muscle contractile function was not significantly impaired by P. Histologically, P generally increased cardiac damage, estimated by percentage area infiltrated by IgG as well as by collagen staining. In general, groups that only differed in the presence or absence of P (i.e. mdx vs. P, LS vs. LSP, and TS vs. TSP) demonstrated a significant detrimental impact of P on many assessed parameters, with the most profound impact on cardiac pathology.

  20. Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy.

    PubMed

    Hibaoui, Youssef; Reutenauer-Patte, Julie; Patthey-Vuadens, Ophélie; Ruegg, Urs T; Dorchies, Olivier M

    2011-09-01

    Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine® implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.

  1. Transgenic mice overexpressing glia maturation factor-β, an oxidative stress inducible gene, show premature aging due to Zmpste24 down-regulation.

    PubMed

    Imai, Rika; Asai, Kanae; Hanai, Jun-ichi; Takenaka, Masaru

    2015-07-01

    Glia Maturation Factor-β (GMF), a brain specific protein, is induced by proteinuria in renal tubules. Ectopic GMF overexpression causes apoptosisin vitro via cellular vulnerability to oxidative stress. In order to examine the roles of GMF in non-brain tissue, we constructed transgenic mice overexpressing GMF (GMF-TG). The GMF-TG mice exhibited appearance phenotypes associated with premature aging. The GMF-TG mice also demonstrated short lifespans and reduced hair regrowth, suggesting an accelerated aging process. The production of an abnormal lamin A, a nuclear envelope protein, plays a causal role in both normal aging and accelerated aging diseases, known as laminopathies. Importantly, we identified the abnormal lamin A (prelamin A), accompanied by a down-regulation of a lamin A processing enzyme (Zmpste24) in the kidney of the GMF-TG mice. The GMF-TG mice showed accelerated aging in the kidney, compared with wild-type mice, showing increased TGF-β1, CTGF gene and serum creatinine. The gene expression of p21/waf1 was increased at an earlier stage of life, at 10 weeks, which was in turn down-regulated at a later stage, at 60 weeks. In conclusion, we propose that GMF-TG mice might be a novel mouse model of accelerated aging, due to the abnormal lamin A.

  2. Self-Improvement of Keratinocyte Differentiation Defects During Skin Maturation in ABCA12-Deficient Harlequin Ichthyosis Model Mice

    PubMed Central

    Yanagi, Teruki; Akiyama, Masashi; Nishihara, Hiroshi; Ishikawa, Junko; Sakai, Kaori; Miyamura, Yuki; Naoe, Ayano; Kitahara, Takashi; Tanaka, Shinya; Shimizu, Hiroshi

    2010-01-01

    Harlequin ichthyosis (HI) is caused by loss-of-function mutations in the keratinocyte lipid transporter ABCA12. The patients often die in the first 1 or 2 weeks of life, although HI survivors’ phenotypes improve within several weeks after birth. In order to clarify the mechanisms of phenotypic recovery, we studied grafted skin and keratinocytes from Abca12-disrupted (Abca12−/−) mice showing abnormal lipid transport. Abca12−/− neonatal epidermis showed significantly reduced total ceramide amounts and aberrant ceramide composition. Immunofluorescence and immunoblotting of Abca12−/− neonatal epidermis revealed defective profilaggrin/filaggrin conversion and reduced protein expression of the differentiation-specific molecules, loricrin, kallikrein 5, and transglutaminase 1, although their mRNA expression was up-regulated. In contrast, Abca12−/− skin grafts kept in a dry environment exhibited dramatic improvements in all these abnormalities. Increased transepidermal water loss, a parameter representing barrier defect, was remarkably decreased in grafted Abca12−/− skin. Ten-passage sub-cultured Abca12−/− keratinocytes showed restoration of intact ceramide distribution, differentiation-specific protein expression and profilaggrin/filaggrin conversion, which were defective in primary-cultures. Using cDNA microarray analysis, lipid transporters including four ATP-binding cassette transporters were up-regulated after sub-culture of Abca12−/− keratinocytes compared with primary-culture. These results indicate that disrupted keratinocyte differentiation during the fetal development is involved in the pathomechanism of HI and, during maturation, Abca12−/− epidermal keratinocytes regain normal differentiation processes. This restoration may account for the skin phenotype improvement observed in HI survivors. PMID:20489143

  3. Resveratrol accelerates erythroid maturation by activation of FoxO3 and ameliorates anemia in beta-thalassemic mice

    PubMed Central

    Franco, Sara Santos; De Falco, Luigia; Ghaffari, Saghi; Brugnara, Carlo; Sinclair, David A.; Matte’, Alessandro; Iolascon, Achille; Mohandas, Narla; Bertoldi, Mariarita; An, Xiuli; Siciliano, Angela; Rimmelé, Pauline; Cappellini, Maria Domenica; Michan, Shaday; Zoratti, Elisa; Anne, Janin; De Franceschi, Lucia

    2014-01-01

    Resveratrol, a polyphenolic-stilbene, has received increased attention in the last decade due to its wide range of biological activities. Beta(β)-thalassemias are inherited red cell disorders, found worldwide, characterized by ineffective erythropoiesis and red cell oxidative damage with reduced survival. We evaluated the effects of low-dose-resveratrol (5 μM) on in vitro human erythroid differentiation of CD34+ from normal and β-thalassemic subjects. We found that resveratrol induces accelerated erythroid-maturation, resulting in the reduction of colony-forming units of erythroid cells and increased intermediate and late erythroblasts. In sorted colony-forming units of erythroid cells resveratrol activates Forkhead-box-class-O3, decreases Akt activity and up-regulates anti-oxidant enzymes as catalase. In an in vivo murine model for β-thalassemia, resveratrol (2.4 mg/kg) reduces ineffective erythropoiesis, increases hemoglobin levels, reduces reticulocyte count and ameliorates red cell survival. In both wild-type and β-thalassemic mice, resveratrol up-regulates scavenging enzymes such as catalase and peroxiredoxin-2 through Forkhead-box-class-O3 activation. These data indicate that resveratrol inhibits Akt resulting in FoxO3 activation with upregulation of cytoprotective systems enabling the pathological erythroid precursors to resist the oxidative damage and continue to differentiate. Our data suggest that the dual effect of resveratrol on erythropoiesis through activation of FoxO3 transcriptional factor combined with the amelioration of oxidative stress in circulating red cells may be considered as a potential novel therapeutic strategy in treating β-thalassemia. PMID:23975182

  4. Expansion of revertant fibers in dystrophic mdx muscles reflects activity of muscle precursor cells and serves as an index of muscle regeneration.

    PubMed

    Yokota, Toshifumi; Lu, Qi-Long; Morgan, Jennifer E; Davies, Kay E; Fisher, Rosie; Takeda, Shin'ichi; Partridge, Terence A

    2006-07-01

    Duchenne muscular dystrophy and the mdx mouse myopathies reflect a lack of dystrophin in muscles. However, both contain sporadic clusters of revertant fibers (RFs) that express dystrophin. RF clusters expand in size with age in mdx mice. To test the hypothesis that the expansion of clusters is achieved through the process of muscle degeneration and regeneration, we analyzed muscles of mdx mice in which degeneration and regeneration were inhibited by the expression of micro-dystrophins or utrophin transgenes. Postnatal RF expansion was diminished in direct correlation to the protective effect of the transgene expression. Similarly, expansion of RFs was inhibited when muscle regeneration was blocked by irradiation. However, in irradiated muscles, irradiation-tolerant quiescent muscle precursor cells reactivated by notexin effectively restored RF expansion. Our observations demonstrate that revertant events occur initially within a subset of muscle precursor cells. The proliferation of these cells, as part of the regeneration process, leads to the expansion of RF clusters within degenerating muscles. This expansion of revertant clusters depicts the cumulative history of regeneration, thus providing a useful index for functional evaluation of therapies that counteract muscle degeneration.

  5. Visualizing and quantifying oxidized protein thiols in tissue sections: a comparison of dystrophic mdx and normal skeletal mouse muscles.

    PubMed

    Iwasaki, Tomohito; Terrill, Jessica; Shavlakadze, Tea; Grounds, Miranda D; Arthur, Peter G

    2013-12-01

    Reactive oxygen species (ROS) are not only a cause of oxidative stress in a range of disease conditions but are also important regulators of physiological pathways in vivo. One mechanism whereby ROS can regulate cell function is by modification of proteins through the reversible oxidation of their thiol groups. An experimental challenge has been the relative lack of techniques to probe the biological significance of protein thiol oxidation in complex multicellular tissues and organs. We have developed a sensitive and quantitative fluorescence labeling technique to detect and localize protein thiol oxidation in histological tissue sections. In our technique, reduced and oxidized protein thiols are visualized and quantified on two consecutive tissue sections and the extent of protein thiol oxidation is expressed as a percentage of total protein thiols (reduced plus oxidized). We tested the application of this new technique using muscles of dystrophic (mdx) and wild-type C57Bl/10Scsn (C57) mice. In mdx myofibers, protein thiols were consistently more oxidized (19 ± 3%) compared with healthy myofibers (10 ± 1%) in C57 mice. A striking observation was the localization of intensive protein thiol oxidation (70 ± 9%) within myofibers associated with necrotic damage. Oxidative stress is an area of active investigation in many fields of research, and this technique provides a useful tool for locating and further understanding protein thiol oxidation in normal, damaged, and diseased tissues.

  6. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

    PubMed Central

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  7. Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse.

    PubMed

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

    2010-04-01

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

  8. Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis.

    PubMed

    Ljubicic, Vladimir; Burt, Matthew; Lunde, John A; Jasmin, Bernard J

    2014-07-01

    Slower, more oxidative muscle fibers are more resistant to the dystrophic pathology in Duchenne muscular dystrophy (DMD) patients as well as in the preclinical mdx mouse model of DMD. Therefore, one therapeutic strategy for DMD focuses on promoting expression of the slow, oxidative myogenic program. In the current study, we explored the therapeutic potential of stimulating the slow, oxidative phenotype in mdx mice by feeding 6-wk-old animals with the natural phenol resveratrol (RSV; ~100 mg·kg(-1)·day(-1)) for 6 wk. Sirtuin 1 (SIRT1) activity and protein levels increased significantly, as well as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) activity, in the absence of alterations in AMPK signaling. These adaptations occurred concomitant with evidence of a fast, glycolytic, to slower, more oxidative fiber type conversion, including mitochondrial biogenesis and increased expression of slower myosin heavy chain isoforms. These positive findings raised the question of whether increased exposure to RSV would result in greater therapeutic benefits. We discovered that an elevated RSV dose of ~500 mg·kg(-1)·day(-1) across a duration of 12 wk was clearly less effective at muscle remodeling in mdx mice. This treatment protocol failed to influence SIRT1 or AMPK signaling and did not result in a shift towards a slower, more oxidative phenotype. Taken together, this study demonstrates that RSV can stimulate SIRT1 and PGC-1α activation, which in turn may promote expression of the slow, oxidative myogenic program in mdx mouse muscle. The data also highlight the importance of selecting an appropriate dosage regimen of RSV to maximize its potential therapeutic effectiveness for future application in DMD patients.

  9. Deletion of Galgt2 (B4Galnt2) Reduces Muscle Growth in Response to Acute Injury and Increases Muscle Inflammation and Pathology in Dystrophin-Deficient Mice

    PubMed Central

    Xu, Rui; Singhal, Neha; Serinagaoglu, Yelda; Chandrasekharan, Kumaran; Joshi, Mandar; Bauer, John A.; Janssen, Paulus M.L.; Martin, Paul T.

    2016-01-01

    Transgenic overexpression of Galgt2 (official name B4Galnt2) in skeletal muscle stimulates the glycosylation of α dystroglycan (αDG) and the up-regulation of laminin α2 and dystrophin surrogates known to inhibit muscle pathology in mouse models of congenital muscular dystrophy 1A and Duchenne muscular dystrophy. Skeletal muscle Galgt2 gene expression is also normally increased in the mdx mouse model of Duchenne muscular dystrophy compared with the wild-type mice. To assess whether this increased endogenous Galgt2 expression could affect disease, we quantified muscular dystrophy measures in mdx mice deleted for Galgt2 (Galgt2−/−mdx). Galgt2−/− mdx mice had increased heart and skeletal muscle pathology and inflammation, and also worsened cardiac function, relative to age-matched mdx mice. Deletion of Galgt2 in wild-type mice also slowed skeletal muscle growth in response to acute muscle injury. In each instance where Galgt2 expression was elevated (developing muscle, regenerating muscle, and dystrophic muscle), Galgt2-dependent glycosylation of αDG was also increased. Overexpression of Galgt2 failed to inhibit skeletal muscle pathology in dystroglycan-deficient muscles, in contrast to previous studies in dystrophin-deficient mdx muscles. This study demonstrates that Galgt2 gene expression and glycosylation of αDG are dynamically regulated in muscle and that endogenous Galgt2 gene expression can ameliorate the extent of muscle pathology, inflammation, and dysfunction in mdx mice. PMID:26435413

  10. Physiology of B cells in mice with X-linked immunodeficiency. II. Influence of the thymus and mature T cells on B cell differentiation

    SciTech Connect

    Sprent, J.; Bruce, J.

    1984-07-01

    Evidence is presented that the in vivo differentiation of B cells expressing X-linked immunodeficiency (xid) is controlled by mature T cells. Normal (C57BL/6 X CBA/J)F1 mice were thymectomized (ATx), heavily irradiated, and reconstituted with CBA/N (xid) or CBA/Ca (nondefective) marrow. In contrast to sham-operated mice, ATx recipients of xid marrow showed an almost total absence of Ig+ B cells in lymph nodes (LN) and thoracic duct lymph at 2 mo post-reconstitution; B cells were markedly reduced in the spleen in some mice but only moderately in others. Addition of mature T cells soon after marrow reconstitution substantially abrogated the B cell depletion. In control experiments with nondefective B cells, the number of B cells developing in ATx irradiated recipients of normal (xid-) marrow cells was not detectably lower than in sham-operated recipients. These data imply that a subset of T-dependent B cells is either missing in normal mice or present in only very small numbers.

  11. The study of mechanisms of protective effect of Rg1 against arthritis by inhibiting osteoclast differentiation and maturation in CIA mice.

    PubMed

    Gu, Yanqing; Fan, Weimin; Yin, Guoyong

    2014-01-01

    Ginsenoside Rg1 is a natural product extracted from Panax ginseng C.A. Although Rg1 protects tissue structure and functions by inhibiting local inflammatory reaction, the mechanism remains poorly understood. In vitro, Rg1 dose-dependently inhibited TRAP activity in receptor activator of nuclear factor-κB ligand- (RANKL-) induced osteoclasts and decreased the number of osteoclasts and osteoclast resorption area. Rg1 also significantly inhibited the RANK signaling pathway, including suppressing the expression of Trap, cathepsin K, matrix metalloproteinase 9 (MMP9), and calcitonin receptor (CTR). In vivo, Rg1 dramatically decreased arthritis scores in CIA mice and effectively controlled symptoms of inflammatory arthritis. Pathologic analysis demonstrated that Rg1 significantly attenuated pathological changes in CIA mice. Pronounced reduction in synovial hyperplasia and inflammatory cell invasion were observed in CIA mice after Rg1 therapy. Alcian blue staining results illustrated that mice treated with Rg1 had significantly reduced destruction in the articular cartilage. TRAP and cathepsin K staining results demonstrated a significant reduction of numbers of OCs in the articular cartilage in proximal interphalangeal joints and ankle joints in Rg1-treated mice. In summary, this study revealed that Rg1 reduced the inflammatory destruction of periarticular bone by inhibiting differentiation and maturation of osteoclasts in CIA mice.

  12. Defects in dendrite and spine maturation and synaptogenesis associated with an anxious-depressive-like phenotype of GABAA receptor-deficient mice.

    PubMed

    Ren, Zhen; Sahir, Nadia; Murakami, Shoko; Luellen, Beth A; Earnheart, John C; Lal, Rachnanjali; Kim, Ju Young; Song, Hongjun; Luscher, Bernhard

    2015-01-01

    Mice that were rendered heterozygous for the γ2 subunit of GABAA receptors (γ2(+/-) mice) have been characterized extensively as a model for major depressive disorder. The phenotype of these mice includes behavior indicative of heightened anxiety, despair, and anhedonia, as well as defects in hippocampus-dependent pattern separation, HPA axis hyperactivity and increased responsiveness to antidepressant drugs. The γ2(+/-) model thereby provides strong support for the GABAergic deficit hypothesis of major depressive disorder. Here we show that γ2(+/-) mice additionally exhibit specific defects in late stage survival of adult-born hippocampal granule cells, including reduced complexity of dendritic arbors and impaired maturation of synaptic spines. Moreover, cortical γ2(+/-) neurons cultured in vitro show marked deficits in GABAergic innervation selectively when grown under competitive conditions that may mimic the environment of adult-born hippocampal granule cells. Finally, brain extracts of γ2(+/-) mice show a numerical but insignificant trend (p = 0.06) for transiently reduced expression of brain derived neurotrophic factor (BDNF) at three weeks of age, which might contribute to the previously reported developmental origin of the behavioral phenotype of γ2(+/-) mice. The data indicate increasing congruence of the GABAergic, glutamatergic, stress-based and neurotrophic deficit hypotheses of major depressive disorder.

  13. Altered Affinity Maturation in Primary Response to (4-hydroxy-3-nitrophenyl) Acetyl (NP) after Autologous Reconstitution of Irradiated C57BL/6 Mice

    PubMed Central

    De Trez, Carl; Van Acker, Annette; Vansanten, Georgette; Urbain, Jacques; Brait, Maryse

    2002-01-01

    Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed. The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the λ1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by λ1 positive antibodies but fail to produce high affinity NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected. PMID:12885152

  14. FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF.

    PubMed

    Vidal-Martínez, Guadalupe; Vargas-Medrano, Javier; Gil-Tommee, Carolina; Medina, David; Garza, Nathan T; Yang, Barbara; Segura-Ulate, Ismael; Dominguez, Samantha J; Perez, Ruth G

    2016-09-23

    Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.

  15. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    PubMed

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P < 0.01; mean ± standard error of the mean (SEM), Student's unpaired t-test], as well as in the tongue muscle (65.7 versus 73.8 per cent; P < 0.05). Similarly, the content of MyHC-2x isoforms in mdx tongue muscle was lower than in the controls (25.9 versus 30.8 per cent; P < 0.05). The observed down-regulation of the MyHC-2x and MyHC-2b mRNA in the masticatory muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  16. Extracellular Vesicles Containing P301L Mutant Tau Accelerate Pathological Tau Phosphorylation and Oligomer Formation but Do Not Seed Mature Neurofibrillary Tangles in ALZ17 Mice.

    PubMed

    Baker, Siân; Polanco, Juan Carlos; Götz, Jϋrgen

    2016-10-04

    In Alzheimer's disease, the distribution of neurofibrillary tangles, a histological hallmark comprised of phosphorylated forms of the protein tau, follows a distinct pattern through anatomically connected brain regions. The well-documented correlation between the severity of tau pathology and disease progression implies a prion-like seeding and spreading mechanism for tau. Experimentally, this has been addressed in transgenic mice by the injection of protein lysates isolated from brains of transgenic mice or patients with tauopathies, including AD, that were shown to behave like seeds, accelerating tau pathology and tangle formation in predisposed mice. More specifically, in vivo data suggest that brain lysates from mice harboring the P301S mutation of tau can seed protein aggregation when injected into the hippocampi of human wild-type tau transgenic ALZ17 mice. Here, we compared the seeding potential of lysates and extracellular vesicles enriched for exosomes (EVs) from wild-type and human P301L tau transgenic rTg4510 mouse brains. We show that transgenic EVs cause increased tau phosphorylation and soluble oligomer formation in a manner comparable to that of freely available proteins in brain lysates, a prerequisite for the formation of mature protein aggregates.

  17. Increased calcium in neurons in the cerebral cortex and cerebellum is not associated with cell loss in the mdx mouse model of Duchenne muscular dystrophy.

    PubMed

    Tuckett, Emma; Gosetti, Troy; Hayes, Alan; Rybalka, Emma; Verghese, Elizabeth

    2015-09-09

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease resulting from mutation of the X-linked dystrophin gene. In addition to skeletal muscle pathology, cognitive deficits have been identified in patients with DMD. There is a lack of research investigating the pathological mechanisms underlying the neurological deficits apparent in DMD. The current study assessed whether increases in calcium contributed towards neuronal cell loss or histopathological changes in the genetically homologous mdx mouse model of DMD in sections from the cerebral cortex, hippocampus and cerebellum at 24 days, 12 weeks and 9 months of age. Alizarin S staining showed a significant increase in calcium-positive neurons in the mdx cerebral cortex at 24 days and 9 months and the cerebellum at 24 days, 12 weeks and 9 months compared with age-matched controls. However, neuronal cell counts of haemotoxylin and eosin-stained sections showed that altered calcium levels did not lead to neuronal cell loss. A better understanding of how the disruption of calcium regulation affects the function of neurons may explain the neurological deficits apparent in mdx mice and patients with DMD.

  18. Membrane Sealant Poloxamer P188 Protects Against Isoproterenol Induced Cardiomyopathy in Dystrophin Deficient Mice

    PubMed Central

    2011-01-01

    Background Cardiomyopathy in Duchenne muscular dystrophy (DMD) is an increasing cause of death in patients. The absence of dystrophin leads to loss of membrane integrity, cell death and fibrosis in cardiac muscle. Treatment of cardiomyocyte membrane instability could help prevent cardiomyopathy. Methods Three month old female mdx mice were exposed to the β1 receptor agonist isoproterenol subcutaneously and treated with the non-ionic tri-block copolymer Poloxamer P188 (P188) (460 mg/kg/dose i.p. daily). Cardiac function was assessed using high frequency echocardiography. Tissue was evaluated with Evans Blue Dye (EBD) and picrosirius red staining. Results BL10 control mice tolerated 30 mg/kg/day of isoproterenol for 4 weeks while death occurred in mdx mice at 30, 15, 10, 5 and 1 mg/kg/day within 24 hours. Mdx mice tolerated a low dose of 0.5 mg/kg/day. Isoproterenol exposed mdx mice showed significantly increased heart rates (p < 0.02) and cardiac fibrosis (p < 0.01) over 4 weeks compared to unexposed controls. P188 treatment of mdx mice significantly increased heart rate (median 593 vs. 667 bpm; p < 0.001) after 2 weeks and prevented a decrease in cardiac function in isoproterenol exposed mice (Shortening Fraction = 46 ± 6% vs. 35 ± 6%; p = 0.007) after 4 weeks. P188 treated mdx mice did not show significant differences in cardiac fibrosis, but demonstrated significantly increased EBD positive fibers. Conclusions This model suggests that chronic intermittent intraperitoneal P188 treatment can prevent isoproterenol induced cardiomyopathy in dystrophin deficient mdx mice. PMID:21575230

  19. Improvement of the mdx mouse dystrophic phenotype by systemic in utero AAV8 delivery of a minidystrophin gene.

    PubMed

    Koppanati, B M; Li, J; Reay, D P; Wang, B; Daood, M; Zheng, H; Xiao, X; Watchko, J F; Clemens, P R

    2010-11-01

    Duchenne muscular dystrophy (DMD) is a devastating primary muscle disease with pathological changes in skeletal muscle that are ongoing at the time of birth. Progressive deterioration in striated muscle function in affected individuals ultimately results in early death due to cardio-pulmonary failure. As affected individuals can be identified before birth by prenatal genetic testing for DMD, gene replacement treatment can be started in utero. This approach offers the possibility of preventing pathological changes in muscle that begin early in life. To test in utero gene transfer in the mdx mouse model of DMD, a minidystrophin gene driven by the human cytomegalovirus promoter was delivered systemically by an intraperitoneal injection to the fetus at embryonic day 16. Treated mdx mice studied at 9 weeks after birth showed widespread expression of recombinant dystrophin in skeletal muscle, restoration of the dystrophin-associated glycoprotein complex in dystrophin-expressing muscle fibers, improved muscle pathology, and functional benefit to the transduced diaphragm compared with untreated littermate controls. These results support the potential of the AAV8 vector to efficiently cross the blood vessel barrier to achieve systemic gene transfer to skeletal muscle in utero in a mouse model of muscular dystrophy, to significantly improve the dystrophic phenotype and to ameliorate the processes that lead to exhaustion of the skeletal muscle regenerative capacity.

  20. Functional and Molecular Effects of Arginine Butyrate and Prednisone on Muscle and Heart in the mdx Mouse Model of Duchenne Muscular Dystrophy

    PubMed Central

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

    2010-01-01

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

  1. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men

    PubMed Central

    O’Hara, Laura; McInnes, Kerry; Simitsidellis, Ioannis; Morgan, Stephanie; Atanassova, Nina; Slowikowska-Hilczer, Jolanta; Kula, Krzysztof; Szarras-Czapnik, Maria; Milne, Laura; Mitchell, Rod T.; Smith, Lee B.

    2015-01-01

    Leydig cell number and function decline as men age, and low testosterone is associated with all “Western” cardio-metabolic disorders. However, whether perturbed androgen action within the adult Leydig cell lineage predisposes individuals to this late-onset degeneration remains unknown. To address this, we generated a novel mouse model in which androgen receptor (AR) is ablated from ∼75% of adult Leydig stem cell/cell progenitors, from fetal life onward (Leydig cell AR knockout mice), permitting interrogation of the specific roles of autocrine Leydig cell AR signaling through comparison to adjacent AR-retaining Leydig cells, testes from littermate controls, and to human testes, including from patients with complete androgen insensitivity syndrome (CAIS). This revealed that autocrine AR signaling is dispensable for the attainment of final Leydig cell number but is essential for Leydig cell maturation and regulation of steroidogenic enzymes in adulthood. Furthermore, these studies reveal that autocrine AR signaling in Leydig cells protects against late-onset degeneration of the seminiferous epithelium in mice and inhibits Leydig cell apoptosis in both adult mice and patients with CAIS, possibly via opposing aberrant estrogen signaling. We conclude that autocrine androgen action within Leydig cells is essential for the lifelong support of spermatogenesis and the development and lifelong health of Leydig cells.—O’Hara, L., McInnes, K., Simitsidellis, I., Morgan, S., Atanassova, N., Slowikowska-Hilczer, J., Kula, K., Szarras-Czapnik, M., Milne, L., Mitchell, R. T., Smith, L. B. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men. PMID:25404712

  2. Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice

    SciTech Connect

    Nair, Ramya; Singh, Vikram Jeet; Salian, Sujith Raj; Kalthur, Sneha Guruprasad; D'Souza, Antony Sylvan; Shetty, Pallavi K.; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

    2014-09-15

    Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20 mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5 mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20 mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome. - Highlights: • Methyl parathion induces severe cytoplasmic abnormalities in oocytes. • Inhibits nuclear maturation and spindle damage • Poor blastocyst quality and high DNA

  3. Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice.

    PubMed

    Yamada, Hiromi; Ito, Daisuke; Oki, Yoshinao; Kitagawa, Masato; Matsumoto, Taro; Watari, Tosihiro; Kano, Koichiro

    2014-11-14

    Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.

  4. Molecular, cellular, and muscle strip mechanics of the mdx mouse diaphragm.

    PubMed

    Bates, Genevieve; Sigurdardottir, Sara; Kachmar, Linda; Zitouni, Nedjma B; Benedetti, Andrea; Petrof, Basil J; Rassier, Dilson; Lauzon, Anne-Marie

    2013-05-01

    Duchenne muscular dystrophy (DMD) is a lethal disorder caused by defects in the dystrophin gene, which leads to respiratory or cardiac muscle failure. Lack of dystrophin predisposes the muscle cell sarcolemmal membrane to mechanical damage. However, the role of myosin in this muscle weakness has been poorly addressed. In the current study, in addition to measuring the velocity of actin filament propulsion (υmax) of mdx myosin molecules purified from 3- and 12-mo-old control (C57Bl/10) and mdx (C57Bl/10mdx) mouse diaphragms, we also measured myosin force production. Furthermore, we measured cellular and muscle strip force production at three mo of age. Stress (force/cross-sectional area) was smaller for mdx than control at the muscle strip level but was not different at the single fiber level. υmax of mdx myosin was not different from control at either 3 or 12 mo nor was their relative myosin force. The type I and IIb myosin heavy chain composition was not different between control and mdx diaphragms at 3 or 12 mo. These results suggest that the myosin function, as well as the single fiber mechanics, do not underlie the weakness of the mdx diaphragm. This weakness was only observed at the level of the intact muscle bundle and could not be narrowed down to a specific mechanical impairment of its individual fibers or myosin molecules.

  5. Deletion of Mylk1 in oocytes causes delayed morula-to-blastocyst transition and reduced fertility without affecting folliculogenesis and oocyte maturation in mice.

    PubMed

    Liang, Qiu-Xia; Zhang, Qing-Hua; Qi, Shu-Tao; Wang, Zhong-Wei; Hu, Meng-Wen; Ma, Xue-Shan; Zhu, Min-Sheng; Schatten, Heide; Wang, Zhen-Bo; Sun, Qing-Yuan

    2015-04-01

    The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation.

  6. Correlated NOS-Imu and myf5 expression by satellite cells in mdx mouse muscle regeneration during NOS manipulation and deflazacort treatment.

    PubMed

    Anderson, Judy E; Vargas, Cinthya

    2003-06-01

    Satellite cells, muscle precursor cells in skeletal muscle, are normally quiescent and become activated by disease or injury. A lack of dystrophin and changes in the expression or activity of neuronal nitric oxide synthase (NOS-I) affect the timing of activation in vivo. Nitric oxide synthase inhibition delays muscle repair in normal mice, and worsens muscular dystrophy in the mdx mouse, a genetic homologue of Duchenne muscular dystrophy. However, the potential role of activation and repair events mediated by nitric oxide in determining the outcome of steroid or other treatments for muscular dystrophy is not clear. We tested the hypothesis that the extent of repair in dystrophic muscles of mdx mice is partly dependent on NOS-Imu expression and activity. Myotube formation in regenerating muscle was promoted by deflazacort treatment of mdx dystrophic mice (P<0.05), and improved by combination with the nitric oxide synthase substrate, L-arginine, especially in the diaphragm. NOS-Imu mRNA expression and activity were present in satellite cells and very new myotubes of regenerating and dystrophic muscle. Deflazacort treatment resulted in increased NOS-Imu expression in regenerating muscles in a strong and specific correlation with myf5 expression (r=0.95, P<0.01), a marker for muscle repair. Nitric oxide synthase inhibition prevented the deflazacort-induced rise in NOS-Imu and myf5 expression in the diaphragm without affecting the diameter of non-regenerating fibres. These in vivo studies suggest that gains in NOS-Imu expression and nitric oxide synthase activity in satellite cells can increase the extent and speed of repair, even in the absence of dystrophin in muscle fibres. NOS-Imu may be a useful therapeutic target to augment the effects of steroidal or other treatments of muscular dystrophy.

  7. Lengthening-contractions in isolated myocardium impact force development and worsen cardiac contractile function in the mdx mouse model of muscular dystrophy.

    PubMed

    Xu, Ying; Delfín, Dawn A; Rafael-Fortney, Jill A; Janssen, Paul M L

    2011-02-01

    Lengthening-contractions exert eccentric stress on myofibers in normal myocardium. In congestive heart failure caused by a variety of diseases, the impact of lengthening-contractions of myocardium likely becomes more prevalent and severe. The present study introduces a method to investigate the role of stretching imposed by repetitive lengthening-contractions in myocardium under near-physiological conditions. By exerting various stretch-release ramps while the muscle is contracting, consecutive lengthening-contractions and their potential detrimental effect on cardiac function can be studied. We tested our model and hypothesis in age-matched (young and adult) mdx and wild-type mouse right ventricular trabeculae. These linear and ultrathin muscles possess all major cardiac cell types, and their contractile behavior very closely mimics that of the whole myocardium. In the first group of experiments, 10 lengthening-contractions at various magnitudes of stretch were performed in trabeculae from 10-wk-old mdx and wild-type mice. In the second group, 100 lengthening-contractions at various magnitudes were conducted in trabeculae from 10- and 20-wk-old mice. The peak isometric active developed tension (F(dev), in mN/mm(2)) and kinetic parameters time to peak tension (TTP, in ms) and time from peak tension to half-relaxation (RT50, in ms) were measured. Our results indicate lengthening-contractions significantly impact contractile behavior, and that dystrophin-deficient myocardium in mdx mice is significantly more susceptible to these damaging lengthening-contractions. The results indicate that lengthening-contractions in intact myocardium can be used in vitro to study this emerging contributor to cardiomyopathy.

  8. Disruption of Coordinated Presynaptic and Postsynaptic Maturation Underlies the Defects in Hippocampal Synapse Stability and Plasticity in Abl2/Arg-Deficient Mice

    PubMed Central

    Xiao, Xiao; Levy, Aaron D.; Rosenberg, Brian J.; Higley, Michael J.

    2016-01-01

    Immature glutamatergic synapses in cultured neurons contain high-release probability (Pr) presynaptic sites coupled to postsynaptic sites bearing GluN2B-containing NMDA receptors (NMDARs), which mature into low-Pr, GluN2B-deficient synapses. Whether this coordinated maturation of high-Pr, GluN2B+ synapses to low-Pr, GluN2B-deficient synapses actually occurs in vivo, and if so, what factors regulate it and what role it might play in long-term synapse function and plasticity are unknown. We report that loss of the integrin-regulated Abl2/Arg kinase in vivo yields a subpopulation of “immature” high-Pr, GluN2B+ hippocampal synapses that are maintained throughout late postnatal development and early adulthood. These high-Pr, GluN2B+ synapses are evident in arg−/− animals as early as postnatal day 21 (P21), a time that precedes any observable defects in synapse or dendritic spine number or structure in arg−/− mice. Using focal glutamate uncaging at individual synapses, we find only a subpopulation of arg−/− spines exhibits increased GluN2B-mediated responses at P21. As arg−/− mice age, these synapses increase in proportion, and their associated spines enlarge. These changes coincide with an overall loss of spines and synapses in the Arg-deficient mice. We also demonstrate that, although LTP and LTD are normal in P21 arg−/− slices, both forms of plasticity are significantly altered by P42. These data demonstrate that the integrin-regulated Arg kinase coordinates the maturation of presynaptic and postsynaptic compartments in a subset of hippocampal synapses in vivo, and this coordination is critical for NMDAR-dependent long-term synaptic stability and plasticity. SIGNIFICANCE STATEMENT Synapses mature in vitro from high-release probability (Pr) GluN2B+ to low-Pr, GluN2B−, but it is unknown why this happens or whether it occurs in vivo. High-Pr, GluN2B+ synapses persist into early adulthood in Arg-deficient mice in vivo and have elevated NMDA

  9. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men.

    PubMed

    O'Hara, Laura; McInnes, Kerry; Simitsidellis, Ioannis; Morgan, Stephanie; Atanassova, Nina; Slowikowska-Hilczer, Jolanta; Kula, Krzysztof; Szarras-Czapnik, Maria; Milne, Laura; Mitchell, Rod T; Smith, Lee B

    2015-03-01

    Leydig cell number and function decline as men age, and low testosterone is associated with all "Western" cardio-metabolic disorders. However, whether perturbed androgen action within the adult Leydig cell lineage predisposes individuals to this late-onset degeneration remains unknown. To address this, we generated a novel mouse model in which androgen receptor (AR) is ablated from ∼75% of adult Leydig stem cell/cell progenitors, from fetal life onward (Leydig cell AR knockout mice), permitting interrogation of the specific roles of autocrine Leydig cell AR signaling through comparison to adjacent AR-retaining Leydig cells, testes from littermate controls, and to human testes, including from patients with complete androgen insensitivity syndrome (CAIS). This revealed that autocrine AR signaling is dispensable for the attainment of final Leydig cell number but is essential for Leydig cell maturation and regulation of steroidogenic enzymes in adulthood. Furthermore, these studies reveal that autocrine AR signaling in Leydig cells protects against late-onset degeneration of the seminiferous epithelium in mice and inhibits Leydig cell apoptosis in both adult mice and patients with CAIS, possibly via opposing aberrant estrogen signaling. We conclude that autocrine androgen action within Leydig cells is essential for the lifelong support of spermatogenesis and the development and lifelong health of Leydig cells.

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

    SciTech Connect

    Love, D.R.; Marsden, R.F.; Bloomfield, J.F.; Davies, K.E. ); Morris, G.E.; Ellis, J.M. ); Fairbrother, U.; Edwards, Y.H. ); Slater, C.P. ); Parry, D.J. )

    1991-04-15

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

  11. Targeting the Activin Type IIB Receptor to Improve Muscle Mass and Function in the mdx Mouse Model of Duchenne Muscular Dystrophy

    PubMed Central

    Pistilli, Emidio E.; Bogdanovich, Sasha; Goncalves, Marcus D.; Ahima, Rexford S.; Lachey, Jennifer; Seehra, Jasbir; Khurana, Tejvir

    2011-01-01

    The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg−1 body weight. After 12 weeks of treatment, the 10.0 mg/kg−1 dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg−1), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies. PMID:21356379

  12. Targeting the activin type IIB receptor to improve muscle mass and function in the mdx mouse model of Duchenne muscular dystrophy.

    PubMed

    Pistilli, Emidio E; Bogdanovich, Sasha; Goncalves, Marcus D; Ahima, Rexford S; Lachey, Jennifer; Seehra, Jasbir; Khurana, Tejvir

    2011-03-01

    The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg(-1) body weight. After 12 weeks of treatment, the 10.0 mg/kg(-1) dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg(-1)), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies.

  13. Effect of proton irradiation followed by hindlimb unloading on bone in mature mice: a model of long-duration spaceflight.

    PubMed

    Lloyd, Shane A; Bandstra, Eric R; Willey, Jeffrey S; Riffle, Stephanie E; Tirado-Lee, Leidamarie; Nelson, Gregory A; Pecaut, Michael J; Bateman, Ted A

    2012-10-01

    Bone loss associated with microgravity unloading is well documented; however, the effects of spaceflight-relevant types and doses of radiation on the skeletal system are not well defined. In addition, the combined effect of unloading and radiation has not received much attention. In the present study, we investigated the effect of proton irradiation followed by mechanical unloading via hindlimb suspension (HLS) in mice. Sixteen-week-old female C57BL/6 mice were either exposed to 1 Gy of protons or a sham irradiation procedure (n=30/group). One day later, half of the mice in each group were subjected to four weeks of HLS or normal loading conditions. Radiation treatment alone (IRR) resulted in approximately 20% loss of trabecular bone volume fraction (BV/TV) in the tibia and femur, with no effect in the cortical bone compartment. Conversely, unloading induced substantially greater loss of both trabecular bone (60-70% loss of BV/TV) and cortical bone (approximately 20% loss of cortical bone volume) in both the tibia and femur, with corresponding decreases in cortical bone strength. Histological analyses and serum chemistry data demonstrated increased levels of osteoclast-mediated bone resorption in unloaded mice, but not IRR. HLS+IRR mice generally experienced greater loss of trabecular bone volume fraction, connectivity density, and trabecular number than either unloading or irradiation alone. Although the duration of unloading may have masked certain effects, the skeletal response to irradiation and unloading appears to be additive for certain parameters. Appropriate modeling of the environmental challenges of long duration spaceflight will allow for a better understanding of the underlying mechanisms mediating spaceflight-associated bone loss and for the development of effective countermeasures.

  14. Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice.

    PubMed

    Nair, Ramya; Singh, Vikram Jeet; Salian, Sujith Raj; Kalthur, Sneha Guruprasad; D'Souza, Antony Sylvan; Shetty, Pallavi K; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

    2014-09-15

    Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome.

  15. TRAF6 regulates the effects of polarized maturation of tolerability: Marrow-derived dendritic cells on collagen-induced arthritis in mice

    PubMed Central

    Zhuang, Chenchen; Hong, Xuezhi; Liu, Jia; Luo, Xiaohong; Mo, Hanyou

    2017-01-01

    The study aimed to investigate the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and a differentially mature dendritic cell (mDC) in collagen-induced arthritis (CIA) mice and to determine whether or not TRAF6 regulates the activation of an immature dendritic cell (iDC) and inhibits iDC maturation to induce immune tolerance. The mouse bone marrow stem cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to differentiate immature dendritic cells (DCs), which were divided into four groups with different maturation states: rmGM-CSF, rmIL-4; TNF-α; LPS; and FK506 group. The levels of the cell surfaces of CD80, CD86, and MHI-II were analyzed by flow cytometry to prove DCs at different levels of maturity. The expression of IL-12 in DCs at different maturation states was detected by enzyme-linked immunosorbent assay (ELISA). The expression of TRAF6 mRNA and protein in each group of DCs was detected by a reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The results revealed that the differentiation of bone marrow cells into iDCs was significantly induced by cytokines (rmGM-CSF, IL-4). CD80, CD86, MHC-II were expressed in the four groups, and the difference between them was statistically significant (P<0.05). A higher degree of DC differentiation led to a gradual increase of IL-12 secretion in the four groups. The difference was statistically significant (P<0.05) for this secretion (group D, 10,620.73±276.73 pg/ml). The expression levels of TRAF6 mRNA were significantly higher in group D than those in the other three groups (P<0.01). Although there was no significant difference in the expression levels of TRAF6 mRNA between groups B and C, the expression levels of TRAF6 mRNA between groups B and C were higher than those of the control group. The TRAF6 protein expression was higher in group D than that in the other three

  16. Plasmacytoid DC from Aged Mice Down-Regulate CD8 T Cell Responses by Inhibiting cDC Maturation after Encephalitozoon cuniculi Infection

    PubMed Central

    Gigley, Jason P.; Khan, Imtiaz A.

    2011-01-01

    Age associated impairment of immune function results in inefficient vaccination, tumor surveillance and increased severity of infections. Several alterations in adaptive immunity have been observed and recent studies report age related declines in innate immune responses to opportunistic pathogens including Encephalitozoon cuniculi. We previously demonstrated that conventional dendritic cells (cDC) from 9-month-old animals exhibit sub-optimal response to E. cuniculi infection, suggesting that age associated immune senescence begins earlier than expected. We focused this study on how age affects plasmacytoid DC (pDC) function. More specifically how aged pDC affect cDC function as we observed that the latter are the predominant activators of CD8 T cells during this infection. Our present study demonstrates that pDC from middle-aged mice (12 months) suppress young (8 week old) cDC driven CD8 T cell priming against E. cuniculi infection. The suppressive effect of pDC from older mice decreased maturation of young cDC via cell contact. Aged mouse pDC exhibited higher expression of PD-L1 and blockade of their interaction with cDC via this molecule restored cDC maturation and T cell priming. Furthermore, the PD-L1 dependent suppression of cDC T cell priming was restricted to effector function of antigen-specific CD8 T cells not their expansion. To the best of our knowledge, the data presented here is the first report highlighting a cell contact dependent, PD-L1 regulated, age associated defect in a DC subpopulation that results in a sub-optimal immune response against E. cuniculi infection. These results have broad implications for design of immunotherapeutic approaches to enhance immunity for aging populations. PMID:21695169

  17. Plasmacytoid DC from aged mice down-regulate CD8 T cell responses by inhibiting cDC maturation after Encephalitozoon cuniculi infection.

    PubMed

    Gigley, Jason P; Khan, Imtiaz A

    2011-01-01

    Age associated impairment of immune function results in inefficient vaccination, tumor surveillance and increased severity of infections. Several alterations in adaptive immunity have been observed and recent studies report age related declines in innate immune responses to opportunistic pathogens including Encephalitozoon cuniculi. We previously demonstrated that conventional dendritic cells (cDC) from 9-month-old animals exhibit sub-optimal response to E. cuniculi infection, suggesting that age associated immune senescence begins earlier than expected. We focused this study on how age affects plasmacytoid DC (pDC) function. More specifically how aged pDC affect cDC function as we observed that the latter are the predominant activators of CD8 T cells during this infection. Our present study demonstrates that pDC from middle-aged mice (12 months) suppress young (8 week old) cDC driven CD8 T cell priming against E. cuniculi infection. The suppressive effect of pDC from older mice decreased maturation of young cDC via cell contact. Aged mouse pDC exhibited higher expression of PD-L1 and blockade of their interaction with cDC via this molecule restored cDC maturation and T cell priming. Furthermore, the PD-L1 dependent suppression of cDC T cell priming was restricted to effector function of antigen-specific CD8 T cells not their expansion. To the best of our knowledge, the data presented here is the first report highlighting a cell contact dependent, PD-L1 regulated, age associated defect in a DC subpopulation that results in a sub-optimal immune response against E. cuniculi infection. These results have broad implications for design of immunotherapeutic approaches to enhance immunity for aging populations.

  18. Immunization of Newborn Mice Accelerates the Architectural Maturation of Lymph Nodes, But AID-Dependent IgG Responses Are Still Delayed Compared to the Adult

    PubMed Central

    Munguía-Fuentes, Rosario; Yam-Puc, Juan Carlos; Silva-Sánchez, Aarón; Marcial-Juárez, Edith; Gallegos-Hernández, Isis Amara; Calderón-Amador, Juana; Randall, Troy D.; Flores-Romo, Leopoldo

    2017-01-01

    Lymph nodes (LNs) have evolved to maximize antigen (Ag) collection and presentation as well as lymphocyte proliferation and differentiation—processes that are spatially regulated by stromal cell subsets, including fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs). Here, we showed that naïve neonatal mice have poorly organized LNs with few B and T cells and undetectable FDCs, whereas adult LNs have numerous B cells and large FDC networks. Interestingly, immunization on the day of birth accelerated B cell accumulation and T cell recruitment into follicles as well as FDC maturation and FRC organization in neonatal LNs. However, compared to adults, the formation of germinal centers was both delayed and reduced following immunization of neonatal mice. Although immunized neonates poorly expressed activation-induced cytidine deaminase (AID), they were able to produce Ag-specific IgGs, but with lower titers than adults. Interestingly, the Ag-specific IgM response in neonates was similar to that in adults. These results suggest that despite an accelerated structural maturation of LNs in neonates following vaccination, the B cell response is still delayed and reduced in its ability to isotype switch most likely due to poor AID expression. Of note, naïve pups born to Ag-immunized mothers had high titers of Ag-specific IgGs from day 0 (at birth). These transferred antibodies confirm a mother-derived coverage to neonates for Ags to which mothers (and most likely neonates) are exposed, thus protecting the neonates while they produce their own antibodies. Finally, the type of Ag used in this study and the results obtained also indicate that T cell help would be operating at this stage of life. Thus, neonatal immune system might not be intrinsically immature but rather evolutionary adapted to cope with Ags at birth. PMID:28154564

  19. Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice

    PubMed Central

    Meurer, Steffen K.; Neß, Melanie; Weiskirchen, Sabine; Kim, Philipp; Tag, Carmen G.; Kauffmann, Marlies; Huber, Michael; Weiskirchen, Ralf

    2016-01-01

    Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research. PMID:27337047

  20. Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

    PubMed

    Kübler, Ayline; Woiterski, Jeanette; Witte, Kai-Erik; Bühring, Hans-Jörg; Hartwig, Udo F; Ebinger, Martin; Oevermann, Lena; Mezger, Markus; Herr, Wolfgang; Lang, Peter; Handgretinger, Rupert; Münz, Christian; André, Maya C

    2014-12-18

    Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+) NK cells and immature KIR(-) NK cells arising early posttransplantation in humanized NSG mice exerted substantial antileukemic activity. Low-dose and long-term treatment of humanized NSG mice with the DNA-demethylating agent 5-aza-cytidine distinctly enhanced the antitumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK-cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK-cell-mediated immune responses for poor prognosis pediatric BCP-ALL patients.

  1. Effects of prenatal stress and exercise on dentate granule cells maturation and spatial memory in adolescent mice.

    PubMed

    Bustamante, Carlos; Bilbao, Pamela; Contreras, William; Martínez, Mauricio; Mendoza, Antonio; Reyes, Alvaro; Pascual, Rodrigo

    2010-11-01

    Exposure to prenatal stress (PS) increases the risk of developing neurobehavioral disturbances later in life. Previous work has shown that exercise can exert beneficial effects on brain damage; however, it is unknown whether voluntary wheel running (VWR) can ameliorate the neurobehavioral impairments induced by PS in adolescent offspring. Pregnant CF-1 mice were randomly assigned to control (n=5) or stressed (n=5) groups. Pregnant dams were subjected to restraint stress between gestational days 14 and 21 (G14-21), whereas controls remained undisturbed in their home cages. On postnatal day 21 (P21), male pups were randomly assigned to the following experimental groups: control (n=5), stressed (n=5), and stressed mice+daily submitted to VWR (n=4). At P52, all groups were behaviorally evaluated in the Morris water maze. Animals were then sacrificed, and Golgi-impregnated granule cells were morphometrically analyzed. The results indicate that PS produced significant behavioral and neuronal impairments in adolescent offspring and that VWR significantly offset these deleterious effects.

  2. Pre- and postsynaptic changes in the neuromuscular junction in dystrophic mice

    PubMed Central

    Pratt, Stephen J. P.; Valencia, Ana P.; Le, Gloribel K.; Shah, Sameer B.; Lovering, Richard M.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated in mdx mice (murine model for DMD) significant morphologic alterations at the motor endplate of the neuromuscular junction (NMJ) and corresponding NMJ transmission failure after injury. Here we extend these initial observations at the motor endplate to gain insight into the pre- vs. postsynaptic morphology, as well as the subsynaptic nuclei in healthy (WT) vs. mdx mice. We quantified the discontinuity and branching of the terminal nerve in adult mice. We report mdx- and age-dependent changes for discontinuity and an increase in branching when compared to WT. To examine mdx- and age-dependent changes in the relative localization of pre- and postsynaptic structures, we calculated NMJ occupancy, defined as the ratio of the footprint occupied by presynaptic vesicles vs. that of the underlying motor endplate. The normally congruent coupling between presynaptic and postsynaptic morphology was altered in mdx mice, independent of age. Finally we found an almost two-fold increase in the number of nuclei and an increase in density (nuclei/area) underlying the NMJ. These outcomes suggest substantial remodeling of the NMJ during dystrophic progression. This remodeling reflects plasticity in both pre- and postsynaptic contributors to NMJ structure, and thus perhaps also NM transmission and muscle function. PMID:26441672

  3. Mice lacking doublecortin and doublecortin-like kinase 2 display altered hippocampal neuronal maturation and spontaneous seizures.

    PubMed

    Kerjan, Géraldine; Koizumi, Hiroyuki; Han, Edward B; Dubé, Celine M; Djakovic, Stevan N; Patrick, Gentry N; Baram, Tallie Z; Heinemann, Stephen F; Gleeson, Joseph G

    2009-04-21

    Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.

  4. Maturation of responsiveness to cardioactive drugs. Differential effects of acetylcholine, norepinephrine, theophylline, tyramine, glucagon, and dibutyryl cyclic AMP on atrial rate in hearts of fetal mice.

    PubMed

    Wildenthal, K

    1973-09-01

    Freshly isolated hearts of fetal mice of gestational ages ranging between 12 and 22 days (term) were exposed to several concentrations of a variety of chronotropic agents. Acetylcholine (10(-4)-10(-2) M) caused marked bradycardia in all hearts, even after only 12-14 days' gestation (i.e., even before cardiac innervation had occurred), and the intensity of the response increased steadily with advancing age throughout gestation. Responsiveness to norepinephrine was present but minimal at 12-14 days, so that mean atrial rate rose by < 10% with a maximal concentration of the drug (10(-5) M); responsiveness became more marked by 15-16 days (just after the time atrial innervation is thought to begin) and still greater effects appeared just before term. Glucagon had no effect in hearts of < 17 days' gestational age, but caused tachycardia thereafter, indicating that cardiac responsiveness to glucagon differentiates later than does responsiveness to norepinephrine. Responses to theophyl-line in 12-14 day hearts exceeded those to norepinephrine, indicating that the drug can affect heart rate independently of its ability to cause release of endogenous catecholamines. In contrast, tyramine caused no response until 21-22 days, well after the time the beta-receptor has differentiated and after innervation is fairly well developed, suggesting that the drug's primary sympathomimetic effect is indirect rather than direct. Dibutyryl cyclic AMP did not cause tachycardia at any fetal age. It is concluded that maturation of responsiveness of the mouse heart to cardioactive drugs develops in specific patterns for different agents. The identification of differential patterns of maturation for various drugs may provide valuable means for characterizing the differentiation of specific receptors and for investigating possible mechanisms of action of the drugs.

  5. Unilateral T cell maturation arrest in the thymus of CBA/H mice as a long-term effect after neutron irradiation

    SciTech Connect

    Huiskamp, R.; van Ewijk, W.

    1987-04-01

    Thymuses of CBA/H mice were investigated up to 570 days after whole-body irradiation with 2.5 Gy fast fission neutrons or 6.0 Gy X rays. A number of these thymuses, observed 220-270 days after neutron irradiation, have two equal sized lobes, one of which has an abnormal T cell distribution. The present paper reports on the distribution of lymphoid and stromal cell types in these thymuses. For this purpose, we employed immunohistology using the indirect immunoperoxidase method. We incubated frozen sections of these aberrant thymuses with monoclonal antibodies directed to cell surface differentiation antigens on lymphoid cells, such as Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14; monoclonal antibodies directed to major histocompatibility complex (MHC) antigens, such as I-A and H-2K; and monoclonal antibodies directed to determinants in various thymic stromal cell types. The results of this study show a T cell differentiation arrest in only one of the two thymic lobes. T cells in the aberrant lobe express Thy-1, T-200, and MEL-14 antigens but are MT-4- and Lyt-1-. In some lobes, a weak Lyt-2 expression was observed. The observed T cell maturation arrest is mainly restricted to the cortex since in the medulla, in addition to cells with an aberrant cortical phenotype, normal T cell phenotypes are observed. This indicates that cortex and medulla have independent generation kinetics in T cell maturation. The stromal cell composition in these abnormal lobes is not different from that in the normal lobe, but the size of the medulla tends to be smaller. Furthermore, the I-A expression on the cortical epithelial cells does not reveal the characteristic reticular staining pattern that is observed in the normal lobe, since the I-A determinants are not strictly confined to the epithelial cells.

  6. Assessing Functional Performance in the Mdx Mouse Model

    PubMed Central

    Aartsma-Rus, Annemieke; van Putten, Maaike

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories. PMID:24747372

  7. Black bear parathyroid hormone has greater anabolic effects on trabecular bone in dystrophin-deficient mice than in wild type mice.

    PubMed

    Gray, Sarah K; McGee-Lawrence, Meghan E; Sanders, Jennifer L; Condon, Keith W; Tsai, Chung-Jui; Donahue, Seth W

    2012-09-01

    Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease that has deleterious consequences in muscle and bone, leading to decreased mobility, progressive osteoporosis, and premature death. Patients with DMD experience a higher-than-average fracture rate, particularly in the proximal and distal femur and proximal tibia. The dystrophin-deficient mdx mouse is a model of DMD that demonstrates muscle degeneration and fibrosis and osteoporosis. Parathyroid hormone, an effective anabolic agent for post-menopausal and glucocorticoid-induced osteoporosis, has not been explored for DMD. Black bear parathyroid hormone (bbPTH) has been implicated in the maintenance of bone properties during extended periods of disuse (hibernation). We cloned bbPTH and found 9 amino acid residue differences from human PTH. Apoptosis was mitigated and cAMP was activated by bbPTH in osteoblast cultures. We administered 28nmol/kg of bbPTH 1-84 to 4-week old male mdx and wild type mice via daily (5×/week) subcutaneous injection for 6 weeks. Vehicle-treated mdx mice had 44% lower trabecular bone volume fraction than wild type mice. No changes were found in femoral cortical bone geometry or mechanical properties with bbPTH treatment in wild type mice, and only medio-lateral moment of inertia changed with bbPTH treatment in mdx femurs. However, μCT analyses of the trabecular regions of the distal femur and proximal tibia showed marked increases in bone volume fraction with bbPTH treatment, with a greater anabolic response (7-fold increase) in mdx mice than wild type mice (2-fold increase). Trabecular number increased in mdx long bone, but not wild type bone. Additionally, greater osteoblast area and decreased osteoclast area were observed with bbPTH treatment in mdx mice. The heightened response to PTH in mdx bone compared to wild type suggests a link between dystrophin deficiency, altered calcium signaling, and bone. These findings support further investigation of PTH as an anabolic

  8. Evaluation of Electrical Impedance as a Biomarker of Myostatin Inhibition in Wild Type and Muscular Dystrophy Mice

    PubMed Central

    Sanchez, Benjamin; Li, Jia; Yim, Sung; Pacheck, Adam; Widrick, Jeffrey J.; Rutkove, Seward B.

    2015-01-01

    Objectives Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx). Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters. Methods Eight wild-type (wt) and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg−1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed. Results As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p < 0.001 respectively) and muscle mass (25% p < 0.05 and 22% p < 0.001, respectively). The Cole impedance parameters in treated wt mice, showed a 24% lower central frequency (p < 0.05) and 19% higher resistance ratio (p < 0.05); no significant differences were observed in the mdx mice. These differences were consistent with those seen in maximum isometric force, which was greater in the wt animals (p < 0.05 at > 70 Hz), but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01). Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively). Conclusions Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials. PMID:26485280

  9. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice.

    PubMed

    Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang; Liang, Cheng-Guang

    2016-01-01

    Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

  10. Upregulation of store-operated Ca2+ entry in dystrophic mdx mouse muscle.

    PubMed

    Edwards, Joshua N; Friedrich, Oliver; Cully, Tanya R; von Wegner, Frederic; Murphy, Robyn M; Launikonis, Bradley S

    2010-07-01

    Store-operated Ca(2+) entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca(2+) from the transverse tubule into the junctional cleft during periods of depleting Ca(2+) release. In dystrophic muscle fibers, SOCE may be a source of Ca(2+) overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca(2+) release. To examine this, Ca(2+) in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca(2+) from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca(2+) transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca(2+) concentrations ([Ca(2+)](SR)). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca(2+) into the fiber for an equivalent depletion of [Ca(2+)](SR) via SOCE, it remains unclear whether this is deleterious.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2012-09-01

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

  13. Intestine of dystrophic mice presents enhanced contractile resistance to stretching despite morphological impairment.

    PubMed

    Alves, Gabriel A; Silva, Luisa R; Rosa, Eloi F; Aboulafia, Jeannine; Freymüller-Haapalainen, Edna; Souccar, Caden; Nouailhetas, Viviane L A

    2014-02-01

    Protein dystrophin is a component of the dystrophin-associated protein complex, which links the contractile machinery to the plasma membrane and to the extracellular matrix. Its absence leads to a condition known as Duchenne muscular dystrophy (DMD), a disease characterized by progressive skeletal muscle degeneration, motor disability, and early death. In mdx mice, the most common DMD animal model, loss of muscle cells is observed, but the overall disease alterations are less intense than in DMD patients. Alterations in gastrointestinal tissues from DMD patients and mdx mice are not yet completely understood. Thus, we investigated the possible relationships between morphological (light and electron microscopy) and contractile function (by recording the isometric contractile response) with alterations in Ca²⁺ handling in the ileum of mdx mice. We evidenced a 27% reduction in the ileal muscular layer thickness, a partial damage to the mucosal layer, and a partial damage to mitochondria of the intestinal myocytes. Functionally, the ileum from mdx presented an enhanced responsiveness during stretch, a mild impairment in both the electromechanical and pharmacomechanical signaling associated with altered calcium influx-induced contraction, with no alterations in the sarcoplasmic reticulum Ca²⁺ storage (maintenance of the caffeine and thapsigargin-induced contraction) compared with control animals. Thus, it is evidenced that the protein dystrophin plays an important role in the preservation of both the microstructure and ultrastructure of mice intestine, while exerting a minor but important role concerning the intestinal contractile responsiveness and calcium handling.

  14. Altered expression of Armet and Mrlp51 in the oocyte, preimplantation embryo, and brain of mice following oocyte in vitro maturation but postnatal brain development and cognitive function are normal.

    PubMed

    Wang, Ning; Wang, Liya; Le, Fang; Zhan, Qitao; Zheng, Yingming; Ding, Guolian; Chen, Xijing; Sheng, Jianzhong; Dong, Minyue; Huang, Hefeng; Jin, Fan

    2011-09-01

    Despite the efforts to recapitulate the follicle environment, oocytes from in vitro maturation (IVM) have poorer developmental potential than those matured in vivo and the effects on the resultant offspring are of concern. The aim of this study was to determine altered gene expression in oocytes following IVM and to evaluate the expression of the arginine rich, mutated in early stage of tumors gene (Armet) and mitochondrial ribosomal protein L51 (Mrpl51) in embryos and brains of fetal/postnatal mice and the brain development of IVM offspring. An IVM mouse model was established while oocytes matured in vivo were used as the controls. Suppressive subtractive hybridization (SSH) and RT-PCR/western blot were used to analyze the differential expression of genes/proteins between IVM and the control group. HE staining and water maze were used to assess the histological changes in brain tissue and cognition of the offspring. The rates of fertilization, cleavage, and live birth were significantly decreased in IVM group. Thirteen genes were upregulated in IVM oocytes compared with the control, including Armet and Mrpl51. The higher level of Armet in IVM oocytes was retained in brain of newborn mice, which could be related to the upregulation of activating transcription factor 6 (Atf6) and X-box binding protein 1 (Xbp1), while Mrpl51 was expressed normally in brain of postnatal mice. No significant differences were detected in brain weight, neuronal counts, and the cognition in the offspring between the two groups. The present results suggested that IVM could affect the pregnancy outcome and the Armet and Mrpl51 gene/protein expression. The change in Armet expression lasted while the change of Mrpl51 disappeared after birth. However, the brain development of the offspring seemed to be unaffected by IVM.

  15. RhoA mediates defective stem cell function and heterotopic ossification in dystrophic muscle of mice.

    PubMed

    Mu, Xiaodong; Usas, Arvydas; Tang, Ying; Lu, Aiping; Wang, Bing; Weiss, Kurt; Huard, Johnny

    2013-09-01

    Heterotopic ossification (HO) and fatty infiltration (FI) often occur in diseased skeletal muscle and have been previously described in various animal models of Duchenne muscular dystrophy (DMD); however, the pathological mechanisms remain largely unknown. Dystrophin-deficient mdx mice and dystrophin/utrophin double-knockout (dKO) mice are mouse models of DMD; however, mdx mice display a strong muscle regeneration capacity, while dKO mice exhibit a much more severe phenotype, which is similar to patients with DMD. Our results revealed that more extensive HO, but not FI, occurred in the skeletal muscle of dKO mice versus mdx mice, and RhoA activation specifically occurred at the sites of HO. Moreover, the gene expression of RhoA, BMPs, and several inflammatory factors were significantly up-regulated in muscle stem cells isolated from dKO mice; while inactivation of RhoA in the cells with RhoA/ROCK inhibitor Y-27632 led to reduced osteogenic potential and improved myogenic potential. Finally, inactivation of RhoA signaling in the dKO mice with Y-27632 improved muscle regeneration and reduced the expression of BMPs, inflammation, HO, and intramyocellular lipid accumulation in both skeletal and cardiac muscle. Our results revealed that RhoA represents a major molecular switch in the regulation of HO and muscle regeneration in dystrophic skeletal muscle of mice.

  16. Proteasomal Activity Is Required to Initiate and to Sustain Translational Activation of Messenger RNA Encoding the Stem-Loop-Binding Protein During Meiotic Maturation in Mice1

    PubMed Central

    Yang, Qin; Allard, Patrick; Huang, Michael; Zhang, Wenling; Clarke, Hugh J.

    2009-01-01

    Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3′-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3′-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation. PMID:19759367

  17. Microarchitecture is severely compromised but motor protein function is preserved in dystrophic mdx skeletal muscle.

    PubMed

    Friedrich, O; Both, M; Weber, C; Schürmann, S; Teichmann, M D H; von Wegner, F; Fink, R H A; Vogel, M; Chamberlain, J S; Garbe, C

    2010-02-17

    Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.

  18. Microarchitecture Is Severely Compromised but Motor Protein Function Is Preserved in Dystrophic mdx Skeletal Muscle

    PubMed Central

    Friedrich, O.; Both, M.; Weber, C.; Schürmann, S.; Teichmann, M.D.H.; von Wegner, F.; Fink, R.H.A.; Vogel, M.; Chamberlain, J.S.; Garbe, C.

    2010-01-01

    Abstract Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (∼1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities (“verniers”) than wild-type fibers (∼130/1000 μm3 vs. ∼36/1000 μm3). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (∼38/1000 μm3 counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies. PMID:20159157

  19. Lactational exposure of mice to low levels of non-dioxin-like polychlorinated biphenyls increases susceptibility to neuronal stress at a mature age.

    PubMed

    Elnar, Arpiné Ardzivian; Allouche, Ahmad; Desor, Frédéric; Yen, Frances T; Soulimani, Rachid; Oster, Thierry

    2016-03-01

    Lactational exposure to low levels of the sum of the six indicator polychlorinated biphenyls (Σ6 NDL-PCBs, 10ng/kg/day) is known to lead to persistent anxious behavior in young and adult offspring mice at postnatal days 40 and 160, respectively. At more advanced life stages, we evaluated the effects on the mouse brain of neuronal stress induced by the synaptotoxic amyloid-beta (Aβ) peptide. Perinatal exposure of lactating mice to Σ6 NDL-PCBs did not affect short-term memory performances of their offspring male mice aged 14 months as compared to control PCB-naive mice. However, following intracerebroventricular injection of soluble Aβ oligomers, significant impairments in long-term memory were detected in the mice that had been lactationally treated with Σ6 NDL-PCBs. In addition, immunoblot analyses of the synaptosomal fraction of hippocampal tissues from treated mice revealed a lower expression of the synaptic proteins synaptophysin and PSD-95. Though preliminary, our findings suggest for the first time that early exposure to low levels of NDL-PCBs induce late neuronal vulnerability to amyloid stress. Additional experiments are needed to confirm whether early environmental influences are involved in the etiology of brain aging and cognitive decline.

  20. Cutting Edge: Ikaros null thymocytes mature into the CD4 lineage with reduced TCR signal: A study using CD3{zeta} immunoreceptor tyrosine-based activation motif transgenic mice.

    PubMed

    Urban, Julie A; Brugmann, William; Winandy, Susan

    2009-04-01

    Positive selection is a critical T cell developmental checkpoint that is driven by TCR signals. Enhanced positive selection toward the CD4 lineage occurs in the absence of Ikaros. One explanation for this phenotype is that Ikaros establishes the TCR signaling threshold that must be overcome for positive selection to occur. In the current study, this possibility is explored through the use of CD3zeta ITAM transgenic mice that express a CD3 zeta-chain with zero, one, or three ITAMs and an MHC class II (DO11.10)- or MHC class I (H-Y)-restricted TCR transgene. Using this system, we demonstrate that in the absence of Ikaros, thymocytes are able to mature into the CD4 lineage with reduced TCR signaling potential compared with that required to drive the maturation of wild-type thymocytes. We also demonstrate that maturation into the CD8 lineage is enhanced under conditions of reduced TCR signaling potential in the absence of Ikaros.

  1. The mdx Mutation in the 129/Sv Background Results in a Milder Phenotype: Transcriptome Comparative Analysis Searching for the Protective Factors

    PubMed Central

    Calyjur, Priscila Clara; Almeida, Camila de Freitas; Ayub-Guerrieri, Danielle; Ribeiro, Antonio Fernando; Fernandes, Stephanie de Alcântara; Ishiba, Renata; dos Santos, Andre Luis Fernandes; Onofre-Oliveira, Paula; Vainzof, Mariz

    2016-01-01

    The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process. PMID:26954670

  2. Abnormal thymic maturation and lymphoproliferation in MRL-Faslpr/lpr mice can be partially reversed by synthetic oligonucleotides: implications for systemic lupus erythematosus and autoimmune lymphoproliferative syndrome.

    PubMed

    Ashman, R F; Singh, N; Lenert, P S

    2016-11-10

    MRL-Fas (lpr/lpr) mice represent an excellent animal model for studying non-malignant lymphoproliferation, regeneration and systemic autoimmunity. Retro-transposon insertion into the second intron of the pro-apoptotic Fas gene appears to be responsible for both lymphoproliferation and autoimmunity, while other genes are more likely to contribute to the regenerative healing characteristic of this mouse strain. Previous studies have shown that neonatal thymectomy can halt the development of abnormal lymphoproliferation. Whereas at four weeks of age primary and secondary lymphoid organs appear to be grossly intact, vigorous lymphoproliferation and autoantibody production subsequently ensues. This is first noticeable at six weeks of age, at which time lymph nodes, spleens and thymuses, but not the bone marrow, become infiltrated with abnormal B220(+)CD3(+)CD4(-)CD8(-) T cells. Around the same time, thymuses show a significant drop in CD4(+)CD8(+)double-positive T cells generating an abnormal ratio between double-positive and single-positive thymocytes. The objective of current study was to evaluate the effect of synthetic oligonucleotides-toll-like receptor antagonists on early lymphoid development in this strain of mice. Herein, we demonstrate the ability of synthetic oligonucleotides made with the nuclease-resistant phosphorothioate backbone to partially reverse abnormal lymphoproliferation and thymic involution in pre-diseased MRL-Fas (lpr/lpr) mice when administered intraperitoneally starting from week four of age. This curative effect of oligonucleotides was primary sequence/secondary oligonucleotide structure-independent, suggesting an effect through the toll-like receptor 7. A similar approach may potentially benefit patients with autoimmune lymphoproliferative syndrome who, like MRL-Fas (lpr/lpr) mice, carry a mutation in the Fas gene.

  3. Exposure to ethinyl estradiol prenatally and/or after sexual maturity induces endometriotic and precancerous lesions in uteri and ovaries of mice.

    PubMed

    Koike, Eiji; Yasuda, Yoshiko; Shiota, Mitsuru; Shimaoka, Masao; Tsuritani, Mitsuhiro; Konishi, Hiroyoshi; Yamasaki, Harufumi; Okumoto, Katsumi; Hoshiai, Hiroshi

    2013-03-01

    Unrecognizable exposure to estrogenic substance may cause estrogen-dependent diseases, endometriosis and cancer. Pregnant mice (ICR/Jcl, CLEA) were exposed to 0.01 mg ethinyl estradiol (EE2 )/kg per day or vehicle (olive oil) through oral intubation from day 11 to 17 of gestation. They delivered their offspring and raised them. When the experimental female F1 mice were at 8 weeks of age, they were not exposed to EE2 or to the same dose of EE2 or to vehicle twice a week until 20 weeks of age. The control female F1 mice were exposed to the same dose of EE2 or vehicle alone, similarly. All mice were killed at 28 weeks of age. The resected uteri and ovaries were processed for microscopic examinations and for determination of the aromatase mRNA levels and aromatase protein through quantitative RT-PCR and Western blotting, respectively. Adenomyosis and adenocarcinomatous changes were significantly discernible in the EE2 -exposed uteri, and incidence of ectopic glands and serous cysts were significantly increased in the prenatally EE2 -exposed ovaries as compared with respective controls. Significant upregulation of the aromatase mRNA was seen in the prenatally EE2 -exposed uteri and in the EE2 -exposed ovaries. The aromatase protein was identified in all ovaries examined, and in EE2 -exposed uteri but not in controls and confirmed its localization in eutopic and ectopic glands, abnormally proliferated lesions and the lining of the cysts. Taken together, continuous EE2 exposure may cause endometriotic and precancerous lesions due to excessive estrogen synthesis in both target organs.

  4. Proteomic Profiling of the Dystrophin-Deficient mdx Phenocopy of Dystrophinopathy-Associated Cardiomyopathy

    PubMed Central

    2014-01-01

    Cardiorespiratory complications are frequent symptoms of Duchenne muscular dystrophy, a neuromuscular disorder caused by primary abnormalities in the dystrophin gene. Loss of cardiac dystrophin initially leads to changes in dystrophin-associated glycoproteins and subsequently triggers secondarily sarcolemmal disintegration, fibre necrosis, fibrosis, fatty tissue replacement, and interstitial inflammation. This results in progressive cardiac disease, which is the cause of death in a considerable number of patients afflicted with X-linked muscular dystrophy. In order to better define the molecular pathogenesis of this type of cardiomyopathy, several studies have applied mass spectrometry-based proteomics to determine proteome-wide alterations in dystrophinopathy-associated cardiomyopathy. Proteomic studies included both gel-based and label-free mass spectrometric surveys of dystrophin-deficient heart muscle from the established mdx animal model of dystrophinopathy. Comparative cardiac proteomics revealed novel changes in proteins associated with mitochondrial energy metabolism, glycolysis, signaling, iron binding, antibody response, fibre contraction, basal lamina stabilisation, and cytoskeletal organisation. This review summarizes the importance of studying cardiomyopathy within the field of muscular dystrophy research, outlines key features of the mdx heart and its suitability as a model system for studying cardiac pathogenesis, and discusses the impact of recent proteomic findings for exploring molecular and cellular aspects of cardiac abnormalities in inherited muscular dystrophies. PMID:24772416

  5. Pre-clinical drug tests in the mdx mouse as a model of dystrophinopathies: an overview.

    PubMed

    De Luca, Annamaria

    2012-05-01

    Duchenne muscular dystrophy is a lethal X-linked muscle disease affecting 1/3500 live male birth. It results from defects in the subsarcolemmal protein dystrophin, a component of the dystrophin-glycoprotein complex (DGC) which links the intracellular cytoskeleton to the extracellular matrix. The absence of dystrophin leads to muscle membrane fragility, muscle necrosis and gradual replacement of skeletal muscle by fat and connective tissue, through a complex and still unclear cascade of interconnecting events. No cure is currently available, with glucocorticoids being the sole drugs in clinical use in spite of their remarkable side effects. A great effort is devoted at performing pre-clinical tests on the mdx mouse, the mostly used homologous animal model for DMD, with the final aim to identify drugs safer than steroids and able to target the pathogenic mechanisms so to delay pathology progression. This review updates the efforts on this topic, focusing on the open issues about the animal model and highlighting the classes of pharmaceuticals that are more promising as disease-modifiers, while awaiting for more corrective therapies. Although caution is necessary in data transfer from mdx model to DMD patients, the implementation of standard operating procedures and the growing understanding of the pathology may allow a more accurate evaluation of therapeutics, alone or in combination, in pre-clinical settings. A continuous cross-talk with clinicians and patients associations are also crucial points for proper translation of data from mouse to bedside.

  6. Store-operated calcium entry contributes to abnormal Ca²⁺ signalling in dystrophic mdx mouse myoblasts.

    PubMed

    Onopiuk, Marta; Brutkowski, Wojciech; Young, Christopher; Krasowska, Elżbieta; Róg, Justyna; Ritso, Morten; Wojciechowska, Sylwia; Arkle, Stephen; Zabłocki, Krzysztof; Górecki, Dariusz C

    2015-03-01

    Sarcolemma damage and activation of various calcium channels are implicated in altered Ca(2+) homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca(2+) concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease.

  7. Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats.

    PubMed

    Ramaswamy, Krishnan S; Palmer, Mark L; van der Meulen, Jack H; Renoux, Abigail; Kostrominova, Tatiana Y; Michele, Daniel E; Faulkner, John A

    2011-03-01

    The dystrophin–glycoprotein complex (DGC) provides an essential link from the muscle fibre cytoskeleton to the extracellular matrix. In dystrophic humans and mdx mice, mutations in the dystrophin gene disrupt the structure of the DGC causing severe damage to muscle fibres. In frog muscles, transmission of force laterally from an activated fibre to the muscle surface occurs without attenuation, but lateral transmission of force has not been demonstrated in mammalian muscles. A unique ‘yoke' apparatus was developed that attached to the epimysium of muscles midway between the tendons and enabled the measurement of lateral force. We now report that in muscles of young wild-type (WT) mice and rats, compared over a wide range of longitudinal forces, forces transmitted laterally showed little or no decrement. In contrast, for muscles of mdx mice and very old rats, forces transmitted laterally were impaired severely. Muscles of both mdx mice and very old rats showed major reductions in the expression of dystrophin. We conclude that during contractions, forces developed by skeletal muscles of young WT mice and rats are transmitted laterally from fibre to fibre through the DGC without decrement. In contrast, in muscles of dystrophic or very old animals, disruptions in DGC structure and function impair lateral transmission of force causing instability and increased susceptibility of fibres to contraction-induced injury.

  8. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the "hairy" skin of newborn mice: early maturation of hair follicle afferents.

    PubMed

    Woodbury, C J; Ritter, A M; Koerber, H R

    2001-07-30

    Adult skin sensory neurons exhibit characteristic projection patterns in the dorsal horn of the spinal gray matter that are tightly correlated with modality. However, little is known about how these patterns come about during the ontogeny of the distinct subclasses of skin sensory neurons. To this end, we have developed an intact ex vivo somatosensory system preparation in neonatal mice, allowing single, physiologically identified cutaneous afferents to be iontophoretically injected with Neurobiotin for subsequent histological analyses. The present report, centered on rapidly adapting mechanoreceptors, represents the first study of the central projections of identified skin sensory neurons in neonatal animals. Cutaneous afferents exhibiting rapidly adapting responses to sustained natural stimuli were encountered as early as recordings were made. Well-stained representatives of coarse (tylotrich and guard) and fine-diameter (down) hair follicle afferents, along with a putative Pacinian corpuscle afferent, were recovered from 2-7-day-old neonates. All were characterized by narrow, uninflected somal action potentials and generally low mechanical thresholds, and many could be activated via deflection of recently erupted hairs. The central collaterals of hair follicle afferents formed recurrent, flame-shaped arbors that were essentially miniaturized replicas of their adult counterparts, with identical laminar terminations. The terminal arbors of down hair afferents, previously undescribed in rodents, were distinct and consistently occupied a more superficial position than tylotrich and guard hair afferents. Nevertheless, the former extended no higher than the middle of the incipient substantia gelatinosa, leaving a clear gap more dorsally. In all major respects, therefore, hair follicle afferents display the same laminar specificity in neonates as they do in adults. The widely held misperception that their collaterals extend exuberant projections into pain

  9. Toward Integrated Molecular Diagnostic System (iMDx): Principles and Applications

    PubMed Central

    Park, Seung-min; Sabour, Andrew F.; Son, Jun Ho; Lee, Sang Hun

    2014-01-01

    Integrated molecular diagnostic systems (iMDx), which are automated, sensitive, specific, user-friendly, robust, rapid, easy-to-use, and portable, can revolutionize future medicine. This review will first focus on the components of sample extraction, preservation, and filtration necessary for all point-of-care devices to include for practical use. Subsequently, we will look for low-powered and precise methods for both sample amplification and signal transduction, going in-depth to the details behind their principles. The final field of total device integration and its application to the clinical field will also be addressed to discuss the practicality for future patient care. We envision that microfluidic systems hold the potential to breakthrough the number of problems brought into the field of medical diagnosis today. PMID:24759281

  10. Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice.

    PubMed

    van Putten, Maaike; Hulsker, Margriet; Young, Courtney; Nadarajah, Vishna D; Heemskerk, Hans; van der Weerd, Louise; 't Hoen, Peter A C; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M

    2013-06-01

    Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by the lack of functional dystrophin. There is no cure, but several clinical trials aimed to restore the synthesis of functional dystrophin are underway. The dystrophin levels needed for improvement of muscle pathology, function, and overall vitality are not known. Here, we describe the mdx/utrn(-/-)/Xist(Δhs) mouse model, which expresses a range of low dystrophin levels, depending on the degree of skewing of X inactivation in a utrophin-negative background. Mdx/utrn(-/-) mice develop severe muscle weakness, kyphosis, respiratory and heart failure, and premature death closely resembling DMD pathology. We show that at dystrophin levels < 4%, survival and motor function in these animals are greatly improved. In mice expressing >4% dystrophin, histopathology is ameliorated, as well. These findings suggest that the dystrophin levels needed to benefit vitality and functioning of patients with DMD might be lower than those needed for full protection against muscle damage.

  11. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  12. Spontaneous opening of the acetylcholine receptor channel in developing muscle cells from normal and dystrophic mice

    SciTech Connect

    Franco-Obregon, A.; Lansman, J.B.

    1995-12-31

    Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either 4v/dy or dyl + myotubes. but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes. but not in wild- type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.

  13. Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

    PubMed

    Tjondrokoesoemo, Andoria; Schips, Tobias G; Sargent, Michelle A; Vanhoutte, Davy; Kanisicak, Onur; Prasad, Vikram; Lin, Suh-Chin J; Maillet, Marjorie; Molkentin, Jeffery D

    2016-05-06

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and β-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD.

  14. Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes*

    PubMed Central

    Altamirano, Francisco; López, Jose R.; Henríquez, Carlos; Molinski, Tadeusz; Allen, Paul D.; Jaimovich, Enrique

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca2+]rest) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca2+]rest was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca2+ entry (low Ca2+ solution, Ca2+-free solution, and Gd3+) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca2+]rest. Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca2+]rest was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca2+]rest using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca2+]rest, is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells. PMID:22549782

  15. NAD⁺ repletion improves mitochondrial and stem cell function and enhances life span in mice.

    PubMed

    Zhang, Hongbo; Ryu, Dongryeol; Wu, Yibo; Gariani, Karim; Wang, Xu; Luan, Peiling; D'Amico, Davide; Ropelle, Eduardo R; Lutolf, Matthias P; Aebersold, Ruedi; Schoonjans, Kristina; Menzies, Keir J; Auwerx, Johan

    2016-06-17

    Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD(+)) and its effect on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD(+) precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the mdx (C57BL/10ScSn-Dmd(mdx)/J) mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs and melanocyte SCs and increases mouse life span. Strategies that conserve cellular NAD(+) may reprogram dysfunctional SCs and improve life span in mammals.

  16. The Dietary Polysaccharide Maltodextrin Promotes Salmonella Survival and Mucosal Colonization in Mice

    PubMed Central

    Nickerson, Kourtney P.; Homer, Craig R.; Kessler, Sean P.; Dixon, Laura J.; Kabi, Amrita; Gordon, Ilyssa O.; Johnson, Erin E.; de la Motte, Carol A.; McDonald, Christine

    2014-01-01

    In the latter half of the 20th century, societal and technological changes led to a shift in the composition of the American diet to include a greater proportion of processed, pre-packaged foods high in fat and carbohydrates, and low in dietary fiber (a “Western diet”). Over the same time period, there have been parallel increases in Salmonella gastroenteritis cases and a broad range of chronic inflammatory diseases associated with intestinal dysbiosis. Several polysaccharide food additives are linked to bacterially-driven intestinal inflammation and may contribute to the pathogenic effects of a Western diet. Therefore, we examined the effect of a ubiquitous polysaccharide food additive, maltodextrin (MDX), on clearance of the enteric pathogen Salmonella using both in vitro and in vivo infection models. When examined in vitro, murine bone marrow-derived macrophages exposed to MDX had altered vesicular trafficking, suppressed NAPDH oxidase expression, and reduced recruitment of NADPH oxidase to Salmonella-containing vesicles, which resulted in persistence of Salmonella in enlarged Rab7+ late endosomal vesicles. In vivo, mice consuming MDX-supplemented water had a breakdown of the anti-microbial mucous layer separating gut bacteria from the intestinal epithelium surface. Additionally, oral infection of these mice with Salmonella resulted in increased cecal bacterial loads and enrichment of lamina propria cells harboring large Rab7+ vesicles. These findings indicate that consumption of processed foods containing the polysaccharide MDX contributes to suppression of intestinal anti-microbial defense mechanisms and may be an environmental priming factor for the development of chronic inflammatory disease. PMID:25000398

  17. What has the mdx mouse model of Duchenne muscular dystrophy contributed to our understanding of this disease?

    PubMed

    Manning, Jennifer; O'Malley, Dervla

    2015-04-01

    Duchenne muscular dystrophy (DMD) is a fatal X-chromosome linked recessive disorder caused by the truncation or deletion of the dystrophin gene. The most widely used animal model of this disease is the dystrophin-deficient mdx mouse which was first discovered 30 years ago. Despite its extensive use in DMD research, no effective treatment has yet been developed for this devastating disease. This review explores what we have learned from this mouse model regarding the pathophysiology of DMD and asks if it has a future in providing a better more thorough understanding of this disease or if it will bring us any closer to improving the outlook for DMD patients.

  18. How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse

    PubMed Central

    Godfrey, Caroline; Muses, Sofia; McClorey, Graham; Wells, Kim E.; Coursindel, Thibault; Terry, Rebecca L.; Betts, Corinne; Hammond, Suzan; O'Donovan, Liz; Hildyard, John; El Andaloussi, Samir; Gait, Michael J.; Wood, Matthew J.; Wells, Dominic J.

    2015-01-01

    Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model. PMID:25935000

  19. How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

    PubMed

    Godfrey, Caroline; Muses, Sofia; McClorey, Graham; Wells, Kim E; Coursindel, Thibault; Terry, Rebecca L; Betts, Corinne; Hammond, Suzan; O'Donovan, Liz; Hildyard, John; El Andaloussi, Samir; Gait, Michael J; Wood, Matthew J; Wells, Dominic J

    2015-08-01

    Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.

  20. Comparative Label-Free Mass Spectrometric Analysis of Mildly versus Severely Affected mdx Mouse Skeletal Muscles Identifies Annexin, Lamin, and Vimentin as Universal Dystrophic Markers.

    PubMed

    Holland, Ashling; Henry, Michael; Meleady, Paula; Winkler, Claudia K; Krautwald, Mirjam; Brinkmeier, Heinrich; Ohlendieck, Kay

    2015-06-19

    The primary deficiency in the membrane cytoskeletal protein dystrophin results in complex changes in dystrophic muscles. In order to compare the degree of secondary alterations in differently affected subtypes of skeletal muscles, we have conducted a global analysis of proteome-wide changes in various dystrophin-deficient muscles. In contrast to the highly degenerative mdx diaphragm muscle, which showed considerable alterations in 35 distinct proteins, the spectrum of mildly to moderately dystrophic skeletal muscles, including interosseus, flexor digitorum brevis, soleus, and extensor digitorum longus muscle, exhibited a smaller number of changed proteins. Compensatory mechanisms and/or cellular variances may be responsible for differing secondary changes in individual mdx muscles. Label-free mass spectrometry established altered expression levels for diaphragm proteins associated with contraction, energy metabolism, the cytoskeleton, the extracellular matrix and the cellular stress response. Comparative immunoblotting verified the differences in the degree of secondary changes in dystrophin-deficient muscles and showed that the up-regulation of molecular chaperones, the compensatory increase in proteins of the intermediate filaments, the fibrosis-related increase in collagen levels and the pathophysiological decrease in calcium binding proteins is more pronounced in mdx diaphragm as compared to the less severely affected mdx leg muscles. Annexin, lamin, and vimentin were identified as universal dystrophic markers.

  1. Myostatin inhibition by a follistatin-derived peptide ameliorates the pathophysiology of muscular dystrophy model mice.

    PubMed

    Tsuchida, K

    2008-07-01

    Gene-targeted therapies, such as adeno-associated viral vector (AAV)-mediated gene therapy and cell-mediated therapy using myogenic stem cells, are hopeful molecular strategies for muscular dystrophy. In addition, drug therapies based on the pathophysiology of muscular dystrophy patients are desirable. Multidisciplinary approaches to drug design would offer promising therapeutic strategies. Myostatin, a member of the transforming growth factor-beta superfamily, is predominantly produced by skeletal muscle and negatively regulates the growth and differentiation of cells of the skeletal muscle lineage. Myostatin inhibition would increase the skeletal muscle mass and prevent muscle degeneration, regardless of the type of muscular dystrophy. Myostatin inhibitors include myostatin antibodies, myostatin propeptide, follistatin and follistatin-related protein. Although follistatin possesses potent myostatin-inhibiting activity, it works as an efficient inhibitor of activins. Unlike myostatin, activins regulate the growth and differentiation of nearly all cell types, including cells of the gonads, pituitary gland and skeletal muscle. We have developed a myostatin-specific inhibitor derived from follistatin, designated FS I-I. Transgenic mice expressing this myostatin-inhibiting peptide under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. mdx mice were crossed with FS I-I transgenic mice and any improvement of the pathological signs was investigated. The resulting mdx/FS I-I mice exhibited increased skeletal muscle mass and reduced cell infiltration in muscles. Muscle strength was also recovered in mdx/FS I-I mice. Our data indicate that myostatin inhibition by this follistatin-derived peptide has therapeutic potential for muscular dystrophy.

  2. [Adolescent brain maturation].

    PubMed

    Holzer, L; Halfon, O; Thoua, V

    2011-05-01

    Recent progress in neuroscience has yielded major findings regarding brain maturation during adolescence. Unlike the body, which reaches adult size and morphology during this period, the adolescent brain is still maturing. The prefrontal cortex appears to be an important locus of maturational change subserving executive functions that may regulate emotional and motivational issues. The recent expansion of the adolescent period has increased the lag between the onset of emotional and motivational changes activated by puberty and the completion of cognitive development-the maturation of self-regulatory capacities and skills that are continuing to develop long after puberty has occurred. This "disconnect" predicts risk for a broad set of behavioral and emotional problems. Adolescence is a critical period for high-level cognitive functions such as socialization that rely on maturation of the prefrontal cortex. Intervention during the period of adolescent brain development provides opportunities and requires an interdisciplinary approach.

  3. Myostatin DNA vaccine increases skeletal muscle mass and endurance in mice.

    PubMed

    Tang, Liang; Yan, Zhen; Wan, Yi; Han, Wei; Zhang, Yingqi

    2007-09-01

    Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle growth. In mice, genetic disruption of the myostatin gene leads to a marked increase in body weight and muscle mass. Similarly, pharmacological interference with myostatin in vivo in mdx knockout mice results in a functional improvement of the dystrophic phenotype. Consequently, myostatin is an important therapeutic target for treatment of diseases associated with muscle wasting. To construct a therapeutic DNA vaccine against myostatin, we coupled the foreign, immunodominant T-helper epitope of tetanus toxin to the N terminus of myostatin, and BALB/c mice were immunized with the recombinant vector. Sera from vaccinated mice showed the presence of specific antibodies against the recombinant protein. In addition, body weight, muscle mass, and grip endurance of vaccinated mice were significantly increased. Our study provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting.

  4. Toward Teacher Maturity.

    ERIC Educational Resources Information Center

    Pickle, Judy

    1985-01-01

    The essence of teacher maturity can be synthesized into personal, professional, and process domains. Although overlapping, these categories add a multidimensional approach to the search for what is good in teaching and provide a model for professional development. (MT)

  5. Macromegakaryocytosis after hydroxyurea. [Mice

    SciTech Connect

    Ebbe, S.; Phalen, E.

    1982-11-01

    A single injection of hydroxyurea (OHU) produced transient megakaryocytopenia in mice. An increase in the average mean size of mature, stage III megakaryocytes coincided with their depopulation. This was due to a selective reduction in numbers of smaller cells. In contrast, the macromegakaryocytosis of immunothrombocytopenia showed substantial increases in numbers of larger cells and reductions in smaller. Further reduction in numbers of smaller cells occurred when OHU was given to mice with immunothrombocytopenia, and the megakaryocytopenia was somewhat more severe than that produced by OHU in normal mice. OHU produced mild thrombocytopenia in normal mice and compromised recovery of the platelet count from immunothrombocytopenia. The most likely explanation for the increase in mean megakaryocyte size in the hypomegakaryocytic state produced by OHU is that the temporary imbalance between a low rate of influx and a normal rate of maturation produced a shift of the age distribution of the cells due to a deficiency of immature cells.

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

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

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

  7. Lysosomal protease expression in mature enamel.

    PubMed

    Tye, Coralee E; Lorenz, Rachel L; Bartlett, John D

    2009-01-01

    The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

  8. Anabolic actions of Notch on mature bone

    PubMed Central

    Liu, Peng; Ping, Yilin; Ma, Meng; Zhang, Demao; Liu, Connie; Zaidi, Samir; Gao, Song; Ji, Yaoting; Lou, Feng; Yu, Fanyuan; Lu, Ping; Stachnik, Agnes; Bai, Mingru; Wei, Chengguo; Zhang, Liaoran; Wang, Ke; Chen, Rong; New, Maria I.; Rowe, David W.; Yuen, Tony; Sun, Li; Zaidi, Mone

    2016-01-01

    Notch controls skeletogenesis, but its role in the remodeling of adult bone remains conflicting. In mature mice, the skeleton can become osteopenic or osteosclerotic depending on the time point at which Notch is activated or inactivated. Using adult EGFP reporter mice, we find that Notch expression is localized to osteocytes embedded within bone matrix. Conditional activation of Notch signaling in osteocytes triggers profound bone formation, mainly due to increased mineralization, which rescues both age-associated and ovariectomy-induced bone loss and promotes bone healing following osteotomy. In parallel, mice rendered haploinsufficient in γ-secretase presenilin-1 (Psen1), which inhibits downstream Notch activation, display almost-absent terminal osteoblast differentiation. Consistent with this finding, pharmacologic or genetic disruption of Notch or its ligand Jagged1 inhibits mineralization. We suggest that stimulation of Notch signaling in osteocytes initiates a profound, therapeutically relevant, anabolic response. PMID:27036007

  9. Dystrophin and dysferlin double mutant mice: a novel model for rhabdomyosarcoma.

    PubMed

    Hosur, Vishnu; Kavirayani, Anoop; Riefler, Jennifer; Carney, Lisa M B; Lyons, Bonnie; Gott, Bruce; Cox, Gregory A; Shultz, Leonard D

    2012-05-01

    Although researchers have yet to establish a link between muscular dystrophy (MD) and sarcomas in human patients, literature suggests that the MD genes dystrophin and dysferlin act as tumor suppressor genes in mouse models of MD. For instance, dystrophin-deficient mdx and dysferlin-deficient A/J mice, models of human Duchenne MD and limb-girdle MD type 2B, respectively, develop mixed sarcomas with variable penetrance and latency. To further establish the correlation between MD and sarcoma development, and to test whether a combined deletion of dystrophin and dysferlin exacerbates MD and augments the incidence of sarcomas, we generated dystrophin and dysferlin double mutant mice (STOCK-Dysf(prmd)Dmd(mdx-5Cv)). Not surprisingly, the double mutant mice develop severe MD symptoms and, moreover, develop rhabdomyosarcoma (RMS) at an average age of 12 months, with an incidence of >90%. Histological and immunohistochemical analyses, using a panel of antibodies against skeletal muscle cell proteins, electron microscopy, cytogenetics, and molecular analysis reveal that the double mutant mice develop RMS. The present finding bolsters the correlation between MD and sarcomas, and provides a model not only to examine the cellular origins but also to identify mechanisms and signal transduction pathways triggering development of RMS.

  10. Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2).

    PubMed

    Morales, María Gabriela; Cabrera, Daniel; Céspedes, Carlos; Vio, Carlos P; Vazquez, Yaneisi; Brandan, Enrique; Cabello-Verrugio, Claudio

    2013-07-01

    The renin-angiotensin system (RAS), through angiotensin II and the angiotensin-converting enzyme (ACE), is involved in the genesis and progression of fibrotic diseases characterized by the replacement of normal tissue by an accumulation of an extracellular matrix (ECM). Duchenne muscular dystrophy (DMD) presents fibrosis and a decrease in muscle strength produced by chronic damage. The mdx mouse is a murine model of DMD and develops the same characteristics as dystrophic patients when subjected to chronic exercise. The connective tissue growth factor (CTGF/CCN2) and transforming growth factor type beta (TGF-β), which are overexpressed in muscular dystrophies, play a major role in many progressive scarring conditions. We have tested the hypothesis that ACE inhibition decreases fibrosis in dystrophic skeletal muscle by treatment of mdx mice with the ACE inhibitor enalapril. Both sedentary and exercised mdx mice treated with enalapril showed improvement in gastrocnemius muscle strength explained by a reduction in both muscle damage and ECM accumulation. ACE inhibition decreased CTGF expression in sedentary or exercised mdx mice and diminished CTGF-induced pro-fibrotic activity in a model of CTGF overexpression by adenoviral infection. Enalapril did not have an effect on TGF-β1 expression or its signaling activity in sedentary or exercised dystrophic mice. Thus, ACE inhibition might improve muscle strength and decrease fibrosis by diminishing specifically CTGF expression and activity without affecting TGF-β1 signaling. Our data provide insights into the pathogenic events in dystrophic muscle. We propose ACE as a target for developing therapies for DMD and related diseases.

  11. B cell function in mice transgenic for mCTLA4-H gamma 1: lack of germinal centers correlated with poor affinity maturation and class switching despite normal priming of CD4+ T cells

    PubMed Central

    1994-01-01

    This report outlines the B cell phenotype of transgenic mice that overexpresses the mouse CTLA-4-human gamma 1 (mCTLA4-H gamma 1) protein. Despite the fact that these mice prime CD4+ T cells (Ronchese, F., B. Housemann, S. Hubele, and P. Lane. 1994. J. Exp. Med. 179:809), antibody responses to T-dependent antigens are severely impaired. In contrast, T-independent responses are normal which suggests mCTLA4-H gamma 1 does not act directly on B cells, but acts indirectly by impairing T cell help. The impaired antibody defect is associated with impaired class switching, with low total immunoglobulin (Ig)G and antigen-specific IgG responses, and an absence of germinal center formation in spleen and lymph nodes but not gut-associated tissues. The defective germinal center formation is associated with a reduction in the degree of somatic mutation in hybridomas made from transgenic mice in comparison with those made from normal mice. It seems likely that mCTLA4-H gamma 1 exerts its effect by blocking an interaction between T and B cells that induce T cell help for B cells. PMID:7509361

  12. Effects of macrophage colony-stimulating factor (M-CSF) on the development, differentiation, and maturation of marginal metallophilic macrophages and marginal zone macrophages in the spleen of osteopetrosis (op) mutant mice lacking functional M-CSF activity.

    PubMed

    Takahashi, K; Umeda, S; Shultz, L D; Hayashi, S; Nishikawa, S

    1994-05-01

    Immunohistochemical techniques using an anti-mouse panmacrophage monoclonal antibody and anti-mouse monoclonal antibodies specific for marginal metallophilic macrophages or marginal zone macrophages were used to detect red pulp macrophages, marginal metallophilic macrophages, and marginal zone macrophages in the spleen of op/op mice. In the mutant mice, the red pulp macrophages were reduced to about 60% of those in the normal littermates and the marginal metallophilic macrophages and marginal zone macrophages were absent. After administration of recombinant human macrophage colony-stimulating factor (rhM-CSF), numbers of red pulp macrophages increased rapidly, reaching levels found in normal littermates 1 week later. In contrast, the marginal metallophilic macrophages as well as the marginal zone macrophages appeared slowly after rhM-CSF administration and their numbers were less than half of the baseline level of normal littermates even at 12 weeks of administration. The distribution of marginal metallophilic macrophages and marginal zone macrophages appearing after M-CSF administration was irregular in the spleen of the op/op mice. These splenic macrophage subpopulations differed in their responses to rhM-CSF, suggesting that distinct mechanisms may be involved in their development and differentiation. The splenic red pulp macrophages present in unmanipulated op/op mice are an M-CSF-independent macrophage population. Although the marginal metallophilic macrophages and marginal zone macrophages are thought to be M-CSF-dependent, their development and differentiation appear to be influenced by locally produced M-CSF or other cytokines.

  13. Phagosome maturation: aging gracefully.

    PubMed Central

    Vieira, Otilia V; Botelho, Roberto J; Grinstein, Sergio

    2002-01-01

    Foreign particles and apoptotic bodies are eliminated from the body by phagocytic leucocytes. The initial stage of the elimination process is the internalization of the particles into a plasma membrane-derived vacuole known as the phagosome. Such nascent phagosomes, however, lack the ability to kill pathogens or to degrade the ingested targets. These properties are acquired during the course of phagosomal maturation, a complex sequence of reactions that result in drastic remodelling of the phagosomal membrane and contents. The determinants and consequences of the fusion and fission reactions that underlie phagosomal maturation are the topic of this review. PMID:12061891

  14. Altered ROS production, NF-κB activation and Interleukin-6 gene expression induced by electrical stimulation of in dystrophic mdx skeletal muscle cells

    PubMed Central

    Henríquez-Olguín, Carlos; Altamirano, Francisco; Valladares, Denisse; López, José R.; Allen, Paul D.; Jaimovich, Enrique

    2015-01-01

    Duchenne Muscular Dystrophy (DMD) is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. DMD pathology is associated with an increased production of reactive oxygen and nitrogen species (ROS and RNS). The aim of this work was to study the alterations in NF-κB activation and Interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation (ES) increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by ES. On the other hand, ES induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits; p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50µM H2O2 increased NF-κB activity; after administration of 100 and 200 µM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 µM and 200 µM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of ES in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide (LPS) on both NF-κB activation and IL-6 expression

  15. Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy

    NASA Astrophysics Data System (ADS)

    Dowling, Paul; Culligan, Kevin; Ohlendieck, Kay

    2002-02-01

    Unique unaffected skeletal muscle fibres, unlike necrotic torso and limb muscles, may pave the way for a more detailed understanding of the molecular pathogenesis of inherited neuromuscular disorders and help to develop new treatment strategies for muscular dystrophies. The sparing of extraocular muscle in Duchenne muscular dystrophy is mostly attributed to the special protective properties of extremely fast-twitching small-diameter fibres, but here we show that distal muscles also represent a particular phenotype that is more resistant to necrosis. Immunoblot analysis of membranes isolated from the well established dystrophic animal model mdx shows that, in contrast to dystrophic limb muscles, the toe musculature exhibits an up-regulation of the autosomal dystrophin homologue utrophin and a concomitant rescue of dystrophin-associated glycoproteins. Thus distal mdx muscle groups provide a cellular system that naturally avoids myofibre degeneration which might be useful in the search for naturally occurring compensatory mechanisms in inherited skeletal muscle diseases.

  16. Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells.

    PubMed

    Henríquez-Olguín, Carlos; Altamirano, Francisco; Valladares, Denisse; López, José R; Allen, Paul D; Jaimovich, Enrique

    2015-07-01

    Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 μM H2O2 increased NF-κB activity; after administration of 100 and 200 μM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 μM and 200 μM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF

  17. Brain maturation and epilepsy.

    PubMed

    Dulac, Olivier; Milh, Mathieu; Holmes, Gregory L

    2013-01-01

    At full term, both glutamate and gamma-amino-butyric acid (GABA) are excitatory; cortical synapses are beginning to appear, there is little myelin in the cerebral hemispheres, and long tracts hardly start to develop. Neonatal myoclonic encephalopathy can result from premature activation of N-methyl-D-aspartate (NMDA) transmission. Benign neonatal seizures and migrating partial seizures in infancy could involve excessive or premature excitability of deep cortical layers. Benign rolandic epilepsy and continuous spike waves in slow sleep are consistent with an excess of both excitatory and inhibitory cortical synapses. West and Lennox-Gastaut syndromes express age-related diffuse cortical hyperexcitability, the pattern depending on the age of occurrence; synchronization of spikes is becoming possible with maturation of the myelin. Idiopathic generalized epilepsy is itself modulated by maturation that causes frontal hyperexcitability generating myoclonic-astatic seizures, between the ages of infantile and juvenile myoclonic epilepsies. Physiological delay of hippocampo-neocortical pathways maturation could account for the delayed occurrence of mesial temporal epilepsy following infantile damage, whereas premature maturation could contribute to fronto-temporal damage characteristic of fever-induced epileptic encephalopathy in school-age children, a dramatic school-age epileptic encephalopathy.

  18. Maturation in Larch 1

    PubMed Central

    Greenwood, Michael S.; Hopper, Catherine A.; Hutchison, Keith W.

    1989-01-01

    The time course of maturation in eastern larch (Larix laricina [Du Roi] K. Koch) was examined by grafting scions from trees of different ages onto 2-year-old root stock and following scion development for several years. Height, diameter, foliar chlorophyll content, and rooting ability of scion-derived cuttings all varied linearly as a function of log10 age. Chlorophyll content (milligrams per gram of dry weight) increased while height, diameter, and ability to root decreased with age (P < 0.01). The tendency toward orthotropic growth and branch formation per centimeter of main stem decreased abruptly between age 1 and 5 years (P < 0.01). Total chlorophyll content of both long and short shoot foliage increased by 30 to 50% with increasing age, but the chlorophyll a/b ratio did not change. Also, juvenile long shoot needles were significantly longer than mature (P < 0.01). Surprisingly, the juvenile scions produced more total strobili over two successive years, but the mature scions produced a significantly higher proportion of male strobili (P < 0.001 year 1; P < 0.02 year 2). The age-related changes in foliar traits were not associated with changes in DNA methylation between juvenile and mature scions. Using HPLC, we found that 20% of foliar DNA cytosine residues were methylated in both scion types. Images Figure 1 PMID:16666785

  19. Mature Students Studying Mathematics.

    ERIC Educational Resources Information Center

    Hirst, Keith

    1999-01-01

    Discusses mature students in the single subject area of mathematics in a single institution and makes comparisons with traditional universities. Reviews some features of the age distribution, entry qualifications, degree-class distribution, non-completion rates and gender distribution. (Author/ASK)

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

    PubMed Central

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

    2015-01-01

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

  1. Delayed visual maturation.

    PubMed Central

    Cole, G F; Hungerford, J; Jones, R B

    1984-01-01

    Sixteen blind babies who were considered to be showing the characteristics of delayed visual maturation were studied prospectively. The diagnosis was made on clinical grounds, and the criteria for this are discussed. All of these infants developed visual responses between 4 and 6 months of age and had normal or near normal visual acuities by 1 year of age. Long term follow up, however, has shown neurological abnormalities in some of these children. PMID:6200080

  2. Differentiation of preadipocytes and mature adipocytes requires PSMB8

    PubMed Central

    Arimochi, Hideki; Sasaki, Yuki; Kitamura, Akiko; Yasutomo, Koji

    2016-01-01

    The differentiation of adipocytes is tightly regulated by a variety of intrinsic molecules and also by extrinsic molecules produced by adjacent cells. Dysfunction of adipocyte differentiation causes lipodystrophy, which impairs glucose and lipid homeostasis. Although dysfunction of immunoproteasomes causes partial lipodystrophy, the detailed molecular mechanisms remain to be determined. Here, we demonstrate that Psmb8, a catalytic subunit for immunoproteasomes, directly regulates the differentiation of preadipocytes and additionally the differentiation of preadipocytes to mature adipocytes. Psmb8−/− mice exhibited slower weight gain than wild-type mice, and this was accompanied by reduced adipose tissue volume and smaller size of mature adipocytes compared with controls. Blockade of Psmb8 activity in 3T3-L1 cells disturbed the differentiation to mature adipocytes. Psmb8−/− mice had fewer preadipocyte precursors, fewer preadipocytes and a reduced ability to differentiate preadipocytes toward mature adipocytes. Our data demonstrate that Psmb8-mediated immunoproteasome activity is a direct regulator of the differentiation of preadipocytes and their ultimate maturation. PMID:27225296

  3. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  4. Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.

    PubMed

    Yeung, Davy; Zablocki, Krzysztof; Lien, Chun-Fu; Jiang, Taiwen; Arkle, Stephen; Brutkowski, Wojciech; Brown, James; Lochmuller, Hanns; Simon, Joseph; Barnard, Eric A; Górecki, Dariusz C

    2006-04-01

    Pathological cellular hallmarks of Duchenne muscular dystrophy (DMD) include, among others, abnormal calcium homeostasis. Changes in the expression of specific receptors for extracellular ATP in dystrophic muscle have been recently documented: here, we demonstrate that at the earliest, myoblast stage of developing dystrophic muscle a purinergic dystrophic phenotype arises. In myoblasts of a dystrophin-negative muscle cell line established from the mdx mouse model of DMD but not in normal myoblasts, exposure to extracellular ATP triggered a strong increase in cytoplasmic Ca2+ concentrations. Influx of extracellular Ca2+ was stimulated by ATP and BzATP and inhibited by zinc, Coomassie Brilliant Blue-G, and KN-62, demonstrating activation of P2X7 receptors. Significant expression of P2X4 and P2X7 proteins was immunodetected in dystrophic myoblasts. Therefore, full-length dystrophin appears, surprisingly, to play an important role in myoblasts in controlling responses to ATP. Our results suggest that altered function of P2X receptors may be an important contributor to pathogenic Ca2+ entry in dystrophic mouse muscle and may have implications for the pathogenesis of muscular dystrophies. Treatments aiming at inhibition of specific ATP receptors could be of a potential therapeutic benefit.

  5. Dissociation of motor maturation.

    PubMed

    DiMario, Francis J

    2003-06-01

    We prospectively acquired clinical data regarding the presentation, evaluation, and developmental progress of all patients identified with dissociated motor maturation to define their clinical outcomes. Children (N = 8) referred for evaluation of suspected cerebral palsy because of delayed sitting or walking and identified to have dissociated motor maturation were followed with serial clinical examination. All displayed the characteristic "sitting on air" posture while held in vertical suspension and had otherwise normal developmental assessments. This posture is composed of the hips held in flexion and abduction with the knees extended and feet plantar or dorsiflexed. Three children were initially evaluated at 10 months of age owing to absence of sitting and five other children were evaluated at a mean of 14 months (range 12-19 months) owing to inability to stand. Follow-up evaluations were conducted over a mean of 10.5 months (range 5-34 months). Five children were born prematurely at 34 to 36 weeks gestation. Denver Developmental Screening Test and general and neurologic examinations were normal except to note hypotonia in six children and the "sitting on air" posture in all of the children. Four children have older siblings or parents who "walked late" (after 15 months). On average, the children attained sitting by 8 months (range 7-10 months). One child did not crawl prior to independent walking, two children scooted rather than crawled, and five children crawled at an average of 13.5 months (range 10-16 months). All children cruised by a mean of 18 months (range 16-21.5 months) and attained independent walking by 20.1 months (range 18-25 months). Neuroimaging and serum creatine kinase enzyme testing were normal in two children who were tested. These eight children conform to the syndrome of dissociated motor maturation. The "sitting on air" posture serves as a diagnostic sign and anticipated excellent prognosis, but follow-up is required to ensure a normal

  6. Maturation in Larch 1

    PubMed Central

    Hutchison, Keith W.; Sherman, Christopher D.; Weber, Jill; Smith, Sandra Schiller; Singer, Patricia B.; Greenwood, Michael S.

    1990-01-01

    The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO2 per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS

  7. Parental source effect of inherited mutations in the dystrophin gene of mice and men

    SciTech Connect

    Kress, W.; Grimm, T.; Mueller, C.R.; Bittner, R.

    1994-09-01

    Skewed X-inactivation has been suspected the genetic cause for some manifesting female carriers of BMD and DMD. To test whether a parental source effect on the protein expression of the dystrophin gene exists, we have set up backcrosses of mdx mice to wild type strains, enabling us to study the effect of the well-defined origin of the mutation on the dystrophin expression. In skeletal muscle sections the immunohistological staining patterns of dystrophin antibodies were showing a significant difference in the proportion of dystrophin positive versus negative fibers, suggesting a lower expression of paternally inherited mdx mutations. These data are in concordance with the pyruvate kinase (PK) levels in the serum: PK levels were much higher when the mutation was of maternal origin as compared to PK levels in paternally derived mutations. In order to test this {open_quotes}paternal source effect{close_quotes} in humans, we checked obligatory carriers of Becker muscular dystrophy (BMD) for the origin of their mutations. Creatin kinase (CK) levels in 21 carriers with maternally derived mutations were compared to CK values from 8 heterozygotes with mutations of paternal origin: CK (mat) = 140.3 IU/1 versus CK (pat) = 48.6 IU/I. The difference is statistically significant at the 5% level. These observations suggest either a differential X-inactivation or an imprinting of the dystrophin gene in mice and men.

  8. Interleukin-6 and neuregulin-1 as regulators of utrophin expression via the activation of NRG-1/ErbB signaling pathway in mdx cells.

    PubMed

    Juretić, Nevenka; Díaz, Josefina; Romero, Felipe; González, Gustavo; Jaimovich, Enrique; Riveros, Nora

    2017-03-01

    Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by mutations in the dystrophin gene. A promising therapeutic approach deals with functional substitution of dystrophin by utrophin, a structural homolog that might be able to compensate dystrophin absence in DMD muscle fibers. It has been described that both interleukin-6 (IL-6) and neuregulin-1 (NRG-1; Heregulin-HRG) induce utrophin expression in skeletal muscle. We investigated a possible functional link among IL-6, NRG-1 and utrophin, in normal (C57) and dystrophic (mdx) skeletal muscle cells. Western Blot analysis allowed us to demonstrate that IL-6 (100ng/mL) induces NRG-1 receptor phosphorylation (ErbB2/ErbB3) in both cell types, in a process that depends on intracellular Ca(2+) and metalloproteinase activity; it also induces a transient increase of ERK1 and GABPα phosphorylation only in dystrophic myotubes. Semiquantitative PCR showed that IL-6 treatment increases utrophin mRNA levels just in mdx myotubes. We observed that utrophin mRNA induction was abolished by BAPTA-AM (an intracellular Ca(2+) chelator), GM6001 (a general metalloproteinase inhibitor), genistein (a general protein tyrosine kinase inhibitor), PD-158780 (an ErbB receptor tyrosine kinase inhibitor) and PD-98059 (a MEK inhibitor), whereas Ly-294002 and wortmannin (PI3K inhibitors) did not affect utrophin induction evoked by IL-6 in dystrophic myotubes. Our results suggest that IL-6 induces utrophin expression in mdx myotubes through activation of a NRG-1/ErbBs signaling cascade. Soluble NRG-1 elicited by proteolytic processing of transmembrane NRG-1 might induce ErbBs phosphorylation and ERK1/2 pathway activation, leading to utrophin up-regulation.

  9. CFD - Mature Technology?

    NASA Technical Reports Server (NTRS)

    Kwak, Dochan

    2005-01-01

    Over the past 30 years, numerical methods and simulation tools for fluid dynamic problems have advanced as a new discipline, namely, computational fluid dynamics (CFD). Although a wide spectrum of flow regimes are encountered in many areas of science and engineering, simulation of compressible flow has been the major driver for developing computational algorithms and tools. This is probably due to a large demand for predicting the aerodynamic performance characteristics of flight vehicles, such as commercial, military, and space vehicles. As flow analysis is required to be more accurate and computationally efficient for both commercial and mission-oriented applications (such as those encountered in meteorology, aerospace vehicle development, general fluid engineering and biofluid analysis) CFD tools for engineering become increasingly important for predicting safety, performance and cost. This paper presents the author's perspective on the maturity of CFD, especially from an aerospace engineering point of view.

  10. Mature monocytic cells enter tissues and engraft

    PubMed Central

    Kennedy, David W.; Abkowitz, Janis L.

    1998-01-01

    The goal of this study was to identify the circulating cell that is the immediate precursor of tissue macrophages. ROSA 26 marrow mononuclear cells (containing the β-geo transgene that encodes β-galactosidase and neomycin resistance activities) were cultured in the presence of macrophage colony-stimulating factor and flt3 Ligand for 6 days to generate monocytic cells at all stages of maturation. Expanded monocyte cells (EMC), the immature (ER-MP12+) and more mature (ER-MP20+) subpopulations, were transplanted into irradiated B6/129 F2 mice. β-gal staining of tissue sections from animals 15 min after transplantation demonstrated that the donor cells landed randomly. By 3 h, donor cells in lung and liver were more frequent in animals transplanted with ER-MP20+ (more mature) EMC than in animals transplanted with unseparated EMC or fresh marrow mononuclear cells, a pattern that persisted at 3 and 7 days. At 3 days, donor cells were found in spleen, liver, lung, and brain (rarely) as clusters as well as individual cells. By 7 and 14 days, the clusters had increased in size, and the cells expressed the macrophage antigen F4/80, suggesting that further replication and differentiation had occurred. PCR for the neogene was used to quantitate the amount of donor DNA in tissues from transplanted animals and confirmed that ER-MP20+ EMC preferentially engrafted. These data demonstrate that a mature monocytic cell gives rise to tissue macrophages. Because these cells can be expanded and manipulated in vitro, they may be a suitable target population for gene therapy of lysosomal storage diseases. PMID:9843995

  11. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  12. Deficits in Sialylation Impair Podocyte Maturation

    PubMed Central

    Weinhold, Birgit; Sellmeier, Melanie; Schaper, Wiebke; Blume, Linda; Philippens, Brigitte; Kats, Elina; Bernard, Ulrike; Galuska, Sebastian P.; Geyer, Hildegard; Geyer, Rudolf; Worthmann, Kirstin; Schiffer, Mario; Groos, Stephanie; Gerardy-Schahn, Rita

    2012-01-01

    The role of sialylation in kidney biology is not fully understood. The synthesis of sialoglycoconjugates, which form the outermost structures of animal cells, requires CMP-sialic acid, which is a product of the nuclear enzyme CMAS. We used a knock-in strategy to create a mouse with point mutations in the canonical nuclear localization signal of CMAS, which relocated the enzyme to the cytoplasm of transfected cells without affecting its activity. Although insufficient to prevent nuclear entry in mice, the mutation led to a drastically reduced concentration of nuclear-expressed enzyme. Mice homozygous for the mutation died from kidney failure within 72 hours after birth. The Cmasnls mouse exhibited podocyte foot process effacement, absence of slit diaphragms, and massive proteinuria, recapitulating features of nephrin-knockout mice and of patients with Finnish-type congenital nephrotic syndrome. Although the Cmasnls mouse displayed normal sialylation in all organs including kidney, a critical shortage of CMP-sialic acid prevented sialylation of nephrin and podocalyxin in the maturing podocyte where it is required during the formation of foot processes. Accordingly, the sialylation defects progressed with time and paralleled the morphologic changes. In summary, sialylation is critical during the development of the glomerular filtration barrier and required for the proper function of nephrin. Whether altered sialylation impairs nephrin function in human disease requires further study. PMID:22745475

  13. BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex.

    PubMed

    Huang, Z J; Kirkwood, A; Pizzorusso, T; Porciatti, V; Morales, B; Bear, M F; Maffei, L; Tonegawa, S

    1999-09-17

    Maturation of the visual cortex is influenced by visual experience during an early postnatal period. The factors that regulate such a critical period remain unclear. We examined the maturation and plasticity of the visual cortex in transgenic mice in which the postnatal rise of brain-derived neurotrophic factor (BDNF) was accelerated. In these mice, the maturation of GABAergic innervation and inhibition was accelerated. Furthermore, the age-dependent decline of cortical long-term potentiation induced by white matter stimulation, a form of synaptic plasticity sensitive to cortical inhibition, occurred earlier. Finally, transgenic mice showed a precocious development of visual acuity and an earlier termination of the critical period for ocular dominance plasticity. We propose that BDNF promotes the maturation of cortical inhibition during early postnatal life, thereby regulating the critical period for visual cortical plasticity.

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

    PubMed

    Wuebbles, Ryan D; Sarathy, Apurva; Kornegay, Joe N; Burkin, Dean J

    2013-09-01

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

  15. Career Maturity of Welfare Recipients.

    ERIC Educational Resources Information Center

    Beckman, Carol M.

    To investigate the career maturity of welfare recipients, this thesis examines six independent variables: (1) race; (2) sex; (3) age; (4) level of formal education; (5) general intelligence; and (6) locus of control. Scales taken from the Career Maturity Inventory served as the dependent variables. The sample consisted of 83 welfare recipients who…

  16. Career Education and Career Maturity.

    ERIC Educational Resources Information Center

    Trebilco, Geoffrey R.

    1984-01-01

    Investigated the relationships between career maturity and career curriculum in 38 Melbourne metropolitan secondary schools (N=2280 students) using an Australian adaption of the Career Development Inventory. Results confirmed that schools with career education programs achieved higher gains in student career maturity. (JAC)

  17. Megakaryopoiesis: transcriptional insights into megakaryocyte maturation.

    PubMed

    Kostyak, John Creigh; Naik, Ulhas Pandurang

    2007-01-01

    Platelets are small anucleate cells that travel near the vessel wall during laminar flow. In response to vascular injury, platelets undergo alterations in morphology which allow them to aggregate and cover the injured site. Platelets are produced by megakaryocytes in a process that involves the formation of platelet precursors called proplatelets and subsequent release of these proplatelets into the circulation. By forming a demarcation membrane system within the cytosol, megakaryocytes contain a membrane reservoir which allows for the production of thousands of platelets per mature megakaryocyte. Interestingly, the above process known as megakaryopoiesis is not yet fully understood. However, several groups have contributed evidence to unveil the role of thrombopoietin (TPO), the principal regulator of megakaryopoiesis in vivo. TPO is necessary for megakaryocyte maturation in that TPO deficient mice display greatly reduced megakaryocyte production as well as reduced numbers of mature megakaryocytes. Several transcription factors have also been implicated in megakaryopoiesis including, GATA-1, friend of GATA-1 (FOG-1), nuclear factor-erythroid 2 (NF-E2), and Fli-1. In fact, interactions among some of the transcription factors have been reported to produce synergistic effects. GATA-1 and Fli-1 interactions result in heightened GPIX and GPIb (2 components of von Willebrand Factor (vWF) receptor) expression, while GATA-1, RUNX1 and core-binding factor beta interactions result in improved alphaIIb promoter activity. Mutations in the vWF complex and alphaIIb beta3 have been linked to disorders such as Bernard-Soulier syndrome and Glazmann thrombasthenia respectively. Therefore, a more comprehensive understanding of the transcriptional control of megakaryopoiesis may lead to more effective treatments of platelet-related disorders.

  18. Bone is functionally impaired in dystrophic mice but less so than skeletal muscle

    PubMed Central

    Novotny, Susan A.; Warren, Gordon L.; Lin, Angela S.; Gulderg, Robert E.; Baltgalvis, Kristen A.; Lowe, Dawn A.

    2011-01-01

    The primary purpose of this study was to determine if tibial bone strength is compromised in dystrophic mice and if so, what geometric and material properties contribute. Results of three-point ending tests showed that tibia of mdx and dko (dystrophin- and utrophin-deficient) mice had up to 50% lower strength and stiffness compared to wild-type mice. Micro-computed tomography indicated that dystrophic tibia had reductions of 6–57% in cortical cross-sectional moment of inertia and cross-sectional area. Metaphyseal trabecular bone morphometry was also altered up to 78% in dystrophic mice. Bone-to-muscle functional ratios (i.e., three-point bending measures:muscle strength) indicated that bone strength was relatively high in 7-week-old dystrophic mice compared to muscle strength, but ratios were similar to wild-type mice by 24-months of age. Thus, as in boys with Duchenne muscular dystrophy, young dystrophic mice have compromised bone strength; these models may be useful for designing therapeutic regimens aimed at improving the skeleton. PMID:21256750

  19. Bone is functionally impaired in dystrophic mice but less so than skeletal muscle.

    PubMed

    Novotny, Susan A; Warren, Gordon L; Lin, Angela S; Guldberg, Robert E; Baltgalvis, Kristen A; Lowe, Dawn A

    2011-03-01

    The primary purpose of this study was to determine if tibial bone strength is compromised in dystrophic mice and if so, what geometric and material properties contribute. Results of three-point bending tests showed that tibia of mdx and dko (dystrophin- and utrophin-deficient) mice had up to 50% lower strength and stiffness compared to wild-type mice. Micro-computed tomography indicated that dystrophic tibia had reductions of 6-57% in cortical cross-sectional moment of inertia and cross-sectional area. Metaphyseal trabecular bone morphometry was also altered up to 78% in dystrophic mice. Bone-to-muscle functional ratios (i.e., three-point bending measures:muscle strength) indicated that bone strength was relatively high in 7-week-old dystrophic mice compared to muscle strength, but ratios were similar to wild-type mice by 24 months of age. Young dystrophic mice have compromised bone strength; these models may be useful for designing therapeutic regimens aimed at improving the skeleton.

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

    SciTech Connect

    Bhagavati, Satyakam . E-mail: satyakamb@hotmail.com; Xu Weimin

    2005-07-29

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

  1. In vitro maturation and in vitro fertilization of mouse oocytes and preimplantation embryo culture.

    PubMed

    Kidder, Benjamin L

    2014-01-01

    Epigenetic regulation of gene expression in the germline is important for reproductive success of mammals. Misregulation of genes whose expression is correlated with reproductive success may result in subfertility or infertility. To study epigenetic events that occur during oocyte maturation and preimplantation embryo development, it is important to generate large numbers of ovarian follicles and embryos. Oocyte maturation can be modeled using in vitro maturation (IVM), which is a system of maturing ovarian follicles in a culture dish. In addition, fertilization and early embryogenesis can be modeled using in vitro fertilization (IVF), which involves the fertilization of mature oocytes with capacitated sperm in a culture dish. Here, we describe protocols for in vitro maturation (IVM) and in vitro fertilization (IVF) of mouse oocytes and preimplantation embryo culture. These protocols are suitable for the study of oocyte and embryo biology and the production of embryonic mice.

  2. Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

    PubMed Central

    Zasłona, Zbigniew; Serezani, Carlos H.; Okunishi, Katsuhide; Aronoff, David M.

    2012-01-01

    Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation. PMID:22234697

  3. Cutaneous mast cell maturation does not depend on an intact bone marrow microenvironment

    SciTech Connect

    Charley, M.R.; Mikhael, A.; Sontheimer, R.D.; Gilliam, J.N.; Bennett, M.

    1984-01-01

    A study was made to determine whether the maturation of murine cutaneous mast cells from stem cells depends on an intact bone marrow microenvironment. Normal bone marrow cells (+/+) were infused into 2 groups of mast cell-deficient mice: WBB6F1-W/Wv mice and /sup 89/Sr-pretreated W/Wv mice. /sup 89/Sr is a long-lived bone-seeking radioisotope which provides continuous irradiation of the marrow and thereby ablates the marrow microenvironment. Skin biopsies revealed that the /sup 89/Sr-pretreated mice and the controls had repopulated their skin with mast cells equally well. Natural killer cell function was significantly depressed in the /sup 89/Sr-treated mice, confirming that the marrow microenvironment had been functionally altered. It appears that, although the precursors for cutaneous mast cells are marrow derived, they do not need an intact marrow microenvironment for maturation.

  4. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will...

  5. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the...

  6. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the...

  7. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will...

  8. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the...

  9. Epigenetic maturation in colonic mucosa continues beyond infancy in mice.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monozygotic twin and other epidemiologic studies indicate that epigenetic processes may play an important role in the pathogenesis of inflammatory bowel diseases that commonly affect the colonic mucosa. The peak onset of these disorders in young adulthood, suggests that epigenetic changes normally o...

  10. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  11. Survival of mature T cells depends on signaling through HOIP

    PubMed Central

    Okamura, Kazumi; Kitamura, Akiko; Sasaki, Yoshiteru; Chung, Doo Hyun; Kagami, Shoji; Iwai, Kazuhiro; Yasutomo, Koji

    2016-01-01

    T cell development in the thymus is controlled by a multistep process. The NF-κB pathway regulates T cell development as well as T cell activation at multiple differentiation stages. The linear ubiquitin chain assembly complex (LUBAC) is composed of Sharpin, HOIL-1L and HOIP, and it is crucial for regulating the NF-κB and cell death pathways. However, little is known about the roles of LUBAC in T-cell development and activation. Here, we show that in T-HOIPΔlinear mice lacking the ubiquitin ligase activity of LUBAC, thymic CD4+ or CD8+ T cell numbers were markedly reduced with severe defects in NKT cell development. HOIPΔlinear CD4+ T cells failed to phosphorylate IκBα and JNK through T cell receptor-mediated stimulation. Mature CD4+ and CD8+ T cells in T-HOIPΔlinear mice underwent apoptosis more rapidly than control T cells, and it was accompanied by lower CD127 expression on CD4+CD24low and CD8+CD24low T cells in the thymus. The enforced expression of CD127 in T-HOIPΔlinear thymocytes rescued the development of mature CD8+ T cells. Collectively, our results showed that LUBAC ligase activity is key for the survival of mature T cells, and suggest multiple roles of the NF-κB and cell death pathways in activating or maintaining T cell-mediated adaptive immune responses. PMID:27786304

  12. Adolescent Maturation in Transitioning Cultures.

    ERIC Educational Resources Information Center

    Mulroy, Kevin; Palacios, Anna; Reid, Robert E.

    This is a theoretical study of adolescent maturation within a cultural context. Personality development and disintegration due to the pressure of a dominant culture on a minority culture is considered. An attempt is made to understand how teachers might assist students to work out their psychological growth by story telling. The need for cultural…

  13. Psychosocial Maturity or Social Desirability?

    ERIC Educational Resources Information Center

    Greenberger, Ellen

    The psychosocial maturity scale (PSM) described in several earlier papers is a self-report questionnaire. It is vulnerable, as are other questionnaires of this type, to respondents' wishes to present themselves in a socially desirable light. In this study, scores on two social desirability scales are examined in relation to PSM. Correlations…

  14. Enticing Mature Females into College.

    ERIC Educational Resources Information Center

    Loseth, Lexie; Moreau, Linda

    Following a review of the literature on mature female students, this paper examines enrollment trends in a selection of colleges in Alberta (Canada) and presents the findings of a survey of returning women students at Red Deer College. The literature review highlights factors related to the personal and professional development of women graduates…

  15. Human oocyte maturation in vitro.

    PubMed

    Coticchio, Giovanni; Dal-Canto, Mariabeatrice; Guglielmo, Maria-Cristina; Mignini-Renzini, Mario; Fadini, Rubens

    2012-01-01

    Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

  16. Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity.

    PubMed

    Kraft, David C E; Bindslev, Dorthe A; Melsen, Birte; Abdallah, Basem M; Kassem, Moustapha; Klein-Nulend, Jenneke

    2010-02-01

    For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC-mature exhibited higher osteocalcin and alkaline phosphatase gene expression and activity than PDSC-immature. Pulsating fluid flow (PFF) stimulated nitric oxide production within 5 min by PDSC-mature but not by PDSC-immature. In PDSC-mature, PFF induced prostaglandin E(2) production, and cyclooxygenase 2 gene expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs and produce more bone in vivo. These data suggest that PDSCs with a mature osteogenic phenotype might be preferable for bone tissue engineering to restore, for example, maxillofacial defects, because they might be able to perform mature bone cell-specific functions during bone adaptation to mechanical loading in vivo.

  17. CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation.

    PubMed

    Hertwig, Laura; Hamann, Isabell; Romero-Suarez, Silvina; Millward, Jason M; Pietrek, Rebekka; Chanvillard, Coralie; Stuis, Hanna; Pollok, Karolin; Ransohoff, Richard M; Cardona, Astrid E; Infante-Duarte, Carmen

    2016-08-01

    Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals.

  18. Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

    PubMed Central

    Burr, Adam R.; Millay, Douglas P.; Goonasekera, Sanjeewa A.; Park, Ki Ho; Sargent, Michelle A.; Collins, James; Altamirano, Francisco; Philipson, Kenneth D.; Allen, Paul D.; Ma, Jianjie; López, José Rafael

    2014-01-01

    Unregulated Ca2+ entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na+-Ca2+ exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd−/−), Dysf−/−, and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd−/− mice. Measured increases in baseline Na+ and Ca2+ in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca2+ influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca2+ levels. Indeed, Atp1a2+/− (encoding Na+-K+ ATPase α2) mice, which have reduced Na+ clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na+-K+ ATPase inhibitor digoxin. Treatment of Sgcd−/− mice with ranolazine, a broadly acting Na+ channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology. PMID:24662047

  19. CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

    PubMed

    Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O'Keefe, Regis J

    2016-03-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis.

  20. CCN1 Regulates Chondrocyte Maturation and Cartilage Development

    PubMed Central

    Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna; Shen, Jie; Hilton, Matthew J; Zuscik, Michael J; Jonason, Jennifer H; O’Keefe, Regis J

    2016-01-01

    WNT/β-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of β-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which β-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/β-CATENIN signaling in chondrocytes. Specifically, we found that β-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis. PMID:26363286

  1. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-01-01

    Only a fraction of oocytes present in the ovaries at birth are ever ovulated during the lifetime of a female mammal. In vitro maturation (IVM) offers the possibility to exploit what is a largely untapped biological resource. Although IVM is used routinely for the in vitro production of embryos in domestic species, especially cattle, its clinical use in human-assisted reproduction is still evolving. The successful recapitulation in vitro of the events associated with successful oocyte maturation is not always achieved, with the majority of immature oocytes typically failing to develop to the blastocyst stage. Evidence suggests that although culture conditions throughout in vitro embryo production may have a modest influence on the developmental potential of the early embryo, the quality of the oocyte at the start of the process is the key factor determining the proportion of oocytes developing to the blastocyst stage.

  2. Neuroligin1 drives synaptic and behavioral maturation through intracellular interactions

    PubMed Central

    Hoy, Jennifer L.; Haeger, Paola A.; Constable, John R. L.; Arias, Renee J.; McCallum, Raluca; Kyweriga, Michael; Davis, Lawrence; Schnell, Eric; Wehr, Michael; Castillo, Pablo E.; Washbourne, Philip

    2013-01-01

    In vitro studies suggest that the intracellular C-terminus of Neuroligin1 (NL1) could play a central role in the maturation of excitatory synapses. However, it is unknown how this activity affects synapses in vivo, and whether it may impact the development of complex behaviors. To determine how NL1 influences the state of glutamatergic synapses in vivo, we compared the synaptic and behavioral phenotypes of mice overexpressing a full length version of NL1 (NL1FL) with mice overexpressing a version missing part of the intracellular domain (NL1ΔC). We show that overexpression of full length NL1 yielded an increase in the proportion of synapses with mature characteristics and impaired learning and flexibility. In contrast, the overexpression of NL1ΔC increased the number of excitatory postsynaptic structures and led to enhanced flexibility in mnemonic and social behaviors. Transient overexpression of NL1FL revealed that elevated levels are not necessary to maintain synaptic and behavioral states altered earlier in development. In contrast, overexpression of NL1FL in the fully mature adult was able to impair normal learning behavior after one month of expression. These results provide the first evidence that NL1 significantly impacts key developmental processes that permanently shape circuit function and behavior, as well as the function of fully developed neural circuits. Overall, these manipulations of NL1 function illuminate the significance of NL1 intracellular signaling in vivo, and enhance our understanding of the factors that gate the maturation of glutamatergic synapses and complex behavior. This has significant implications for our ability to address disorders such as ASD. PMID:23719805

  3. Maturation of the adolescent brain

    PubMed Central

    Arain, Mariam; Haque, Maliha; Johal, Lina; Mathur, Puja; Nel, Wynand; Rais, Afsha; Sandhu, Ranbir; Sharma, Sushil

    2013-01-01

    Adolescence is the developmental epoch during which children become adults – intellectually, physically, hormonally, and socially. Adolescence is a tumultuous time, full of changes and transformations. The pubertal transition to adulthood involves both gonadal and behavioral maturation. Magnetic resonance imaging studies have discovered that myelinogenesis, required for proper insulation and efficient neurocybernetics, continues from childhood and the brain’s region-specific neurocircuitry remains structurally and functionally vulnerable to impulsive sex, food, and sleep habits. The maturation of the adolescent brain is also influenced by heredity, environment, and sex hormones (estrogen, progesterone, and testosterone), which play a crucial role in myelination. Furthermore, glutamatergic neurotransmission predominates, whereas gamma-aminobutyric acid neurotransmission remains under construction, and this might be responsible for immature and impulsive behavior and neurobehavioral excitement during adolescent life. The adolescent population is highly vulnerable to driving under the influence of alcohol and social maladjustments due to an immature limbic system and prefrontal cortex. Synaptic plasticity and the release of neurotransmitters may also be influenced by environmental neurotoxins and drugs of abuse including cigarettes, caffeine, and alcohol during adolescence. Adolescents may become involved with offensive crimes, irresponsible behavior, unprotected sex, juvenile courts, or even prison. According to a report by the Centers for Disease Control and Prevention, the major cause of death among the teenage population is due to injury and violence related to sex and substance abuse. Prenatal neglect, cigarette smoking, and alcohol consumption may also significantly impact maturation of the adolescent brain. Pharmacological interventions to regulate adolescent behavior have been attempted with limited success. Since several factors, including age, sex

  4. Maturity model for enterprise interoperability

    NASA Astrophysics Data System (ADS)

    Guédria, Wided; Naudet, Yannick; Chen, David

    2015-01-01

    Historically, progress occurs when entities communicate, share information and together create something that no one individually could do alone. Moving beyond people to machines and systems, interoperability is becoming a key factor of success in all domains. In particular, interoperability has become a challenge for enterprises, to exploit market opportunities, to meet their own objectives of cooperation or simply to survive in a growing competitive world where the networked enterprise is becoming a standard. Within this context, many research works have been conducted over the past few years and enterprise interoperability has become an important area of research, ensuring the competitiveness and growth of European enterprises. Among others, enterprises have to control their interoperability strategy and enhance their ability to interoperate. This is the purpose of the interoperability assessment. Assessing interoperability maturity allows a company to know its strengths and weaknesses in terms of interoperability with its current and potential partners, and to prioritise actions for improvement. The objective of this paper is to define a maturity model for enterprise interoperability that takes into account existing maturity models while extending the coverage of the interoperability domain. The assessment methodology is also presented. Both are demonstrated with a real case study.

  5. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  6. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a stage of growth which will insure a proper completion of...

  7. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  8. Career Maturity: The Construct and its Measurement.

    ERIC Educational Resources Information Center

    Savickas, Mark L.

    1984-01-01

    Describes vocational maturity and assists counselors in identifying what the various career maturity instruments measure. Discusses task variable measures, intervening variable measures, response variable measures, and methods of choosing an instrument. (JAC)

  9. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These orange maturity requirements are contained in the Florida Citrus Code, Chapter...

  10. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These tangerine maturity requirements are contained in the Florida Citrus Code, Chapter...

  11. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These orange maturity requirements are contained in the Florida Citrus Code, Chapter...

  12. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These tangerine maturity requirements are contained in the Florida Citrus Code, Chapter...

  13. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These grapefruit maturity requirements are contained in the Florida Citrus Code, Chapter...

  14. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These grapefruit maturity requirements are contained in the Florida Citrus Code, Chapter...

  15. Career Maturity in High School Age Females.

    ERIC Educational Resources Information Center

    Pedro, Joan Daniels

    1982-01-01

    Examined career maturity in high school females by using a set of general career-maturity and gender-specific, career-related measures, and an alternate career-maturity criterion measure, career-planning involvement. Results indicated significant relationships between achievement orientation and occupational information and knowledge of women's…

  16. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity...

  17. Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy

    PubMed Central

    Kayali, Refik; Ku, Jin-Mo; Khitrov, Gregory; Jung, Michael E.; Prikhodko, Olga; Bertoni, Carmen

    2012-01-01

    Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD. PMID:22692682

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

    PubMed Central

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

    2015-01-01

    In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue. PMID:26067837

  19. [Arrest of maturation in spermatogenesis].

    PubMed

    Francavilla, S; Bellocci, M; Martini, M; Bruno, B; Moscardelli, S; Fabbrini, A; Properzi, G

    1982-07-30

    The ultrastructural aspects of the germinal epithelium of 10 infertile men affected by maturative arrest of spermatogenesis were studied. We noted an increased number of malformed germinal cells. Marginal nuclear vescicles were present in spermatogonia of patients affected by spermatogonial arrest. The few spermatid present in the germinal epithelium of the patients affected by a spermatidic arrest presented changes of the nuclear condensation, the acrosome, and the tail. The Sertoli cells presented an immature aspect of the nucleus and changes of the "mantle". A possible correlation between the Sertoli cells changes and the altered spermatogenesis was proposed.

  20. Phenotype of normal hairline maturation.

    PubMed

    Rassman, William R; Pak, Jae P; Kim, Jino

    2013-08-01

    Hairlines change shape with age, starting at birth. A good head of hair is frequently present some time after ages 3 to 5 years. The look of childhood has its corresponding hairline, and, as the child grows and develops into adulthood, facial morphology migrate changes from a childlike look to a more mature look. This article discusses the dynamics of hairline evolution and the phenotypic variations of the front and side hairlines in men and women. A modeling system is introduced that provides a common language to define the various anatomic points of the full range of hairlines.

  1. TGF-ß Regulates Enamel Mineralization and Maturation through KLK4 Expression

    PubMed Central

    Cho, Andrew; Haruyama, Naoto; Hall, Bradford; Danton, Mary Jo S.; Zhang, Lu; Arany, Praveen; Mooney, David J.; Harichane, Yassine; Goldberg, Michel; Gibson, Carolyn W.; Kulkarni, Ashok B.

    2013-01-01

    Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation. PMID:24278477

  2. Academic Achievement of High School Students in Relation to Their Anxiety, Emotional Maturity and Social Maturity

    ERIC Educational Resources Information Center

    Puar, Surjit Singh

    2013-01-01

    The present study has been designed to investigate the non-cognitive variables like anxiety, emotional maturity and social maturity and their relationship with academic achievement and also to see the locale-wise differences on the basis of their anxiety, emotional maturity and social maturity. The study was conducted over a sample of 400 (200…

  3. The Relationship Between Cognitive Career Maturity and Self-Reported Career Maturity of High School Students.

    ERIC Educational Resources Information Center

    Westbrook, Bert W.; And Others

    1987-01-01

    Investigated relationship between scores on measures of cognitive career maturity and self-reported career maturity in high school sophomores (N=391) and juniors (N=283). Results suggest that there is no relationship between measured career maturity competencies and self-reported career maturity competencies of high school students. (Author/NB)

  4. Sexual maturation of female Saguinus oedipus oedipus

    SciTech Connect

    Tardif, S.D.

    1982-01-01

    This study is an examination of the process of female sexual maturation in the cotton-top tamarin, Saguinus oedipus oedipus, a South-American primate of the family, Callitrichidae. Two types of questions are addressed. The first question is whether the type of social grouping in which a young female lives affects the rate of her sexual maturation. Specifically, is there a difference between the maturation rate of a female housed with a strange adult male and a female housed with her natal group (i.e., her parents and various siblings). Second, the effect of sexual maturation on various social interactions is examined. Specifically are male-female interactions in mated pairs and mother-daughter interactions in natal groups changed by the sexual maturation of the young females. The mother's presence was not related to the daughter's maturation age. However, whether the natal group, as a whole, inhibited maturation, or unrelated males accelerated maturation, or both, remains unknown. Most of the behavioral interactions involving maturing females were unchanged by maturation. There was some indication that certain behaviors were affected by maturation, but only if a strange unrelated male was present.

  5. Neural networks predict tomato maturity stage

    NASA Astrophysics Data System (ADS)

    Hahn, Federico

    1999-03-01

    Almost 40% of the total horticultural produce exported from Mexico the USA is tomato, and quality is fundamental for maintaining the market. Many fruits packed at the green-mature stage do not mature towards a red color as they were harvested before achieving its physiological maturity. Tomato gassed for advancing maturation does not respond on those fruits, and repacking is necessary at terminal markets, causing losses to the producer. Tomato spectral signatures are different on each maturity stage and tomato size was poorly correlated against peak wavelengths. A back-propagation neural network was used to predict tomato maturity using reflectance ratios as inputs. Higher success rates were achieved on tomato maturity stage recognition with neural networks than with discriminant analysis.

  6. Physiological thermoregulation of mature alligators.

    PubMed

    Smith, E N; Standora, E A; Robertson, S L

    1984-01-01

    A 67.1 kg alligator (Alligator mississippiensis), tested in air, heated twice as fast as it cooled. The cooling thermal time constant was 425 min while alive. Warming and cooling thermal time constants were 421 min after death. The thermal time constant was not appropriate in describing warming in air of mature alligators. Surface and subdermal heat flow measurements of the 67.1 kg animal indicate greater blood flow in the skin during warming compared to cooling. Two mature alligators, 49.9 and 103 kg, were heated and cooled in water. Warming time constants were 67 and 110 min respectively. Cooling time constants were 180 and 246 min. Data from this study were combined with previously published thermal time constants for alligators providing regression equations for alligators ranging from 37 g to 103 kg. Regression equations for alligators tested in water are: tau w = 8.81 M050 tau c = 12.6 M0.62. Time constants (tau) are in minutes (w = warming, c = cooling) with all measurements in stirred water; mass, M, is in kg. Thermal conductance and metabolism data are combined to provide an estimate of the amount the body temperature of theoretical alligators ranging from 50 g to 1000 kg would be elevated by metabolism. A body temperature of 34.2 degrees C is predicted for a 1000 kg theoretical alligator in 30 degrees C water.

  7. Membrane remodeling during reticulocyte maturation

    PubMed Central

    Liu, Jing; Guo, Xinhua; Mohandas, Narla; Chasis, Joel A.

    2010-01-01

    The transition of reticulocytes into erythrocytes is accompanied by extensive changes in the structure and properties of the plasma membrane. These changes include an increase in shear resistance, loss of surface area, and acquisition of a biconcave shape. The processes by which these changes are effected have remained largely undefined. Here we examine how the expression of 30 distinct membrane proteins and their interactions change during murine reticulocyte maturation. We show that tubulin and cytosolic actin are lost, whereas the membrane content of myosin, tropomyosin, intercellular adhesion molecule-4, glucose transporter-4, Na-K-ATPase, sodium/hydrogen exchanger 1, glycophorin A, CD47, Duffy, and Kell is reduced. The degradation of tubulin and actin is, at least in part, through the ubiquitin-proteasome degradation pathway. In regard to the protein-protein interactions, the formation of membrane-associated spectrin tetramers from dimers is unperturbed, whereas the interactions responsible for the formation of the membrane-skeletal junctions are weaker in reticulocytes, as is the attachment of transmembrane proteins to these structures. This weakness, in part, results from the elevated phosphorylation of 4.1R in reticulocytes, which leads to a decrease in shear resistance by reducing its interaction with spectrin and actin. These observations begin to unravel the mechanistic basis of crucial changes accompanying reticulocyte maturation. PMID:20038785

  8. Sexual maturation in kokanee Oncorhynchus nerka

    USGS Publications Warehouse

    Patterson, S.D.; Scarnecchia, D.L.; Congleton, J.L.

    2008-01-01

    We used observational and experimental approaches to obtain information on factors affecting the timing of maturation of kokanee Oncorhynchus nerka, a semelparous, landlocked salmon. Gonadal staging criteria were developed and applied to three kokanee populations in Idaho lakes and reservoirs. Testes were classified into three stages: immature (stage one, S1), maturing (S2), and mature (S3). Ovaries were classified into eight stages: immature (S1-S3), transitional (stage S4), maturing (S5-S7), and mature (S8). Males entered the maturing stage (S2) in February through April of the spawning year. Females entered maturing stage (S5) as early as July of the year before the spawning year, and as late as March of the spawning year. Three hatchery experiments demonstrated that attainment of a larger body size 10 to 16 months before spawning increased the likelihood of initiation of maturation in both sexes. No gonads in a state of regression were observed. A gonadosomatic index above 0.1 by early July was a good indicator of a maturing male, and a gonadosomatic index above 1.0 by early July was a good indicator of a maturing female. Instantaneous growth rates were not good predictors of maturation, but attaining a size threshold of 18 to 19 cm in the fall was a good predictor of maturation the following year. This improved knowledge of kokanee maturation will permit more effectively management of the species for age, growth and size at maturity as well as for contributions to fisheries. ?? 2008 by the Northwest Scientific Association. All rights reserved.

  9. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

    PubMed Central

    Shea, Jeremy M.; Boskovic, Ana; Derr, Alan G.; Bing, Xin Y.; Belleannee, Clemence; Kucukural, Alper; Serra, Ryan W.; Sun, Fengyun; Song, Lina; Carone, Benjamin R.; Ricci, Emiliano P.; Li, Xin Z.; Fauquier, Lucas; Moore, Melissa J.; Sullivan, Robert; Mello, Craig C.; Garber, Manuel; Rando, Oliver J.

    2016-01-01

    Several recent studies link parental environments to phenotypes in subsequent generations. Here, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA levels in mature sperm, with decreased let-7 levels and increased levels of 5’ fragments of glycine tRNAs. tRNA fragments are scarce in testicular sperm, but are gained as sperm mature in the epididymis. Epididymosomes – vesicles that fuse with sperm during epididymal transit – carry RNA payloads matching those of mature sperm, and deliver RNAs to immature sperm in vitro. Functionally, tRNA-Gly-GCC fragments repress genes associated with the endogenous retroelement MERVL, both in ES cells and embryos. Our results shed light on small RNA biogenesis, and its dietary regulation, during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo. PMID:26721685

  10. CCL-34, a synthetic toll-like receptor 4 activator, modulates differentiation and maturation of myeloid dendritic cells.

    PubMed

    Fu, Shu-Ling; Lin, Chun-Cheng; Hsu, Ming-Ling; Liu, Sheng-Hung; Huang, Yu-Chuen; Chen, Yu-Jen

    2016-03-08

    CCL-34, a synthetic α-galactosylceramide analog, has been reported as an activator of toll-like receptor 4 (TLR4) in macrophages. TLR4 is highly expressed in dendritic cell (DC) and several TLR4 agonists are known to trigger DC maturation. We herein evaluated the effect of CCL-34 on DC maturation. Human CD14+ monocyte-derived immature DC were treated with CCL-34, its inactive structural analog CCL-44, or LPS to assess the DC maturation. CCL-34 induced DC maturation according to their characteristically dendrite-forming morphology, CD83 expression and IL-12p70 production. The allostimulatory activity of DC on proliferation of naive CD4+CD45+RA+ T cells and their secretion of interferon-γ was increased by CCL-34. Phagocytosis, an important function of immature DC, was reduced after CCL-34 treatment. All these effects related to DC maturation were evidently induced by positive control LPS but not by CCL-44 treatment. TLR4 neutralization impaired human DC maturation triggered by CCL-34. The induction of IL-12, a hallmark of DC maturation, by CCL-34 and LPS was only evident in TLR4-competent C3H/HeN, but not in TLR4-defective C3H/HeJ mice. CCL-34 could further elicit the antigen presentation capability in mice inoculated with doxorubicin-treated colorectal cancer cells. In summary, CCL-34 triggers DC maturation via a TLR4-dependent manner, which supports its potential application as an immunostimulator.

  11. Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.

    PubMed

    Matsumoto, Taro; Kano, Koichiro; Kondo, Daisuke; Fukuda, Noboru; Iribe, Yuji; Tanaka, Nobuaki; Matsubara, Yoshiyuki; Sakuma, Takahiro; Satomi, Aya; Otaki, Munenori; Ryu, Jyunnosuke; Mugishima, Hideo

    2008-04-01

    When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.

  12. Augmentation of CFTR maturation by S-nitrosoglutathione reductase

    PubMed Central

    Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J.; Lewis, Stephen J.

    2015-01-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions. PMID:26637637

  13. Maturation of the MOUTh Intervention

    PubMed Central

    Jablonski-Jaudon, Rita A.; Kolanowski, Ann M.; Winstead, Vicki; Jones-Townsend, Corteza; Azuero, Andres

    2016-01-01

    The purpose of the current article is to describe a personalized practice originally conceived as a way to prevent and minimize care-resistant behavior to provide mouth care to older adult with dementia. The original intervention, Managing Oral Hygiene Using Threat Reduction Strategies (MOUTh), matured during the clinical trial study into a relationship-centered intervention with emphasis on developing strategies that support residents behavioral health and staff involved in care. Relationships that were initially pragmatic (i.e., focused on the task of completing mouth care) developed into more personal and responsive relationships that involved deeper engagement between mouth care providers and nursing home (NH) residents. Mouth care was accomplished and completed in a manner enjoyable to NH residents and mouth care providers. The MOUTh intervention may also concurrently affirm the dignity and personhood of the care recipient because of its emphasis on connecting with older adults. PMID:26934969

  14. Developmental maturation of innate immune cell function correlates with susceptibility to central nervous system autoimmunity.

    PubMed

    Hertzenberg, Deetje; Lehmann-Horn, Klaus; Kinzel, Silke; Husterer, Veronika; Cravens, Petra D; Kieseier, Bernd C; Hemmer, Bernhard; Brück, Wolfgang; Zamvil, Scott S; Stüve, Olaf; Weber, Martin S

    2013-08-01

    MS is an inflammatory CNS disorder, which typically occurs in early adulthood and rarely in children. Here we tested whether functional maturation of innate immune cells may determine susceptibility to CNS autoimmune disease in EAE. Two-week-old mice were resistant to active EAE, which causes fulminant paralysis in adult mice; this resistance was associated with an impaired development of Th1 and Th17 cells. Resistant, young mice had higher frequencies of myeloid-derived suppressor cells and plasma-cytoid DCs. Furthermore, myeloid APCs and B cells from young mice expressed lower levels of MHC class II and CD40, produced decreased amounts of proinflammatory cytokines, and released enhanced levels of anti-inflammatory IL-10. When used as APCs, splenocytes from 2-week-old mice failed to differentiate naive T cells into Th1 and Th17 cells irrespective of the T-cell donor's age, and promoted development of Treg cells and Th2 cells instead. Adoptive transfer of adult APCs restored the ability of 2-week-old mice to generate encephalitogenic T cells and develop EAE. Collectively, these findings indicate that the innate immune compartment functionally matures during development, which may be a prerequisite for development of T-cell-mediated CNS autoimmune disease.

  15. Hyperpolarization-independent maturation and refinement of GABA/glycinergic connections in the auditory brain stem

    PubMed Central

    Lee, Hanmi; Bach, Eva; Noh, Jihyun; Delpire, Eric

    2015-01-01

    During development GABA and glycine synapses are initially excitatory before they gradually become inhibitory. This transition is due to a developmental increase in the activity of neuronal potassium-chloride cotransporter 2 (KCC2), which shifts the chloride equilibrium potential (ECl) to values more negative than the resting membrane potential. While the role of early GABA and glycine depolarizations in neuronal development has become increasingly clear, the role of the transition to hyperpolarization in synapse maturation and circuit refinement has remained an open question. Here we investigated this question by examining the maturation and developmental refinement of GABA/glycinergic and glutamatergic synapses in the lateral superior olive (LSO), a binaural auditory brain stem nucleus, in KCC2-knockdown mice, in which GABA and glycine remain depolarizing. We found that many key events in the development of synaptic inputs to the LSO, such as changes in neurotransmitter phenotype, strengthening and elimination of GABA/glycinergic connection, and maturation of glutamatergic synapses, occur undisturbed in KCC2-knockdown mice compared with wild-type mice. These results indicate that maturation of inhibitory and excitatory synapses in the LSO is independent of the GABA and glycine depolarization-to-hyperpolarization transition. PMID:26655825

  16. Optimizing IV and V for Mature Organizations

    NASA Technical Reports Server (NTRS)

    Fuhman, Christopher

    2003-01-01

    NASA is intending for its future software development agencies to have at least a Level 3 rating in the Carnegie Mellon University Capability Maturity Model (CMM). The CMM has built-in Verification and Validation (V&V) processes that support higher software quality. Independent Verification and Validation (IV&V) of software developed by mature agencies can be therefore more effective than for software developed by less mature organizations. How is Independent V&V different with respect to the maturity of an organization? Knowing a priori the maturity of an organization's processes, how can IV&V planners better identify areas of need choose IV&V activities, etc? The objective of this research is to provide a complementary set of guidelines and criteria to assist the planning of IV&V activities on a project using a priori knowledge of the measurable levels of maturity of the organization developing the software.

  17. Plasma Cholesterol–Induced Lesion Networks Activated before Regression of Early, Mature, and Advanced Atherosclerosis

    PubMed Central

    Björkegren, Johan L. M.; Hägg, Sara; Jain, Rajeev K.; Cedergren, Cecilia; Shang, Ming-Mei; Rossignoli, Aránzazu; Takolander, Rabbe; Melander, Olle; Hamsten, Anders; Michoel, Tom; Skogsberg, Josefin

    2014-01-01

    Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr−/−Apob 100/100 Mttp flox/floxMx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions. PMID:24586211

  18. Plasma cholesterol-induced lesion networks activated before regression of early, mature, and advanced atherosclerosis.

    PubMed

    Björkegren, Johan L M; Hägg, Sara; Talukdar, Husain A; Foroughi Asl, Hassan; Jain, Rajeev K; Cedergren, Cecilia; Shang, Ming-Mei; Rossignoli, Aránzazu; Takolander, Rabbe; Melander, Olle; Hamsten, Anders; Michoel, Tom; Skogsberg, Josefin

    2014-02-01

    Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-)Apob (100/100) Mttp (flox/flox)Mx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.

  19. Skeletal maturation determined by cervical vertebrae development.

    PubMed

    San Román, Paloma; Palma, Juan Carlos; Oteo, M Dolores; Nevado, Esther

    2002-06-01

    The aim of this study was to determine the validity of cervical vertebrae radiographic assessment to predict skeletal maturation. Left hand-wrist and lateral cephalometric radiographs of 958 Spanish children from 5 to 18 years of age were measured. On the left hand-wrist radiographs the classification of Grave and Brown was used to assess skeletal maturation. Cervical vertebrae maturation was evaluated with lateral cephalometric radiographs using the stages described by Lamparski and by Hassel and Farman. A new method to evaluate the cervical maturation by studying the changes in the concavity of the lower border, height, and shape of the vertebral body was created. Correlation coefficients were calculated to establish the relationship between skeletal maturation values obtained by the three classifications of vertebral and skeletal maturation measured at the wrist. All correlation values obtained were statistically significant (P < 0.001). The results suggest that this new method to determine skeletal maturation is very reliable. A simple method based on morphological characteristics of the cervical vertebral bodies to evaluate the maturation stage has been designed. In the population investigated, this method is as accurate as the Hassel and Farman classification and superior to the Lamparski classification. The morphological vertebral parameter best able to estimate the maturation is the concavity of the lower border of the body.

  20. Glutathione Peroxidase 4 Is Required for Maturation of Photoreceptor Cells*

    PubMed Central

    Ueta, Takashi; Inoue, Tatsuya; Furukawa, Takahisa; Tamaki, Yasuhiro; Nakagawa, Yasuhito; Imai, Hirotaka; Yanagi, Yasuo

    2012-01-01

    Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells. PMID:22207760

  1. Smart Grid Interoperability Maturity Model

    SciTech Connect

    Widergren, Steven E.; Levinson, Alex; Mater, J.; Drummond, R.

    2010-04-28

    The integration of automation associated with electricity resources (including transmission and distribution automation and demand-side resources operated by end-users) is key to supporting greater efficiencies and incorporating variable renewable resources and electric vehicles into the power system. The integration problems faced by this community are analogous to those faced in the health industry, emergency services, and other complex communities with many stakeholders. To highlight this issue and encourage communication and the development of a smart grid interoperability community, the GridWise Architecture Council (GWAC) created an Interoperability Context-Setting Framework. This "conceptual model" has been helpful to explain the importance of organizational alignment in addition to technical and informational interface specifications for "smart grid" devices and systems. As a next step to building a community sensitive to interoperability, the GWAC is investigating an interoperability maturity model (IMM) based on work done by others to address similar circumstances. The objective is to create a tool or set of tools that encourages a culture of interoperability in this emerging community. The tools would measure status and progress, analyze gaps, and prioritize efforts to improve the situation.

  2. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of...

  3. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of...

  4. The Mature Woman and the Community College

    ERIC Educational Resources Information Center

    Elliot, Jeffrey M.; Mantz, Concetta M.

    1976-01-01

    The factors and motivations contributing to the presence of increasing numbers of mature women in college are examined, and seven proposals are offered, representing an attempt to develop a total community college program which will meet the needs of mature women students. (NHM)

  5. Quantifying Semantic Linguistic Maturity in Children

    ERIC Educational Resources Information Center

    Hansson, Kristina; Bååth, Rasmus; Löhndorf, Simone; Sahlén, Birgitta; Sikström, Sverker

    2016-01-01

    We propose a method to quantify "semantic linguistic maturity" (SELMA) based on a high dimensional semantic representation of words created from the co-occurrence of words in a large text corpus. The method was applied to oral narratives from 108 children aged 4;0-12;10. By comparing the SELMA measure with maturity ratings made by human…

  6. 7 CFR 29.6026 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.6026 Section 29.6026 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6026 Maturity. The degree of ripeness. (See chart.)...

  7. New definitions for cotton fiber maturity ratio

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton fiber maturity affects fiber physical, mechanical, and chemical properties, as well as the processability and qualities of yarn and fabrics. New definitions of cotton fiber maturity ratio are introduced. The influences of sampling, sample preparation, measurement method, and correlations am...

  8. Thinned Mature Deciduous Forest Silvopastures for Appalachia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little information is available on effective management and utilization of silvopastures developed from the ubiquitous mature woodlots which comprise 40-50% of small Appalachian farm acreage. We thinned a white oak dominated mature second growth forested area establishing two 0.5 ha, eight-paddock,...

  9. Canopy temperature and maturity in cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat units are a widely used indicator of maturity in cotton. It is generally assumed that it takes approximately 2200°F (1222°C) heat units for a cotton plant on the South High Plains of Texas to mature. This value is based on a typical planting date of May 15 with ample irrigation. As water for c...

  10. Motivation and Maturity Patterns in Marital Success.

    ERIC Educational Resources Information Center

    McClelland, David C.; And Others

    1978-01-01

    Married couples rated their marital satisfaction and played interpersonal competitive games which revealed the success with which they interacted. Younger husbands who scored more maturely on the Stewart measure of psychosocial maturity belonged to more successful marriages, as did college-educated wives who showed less immaturity and more phallic…

  11. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... GENERAL INTRODUCTION TO FHA PROGRAMS Requirements for Application, Commitment, and Endorsement Generally... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity... included under the applicable section of the Act. (2) Thirty-five years for existing projects, except...

  12. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... GENERAL INTRODUCTION TO FHA PROGRAMS Requirements for Application, Commitment, and Endorsement Generally... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity... included under the applicable section of the Act. (2) Thirty-five years for existing projects, except...

  13. Toward the Measurement of Psychosocial Maturity.

    ERIC Educational Resources Information Center

    Greenberger, Ellen; And Others

    The concept of psychosocial maturity is reviewed in preparation for the exploration of the feasibility of constructing a scale that measures maturity. Investigation produced a preliminary 54-item scale with high reliability and moderate validity, which is appended. A factor analysis of the scale supports the a priori structure by the theoretical…

  14. The FMI: Dimensions of Follower Maturity

    ERIC Educational Resources Information Center

    Moore, Loren I.

    1976-01-01

    The Follower Maturity Index (FMI) is an instrument derived from leadership theory and based on observations of verbal and nonverbal behavior of followers in task groups. Dimensions of follower maturity--achievement, responsibility, experience, activity, dependence, variety, interests, perspective, position, and awareness--are discussed. For…

  15. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS GRAINS AND SIMILARLY HANDLED COMMODITIES-MARKETING ASSISTANCE LOANS AND LOAN DEFICIENCY PAYMENTS FOR 2008 THROUGH 2012 Marketing Assistance Loans § 1421.101 Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than...

  16. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS GRAINS AND SIMILARLY HANDLED COMMODITIES-MARKETING ASSISTANCE LOANS AND LOAN DEFICIENCY PAYMENTS FOR 2008 THROUGH 2012 Marketing Assistance Loans § 1421.101 Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than...

  17. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS GRAINS AND SIMILARLY HANDLED COMMODITIES-MARKETING ASSISTANCE LOANS AND LOAN DEFICIENCY PAYMENTS FOR 2008 THROUGH 2012 Marketing Assistance Loans § 1421.101 Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than...

  18. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS GRAINS AND SIMILARLY HANDLED COMMODITIES-MARKETING ASSISTANCE LOANS AND LOAN DEFICIENCY PAYMENTS FOR 2008 THROUGH 2012 Marketing Assistance Loans § 1421.101 Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than...

  19. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  20. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  1. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  2. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  3. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946... Standards for Cleaned Virginia Type Peanuts in the Shell Definitions § 51.1238 Mature. Mature means that...

  4. Targeted Disruption of Nrg1 in Granulosa Cells Alters the Temporal Progression of Oocyte Maturation

    PubMed Central

    Kawashima, Ikko; Umehara, Takashi; Noma, Noritaka; Kawai, Tomoko; Shitanaka, Manami

    2014-01-01

    Neuregulin 1 (NRG1) is induced in granulosa cells by LH and acts on granulosa and cumulus cells during ovulation. In this study, we sought to determine the role of NRG1 in oocyte maturation by generating a granulosa cell–specific Nrg1 knockout mouse (Nrg1flox/flox;Cyp19a1Cre mice [gcNrg1KO]). In the gcNrg1KO mice, meiosis was induced 2 hours earlier than in control mice. More than 60% of the oocytes in the mutant mice spontaneously re-resumed meiosis beyond the MII stage. The percentage of successful fertilization was comparable in oocytes of both genotypes collected at 14 or 16 hours after human chorionic gonadotropin injection but was significantly lower in oocytes of the gcNrg1KO mice at 18 or 20 hours. The number of pups per litter was significantly decreased in gcNrg1KO mice. To determine the molecular events associated with the abnormal progression of meiosis in the gcNrg1KO mouse oocytes, the defects of cumulus/granulosa cell functions were analyzed. The expression of genes involved in luteinization and cumulus expansion was significantly higher at 2 hours after human chorionic gonadotropin injection in the gcNrg1KO mice; this was related to abnormal activation of protein kinase C (PKC) and phosphorylation of connexin-43 in cumulus cells. Changes in connexin-43 by PKC might lead to early meiotic resumption of oocytes in gcNrg1KO mice. We conclude that NRG1 is induced by LH in mural granulosa cells and exerts an important regulatory role in oocyte meiotic maturation and competence by reducing PKC activation in cumulus cells and preventing premature progression to the MII stage that leads to abnormal fertilization and fertility. PMID:24650175

  5. Transcriptional coactivators PGC-1α and PGC-lβ control overlapping programs required for perinatal maturation of the heart

    PubMed Central

    Lai, Ling; Leone, Teresa C.; Zechner, Christoph; Schaeffer, Paul J.; Kelly, Sean M.; Flanagan, Daniel P.; Medeiros, Denis M.; Kovacs, Attila; Kelly, Daniel P.

    2008-01-01

    Oxidative tissues such as heart undergo a dramatic perinatal mitochondrial biogenesis to meet the high-energy demands after birth. PPARγ coactivator-1 (PGC-1) α and β have been implicated in the transcriptional control of cellular energy metabolism. Mice with combined deficiency of PGC-1α and PGC-1β (PGC-1αβ−/− mice) were generated to investigate the convergence of their functions in vivo. The phenotype of PGC-1β−/− mice was minimal under nonstressed conditions, including normal heart function, similar to that of PGC-1α−/− mice generated previously. In striking contrast to the singly deficient PGC-1 lines, PGC-1αβ−/− mice died shortly after birth with small hearts, bradycardia, intermittent heart block, and a markedly reduced cardiac output. Cardiac-specific ablation of the PGC-1β gene on a PGC-1α-deficient background phenocopied the generalized PGC-1αβ−/− mice. The hearts of the PGC-1αβ−/− mice exhibited signatures of a maturational defect including reduced growth, a late fetal arrest in mitochondrial biogenesis, and persistence of a fetal pattern of gene expression. Brown adipose tissue (BAT) of PGC-1αβ−/− mice also exhibited a severe abnormality in function and mitochondrial density. We conclude that PGC-1α and PGC-1β share roles that collectively are necessary for the postnatal metabolic and functional maturation