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Sample records for mature myostatin protein

  1. Testosterone and trenbolone enanthate increase mature myostatin protein expression despite increasing skeletal muscle hypertrophy and satellite cell number in rodent muscle.

    PubMed

    Dalbo, V J; Roberts, M D; Mobley, C B; Ballmann, C; Kephart, W C; Fox, C D; Santucci, V A; Conover, C F; Beggs, L A; Balaez, A; Hoerr, F J; Yarrow, J F; Borst, S E; Beck, D T

    2017-04-01

    The androgen-induced alterations in adult rodent skeletal muscle fibre cross-sectional area (fCSA), satellite cell content and myostatin (Mstn) were examined in 10-month-old Fisher 344 rats (n = 41) assigned to Sham surgery, orchiectomy (ORX), ORX + testosterone (TEST; 7.0 mg week(-1) ) or ORX + trenbolone (TREN; 1.0 mg week(-1) ). After 29 days, animals were euthanised and the levator ani/bulbocavernosus (LABC) muscle complex was harvested for analyses. LABC muscle fCSA was 102% and 94% higher in ORX + TEST and ORX + TREN compared to ORX (p < .001). ORX + TEST and ORX + TREN increased satellite cell numbers by 181% and 178% compared to ORX, respectively (p < .01), with no differences between conditions for myonuclear number per muscle fibre (p = .948). Mstn protein was increased 159% and 169% in the ORX + TEST and ORX + TREN compared to ORX (p < .01). pan-SMAD2/3 protein was ~30-50% greater in ORX compared to SHAM (p = .006), ORX + TEST (p = .037) and ORX + TREN (p = .043), although there were no between-treatment effects regarding phosphorylated SMAD2/3. Mstn, ActrIIb and Mighty mRNAs were lower in ORX, ORX + TEST and ORX + TREN compared to SHAM (p < .05). Testosterone and trenbolone administration increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Several genes and signalling proteins related to myostatin signalling were differentially regulated by ORX or androgen therapy.

  2. Placental expression of myostatin and follistatin-like-3 protein in a model of developmental programming.

    PubMed

    Peiris, Hassendrini N; Ponnampalam, Anna P; Osepchook, Claire C; Mitchell, Murray D; Green, Mark P

    2010-04-01

    Maternal undernutrition during gestation is known to be detrimental to fetal development, leading to a propensity for metabolic disorders later in the adult lives of the offspring. Identifying possible mediators and physiological processes involved in modulating nutrient transport within the placenta is essential to prevent and/or develop treatments for the effects of aberrant nutrition, nutrient transfer, and detrimental changes to fetal development. A potential role for myostatin as a mediator of nutrient uptake and transport from the mother to the fetus was shown through the recent finding that myostatin acts within the human placenta to modulate glucose uptake and therefore homeostasis. The mRNA and protein expression of myostatin and its inhibitor, follistatin-like-3 (FSTL3), was studied in the placenta and skeletal muscle of a transgenerational Wistar rat model of gestational maternal undernutrition in which the F2 offspring postweaning consumed a high-fat (HF) diet. Alterations in placental characteristics and offspring phenotype, specifically glucose homeostasis, were evident in the transgenerationally undernourished (UNAD) group. Myostatin and FSTL3 protein expression were also higher (P < 0.05) in the placentae of the UNAD compared with the control group. At maturity, UNAD HF-fed animals had higher (P < 0.05) skeletal muscle expression of FSTL3 than control animals. In summary, maternal undernutrition during gestation results in the aberrant regulation of myostatin and FSTL3 in the placenta and skeletal muscle of subsequent generations. Myostatin, through the disruption of maternal nutrient supply to the fetus, may thus be a potential mediator of offspring phenotype.

  3. Laminin binds to myostatin and attenuates its signaling.

    PubMed

    Yasaka, Naofumi; Suzuki, Keisuke; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Nishimura, Takanori

    2013-09-01

    Myostatin is a growth and differentiation factor and acts as a negative regulator of skeletal muscle mass. Although the mechanism whereby myostatin controls muscle cell growth is mostly clarified, the regulation of myostatin activity after its secretion into the extracellular matrix (ECM) is still unclear. In the present study, we investigated the interaction between laminin and myostatin and the effect of laminin on myostatin signaling in vitro. The surface plasmon resonance assay showed that laminin bound to mature myostatin and activin receptor type IIB (ActRIIB), but did not bind to latency-associated protein, which remains non-covalently linked to mature myostatin. Furthermore, kinetic analysis demonstrated that the affinity of mature myostatin for laminin was similar to that for ActRIIB. Next, we examined the action of laminin on the myostatin signaling pathway using a conventional reporter assay. The luciferase activity of myostatin-treated cells was repressed significantly (P < 0.05) by coincubation of laminin. These results suggest that laminin has a potential to regulate myostatin activity through binding to mature myostatin and/or its receptor ActRIIB.

  4. Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ.

    PubMed

    Lamar, Kay-Marie; Bogdanovich, Sasha; Gardner, Brandon B; Gao, Quan Q; Miller, Tamari; Earley, Judy U; Hadhazy, Michele; Vo, Andy H; Wren, Lisa; Molkentin, Jeffery D; McNally, Elizabeth M

    2016-05-01

    Latent TGFβ binding proteins (LTBPs) regulate the extracellular availability of latent TGFβ. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFβ family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFβ and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFβ family ligand binding protein with the capacity to modify muscle disease through overexpression.

  5. Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

    PubMed Central

    Gardner, Brandon B.; Gao, Quan Q.; Hadhazy, Michele; Vo, Andy H.; Wren, Lisa; Molkentin, Jeffery D.; McNally, Elizabeth M.

    2016-01-01

    Latent TGFβ binding proteins (LTBPs) regulate the extracellular availability of latent TGFβ. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFβ family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFβ and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFβ family ligand binding protein with the capacity to modify muscle disease through overexpression. PMID:27148972

  6. Myostatin Regulates Tissue Potency and Cardiac Calcium-Handling Proteins

    PubMed Central

    Jackson, Melissa F.; Li, Naisi

    2014-01-01

    Attenuating myostatin enhances striated muscle growth, reduces adiposity, and improves cardiac contractility. To determine whether myostatin influences tissue potency in a manner that could control such pleiotropic actions, we generated label-retaining mice with wild-type and mstn−/− (Jekyll) backgrounds in which slow-cycling stem, transit-amplifying, and progenitor cells are preferentially labeled by histone 2B/green fluorescent protein. Jekyll mice were born with fewer label-retaining cells (LRCs) in muscle and heart, consistent with increased stem/progenitor cell contributions to embryonic growth of both tissues. Cardiac LRC recruitment from noncardiac sources occurred in both groups, but lasted longer in Jekyll hearts, whereas heightened β-adrenergic sensitivity of mstn−/− hearts was explained by elevated SERCA2a, phospholamban, and β2-adrenergic receptor levels. Jekyll mice were also born with more adipose LRCs despite significantly smaller tissue weights. Reduced adiposity in mstn−/− animals is therefore due to reduced lipid deposition as adipoprogenitor pools appear to be enhanced. By contrast, increased bone densities of mstn−/− mice are likely compensatory to hypermuscularity because LRC counts were similar in Jekyll and wild-type tibia. Myostatin therefore significantly influences the potency of different tissues, not just muscle, as well as cardiac Ca2+-handling proteins. Thus, the pleiotropic phenotype of mstn−/− animals may not be due to enhanced muscle development per se, but also to altered stem/progenitor cell pools that ultimately influence tissue potency. PMID:24517228

  7. Method Development for the Detection of Human Myostatin by High-Resolution and Targeted Mass Spectrometry.

    PubMed

    Peiris, Hassendrini Nileishika; Ashman, Keith; Vaswani, Kanchan; Kvaskoff, David; Rice, Gregory Edward; Mitchell, Murray David

    2014-06-30

    Myostatin, a highly conserved secretory protein, negatively regulates muscle development, affecting both the proliferation and differentiation of muscle cells. Proteolytic processing of the myostatin precursor protein generates a myostatin pro-peptide and mature protein. Dimerization of the mature myostatin protein creates the active form of myostatin. Myostatin dimer activity can be inhibited by noncovalent binding of two monomeric myostatin pro-peptides. This ability for myostatin to self-regulate as well as the altered expression of myostatin in states of abnormal health (e.g., muscle wasting) support the need for specific detection of myostatin forms. Current protein detection methods (e.g., Western blot) rely greatly on antibodies and are semiquantitative at best. Tandem mass spectometry (as in this study) provides a highly specific method of detection, enabling the characterization of myostatin protein forms through the analysis of discrete peptides fragments. Utilizing the scheduled high-resolution multiple reaction monitoring paradigm (sMRM(HR); AB SCIEX 5600 TripleTOF) we identified the lower limit of quantitation (LLOQ) of both mature (DFGLDCDEHSTESR) and pro-peptide regions (ELIDQYDVQR) as 0.19 nmol/L. Furthermore, scheduled multiple reaction monitoring (sMRM; AB SCIEX QTRAP 5500) identified a LLOQ for a peptide of the pro-peptide region (LETAPNISK) as 0.16 nmol/L and a peptide of the mature region (EQIIYGK) as 0.25 nmol/L.

  8. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.

    PubMed

    Taylor, W E; Bhasin, S; Artaza, J; Byhower, F; Azam, M; Willard, D H; Kull, F C; Gonzalez-Cadavid, N

    2001-02-01

    Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

  9. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

    SciTech Connect

    Benny Klimek, Margaret E.; Aydogdu, Tufan; Link, Majik J.; Pons, Marianne; Koniaris, Leonidas G.; Zimmers, Teresa A.

    2010-01-15

    Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

  10. Decorin binds myostatin and modulates its activity to muscle cells

    SciTech Connect

    Miura, Takayuki; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito; Hennebry, Alex; Berry, Carole J.; Sharma, Mridula; Kambadur, Ravi; Nishimura, Takanori . E-mail: nishi@anim.agr.hokudai.ac.jp

    2006-02-10

    Myostatin, a member of TGF-{beta} superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-{beta} and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn{sup 2+} greater than 10 {mu}M, but not in the absence of Zn{sup 2+}. Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K {sub D}) of 2.02 x 10{sup -8} M and 9.36 x 10{sup -9} M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM.

  11. [Altered expression of myostatin gene in the progressive muscular dystrophy patients].

    PubMed

    Zhang, Yong; Chen, Yan; Chen, Jia-Wei; Zhu, Da-Hai

    2005-08-01

    Progressive muscular dystrophy is a group of inherited disorders characterized by progressive skeletal muscle wasting and weakness, which is not of neurogenic origin. Myostatin, a new member of the TGF-beta super-family, is a negative regulator of skeletal muscle growth. To investigate the possible involvement of myostatin in the development of progressive muscular dystrophy, we cloned and sequenced myostatin cDNAs from the progressive muscular dystrophy patients by RT-PCR. Levels of myostatin mRNA and protein in the patients were analyzed by semi-quantitative RT-PCR and Western blot,respectively. We did not find any mutations in the myostatin cDNA sequences from the progressive muscular dystrophy patients in this study. However, we found that the levels of myostatin transcripts were reduced in some patients and the processing and maturation of myostatin protein were inhibited in some patients. Our data demonstrated that the pathogenesis of some types or subtypes of progressive muscular dystrophy is probably associated with the altered myostatin expression and the processing inhibition of myostatin protein.

  12. Myostatin dysfunction impairs force generation in extensor digitorum longus muscle and increases exercise-induced protein efflux from extensor digitorum longus and soleus muscles.

    PubMed

    Baltusnikas, Juozas; Kilikevicius, Audrius; Venckunas, Tomas; Fokin, Andrej; Bünger, Lutz; Lionikas, Arimantas; Ratkevicius, Aivaras

    2015-08-01

    Myostatin dysfunction promotes muscle hypertrophy, which can complicate assessment of muscle properties. We examined force generating capacity and creatine kinase (CK) efflux from skeletal muscles of young mice before they reach adult body and muscle size. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of Berlin high (BEH) mice with dysfunctional myostatin, i.e., homozygous for inactivating myostatin mutation, and with a wild-type myostatin (BEH+/+) were studied. The muscles of BEH mice showed faster (P < 0.01) twitch and tetanus contraction times compared with BEH+/+ mice, but only EDL displayed lower (P < 0.05) specific force. SOL and EDL of age-matched but not younger BEH mice showed greater exercise-induced CK efflux compared with BEH+/+ mice. In summary, myostatin dysfunction leads to impairment in muscle force generating capacity in EDL and increases susceptibility of SOL and EDL to protein loss after exercise.

  13. Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle.

    PubMed

    Petersen, Anne Marie Winther; Magkos, Faidon; Atherton, Philip; Selby, Anna; Smith, Kenneth; Rennie, Michael J; Pedersen, Bente Klarlund; Mittendorfer, Bettina

    2007-09-01

    Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.

  14. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  15. Proteolytic processing of myostatin is auto-regulated during myogenesis.

    PubMed

    McFarlane, Craig; Langley, Brett; Thomas, Mark; Hennebry, Alex; Plummer, Erin; Nicholas, Gina; McMahon, Chris; Sharma, Mridula; Kambadur, Ravi

    2005-07-01

    Myostatin, a potent negative regulator of myogenesis, is proteolytically processed by furin proteases into active mature myostatin before secretion from myoblasts. Here, we show that mature myostatin auto-regulates its processing during myogenesis. In a cell culture model of myogenesis, Northern blot analysis revealed no appreciable change in myostatin mRNA levels between proliferating myoblasts and differentiated myotubes. However, Western blot analysis confirmed a relative reduction in myostatin processing and secretion by differentiated myotubes as compared to proliferating myoblasts. Furthermore, in vivo results demonstrate a lower level of myostatin processing during fetal muscle development when compared to postnatal adult muscle. Consequently, high levels of circulatory mature myostatin were detected in postnatal serum, while fetal circulatory myostatin levels were undetectable. Since Furin proteases are important for proteolytically processing members of the TGF-beta superfamily, we therefore investigated the ability of myostatin to control the transcription of furin and auto-regulate the extent of its processing. Transfection experiments indicate that mature myostatin indeed regulates furin protease promoter activity. Based on these results, we propose a mechanism whereby myostatin negatively regulates its proteolytic processing during fetal development, ultimately facilitating the differentiation of myoblasts by controlling both furin protease gene expression and subsequent active concentrations of mature myostatin peptide.

  16. Myostatin signaling is up-regulated in female patients with advanced heart failure.

    PubMed

    Ishida, Junichi; Konishi, Masaaki; Saitoh, Masakazu; Anker, Markus; Anker, Stefan D; Springer, Jochen

    2017-07-01

    Myostatin, a negative regulator of skeletal muscle mass, is up-regulated in the myocardium of heart failure (HF) and increased myostatin is associated with weight loss in animal models with HF. Although there are disparities in pathophysiology and epidemiology between male and female patients with HF, it remains unclear whether there is gender difference in myostatin expression and whether it is associated with weight loss in HF patients. Heart tissue samples were collected from patients with advanced heart failure (n=31, female n=5) as well as healthy control donors (n=14, female n=6). Expression levels of myostatin and its related proteins in the heart were evaluated by western blotting analysis. Body mass index was significantly lower in female HF patients than in male counterparts (20.0±4.2 in female vs 25.2±3.8 in male, p=0.04). In female HF patients, both mature myostatin and pSmad2 were significantly up-regulated by 1.9 fold (p=0.05) and 2.5 fold (p<0.01) respectively compared to female donors, while expression of pSmad2 was increased by 2.8 times in male HF patients compared to male healthy subjects, but that of myostatin was not. There was no significant difference in protein expression related to myostatin signaling between male and female patients. In this study, myostatin and pSmad2 were significantly up-regulated in the failing heart of female patients, but not male patients, and female patients displayed lower body mass index. Enhanced myostatin signaling in female failing heart may causally contribute to pathogenesis of HF and cardiac cachexia. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Myostatin protein and RNA transcript levels in adult and developing brook trout.

    PubMed

    Roberts, Steven B; Goetz, Frederick W

    2003-11-28

    Quantitative real-time RT-PCR and Western analysis were used to measure RNA expression of the two brook trout myostatin (MSTN) genes ("ovarian", ov and "brain/muscle", b/m), and levels of MSTN immunoreactive protein (MIP) in developing embryos and muscle of brook trout adults. In developing brook trout embryos, ov and b/m MSTN RNAs and MIP significantly increased 45 days post-fertilization. In adult brook trout, the b/m MSTN form was expressed at higher levels in red versus white muscle regardless of gender or time of year. While few changes were observed in MSTN transcripts in fish sampled throughout the year, a significant increase in the processed 14 kDa MIP was observed at spawning in a tissue specific manner, and differences were observed between males and females. These data, along with promoter sequence analysis of the of b/m and ov genes, support a role for MSTN in muscle growth and development in fish.

  18. Endoplasmic reticulum stress induces myostatin precursor protein and NF-kappaB in cultured human muscle fibers: relevance to inclusion body myositis.

    PubMed

    Nogalska, Anna; Wojcik, Slawomir; Engel, W King; McFerrin, Janis; Askanas, Valerie

    2007-04-01

    Sporadic-inclusion body myositis (s-IBM) is the most common progressive muscle disease of older persons. It leads to pronounced muscle fiber atrophy and weakness, and there is no successful treatment. We have previously shown that myostatin precursor protein (MstnPP) and myostatin (Mstn) dimer are increased in biopsied s-IBM muscle fibers, and proposed that MstnPP/Mstn increase may contribute to muscle fiber atrophy and weakness in s-IBM patients. Mstn is known to be a negative regulator of muscle fiber mass. It is synthesized as MstnPP, which undergoes posttranslational processing in the muscle fiber to produce mature, active Mstn. To explore possible mechanisms involved in Mstn abnormalities in s-IBM, in the present study we utilized primary cultures of normal human muscle fibers and experimentally modified the intracellular micro-environment to induce endoplasmic-reticulum (ER)-stress, thereby mimicking an important aspect of the s-IBM muscle fiber milieu. ER stress was induced by treating well-differentiated cultured muscle fibers with either tunicamycin or thapsigargin, both well-established ER stress inducers. Our results indicate for the first time that the ER stress significantly increased MstnPP mRNA and protein. The results also suggest that in our system ER stress activates NF-kappaB, and we suggest that MstnPP increase occurred through the ER-stress-activated NF-kappaB. We therefore propose a novel mechanism leading to the Mstn increase in s-IBM. Accordingly, interfering with pathways inducing ER stress, NF-kappaB activation or its action on the MstnPP gene promoter might prevent Mstn increase and provide a new therapeutic approach for s-IBM and, possibly, for muscle atrophy in other neuromuscular diseases.

  19. Myostatin: expanding horizons.

    PubMed

    Sharma, Mridula; McFarlane, Craig; Kambadur, Ravi; Kukreti, Himani; Bonala, Sabeera; Srinivasan, Shruti

    2015-08-01

    Myostatin is a secreted growth and differentiation factor that belongs to the TGF-β superfamily. Myostatin is predominantly synthesized and expressed in skeletal muscle and thus exerts a huge impact on muscle growth and function. In keeping with its negative role in myogenesis, myostatin expression is tightly regulated at several levels including epigenetic, transcriptional, post-transcriptional, and post-translational. New revelations regarding myostatin regulation also offer mechanisms that could be exploited for developing myostatin antagonists. Increasingly, it is becoming clearer that besides its conventional role in muscle, myostatin plays a critical role in metabolism. Hence, molecular mechanisms by which myostatin regulates several key metabolic processes need to be further explored.

  20. Analysis of myostatin gene structure, expression and function in zebrafish.

    PubMed

    Xu, Cheng; Wu, Gang; Zohar, Yonathan; Du, Shao-Jun

    2003-11-01

    Myostatin is a member of the TGF-beta family that functions as a negative regulator of skeletal muscle development and growth in mammals. Recently, Myostatin has also been identified in fish; however, its role in fish muscle development and growth remains unknown. We have reported here the isolation and characterization of myostatin genomic gene from zebrafish and analysis of its expression in zebrafish embryos, larvae and adult skeletal muscles. Our data showed that myostatin was weakly expressed in early stage zebrafish embryos, and strongly expressed in swimming larvae, juvenile and skeletal muscles of adult zebrafish. Transient expression analysis revealed that the 1.2 kb zebrafish myostatin 5' flanking sequence could direct green fluorescent protein (GFP) expression predominantly in muscle cells, suggesting that the myostatin 5' flanking sequence contained regulatory elements required for muscle expression. To determine the biological function of Myostatin in fish, we generated a transgenic line that overexpresses the Myostatin prodomain in zebrafish skeletal muscles using a muscle-specific promoter. The Myostatin prodomain could act as a dominant negative and inhibit Myostatin function in skeletal muscles. Transgenic zebrafish expressing the Myostatin prodomain exhibited no significant change in myogenic gene expression and differentiation of slow and fast muscle cells at their embryonic stage. The transgenic fish, however, exhibited an increased number of myofibers in skeletal muscles, but no significant difference in fiber size. Together, these data demonstrate that Myostatin plays an inhibitory role in hyperplastic muscle growth in zebrafish.

  1. Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

    PubMed

    Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

    2015-08-24

    Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

  2. Regulation of myostatin activity and muscle growth.

    PubMed

    Lee, S J; McPherron, A C

    2001-07-31

    Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may have applications for promoting muscle growth, we investigated the regulation of myostatin signaling. Myostatin protein purified from mammalian cells consisted of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal myostatin dimer was capable of binding the activin type II receptors, Act RIIB and, to a lesser extent, Act RIIA. Binding of myostatin to Act RIIB could be inhibited by the activin-binding protein follistatin and, at higher concentrations, by the myostatin propeptide. To determine the functional significance of these interactions in vivo, we generated transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB by using a skeletal muscle-specific promoter. Independent transgenic mouse lines for each construct exhibited dramatic increases in muscle mass comparable to those seen in myostatin knockout mice. Our findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful agents for enhancing muscle growth for both human therapeutic and agricultural applications.

  3. Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide.

    PubMed

    Zhao, Baoping; Li, Eileena J; Wall, Robert J; Yang, Jinzeng

    2009-07-08

    Skeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-beta, plays a dominant role in the genetic control of muscle mass. Myostatin is synthesized as a precursor protein, which generates the N-terminal propeptide and the C-terminal mature myostatin peptide by a post-translational cleavage event. Previously, transgenic over-expression of myostatin propeptide in skeletal muscle results in significant muscle growth in early stages of development. The objectives of present study were to further characterize muscle growth in later stages of life and to identify genes and their expression patterns that are responsible for adult muscle build-up by myostatin propeptide. Immunohistochemical staining with an antibody to the N-terminus indicates a high level of myostatin propeptide present in the muscles of transgenic mice while there were no apparent differences in myostatin protein distribution in the muscle fibers between the transgenic and wild-type mice. Main individual muscles increased by 76-152% in the transgenic mice over their wild-type littermate mice at 12 months of age. A large number of nuclei were localized in the central and basal lamina of the myofibers in the transgenic mice as the number of nuclei per fiber and 100 microm(2) area was significantly higher in transgenic mice than wild-type mice. By systemic comparisons of global mRNA expression patterns between transgenic mice and wild-type littermates using microarray and qRT-PCR techniques, we have identified distinct gene expression patterns to support adult muscle build-up by myostatin propeptide, which are comprised of enhanced expressions of myogenic regulatory factors and extracelullar matrix components, and differentially down-regulated expressions of genes related to protein degradation and mitochondrial ATP synthesis. The results present a coordinated pattern of gene expressions for reduced

  4. Coordinated patterns of gene expressions for adult muscle build-up in transgenic mice expressing myostatin propeptide

    PubMed Central

    Zhao, Baoping; Li, Eileena J; Wall, Robert J; Yang, Jinzeng

    2009-01-01

    Background Skeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-β, plays a dominant role in the genetic control of muscle mass. Myostatin is synthesized as a precursor protein, which generates the N-terminal propeptide and the C-terminal mature myostatin peptide by a post-translational cleavage event. Previously, transgenic over-expression of myostatin propeptide in skeletal muscle results in significant muscle growth in early stages of development. The objectives of present study were to further characterize muscle growth in later stages of life and to identify genes and their expression patterns that are responsible for adult muscle build-up by myostatin propeptide. Results Immunohistochemical staining with an antibody to the N-terminus indicates a high level of myostatin propeptide present in the muscles of transgenic mice while there were no apparent differences in myostatin protein distribution in the muscle fibers between the transgenic and wild-type mice. Main individual muscles increased by 76–152% in the transgenic mice over their wild-type littermate mice at 12 months of age. A large number of nuclei were localized in the central and basal lamina of the myofibers in the transgenic mice as the number of nuclei per fiber and 100 μm2 area was significantly higher in transgenic mice than wild-type mice. By systemic comparisons of global mRNA expression patterns between transgenic mice and wild-type littermates using microarray and qRT-PCR techniques, we have identified distinct gene expression patterns to support adult muscle build-up by myostatin propeptide, which are comprised of enhanced expressions of myogenic regulatory factors and extracelullar matrix components, and differentially down-regulated expressions of genes related to protein degradation and mitochondrial ATP synthesis. Conclusion The results present a coordinated pattern of gene

  5. Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt

    PubMed Central

    Morissette, Michael R.; Cook, Stuart A.; Buranasombati, Cattleya; Rosenberg, Michael A.

    2009-01-01

    Myostatin is a highly conserved negative regulator of skeletal muscle growth. Loss of functional myostatin in cattle, mice, sheep, dogs, and humans results in increased muscle mass. The molecular mechanisms responsible for this increase in muscle growth are not fully understood. Previously, we have reported that phenylephrine-induced cardiac muscle growth and Akt activation are enhanced in myostatin knockout mice compared with controls. Here we report that skeletal muscle from myostatin knockout mice show increased Akt protein expression and overall activity at baseline secondary to an increase in Akt mRNA. We examined the functional role of myostatin modulation of Akt in C2C12 myotubes, a well-established in vitro model of skeletal muscle hypertrophy. Adenoviral overexpression of myostatin attenuated the insulin-like growth factor-I (IGF-I)-mediated increase in myotube diameter, as well as IGF-I-stimulated Akt phosphorylation. Inhibition of myostatin by overexpression of the NH2-terminal portion of myostatin was sufficient to increase myotube diameter and Akt phosphorylation. Coexpression of myostatin and constitutively active Akt (myr-Akt) restored the increase in myotube diameter. Conversely, expression of dominant negative Akt (dn-Akt) with the inhibitory myostatin propeptide blocked the increase in myotube diameter. Of note, ribosomal protein S6 phosphorylation and atrogin-1/muscle atrophy F box mRNA were increased in skeletal muscle from myostain knockout mice. Together, these data suggest myostatin regulates muscle growth at least in part through regulation of Akt. PMID:19759331

  6. Myostatin: a modulator of skeletal-muscle stem cells.

    PubMed

    Walsh, F S; Celeste, A J

    2005-12-01

    Myostatin, or GDF-8 (growth and differentiation factor-8), was first identified through sequence identity with members of the BMP (bone morphogenetic protein)/TGF-beta (transforming growth factor-beta) superfamily. The skeletal-muscle-specific expression pattern of myostatin suggested a role in muscle development. Mice with a targeted deletion of the myostatin gene exhibit a hypermuscular phenotype. In addition, inactivating mutations in the myostatin gene have been identified in 'double muscled' cattle breeds, such as the Belgian Blue and Piedmontese, as well as in a hypermuscular child. These findings define myostatin as a negative regulator of skeletal-muscle development. Myostatin binds with high affinity to the receptor serine threonine kinase ActRIIB (activin type IIB receptor), which initiates signalling through a smad2/3-dependent pathway. In an effort to validate myostatin as a therapeutic target in a post-embryonic setting, a neutralizing antibody was developed by screening for inhibition of myostatin binding to ActRIIB. Administration of this antimyostatin antibody to adult mice resulted in a significant increase in both muscle mass and functional strength. Importantly, similar results were obtained in a murine model of muscular dystrophy, the mdx mouse. Unlike the myostatin-deficient animals, which exhibit both muscle hypertrophy and hyperplasia, the antibody-treated mice demonstrate increased musculature through a hypertrophic mechanism. These results validate myostatin inhibition as a therapeutic approach to muscle wasting diseases such as muscular dystrophy, sarcopenic frailty of the elderly and amylotrophic lateral sclerosis.

  7. Activation of AMP-activated protein kinase induce expression of FoxO1, FoxO3a, and myostatin after exercise-induced muscle damage.

    PubMed

    Lee, Kihyuk; Ochi, Eisuke; Song, Hongsun; Nakazato, Koichi

    2015-10-23

    AMP-activated protein kinase (AMPK) has been shown to regulate protein metabolism in skeletal muscle. We previously found that levels of Forkhead box proteins, FoxO1 and FoxO3a, and myostatin in rat gastrocnemius increased after exercise-induced muscle damage (EIMD). Eccentric muscle contractions (ECs), defined as elongation of muscle under tension, were used for inducing EIMD. The objective of this study was to clarify whether AMPK participates in activation and expression of FoxO proteins and myostatin in rat gastrocnemius muscle after EIMD. Wistar rats were randomly assigned into the following three groups; CON (n = 6), 180ECs group (ankle angular velocity, 180°/s; n = 6), and 30ECs group (ankle angular velocity, 30°/s; n = 6). 20 ECs were conducted with percutaneous electrical stimulation of gastrocnemius and simultaneous forced dorsiflexion of ankle joint (from 0° to 45°). To evaluate activation of AMPK, we measured the phosphorylated states of AMPK and acetyl CoA carboxylase. For evaluation of the direct relationships of AMPK and other proteins, we also examined contents of FoxOs and myostatin with stimulation of L6 myotube with AMPK agonist, 5 -aminoimidazole -4 -carboxamide -1-β-d-ribofuranoside (AICAR) (0.1, 0.5, 1, 1.5, and 2 mM). Western blotting was employed for protein analysis. Significant torque deficit was only observed in the 180ECs, suggesting EIMD. We also observed that phosphorylated AMPKα was induced in response to 180ECs (p < 0.01 vs. CON). Additionally, the level of phosphorylated acetyl CoA carboxylase was significantly higher in response to 180ECs and 30ECs. The phosphorylated states of FoxO1, FoxO3a, and myostatin expression were increased significantly in response to 180ECs. Furthermore, treatment of L6 myotubes with AICAR showed similar tendencies to that observed in in vivo gastrocnemius muscle treated with 180ECs. Therefore, we conclude that activation of AMPK plays a key role in increasing the level of FoxO1, FoxO3a

  8. FHL1 activates myostatin signalling in skeletal muscle and promotes atrophy

    PubMed Central

    Lee, Jen Y.; Lori, Dede; Wells, Dominic J.; Kemp, Paul R.

    2015-01-01

    Myostatin is a TGFβ family ligand that reduces muscle mass. In cancer cells, TGFβ signalling is increased by the protein FHL1. Consequently, FHL1 may promote signalling by myostatin. We therefore tested the ability of FHL1 to regulate myostatin function. FHL1 increased the myostatin activity on a SMAD reporter and increased myostatin dependent myotube wasting. In mice, independent expression of myostatin reduced fibre diameter whereas FHL1 increased fibre diameter, both consistent with previously identified effects of these proteins. However, co-expression of FHL1 and myostatin reduced fibre diameter to a greater extent than myostatin alone. Together, these data suggest that the expression of FHL1 may exacerbate muscle wasting under the appropriate conditions. PMID:26504741

  9. Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-{beta}- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

    SciTech Connect

    Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.; Hathaway, M.R.; Dayton, W.R. . E-mail: wdayton@umn.edu

    2005-11-15

    We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-{beta} superfamily members myostatin and TGF-{beta}{sub 1} have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-{beta}{sub 1} or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-{beta}{sub 1} and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-{beta}{sub 1} or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-{beta} and myostatin to suppress proliferation of PEMC.

  10. Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli

    PubMed Central

    Lee, Sang Beum; Park, Sung Kwon

    2017-01-01

    Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity. PMID:28369115

  11. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression.

    PubMed

    Langley, Brett; Thomas, Mark; Bishop, Amy; Sharma, Mridula; Gilmour, Stewart; Kambadur, Ravi

    2002-12-20

    Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

  12. Functional analysis of the Myostatin gene promoter in sheep.

    PubMed

    Du, Rong; An, XiaoRong; Chen, YongFu; Qin, Jian

    2007-10-01

    Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNPro(W)-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNPro(E(3+5+7)M)-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3-1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin

  13. [Functional characterization of recombinant myostatin and its inhibitory role to chicken muscle development].

    PubMed

    Yang, Wei; Wang, Kun; Chen, Yan; Zhang, Yong; Huang, Bo; Zhu, Da-Hai

    2003-11-01

    Myostatin is a recently discovered member of transforming growth factor beta (TGFbeta) superfamily and shares similar structure features with other members of TGFbeta superfamily. For a better understanding of molecular mechanism of myostatin function, the production of C-terminal truncated form of recombinant myostatin protein (rMSTN) in E. coli was previously reported. Herein, the functional role of the recombinant myostatin in regulating myogenesis in a chicken embryonic myoblasts (CEMs) system was determined. By using flow cytometric analysis, the myostatin was found to inhibit cell cycle transition from G1 to S phase and result in a cell cycle arrest at G1. In addition, myostatin blocked the multi-nucleus myotube formation and caused a decreased expression of the muscle cell differentiation markers (myogenin and MHC) in CEMs. In this study, a rabbit polyclonal antibody against myostatin was produced and high affinity and specificity of this anti-myostatin antibody to recombinant and endogenous myostatin were assayed by Western blot analysis. Further studies showed that the antibody could also recognize the tissue endogenous myostatin of human, mouse and rat. A specific 40 kD band was detected in chicken muscle, which suggested that chicken myostatin might have different splicing pattern. Immunofluorescence assay indicated that myostatin predominantly existed in the cytosol in C2C12 cells. Taken together, the results show that myostatin inhibits chicken muscle cells proliferation and differentiation and down-regulates expression of two differentiation marker gene in CEMs. Remarkably, production of functional recombinant myostatin protein and its specific antibody provides important reagents for unraveling molecular mechanisms underlying myostatin action during myogenesis.

  14. Molecular analysis of fiber type-specific expression of murine myostatin promoter.

    PubMed

    Salerno, Mônica Senna; Thomas, Mark; Forbes, Davanea; Watson, Trevor; Kambadur, Ravi; Sharma, Mridula

    2004-10-01

    Myostatin is a negative regulator of muscle growth, and absence of the functional myostatin protein leads to the heavy muscle phenotype in both mouse and cattle. Although the role of myostatin in controlling muscle mass is established, little is known of the mechanisms regulating the expression of the myostatin gene. In this study, we have characterized the murine myostatin promoter in vivo. Various constructs of the murine myostatin promoter were injected into the quadriceps muscle of mice, and the reporter luciferase activity was analyzed. The results indicate that of the seven E-boxes present in the 2.5-kb fragment of the murine myostatin promoter, the E5 E-box plays an important role in the regulation of promoter activity in vivo. Furthermore, the in vitro studies demonstrated that MyoD preferentially binds and upregulates the murine myostatin promoter activity. We also analyzed the activity of the bovine and murine promoters in murine skeletal muscle and showed that, despite displaying comparable levels of activity in murine myoblast cultures, bovine myostatin promoter activity is much weaker than murine myostatin promoter in mice. Finally, we demonstrate that in vivo, the 2.5-kb region of the murine myostatin promoter is sufficient to drive the activity of the reporter gene in a fiber type-specific manner.

  15. Maternal dietary protein affects transcriptional regulation of myostatin gene distinctively at weaning and finishing stages in skeletal muscle of Meishan pigs.

    PubMed

    Liu, Xiujuan; Wang, Jinquan; Li, Runsheng; Yang, Xiaojing; Sun, Qinwei; Albrecht, Elke; Zhao, Ruqian

    2011-07-01

    Myostatin (MSTN) is suggested to mediate the effect of maternal nutrition on offspring phenotype, yet the mechanisms underlying such adaptive gene regulation is elusive. In this study, we determined the effects of maternal dietary protein on transcriptional regulation of MSTN in skeletal muscle of pig offspring. Fourteen Meishan sows were fed either low-protein (LP) or standard-protein (SP) diets throughout gestation and lactation. MSTN expression in the longissimus dorsi muscle was determined both at weaning and finishing stages. Myostatin mRNA abundance was downregulated at weaning, but upregulated at finishing in LP pigs, indicating stage-specific transcriptional regulation. At weaning, CCAAT/enhancer-binding protein beta (C/EBPβ) in muscle nuclear lysate was decreased in LP piglets, associated with diminished binding of C/EBPβ to all the 3 putative binding sites in MSTN promoter. None of the four histone modification marks investigated showed differences between SP and LP piglets. Among 12 microRNAs predicted to target MSTN, none was differently expressed. At finishing stage, C/EBPβ content remained unchanged, but the binding of C/EBPβ to two of the 3 putative binding sites increased in LP pigs. Histone H3 acetylation and histone H3 lysine 27 trimethylation on MSTN promoter were increased, while histone H3 lysine 9 monomethylation was decreased in LP pigs. Moreover, expression of ssc-miR-136 and ssc-miR-500 was significantly reduced. These results indicate that maternal dietary protein affects MSTN expression through distinct regulatory mechanisms at different stages. The immediate effect at weaning is mediated by C/EBPβ binding without epigenetic modifications, whereas the long-term effect at finishing stage involves both C/EBPβ binding and epigenetic regulations, including histone modification and microRNA expression.

  16. Myostatin up-regulation is associated with the skeletal muscle response to hypoxic stimuli.

    PubMed

    Hayot, Maurice; Rodriguez, Julie; Vernus, Barbara; Carnac, Gilles; Jean, Elise; Allen, David; Goret, Lucie; Obert, Philippe; Candau, Robin; Bonnieu, Anne

    2011-01-30

    Myostatin and hypoxia signalling pathways are able to induce skeletal muscle atrophy, but whether a relationship between these two pathways exists is currently unknown. Here, we tested the hypothesis that a potential mechanism for hypoxia effect on skeletal muscle may be through regulation of myostatin. We reported an induction of myostatin expression in muscles of rats exposed to chronic hypoxia. Interestingly, we also demonstrated increased skeletal muscle myostatin protein expression in skeletal muscle of hypoxemic patients with severe chronic obstructive pulmonary disease (COPD). Parallel studies in human skeletal muscle cell cultures showed that induction of myostatin expression in myotubes treated with hypoxia-mimicking agent such as cobalt chloride (CoCl(2)) is associated with myotube atrophy. Furthermore, we demonstrated that inhibition of myostatin by means of genetic deletion of myostatin or treatment with blocking antimyostatin antibodies inhibits the CoCl(2)-induced atrophy in muscle cells. Finally, addition of recombinant myostatin restored the CoCl(2)-induced atrophy in myostatin deficient myotubes. These results strongly suggest that myostatin can play an essential role in the adaptation of skeletal muscle to hypoxic environment.

  17. MatureP: prediction of secreted proteins with exclusive information from their mature regions.

    PubMed

    Orfanoudaki, Georgia; Markaki, Maria; Chatzi, Katerina; Tsamardinos, Ioannis; Economou, Anastassios

    2017-06-12

    More than a third of the cellular proteome is non-cytoplasmic. Most secretory proteins use the Sec system for export and are targeted to membranes using signal peptides and mature domains. To specifically analyze bacterial mature domain features, we developed MatureP, a classifier that predicts secretory sequences through features exclusively computed from their mature domains. MatureP was trained using Just Add Data Bio, an automated machine learning tool. Mature domains are predicted efficiently with ~92% success, as measured by the Area Under the Receiver Operating Characteristic Curve (AUC). Predictions were validated using experimental datasets of mutated secretory proteins. The features selected by MatureP reveal prominent differences in amino acid content between secreted and cytoplasmic proteins. Amino-terminal mature domain sequences have enhanced disorder, more hydroxyl and polar residues and less hydrophobics. Cytoplasmic proteins have prominent amino-terminal hydrophobic stretches and charged regions downstream. Presumably, secretory mature domains comprise a distinct protein class. They balance properties that promote the necessary flexibility required for the maintenance of non-folded states during targeting and secretion with the ability of post-secretion folding. These findings provide novel insight in protein trafficking, sorting and folding mechanisms and may benefit protein secretion biotechnology.

  18. 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.

  19. Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle

    PubMed Central

    Hitachi, Keisuke; Tsuchida, Kunihiro

    2017-01-01

    Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle. PMID:27992376

  20. Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle.

    PubMed

    Hitachi, Keisuke; Tsuchida, Kunihiro

    2017-01-24

    Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle.

  1. Structural and Dynamic Characterization of the C313Y Mutation in Myostatin Dimeric Protein, Responsible for the “Double Muscle” Phenotype in Piedmontese Cattle

    PubMed Central

    Bongiorni, Silvia; Valentini, Alessio; Chillemi, Giovanni

    2016-01-01

    The knowledge of the molecular effects of the C313Y mutation, responsible for the “double muscle” phenotype in Piedmontese cattle, can help understanding the actual mechanism of phenotype determination and paves the route for a better modulation of the positive effects of this economic important phenotype in the beef industry, while minimizing the negative side effects, now inevitably intersected. The structure and dynamic behavior of the active dimeric form of Myostatin in cattle was analyzed by means of three state-of-the-art Molecular Dynamics simulations, 200-ns long, of wild-type and C313Y mutants. Our results highlight a role for the conserved Arg333 in establishing a network of short and long range interactions between the two monomers in the wild-type protein that is destroyed upon the C313Y mutation even in a single monomer. Furthermore, the native protein shows an asymmetry in residue fluctuation that is absent in the double monomer mutant. Time window analysis on further 200-ns of simulation demonstrates that this is a characteristic behavior of the protein, likely dependent on long range communications between monomers. The same behavior, in fact, has already been observed in other mutated dimers. Finally, the mutation does not produce alterations in the secondary structure elements that compose the characteristic TGF-β cystine-knot motif. PMID:26904102

  2. Low-protein diet fed to crossbred sows during pregnancy and lactation enhances myostatin gene expression through epigenetic regulation in skeletal muscle of weaning piglets.

    PubMed

    Jia, Yimin; Gao, Guichao; Song, Haogang; Cai, Demin; Yang, Xiaojing; Zhao, Ruqian

    2016-04-01

    This study was aimed to investigate the effects of a maternal low-protein diet on transcriptional regulation of the myostatin (MSTN) gene in skeletal muscle of weaning piglets. Sows were fed either a standard-protein (SP, 15 and 18 % crude protein) or a low-protein (LP, 50 % protein level of SP) diet throughout pregnancy and lactation. Longissimus dorsi muscle was sampled from male piglets at 28 days of age. The mRNA was determined by RT-PCR, and protein was measured by Western blot. Chromatin immunoprecipitation assay was used to determine the binding of transcription factors and histone H3 modifications on the MSTN gene promoter. The maternal LP diet significantly decreased body weight and average daily gain (P < 0.05), which was associated with significantly lower plasma concentration of urea nitrogen and total protein (P < 0.05), as well as decreased muscle RNA content (P < 0.05). MSTN mRNA (P < 0.05) was significantly increased, together with enhanced (P < 0.05) mRNA and protein expression of forkhead box class O family member protein 3 (FoxO3), and a tendency of an increase (P = 0.10) in glucocorticoid receptor (GR) mRNA in the muscle of LP piglets. Furthermore, the binding of both FoxO3 and GR to the MSTN gene promoter was significantly higher (P < 0.05) in muscle of LP piglets, together with significantly enriched (P < 0.05) gene activation markers, H3K9Ac and H3K4me3. These results indicate that MSTN mediates maternal LP diet-induced growth retardation, through epigenetic regulation involving FoxO3 and GR binding to its promoter.

  3. PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

    SciTech Connect

    Zhang, Feng; Deng, Bing; Wen, Jianghui; Chen, Kun; Liu, Wu; Ye, Shengqiang; Huang, Haijun; Jiang, Siwen; Xiong, Yuanzhu

    2015-03-06

    Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoD expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression. - Highlights: • PPARγ and MyoD mRNA and protein levels are upregulated by myostatin in ADSCs. • PPARγ and MyoD mRNA and protein levels are downregulated by myostatin in MSCs. • PPARγ exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • MyoD exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • PPARγ and MyoD are differentially regulated by myostatin in ADSCs and MSCs.

  4. Myostatin inhibition therapy for insulin-deficient type 1 diabetes

    PubMed Central

    Coleman, Samantha K.; Rebalka, Irena A.; D’Souza, Donna M.; Deodhare, Namita; Desjardins, Eric M.; Hawke, Thomas J.

    2016-01-01

    While Type 1 Diabetes Mellitus (T1DM) is characterized by hypoinsulinemia and hyperglycemia, persons with T1DM also develop insulin resistance. Recent studies have demonstrated that insulin resistance in T1DM is a primary mediator of the micro and macrovascular complications that invariably develop in this chronic disease. Myostatin acts to attenuate muscle growth and has been demonstrated to be elevated in streptozotocin-induced diabetic models. We hypothesized that a reduction in mRNA expression of myostatin within a genetic T1DM mouse model would improve skeletal muscle health, resulting in a larger, more insulin sensitive muscle mass. To that end, Akita diabetic mice were crossed with MyostatinLn/Ln mice to ultimately generate a novel mouse line. Our data support the hypothesis that decreased skeletal muscle expression of myostatin mRNA prevented the loss of muscle mass observed in T1DM. Furthermore, reductions in myostatin mRNA increased Glut1 and Glut4 protein expression and glucose uptake in response to an insulin tolerance test (ITT). These positive changes lead to significant reductions in resting blood glucose levels as well as pronounced reductions in associated diabetic symptoms, even in the absence of exogenous insulin. Taken together, this study provides a foundation for considering myostatin inhibition as an adjuvant therapy in T1DM as a means to improve insulin sensitivity and blood glucose management. PMID:27581061

  5. Myostatin negatively regulates satellite cell activation and self-renewal.

    PubMed

    McCroskery, Seumas; Thomas, Mark; Maxwell, Linda; Sharma, Mridula; Kambadur, Ravi

    2003-09-15

    Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

  6. Modulation of follistatin and myostatin propeptide by anabolic steroids and gender.

    PubMed

    Mosler, S; Geisler, S; Hengevoss, J; Schiffer, T; Piechotta, M; Adler, M; Diel, P

    2013-07-01

    The purpose of this pilot study was to investigate the impact of training, anabolic steroids and endogenous hormones on myostatin-interacting proteins in order to identify manipulations of myostatin signalling. To identify whether analysis of the myostatin interacting proteins follistatin and myostatin propeptide is suitable to detect the abuse of anabolic steroids, their serum concentrations were monitored in untrained males, bodybuilders using anabolic steroids and natural bodybuilders. In addition, we analysed follistatin and myostatin propeptide serum proteins in females during menstrual cycle. Our results showed increased follistatin concentrations in response to anabolic steroids. Furthermore, variations of sex steroid levels during the menstrual cycle had no impact on the expression of follistatin and myostatin propetide. In addition, we identified gender differences in the basal expression of the investigated proteins. In general, follistatin and myostatin propeptide concentrations were relatively stable within the same individual both in males and females. In conclusion, the current findings provide an insight into gender differences in myostatin-interacting proteins and their regulation in response to anabolic steroids and endogenous hormones. Therefore our data provide new aspects for the development of doping prevention strategies. © Georg Thieme Verlag KG Stuttgart · New York.

  7. Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle.

    PubMed

    Sriram, Sandhya; Subramanian, Subha; Juvvuna, Prasanna Kumar; Ge, Xiaojia; Lokireddy, Sudarsanareddy; McFarlane, Craig Desmond; Wahli, Walter; Kambadur, Ravi; Sharma, Mridula

    2014-03-01

    Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-β and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3(-/-) mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3(-/-) mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3(-/-) muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3(-/-) skeletal muscle. The exaggerated ROS in the Smad3(-/-) muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy.

  8. Brief Communication: Sexual dimorphic expression of myostatin and follistatin like-3 in a rat trans-generational under-nutrition model

    PubMed Central

    2010-01-01

    The detrimental effects of maternal under-nutrition during gestation on fetal development are well known with an increased propensity of metabolic disorders identified in the adult offspring. Understanding exactly how and by which molecular pathways inadequate nutrition can impact upon offspring phenotype is critical and necessary for the development of treatment methods and ultimately prevention of any negative health effects. Myostatin, a negative regulator of muscle development, has recently been shown to effect glucose homeostasis and fat deposition. The involvement of myostatin in glucose metabolism and adipogenesis thus supports its ability to act in the continued alterations to the postnatal phenotype of the offspring. This hypothesis was examined in the current study using a trans-generational gestationally under-nourished rat model exposed to a high-fat (HF) diet post-weaning. The body weight, body fat, plasma glucose and insulin concentrations of the offspring, both male and female, were investigated in relation to the protein expression of myostatin and its main inhibitor; follistatin like-3 (FSTL-3), in skeletal muscle of mature offspring. Sexual dimorphism was clearly evident in the majority of these measures, including myostatin and FSTL-3 expression. Generally males displayed higher (P < 0.05) myostatin precursor and dimer expression than females, which was especially apparent (P < 0.01) in both chow and HF trans-generationally undernourished (UNAD) groups. In females only, myostatin precursor and dimer expression was altered by both trans-generational under-nutrition and postnatal diet. Overall FSTL-3 expression did not differ between sexes, although difference between sexes within certain treatments and diets were evident. Most notably, HF fed UNAD females had higher (P < 0.05) FSTL-3 expression than HF fed UNAD males. The former group also displayed higher (P < 0.01) FSTL-3 expression compared to all other female groups. In summary, myostatin may

  9. Brief Communication: Sexual dimorphic expression of myostatin and follistatin like-3 in a rat trans-generational under-nutrition model.

    PubMed

    Peiris, Hassendrini N; Ponnampalam, Anna P; Mitchell, Murray D; Green, Mark P

    2010-05-20

    The detrimental effects of maternal under-nutrition during gestation on fetal development are well known with an increased propensity of metabolic disorders identified in the adult offspring. Understanding exactly how and by which molecular pathways inadequate nutrition can impact upon offspring phenotype is critical and necessary for the development of treatment methods and ultimately prevention of any negative health effects. Myostatin, a negative regulator of muscle development, has recently been shown to effect glucose homeostasis and fat deposition. The involvement of myostatin in glucose metabolism and adipogenesis thus supports its ability to act in the continued alterations to the postnatal phenotype of the offspring. This hypothesis was examined in the current study using a trans-generational gestationally under-nourished rat model exposed to a high-fat (HF) diet post-weaning. The body weight, body fat, plasma glucose and insulin concentrations of the offspring, both male and female, were investigated in relation to the protein expression of myostatin and its main inhibitor; follistatin like-3 (FSTL-3), in skeletal muscle of mature offspring. Sexual dimorphism was clearly evident in the majority of these measures, including myostatin and FSTL-3 expression. Generally males displayed higher (P < 0.05) myostatin precursor and dimer expression than females, which was especially apparent (P < 0.01) in both chow and HF trans-generationally undernourished (UNAD) groups. In females only, myostatin precursor and dimer expression was altered by both trans-generational under-nutrition and postnatal diet. Overall FSTL-3 expression did not differ between sexes, although difference between sexes within certain treatments and diets were evident. Most notably, HF fed UNAD females had higher (P < 0.05) FSTL-3 expression than HF fed UNAD males. The former group also displayed higher (P < 0.01) FSTL-3 expression compared to all other female groups. In summary, myostatin may

  10. IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice

    USDA-ARS?s Scientific Manuscript database

    Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue...

  11. Sequence analysis of myostatin promoter in cattle.

    PubMed

    Crisà, A; Marchitelli, C; Savarese, M C; Valentini, A

    2003-01-01

    Myostatin (GDF8) acts as a negative regulator of muscle growth. Mutations in the gene are responsible for the double muscling phenotype in several European cattle breeds. Here we describe the sequence of the upstream 5' region of the myostatin gene. The sequence analysis was carried out on three animals of nine European cattle breeds, with the aim to search for polymorphisms. A T/A polymorphism at -371 and a G/C polymorphism at -805 (relative to ATG) were found. PCR- RFLP was used to further screen 353 animals of the nine breeds studied and to assess the frequencies of the SNPs. The promoter region of the gene contains several binding sites for transcription factors found also in other myogenic genes. This may play an important role in the regulation of the protein and consequently on muscular development.

  12. Structure of Myostatin·Follistatin-like 3

    PubMed Central

    Cash, Jennifer N.; Angerman, Elizabeth B.; Kattamuri, Chandramohan; Nolan, Kristof; Zhao, Huaying; Sidis, Yisrael; Keutmann, Henry T.; Thompson, Thomas B.

    2012-01-01

    TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes. PMID:22052913

  13. Cloning and sequence analysis of myostatin promoter in sheep.

    PubMed

    Du, Rong; Chen, Yong-Fu; An, Xiao-Rong; Yang, Xing-Yuan; Ma, Yi; Zhang, Lei; Yuan, Xiao-Li; Chen, Li-Mei; Qin, Jian

    2005-12-01

    To better understand the structure and function of the myostatin's gene promoter region in sheep, we cloned and sequenced a 1.517 kb fragment containing the 5'-regulatory region of the sheep myostatin gene (GenBank accession number is AY918121). The promoter sequence consists of three TATA boxes, one CAAT box, and eight putative E-boxes. Some putative muscle growth response elements for Octamer-binding factor 1(Octamer), Activator protein 1(AP1), Growth factor independence 1 zinc finger protein (Gfi-1B), Myocyte enhancer factor 2 (MEF2), Muscle-specific Mt binding site (MTBF), Glucocorticoid response elements (GRE) and Progesterone receptor binding site (PRE) were detected. Some of the motifs are conserved as compared to with that in the goat, bovine and porcine myostatin promoters. However, some differences were also found.

  14. Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation.

    PubMed

    Walker, Ryan G; Poggioli, Tommaso; Katsimpardi, Lida; Buchanan, Sean M; Oh, Juhyun; Wattrus, Sam; Heidecker, Bettina; Fong, Yick W; Rubin, Lee L; Ganz, Peter; Thompson, Thomas B; Wagers, Amy J; Lee, Richard T

    2016-04-01

    Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor β superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.

  15. Expression and function of myostatin in obesity, diabetes, and exercise adaptation.

    PubMed

    Allen, David L; Hittel, Dustin S; McPherron, Alexandra C

    2011-10-01

    Myostatin is a member of the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) superfamily of secreted factors that functions as a potent inhibitor of skeletal muscle growth. Moreover, considerable evidence has accumulated that myostatin also regulates metabolism and that its inhibition can significantly attenuate the progression of obesity and diabetes. Although at least part of these effects on metabolism can be attributable to myostatin's influence over skeletal muscle growth and therefore on the total volume of metabolically active lean body mass, there is mounting evidence that myostatin affects the growth and metabolic state of other tissues, including the adipose and the liver. In addition, recent work has explored the role of myostatin in substrate mobilization, uptake, and/or utilization of muscle independent of its effects on body composition. Finally, the effects of both endurance and resistance exercise on myostatin expression, as well as the potential role of myostatin in the beneficial metabolic adaptations occurring in response to exercise, have also begun to be delineated in greater detail. The purpose of this review was to summarize the work to date on the expression and function of myostatin in obesity, diabetes, and exercise adaptation.

  16. Myostatin induces p300 degradation to silence cyclin D1 expression through the PI3K/PTEN/Akt pathway.

    PubMed

    Ji, Ming; Zhang, Qiang; Ye, Jianwei; Wang, Xueyan; Yang, Wei; Zhu, Dahai

    2008-08-01

    Myostatin is a negative regulator of skeletal muscle growth and affects numerous genes expression involved in cell proliferation, differentiation and metabolism. However, the molecular mechanisms underlying myostatin-regulated genes expression remain to be elucidated. In this study, we showed that myostatin blocked the recruitment of p300 to the cyclin D1 promoter, resulting in the silence of cyclin D1 expression. Our data further demonstrated that myostatin decreased the protein level of p300 by inducing p300 degradation via the ubiquitin-proteasome system. In addition, we provided experimental evidence to show that myostatin-induced p300 degradation was mediated by the phosphatidylinositol 3-kinase/PTEN/Akt signaling pathway and this could be antagonized by IGF-1 or insulin. Results presented in this study uncovered an epigenetic control of genes expression in response to myostatin.

  17. Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation

    PubMed Central

    Kayupov, Erdan; Bradley, Joshua R.; Brooks, Susan V.; Claflin, Dennis R.

    2011-01-01

    Myostatin (MSTN) is a member of the transforming growth factor-β superfamily of cytokines and is a negative regulator of skeletal muscle mass. Compared with MSTN+/+ mice, the extensor digitorum longus muscles of MSTN−/− mice exhibit hypertrophy, hyperplasia, and greater maximum isometric force production (Fo), but decreased specific maximum isometric force (sFo; Fo normalized by muscle cross-sectional area). The reason for the reduction in sFo was not known. Studies in myotubes indicate that inhibiting myostatin may increase muscle mass by decreasing the expression of the E3 ubiquitin ligase atrogin-1, which could impact the force-generating capacity and size of muscle fibers. To gain a greater understanding of the influence of myostatin on muscle contractility, we determined the impact of myostatin deficiency on the contractility of permeabilized muscle fibers and on the levels of atrogin-1 and ubiquitinated myosin heavy chain in whole muscle. We hypothesized that single fibers from MSTN−/− mice have a greater Fo, but no difference in sFo, and a decrease in atrogin-1 and ubiquitin-tagged myosin heavy chain levels. The results indicated that fibers from MSTN−/− mice have a greater cross-sectional area, but do not have a greater Fo and have a sFo that is significantly lower than fibers from MSTN+/+ mice. The extensor digitorum longus muscles from MSTN−/− mice also have reduced levels of atrogin-1 and ubiquitinated myosin heavy chain. These findings suggest that myostatin inhibition in otherwise healthy muscle increases the size of muscle fibers and decreases atrogin-1 levels, but does not increase the force production of individual muscle fibers. PMID:21565991

  18. Synthesis of Endosperm Proteins in Wheat Seed during Maturation 1

    PubMed Central

    Flint, Dennis; Ayers, George S.; Ries, Stanley K.

    1975-01-01

    The time of synthesis, the molecular weight, and the relative glutamine-glutamate and proline to leucine ratios of the endosperm proteins of wheat (Triticum aestivum L. cv. Logan) were determined using a sodium dodecyl sulfate-polyacrylamide gel technique. In general, synthesis of most proteins occurred through much of the maturation of the seed, but past 20 days the rate of synthesis of the high molecular weight proteins declined more rapidly than those of lower molecular weight. The synthesis of at least one protein occurred only late in seed maturation. Several of the high molecular weight proteins had glutamate-glutamine to leucine ratios higher than the remainder of the proteins. No evidence for proteins of a polyglutamine-glutamate and/or proline nature was found. PMID:16659308

  19. Myostatin as a therapeutic target in Amyotrophic lateral sclerosis.

    PubMed

    Walsh, Frank S; Rutkowski, Julia Lynn

    2012-11-01

    Amyotrophic Lateral Sclerosis is a devastating neurological disease that is inevitably fatal after 3-5years duration. Treatment options are minimal and as such new therapeutic modalities are required. In this review, we discuss the role of the myostatin pathway as a modulator of skeletal muscle mass and therapeutic approaches using biological based therapies. Both monoclonal antibodies to myostatin and a soluble receptor decoy to its high affinity receptor have been used in clinical trials of neuromuscular diseases and while there have been efficacy signals with the latter approach there have also been safety issues. Our approach is to target the high affinity receptor-binding site on myostatin and to develop a next generation set of therapeutic reagents built on a novel protein scaffold. This is the natural single domain VNAR found in sharks which is extremely versatile and has the ability to develop products with superior properties compared to existing therapeutics.

  20. Fluorescent proteins: maturation, photochemistry and photophysics.

    PubMed

    Remington, S James

    2006-12-01

    It has long been appreciated that green fluorescent protein (GFP) autocatalytically forms its chromophore in a host-independent process; several of the initial steps in the reaction have recently been elucidated. Nevertheless, the end points of the process are unexpectedly diverse, as six chemically distinct chromophores, including two with three rings, have been identified. All fluorescent proteins continuously produce a low level of reactive oxygen species under illumination, which, in some cases, can lead to host cell death. In one extreme but useful example, the red fluorescent protein KillerRed can be used to selectively destroy cells upon brief illumination. Finally, when photophysical processes such as excited-state proton transfer, reversible photobleaching and photoactivation are understood, useful research tools, for example, real-time biosensors and optical highlighters, can result; however, side effects of their use may lead to significant artifacts in time-dependent microscopy experiments.

  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. Glucocorticoids Enhance Muscle Proteolysis through a Myostatin-Dependent Pathway at the Early Stage

    PubMed Central

    Wang, Ruxia; Jiao, Hongchao; Zhao, Jingpeng; Wang, Xiaojuan; Lin, Hai

    2016-01-01

    Myostatin, a member of the TGF-β superfamily of secreted proteins, is expressed primarily in skeletal muscle. It negatively regulates muscle mass and is associated with glucocorticoid-induced muscle atrophy. However, it remains unclear whether myostatin is involved in glucocorticoid-induced muscle protein turnover. The aim of the present study was to investigate the role of myostatin in protein metabolism during dexamethasone (DEX) treatment. Protein synthesis rates and the expression of the genes for myostatin, ubiquitin-proteasome atrogin-1, MuRF1, FoxO1/3a and mTOR/p70S6K were determined. The results show that DEX decreased (P<0.05) protein synthesis rates while increasing the abundance of myostatin. DEX increased (P<0.05) the level of phospho-FoxO1/3a (Thr 24/32) and the expression of MuRF1. In contrast, DEX treatment had no detectable effect on atrogin-1 protein levels (P>0.05). The phosphorylation levels of mTOR and p70S6K were decreased by DEX treatment (P<0.05). Follistatin treatment inhibited the DEX-induced increase in myostatin (P<0.05) and the activation of phosphor-FoxO1/3a (Thr 24/32) (P< 0.05) and MuRF1 (P<0.05). Follistatin treatment had no influence on the protein synthesis rate or on the phosphorylation levels of mTOR (Ser 2448) and p70S6K (Thr 389) (P> 0.05). In conclusion, the present study suggests that the myostatin signalling pathway is associated with glucocorticoid-induced muscle protein catabolism at the beginning of exposure. Myostatin is not a main pathway associated with the suppression of muscle protein synthesis by glucocorticoids. PMID:27227776

  3. Loss of Myostatin (GDF8) Function Increases Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells but the Osteogenic Effect is Ablated with Unloading

    PubMed Central

    Hamrick, M.; Shi, X.; Zhang, W.; Pennington, C.; Thakore, H.; Haque, M.; Kang, B.; Isales, C.M.; Fulzele, S.; Wenger, K.

    2007-01-01

    Myostatin (GDF8) is a negative regulator of skeletal muscle growth and mice lacking myostatin show a significant increase in muscle mass and bone density compared to normal mice. In order to further define the role of myostatin in regulating bone mass we sought to determine if loss of myostatin function significantly altered the potential for osteogenic differentiation in bone marrow-derived mesenchymal stem cells in vitro and in vivo. We first examined expression of the myostatin receptor, the type IIB activin receptor (AcvrIIB), in bone marrow-derived mesenchymal stem cells (BMSCs) isolated from mouse long bones. This receptor was found to be expressed at high levels in BMSCs, and we were also able to detect AcvrIIB protein in BMSCs in situ using immunofluorescence. BMSCs isolated from myostatin-deficient mice showed increased osteogenic differentiation compared to wild-type mice; however, treatment of BMSCs from myostatin-deficient mice with recombinant myostatin did not attenuate the osteogenic differentiation of these cells. Loading of BMSCs in vitro increased the expression of osteogenic factors such as BMP-2 and IGF-1, but treatment of BMSCs with recombinant myostatin was found to decrease the expression of these factors. We investigated the effects of myostatin loss-of-function on the differentiation of BMSCs in vivo using hindlimb unloading (7 days tail suspension). Unloading caused a greater increase in marrow adipocyte number, and a greater decrease in osteoblast number, in myostatin-deficient mice than in normal mice. These data suggest that the increased osteogenic differentiation of BMSCs from mice lacking myostatin is load-dependent, and that myostatin may alter the mechanosensitivity of BMSCs by suppressing the expression of osteogenic factors during mechanical stimulation. Furthermore, although myostatin deficiency increases muscle mass and bone strength, it does not prevent muscle and bone catabolism with unloading. PMID:17383950

  4. Myostatin Attenuation In Vivo Reduces Adiposity, but Activates Adipogenesis

    PubMed Central

    Li, Naisi; Yang, Qiyuan; Walker, Ryan G.; Thompson, Thomas B.; Du, Min

    2016-01-01

    A potentially novel approach for treating obesity includes attenuating myostatin as this increases muscle mass and decreases fat mass. Notwithstanding, conflicting studies report that myostatin stimulates or inhibits adipogenesis and it is unknown whether reduced adiposity with myostatin attenuation results from changes in fat deposition or adipogenesis. We therefore quantified changes in the stem, transit amplifying and progenitor cell pool in white adipose tissue (WAT) and brown adipose tissue (BAT) using label-retaining wild-type and mstn−/− (Jekyll) mice. Muscle mass was larger in Jekyll mice, WAT and BAT mass was smaller and label induction was equal in all tissues from both wild-type and Jekyll mice. The number of label-retaining cells, however, dissipated quicker in WAT and BAT of Jekyll mice and was only 25% and 17%, respectively, of wild-type cell counts 1 month after induction. Adipose cell density was significantly higher in Jekyll mice and increased over time concomitant with label-retaining cell disappearance, which is consistent with enhanced expansion and differentiation of the stem, transit amplifying and progenitor pool. Stromal vascular cells from Jekyll WAT and BAT differentiated into mature adipocytes at a faster rate than wild-type cells and although Jekyll WAT cells also proliferated quicker in vitro, those from BAT did not. Differentiation marker expression in vitro, however, suggests that mstn−/− BAT preadipocytes are far more sensitive to the suppressive effects of myostatin. These results suggest that myostatin attenuation stimulates adipogenesis in vivo and that the reduced adiposity in mstn−/− animals results from nutrient partitioning away from fat and in support of muscle. PMID:26580671

  5. Myostatin signaling regulates Akt activity via the regulation of miR-486 expression.

    PubMed

    Hitachi, Keisuke; Nakatani, Masashi; Tsuchida, Kunihiro

    2014-02-01

    Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size.

  6. Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

    PubMed

    Kornegay, Joe N; Bogan, Daniel J; Bogan, Janet R; Dow, Jennifer L; Wang, Jiahui; Fan, Zheng; Liu, Naili; Warsing, Leigh C; Grange, Robert W; Ahn, Mihye; Balog-Alvarez, Cynthia J; Cotten, Steven W; Willis, Monte S; Brinkmeyer-Langford, Candice; Zhu, Hongtu; Palandra, Joe; Morris, Carl A; Styner, Martin A; Wagner, Kathryn R

    2016-01-01

    Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn (+/-)) whippets. A total of four GRippets (dystrophic and Mstn (+/-)), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no

  7. Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

    PubMed Central

    Jeanplong, Ferenc; Falconer, Shelley J.; Oldham, Jenny M.; Thomas, Mark; Gray, Tarra S.; Hennebry, Alex; Matthews, Kenneth G.; Kemp, Frederick C.; Patel, Ketan; Berry, Carole; Nicholas, Gina; McMahon, Christopher D.

    2013-01-01

    Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant

  8. Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

    PubMed

    Jeanplong, Ferenc; Falconer, Shelley J; Oldham, Jenny M; Thomas, Mark; Gray, Tarra S; Hennebry, Alex; Matthews, Kenneth G; Kemp, Frederick C; Patel, Ketan; Berry, Carole; Nicholas, Gina; McMahon, Christopher D

    2013-01-01

    Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant

  9. 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.

  10. Protein profile of mature soybean seeds and prepared soybean milk.

    PubMed

    Capriotti, Anna Laura; Caruso, Giuseppe; Cavaliere, Chiara; Samperi, Roberto; Stampachiacchiere, Serena; Zenezini Chiozzi, Riccardo; Laganà, Aldo

    2014-10-08

    The soybean (Glycine max (L.) Merrill) is economically the most important bean in the world, providing a wide range of vegetable proteins. Soybean milk is a colloidal solution obtained as water extract from swelled and ground soybean seeds. Soybean proteins represent about 35-40% on a dry weight basis and they are receiving increasing attention with respect to their health effects. However, the soybean is a well-recognized allergenic food, and therefore, it is urgent to define its protein components responsible for the allergenicity in order to develop hypoallergenic soybean products for sensitive people. The main aim of this work was the characterization of seed and milk soybean proteome and their comparison in terms of protein content and specific proteins. Using a shotgun proteomics approach, 243 nonredundant proteins were identified in mature soybean seeds.

  11. Protein Composition of the Vaccinia Virus Mature Virion

    SciTech Connect

    Resch, Wolfgang; Hixson, Kim K.; Moore, Ronald J.; Lipton, Mary S.; Moss, Bernard

    2007-02-05

    The protein content of vaccinia virus mature virions, purified by rate zonal and isopycnic centrifugation and solubilized by SDS or a solution of urea and thiourea, was determined by the accurate mass and time tag technology which uses both tandem mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry to detect tryptic peptides separated by high-resolution liquid chromatography. Eighty vaccinia virus-encoded proteins representing 37% of the 218 genes annotated in the complete genome sequence were detected in at least three analyses. Ten proteins accounted for approximately 80% of the mass, while the least abundant proteins made up 1% or less of the mass. Thirteen identified proteins were not previously reported as components of virions. On the other hand, 8 previously described virion proteins were not detected here, presumably due to technical reasons including small size and hydrophobicity. In addition to vaccinia virus-encoded proteins, 24 host proteins omitting isoforms were detected. The most abundant of these were cytoskeletal proteins, heat shock proteins, and proteins involved in translation.

  12. Induction of dorsal mesoderm by soluble, mature Vg1 protein.

    PubMed

    Kessler, D S; Melton, D A

    1995-07-01

    Mesoderm induction during Xenopus development has been extensively studied, and two members of the transforming growth factor-beta family, activin beta B and Vg1, have emerged as candidates for a natural inducer of dorsal mesoderm. Heretofore, analysis of Vg1 activity has relied on injection of hybrid Vg1 mRNAs, which have not been shown to direct efficient secretion of ligand and, therefore, the mechanism of mesoderm induction by processed Vg1 protein is unclear. This report describes injection of Xenopus oocytes with a chimeric activin-Vg1 mRNA, encoding the pro-region of activin beta B fused to the mature region of Vg1, resulting in the processing and secretion of mature Vg1. Treatment of animal pole explants with mature Vg1 protein resulted in differentiation of dorsal, but not ventral, mesodermal tissues and dose-dependent activation of both dorsal and ventrolateral mesodermal markers. At high doses, mature Vg1 induced formation of 'embryoids' with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system. Furthermore, truncated forms of the activin and FGF receptors, which block mesoderm induction in the intact embryo, fully inhibited mature Vg1 activity. To examine the mechanism of inhibition, we have performed receptor-binding assays with radiolabeled Vg1. Finally, follistatin, a specific inhibitor of activin beta B which is shown not to block endogenous dorsal mesoderm induction, failed to inhibit Vg1. The results support a role for endogenous Vg1 in dorsal mesoderm induction during Xenopus development.

  13. Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease

    PubMed Central

    Wang, Dong-Tao; Yang, Ya-Jun; Huang, Ren-Hua; Zhang, Zhi-Hua; Lin, Xin

    2015-01-01

    Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems. PMID:26448817

  14. Effect of swimming on myostatin expression in white and red gastrocnemius muscle and in cardiac muscle of rats.

    PubMed

    Matsakas, Antonios; Bozzo, Cyrille; Cacciani, Nicola; Caliaro, Francesca; Reggiani, Carlo; Mascarello, Francesco; Patruno, Marco

    2006-11-01

    The aim of this study was to test the hypothesis that swimming training might impact differentially myostatin expression in skeletal muscles, depending on fibre type composition, and in cardiac muscle of rats. Myostatin expression was analysed by real time reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry of the red deep portion (mainly composed of slow and type II A fibres) and in the superficial, white portion (composed of fast type II X and II B fibres) of the gastrocnemius muscle in adult male Wistar rats: (i) subjected to two consecutive swimming bouts for 3 h; (ii) subjected to intensive swimming training for 4 weeks; and (iii) sedentary control rats. Myostatin mRNA content was in all cases higher in white than in red muscles. Two bouts of swimming did not alter myostatin expression, whereas swimming training for 4 weeks resulted in a significant reduction of myostatin mRNA contents, significant both in white and red muscles but more pronounced in white muscles. Western blot did not detect any change in the amount of myostatin protein. Immunohistochemistry showed that, in control rats, myostatin was localized in presumptive satellite cells of a few muscle fibres. After training, the number of myostatin-positive spots decreased significantly. Myostatin mRNA content in cardiac muscle was lower than in skeletal muscle and was significantly increased by swimming training. In conclusion, the results obtained showed that intense training caused a decreased expression of myostatin mRNA in white and red skeletal muscles but an increase in cardiac muscle.

  15. Contractile Properties of EDL and Soleus Muscles of Myostatin-Deficient Mice

    PubMed Central

    Mendias, Christopher L; Marcin, James E; Calerdon, Daniel R; Faulkner, John A

    2014-01-01

    Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (Po), but decrease the specific Po (sPo) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of EDL and soleus muscles from wild type (MSTN+/+), heterozygous-null (MSTN+/−) and homozygous-null (MSTN−/−) adult male mice were determined. For EDL muscles, the Po of both MSTN+/− and MSTN−/− mice were greater than the Po of MSTN+/+ mice. For soleus muscles, the Po of MSTN−/− mice was greater than that of MSTN+/+ mice. The sPo of EDL muscles of MSTN−/− mice was less than MSTN+/+ mice. For soleus muscles, however, no difference in sPo was observed. Following two lengthening contractions, EDL muscles from MSTN−/− mice had a greater force deficit than MSTN+/+ or MSTN+/− mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, ActRIIB. Compared with the soleus, the amount of ActRIIB was approximately two-fold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles, but also in the contractility and the composition of the ECM of muscles. PMID:16709649

  16. Inhibition of myostatin signaling through Notch activation following acute resistance exercise.

    PubMed

    MacKenzie, Matthew G; Hamilton, David Lee; Pepin, Mark; Patton, Amy; Baar, Keith

    2013-01-01

    Myostatin is a TGFβ family member and negative regulator of muscle size. Due to the complexity of the molecular pathway between myostatin mRNA/protein and changes in transcription, it has been difficult to understand whether myostatin plays a role in resistance exercise-induced skeletal muscle hypertrophy. To circumvent this problem, we determined the expression of a unique myostatin target gene, Mighty, following resistance exercise. Mighty mRNA increased by 6 h (82.9 ± 24.21%) and remained high out to 48 h (56.5 ± 19.67%) after resistance exercise. Further examination of the soleus, plantaris and tibialis anterior muscles showed that the change in Mighty mRNA at 6 h correlated with the increase in muscle size associated with this protocol (R(2) = 0.9996). The increase in Mighty mRNA occurred both independent of Smad2 phosphorylation and in spite of an increase in myostatin mRNA (341.8 ± 147.14% at 3 h). The myostatin inhibitor SKI remained unchanged. However, activated Notch, another potential inhibitor of TGFβ signaling, increased immediately following resistance exercise (83 ± 11.2%) and stayed elevated out to 6 h (78 ± 16.6%). Electroportion of the Notch intracellular domain into the tibialis anterior resulted in an increase in Mighty mRNA (63 ± 13.4%) that was equivalent to the canonical Notch target HES-1 (94.4 ± 7.32%). These data suggest that acute resistance exercise decreases myostatin signaling through the activation of the TGFβ inhibitor Notch resulting in a decrease in myostatin transcriptional activity that correlates well with muscle hypertrophy.

  17. Effects of oral creatine and resistance training on serum myostatin and GASP-1.

    PubMed

    Saremi, A; Gharakhanloo, R; Sharghi, S; Gharaati, M R; Larijani, B; Omidfar, K

    2010-04-12

    Myostatin is a catabolic regulator of skeletal muscle mass. The purpose of this study was to determine the effect of resistance training for 8 weeks in conjunction with creatine supplementation on muscle strength, lean body mass, and serum levels of myostatin and growth and differentiation factor-associated serum protein-1 (GASP-1). In a double-blinded design 27 healthy male subjects (23.42+/-2.2 years) were assigned to control (CON), resistance training+placebo (RT+PL) and resistance training+creatine supplementation (RT+CR) groups. The protocol consisted of 3 days per week of training for 8 weeks, each session including three sets of 8-10 repetitions at 60-70% of 1 RM for whole-body exercise. Blood sampling, muscular strength testing and body composition analysis (full body DEXA) were performed at 0, 4th and 8th weeks. Myostatin and GASP-1 was measured. Resistance training caused significant decrease in serum levels of myostatin and increase in that of GASP-1. Creatine supplementation in conjunction with resistance training lead to greater decreases in serum myostatin (p<0.05), but had not additional effect on GASP-1 (p>0.05). The effects of resistance training on serum levels of myostatin and GASP-1, may explain the increased muscle mass that is amplified by creatine supplementation.

  18. Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

    NASA Astrophysics Data System (ADS)

    Li, Shengjie; Bai, Junjie; Wang, Lin

    2008-08-01

    Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

  19. Myostatin inhibitors as therapies for muscle wasting associated with cancer and other disorders

    PubMed Central

    Smith, Rosamund C.; Lin, Boris K.

    2013-01-01

    Purpose of review This review summarizes recent progress in the development of myostatin inhibitors for the treatment of muscle wasting disorders. It also focuses on findings in myostatin biology that may have implications for the development of antimyostatin therapies. Recent findings There has been progress in evaluating antimyostatin therapies in animal models of muscle wasting disorders. Some programs have progressed into clinical development with initial results showing positive impact on muscle volume. In normal mice myostatin deficiency results in enlarged muscles with increased total force but decreased specific force (total force/total mass). An increase in myofibrillar protein synthesis without concomitant satellite cell proliferation and fusion leads to muscle hypertrophy with unchanged myonuclear number. A specific force reduction is not observed when atrophied muscle, the predominant therapeutic target of myostatin inhibitor therapy, is made myostatindeficient. Myostatin has been shown to be expressed by a number of tumor cell lines in mice and man. Summary Myostatin inhibition remains a promising therapeutic strategy for a range of muscle wasting disorders. PMID:24157714

  20. Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin.

    PubMed

    Reisz-Porszasz, Suzanne; Bhasin, Shalender; Artaza, Jorge N; Shen, Ruoqing; Sinha-Hikim, Indrani; Hogue, Aimee; Fielder, Thomas J; Gonzalez-Cadavid, Nestor F

    2003-10-01

    Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.

  1. Ultrastructure of mature protein body in the starchy endosperm of dry cereal grain.

    PubMed

    Saito, Yuhi; Shigemitsu, Takanari; Tanaka, Kunisuke; Morita, Shigeto; Satoh, Shigeru; Masumura, Takehiro

    2010-01-01

    The development of the protein body in the late stage of seed maturation is poorly understood, because electron-microscopy of mature cereal endosperm is technically difficult. In this study, we attempted to modify the existing method of embedding rice grain in resin. The modified method revealed the ultrastructures of the mature protein body in dry cereal grains.

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

    PubMed Central

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

    2015-01-01

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

  3. A secreted protein promotes cleavage furrow maturation during cytokinesis.

    PubMed

    Xu, Xuehong; Vogel, Bruce E

    2011-01-25

    Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1-3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4-9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. A Secreted Protein Promotes Cleavage Furrow Maturation during Cytokinesis

    PubMed Central

    Xu, Xuehong; Vogel, Bruce E.

    2010-01-01

    Summary Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1–3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4–9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis. PMID:21215633

  5. Muscle regeneration in the prolonged absence of myostatin.

    PubMed

    Wagner, Kathryn R; Liu, Xiaosong; Chang, Xiaoli; Allen, Ronald E

    2005-02-15

    Myostatin is an endogenous inhibitor of muscle conserved across diverse species. In the absence of myostatin, there is massive muscle growth in mice, cattle, and humans. Previous studies in the mdx mouse model of muscular dystrophy demonstrate that inhibiting myostatin attenuates several features of dystrophic muscle. These findings have encouraged the development of human therapies to block myostatin. However, little is known of the long-term effects on muscle of myostatin blockade. To evaluate potential sequelae from the prolonged absence of myostatin, senescent myostatin null (mstn-/-) mice were studied. Senescent mstn-/- mice continue to have normal muscle with increased mass and strength relative to controls. Muscles of senescent mstn-/- mice regenerate robustly from both chronic and acute injury. Early markers of regeneration are enhanced in the absence of myostatin, suggesting a mechanism for the attenuation of dystrophic features found in mdx mice lacking myostatin.

  6. INVITED REVIEW: Inhibitors of myostatin as methods of enhancing muscle growth and development.

    PubMed

    Chen, P R; Lee, K

    2016-08-01

    With the increasing demand for affordable, high-quality meat, livestock and poultry producers must continually find ways to maximize muscle growth in their animals without compromising palatability of the meat products. Muscle mass relies on myoblast proliferation during prenatal or prehatch stages and fiber hypertrophy through protein synthesis and nuclei donation by satellite cells after birth or hatch. Therefore, understanding the cellular and molecular mechanisms of myogenesis and muscle development is of great interest. Myostatin is a well-known negative regulator of muscle growth and development that inhibits proliferation and differentiation in myogenic cells as well as protein synthesis in existing muscle fibers. In this review, various inhibitors of myostatin activity or signaling are examined that may be used in animal agriculture for enhancing muscle growth. Myostatin inhibitors are relevant as potential therapies for muscle-wasting diseases and muscle weakness in humans and animals. Currently, there are no commercial myostatin inhibitors for agriculture or biomedical purposes because the safest and most effective option has yet to be identified. Further investigation of myostatin inhibitors and administration strategies may revolutionize animal production and the medical field.

  7. Quantitative proteomic reveals the dynamic of protein profile during final oocyte maturation in zebrafish.

    PubMed

    Ge, Chunmei; Lu, Weiqun; Chen, Aqin

    2017-08-26

    The molecular mechanisms underlying final oocyte maturation in zebrafish (Danio rerio) remain poorly understood. The present study aimed to employ iTRAQ approach for a comprehensive characterization of during zebrafish oocyte maturation proteome and for comparison between fully-grow immature and mature oocytes prior to ovulation. A total of 1568 proteins were identified, which was representing the largest zebrafish isolated oocytes proteome dataset to date. Differential expression analysis revealed 190 proteins significantly changes between immature and mature oocytes, which 136 proteins were up-regulated and 54 proteins were down-regulated in mature oocytes comparison with immature oocytes. Functional analysis revealed that these differential proteins were mostly involved in cellular response to estrogen stimulus, cellular components, extracellular region, and enzyme regulator activity, etc. The revealed differentially changes in protein expression patterns associated with oocyte maturation suggest that several of the examined proteins, such as vitellogenin(Vtg3), protein S100(S100A10), 17-beta hydroxysteroid dehydrogenase(HSD17B1), pentaxin, zona pellucida (ZP3.2), elongation factor1-alpha, caluemnin B, and 14-3-3 protein may play a specific role during zebrafish final oocyte maturation. These data will provide powerful information for understanding the molecular mechanism underlying zebrafish oocyte maturation, and these proteins may potentially act as markers to predict control oocyte maturation of zebrafish oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Dominant negative myostatin produces hypertrophy without hyperplasia in muscle.

    PubMed

    Zhu, X; Hadhazy, M; Wehling, M; Tidball, J G; McNally, E M

    2000-05-26

    Myostatin, a TGF-beta family member, is a negative regulator of muscle growth. Here, we generated transgenic mice that expressed myostatin mutated at its cleavage site under the control of a muscle specific promoter creating a dominant negative myostatin. These mice exhibited a significant (20-35%) increase in muscle mass that resulted from myofiber hypertrophy and not from myofiber hyperplasia. We also evaluated the role of myostatin in muscle degenerative states, such as muscular dystrophy, and found significant downregulation of myostatin. Thus, further inhibition of myostatin may permit increased muscle growth in muscle degenerative disorders.

  9. Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization

    PubMed Central

    McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.

    2011-01-01

    The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

  10. Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression.

    PubMed

    Rossi, Giuliana; Antonini, Stefania; Bonfanti, Chiara; Monteverde, Stefania; Vezzali, Chiara; Tajbakhsh, Shahragim; Cossu, Giulio; Messina, Graziella

    2016-03-08

    Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway.

  11. Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression

    PubMed Central

    Rossi, Giuliana; Antonini, Stefania; Bonfanti, Chiara; Monteverde, Stefania; Vezzali, Chiara; Tajbakhsh, Shahragim; Cossu, Giulio; Messina, Graziella

    2016-01-01

    Summary Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway. PMID:26923583

  12. Effects of maturation, diet, and estradiol on indices of protein degradation in rainbow trout (Oncorhychus mykiss)

    USDA-ARS?s Scientific Manuscript database

    Sexual maturation in salmonids requires mobilization of proteins from muscle tissue as evidenced by increased expression of proteolytic genes and decreased muscle protein content. However, it is unknown how ration level affects this proteolytic response. Female diploid rainbow trout (Oncorhynchus ...

  13. Purification and Crystallization of Murine Myostatin: A Negative Regulator of Muscle Mass

    NASA Technical Reports Server (NTRS)

    Hong, Young S.; Adamek, Daniel; Bridge, Kristi; Malone, Christine C.; Young, Ronald B.; Miller, Teresa; Karr, Laurel

    2004-01-01

    Myostatin (MSTN) has been crystallized and its preliminary X-ray diffraction data were collected. MSTN is a negative regulator of muscle growt/differentiation and suppressor of fat accumulation. It is a member of TGF-b family of proteins. Like other members of this family, the regulation of MSTN is critically tied to its process of maturation. This process involves the formation of a homodimer followed by two proteolytic steps. The first proteolytic cleavage produces a species where the n-terminal portion of the dimer is covalently separated from, but remains non-covalently bound to, the c-terminal, functional, portion of the protein. The protein is activated upon removal of the n-terminal "pro-segment" by a second n-terminal proteolytic cut by BMP-1 in vivo, or by acid treatment in vitro. Understanding the structural nature and physical interactions involved in these regulatory processes is the objective of our studies. Murine MSTN was purified from culture media of genetically engineered Chinese Hamster Ovary cells by multicolumn purification process and crystallized using the vapor diffusion method.

  14. Purification and Crystallization of Murine Myostatin: A Negative Regulator of Muscle Mass

    NASA Technical Reports Server (NTRS)

    Hong, Young S.; Adamek, Daniel; Bridge, Kristi; Malone, Christine C.; Young, Ronald B.; Miller, Teresa; Karr, Laurel

    2004-01-01

    Myostatin (MSTN) has been crystallized and its preliminary X-ray diffraction data were collected. MSTN is a negative regulator of muscle growt/differentiation and suppressor of fat accumulation. It is a member of TGF-b family of proteins. Like other members of this family, the regulation of MSTN is critically tied to its process of maturation. This process involves the formation of a homodimer followed by two proteolytic steps. The first proteolytic cleavage produces a species where the n-terminal portion of the dimer is covalently separated from, but remains non-covalently bound to, the c-terminal, functional, portion of the protein. The protein is activated upon removal of the n-terminal "pro-segment" by a second n-terminal proteolytic cut by BMP-1 in vivo, or by acid treatment in vitro. Understanding the structural nature and physical interactions involved in these regulatory processes is the objective of our studies. Murine MSTN was purified from culture media of genetically engineered Chinese Hamster Ovary cells by multicolumn purification process and crystallized using the vapor diffusion method.

  15. Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas.

    PubMed

    Kingtong, Sutin; Kellner, Kristell; Bernay, Benoît; Goux, Didier; Sourdaine, Pascal; Berthelin, Clothilde Heude

    2013-04-26

    Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P<0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries. This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa

  16. Myostatin--the holy grail for muscle, bone, and fat?

    PubMed

    Buehring, B; Binkley, N

    2013-12-01

    Myostatin, a member of the transforming growth factor beta (TGF-β) superfamily, was first described in 1997. Since then, myostatin has gained growing attention because of the discovery that myostatin inhibition leads to muscle mass accrual. Myostatin not only plays a key role in muscle homeostasis, but also affects fat and bone. This review will focus on the impact of myostatin and its inhibition on muscle mass/function, adipose tissue and bone density/geometry in humans. Although existing data are sparse, myostatin inhibition leads to increased lean mass and 1 study found a decrease in fat mass and increase in bone formation. In addition, myostatin levels are increased in sarcopenia, cachexia and bed rest whereas they are increased after resistance training, suggesting physiological regulatory of myostatin. Increased myostatin levels have also been found in obesity and levels decrease after weight loss from caloric restriction. Knowledge on the relationship of myostatin with bone is largely based on animal data where elevated myostatin levels lead to decreased BMD and myostatin inhibition improved BMD. In summary, myostatin appears to be a key factor in the integrated physiology of muscle, fat, and bone. It is unclear whether myostatin directly affects fat and bone, or indirectly via muscle. Whether via direct or indirect effects, myostatin inhibition appears to increase muscle and bone mass and decrease fat tissue-a combination that truly appears to be a holy grail. However, at this time, human data for both efficacy and safety are extremely limited. Moreover, whether increased muscle mass also leads to improved function remains to be determined. Ultimately potential beneficial effects of myostatin inhibition will need to be determined based on hard outcomes such as falls and fractures.

  17. Molecular characterization of myostatin from the skeletal muscle of the African lungfish, Protopterus annectens, and changes in its mRNA and protein expression levels during three phases of aestivation.

    PubMed

    Ong, Jasmine L Y; Chng, You R; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2017-05-01

    African lungfishes can aestivate and remain torpid without food and water for years, but disuse muscle atrophy is not prominent during aestivation. This study aimed to clone myostatin (mstn/Mstn), a factor associated with disuse muscle atrophy in mammals, from the skeletal muscle of the African lungfish Protopterus annectens, and to determine its mRNA expression level and protein abundance therein during the induction, maintenance, and arousal phases of aestivation. The complete coding cDNA sequence of mstn comprised 1128 bp, encoding for 376 amino acids with an estimated molecular mass of 42.9 kDa. It was grouped together with Mstn/MSTN of coelacanth and tetrapods in a clade separated from teleost Mstn. After 6 days (the induction phase) of aestivation, the mstn transcript level in the muscle increased significantly, while the protein abundance of Mstn remained comparable to the control. Following that, a significant increase in the expression levels of mstn/Mstn occurred on day 12 (the early maintenance phase) of aestivation. After 6 months of aestivation (the prolonged maintenance phase), the expression levels of mstn/Mstn returned to control levels, indicating the possible impediment of a drastic increase in muscle degradation to prevent muscle atrophy. During 1-3 days of arousal from aestivation, the expression levels of mstn/Mstn in the muscle remained comparable to the control. Hence, tissue reconstruction/regeneration of certain organs might not involve the mobilization of amino acids from the muscle during the early arousal. These results provide insights into how aestivating P. annectens regulates the expression of mstn/Mstn possibly to ameliorate disuse muscle atrophy.

  18. Cell wall acyl-lipids, proteins and polysaccharides in mature and germinated olive pollen.

    PubMed

    Rodríguez-Rosales, M P; Ferrol, N; Roldán, M; Belver, A; Donaire, J P

    1990-12-01

    Proteins, acyl-lipids and polysaccharides from cell walls of mature and germinated olive pollen were studied. In general, hemicelluloses are the most abundant polysaccharides, arabinose in mature and glucose in germinated pollen being the main components of these macromolecules. Protein content and its amino acid composition are very similar in walls from mature and germinated pollen, these compounds showing a weak acid character. Free-fatty acids are the most abundant lipid molecules in mature and germinated pollen walls and a decrease in acyl-lipids, especially in polar lipids, as well as a higher unsaturation of their fatty acid components are observed after germination.

  19. Genome walk of an unknown upstream region of myostatin gene in Spanish goats

    USDA-ARS?s Scientific Manuscript database

    Myostatin (MSTN) gene product also known as growth differentiation factor (GDF8) is a member of the TGF-ß family of secreted proteins. It is shown to be a negative regulator of muscle mass development. Mutations in the MSTN gene have been reported in mice, cattle and humans that lead to muscular hyp...

  20. Myostatin, a profibrotic factor and the main inhibitor of striated muscle mass, is present in the penile and vascular smooth muscle.

    PubMed

    Kovanecz, I; Masouminia, M; Gelfand, R; Vernet, D; Rajfer, J; Gonzalez-Cadavid, N F

    2017-09-01

    Myostatin is present in striated myofibers but, except for myometrial cells, has not been reported within smooth muscle cells (SMC). We investigated in the rat whether myostatin is present in SMC within the penis and the vascular wall and, if so, whether it is transcriptionally expressed and associated with the loss of corporal SMC occurring in certain forms of erectile dysfunction (ED). Myostatin protein was detected by immunohistochemistry/fluorescence and western blots in the perineal striated muscles, and also in the SMC of the penile corpora, arteries and veins, and aorta. Myostatin was found in corporal SMC cultures, and its transcriptional expression (and its receptor) was shown there by DNA microarrays. Myostatin protein was measured by western blots in the penile shaft of rats subjected to bilateral cavernosal nerve resection (BCNR), that were left untreated, or treated (45 days) with muscle-derived stem cells (MDSC), or concurrent daily low-dose sildenafil. Myostatin was not increased by BCNR (compared with sham operated animals), but over expressed after treatment with MDSC. This was reduced by concurrent sildenafil. The presence of myostatin in corporal and vascular SMC, and its overexpression in the corpora by MDSC therapy, may have relevance for the stem cell treatment of corporal fibrosis and ED.

  1. Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development.

    PubMed

    Yi, Young-Joo; Sutovsky, Miriam; Song, Won-Hee; Sutovsky, Peter

    2015-11-01

    Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation.

  2. Substrate-induced protein stabilization reveals a predominant contribution from mature protein to peptides presented on MHC class I

    PubMed Central

    Colbert, Jeff D.; Farfán-Arribas, Diego J.; Rock, Kenneth L.

    2013-01-01

    The origin of the MHC class I-presented peptides are thought to be primarily from newly synthesized but defective proteins, termed DRiPs. Most of the data supporting this concept come from studies where inhibitors of protein synthesis were found to rapidly block antigen presentation even when cells contained a pool of mature protein. However, these data only indirectly address the origin of presented peptides and in most studies the contribution of mature functional protein to the class I peptide pool has not been directly quantified. In this report we address the efficiency and contribution of mature protein by using a tetracycline-inducible system to express antigen that is conditionally stabilized upon ligand binding. This system circumvents the use of general inhibitors of protein synthesis to control antigen expression. Moreover, by controlling antigen stabilization, we could investigate whether the degradation of mature antigen contributed to antigen presentation at early and/or late time points. We show that mature protein is the major contributor of peptides presented on class I for two distinct antigenic constructs. Furthermore our data show that the protein synthesis inhibitors used previously to test the contribution of defective proteins actually block antigen presentation in ways that are independent from blocking antigen synthesis. These data suggest that for the constructs we have analyzed, mature functional protein rather than DRiPs are the predominant source of MHC class I presented-peptides PMID:24174619

  3. miRNA Transcriptome of Hypertrophic Skeletal Muscle with Overexpressed Myostatin Propeptide

    PubMed Central

    Jing, Lu; Li, Xinyun; Cao, Jianhua; Zhao, Shuhong

    2014-01-01

    MicroRNAs (miRNAs) play an imperative role in cell proliferation, differentiation, and cell metabolism through regulation of gene expression. Skeletal muscle hypertrophy that results from myostatin depression by its propeptide provides an interesting model to understand how miRNA transcriptome is involved in myostatin-based fiber hypertrophy. This study employed Solexa deep sequencing followed by Q-PCR methods to analyze miRNA transcriptome of skeletal muscle of myostatin propeptide transgenic mice in comparison with their littermate controls. A total of 461 mature known and 69 novel miRNAs were reported from this study. Fifty-seven miRNAs were expressed differentially between transgenic and littermate controls, of which most abundant miRNAs, miR-133a and 378a, were significantly differentially expressed. Expression profiling was validated on 8 known and 2 novel miRNAs. The miRNA targets prediction and pathway analysis showed that FST, SMAD3, TGFBR1, and AcvR1a genes play a vital role in skeletal muscle hypertrophy in the myostatin propeptide transgenic mice. It is predicted that miR-101 targeted to TGFBR1 and SMAD3, miR-425 to TGFBR2 and FST, and miR-199a to AcvR2a and TGF-β genes. In conclusion, the study offers initial miRNA profiling and methodology of miRNA targets prediction for myostatin-based hypertrophy. These differentially expressed miRNAs are proposed as candidate miRNAs for skeletal muscle hypertrophy. PMID:25147795

  4. Growth hormone differentially regulates muscle myostatin1 and -2 and increases circulating cortisol in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Biga, Peggy R; Cain, Kenneth D; Hardy, Ronald W; Schelling, Gerald T; Overturf, Kenneth; Roberts, Steven B; Goetz, Frederick W; Ott, Troy L

    2004-08-01

    Myostatin (MSTN) negatively regulates muscle growth in vertebrates. Salmonids produce two myostatin transcripts from separate genes. Surprisingly, quantitative analyses indicate different regulatory mechanisms for the two myostatin genes in rainbow trout. MSTN1 mRNA levels were elevated 26% following recombinant bovine growth hormone (rbGH) treatment, while MSTN2 mRNA levels were reduced 74% compared to controls. MSTN precursor protein (42kDa) levels were elevated in rbGH treated fish compared to controls. In addition, circulating cortisol levels were elevated 71% following rbGH treatment compared to controls. In treated and control fish, cortisol levels were elevated 245% at day 0 compared to subsequent days. Treated fish exhibited cortisol levels 207% higher than controls at 0.5 day, and remained at least 50% higher for 7 days following treatment. This pattern of change was positively correlated to MSTN1 mRNA levels. This is the first time a direct relationship has been reported between GH, cortisol, and myostatin. In addition, following rbGH administration, myosin protein concentrations in skeletal muscle samples increased, suggesting that GH regulates expression of the most abundant muscle protein. These results indicate the two myostatin genes are differentially regulated and may possess different functions in rainbow trout muscle, and suggests a possible interaction between GH, cortisol, and muscle growth.

  5. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

  6. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

    PubMed

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

  7. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

    PubMed Central

    Nielsen, P J; Thomas, G; Maller, J L

    1982-01-01

    A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor. Images PMID:7045876

  8. Upregulation of the plant protein remorin correlates with dehiscence and cell maturation

    PubMed Central

    Raffaele, Sylvain; Bayer, Emmanuelle

    2009-01-01

    Remorins are plant-specific proteins found associated with plasma membrane microdomains, called lipid rafts. Recently, we have shown that this lipid raft marker also accumulated at plasmodesmata, likely within the plasma membrane lining these structures. Here, we have investigated the gene expression and protein accumulation patterns of remorin at the organ and cell type levels. We show that remorin level is significantly increased in dehiscent, mature and ageing tissues, as well as in source parts of the leaves, where mature branched plasmodesmata are in majority. these results suggest that remorin predominantly associates with mature branched plasmodesmata. PMID:19826231

  9. Some motifs were important for myostatin transcriptional regulation in sheep (Ovis aries).

    PubMed

    Du, Rong; An, Xiao-Rong; Chen, Yong-Fu; Qin, Jian

    2007-07-31

    Many motifs along the 1.2 kb myostatin promoter (MSTNpro) in sheep have been found by the MatInspecter program in our recent study. To further verify the role of the motifs and better understand the transcriptional regulation mechanism of the myostatin gene in sheep, the reporter gene EGFP (enhanced green fluorescent protein) was selected and the wild-type (W) vector MSTNPro(W)-EGFP or motif-mutational (M) vector MSTNPro(M)-EGFP were constructed. The transcriptional regulation activities were analyzed by detecting the fluorescence strength of EGFP in C2C12 myoblasts transfected with the vectors. The results showed that E-box (E) 3, E4, E5 and E7, particularly E3, E5 and E7, had important effects on the activity of the 1.2 kb sheep myostatin promoter. In addition, we also detected several other important motifs such as MTBF (muscle-specific Mt binding factor), MEF2 (myocyte enhancer factor 2), GRE (glucocorticoid response elements) and PRE (progesterone response elements) along the sheep myostatin promoter by the mutational analysis.

  10. [Regulation of myostatin promoter activity by myocyte enhancer factor 2].

    PubMed

    Li, Jia; Deng, Jie; Zhang, Junlin; Cheng, De; Wang, Huayan

    2012-08-01

    Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

  11. High Variation of Fluorescence Protein Maturation Times in Closely Related Escherichia coli Strains

    PubMed Central

    Hebisch, Elke; Knebel, Johannes; Landsberg, Janek; Frey, Erwin; Leisner, Madeleine

    2013-01-01

    Fluorescent proteins (FPs) are widely used in biochemistry, biology and biophysics. For quantitative analysis of gene expression FPs are often used as marking molecules. Therefore, sufficient knowledge of maturation times and their affecting factors is of high interest. Here, we investigate the maturation process of the FPs GFP and mCherry expressed by the three closely related Escherichia coli strains of the Colicin E2 system, a model system for colicinogenic interaction. One strain, the C strain produces Colicin, a toxin to which the S strain is sensitive, and against which the R strain is resistant. Under the growth conditions used in this study, the S and R strain have similar growth rates, as opposed to the C strain whose growth rate is significantly reduced due to the toxin production. In combination with theoretical modelling we studied the maturation kinetics of the two FPs in these strains and could confirm an exponential and sigmoidal maturation kinetic for GFP and mCherry, respectively. Our subsequent quantitative experimental analysis revealed a high variance in maturation times independent of the strain studied. In addition, we determined strain dependent maturation times and maturation behaviour. Firstly, FPs expressed by the S and R strain mature on similar average time-scales as opposed to FPs expressed by the C strain. Secondly, dependencies of maturation time with growth conditions are most pronounced in the GFP expressing C strain: Doubling the growth rate of this C strain results in an increased maturation time by a factor of 1.4. As maturation times can vary even between closely related strains, our data emphasize the importance of profound knowledge of individual strains' maturation times for accurate interpretation of gene expression data. PMID:24155882

  12. Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

    PubMed

    Iwai, Chisato; Li, Peili; Kurata, Yasutaka; Hoshikawa, Yoshiko; Morikawa, Kumi; Maharani, Nani; Higaki, Katsumi; Sasano, Tetsuro; Notsu, Tomomi; Ishido, Yuko; Miake, Junichiro; Yamamoto, Yasutaka; Shirayoshi, Yasuaki; Ninomiya, Haruaki; Nakai, Akira; Murata, Shigeo; Yoshida, Akio; Yamamoto, Kazuhiro; Hiraoka, Masayasu; Hisatome, Ichiro

    2013-12-01

    We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

  13. Protein composition of oil bodies from mature Brassica napus seeds.

    PubMed

    Jolivet, Pascale; Boulard, Céline; Bellamy, Annick; Larré, Colette; Barre, Marion; Rogniaux, Hélène; d'Andréa, Sabine; Chardot, Thierry; Nesi, Nathalie

    2009-06-01

    Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies and/or myrosin cells, they must be purified step by step using floatation technique in order to remove non-specifically trapped proteins. An exhaustive description of the protein composition of rapeseed OBs from two double-zero varieties was achieved by a combination of proteomic and genomic tools. Genomic analysis led to the identification of sequences coding for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of these proteins were also identified through proteomic analysis and displayed a high level of sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin orthologs appeared acetylated on their N-terminal alanine residue and both caleosins and steroleosins displayed a low level of phosphorylation.

  14. Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process.

    PubMed

    Akiyama, Y; Griffith, R; Miller, P; Stevenson, G W; Lund, S; Kanapa, D J; Stevenson, H C

    1988-03-01

    Elutriator-purified human monocytes were cultured in a serum-free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate. We attempted to characterize certain of the serum protein(s) found in HuAB which promoted the monocyte maturation process. Human immunoglobulin G (IgG) was found to be the most potent serum protein in increasing 5'-N activity and decreasing peroxidase activity of suspension cultured monocytes. Immunoglobulin M (IgM) and albumin (Alb) were shown not to have significant monocyte maturation activity. Heat-treated human gamma globulin and IgG purified by high-performance liquid chromatography (HPLC) were shown to have patterns identical with that of untreated HGG and IgG with regard to promoting monocyte maturation; F(ab')2 was not an active maturation promoter, indicating the need for an intact Fc portion of the IgG molecule. Fibrinogen and fibronectin also had maturation promoting activity. Finally, addition of the potent monocyte functional activators, muramyl dipeptide (MDP), polyriboinosinic:polyribocytidilic acid (Poly I:C), and lipopolysaccharide (LPS) had no effect on the monocyte

  15. Seasonal variation in pectoralis muscle and heart myostatin and tolloid-like proteinases in small birds: a regulatory role for seasonal phenotypic flexibility?

    PubMed

    Swanson, David L; King, Marisa O; Harmon, Erin

    2014-02-01

    Seasonally variable environments produce seasonal phenotypes in small birds such that winter birds have higher thermogenic capacities and pectoralis and heart masses. One potential regulator of these seasonal phenotypes is myostatin, a muscle growth inhibitor, which may be downregulated under conditions promoting increased energy demand. We examined summer-to-winter variation in skeletal muscle and heart masses and used qPCR and Western blots to measure levels of myostatin and its metalloproteinase activators TLL-1 and TLL-2 for two small temperate-zone resident birds, American goldfinches (Spinus tristis) and black-capped chickadees (Poecile atricapillus). Winter pectoralis and heart masses were significantly greater than in summer for American goldfinches. Neither myostatin expression nor protein levels differed significantly between seasons for goldfinch pectoralis. However, myostatin levels in goldfinch heart were significantly greater in summer than in winter, although heart myostatin expression was seasonally stable. In addition, expression of both metalloproteinase activators was greater in summer than in winter goldfinches for both pectoralis and heart, significantly so except for heart TLL-2 (P = 0.083). Black-capped chickadees showed no significant seasonal variation in muscle or heart masses. Seasonal patterns of pectoralis and heart expression and/or protein levels for myostatin and its metalloproteinase activators in chickadees showed no consistent seasonal trends, which may help explain the absence of significant seasonal variation in muscle or heart masses for chickadees in this study. These data are partially consistent with a regulatory role for myostatin, and especially myostatin processing capacity, in mediating seasonal metabolic phenotypes of small birds.

  16. G protein-coupled receptors in child development, growth, and maturation.

    PubMed

    Latronico, Ana Claudia; Hochberg, Ze'ev

    2010-10-12

    G protein-coupled receptors (GPCRs) constitute a large family of cell membrane receptors that affect embryogenesis, development, and child physiology, and they are targets for approved drugs and those still in development. The sensitivity of GPCRs to their respective extracellular hormones, neurotransmitters, and environmental stimulants, as well as their interaction with other receptors and intracellular signaling proteins (such as receptor activity-modifying proteins), contribute to variations in child development, growth, and maturation. Here, we summarize current knowledge about the mechanisms of activation (in either the presence or absence of ligands) that lead to the sensitivities of GPCRs and their respective effects as seen throughout human developmental and maturational phases.

  17. The growth factor myostatin, a key regulator in skeletal muscle growth and homeostasis.

    PubMed

    Matsakas, A; Diel, P

    2005-03-01

    Skeletal muscle possesses the ability to both respond and adapt to changing environmental stimuli, leading to a set of metabolic and morphological adaptations, which allow it to better meet the energy demands of sustained physical activity. Great progress has been achieved over the past years by means of innovative molecular techniques, which has led to the discovery of new growth factors and the identification of molecular mechanisms involved in the regulation of muscle development. These findings provide new starting points to understand the molecular mechanisms involved in the adaptation of skeletal muscle to exercise training. One of these new identified growth factors is myostatin, a member of the transforming growth factor-beta family of proteins that has been demonstrated to play a fundamental role in the regulation of skeletal muscle growth during embryogenesis. Blocking of the myostatin signalling transduction pathway by specific inhibitors and genetic manipulations has been shown to result in a dramatic increase of skeletal muscle mass. This review focuses on the importance of myostatin in mediating skeletal muscle homeostasis in response to training as well as during the progress of myogenic disease, like atrophy or dystrophy. Manipulations of myostatin signalling may be useful for agriculture applications, treatment of muscle diseases, inhibition of muscle atrophy and last but not least as life style drugs in antiaging therapies or manipulations of the muscle to fat ratio. Drugs with the ability to modulate myostatin signalling may have the potential to enhance physical performance in athletes and therefore they probably represent a new class of doping substances.

  18. 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

  19. Myostatin - From the Mighty Mouse to cardiovascular disease and cachexia.

    PubMed

    Dschietzig, Thomas Bernd

    2014-06-10

    In 1997, McPherron et al. created the so-called Mighty Mouse: owing to the knock-out of a new member of the TGF-β superfamily of peptides, this mouse line was extremely hypermuscular and also characterized by very low body fat. The new peptide, a powerful negative muscle regulator, was named myostatin. Apart from regulating skeletal muscle growth, myostatin has recently been reported to be significantly involved in different cardio-vascular and metabolic pathologies. This review is focused on these non-muscular myostatin actions. First, myostatin is intricately involved in regulating metabolism: it causes insulin resistance, and the advantageous metabolic profile achieved by myostatin inhibition is mainly attributable to its effects on skeletal muscle. Myostatin is further expressed in myocardium where it exerts anti-hypertrophic, but pro-fibrotic effects. Circulating and local myostatin is elevated in chronic heart failure and poses a major player in cardiac cachexia. Eventually, the current body of evidence regarding myostatin's significant involvement in different entities of the cachexia syndrome is summarized. Activin type-2 receptor antagonism and/or inhibitory myostatin antibodies have emerged as a promising therapeutic approach to treat the cachexia syndrome although the general applicability of this therapeutic approach to the human clinical situation has still to be demonstrated.

  20. Myostatin inhibits proliferation of human urethral rhabdosphincter satellite cells.

    PubMed

    Akita, Yasuyuki; Sumino, Yasuhiro; Mori, Ken-ichi; Nomura, Takeo; Sato, Fuminori; Mimata, Hiromitsu

    2013-05-01

    Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of myogenesis in skeletal muscle. We examined the effect of myostatin and myostatin inhibition by an antagonistic agent, follistatin, on growth of human urethral rhabdosphincter satellite cells (muscle stem cells) to develop a new strategy for treatment of stress urinary incontinence. Rhabdosphincter satellite cells were cultured and selected by magnetic affinity cell sorting using an anti-neural cell adhesion molecule antibody. The cells were transfected with simian virus-40 antigen to extend their lifespan. A cell proliferation assay, a cell cycle analysis and an investigation of signal transduction were carried out. The autocrine action of endogenous myostatin by western blotting, real-time reverse transcription polymerase chain reaction and immunoneutralization using an anti-myostatin antibody was also evaluated. Selectively cultured cells expressed markers of striated muscles and successfully differentiated into myotubes. Myostatin inhibited proliferation of these cells through Smad2 phosphorylation and cell cycle arrest. Inhibitory effects of myostatin were reversed by addition of follistatin. However, rhabdosphincter satellite cells did not appear to use autocrine secretion of myostatin to regulate their proliferation. Inhibition of myostatin function might be a useful pathway in the development of novel strategies for stimulating rhabdosphincter cells regeneration to treat stress urinary incontinence. © 2012 The Japanese Urological Association.

  1. Polybromo protein BAF180 functions in mammalian cardiac chamber maturation

    PubMed Central

    Wang, Zhong; Zhai, Weiguo; Richardson, James A.; Olson, Eric N.; Meneses, Juanito J.; Firpo, Meri T.; Kang, Chulho; Skarnes, William C.; Tjian, Robert

    2004-01-01

    BAF and PBAF are two related mammalian chromatin remodeling complexes essential for gene expression and development. PBAF, but not BAF, is able to potentiate transcriptional activation in vitro mediated by nuclear receptors, such as RXRα, VDR, and PPARγ. Here we show that the ablation of PBAF-specific subunit BAF180 in mouse embryos results in severe hypoplastic ventricle development and trophoblast placental defects, similar to those found in mice lacking RXRα and PPARγ. Embryonic aggregation analyses reveal that in contrast to PPARγ-deficient mice, the heart defects are likely a direct result of BAF180 ablation, rather than an indirect consequence of trophoblast placental defects. We identified potential target genes for BAF180 in heart development, such as S100A13 as well as retinoic acid (RA)-induced targets RARβ2 and CRABPII. Importantly, BAF180 is recruited to the promoter of these target genes and BAF180 deficiency affects the RA response for CRABPII and RARβ2. These studies reveal unique functions of PBAF in cardiac chamber maturation. PMID:15601824

  2. The Role of RING Box Protein 1 in Mouse Oocyte Meiotic Maturation

    PubMed Central

    Zhou, Lin; Yang, Ye; Zhang, Juanjuan; Guo, Xuejiang; Bi, Ye; Li, Xin; Zhang, Ping; Zhang, Junqiang; Lin, Min; Zhou, Zuomin; Shen, Rong; Guo, Xirong; Huo, Ran; Ling, Xiufeng; Sha, Jiahao

    2013-01-01

    RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCFβTrCP-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation. PMID:23874827

  3. Requirement of enhanced Survival Motoneuron protein imposed during neuromuscular junction maturation

    PubMed Central

    Kariya, Shingo; Obis, Teresa; Garone, Caterina; Akay, Turgay; Sera, Fusako; Iwata, Shinichi; Homma, Shunichi; Monani, Umrao R.

    2014-01-01

    Spinal muscular atrophy is a common motor neuron disease caused by low survival motoneuron (SMN), a key protein in the proper splicing of genes. Restoring the protein is therefore a promising therapeutic strategy. Implementation of this strategy, however, depends on defining the temporal requirements for SMN. Here, we used controlled knockdown of SMN in transgenic mice to determine the precise postnatal stage requirements for this protein. Reducing SMN in neonatal mice resulted in a classic SMA-like phenotype. Unexpectedly, depletion of SMN in adults had relatively little effect. Insensitivity to low SMN emerged abruptly at postnatal day 17, which coincided with establishment of the fully mature neuromuscular junction (NMJ). Mature animals depleted of SMN eventually exhibited evidence of selective neuromuscular pathology that was made worse by traumatic injury. The ability to regenerate the mature NMJ in aged or injured SMN-depleted mice was grossly impaired, a likely consequence of the inability to meet the surge in demand for motoneuronal SMN that was seen in controls. Our results demonstrate that relative maturity of the NMJ determines the temporal requirement for the SMN protein. These observations suggest that the use of potent but potentially deleterious SMN-enhancing agents could be tapered in human patients once the neuromuscular system matures and reintroduced as needed to enhance SMN for remodeling aged or injured NMJs. PMID:24463453

  4. Target genes of myostatin loss-of-function in muscles of late bovine fetuses

    PubMed Central

    Cassar-Malek, Isabelle; Passelaigue, Florent; Bernard, Carine; Léger, Jean; Hocquette, Jean-François

    2007-01-01

    Background Myostatin, a muscle-specific member of the Transforming Growth Factor beta family, negatively regulates muscle development. Double-muscled (DM) cattle have a loss-of-function mutation in their myostatin gene responsible for the hypermuscular phenotype. Thus, these animals are a good model for understanding the mechanisms underpinning muscular hypertrophy. In order to identify individual genes or networks that may be myostatin targets, we looked for genes that were differentially expressed between DM and normal (NM) animals (n = 3 per group) in the semitendinosus muscle (hypertrophied in DM animals) at 260 days of fetal development (when the biochemical differentiation of muscle is intensive). A heterologous microarray (human and murine oligonucleotide sequences) of around 6,000 genes expressed in muscle was used. Results Many genes were found to be differentially expressed according to genetic type (some with a more than 5-fold change), and according to the presence of one or two functional myostatin allele(s). They belonged to various functional categories. The genes down-regulated in DM fetuses were mainly those encoding extracellular matrix proteins, slow contractile proteins and ribosomal proteins. The genes up-regulated in DM fetuses were mainly involved in the regulation of transcription, cell cycle/apoptosis, translation or DNA metabolism. These data highlight features indicating that DM muscle is shifted towards a more glycolytic metabolism, and has an altered extracellular matrix composition (e.g. down-regulation of COL1A1 and COL1A2, and up-regulation of COL4A2) and decreased adipocyte differentiation (down-regulation of C1QTNF3). The altered gene expression in the three major muscle compartments (fibers, connective tissue and intramuscular adipose tissue) is consistent with the well-known characteristics of DM cattle. In addition, novel potential targets of the myostatin gene were identified (MB, PLN, troponins, ZFHX1B). Conclusion Thus, the

  5. Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

    SciTech Connect

    Retamales, A.; Zuloaga, R.; Valenzuela, C.A.; Gallardo-Escarate, C.; Molina, A.; Valdés, J.A.

    2015-08-21

    Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletal myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.

  6. Evolution of green coffee protein profiles with maturation and relationship to coffee cup quality.

    PubMed

    Montavon, Philippe; Duruz, Eliane; Rumo, Gilbert; Pratz, Gudrun

    2003-04-09

    Coffee flavor is the product of a complex chain of chemical transformations. The green bean has only a faint odor that is not at all reminiscent of coffee aroma. It contains, however, all of the necessary precursors to generate the unmistakable coffee flavor during roasting. The levels and biochemical status of these precursors may vary in relation to genetic traits, environmental factors, maturation level, postharvest treatment, and storage. To improve our understanding of coffee flavor generation, the sensory and biochemical impact of maturation was assessed. Maturation clearly favored the development of high-quality flavor in the coffee brew. A specific subclass of green coffee beans, however, generated high-quality coffee flavor irrespective of maturation. Biochemical aspects were examined using a dynamic system: immature and mature green coffee suspensions were incubated under air or argon. On the analytical side, a specific pool of flavor precursors was monitored: chlorogenic acids, green coffee proteins, and free amino acids. A link between maturation, the redox behavior of green coffee suspensions, and their sensory scores was identified. Compared to ripe beans, unripe beans were found to be more sensitive to oxidation of chlorogenic acids. Aerobic incubation also triggered the fragmentation or digestion of the 11S seed storage protein and the release of free amino acids.

  7. Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

    PubMed Central

    Moore, Matthew M.; Oteng-Pabi, Samuel K.; Pandelieva, Antonia T.; Mayo, Stephen L.; Chica, Roberto A.

    2012-01-01

    Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore. PMID:23285050

  8. Myostatin gene targeting in cultured China Han ovine myoblast cells.

    PubMed

    Zhang, L; Yang, X; An, X; Chen, Y

    2007-11-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, has been shown to be a negative regulator of myogenesis. Natural mutation in beef cattle causes double-muscling phenotypes. We report an investigation designed to knockout the MSTN gene by gene targeting in ovine myoblast cells. Two promoter-trap targeting vectors MSTN-green fluorescent protein (GFP) and MSTN-neo were constructed and used to transfect foetal and neonatal ovine primary myoblast cells. Both GFP-expressing cells and drug-resistant cells were obtained. Targeted cells expressing GFP were confirmed by polymerase chain reaction (PCR) assay and drug-resistant cells were characterised by PCR and Southern blot after growing into cell clones.

  9. Synergistic and Antagonistic Interplay between Myostatin Gene Expression and Physical Activity Levels on Gene Expression Patterns in Triceps Brachii Muscles of C57/BL6 Mice

    PubMed Central

    Caetano-Anollés, Kelsey; Mishra, Sanjibita; Rodriguez-Zas, Sandra L.

    2015-01-01

    Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current

  10. Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function

    PubMed Central

    Islam, Md Soriful; Catherino, William H.; Protic, Olga; Janjusevic, Milijana; Gray, Peter Clarke; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Lamanna, Pasquale; Tranquilli, Andrea Luigi; Petraglia, Felice

    2014-01-01

    Context: Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. Objective: The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. Design: This was a laboratory study. Setting: Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. Patients: The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Interventions: Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. Results: We found that activin-A and myostatin significantly reduce cell proliferation in primary myometrial cells but not in leiomyoma cells as measured by a CyQUANT cell proliferation assay kit. Reduced expression of proliferating cell nuclear antigen and Ki-67 were also observed in myometrial cells in response to activin-A and myostatin treatment. Activin-A also significantly increased mRNA expression of fibronectin, collagen1A1, and versican in primary leiomyoma cells. Finally, we found that activin-A and myostatin activate Smad-2/3 signaling but do not affect ERK or p38 signaling in both myometrial and leiomyoma cells. Conclusions: This study results suggest that activin-A and myostatin can exert antiproliferative and/or fibrotic effects on these cell types via Smad-2/3 signaling. PMID:24606069

  11. Myostatin promotes the terminal differentiation of embryonic muscle progenitors.

    PubMed

    Manceau, Marie; Gros, Jérôme; Savage, Kathleen; Thomé, Virginie; McPherron, Alexandra; Paterson, Bruce; Marcelle, Christophe

    2008-03-01

    Myostatin, a TGF-beta family member, is an important regulator of adult muscle size. While extensively studied in vitro, the mechanisms by which this molecule mediates its effect in vivo are poorly understood. We addressed this question using chick and mouse embryos. We show that while myostatin overexpression in chick leads to an exhaustion of the muscle progenitor population that ultimately results in muscle hypotrophy, myostatin loss of function in chick and mouse provokes an expansion of this population. Our data demonstrate that myostatin acts in vivo to regulate the balance between proliferation and differentiation of embryonic muscle progenitors by promoting their terminal differentiation through the activation of p21 and MyoD. Previous studies have suggested that myostatin imposes quiescence on muscle progenitors. Our data suggest that myostatin's effect on muscle progenitors is more complex than previously realized and is likely to be context-dependent. We propose a novel model for myostatin mode of action in vivo, in which myostatin affects the balance between proliferation and differentiation of embryonic muscle progenitors by enhancing their differentiation.

  12. The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

    SciTech Connect

    Li, Long; Lok, Shee-Mei; Yu, I-Mei; Zhang, Ying; Kuhn, Richard J.; Chen, Jue; Rossmann, Michael G.

    2008-09-17

    Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

  13. The LGI1-ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function.

    PubMed

    Lovero, Kathryn L; Fukata, Yuko; Granger, Adam J; Fukata, Masaki; Nicoll, Roger A

    2015-07-28

    Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

  14. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    SciTech Connect

    Downs, S.M. )

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  15. Serum irisin and myostatin levels after 2 weeks of high-altitude climbing.

    PubMed

    Śliwicka, Ewa; Cisoń, Tomasz; Kasprzak, Zbigniew; Nowak, Alicja; Pilaczyńska-Szcześniak, Łucja

    2017-01-01

    Exposure to high-altitude hypoxia causes physiological and metabolic adaptive changes by disturbing homeostasis. Hypoxia-related changes in skeletal muscle affect the closely interconnected energy and regeneration processes. The balance between protein synthesis and degradation in the skeletal muscle is regulated by several molecules such as myostatin, cytokines, vitamin D, and irisin. This study investigates changes in irisin and myostatin levels in male climbers after a 2-week high-altitude expedition, and their association with 25(OH)D and indices of inflammatory processes. The study was performed in 8 men aged between 23 and 31 years, who participated in a 2-week climbing expedition in the Alps. The measurements of body composition and serum concentrations of irisin, myostatin, 25(OH)D, interleukin-6, myoglobin, high-sensitivity C-reactive protein, osteoprotegerin, and high-sensitivity soluble receptor activator of NF-κB ligand (sRANKL) were performed before and after expedition. A 2-week exposure to hypobaric hypoxia caused significant decrease in body mass, body mass index (BMI), free fat mass and irisin, 25-Hydroxyvitamin D levels. On the other hand, significant increase in the levels of myoglobin, high-sensitivity C-reactive protein, interleukin-6, and osteoprotegerin were noted. The observed correlations of irisin with 25(OH)D levels, as well as myostatin levels with inflammatory markers and the OPG/RANKL ratio indicate that these myokines may be involved in the energy-related processes and skeletal muscle regeneration in response to 2-week exposure to hypobaric hypoxia.

  16. MicroRNA-208b progressively declines after spinal cord injury in humans and is inversely related to myostatin expression

    PubMed Central

    Boon, Hanneke; Sjögren, Rasmus J O; Massart, Julie; Egan, Brendan; Kostovski, Emil; Iversen, Per O; Hjeltnes, Nils; Chibalin, Alexander V; Widegren, Ulrika; Zierath, Juleen R

    2015-01-01

    The effects of long-term physical inactivity on the expression of microRNAs involved in the regulation of skeletal muscle mass in humans are largely unknown. MicroRNAs are short, noncoding RNAs that fine-tune target expression through mRNA degradation or by inhibiting protein translation. Intronic to the slow, type I, muscle fiber type genes MYH7 and MYH7b, microRNA-208b and microRNA-499-5p are thought to fine-tune the expression of genes important for muscle growth, such as myostatin. Spinal cord injured humans are characterized by both skeletal muscle atrophy and transformation toward fast-twitch, type II fibers. We determined the expression of microRNA-208b, microRNA-499-5p, and myostatin in human skeletal muscle after complete cervical spinal cord injury. We also determined whether these microRNAs altered myostatin expression in rodent skeletal muscle. A progressive decline in skeletal muscle microRNA-208b and microRNA-499-5p expression occurred in humans during the first year after spinal cord injury and with long-standing spinal cord injury. Expression of myostatin was inversely correlated with microRNA-208b and microRNA-499-5p in human skeletal muscle after spinal cord injury. Overexpression of microRNA-208b in intact mouse skeletal muscle decreased myostatin expression, whereas microRNA-499-5p was without effect. In conclusion, we provide evidence for an inverse relationship between expression of microRNA-208b and its previously validated target myostatin in humans with severe skeletal muscle atrophy. Moreover, we provide direct evidence that microRNA-208b overexpression decreases myostatin gene expression in intact rodent muscle. Our results implicate that microRNA-208b modulates myostatin expression and this may play a role in the regulation of skeletal muscle mass following spinal cord injury. PMID:26603456

  17. MicroRNA-208b progressively declines after spinal cord injury in humans and is inversely related to myostatin expression.

    PubMed

    Boon, Hanneke; Sjögren, Rasmus J O; Massart, Julie; Egan, Brendan; Kostovski, Emil; Iversen, Per O; Hjeltnes, Nils; Chibalin, Alexander V; Widegren, Ulrika; Zierath, Juleen R

    2015-11-01

    The effects of long-term physical inactivity on the expression of microRNAs involved in the regulation of skeletal muscle mass in humans are largely unknown. MicroRNAs are short, noncoding RNAs that fine-tune target expression through mRNA degradation or by inhibiting protein translation. Intronic to the slow, type I, muscle fiber type genes MYH7 and MYH7b, microRNA-208b and microRNA-499-5p are thought to fine-tune the expression of genes important for muscle growth, such as myostatin. Spinal cord injured humans are characterized by both skeletal muscle atrophy and transformation toward fast-twitch, type II fibers. We determined the expression of microRNA-208b, microRNA-499-5p, and myostatin in human skeletal muscle after complete cervical spinal cord injury. We also determined whether these microRNAs altered myostatin expression in rodent skeletal muscle. A progressive decline in skeletal muscle microRNA-208b and microRNA-499-5p expression occurred in humans during the first year after spinal cord injury and with long-standing spinal cord injury. Expression of myostatin was inversely correlated with microRNA-208b and microRNA-499-5p in human skeletal muscle after spinal cord injury. Overexpression of microRNA-208b in intact mouse skeletal muscle decreased myostatin expression, whereas microRNA-499-5p was without effect. In conclusion, we provide evidence for an inverse relationship between expression of microRNA-208b and its previously validated target myostatin in humans with severe skeletal muscle atrophy. Moreover, we provide direct evidence that microRNA-208b overexpression decreases myostatin gene expression in intact rodent muscle. Our results implicate that microRNA-208b modulates myostatin expression and this may play a role in the regulation of skeletal muscle mass following spinal cord injury. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  18. Hypoxia inhibits maturation and trafficking of HERG K+ channel protein: Role of Hsp90 and ROS

    PubMed Central

    Nanduri, Jayasri; Bergson, Pamela; Wang, Ning; Ficker, Eckhard; Prabhakar, Nanduri R.

    2009-01-01

    We previously reported that reactive oxygen species (ROS) generated during hypoxia decrease hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we investigated the molecular mechanisms involved in hypoxia induced downregulation of hERG protein. Culturing cells at low temperatures and addition of chemical chaperones during hypoxia restored hERG expression and currents to normoxic levels while antiarrhythmic drugs, which selectively block hERG channels, had no effect on hERG protein levels. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Co-immunoprecipitation experiments revealed that hypoxia inhibited interaction of hERG with Hsp90 chaperone required for maturation, which was restored in the presence of ROS scavengers. These results demonstrate that ROS generated during hypoxia prevents maturation of the hERG protein by inhibiting Hsp90 interaction resulting in decreased protein expression and currents. PMID:19654002

  19. Critical roles for WDR72 in calcium transport and matrix protein removal during enamel maturation

    PubMed Central

    Wang, Shih-Kai; Hu, Yuanyuan; Yang, Jie; Smith, Charles E; Nunez, Stephanie M; Richardson, Amelia S; Pal, Soumya; Samann, Andrew C; Hu, Jan C-C; Simmer, James P

    2015-01-01

    Defects in WDR72 (WD repeat-containing protein 72) cause autosomal recessive hypomaturation amelogenesis imperfecta. We generated and characterized Wdr72-knockout/lacZ-knockin mice to investigate the role of WDR72 in enamel formation. In all analyses, enamel formed by Wdr72 heterozygous mice was indistinguishable from wild-type enamel. Without WDR72, enamel mineral density increased early during the maturation stage but soon arrested. The null enamel layer was only a tenth as hard as wild-type enamel and underwent rapid attrition following eruption. Despite the failure to further mineralize enamel deposited during the secretory stage, ectopic mineral formed on the enamel surface and penetrated into the overlying soft tissue. While the proteins in the enamel matrix were successfully degraded, the digestion products remained inside the enamel. Interactome analysis of WDR72 protein revealed potential interactions with clathrin-associated proteins and involvement in ameloblastic endocytosis. The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts. Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane. Fewer blood vessels were observed in the papillary layer supporting ameloblasts. Specific WDR72 expression by maturation stage ameloblasts explained the observation that enamel thickness and rod decussation (established during the secretory stage) are normal in the Wdr72 null mice. We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization. PMID:26247047

  20. Peroxisome proliferator-activated receptor β/δ induces myogenesis by modulating myostatin activity.

    PubMed

    Bonala, Sabeera; Lokireddy, Sudarsanareddy; Arigela, Harikumar; Teng, Serena; Wahli, Walter; Sharma, Mridula; McFarlane, Craig; Kambadur, Ravi

    2012-04-13

    Classically, peroxisome proliferator-activated receptor β/δ (PPARβ/δ) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARβ/δ during skeletal muscle growth and regeneration. Although PPARβ/δ has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPARβ/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARβ/δ-specific ligand (L165041) and the PPARβ/δ-null mouse model, that PPARβ/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPARβ/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARβ/δ-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPARβ/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPARβ/δ. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPARβ/δ activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPARβ/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1.

  1. Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits.

    PubMed

    Liu, Ying; Bouhenni, Rachida A; Dufresne, Craig P; Semba, Richard D; Edward, Deepak P

    2016-01-01

    Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.

  2. Structural basis for the fast maturation of Arthropoda green fluorescent protein.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew E; Egorov, Nikolay S; Zaraisky, Andrey G; Yampolsky, Ilya V; Merzlyak, Ekaterina M; Shkoporov, Andrey N; Sander, Ian; Lukyanov, Konstantin A; Chudakov, Dmitriy M

    2006-10-01

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

  3. 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

  4. Generation of a fast maturating red fluorescent protein by a combined approach of elongation mutagenesis and functional salvage screening

    SciTech Connect

    Choi, Eun-Sil; Han, Sang-Soo; Cheong, Dea-Eun; Park, Mi-Young; Kim, Jeong-Sun; Kim, Geun-Joong

    2010-01-01

    Fluorescent proteins that can be useful as indicators or reporters must have rapid maturation time, high quantum yield and photobleaching stability. A red fluorescent protein DsRed that has a high quantum yield and photostability has an innately slow maturation time when compared to other fluorescence proteins. In this study, we combined a functional salvage screen (FSS) and elongation mutagenesis to obtain a DsRed variant that maintained structural features closely linked with a high quantum yield and photostability and evolved to have a rapid maturation time. It is expected that the variant generated here, FmRed (fast maturating red fluorescent protein), will be widely used as an indicator or reporter because it maintained traits superior to that of the wild-type protein and also matured rapidly.

  5. Myostatin promotes the terminal differentiation of embryonic muscle progenitors

    PubMed Central

    Manceau, Marie; Gros, Jérôme; Savage, Kathleen; Thomé, Virginie; McPherron, Alexandra; Paterson, Bruce; Marcelle, Christophe

    2008-01-01

    Myostatin, a TGF-β family member, is an important regulator of adult muscle size. While extensively studied in vitro, the mechanisms by which this molecule mediates its effect in vivo are poorly understood. We addressed this question using chick and mouse embryos. We show that while myostatin overexpression in chick leads to an exhaustion of the muscle progenitor population that ultimately results in muscle hypotrophy, myostatin loss of function in chick and mouse provokes an expansion of this population. Our data demonstrate that myostatin acts in vivo to regulate the balance between proliferation and differentiation of embryonic muscle progenitors by promoting their terminal differentiation through the activation of p21 and MyoD. Previous studies have suggested that myostatin imposes quiescence on muscle progenitors. Our data suggest that myostatin’s effect on muscle progenitors is more complex than previously realized and is likely to be context-dependent. We propose a novel model for myostatin mode of action in vivo, in which myostatin affects the balance between proliferation and differentiation of embryonic muscle progenitors by enhancing their differentiation. PMID:18316481

  6. Selection of dietary protein and carbohydrate by rats: Changes with maturation

    NASA Technical Reports Server (NTRS)

    Yokogoshi, Hidehiko; Theall, Cynthia L.; Wurtman, Richard J.

    1985-01-01

    Weaning (21-day-old; 40-50 g) male rats given simultaneous access to foods, containing 18 percent casein and 15 or 70 percent carbohydrate (dextrin), tended to consume only 29-35 percent as much protein as carbohydrate (i.e., protein/carbohydrate ratios were 0.29-0.35). With maturation, when animals weighed 100 g or more, about half continued this pattern of nutrient choice, but the others abruptly began to consume considerably larger proportions of protein, exhibiting protein/carbohydrate ratios as high as 0.80-1.00. Each adult animal's protein/carbohydrate ratio tended to vary only slightly (s.e. = 3 percent of means). Adult protein/carbohydrate ratios were not correlated with fasting brain 5-HT or 5-HIAA levels. These marked differences among rats in eating behavior would not be observed when--as is usually the case--animals are given access to only one diet.

  7. Selection of dietary protein and carbohydrate by rats: Changes with maturation

    NASA Technical Reports Server (NTRS)

    Yokogoshi, Hidehiko; Theall, Cynthia L.; Wurtman, Richard J.

    1985-01-01

    Weaning (21-day-old; 40-50 g) male rats given simultaneous access to foods, containing 18 percent casein and 15 or 70 percent carbohydrate (dextrin), tended to consume only 29-35 percent as much protein as carbohydrate (i.e., protein/carbohydrate ratios were 0.29-0.35). With maturation, when animals weighed 100 g or more, about half continued this pattern of nutrient choice, but the others abruptly began to consume considerably larger proportions of protein, exhibiting protein/carbohydrate ratios as high as 0.80-1.00. Each adult animal's protein/carbohydrate ratio tended to vary only slightly (s.e. = 3 percent of means). Adult protein/carbohydrate ratios were not correlated with fasting brain 5-HT or 5-HIAA levels. These marked differences among rats in eating behavior would not be observed when--as is usually the case--animals are given access to only one diet.

  8. Functional effect of mir-27b on myostatin expression: a relationship in piedmontese cattle with double-muscled phenotype

    PubMed Central

    2013-01-01

    Background In Piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the myostatin (MSTN) gene. The Piedmontese MSTN missense mutation G938A is translated to C313Y myostatin protein. This mutation alters MSTN function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. MiRNAs could play a role in skeletal muscle hypertrophy modulation by down-regulating gene expression. Results After identifying a 3′-UTR consensus sequence of several negative and positive modulator genes involved in the skeletal muscle hypertrophy pathway, such as IGF1, IGF1R, PPP3CA, NFATc1, MEF2C, GSK3b, TEAD1 and MSTN, we screened miRNAs matching to it. This analysis led to the identification of miR-27b, miR-132, miR-186 and miR-199b-5p as possible candidates. We collected samples of longissimus thoracis from twenty Piedmontese and twenty Friesian male bovines. In Piedmontese group miR-27b was up-regulated 7.4-fold (p < 0.05). Further, we report that the level of MSTN mRNA was about 5-fold lower in Piedmontese cattle vs Friesian cattle (p < 0.0001) and that less mature MSTN protein was detected in the Piedmontese one (p < 0.0001). Cotransfection of miR-27b and psi-check2 vector with the luciferase reporter gene linked to the bovine wild-type 3′-UTR of MSTN strongly inhibited the luciferase activity (79%, p < 0.0001). Conclusions These data demonstrate that bovine MSTN is a specific target of miR-27b and that miRNAs contribute to explain additive phenotypic hypertrophy in Piedmontese cattle selected for the MSTN gene mutation, possibly outlining a more precise genetic signature able to elucidate differences in muscle conformation. PMID:23510267

  9. Generation of myostatin B knockout yellow catfish (Tachysurus fulvidraco) using transcription activator-like effector nucleases.

    PubMed

    Dong, Zhangji; Ge, Jiachun; Xu, Zhiqiang; Dong, Xiaohua; Cao, Shasha; Pan, Jianlin; Zhao, Qingshun

    2014-06-01

    Myostatin (Mstn), a member of the transforming growth factor β superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

  10. Migration- and exercise-induced changes to flight muscle size in migratory birds and association with IGF1 and myostatin mRNA expression.

    PubMed

    Price, Edwin R; Bauchinger, Ulf; Zajac, Daria M; Cerasale, David J; McFarlan, Jay T; Gerson, Alexander R; McWilliams, Scott R; Guglielmo, Christopher G

    2011-09-01

    Seasonal adjustments to muscle size in migratory birds may result from preparatory physiological changes or responses to changed workloads. The mechanisms controlling these changes in size are poorly understood. We investigated some potential mediators of flight muscle size (myostatin and insulin-like growth factor, IGF1) in pectoralis muscles of wild wintering or migrating white-throated sparrows (Zonotrichia albicollis), captive white-throated sparrows that were photoperiod manipulated to be in a `wintering' or `migratory' (Zugunruhe) state, and captive European starlings (Sturnus vulgaris) that were either exercised for 2 weeks in a wind tunnel or untrained. Flight muscle size increased in photo-stimulated `migrants' and in exercised starlings. Acute exercise but not long-term training caused increased expression of IGF1, but neither caused a change in expression of myostatin or its metalloprotease activator TLL1. Photo-stimulated `migrant' sparrows demonstrated increased expression of both myostatin and IGF1, but wild sparrows exhibited no significant seasonal changes in expression of either myostatin or IGF1. Additionally, in both study species we describe several splice variants of myostatin that are shared with distantly related bird species. We demonstrate that their expression patterns are not different from those of the typical myostatin, suggesting that they have no functional importance and may be mistakes of the splicing machinery. We conclude that IGF1 is likely to be an important mediator of muscle phenotypic flexibility during acute exercise and during endogenous, seasonal preparation for migration. The role of myostatin is less clear, but its paradoxical increase in photo-stimulated `migrants' may indicate a role in seasonal adjustments of protein turnover.

  11. Cross-training in birds: cold and exercise training produce similar changes in maximal metabolic output, muscle masses and myostatin expression in house sparrows (Passer domesticus)

    PubMed Central

    Zhang, Yufeng; Eyster, Kathleen; Liu, Jin-Song; Swanson, David L.

    2015-01-01

    ABSTRACT Maximal metabolic outputs for exercise and thermogenesis in birds presumably influence fitness through effects on flight and shivering performance. Because both summit (Msum, maximum thermoregulatory metabolic rate) and maximum (MMR, maximum exercise metabolic rate) metabolic rates are functions of skeletal muscle activity, correlations between these measurements and their mechanistic underpinnings might occur. To examine whether such correlations occur, we measured the effects of experimental cold and exercise training protocols for 3 weeks on body (Mb) and muscle (Mpec) masses, basal metabolic rate (BMR), Msum, MMR, pectoralis mRNA and protein expression for myostatin, and mRNA expression of TLL-1 and TLL-2 (metalloproteinase activators of myostatin) in house sparrows (Passer domesticus). Both training protocols increased Msum, MMR, Mb and Mpec, but BMR increased with cold training and decreased with exercise training. No significant differences occurred for pectoralis myostatin mRNA expression, but cold and exercise increased the expression of TLL-1 and TLL-2. Pectoralis myostatin protein levels were generally reduced for both training groups. These data clearly demonstrate cross-training effects of cold and exercise in birds, and are consistent with a role for myostatin in increasing pectoralis muscle mass and driving organismal increases in metabolic capacities. PMID:25987736

  12. Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs.

    PubMed

    Cai, Chunbo; Qian, Lili; Jiang, Shengwang; Sun, Youde; Wang, Qingqing; Ma, Dezun; Xiao, Gaojun; Li, Biao; Xie, Shanshan; Gao, Ting; Chen, Yaoxing; Liu, Jie; An, Xiaorong; Cui, Wentao; Li, Kui

    2017-05-23

    Myostatin-deficient mice showed a remarkable hypertrophy of skeletal muscle, with a decreased fat mass and enhanced insulin sensitivity. Currently, it is unclear if the inhibition of myostatin could be used as an approach to treat human obesity and insulin resistance. In this study, we investigated if the inhibition of porcine myostatin has any effect on fat deposition and insulin sensitivity using genetically engineered Meishan pigs containing a myostatin loss-of-function mutation (Mstn -/- ). Our results indicated that, when compared with wild-type pigs, the amount of subcutaneous fat and leaf fat of Mstn -/- pigs were significantly decreased mainly due to the browning of subcutaneous adipose tissue. Additionally, the serum insulin level decreased and the insulin sensitivity increased significantly in Mstn -/- pigs. Moreover, we found a significant increase in levels of insulin receptor and insulin receptor substrate proteins in skeletal muscle of Mstn -/- pigs, which then activating the insulin signaling pathway. Irisin-mediated regulation is not the only pathway for the activation of insulin signal in Mstn -/- skeletal muscle. This study provides valuable insight for the treatment of human obesity and diabetes mellitus.

  13. Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs

    PubMed Central

    Cai, Chunbo; Qian, Lili; Jiang, Shengwang; Sun, Youde; Wang, Qingqing; Ma, Dezun; Xiao, Gaojun; Li, Biao; Xie, Shanshan; Gao, Ting; Chen, Yaoxing; Liu, Jie; An, Xiaorong; Cui, Wentao; Li, Kui

    2017-01-01

    Myostatin-deficient mice showed a remarkable hypertrophy of skeletal muscle, with a decreased fat mass and enhanced insulin sensitivity. Currently, it is unclear if the inhibition of myostatin could be used as an approach to treat human obesity and insulin resistance. In this study, we investigated if the inhibition of porcine myostatin has any effect on fat deposition and insulin sensitivity using genetically engineered Meishan pigs containing a myostatin loss-of-function mutation (Mstn −/− ). Our results indicated that, when compared with wild-type pigs, the amount of subcutaneous fat and leaf fat of Mstn −/− pigs were significantly decreased mainly due to the browning of subcutaneous adipose tissue. Additionally, the serum insulin level decreased and the insulin sensitivity increased significantly in Mstn −/− pigs. Moreover, we found a significant increase in levels of insulin receptor and insulin receptor substrate proteins in skeletal muscle of Mstn −/− pigs, which then activating the insulin signaling pathway. Irisin-mediated regulation is not the only pathway for the activation of insulin signal in Mstn −/− skeletal muscle. This study provides valuable insight for the treatment of human obesity and diabetes mellitus. PMID:28432282

  14. Atomic-resolution monitoring of protein maturation in live human cells by NMR

    PubMed Central

    Banci, Lucia; Barbieri, Letizia; Luchinat, Enrico; Secci, Erica; Zhao, Yuguang; Aricescu, A. Radu

    2014-01-01

    We used NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We could follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 (CCS) oxidation of the SOD1 intrasubunit disulfide bond occurs through both copper-dependent and independent mechanisms. Our approach represents a new strategy for structural investigation of endogeneously expressed proteins within a physiological (cellular) environment. PMID:23455544

  15. Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway

    SciTech Connect

    Ortego, Javier; Ceriani, Juan E.; Patino, Cristina; Plana, Juan; Enjuanes, Luis

    2007-11-25

    A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-{delta}E) has been engineered. This deletion mutant only grows in cells expressing E protein (E{sup +} cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-{delta}E infected BHK-pAPN-E{sup -} cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E{sup -} cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-{delta}E virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-{delta}E subcellular localization by confocal and immunoelectron microscopy in infected E{sup -} cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation.

  16. Specialized Function of Yeast Isa1 and Isa2 Proteins in the Maturation of Mitochondrial [4Fe-4S] Proteins*

    PubMed Central

    Mühlenhoff, Ulrich; Richter, Nadine; Pines, Ophry; Pierik, Antonio J.; Lill, Roland

    2011-01-01

    Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these “A-type” ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins. PMID:21987576

  17. Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

    PubMed

    Bourgeois, Michael; Jacquin, Françoise; Savois, Vincent; Sommerer, Nicolas; Labas, Valérie; Henry, Céline; Burstin, Judith

    2009-01-01

    Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post-translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

  18. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

    PubMed

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H; van Wijk, Klaas J

    2015-11-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

  19. Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C. elegans.

    PubMed

    Detwiler, M R; Reuben, M; Li, X; Rogers, E; Lin, R

    2001-08-01

    Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation. We present here a genetic characterization of oocyte maturation, using C. elegans as a model system. We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation. Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I. Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes. The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation.

  20. The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking

    PubMed Central

    Dumas, Audrey; Lê-Bury, Gabrielle; Marie-Anaïs, Florence; Herit, Floriane; Mazzolini, Julie; Guilbert, Thomas; Bourdoncle, Pierre; Russell, David G.; Benichou, Serge; Zahraoui, Ahmed

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1–infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150Glued, and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150Glued, hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1–infected macrophages, leading to strong alterations in phagolysosome biogenesis. PMID:26504171

  1. A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.

    PubMed

    Hu, Jian; Qin, Huajun; Gao, Fei Philip; Cross, Timothy A

    2011-11-01

    Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.

  2. Axon and muscle spindle hyperplasia in the myostatin null mouse

    PubMed Central

    Elashry, Mohamed I; Otto, Anthony; Matsakas, Antonios; El-Morsy, Salah E; Jones, Lisa; Anderson, Bethan; Patel, Ketan

    2011-01-01

    Germline deletion of the myostatin gene results in hyperplasia and hypertrophy of the tension-generating (extrafusal) fibres in skeletal muscle. As this gene is expressed predominantly in myogenic tissues it offers an excellent model with which to investigate the quantitative relationship between muscle and axonal development. Here we show that skeletal muscle hyperplasia in myostatin null mouse is accompanied by an increase in nerve fibres in major nerves of both the fore- and hindlimbs. We show that axons within these nerves undergo hypertrophy. Furthermore, we provide evidence that the age-related neural atrophic process is delayed in the absence of myostatin. Finally, we show that skeletal muscle hyperplasia in the myostatin null mouse is accompanied by an increase in the number of muscle spindles (also called stretch receptors or proprioceptors). However, our work demonstrates that the mechanisms regulating intrafusal fibre hyperplasia and hypertrophy differ from those that control the aetiology of extrafusal fibres. PMID:21208206

  3. Axon and muscle spindle hyperplasia in the myostatin null mouse.

    PubMed

    Elashry, Mohamed I; Otto, Anthony; Matsakas, Antonios; El-Morsy, Salah E; Jones, Lisa; Anderson, Bethan; Patel, Ketan

    2011-02-01

    Germline deletion of the myostatin gene results in hyperplasia and hypertrophy of the tension-generating (extrafusal) fibres in skeletal muscle. As this gene is expressed predominantly in myogenic tissues it offers an excellent model with which to investigate the quantitative relationship between muscle and axonal development. Here we show that skeletal muscle hyperplasia in myostatin null mouse is accompanied by an increase in nerve fibres in major nerves of both the fore- and hindlimbs. We show that axons within these nerves undergo hypertrophy. Furthermore, we provide evidence that the age-related neural atrophic process is delayed in the absence of myostatin. Finally, we show that skeletal muscle hyperplasia in the myostatin null mouse is accompanied by an increase in the number of muscle spindles (also called stretch receptors or proprioceptors). However, our work demonstrates that the mechanisms regulating intrafusal fibre hyperplasia and hypertrophy differ from those that control the aetiology of extrafusal fibres.

  4. Xanthohumol impairs autophagosome maturation through direct inhibition of valosin-containing protein.

    PubMed

    Sasazawa, Yukiko; Kanagaki, Shuhei; Tashiro, Etsu; Nogawa, Toshihiko; Muroi, Makoto; Kondoh, Yasumitsu; Osada, Hiroyuki; Imoto, Masaya

    2012-05-18

    Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupulus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate cofactor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II). This is the first report demonstrating XN as a VCP inhibitor that binds directly to the N domain of VCP. Our finding that XN bound to and inactivated VCP not only reveals the molecular mechanism of XN-modulated autophagy but may also explain how XN exhibits various biological activities that have been reported previously.

  5. Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form.

    PubMed

    Gould, Christine M; Antal, Corina E; Reyes, Gloria; Kunkel, Maya T; Adams, Ryan A; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C

    2011-08-19

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.

  6. The Native Form and Maturation Process of Hepatitis C Virus Core Protein

    PubMed Central

    Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

    1998-01-01

    The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

  7. Characterization of a molt-related myostatin gene (FmMstn) from the banana shrimp Fenneropenaeus merguiensis.

    PubMed

    Zhuo, Rui Qun; Zhou, Ting Ting; Yang, Shi Ping; Chan, Siuming Francis

    2017-07-01

    Myostatin is an important member of the transforming growth factor (TGF) family that functions to regulate muscle growth in animals. In this study, the myostatin gene (FmMstn) and two slightly different (short and long forms) cDNAs of the banana shrimp Fenneropenaeus merguiensis were cloned and characterized. Similar to Mstn gene of the scallop, fish and mammal, FmMstn gene consists of 3 exons and 2 introns. The 2kb upstream promoter region of the FmMstn gene consists of putative response elements for myocyte enhancing factor (MEF2) and E-box factors. The longest open reading frame of the short Mstn consists of 1260bp encoding for a protein with 420 amino acid residues. The long FmMstn is almost identical to the short FmMstn with the exception of 8 amino acid insertions. FmMstn is most similar to the Mstn of Litopenaeus vannamei and Penaeus monodon sharing >92-98% amino acid sequence identity. Multiple sequence alignment results revealed high degree of amino acid conservation of the cysteine residues and mature peptide of the FmMstn with Mstn from other animals. FmMstn transcript was detected in the heart, muscle, optic nerve and thoracic ganglion. FmMstn transcript level in muscle is higher in early postmolt, decreases in intermolt and increases again towards ecdysis. Higher expression level of FmMstn is also observed in smaller shrimp of the same age. Knock-down of FmMstn gene by RNAi can cause a significant increase in molt cycle duration and failure of some shrimp to undergo ecdysis. Direct DNA sequencing results revealed that FmMstn gene is highly polymorphic and several potential SNPs have been identified. Some SNPs are associated with the size difference of the shrimp. In summary, the result of this study indicates that shrimp FmMstn gene is molt/growth-related and the presence of SNP suggests that it could be a candidate gene for shrimp genetic improvement research. Copyright © 2017. Published by Elsevier Inc.

  8. Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveolata (polychaete annelid).

    PubMed

    Peaucellier, G; Dorée, M; Picard, A

    1984-12-01

    Incorporation of [32P]phosphate into proteins was monitored, in preloaded Sabellaria oocytes, during meiosis. After a fourfold increase during the transition from prophase to metaphase I, the incorporated radioactivity decreased steadily by 25% during completion of meiosis, while it slowly increased in metaphase I-blocked oocytes. Measurements of the amount and specific activity of nucleotide pools showed no variation, while total alkali-labile protein-bound phosphate was found to increase and then decrease during meiosis. Autoradiography of sodium dodecyl sulfate slabgels showed that some proteins have peculiar phosphorylation-dephosphorylation kinetics. The changes in the level of phosphorylation of proteins may be related to similar changes in maturation-promoting factor (MPF) activity.

  9. The heat shock protein 70 cochaperone hip enhances functional maturation of glucocorticoid receptor.

    PubMed

    Nelson, Gregory M; Prapapanich, Viravan; Carrigan, Patricia E; Roberts, Patricia J; Riggs, Daniel L; Smith, David F

    2004-07-01

    Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes. The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established. Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo. Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR). Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded. Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein. Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone. In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells.

  10. Myostatin regulates cardiomyocyte growth through modulation of Akt signaling.

    PubMed

    Morissette, Michael R; Cook, Stuart A; Foo, ShiYin; McKoy, Godfrina; Ashida, Noboru; Novikov, Mikhail; Scherrer-Crosbie, Marielle; Li, Ling; Matsui, Takashi; Brooks, Gavin; Rosenzweig, Anthony

    2006-07-07

    Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine-threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

  11. Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

    PubMed Central

    Drews, R; Paleyanda, R K; Lee, T K; Chang, R R; Rehemtulla, A; Kaufman, R J; Drohan, W N; Luboń, H

    1995-01-01

    Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7479820

  12. Analysis of protein thiol changes occurring during rat sperm epididymal maturation.

    PubMed

    Baker, Mark A; Weinberg, Anita; Hetherington, Louise; Villaverde, Ana Izabel S B; Velkov, Tony

    2015-01-01

    The maturation of spermatozoa throughout the epididymal environment occurs in the complete absence of nuclear protein biosynthesis. As such, these cells rely heavily on posttranslational modifications of existing proteins in order to obtain the potential for fertilization. We have used an OxiCat approach to label both free and oxidized cysteine residues in rat sperm proteins and compared the ratio of reduced:oxidized peptides as these cells undergo epididymal transit. In all, 20 peptides, corresponding to 15 proteins, underwent a change in their redox status. Included in this list were A-kinase anchoring protein 4 and fatty acid-binding protein 9. Both of these proteins undergo intradisulfide bonding, leading to reduced solubility and, in the case of the latter, is likely to cause a loss of protein function. Interestingly, two glycolytic enzymes, hexokinase-1 and lactate dehydrogenase, also display increased cysteine oxidation during epididymal transit, which may be involved in the regulation of the enzyme activities. © 2015 by the Society for the Study of Reproduction, Inc.

  13. Glycoproteomics Reveals Decorin Peptides with Anti-Myostatin Activity in Human Atrial Fibrillation

    PubMed Central

    Barallobre-Barreiro, Javier; Gupta, Shashi K.; Zoccarato, Anna; Kitazume-Taneike, Rika; Fava, Marika; Yin, Xiaoke; Werner, Tessa; Hirt, Marc N; Zampetaki, Anna; Viviano, Alessandro; Chong, Mei; Bern, Marshall; Kourliouros, Antonios; Domenech, Nieves; Willeit, Peter; Shah, Ajay M; Jahangiri, Marjan; Schaefer, Liliana; Fischer, Jens W.; Iozzo, Renato V.; Viner, Rosa; Thum, Thomas; Heineke, Joerg; Kichler, Antoine; Otsu, Kinya; Mayr, Manuel

    2016-01-01

    Background Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). Methods Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. Results ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. Upon enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were consistently identified in the flow through. Out of all ECM proteins identified, decorin was found to be most fragmented. Within its protein core, eighteen different cleavage sites were identified. In contrast, no cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was predominantly detected in ventricles compared to atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had higher levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. Conclusions This proteomics study is the first to analyse the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages can regulate

  14. Small RNA-Mediated Epigenetic Myostatin Silencing.

    PubMed

    Roberts, Thomas C; Andaloussi, Samir El; Morris, Kevin V; McClorey, Graham; Wood, Matthew Ja

    2012-05-15

    Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

  15. Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

    NASA Astrophysics Data System (ADS)

    Lubarsky, Gennady V.; Lemoine, Patrick; Meenan, Brian J.; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

    2014-04-01

    A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix.

  16. Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

    PubMed Central

    Mitterer, Valentin; Murat, Guillaume; Réty, Stéphane; Blaud, Magali; Delbos, Lila; Stanborough, Tamsyn; Bergler, Helmut; Leulliot, Nicolas; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. PMID:26831757

  17. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

    PubMed Central

    Malerba, Alberto; Kang, Jagjeet K; McClorey, Graham; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Wood, Matthew JA; Dickson, George

    2012-01-01

    The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD. PMID:23250360

  18. Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms*

    PubMed Central

    Harte, Níal P.; Klyubin, Igor; McCarthy, Eoin K.; Min, Soyoung; Garrahy, Sarah Ann; Xie, Yongjing; Davey, Gavin P.; Boland, John J.; Rowan, Michael J.; Mok, K. Hun

    2015-01-01

    Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12undiff). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species. PMID:26221033

  19. Mechanisms stimulating muscle wasting in chronic kidney disease: the roles of the ubiquitin-proteasome system and myostatin.

    PubMed

    Thomas, Sandhya S; Mitch, William E

    2013-04-01

    Catabolic conditions including chronic kidney disease (CKD), cancer, and diabetes cause muscle atrophy. The loss of muscle mass worsens the burden of disease because it is associated with increased morbidity and mortality. To avoid these problems or to develop treatment strategies, the mechanisms leading to muscle wasting must be identified. Specific mechanisms uncovered in CKD generally occur in other catabolic conditions. These include stimulation of protein degradation in muscle arising from activation of caspase-3 and the ubiquitin-proteasome system (UPS). These proteases act in a coordinated fashion with caspase-3 initially cleaving the complex structure of proteins in muscle, yielding fragments that are substrates that are degraded by the UPS. Fortunately, the UPS exhibits remarkable specificity for proteins to be degraded because it is the major intracellular proteolytic system. Without a high level of specificity cellular functions would be disrupted. The specificity is accomplished by complex reactions that depend on recognition of a protein substrate by specific E3 ubiquitin ligases. In muscle, the specific ligases are Atrogin-1 and MuRF-1, and their expression has characteristics of a biomarker of accelerated muscle proteolysis. Specific complications of CKD (metabolic acidosis, insulin resistance, inflammation, and angiotensin II) activate caspase-3 and the UPS through mechanisms that include glucocorticoids and impaired insulin or IGF-1 signaling. Mediators activate myostatin, which functions as a negative growth factor in muscle. In models of cancer or CKD, strategies that block myostatin prevent muscle wasting, suggesting that therapies that block myostatin could prevent muscle wasting in catabolic conditions.

  20. Characterization of follistatin-type domains and their contribution to myostatin and activin A antagonism.

    PubMed

    Cash, Jennifer N; Angerman, Elizabeth B; Keutmann, Henry T; Thompson, Thomas B

    2012-07-01

    Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.

  1. Characterization of Follistatin-Type Domains and Their Contribution to Myostatin and Activin A Antagonism

    PubMed Central

    Cash, Jennifer N.; Angerman, Elizabeth B.; Keutmann, Henry T.

    2012-01-01

    Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists. PMID:22593183

  2. Optimum mating systems for the myostatin locus in cattle.

    PubMed

    Keele, J W; Fahrenkrug, S C

    2001-08-01

    Inactive myostatin (one or two copies) results in increased muscularity, increased yield of closely trimmed retail product, reduced fat content, increased lean growth efficiency, reduced quality grade, increased birth weight, and increased dystocia. Even though one or two copies of inactive myostatin reduces quality grade or marbling compared to zero copies, there is no decrease in meat tenderness. It may be possible to use mating systems to make the most of the advantages of inactive myostatin while minimizing the disadvantages. The objective of this study was to develop a method to compare mating systems among genotypes at the myostatin locus. Economic variables that influence the profitability of alternative mating systems are prices per unit of retail product for USDA quality grades Standard, Select, and Choice; cost of an assisted calving; and cost of genotyping. Because of variation in both economic variables and biological parameters, a single mating system is not expected to universally maximize profit. We identified seven mating systems that each yield maximum profit for different combinations of values for biological parameters and economic variables. Use of inactive myostatin was profitable as long as the price for Select was at least 80% of the Choice price and the price for Standard at least 60%. As the price for Select and Standard increase up to the Choice price, mating systems that produce a higher proportion of inactive myostatin alleles become more profitable. Profitable use of inactive myostatin depends either on retaining ownership of beef until it is fabricated into retail product or the development of specialty markets that place greater value on lean yield and less on marbling, unlike conventional U. S. markets.

  3. Modulation of Stem Cells Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration after Injury

    DTIC Science & Technology

    2008-03-01

    cells in the smooth muscle region of the corpora (D). In this case, no overlay was possible since the glutaraldehyde fixation or the beta gal...regulation. J. Biol. Chem 1992; 267: 984-9 35. Ma W, Rogers K, Zbar B, Schmidt L. Effects of different fixatives on beta -galactosidase activity. J...the Scion Image software beta 4.0.2 (Scion Corp., Frederick, MD, USA).Recombinant proteins Myostatin recombinant proteinwas produced inEscherichia

  4. Revisiting the paradigm of myostatin in vertebrates: insights from fishes.

    PubMed

    Gabillard, Jean-Charles; Biga, Peggy R; Rescan, Pierre-Yves; Seiliez, Iban

    2013-12-01

    In the last decade, myostatin (MSTN), a member of the TGFβ superfamily, has emerged as a strong inhibitor of muscle growth in mammals. In fish many studies reveal a strong conservation of mstn gene organization, sequence, and protein structures. Because of ancient genome duplication, teleostei may have retained two copies of mstn genes and even up to four copies in salmonids due to additional genome duplication event. In sharp contrast to mammals, the different fish mstn orthologs are widely expressed with a tissue-specific expression pattern. Quantification of mstn mRNA in fish under different physiological conditions, demonstrates that endogenous expression of mstn paralogs is rarely related to fish muscle growth rate. In addition, attempts to inhibit MSTN activity did not consistently enhance muscle growth as in mammals. In vitro, MSTN stimulates myotube atrophy and inhibits proliferation but not differentiation of myogenic cells as in mammals. In conclusion, given the strong mstn expression non-muscle tissues of fish, we propose a new hypothesis stating that fish MSTN functions as a general inhibitors of cell proliferation and cell growth to control tissue mass but is not specialized into a strong muscle regulator. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Microsomal triglyceride transfer protein contributes to lipid droplet maturation in adipocytes.

    PubMed

    Swift, Larry L; Love, Joseph D; Harris, Carla M; Chang, Benny H; Jerome, W Gray

    2017-01-01

    Previous studies in our laboratory have established the presence of MTP in both white and brown adipose tissue in mice as well as in 3T3-L1 cells. Additional studies demonstrated an increase in MTP levels as 3T3-L1 cells differentiate into adipocytes concurrent with the movement of MTP from the juxtanuclear region of the cell to the surface of lipid droplets. This suggested a role for MTP in lipid droplet biogenesis and/or maturation. To probe the role of MTP in adipocytes, we used a Cre-Lox approach with aP2-Cre and Adipoq-Cre recombinase transgenic mice to knock down MTP expression in brown and white fat of mice. MTP expression was reduced approximately 55% in white fat and 65-80% in brown fat. Reducing MTP expression in adipose tissue had no effect on weight gain or body composition, whether the mice were fed a regular rodent or high fat diet. In addition, serum lipids and unesterified fatty acid levels were not altered in the knockdown mice. Importantly, decreased MTP expression in adipose tissue was associated with smaller lipid droplets in brown fat and smaller adipocytes in white fat. These results combined with our previous studies showing MTP lipid transfer activity is not necessary for lipid droplet initiation or growth in the early stages of differentiation, suggest that a structural feature of the MTP protein is important in lipid droplet maturation. We conclude that MTP protein plays a critical role in lipid droplet maturation, but does not regulate total body fat accumulation.

  6. The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome

    PubMed Central

    Rozanska, Agata; Richter-Dennerlein, Ricarda; Rorbach, Joanna; Gao, Fei; Lewis, Richard J.

    2017-01-01

    Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation. PMID:28512204

  7. Atypical protein kinase C couples cell sorting with primitive endoderm maturation in the mouse blastocyst.

    PubMed

    Saiz, Néstor; Grabarek, Joanna B; Sabherwal, Nitin; Papalopulu, Nancy; Plusa, Berenika

    2013-11-01

    During mouse pre-implantation development, extra-embryonic primitive endoderm (PrE) and pluripotent epiblast precursors are specified in the inner cell mass (ICM) of the early blastocyst in a 'salt and pepper' manner, and are subsequently sorted into two distinct layers. Positional cues provided by the blastocyst cavity are thought to be instrumental for cell sorting; however, the sequence of events and the mechanisms that control this segregation remain unknown. Here, we show that atypical protein kinase C (aPKC), a protein associated with apicobasal polarity, is specifically enriched in PrE precursors in the ICM prior to cell sorting and prior to overt signs of cell polarisation. aPKC adopts a polarised localisation in PrE cells only after they reach the blastocyst cavity and form a mature epithelium, in a process that is dependent on FGF signalling. To assess the role of aPKC in PrE formation, we interfered with its activity using either chemical inhibition or RNAi knockdown. We show that inhibition of aPKC from the mid blastocyst stage not only prevents sorting of PrE precursors into a polarised monolayer but concomitantly affects the maturation of PrE precursors. Our results suggest that the processes of PrE and epiblast segregation, and cell fate progression are interdependent, and place aPKC as a central player in the segregation of epiblast and PrE progenitors in the mouse blastocyst.

  8. Maturation Proteins and Sugars in Desiccation Tolerance of Developing Soybean Seeds 1

    PubMed Central

    Blackman, Sheila A.; Obendorf, Ralph L.; Leopold, A. Carl

    1992-01-01

    The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 μg/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 μg/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance. Images Figure 4 PMID:16652951

  9. A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

    PubMed Central

    Rosenblum, Jonathan S.; Pemberton, Lucy F.; Blobel, Günter

    1997-01-01

    A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing. PMID:9412461

  10. Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches

    PubMed Central

    Rasal, Kiran Dashrath; Chakrapani, Vemulawada; Patra, Swagat Kumar; Mohapatra, Shibani D.; Nayak, Swapnarani; Jena, Sasmita; Sundaray, Jitendra Kumar; Jayasankar, Pallipuram; Barman, Hirak Kumar

    2016-01-01

    The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies. PMID:27019850

  11. Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches.

    PubMed

    Rasal, Kiran Dashrath; Chakrapani, Vemulawada; Patra, Swagat Kumar; Mohapatra, Shibani D; Nayak, Swapnarani; Jena, Sasmita; Sundaray, Jitendra Kumar; Jayasankar, Pallipuram; Barman, Hirak Kumar

    2016-01-01

    The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.

  12. [Reduction of muscle mass mediated by myostatin in an experimental model of pulmonary emphysema].

    PubMed

    Fermoselle, Clara; Sanchez, Francisco; Barreiro, Esther

    2011-12-01

    Among the extrapulmonary manifestations of COPD, dysfunction and loss of muscle mass/weight are those that have the greatest impact on the quality of life of patients. Our objective was to evaluate the molecular mechanisms that are potentially implicated in the limited development of muscle mass in the diaphragm and gastrocnemius of mice with experimentally-induced emphysema. An experimental model in mice, in which emphysema was induced by means of the local instillation of elastase (n=6), while saline was administered to the controls (n=7). We determined the levels of oxidative stress, proteolytic systems, signaling pathways, growth factors and cell differentiation (western-blot) in the diaphragm and gastrocnemius of all the mice after 34 weeks. Upon comparing the mice with emphysema with the controls, the following findings were observed: (1) lower total body weight and lower weight of the diaphragm and gastrocnemius; (2) in the diaphragm, the levels of protein oxidation were increased, the mitochondrial antioxidant systems reduced, the levels of myostatin and of the ERK1/2 and FoxO1 signaling pathways were higher, and the myosin content was lower (67%); and (3) in the gastrocnemius of the emphysematous mice, the cytosolic antioxidants were decreased and the levels of myostatin and of the JNK and NF-kB signaling pathways were increased. The reduction of the myosin content observed in the diaphragm of mice with emphysema could explain their smaller size. Oxidative stress, myostatin and FoxO could be implicated in the loss of this structural protein. Copyright © 2011 SEPAR. Published by Elsevier Espana. All rights reserved.

  13. The RNA-Binding Protein, ZFP36L2, Influences Ovulation and Oocyte Maturation

    PubMed Central

    Ball, Christopher B.; Rodriguez, Karina F.; Stumpo, Deborah J.; Ribeiro-Neto, Fernando; Korach, Kenneth S.; Blackshear, Perry J.; Birnbaumer, Lutz; Ramos, Silvia B. V.

    2014-01-01

    ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3′UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest. PMID:24830504

  14. Functional improvement of dystrophic muscle by myostatin blockade.

    PubMed

    Bogdanovich, Sasha; Krag, Thomas O B; Barton, Elisabeth R; Morris, Linda D; Whittemore, Lisa-Anne; Ahima, Rexford S; Khurana, Tejvir S

    2002-11-28

    Mice and cattle with mutations in the myostatin (GDF8) gene show a marked increase in body weight and muscle mass, indicating that this new member of the TGF-beta superfamily is a negative regulator of skeletal muscle growth. Inhibition of the myostatin gene product is predicted to increase muscle mass and improve the disease phenotype in a variety of primary and secondary myopathies. We tested the ability of inhibition of myostatin in vivo to ameliorate the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). Blockade of endogenous myostatin by using intraperitoneal injections of blocking antibodies for three months resulted in an increase in body weight, muscle mass, muscle size and absolute muscle strength in mdx mouse muscle along with a significant decrease in muscle degeneration and concentrations of serum creatine kinase. The functional improvement of dystrophic muscle by myostatin blockade provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD, and circumvents the major problems associated with conventional gene therapy in these disorders.

  15. MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

    PubMed Central

    Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

    2014-01-01

    Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

  16. Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

    PubMed Central

    Lobo, Vivian; Rao, Parimala; Gajbhiye, Rahul; Kulkarni, Vijay; Parte, Priyanka

    2015-01-01

    GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant

  17. Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS.

    PubMed

    Van Hoewyk, Douglas; Abdel-Ghany, Salah E; Cohu, Christopher M; Herbert, Stephen K; Kugrens, Paul; Pilon, Marinus; Pilon-Smits, Elizabeth A H

    2007-03-27

    NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation. In vitro, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far in planta. To study the role of CpNifS in Fe-S cluster biogenesis in vivo, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.

  18. Effects of feeding level and sexual maturation on carcass and fillet characteristics and indices of protein degradation in rainbow trout (Oncorhynchus mykiss)

    USDA-ARS?s Scientific Manuscript database

    Sexual maturation in many species of fish including salmonids requires mobilization of energy and nutrient resources to support gonad growth. During sexual maturation, particularly vitellogenesis, proteins are mobilized from muscle tissue, which is evidenced by increased expression of proteolytic g...

  19. Myostatin deficiency is associated with an increase in number of total axons and motor axons innervating mouse tibialis anterior muscle.

    PubMed

    Gay, Stephanie; Jublanc, Elodie; Bonnieu, Anne; Bacou, Francis

    2012-05-01

    Myostatin (Mstn) is a secreted protein that acts as a negative regulator of skeletal muscle mass. However, a critical evaluation of neuromuscular aspects of hypertrophied muscles induced by Mstn deficiency has not been done. We compared the tibialis anterior muscle-nerve interrelationships in wild-type and Mstn-null mice of both genders by immunohistochemical analyses, which allowed us to count the number of total axons and motor axons and estimate the size of motor units and the innervation ratio of the tibialis anterior muscle (TAm). There was an increase in the number of total axons and motor axons, and higher values in both the motor unit size and the innervation ratio of Mstn-null TAm compared with those of wild-type TAm. We found that myostatin is involved either directly in the control of neuromuscular interrelationships or indirectly through its effect on muscle size. Copyright © 2012 Wiley Periodicals, Inc.

  20. Acute loading and aging effects on myostatin pathway biomarkers in human skeletal muscle after three sequential bouts of resistance exercise.

    PubMed

    Dalbo, Vincent J; Roberts, Michael D; Sunderland, Kyle L; Poole, Chris N; Stout, Jeff R; Beck, Travis W; Bemben, Mike; Kerksick, Chad M

    2011-08-01

    To determine the influence of age and resistance exercise on myostatin pathway-related genes, younger (n = 10; 28 ± 5 years) and older (n = 10; 68 ± 6 years) men underwent four testing conditions (T1-T4). A baseline (T1) muscle sample was obtained, whereas the second and third biopsies were obtained 48 hours following the first and second training sessions (T2, T3), and a final biopsy was taken 24 hours following T3. The training sessions consisted of 3 sets of 10 repetitions (80% of one repetition maximum) on leg press, hack squat, and leg extension exercises. Follistatin (FST) messenger RNA was greater in older compared with younger men at T1 and T2 (p < .05). Follistatin-like 3 (FSTL3) messenger RNA was greater in older compared with younger men at T1 and T4 (p < .05). In older men, there was a significant decrease in myostatin (MSTN) messenger RNA at T4 (p < .05). Older men contained less active (Ser-425 phosphorylated) SMAD3 (p-SMAD3) protein than younger men at T3 and T4 (p < .05).Although it is well known that younger individuals possess a greater hypertrophic potential to resistance exercise, it appears that older individuals may paradoxically possess a more favorable resistance exercise response regarding myostatin pathway-related genes and a protein marker of pathway activity. Future research is warranted to examine the physiological significance of this age-dependent mechanism.

  1. Combinatory effects of siRNA‐induced myostatin inhibition and exercise on skeletal muscle homeostasis and body composition

    PubMed Central

    Mosler, Stephanie; Relizani, Karima; Mouisel, Etienne; Amthor, Helge; Diel, Patrick

    2014-01-01

    Abstract Inhibition of myostatin (Mstn) stimulates skeletal muscle growth, reduces body fat, and induces a number of metabolic changes. However, it remains unexplored how exercise training modulates the response to Mstn inhibition. The aim of this study was to investigate how siRNA‐mediated Mstn inhibition alone but also in combination with physical activity affects body composition and skeletal muscle homeostasis. Adult mice were treated with Mstn‐targeting siRNA and subjected to a treadmill‐based exercise protocol for 4 weeks. Effects on skeletal muscle and fat tissue, expression of genes, and serum concentration of proteins involved in myostatin signaling, skeletal muscle homeostasis, and lipid metabolism were investigated and compared with Mstn−/− mice. The combination of siRNA‐mediated Mstn knockdown and exercise induced skeletal muscle hypertrophy, which was associated with an upregulation of markers for satellite cell activity. SiRNA‐mediated Mstn knockdown decreased visceral fat and modulated lipid metabolism similar to effects observed in Mstn−/− mice. Myostatin did not regulate its own expression via an autoregulatory loop, however, Mstn knockdown resulted in a decrease in the serum concentrations of myostatin propeptide, leptin, and follistatin. The ratio of these three parameters was distinct between Mstn knockdown, exercise, and their combination. Taken together, siRNA‐mediated Mstn knockdown in combination with exercise stimulated skeletal muscle hypertrophy. Each intervention or their combination induced a specific set of adaptive responses in the skeletal muscle and fat metabolism which could be identified by marker proteins in serum. PMID:24760516

  2. Within-Winter Flexibility in Muscle Masses, Myostatin, and Cellular Aerobic Metabolic Intensity in Passerine Birds.

    PubMed

    Swanson, David L; King, Marisa O; Culver, William; Zhang, Yufeng

    Metabolic rates of passerine birds are flexible traits that vary both seasonally and among and within winters. Seasonal variation in summit metabolic rates (Msum = maximum thermoregulatory metabolism) in birds is consistently correlated with changes in pectoralis muscle and heart masses and sometimes with variation in cellular aerobic metabolic intensity, so these traits might also be associated with shorter-term, within-winter variation in metabolic rates. To determine whether these mechanisms are associated with within-winter variation in Msum, we examined the effects of short-term (ST; 0-7 d), medium-term (MT; 14-30 d), and long-term (LT; 30-yr means) temperature variables on pectoralis muscle and heart masses, pectoralis expression of the muscle-growth inhibitor myostatin and its metalloproteinase activators TLL-1 and TLL-2, and pectoralis and heart citrate synthase (CS; an indicator of cellular aerobic metabolic intensity) activities for two temperate-zone resident passerines, house sparrows (Passer domesticus) and dark-eyed juncos (Junco hyemalis). For both species, pectoralis mass residuals were positively correlated with ST temperature variables, suggesting that cold temperatures resulted in increased turnover of pectoralis muscle, but heart mass showed little within-winter variation for either species. Pectoralis mRNA and protein expression of myostatin and the TLLs were only weakly correlated with ST and MT temperature variables, which is largely consistent with trends in muscle masses for both species. Pectoralis and heart CS activities showed weak and variable trends with ST temperature variables in both species, suggesting only minor effects of temperature variation on cellular aerobic metabolic intensity. Thus, neither muscle or heart masses, regulation by the myostatin system, nor cellular aerobic metabolic intensity varied consistently with winter temperature, suggesting that other factors regulate within-winter metabolic variation in these birds.

  3. Generation of Myostatin b Knockout Yellow Catfish (Tachysurus Fulvidraco) Using Transcription Activator-Like Effector Nucleases

    PubMed Central

    Dong, Zhangji; Ge, Jiachun; Xu, Zhiqiang; Dong, Xiaohua; Cao, Shasha; Pan, Jianlin

    2014-01-01

    Abstract Myostatin (Mstn), a member of the transforming growth factor β superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnbnju22, an 8 bp deletion, and mstnbnju24, a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth. PMID:24813227

  4. Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice.

    PubMed

    Personius, Kirkwood E; Jayaram, Aditi; Krull, David; Brown, Roger; Xu, Tianshun; Han, Bajin; Burgess, Kerri; Storey, Christopher; Shah, Bharati; Tawil, Rabi; Welle, Stephen

    2010-09-01

    There is no consensus about whether making muscles abnormally large by reducing myostatin activity affects force-generating capacity or the ability to perform activities requiring muscular endurance. We therefore examined grip force, contractile properties of extensor digitorum longus (EDL) muscles, and voluntary wheel running in mice in which myostatin was depleted after normal muscle development. Cre recombinase activity was induced to knock out exon 3 of the myostatin gene in 4-mo-old mice in which this exon was flanked by loxP sequences (Mstn[f/f]). Control mice with normal myostatin genes (Mstn[w/w]) received the same Cre-activating treatment. Myostatin depletion increased the mass of all muscles that were examined (gastrocnemius, quadriceps, tibialis anterior, EDL, soleus, triceps) by approximately 20-40%. Grip force, measured multiple times 2-22 wk after myostatin knockout, was not consistently greater in the myostatin-deficient mice. EDL contractile properties were determined 7-13 mo after myostatin knockout. Twitch force tended to be greater in myostatin-deficient muscles (+24%; P=0.09), whereas tetanic force was not consistently elevated (mean +11%; P=0.36), even though EDL mass was greater than normal in all myostatin-deficient mice (mean +36%; P<0.001). The force deficit induced by eccentric contractions was approximately twofold greater in myostatin-deficient than in normal EDL muscles (31% vs. 16% after five eccentric contractions; P=0.02). Myostatin-deficient mice ran 19% less distance (P<0.01) than control mice during the 12 wk following myostatin depletion, primarily because of fewer running bouts per night rather than diminished running speed or bout duration. Reduced specific tension (ratio of force to mass) and reduced running have been observed after muscle hypertrophy was induced by other means, suggesting that they are characteristics generally associated with abnormally large muscles rather than unique effects of myostatin deficiency.

  5. Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system.

    PubMed

    Boyer, Marcus E; Wang, Chia-Wei; Swartz, James R

    2006-05-05

    The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.

  6. The role of ERp44 in maturation of serotonin transporter protein.

    PubMed

    Freyaldenhoven, Samuel; Li, Yicong; Kocabas, Arif M; Ziu, Enrit; Ucer, Serra; Ramanagoudr-Bhojappa, Raman; Miller, Grover P; Kilic, Fusun

    2012-05-18

    In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT

  7. Brucella Control of Dendritic Cell Maturation Is Dependent on the TIR-Containing Protein Btp1

    PubMed Central

    Salcedo, Suzana P; Marchesini, María Ines; Lelouard, Hugues; Fugier, Emilie; Jolly, Gilles; Balor, Stephanie; Muller, Alexandre; Lapaque, Nicolas; Demaria, Olivier; Alexopoulou, Lena; Comerci, Diego J; Ugalde, Rodolfo A; Pierre, Philippe; Gorvel, Jean-Pierre

    2008-01-01

    Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis. PMID:18266466

  8. Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation.

    PubMed

    Tovar, Jorge; León-Avila, Gloria; Sánchez, Lidya B; Sutak, Robert; Tachezy, Jan; van der Giezen, Mark; Hernández, Manuel; Müller, Miklós; Lucocq, John M

    2003-11-13

    Giardia intestinalis (syn. lamblia) is one of the most widespread intestinal protozoan pathogens worldwide, causing hundreds of thousands of cases of diarrhoea each year. Giardia is a member of the diplomonads, often described as an ancient protist group whose primitive nature is suggested by the lack of typical eukaryotic organelles (for example, mitochondria, peroxisomes), the presence of a poorly developed endomembrane system and by their early branching in a number of gene phylogenies. The discovery of nuclear genes of putative mitochondrial ancestry in Giardia and the recent identification of mitochondrial remnant organelles in amitochondrial protists such as Entamoeba histolytica and Trachipleistophora hominis suggest that the eukaryotic amitochondrial state is not a primitive condition but is rather the result of reductive evolution. Using an in vitro protein reconstitution assay and specific antibodies against IscS and IscU--two mitochondrial marker proteins involved in iron-sulphur cluster biosynthesis--here we demonstrate that Giardia contains mitochondrial remnant organelles (mitosomes) bounded by double membranes that function in iron-sulphur protein maturation. Our results indicate that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont.

  9. Pharmacological Analyses of Protein Kinases Regulating Egg Maturation in Marine Nemertean Worms: A Review and Comparison with Mammalian Eggs

    PubMed Central

    Stricker, Stephen A.; Escalona, Jose R.; Abernathy, Samuel; Marquardt, Alicia

    2010-01-01

    For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor. PMID:20948915

  10. Phosphorylation of the Tight Junction Protein Occludin Regulates Epithelial Monolayer Proliferation and Maturation

    NASA Astrophysics Data System (ADS)

    Bolinger, Mark Thomas

    Barriers against the external environment are crucial for sustaining life in multicellular organisms, and form following convergent growth and development of cell-cell junctions. At least four types of epithelial cell-cell junctions exist, the most apical of which is known as the tight junction (TJ). A specific transmembrane protein known as occludin is highly phosphorylated on its C-terminal coiled-coil, and certain sites have been found to regulate specific aspects of TJ function, including the response to certain cytokines. Previously, our lab discovered a novel phosphosite at serine 471 that is located at a contact site with an important central organizer of the TJ, zonula occludens-1. Phosphoinhibitory, serine to alanine (S471A) occludin point mutant MDCK cell lines demonstrate that S471A monolayers are poorly organized compared to WT occludin (WT Occ) or phosphomimetic, serine to aspartic acid (S471D) lines. Additionally, S471A monolayers are composed of fewer, larger cells than controls, and exhibit proliferative arrest almost immediately following confluency, in contrast to control lines, which go through at least one additional round of proliferation. This phenotype can be recapitulated with a cell cycle inhibitor, demonstrating that confluent proliferation or cell packing is necessary for barrier maturation. G-protein coupled receptor kinase (GRK) was confirmed to be an S471 kinase by inhibitor experiments from a bioinformatically compiled candidate kinase list, and GRK inhibitors were able to recapitulate the phenotype of S471A lines. Finally, S471A expression perturbed purified coiled-coil stability as determined by NMR. Modeling of inter-coil interactions identified several possible hydrogen bonds that differ between the phosphorylated and non-phosphorylated forms. Expression of S471N (asparagine) transgenic occludin in vitro demonstrated highly organized border organization despite the lack of a negative charge at the S471 position. This result

  11. Role of protein haptenation in triggering maturation events in the dendritic cell surrogate cell line THP-1

    SciTech Connect

    Megherbi, Rym; Kiorpelidou, Evanthia; Foster, Brian; Rowe, Cliff; Naisbitt, Dean J.; Goldring, Christopher E.; Park, B. Kevin

    2009-07-15

    Dendritic cell (DC) maturation in response to contact sensitizers is a crucial step in the induction of sensitization reactions; however the underlying mechanism of activation remains unknown. To test whether the extent of protein haptenation is a determinant in DC maturation, we tested the effect of five dinitrophenyl (DNP) analogues of different reactivity, on maturation markers in the cell line, THP-1. The potencies of the test compounds in upregulating CD54 levels, inducing IL-8 release and triggering p38 MAPK phosphorylation did not correlate with their ability to deplete intracellular glutathione (GSH) levels or cause cell toxicity. However, the compounds' potency at inducing p38 phosphorylation was significantly associated with the amount of intracellular protein adducts formed (p < 0.05). Inhibition experiments show that, at least for DNFB, p38 MAP kinase signalling controls compound-specific changes in CD54 expression and IL-8 release. 2D-PAGE analysis revealed that all the DNP analogues appeared to bind similar proteins. The analogues failed to activate NFkB, however, they activated Nrf2, which was used as a marker of oxidative stress. Neither GSH depletion, by use of buthionine sulfoximine, nor treatment with the strongly lysine-reactive hapten penicillin elicited maturation. We conclude that protein haptenation, probably through reactive cysteine residues may be a trigger for maturation events in this in vitro model and that p38 activation may be a discriminatory marker for the classification of potency of chemical sensitizers.

  12. Vaccinia Mature Virus Fusion Regulator A26 Protein Binds to A16 and G9 Proteins of the Viral Entry Fusion Complex and Dissociates from Mature Virions at Low pH

    PubMed Central

    Chang, Shu-Jung; Shih, Ao-Chun; Tang, Yin-Liang

    2012-01-01

    Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422–8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive. PMID:22278246

  13. Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH.

    PubMed

    Chang, Shu-Jung; Shih, Ao-Chun; Tang, Yin-Liang; Chang, Wen

    2012-04-01

    Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.

  14. Indoxyl sulfate potentiates skeletal muscle atrophy by inducing the oxidative stress-mediated expression of myostatin and atrogin-1

    PubMed Central

    Enoki, Yuki; Watanabe, Hiroshi; Arake, Riho; Sugimoto, Ryusei; Imafuku, Tadashi; Tominaga, Yuna; Ishima, Yu; Kotani, Shunsuke; Nakajima, Makoto; Tanaka, Motoko; Matsushita, Kazutaka; Fukagawa, Masafumi; Otagiri, Masaki; Maruyama, Toru

    2016-01-01

    Skeletal muscle atrophy, referred to as sarcopenia, is often observed in chronic kidney disease (CKD) patients, especially in patients who are undergoing hemodialysis. The purpose of this study was to determine whether uremic toxins are involved in CKD-related skeletal muscle atrophy. Among six protein-bound uremic toxins, indole containing compounds, indoxyl sulfate (IS) significantly inhibited proliferation and myotube formation in C2C12 myoblast cells. IS increased the factors related to skeletal muscle breakdown, such as reactive oxygen species (ROS) and inflammatory cytokines (TNF-α, IL-6 and TGF-β1) in C2C12 cells. IS also enhanced the production of muscle atrophy-related genes, myostatin and atrogin-1. These effects induced by IS were suppressed in the presence of an antioxidant or inhibitors of the organic anion transporter and aryl hydrocarbon receptor. The administered IS was distributed to skeletal muscle and induced superoxide production in half-nephrectomized (1/2 Nx) mice. The chronic administration of IS significantly reduced the body weights accompanied by skeletal muscle weight loss. Similar to the in vitro data, IS induced the expression of myostatin and atrogin-1 in addition to increasing the production of inflammatory cytokines by enhancing oxidative stress in skeletal muscle. These data suggest that IS has the potential to accelerate skeletal muscle atrophy by inducing oxidative stress-mediated myostatin and atrogin-1 expression. PMID:27549031

  15. Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors.

    PubMed

    Allen, David L; Unterman, Terry G

    2007-01-01

    Myostatin, a member of the transforming growth factor (TGF)-beta family, plays an important role in regulating skeletal muscle growth and differentiation. Here we examined the role of FoxO1 and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C(2)C(12) myotubes in vitro. Both myostatin and FoxO1 mRNA expression were greater in fast- vs. slow-twitch skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C(2)C(12) myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs bind to their respective sites in the myostatin promoter. Treatment with TGF-beta and/or overexpression of SMAD2, -3, or -4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-beta along with overexpression of SMAD2 and FoxO1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-beta treatment and SMAD2 overexpression greatly potentiated FoxO1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO1 and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation.

  16. Mature Surfactant Protein-B Expression by Immunohistochemistry as a Marker for Surfactant System Development in the Fetal Sheep Lung

    PubMed Central

    Lock, Mitchell C.; McGillick, Erin V.; Orgeig, Sandra; Zhang, Song; McMillen, I. Caroline; Morrison, Janna L.

    2015-01-01

    Evaluation of the number of type II alveolar epithelial cells (AECs) is an important measure of the lung’s ability to produce surfactant. Immunohistochemical staining of these cells in lung tissue commonly uses antibodies directed against mature surfactant protein (SP)-C, which is regarded as a reliable SP marker of type II AECs in rodents. There has been no study demonstrating reliable markers for surfactant system maturation by immunohistochemistry in the fetal sheep lung despite being widely used as a model to study lung development. Here we examine staining of a panel of surfactant pro-proteins (pro–SP-B and pro–SP-C) and mature proteins (SP-B and SP-C) in the fetal sheep lung during late gestation in the saccular/alveolar phase of development (120, 130, and 140 days), with term being 150 ± 3 days, to identify the most reliable marker of surfactant producing cells in this species. Results from this study indicate that during late gestation, use of anti-SP-B antibodies in the sheep lung yields significantly higher cell counts in the alveolar epithelium than SP-C antibodies. Furthermore, this study highlights that mature SP-B antibodies are more reliable markers than SP-C antibodies to evaluate surfactant maturation in the fetal sheep lung by immunohistochemistry. PMID:26297137

  17. Mature Surfactant Protein-B Expression by Immunohistochemistry as a Marker for Surfactant System Development in the Fetal Sheep Lung.

    PubMed

    Lock, Mitchell C; McGillick, Erin V; Orgeig, Sandra; Zhang, Song; McMillen, I Caroline; Morrison, Janna L

    2015-11-01

    Evaluation of the number of type II alveolar epithelial cells (AECs) is an important measure of the lung's ability to produce surfactant. Immunohistochemical staining of these cells in lung tissue commonly uses antibodies directed against mature surfactant protein (SP)-C, which is regarded as a reliable SP marker of type II AECs in rodents. There has been no study demonstrating reliable markers for surfactant system maturation by immunohistochemistry in the fetal sheep lung despite being widely used as a model to study lung development. Here we examine staining of a panel of surfactant pro-proteins (pro-SP-B and pro-SP-C) and mature proteins (SP-B and SP-C) in the fetal sheep lung during late gestation in the saccular/alveolar phase of development (120, 130, and 140 days), with term being 150 ± 3 days, to identify the most reliable marker of surfactant producing cells in this species. Results from this study indicate that during late gestation, use of anti-SP-B antibodies in the sheep lung yields significantly higher cell counts in the alveolar epithelium than SP-C antibodies. Furthermore, this study highlights that mature SP-B antibodies are more reliable markers than SP-C antibodies to evaluate surfactant maturation in the fetal sheep lung by immunohistochemistry.

  18. Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus

    PubMed Central

    Mashruwala, Ameya A.; Pang, Yun Y.; Rosario-Cruz, Zuelay; Chahal, Harsimranjit K.; Benson, Meredith A.; Anzaldi-Mike, Laura L.; Skaar, Eric P.; Torres, Victor J.; Nauseef, William M.; Boyd, Jeffrey M.

    2015-01-01

    Summary The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as a Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target. PMID:25388433

  19. Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins

    PubMed Central

    Chen, Yu; Yang, Fan; Zubovic, Lorena; Pavelitz, Tom; Yang, Wen; Godin, Katherine; Walker, Matthew; Zheng, Suxin; Macchi, Paolo; Varani, Gabriele

    2016-01-01

    The RNA Recognition Motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with new specificity would provide valuable tools and an exacting test of our understanding of specificity. We have achieved the first successful re-design of the specificity of an RRM using rational methods and demonstrated re-targeting of activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of miR-21 precursor with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications. PMID:27428511

  20. Intestinal fatty acid-binding protein: a possible marker for gut maturation.

    PubMed

    Reisinger, Kostan W; Elst, Marieke; Derikx, Joep P M; Nikkels, Peter G J; de Vries, Bart; Adriaanse, Marlou P M; Jellema, Reint K; Kramer, Boris W W; Wolfs, Tim G A M

    2014-09-01

    Gut immaturity is linked with postnatal intestinal disorders. However, biomarkers to assess the intestinal developmental stage around birth are lacking. The aim of this study was to gain more insight on intestinal fatty acid-binding protein (I-FABP) as an indicator of gut maturity. Antenatal I-FABP distribution and release was investigated in extremely premature, moderately premature, and term lambs, and these findings were verified in human urinary samples. Ileal I-FABP distribution was confirmed in autopsy material within 24 h postnatally. Median (range) serum I-FABP levels were lower in extremely premature lambs compared with moderately premature lambs (156 (50.0-427) vs. 385 (100-1,387) pg/ml; P = 0.02). Contrarily, median early postnatal urine I-FABP levels in human infants were higher in extremely premature compared with moderately premature and term neonates (1,219 (203-15,044) vs. 256 (50-1,453) and 328 (96-1,749) pg/ml; P = 0.008 and P = 0.04, respectively). I-FABP expression was most prominent in nonvacuolated enterocytes and increased with rising gestational age (GA) in ovine and human tissue samples. The epithelial distribution pattern changed from a phenotype displaying I-FABP-positive enterocytes merely in the crypts early in gestation into a phenotype with I-FABP expressing cells exclusively present in the villus tips at term in ovine and human tissue. In this ovine and human study, increasing GA is accompanied by an increase in I-FABP tissue content. Cord I-FABP levels correlate with gestation in ovine fetuses, identifying I-FABP as a marker for gut maturation. Raised postnatal urine I-FABP levels in preterm human infants may indicate intestinal injury and/or inflammation in utero.

  1. Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth.

    PubMed

    Dai, Shaojun; Li, Lei; Chen, Taotao; Chong, Kang; Xue, Yongbiao; Wang, Tai

    2006-04-01

    As a highly reduced organism, pollen performs specialized functions to generate and carry sperm into the ovule by its polarily growing pollen tube. Yet the molecular genetic basis of these functions is poorly understood. Here, we identified 322 unique proteins, most of which were not reported previously to be in pollen, from mature pollen of Oryza sativa L. ssp japonica using a proteomic approach, 23% of them having more than one isoform. Functional classification reveals that an overrepresentation of the proteins was related to signal transduction (10%), wall remodeling and metabolism (11%), and protein synthesis, assembly and degradation (14%), as well as carbohydrate and energy metabolism (25%). Further, 11% of the identified proteins are functionally unknown and do not contain any conserved domain associated with known activities. These analyses also identified 5 novel proteins by de novo sequencing and revealed several important proteins, mainly involved in signal transduction (such as protein kinases, receptor kinase-interacting proteins, guanosine 5'-diphosphate dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPase), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). The study is the first close investigation, to our knowledge, of protein complement in mature pollen, and presents useful molecular information at the protein level to further understand the mechanisms underlying pollen germination and tube growth.

  2. Genetics Home Reference: myostatin-related muscle hypertrophy

    MedlinePlus

    ... 765-70. Review. Citation on PubMed Schuelke M, Wagner KR, Stolz LE, Hübner C, Riebel T, Kömen ... 24;350(26):2682-8. Citation on PubMed Wagner KR, Cohen JS. Myostatin-Related Muscle Hypertrophy. 2005 ...

  3. The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression.

    PubMed

    Núñez-Acuña, Gustavo; Gallardo-Escárate, Cristian

    2014-02-15

    Myostatin (MSTN) is a protein of the Transforming Growth Factor-β (TGF-β) superfamily and plays a crucial role in muscular development for higher vertebrates. However, its biological function in marine invertebrates remains undiscovered. This study characterizes the full-length sequence of the Mytilus chilensis myostatin gene (Mc-MSTN). Furthermore, tissue transcription patterns and putative single nucleotide polymorphisms (SNPs) were also identified. The Mc-MSTN cDNA sequence showed 3528 base pairs (bp), consisting of 161 bp of 5' UTR, 2,110 bp of 3' UTR, and an open reading frame of 1,257 bp encoding for 418 amino acids and with an RXXR proteolytic site and nine cysteine-conserved residues. Gene transcription analysis revealed that the Mc-MSTN has ubiquitous expression among several tissues, with higher expression in the gonads and mantle than in the digestive gland, gills, and hemolymph. Furthermore, high levels of polymorphisms were detected (28 SNPs in 3'-UTR and 9 SNPs in the coding region). Two SNPs were non-synonymous and involved amino acid changes between Glu/Asp and Thr/Ile. Until now, the MSTN gene has been mainly related to muscle growth in marine bivalves. However, the present study suggests a putative biological function not entirely associated to muscle tissue and contributes molecular evidence to the current debate about the function of the MSTN gene in marine invertebrates.

  4. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

    PubMed

    Lee, Eun Ju; Jan, Arif Tasleem; Baig, Mohammad Hassan; Ashraf, Jalaluddin Mohammad; Nahm, Sang-Soep; Kim, Yong-Woon; Park, So-Young; Choi, Inho

    2016-08-01

    Differentiation of muscle satellite cells (MSCs) involves interaction of the proteins present in the extracellular matrix (ECM) with MSCs to regulate their activity, and therefore phenotype. Herein, we report fibromodulin (FMOD), a member of the proteoglycan family participating in the assembly of ECM, as a novel regulator of myostatin (MSTN) during myoblast differentiation. In addition to having a pronounced effect on the expression of myogenic marker genes [myogenin (MYOG) and myosin light chain 2 (MYL2)], FMOD was found to maintain the transcriptional activity of MSTN Moreover, coimmunoprecipitation and in silico studies performed to investigate the interaction of FMOD helped confirm that it antagonizes MSTN function by distorting its folding and preventing its binding to activin receptor type IIB. Furthermore, in vivo studies revealed that FMOD plays an active role in healing by increasing satellite cell recruitment to sites of injury. Together, these findings disclose a hitherto unrecognized regulatory role for FMOD in MSCs and highlight new mechanisms whereby FMOD circumvents the inhibitory effects of MSTN and triggers myoblast differentiation. These findings offer a basis for the design of novel MSTN inhibitors that promote muscle regeneration after injury or for the development of pharmaceutical agents for the treatment of different muscle atrophies.-Lee, E. J., Jan, A. T., Baig, M. H., Ashraf, J. M., Nahm, S.-S., Kim, Y.-W., Park, S.-Y., Choi, I. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

  5. Expression of a Novel Ciliary Protein, IIIG9, During the Differentiation and Maturation of Ependymal Cells.

    PubMed

    Cifuentes, M; Baeza, V; Arrabal, P M; Visser, R; Grondona, J M; Saldivia, N; Martínez, F; Nualart, F; Salazar, K

    2017-02-13

    IIIG9 is the regulatory subunit 32 of protein phosphatase 1 (PPP1R32), a key phosphatase in the regulation of ciliary movement. IIIG9 localization is restricted to cilia in the trachea, fallopian tube, and testicle, suggesting its involvement in the polarization of ciliary epithelium. In the adult brain, IIIG9 mRNA has only been detected in ciliated ependymal cells that cover the ventricular walls. In this work, we prepared a polyclonal antibody against rat IIIG9 and used this antibody to show for the first time the ciliary localization of this protein in adult ependymal cells. We demonstrated IIIG9 localization at the apical border of the ventricular wall of 17-day-old embryonic (E17) and 1-day-old postnatal (PN1) brains and at the level of ependymal cilia at 10- and 20-day-old postnatal (PN10-20) using temporospatial distribution analysis and comparing the localization with a ciliary marker. Spectral confocal and super-resolution Structured Illumination Microscopy (SIM) analysis allowed us to demonstrate that IIIG9 shows a punctate pattern that is preferentially located at the borders of ependymal cilia in situ and in cultures of ependymocytes obtained from adult rat brains. Finally, by immunogold ultrastructural analysis, we showed that IIIG9 is preferentially located between the axoneme and the ciliary membrane. Taken together, our data allow us to conclude that IIIG9 is localized in the cilia of adult ependymal cells and that its expression is correlated with the process of ependymal differentiation and with the maturation of radial glia. Similarly, its particular localization within ependymal cilia suggests a role of this protein in the regulation of ciliary movement.

  6. Proteomic comparison between maturation drying and prematurely imposed drying of Zea mays seeds reveals a potential role of maturation drying in preparing proteins for seed germination, seedling vigor, and pathogen resistance.

    PubMed

    Wang, Wei-Qing; Ye, Jian-Qing; Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna I; Jensen, Ole Nørregaard; Møller, Ian Max; Song, Song-Quan

    2014-02-07

    We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p < 0.05) in abundance during maturation drying in embryo and endosperm, respectively. Fewer proteins (48 and 59 in embryo and endosperm, respectively) changed in abundance during prematurely imposed drying. A number of proteins, 33 and 38 in embryo and endosperm, respectively, changed similarly in abundance during both maturation and prematurely imposed drying. Storage proteins were abundant in this group and may contribute to the acquisition of seed germinability. However, a relatively large number of proteins changed in the embryo (47 spots) and endosperm (76 spots) specifically during maturation drying. Among these proteins, storage proteins in the embryo and defense proteins in the endosperm may be particularly important for seedling vigor and resistance to fungal infection, respectively.

  7. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    PubMed

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Muscle hypertrophy induced by myostatin inhibition accelerates degeneration in dysferlinopathy.

    PubMed

    Lee, Yun-Sil; Lehar, Adam; Sebald, Suzanne; Liu, Min; Swaggart, Kayleigh A; Talbot, C Conover; Pytel, Peter; Barton, Elisabeth R; McNally, Elizabeth M; Lee, Se-Jin

    2015-10-15

    Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss. One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression. To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf(-/-)) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc). Here, we show that myostatin inhibition by follistatin transgene expression in Dysf(-/-) mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf(-/-) mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8-month-old Dysf(-/-) mice. Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf(-/-) mice, indicating possible muscle damage induced by hypertrophy. These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis. © The Author 2015. Published by Oxford University Press.

  9. Muscle hypertrophy induced by myostatin inhibition accelerates degeneration in dysferlinopathy

    PubMed Central

    Lee, Yun-Sil; Lehar, Adam; Sebald, Suzanne; Liu, Min; Swaggart, Kayleigh A.; Talbot, C. Conover; Pytel, Peter; Barton, Elisabeth R.; McNally, Elizabeth M.; Lee, Se-Jin

    2015-01-01

    Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss. One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression. To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf−/−) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc). Here, we show that myostatin inhibition by follistatin transgene expression in Dysf−/− mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf−/− mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8-month-old Dysf−/− mice. Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf−/− mice, indicating possible muscle damage induced by hypertrophy. These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis. PMID:26206886

  10. Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes

    USDA-ARS?s Scientific Manuscript database

    Myostatin is a potent negative regulator of skeletal muscle growth. Although several cDNA clones have been characterized in different vertebrates, the genomic organization and bioactivity of non-mammalian homologs have not. The intron/exon organization and promoter subsequence analysis of two rainbo...

  11. Proteins of the cauda epididymal fluid associated with fertility of mature dairy bulls.

    PubMed

    Moura, Arlindo A; Chapman, David A; Koc, Hasan; Killian, Gary J

    2006-01-01

    We evaluated the relationships between proteins in cauda epididymis fluid (CEF) and fertility scores of dairy bulls. Fertility was expressed as the percentage point deviation (PD) of bull nonreturn rate from the average fertility of all bulls at an artificial insemination center. The number of services for each bull ranged from 1074 to 52 820, and PD values ranged from +7.7% to -6.6%. CEF from 20 bulls was obtained from vasa deferentia cannulae and was separated from sperm by centrifugation immediately after collection. Samples were evaluated by 2-dimensional (2-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis gels stained with Coomassie blue, and polypeptide maps were analyzed by PDQuest software. Protein quantities, defined as the total integrated optical density of the spots, were compared between groups of high-fertility sires (n = 12; PD >or= 0) and low-fertility sires (n = 8; PD < 0) and were also used as independent variables in regression analysis. Proteins were identified by capillary liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry. An average of 118 spots was detected in 2-D maps of the CEF, but we were unable to distinguish any protein that was expressed only in high-fertility or in low-fertility bulls. However, the amount of alpha-L-fucosidase 2 and cathepsin D was 2.3- and 2.4-fold greater (P < .05) in high-fertility than in low-fertility bulls, respectively. Conversely, the intensities of 3 isoforms (24-27 kd; pl 6.3-5.8) of prostaglandin D-synthase (PGDS) were from 3.2- to 2.2-fold greater in low-fertility sires (P < .05). An empirical regression model established that a significant proportion (R(2) = 0.72; P < .0001) of the variation in fertility scores (PD values) was explained by the intensities of cathepsin D and 1 isoform of PGDS (24 kd; pl 6.3). Thus, multiple proteins present in the CEF are potential biomarkers of fertility in high-use, mature Holstein bulls.

  12. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation.

    PubMed

    Kim, Sungsu; Wairkar, Yogesh P; Daniels, Richard W; DiAntonio, Aaron

    2010-03-08

    Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.

  13. A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle.

    PubMed

    Nishi, Masumi; Yasue, Akihiro; Nishimatu, Shinichirou; Nohno, Tsutomu; Yamaoka, Takashi; Itakura, Mitsuo; Moriyama, Keiji; Ohuchi, Hideyo; Noji, Sumihare

    2002-04-26

    Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.

  14. Hypoxia inhibits maturation and trafficking of hERG K(+) channel protein: Role of Hsp90 and ROS.

    PubMed

    Nanduri, Jayasri; Bergson, Pamela; Wang, Ning; Ficker, Eckhard; Prabhakar, Nanduri R

    2009-10-16

    We previously reported that reactive oxygen species (ROS) generated during hypoxia decrease hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we investigated the molecular mechanisms involved in hypoxia-induced downregulation of hERG protein. Culturing cells at low temperatures and addition of chemical chaperones during hypoxia restored hERG expression and currents to normoxic levels while antiarrhythmic drugs, which selectively block hERG channels, had no effect on hERG protein levels. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Co-immunoprecipitation experiments revealed that hypoxia inhibited interaction of hERG with Hsp90 chaperone required for maturation, which was restored in the presence of ROS scavengers. These results demonstrate that ROS generated during hypoxia prevents maturation of the hERG protein by inhibiting Hsp90 interaction resulting in decreased protein expression and currents.

  15. Bovine whey protein concentrate supplementation modulates maturation of immune system in suckling rats.

    PubMed

    Pérez-Cano, Francisco J; Marín-Gallén, Silvia; Castell, Margarida; Rodríguez-Palmero, María; Rivero, Montserrat; Franch, Angels; Castellote, Cristina

    2007-10-01

    During neonatal life, challenges from breast milk and microbial flora promote immune system maturation. Immunonutrition in these stages may become an important way to increase natural defence systems. The aim of this study was to determine the effect of a daily bovine milk whey protein concentrate (WPC) supplement on the intestinal and systemic immune systems in suckling rats. The composition of intraepithelial and lamina propria lymphocytes (IEL and LPL) was analysed by flow cytometry. Systemic and intestinal humoral immune responses were determined by sera Ig levels and Ig-secreting cell quantification by ELISA and ELISPOT, respectively. From birth, suckling Wistar rats were supplemented with WPC or standard infant formula (SIF). The WPC group showed the same proportion of most of the main mucosal cell subsets as the reference animals. However, in the first days of life WPC enhanced the innate immunity by increasing the NK cell proportion in both epithelial and lamina propria (LP) compartments. A rise in intestinal CD8alphaalpha+ IEL was also induced by WPC supplementation. A time-course of sera Ig levels and spontaneous IgA, IgM and IgG production by LPL and mononuclear cells from blood and spleen, in the WPC group, exhibited a similar pattern to those pups fed only by dam's milk. In summary, the present results show the effects of WPC on enhancing mucosal innate immunity during early life.

  16. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing

    PubMed Central

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D.; Scharler, Cornelia; Niestrawska, Justyna A.; Holzapfel, Gerhard A.; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-01-01

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc−/− tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc−/− tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons. PMID:27586416

  17. The Wiskott-Aldrich syndrome protein is required for iNKT cell maturation and function

    PubMed Central

    Locci, Michela; Draghici, Elena; Marangoni, Francesco; Bosticardo, Marita; Catucci, Marco; Aiuti, Alessandro; Cancrini, Caterina; Marodi, Laszlo; Espanol, Teresa; Bredius, Robbert G.M.; Thrasher, Adrian J.; Schulz, Ansgar; Litzman, Jiri; Roncarolo, Maria Grazia; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was−/− mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was−/− mice as compared with wild-type controls. Moreover analysis of was−/− iNKT cell maturation revealed a complete arrest at the CD44+ NK1.1− intermediate stage. Notably, generation of BM chimeras demonstrated a was−/− iNKT cell-autonomous developmental defect. was−/− iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon γ upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology. PMID:19307326

  18. Nelfinavir Impairs Glycosylation of Herpes Simplex Virus 1 Envelope Proteins and Blocks Virus Maturation

    PubMed Central

    Gantt, Soren; Gachelet, Eliora; Carlsson, Jacquelyn; Barcy, Serge; Casper, Corey; Lagunoff, Michael

    2015-01-01

    Nelfinavir (NFV) is an HIV-1 aspartyl protease inhibitor that has numerous effects on human cells, which impart attractive antitumor properties. NFV has also been shown to have in vitro inhibitory activity against human herpesviruses (HHVs). Given the apparent absence of an aspartyl protease encoded by HHVs, we investigated the mechanism of action of NFV herpes simplex virus type 1 (HSV-1) in cultured cells. Selection of HSV-1 resistance to NFV was not achieved despite multiple passages under drug pressure. NFV did not significantly affect the level of expression of late HSV-1 gene products. Normal numbers of viral particles appeared to be produced in NFV-treated cells by electron microscopy but remain within the cytoplasm more often than controls. NFV did not inhibit the activity of the HSV-1 serine protease nor could its antiviral activity be attributed to inhibition of Akt phosphorylation. NFV was found to decrease glycosylation of viral glycoproteins B and C and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by NFV. These results demonstrate that NFV causes alterations in HSV-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for HHV replication. PMID:25709648

  19. Nelfinavir impairs glycosylation of herpes simplex virus 1 envelope proteins and blocks virus maturation.

    PubMed

    Gantt, Soren; Gachelet, Eliora; Carlsson, Jacquelyn; Barcy, Serge; Casper, Corey; Lagunoff, Michael

    2015-01-01

    Nelfinavir (NFV) is an HIV-1 aspartyl protease inhibitor that has numerous effects on human cells, which impart attractive antitumor properties. NFV has also been shown to have in vitro inhibitory activity against human herpesviruses (HHVs). Given the apparent absence of an aspartyl protease encoded by HHVs, we investigated the mechanism of action of NFV herpes simplex virus type 1 (HSV-1) in cultured cells. Selection of HSV-1 resistance to NFV was not achieved despite multiple passages under drug pressure. NFV did not significantly affect the level of expression of late HSV-1 gene products. Normal numbers of viral particles appeared to be produced in NFV-treated cells by electron microscopy but remain within the cytoplasm more often than controls. NFV did not inhibit the activity of the HSV-1 serine protease nor could its antiviral activity be attributed to inhibition of Akt phosphorylation. NFV was found to decrease glycosylation of viral glycoproteins B and C and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by NFV. These results demonstrate that NFV causes alterations in HSV-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for HHV replication.

  20. Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18.

    PubMed

    Harms, Robert Z; Creer, Austin J; Lorenzo-Arteaga, Kristina M; Ostlund, Katie R; Sarvetnick, Nora E

    2017-01-01

    The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α(-) NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor.

  1. Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18

    PubMed Central

    Harms, Robert Z.; Creer, Austin J.; Lorenzo-Arteaga, Kristina M.; Ostlund, Katie R.; Sarvetnick, Nora E.

    2017-01-01

    The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α− NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor. PMID:28900426

  2. Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

    PubMed

    Cattelan, Natalia; Villalba, María Inés; Parisi, Gustavo; Arnal, Laura; Serra, Diego Omar; Aguilar, Mario; Yantorno, Osvaldo

    2016-02-01

    Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.

  3. Whey protein processing influences formula-induced gut maturation in preterm pigs.

    PubMed

    Li, Yanqi; Østergaard, Mette V; Jiang, Pingping; Chatterton, Dereck E W; Thymann, Thomas; Kvistgaard, Anne S; Sangild, Per T

    2013-12-01

    Immaturity of the gut predisposes preterm infants to nutritional challenges potentially leading to clinical complications such as necrotizing enterocolitis. Feeding milk formulas is associated with greater risk than fresh colostrum or milk, probably due to loss of bioactive proteins (e.g., immunoglobulins, lactoferrin, insulin-like growth factor, transforming growth factor-β) during industrial processing (e.g., pasteurization, filtration, spray-drying). We hypothesized that the processing method for whey protein concentrate (WPC) would affect gut maturation in formula-fed preterm pigs used as a model for preterm infants. Fifty-five caesarean-delivered preterm pigs were distributed into 4 groups given 1 of 4 isoenergetic diets: formula containing conventional WPC (filtration, multi-pasteurization, standard spray-drying) (CF); formula containing gently treated WPC (reduced filtration and pasteurization, gentle spray-drying) (GF); formula containing minimally treated WPC (rennet precipitation, reduced filtration, heat treatment <40°C, freeze-drying) (MF); and bovine colostrum (used as a positive reference group) (BC). Relative to CF, GF, and MF pigs, BC pigs had greater villus heights, lactose digestion, and absorption and lower gut permeability (P < 0.05). MF and BC pigs had greater plasma citrulline concentrations than CF and GF pigs and intestinal interleukin-8 was lower in BC pigs than in the other groups (P < 0.05). MF pigs had lower concentrations of intestinal claudin-4, cleaved caspase-3, and phosphorylated c-Jun than CF pigs (P < 0.05). The conventional and gently treated WPCs had similar efficacy in stimulating proliferation of porcine intestinal epithelial cells. We conclude that processing of WPC affects intestinal structure, function, and integrity when included in formulas for preterm pigs. Optimization of WPC processing technology may be important to preserve the bioactivity and nutritional value of formulas for sensitive newborns.

  4. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    PubMed Central

    Pavoni, Emiliano; Flego, Michela; Dupuis, Maria Luisa; Barca, Stefano; Petronzelli, Fiorella; Anastasi, Anna Maria; D'Alessio, Valeria; Pelliccia, Angela; Vaccaro, Paola; Monteriù, Giorgia; Ascione, Alessandro; De Santis, Rita; Felici, Franco; Cianfriglia, Maurizio; Minenkova, Olga

    2006-01-01

    Background CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. Methods The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. Results The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. Conclusion The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance. PMID:16504122

  5. The mechanism of dehydration in chromophore maturation of wild-type green fluorescent protein: A theoretical study

    NASA Astrophysics Data System (ADS)

    Ma, Yingying; Yu, Jian-Guo; Sun, Qiao; Li, Zhen; Smith, Sean C.

    2015-07-01

    An interesting aspect of the green fluorescent protein (GFP) is its autocatalytic chromophore maturation. Numerous experimental studies have indicated that dehydration is the last step in the chromophore maturation process of wild-type GFP. Based on the crystal structure of wild-type GFP, the mechanism of the reverse reaction of dehydration was investigated by using density functional theory (DFT) in this study. Our results proposed that the dehydration is exothermic. Moreover, the rate-limiting step of the mechanism is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66, which is consistent with the experimental observation.

  6. LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii-containing vacuole.

    PubMed

    Schulze-Luehrmann, Jan; Eckart, Rita A; Ölke, Martha; Saftig, Paul; Liebler-Tenorio, Elisabeth; Lührmann, Anja

    2016-02-01

    The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal-like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii-containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP-1 and LAMP-2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP-1/2 double-deficient fibroblasts as well as in LAMP-1/2 knock-down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double-deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP-1 or LAMP-2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead-containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.

  7. Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging

    PubMed Central

    Wu, Weimin; Newcomb, William W.; Cheng, Naiqian; Aksyuk, Anastasia; Winkler, Dennis C.

    2016-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids—which develop into infectious virions—are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled. IMPORTANCE In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation

  8. Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle.

    PubMed

    Mouisel, Etienne; Relizani, Karima; Mille-Hamard, Laurence; Denis, Raphaël; Hourdé, Christophe; Agbulut, Onnik; Patel, Ketan; Arandel, Ludovic; Morales-Gonzalez, Susanne; Vignaud, Alban; Garcia, Luis; Ferry, Arnaud; Luquet, Serge; Billat, Véronique; Ventura-Clapier, Renée; Schuelke, Markus; Amthor, Helge

    2014-08-15

    Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn(-/-) mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn(-/-) mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn(-/-) mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn(-/-) mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.

  9. Functional role of the bovine oocyte-specific protein JY-1 in meiotic maturation, cumulus expansion, and subsequent embryonic development.

    PubMed

    Lee, Kyung-Bon; Wee, Gabbine; Zhang, Kun; Folger, Joseph K; Knott, Jason G; Smith, George W

    2014-03-01

    Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine early embryogenesis. Given that JY-1 is present in oocytes throughout folliculogenesis, and oocyte-derived JY-1 mRNA is temporally regulated postfertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 small interfering RNA was microinjected into cumulus-enclosed germinal vesicle-stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation (IVM), was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion, and embryonic development after in vitro fertilization. Depletion of JY-1 protein during IVM effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8- to 16-cell stage, and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion, and early cleavage, but did not rescue development to 8- to 16-cell and blastocyst stages. However, effects of JY-1 depletion postfertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion, and subsequent embryonic development.

  10. Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes.

    PubMed

    Opiela, Jolanta; Latasiewicz, Ewa; Smorag, Zdzisław

    2012-12-01

    With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 microL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn't affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn't have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes' meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells.

  11. Mechanisms Stimulating Muscle Wasting in Chronic Kidney Disease: The Roles of the Ubiquitin-Proteasome System and Myostatin

    PubMed Central

    Thomas, Sandhya S.; Mitch, William E.

    2013-01-01

    Catabolic conditions including chronic kidney disease (CKD), cancer, and diabetes cause muscle atrophy. The loss of muscle mass worsens the burden of disease because it is associated with increased morbidity and mortality. To avoid these problems or to develop treatment strategies, the mechanisms leading to muscle wasting must be identified. Specific mechanisms uncovered in CKD generally occur in other catabolic conditions. These include stimulation of protein degradation in muscle arising from activation of caspase-3 and the ubiquitin-proteasome system (UPS). These proteases act in a coordinated fashion with caspase-3 initially cleaving the complex structure of proteins in muscle yielding fragments that are substrates which are degraded by the UPS. Fortunately, the UPS exhibits remarkable specificity for proteins to be degraded because it is the major intracellular proteolytic system. Without a high level of specificity cellular functions would be disrupted. The specificity is accomplished by complex reactions that depend on recognition of a protein substrate by specific E3 ubiquitin ligases. In muscle, the specific ligases are Atrogin-1 and MuRF1 and their expression has characteristics of a biomarker of accelerated muscle proteolysis. Specific complications of CKD (metabolic acidosis, insulin resistance, inflammation, and angiotensin II) activate caspase-3 and the UPS through mechanisms that include glucocorticoids and impaired insulin or IGF-1 signaling. Mediators activate myostatin which functions as a negative growth factor in muscle. In models of cancer or CKD, strategies that block myostatin prevent muscle wasting suggesting that therapies which block myostatin could prevent muscle wasting in catabolic conditions. PMID:23292175

  12. Activation of Protein Kinase A in Mature Osteoblasts Promotes a Major Bone Anabolic Response.

    PubMed

    Tascau, Liana; Gardner, Thomas; Anan, Hussein; Yongpravat, Charlie; Cardozo, Christopher P; Bauman, William A; Lee, Francis Y; Oh, Daniel S; Tawfeek, Hesham A

    2016-01-01

    Protein kinase A (PKA) regulates osteoblast cell function in vitro and is activated by important bone mass modulating agents. We determined whether PKA activation in osteoblasts is sufficient to mediate a bone anabolic response. Thus, a mouse model conditionally expressing a constitutively active PKA (CA-PKA) in osteoblasts (CA-PKA-OB mouse) was developed by crossing a 2.3-kb α1 (I)-collagen promoter-Cre mouse with a floxed-CA-PKA mouse. Primary osteoblasts from the CA-PKA-OB mice exhibited higher basal PKA activity than those from control mice. Microcomputed tomographic analysis revealed that CA-PKA-OB female mice had an 8.6-fold increase in femoral but only 1.16-fold increase in lumbar 5 vertebral bone volume/total volume. Femur cortical thickness and volume were also higher in the CA-PKA-OB mice. In contrast, alterations in many femoral microcomputed tomographic parameters in male CA-PKA-OB mice were modest. Interestingly, the 3-dimensional structure model index was substantially lower both in femur and lumbar 5 of male and female CA-PKA-OB mice, reflecting an increase in the plate to rod-like structure ratio. In agreement, femurs from female CA-PKA-OB mice had greater load to failure and were stiffer compared with those of control mice. Furthermore, the CA-PKA-OB mice had higher levels of serum bone turnover markers and increased osteoblast and osteoclast numbers per total tissue area compared with control animals. In summary, constitutive activation of PKA in osteoblasts is sufficient to increase bone mass and favorably modify bone architecture and improve mechanical properties. PKA activation in mature osteoblasts is, therefore, an important target for designing anabolic drugs for treating diseases with bone loss.

  13. The protein domains of the Dictyostelium microprocessor that are required for correct subcellular localization and for microRNA maturation.

    PubMed

    Kruse, Janis; Meier, Doreen; Zenk, Fides; Rehders, Maren; Nellen, Wolfgang; Hammann, Christian

    2016-10-02

    The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in Dictyostelium discoideum of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a ΔdrnB gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the Dictyostelium microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.

  14. Genetic deletion of myostatin from the heart prevents skeletal muscle atrophy in heart failure

    PubMed Central

    Heineke, Joerg; Auger-Messier, Mannix; Xu, Jian; Sargent, Michelle; York, Allen; Welle, Stephen; Molkentin, Jeffery D.

    2010-01-01

    Background Cardiac cachexia is characterized by an exaggerated loss of skeletal muscle, weakness, and exercise intolerance, although the etiology of these effects remains unknown. Here we hypothesized that the heart functions as an endocrine organ in promoting systemic cachexia by secreting peptide factors such as myostatin. Myostatin is a cytokine of the transforming growth factor β(TGFβ) superfamily that is known to control muscle wasting. Methods and Results We used a Cre/loxP system to ablate myostatin (Mstn gene) expression in a celltype-specific manner. As expected, elimination of Mstn selectively in skeletal muscle with a myosin light chain 1f (MLC1f)-cre allele induced robust hypertrophy in all skeletal muscle. However, heart-specific deletion of Mstn with a Nkx2.5-cre allele did not alter baseline heart size or secondarily affect skeletal muscle size, but the characteristic wasting and atrophy of skeletal muscle that typifies heart failure was not observed in these heart-specific null mice, indicating that myocardial myostatin expression controls muscle atrophy in heart failure. Indeed, myostatin levels in the plasma were significantly increased in wildtype mice subjected to pressure overload-induced cardiac hypertrophy, but not in Mstn heart-specific deleted mice. Moreover, cardiac-specific overexpression of myostatin, which increased circulating levels of myostatin by 3–4-fold, caused a reduction in weight of the quadriceps, gastrocnemius, soleus, and even the heart itself. Lastly, to investigate myostatin as a potential therapeutic target for the treatment of muscle wasting in heart failure, we infused a myostatin blocking antibody (JA-16), which promoted greater maintenance of muscle mass in heart failure. Conclusions Myostatin released from cardiomyocytes induces skeletal muscle wasting in heart failure. Targeted inhibition of myostatin in cardiac cachexia might be a therapeutic option in the future. PMID:20065166

  15. Genetic deletion of myostatin from the heart prevents skeletal muscle atrophy in heart failure.

    PubMed

    Heineke, Joerg; Auger-Messier, Mannix; Xu, Jian; Sargent, Michelle; York, Allen; Welle, Stephen; Molkentin, Jeffery D

    2010-01-26

    Cardiac cachexia is characterized by an exaggerated loss of skeletal muscle, weakness, and exercise intolerance, although the cause of these effects remains unknown. Here, we hypothesized that the heart functions as an endocrine organ in promoting systemic cachexia by secreting peptide factors such as myostatin. Myostatin is a cytokine of the transforming growth factor-beta superfamily that is known to control muscle wasting. We used a Cre/loxP system to ablate myostatin (Mstn gene) expression in a cell type-specific manner. As expected, elimination of Mstn selectively in skeletal muscle with a myosin light chain 1f (MLC1f)-cre allele induced robust hypertrophy in all skeletal muscle. However, heart-specific deletion of Mstn with an Nkx2.5-cre allele did not alter baseline heart size or secondarily affect skeletal muscle size, but the characteristic wasting and atrophy of skeletal muscle that typify heart failure were not observed in these heart-specific null mice, indicating that myocardial myostatin expression controls muscle atrophy in heart failure. Indeed, myostatin levels in the plasma were significantly increased in wild-type mice subjected to pressure overload-induced cardiac hypertrophy but not in Mstn heart-specific deleted mice. Moreover, cardiac-specific overexpression of myostatin, which increased circulating levels of myostatin by 3- to 4-fold, caused a reduction in weight of the quadriceps, gastrocnemius, soleus, and even the heart itself. Finally, to investigate myostatin as a potential therapeutic target for the treatment of muscle wasting in heart failure, we infused a myostatin blocking antibody (JA-16), which promoted greater maintenance of muscle mass in heart failure. Myostatin released from cardiomyocytes induces skeletal muscle wasting in heart failure. Targeted inhibition of myostatin in cardiac cachexia might be a therapeutic option in the future.

  16. [Positional clonage and characterization of the bovine myostatin gene].

    PubMed

    Grobet, L

    2000-01-01

    The double-muscled condition has been intensively selected for in the Belgian Blue cattle breed, where segregation studies have demonstrated the monogenic, autosomal and recessive determinism. This has been confirmed by genetic linkage which located the gene to the centromeric tip of chromosome 2. Our positional cloning strategy, and the discovery of a positional candidate in the mouse, led us to the identification of the causative gene now referred to as the Myostatin gene, since its product downregulates skeletal muscle mass. Disruptive mutations of the gene in cattle have been shown to be responsible for the muscular hypertrophy found in eight european beef breeds. A 15 Kilobases genomic region, including the myostatin gene, has been sequenced and compared in cattle and mice. The murine gene has undergone a complex genetic engineering in order to test different allelic variants in vivo after gene targeting transgenesis.

  17. Meiotic response of in vitro matured canine oocytes under different proteins and heterologous hormone supplementation.

    PubMed

    Rodrigues, B A; Rodrigues, J L

    2003-02-01

    The impact of TCM-199 supplemented with different proteins and heterologous hormones on the in vitro maturation (IVM) rate of bitch oocytes was evaluated by nuclear staining under fluorescence microscopy. Oocytes were recovered by slicing of ovaries from bitches presented at various stages of oestrous cycle to ovariohysterectomy. The basic culture medium was TCM-199 supplemented with 25 mM Hepes/l, with 10% heat-inactivated oestrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium bicarbonate and 22-microg/ml pyruvic acid, 1.0-microg/ml oestradiol (E 8875; Sigma), 0.5-microg/ml follicle-stimulating hormone (FSH) (Folltropin-V; Vetrepharm Inc., Ontario, Canada) and 0.03 IU/ml human gonadotropin (hCG) (Profasi HP; Serono, Aubonne, Switzerland). Oocytes were distributed randomly between basic culture medium (control) and the corresponding experimental treatment. Hormone treatments were: oocytes cultured in; (1) medium without FSH, (2) control medium supplemented with 20 microg/ml oestradiol, or (3) medium supplemented with 1 microg/ml human somatotropin (hST; Humatrope, Lilly, Saint Cloud, France). The second experiment consisted of oocytes cultured in medium supplemented with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Gibco Grand Island, NY, USA) instead of ECS, or oocytes cultured in medium with 10% inactivated oestrous bitch serum (EBS) instead of ECS. Oocytes were cultured in 100 microl droplets (up to 25 oocytes per drop) under mineral oil at 37 degrees C in a 100% humidified atmosphere containing 5% CO2 in air. After 72 h of IVM, the highest rates (p < 0.05) of meiotic resumption were achieved with the 0.4% BSA supplementation. A positive influence on the metaphase II (MII) acquisition rate was observed with hST supplement. Oocytes cultured with 10% EBS supplementation did not develop to the MII stage. The results in this study show that the protein and hormone supplements to TCM-199 culture medium tested did not promote the final steps of IVM

  18. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    SciTech Connect

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-03-15

    The Nipah virus fusion (F) protein is proteolytically processed to F{sub 1} + F{sub 2} subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.

  19. Immunophotoaffinity labeling of the binding proteins for 1-methyladenine, an oocyte maturation-inducing hormone of starfish.

    PubMed

    Kida, Tetsuo; Matsuda, Shinjiro; Kuyama, Atsushi; Toraya, Tetsuo

    2014-01-01

    Starfish oocytes are naturally arrested at the prophase stage of the first meiotic division and resume meiosis in response to 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative receptors for 1-MeAde have not yet been characterized biochemically, although the specific binding of 1-MeAde to the isolated cortices of starfish oocytes was reported so far. Based on the structure-activity relationship of 1-MeAde analogs, we have designed a photoaffinity labeling reagent. The photoaffinity labeling of oocyte membrane fractions, followed by immunoblotting analysis with anti-1-MeAde antibody, results in the detection of an almost single protein band. This 1-MeAde-binding protein might be a possible candidate of the maturation-inducing hormone receptor of starfish.

  20. Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-{beta} family signaling

    SciTech Connect

    Nomura, Tetsuya; Ueyama, Tomomi; Ashihara, Eishi; Tateishi, Kento; Asada, Satoshi; Nakajima, Norio; Isodono, Koji; Takahashi, Tomosaburo; Matsubara, Hiroaki Oh, Hidemasa

    2008-01-25

    The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-{beta} family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.

  1. Megakaryocytic Maturation in Response to Shear Flow Is Mediated by the Activator Protein 1 (AP-1) Transcription Factor via Mitogen-activated Protein Kinase (MAPK) Mechanotransduction.

    PubMed

    Luff, Stephanie A; Papoutsakis, Eleftherios T

    2016-04-08

    Megakaryocytes (MKs) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation to release pro/preplatelets into circulating blood. Shear forces promote DNA synthesis, polyploidization, and maturation in MKs, and platelet biogenesis. To investigate mechanisms underlying these MK responses to shear, we carried out transcriptional analysis on immature and mature stem cell-derived MKs exposed to physiological shear. In immature (day (d)9) MKs, shear exposure up-regulated genes related to growth and MK maturation, whereas in mature (d12) MKs, it up-regulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, six activator protein 1 (AP-1) transcripts (ATF4,JUNB,JUN,FOSB,FOS, andJUND) were up-regulated at d9 and two AP-1 proteins (JunD and c-Fos) were up-regulated both at d9 and d12. We show that mitogen-activated protein kinase (MAPK) signaling is linked to both the shear stress response and AP-1 up-regulation. c-Jun N-terminal kinase (JNK) phosphorylation increased significantly following shear stimulation, whereas JNK inhibition reduced shear-induced JunD expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos expression. JNK inhibition reduced fibrinogen binding and P-selectin expression of d12 platelet-like particles (PLPs), whereas p38 inhibition reduced fibrinogen binding of d12 PLPs. AP-1 expression correlated with increased MK DNA synthesis and polyploidization, which might explain the observed impact of shear on MKs. To summarize, we show that MK exposure to shear forces results in JNK activation, AP-1 up-regulation, and downstream transcriptional changes that promote maturation of immature MKs and platelet biogenesis in mature MKs.

  2. A p6Pol-protease fusion protein is present in mature particles of human immunodeficiency virus type 1.

    PubMed Central

    Almog, N; Roller, R; Arad, G; Passi-Even, L; Wainberg, M A; Kotler, M

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease (PR) and p6(Pol) are translated as part of the Gag-Pol polyprotein after a ribosomal frameshift. PR is essential to virus replication and is responsible for cleaving Gag and Gag-Pol precursors, but the role of p6(Pol) in HIV-1 infection is poorly understood. Here, we report that (i) PR is present in mature HIV-1 virions primarily as a p6(Pol)-PR fusion protein; (ii) HIV-1 PR cleaves viral precursor proteins expressed in bacterial cells at the Phe-Leu bond (positions 1639 to 1642) located at the junction of the NC and p6(Pol) proteins, releasing the p6(Pol)-PR fusion protein; and (iii) purified p6(Pol)-PR fusion protein undergoes autocleavage in vitro at at least three sites. PMID:8794372

  3. The function of myostatin in the regulation of fat mass in mammals.

    PubMed

    Deng, Bing; Zhang, Feng; Wen, Jianghui; Ye, Shengqiang; Wang, Lixia; Yang, Yu; Gong, Ping; Jiang, Siwen

    2017-01-01

    Myostatin (MSTN), also referred to as growth and differentiation factor-8, is a protein secreted in muscle tissues. Researchers believe that its primary function is in negatively regulating muscle because a mutation in its coding region can lead to the famous double muscle trait in cattle. Muscle and adipose tissue develop from the same mesenchymal stem cells, and researchers have found that MSTN is expressed in fat tissues and plays a key role in adipogenesis. Interestingly, MSTN can exert a dual function, either inhibiting or promoting adipogenesis, according to the situation. Due to its potential function in controlling body fat mass, MSTN has attracted the interest of researchers. In this review, we explore its function in regulating adipogenesis in mammals, including preadipocytes, multipotent stem cells and fat mass.

  4. A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

    PubMed Central

    Al-Nihmi, Faisal Mohammed Abdul; Kolli, Surendra Kumar; Reddy, Segireddy Rameswara; Mastan, Babu S.; Togiri, Jyothi; Maruthi, Mulaka; Gupta, Roshni; Sijwali, Puran Singh; Mishra, Satish; Kumar, Kota Arun

    2017-01-01

    Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus. PMID:28067322

  5. Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein

    SciTech Connect

    Li, H.M.; Sullivan, T.D.; Keegstra, K.

    1992-09-15

    Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize Bt1 locus encodes an amyloplast membrane metabolite translocator protein. The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite tranlocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.

  6. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability1[OPEN

    PubMed Central

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H.; van Wijk, Klaas J.

    2015-01-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. PMID:26371235

  7. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy.

    PubMed

    Malerba, Alberto; Kang, Jagjeet K; McClorey, Graham; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Wood, Matthew Ja; Dickson, George

    2012-12-18

    The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD.Molecular Therapy - Nucleic Acids (2012) 1, e62; doi:10.1038/mtna.2012.54; published online 18 December 2012.

  8. Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting.

    PubMed

    Breitbart, Astrid; Auger-Messier, Mannix; Molkentin, Jeffery D; Heineke, Joerg

    2011-06-01

    A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-β (TGF-β) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future.

  9. Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting

    PubMed Central

    Breitbart, Astrid; Auger-Messier, Mannix; Molkentin, Jeffery D.

    2011-01-01

    A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-β (TGF-β) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future. PMID:21421824

  10. Enhanced Myogenesis in adult skeletal muscle by transgenic expression of Myostatin Propeptide

    USDA-ARS?s Scientific Manuscript database

    Skeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-ß, plays a dominant role in the genetic control of muscle mass. Transgenic expression of myostatin propeptide in skeletal muscle showed ...

  11. The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding

    SciTech Connect

    Cash, Jennifer N.; Rejon, Carlis A.; McPherron, Alexandra C.; Bernard, Daniel J.; Thompson, Thomas B.

    2009-09-29

    Myostatin is a member of the transforming growth factor-{beta} (TGF-{beta}) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-{beta} class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.

  12. In vitro maturation of dromedary (Camelus dromedarius) oocytes: effect of different protein supplementations and epidermal growth factor*.

    PubMed

    Wani, Na; Wernery, U

    2010-10-01

    The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4-well culture plates containing 500 μl of the maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO(2) in air for 32-36 h. The basic maturation medium consisted of TCM-199 supplemented with 0.1 mg/ml L-glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto-orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase-I, anaphase-I (A-I), metaphase-II (M-II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M-II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M-II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any

  13. 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

  14. Bone morphogenetic protein 1 is expressed in porcine ovarian follicles and promotes oocyte maturation and early embryonic development

    PubMed Central

    LEI, Xiaocan; CUI, Kuiqing; CAI, Xiaoyan; REN, Yanping; LIU, Qingyou; SHI, Deshun

    2016-01-01

    In the present study, we tried to determine whether bone morphogenetic protein 1 (BMP1) plays a role in ovarian follicular development and early embryo development. We systematically investigated the expression and influence of BMP1 during porcine follicle and early embryonic development. Immunohistochemistry demonstrated that the BMP1 protein is expressed in granular cells and oocytes during follicular development, from primary to pre-ovulatory follicles, including atretic follicles and the corpus luteum. The mRNA expression of BMP1 significantly increased as the porcine follicles grew. Immunofluorescence analysis indicated that BMP1 was expressed in cumulus-oocyte complexes (COCs), oocytes and porcine embryos during early in vitro culture. qPCR and western blot analysis showed that the expression of BMP1 was significantly up-regulated in mature porcine oocytes and COCs compared to immature oocytes and COCs. BMP1 is expressed in early porcine embryos, and its expression reaches a peak at the 8-cell stage. To determine the effect of BMP1 on the maturation of oocytes and the development of early embryos, various concentrations of BMP1 recombinant protein or antibody were added to the in vitro culture media, respectively. BMP1 significantly affected the porcine oocyte maturation rate, the cleavage rate and the blastocyst development rate of embryos cultured in vitro in a positive way, as well as the blastocyst cell number. In conclusion, BMP1 is expressed throughout porcine ovarian follicle development and early embryogenesis, and it promotes oocyte maturation and the developmental ability of embryos during early in vitro culture. PMID:27890905

  15. Disruption of the myostatin gene in porcine primary fibroblasts and embryos using zinc-finger nucleases.

    PubMed

    Huang, Xian-Ju; Zhang, Hong-Xiao; Wang, Huili; Xiong, Kai; Qin, Ling; Liu, Honglin

    2014-04-01

    Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin lossof- function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos.

  16. Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    PubMed Central

    Huang, Xian-Ju; Zhang, Hong-Xiao; Wang, Huili; Xiong, Kai; Qin, Ling; Liu, Honglin

    2014-01-01

    Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin loss-of-function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos. PMID:24802055

  17. The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin

    PubMed Central

    2016-01-01

    In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals. PMID:27810970

  18. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    PubMed

    Santiquet, Nicolas; Sasseville, Maxime; Laforest, Martin; Guillemette, Christine; Gilchrist, Robert B; Richard, François J

    2014-08-01

    The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian

  19. Protein export by the mycobacterial SecA2 system is determined by the preprotein mature domain.

    PubMed

    Feltcher, Meghan E; Gibbons, Henry S; Ligon, Lauren S; Braunstein, Miriam

    2013-02-01

    At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

  20. Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

    PubMed Central

    Feltcher, Meghan E.; Gibbons, Henry S.; Ligon, Lauren S.

    2013-01-01

    At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm. PMID:23204463

  1. Multiple vitellogenins and product yolk proteins in striped bass, Morone saxatilis: molecular characterization and processing during oocyte growth and maturation.

    PubMed

    Williams, V N; Reading, B J; Hiramatsu, N; Amano, H; Glassbrook, N; Hara, A; Sullivan, C V

    2014-04-01

    The multiple vitellogenin (Vtg) system of striped bass, a perciform species spawning nearly neutrally buoyant eggs in freshwater, was investigated. Vitellogenin cDNA cloning, Western blotting of yolk proteins (YPs) using Vtg and YP type-specific antisera, and tandem mass spectrometry (MS/MS) of the YPs revealed the complex mechanisms of yolk formation and maturation in this species. It was discovered that striped bass possesses a tripartite Vtg system (VtgAa, VtgAb, and VtgC) in which all three forms of Vtg make a substantial contribution to the yolk. The production of Vtg-derived YPs is generally similar to that described for other perciforms. However, novel amino-terminal labeling of oocyte YPs prior to MS/MS identified multiple alternative sites for cleavage of these proteins from their parent Vtg, revealing a YP mixture far more complex than reported previously. This approach also revealed that the major YP product of each form of striped bass Vtg, lipovitellin heavy chain (LvH), undergoes limited degradation to smaller polypeptides during oocyte maturation, unlike the case in marine fishes spawning buoyant eggs in which LvHAa undergoes extensive proteolysis to osmotically active free amino acids. These differences likely reflect the lesser need for hydration of pelagic eggs spawned in freshwater. The detailed characterization of Vtgs and their proteolytic fate(s) during oocyte growth and maturation establishes striped bass as a freshwater model for investigating teleost multiple Vtg systems.

  2. Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes.

    PubMed

    Garikipati, Dilip K; Gahr, Scott A; Rodgers, Buel D

    2006-09-01

    Myostatin is a potent negative regulator of skeletal muscle growth. Although several cDNA clones have been characterized in different vertebrates, the genomic organization and bioactivity of non-mammalian homologs have not. The intron/exon organization and promoter subsequence analysis of two rainbow trout myostatin genes, rtMSTN-1a and rtMSTN-1b (formerly 1 and 2 respectively), as well as a quantitative assessment of their embryonic, larval, and adult tissue expression profiles are reported herein. Each gene was similarly organized into three exons of 490, 368, and 1600 bp for MSTN-1a and 486, 386, and 1419 bp for MSTN-1b. Comparative mapping of coding regions from several vertebrate myostatin genes revealed a common organization between species, including conserved pre-mRNA splice sites; the first among the fishes and the second across all vertebrate species. In silico subsequence analysis of the promoter regions identified E-boxes and other putative myogenic response elements. However, the number and diversity of elements were considerably less than those found in mammalian promoters or in the recently characterized zebrafish MSTN-2 gene. A quantitative analysis of the embryonic expression profile for both genes indicates that rtMSTN-1a expression is consistently greater than that of rtMSTN-1b and neither gene is significantly expressed throughout gastrulation. Expression of both steadily increases fourfold during somitogenesis and subsides as this period ends. After eyeing, however, rtMSTN-1a mRNA levels ultimately rise 20-fold by day 49 and peak before hatching and yolk sac absorption (YSA). Levels of rtMSTN-1b rise similarly, but do not peak before YSA. An analysis of adult (2-year-old fish) tissue expression indicates that both transcripts are present in most tissues although levels are highest in brain, testes, eyes, muscle, and surprisingly spleen. These studies suggest that strong selective pressures have preserved the genomic organization of myostatin

  3. Expression of myostatin, myostatin receptors and follistatin in diabetic rats submitted to exercise.

    PubMed

    Dutra, Daniela B; Bueno, Patrícia G; Silva, Rafaella N; Nakahara, Natália H; Selistre-Araújo, Heloísa S; Nonaka, Keico O; Leal, Angela Mo

    2012-05-01

    Myostatin (MSTN) has been implicated in metabolic adaptation to physiological stimuli, such as physical exercise, which is linked to improved glucose homeostasis. The aim of the present study was to evaluate the influence of exercise on the expression of MSTN, MSTN receptors (ActRIIB and ALK4) and follistatin (FS) in the muscle and fat of streptozotocin-induced diabetic rats. Control and diabetic rats were randomly assigned to a swimming training group (EC and ED, respectively) and a sedentary group (SC and SD, respectively). Exercising animals swam for 45 min at 0900 and 1700 hours, 5 day/week, for 4 weeks. The mRNA expression of MSTN, ActRIIB, ALK4 and FS mRNA was quantified by real-time reverse transcription-polymerase chain reaction. Expression of MSTN and FS mRNA increased in the muscle and subcutaneous fat of SD compared with SC rats. Expression of ActRIIB mRNA was increased in the muscle, mesenteric fat and brown adipose tissue (BAT) of SD compared with SC rats, whereas ALK4 mRNA expression was only increased in the BAT of SD compared with SC rats. After training, MSTN and ActRIIB expression was lower in the BAT of EC compared with SC rats. Expression of MSTN mRNA increased in the mesenteric fat of ED compared with SD rats, whereas FS mRNA expression decreased in the muscle, mesenteric and subcutaneous fat and BAT. Lower ALK4 mRNA expression was noted in the BAT of ED compared with SD rats. These results indicate that MSTN, its receptors and FS expression change in both the muscle and fat of diabetic rats and that the expression of these factors can be modulated by exercise in diabetes.

  4. High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo.

    PubMed

    Yamada, Michiko; Tatsumi, Ryuichi; Yamanouchi, Keitaro; Hosoyama, Tohru; Shiratsuchi, Sei-ichi; Sato, Akiko; Mizunoya, Wataru; Ikeuchi, Yoshihide; Furuse, Mitsuhiro; Allen, Ronald E

    2010-03-01

    Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced

  5. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    SciTech Connect

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  6. Myostatin from the American lobster, Homarus americanus: Cloning and effects of molting on expression in skeletal muscles.

    PubMed

    MacLea, Kyle S; Covi, Joseph A; Kim, Hyun-Woo; Chao, Erica; Medler, Scott; Chang, Ernest S; Mykles, Donald L

    2010-12-01

    A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S₁) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

  7. Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

    PubMed

    Zhang, Lina; de Waard, Marita; Verheijen, Hester; Boeren, Sjef; Hageman, Jos A; van Hooijdonk, Toon; Vervoort, Jacques; van Goudoever, Johannes B; Hettinga, Kasper

    2016-09-16

    To objective of this study was to better understand the biological functions of breast milk proteins in relation to the growth and development of infants over the first six months of life. Breast milk samples from four individual women collected at seven time points in the first six months after delivery were analyzed by filter aided sample preparation and dimethyl labeling combined with liquid chromatography tandem mass spectrometry. A total of 247 and 200 milk serum proteins were identified and quantified, respectively. The milk serum proteome showed a high similarity (80% overlap) on the qualitative level between women and over lactation. The quantitative changes in milk serum proteins were mainly caused by three groups of proteins, enzymes, and transport and immunity proteins. Of these 21 significantly changed proteins, 30% were transport proteins, such as serum albumin and fatty acid binding protein, which are both involved in transporting nutrients to the infant. The decrease of the enzyme bile salt-activated lipase as well as the immunity proteins immunoglobulins and lactoferrin coincide with the gradual maturation of the digestive and immune system of infants. The human milk serum proteome didn't differ qualitatively but it did quantitatively, both between mothers and as lactation advanced. The changes of the breast milk serum proteome over lactation corresponded with the development of the digestive and immune system of infants. Breast milk proteins provide nutrition, but also contribute to healthy development of infants. Despite the previously reported large number of identified breast milk proteins and their changes over lactation, less is known on the changes of these proteins in individual mothers. This study is the first to determine the qualitative and quantitative changes of milk proteome over lactation between individual mothers. The results indicate that the differences in the milk proteome between individual mothers are more related to the

  8. A nine-country study of the protein content and amino acid composition of mature human milk

    PubMed Central

    Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

    2016-01-01

    Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and

  9. Production of Transgenic Calves Expressing an shRNA Targeting Myostatin

    PubMed Central

    Tessanne, K; Golding, MC; Long, CR; Peoples, MD; Hannon, G; Westhusin, ME

    2012-01-01

    Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing. PMID:22139943

  10. Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.

    PubMed Central

    Wormington, M; Searfoss, A M; Hurney, C A

    1996-01-01

    The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability. Images PMID:8631310

  11. Myostatin induces insulin resistance via Casitas B-lineage lymphoma b (Cblb)-mediated degradation of insulin receptor substrate 1 (IRS1) protein in response to high calorie diet intake.

    PubMed

    Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

    2014-03-14

    To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.

  12. Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function.

    PubMed

    Ferreira-Cerca, Sébastien; Pöll, Gisela; Gleizes, Pierre-Emmanuel; Tschochner, Herbert; Milkereit, Philipp

    2005-10-28

    Despite the rising knowledge about ribosome function and structure and how ribosomal subunits assemble in vitro in bacteria, the in vivo role of many ribosomal proteins remains obscure both in pro- and eukaryotes. Our systematic analysis of yeast ribosomal proteins (r-proteins) of the small subunit revealed that most eukaryotic r-proteins fulfill different roles in ribosome biogenesis, making them indispensable for growth. Different r-proteins control distinct steps of nuclear and cytoplasmic pre-18S rRNA processing and, thus, ensure that only properly assembled ribosomes become engaged in translation. Comparative analysis of dynamic and steady-state maturation assays revealed that several r-proteins are required for efficient nuclear export of pre-18S rRNA, suggesting that they form an interaction platform with the export machinery. In contrast, the presence of other r-proteins is mainly required before nuclear export is initiated. Our studies draw a correlation between the in vitro assembly, structural localization, and in vivo function of r-proteins.

  13. Functions of defense-related proteins and dehydrogenases in resistance response induced by salicylic acid in sweet cherry fruits at different maturity stages.

    PubMed

    Chan, Zhulong; Wang, Qing; Xu, Xiangbin; Meng, Xianghong; Qin, Guozheng; Li, Boqiang; Tian, Shiping

    2008-11-01

    We report here a comparative analysis of sweet cherry (Prunus avium) fruits proteome induced by salicylic acid (SA) at different maturity stages. The results demonstrated that SA enhanced the resistance of sweet cherry fruits against Penicillium expansum, resulting in lower disease incidences and smaller lesion diameters, especially at earlier maturity stage. Based on proteomics analysis, 13 and 28 proteins were identified after SA treatment at earlier (A) and later (B) maturity stage, respectively. Seven antioxidant proteins and three pathogenesis related-proteins were identified at both A and B stages, while five heat shock proteins and four dehydrogenases were only detected at B stage. SA treatment also stimulated higher transcript levels of peroxidase, but repressed that of catalase. Moreover, some proteins regulated by SA at B maturity stage were identified as enzymes involved in glycolysis and tricarboxylic acid cycle. These findings indicated that younger sweet cherry fruits showed stronger resistance against pathogen invasion after SA treatment. It further indicated that antioxidant proteins were involved in the resistance response of fruits at every maturity stage, while heat shock proteins and dehydrogenases might potentially act as factors only at later maturity stages.

  14. Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.

    PubMed Central

    Boyartchuk, V L; Rine, J

    1998-01-01

    In eukaryotes small secreted peptides are often proteolytically cleaved from larger precursors. In Saccharomyces cerevisiae multiple proteolytic processing steps are required for production of mature 12-amino-acid a-factor from its 36-amino-acid precursor. This study provides additional genetic data supporting a direct role for Afc1p in cleavage of the carboxyl-terminal tripeptide from the CAAX motif of the prenylated a-factor precursor. In addition, Afc1p had a second role in a-factor processing that was independent of, and in addition to, its role in the carboxyl-terminal processing in vivo. Using ubiquitin-a-factor fusions we confirmed that the pro-region of the a-factor precursor was not required for production of the mature pheromone. However, the pro-region of the a-factor precursor contributed quantitatively to a-factor production. PMID:9725832

  15. Elevated insulin-like growth factor 2 expression may contribute to the hypermuscular phenotype of myostatin null mice.

    PubMed

    Clark, Daniel L; Clark, Diana I; Hogan, Elizabeth K; Kroscher, Kellie A; Dilger, Anna C

    2015-10-01

    Myostatin (Mstn) inhibits while insulin-like growth factors 1 and 2 (Igf1 and Igf2) increase skeletal muscle growth. However, there is little known regarding Mstn regulation of Igf1 and Igf2 expression. Therefore, the objective of this study was to quantify the expression of IGF family members in skeletal muscle and liver throughout the growth phase of Mstn null (MN) mice. Further, differences between male and female mice were investigated. Male and female wild type (WT) and MN mice were euthanized at birth (0 d), 7 days (7 d), weaning (21 d), sexual maturity (42 d), and 70 d. For the neonatal periods, 0 d and 7 d, all muscles from the hind limbs were compiled for RNA extraction. At 21 d, 42 d, and 70 d, biceps femoris (BF), tibialis anterior, triceps brachii (TB), and gastrocnemius-soleus complex were collected. As expected, muscle weights were up to 90% greater in MN mice compared with WT mice at 21 d, 42 d and 70 d. However, Igf1 expression was reduced (P ≤ 0.04) at 7d and 21 d in MN mice compared to WT mice. Expression of Igf2 did not differ between genotypes at 0 d and 7d, but, at 21 d, 42 d and 70 d in BF and TB muscles, Igf2 expression was 1.9-2.9 fold greater (P<0.01) in MN compared to WT mice. Hepatic Igf1 and Igf2 levels were minimally affected by genotype; with the exception of a 1.4-fold reduction (P=0.04) in Igf1 expression in 21 d MN mice compared with WT mice. Though male mice were heavier than females starting at 21 d of age, expression differences in Igf1, Igf2, their receptors and binding proteins do not account for growth differences. In every case, when expression was different between sexes, female expression was increased despite increased growth in male mice. This study is the first to provide evidence that Mstn may negatively regulate Igf2 expression to control postnatal skeletal muscle growth, however differences in growth between male and female mice are not readily explained by changes in expression of Igf family members. Copyright

  16. Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination

    DTIC Science & Technology

    2009-12-01

    exosporium maturation and assembly and suggest a novel role for the exosporium in germination. During starvation, bacteria of the genus Bacillus...collection of information, including suggestions for reducing this burden, to Washington Headquarters Services , Directorate for Information Operations and...homogenized spleens were titrated on LB agar plates to determine the total numbers of bacteria and on LB agar plates containing kanamycin, on which only

  17. Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    Pollen tubes navigate through different female tissues and deliver the sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here we show that truncated versions of a protein disulfide isomerase (PDI), ...

  18. Proteomic analysis of the mature Brassica stigma reveals proteins with diverse roles in vegetative and reproductive development.

    PubMed

    Nazemof, Nazila; Couroux, Philippe; Xing, Tim; Robert, Laurian S

    2016-09-01

    The stigma, the specialized apex of the Brassicaceae gynoecium, plays a role in pollen capture, discrimination, hydration, germination, and guidance. Despite this crucial role in reproduction, the global proteome underlying Brassicaceae stigma development and function remains largely unknown. As a contribution towards the characterization of the Brassicaceae dry stigma global proteome, more than 2500 Brassica napus mature stigma proteins were identified using three different gel-based proteomics approaches. Most stigma proteins participated in Metabolic Processes, Responses to Stimulus or Stress, Cellular or Developmental Processes, and Transport. The stigma was found to express a wide variety of proteins with demonstrated roles in cellular and organ development including proteins known to be involved in cellular expansion and morphogenesis, embryo development, as well as gynoecium and stigma development. Comparisons to a corresponding proteome from a very morphologically different Poaceae dry stigma showed a very similar distribution of proteins among different functional categories, but also revealed evident distinctions in protein composition especially in glucosinolate and carotenoid metabolism, photosynthesis, and self-incompatibility. To our knowledge, this study reports the largest Brassicaceae stigma protein dataset described to date.

  19. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    PubMed Central

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

    2017-01-01

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein. PMID:28134243

  20. The N-Terminus of Murine Leukaemia Virus p12 Protein Is Required for Mature Core Stability

    PubMed Central

    Wight, Darren J.; Boucherit, Virginie C.; Wanaguru, Madushi; Elis, Efrat; Hirst, Elizabeth M. A.; Li, Wilson; Ehrlich, Marcelo; Bacharach, Eran; Bishop, Kate N.

    2014-01-01

    The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices. PMID:25356837

  1. Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

    PubMed Central

    Mullins, Chris; Bonifacino, Juan S.

    2001-01-01

    The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of α-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and α-factor maturation and identify structural determinants that are critical for these functions. PMID:11689690

  2. Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

    PubMed Central

    García-Gómez, Juan J.; Rosado, Iván V.; Tollervey, David; Kressler, Dieter; de la Cruz, Jesús

    2014-01-01

    Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles. PMID:24603549

  3. Bone architecture and disc degeneration in the lumbar spine of mice lacking GDF-8 (myostatin).

    PubMed

    Hamrick, Mark W; Pennington, Catherine; Byron, Craig D

    2003-11-01

    GDF-8, also known as myostatin, is a member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle. Myostatin functions as a negative regulator of skeletal muscle growth and myostatin null mice show a doubling of muscle mass compared to normal mice. We describe here morphology of the lumbar spine in myostatin knockout (Mstn(-/-)) mice using histological and densitometric techniques. The Mstn(-/-) mice examined in this study weigh approximately 10% more than controls (p<0.001) but the iliopsoas muscle is over 50% larger in the knockout mice than in wild-type mice (p<0.001). Peripheral quantitative computed tomography (pQCT) data from the fifth lumbar vertebra show that mice lacking myostatin have approximately 50% greater trabecular bone mineral density (p=0.001) and significantly greater cortical bone mineral content than normal mice. Toluidine blue staining of the intervertebral disc between L4-L5 reveals loss of proteoglycan staining in the hyaline end plates and inner annulus fibrosus of the knockout mice. Loss of cartilage staining in the caudal end plate of L4 is due to ossification of the end plate in the myostatin-deficient animals. Results from this study suggest that increased muscle mass in mice lacking myostatin is associated with increased bone mass as well as degenerative changes in the intervertebral disc.

  4. Myostatin signals through Pax7 to regulate satellite cell self-renewal

    SciTech Connect

    McFarlane, Craig; Hennebry, Alex; Thomas, Mark; Plummer, Erin; Ling, Nicholas; Sharma, Mridula; Kambadur, Ravi

    2008-01-15

    Myostatin, a Transforming Growth Factor-beta (TGF-{beta}) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.

  5. MicroRNA-27a promotes myoblast proliferation by targeting myostatin

    SciTech Connect

    Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun; Chen, Daiwen

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We identified a myogenic role for miR-27a and a new target, myostatin. Black-Right-Pointing-Pointer The miR-27a was confirmed to target myostatin 3 Prime UTR. Black-Right-Pointing-Pointer miR-27a is upregulated and myostatin is downregulated during myoblast proliferation. Black-Right-Pointing-Pointer miR-27a promotes myoblast proliferation by reducing the expression of myostatin. -- Abstract: MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3 Prime UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

  6. Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

    USGS Publications Warehouse

    Yamamoto, Y.; Yoshizaki, G.; Takeuchi, T.; Soyano, K.; Patino, R.

    2008-01-01

    Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18??-glycyrrhetinic acid (??-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20??-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption. ?? 2007 Elsevier Inc. All rights reserved.

  7. Insulin-like growth factor-1 and myostatin mRNA expression in muscle: comparison between 62-77 and 21-31 yr old men.

    PubMed

    Welle, Stephen; Bhatt, Kirti; Shah, Bharati; Thornton, Charles

    2002-06-01

    The present study was done to determine the effect of age on muscle concentrations of mRNAs encoding two growth factors that are thought to be important regulators of muscle mass: insulin-like growth factor-1 (IGF-1) and myostatin. Quantitative RT-PCR assays indicated that the mean IGF-1 mRNA concentration in older muscle (62-77 yr, n=15 men) was approximately 25% less, per ng total RNA (P<0.005), than in young adult muscle (21-31 yr, n=12 men). One third of the older men had IGF-1 mRNA levels below the lowest concentration observed in young muscle. Myostatin mRNA concentrations were similar in young and old muscle. Muscle mass and myofibrillar protein synthesis rates among eight older men did not correlate with either IGF-1 or myostatin mRNA levels. We conclude that IGF-1 gene expression in muscle tends to decline with normal aging. The functional significance is uncertain.

  8. The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana

    PubMed Central

    Moseler, Anna; Aller, Isabel; Wagner, Stephan; Nietzel, Thomas; Przybyla-Toscano, Jonathan; Mühlenhoff, Ulrich; Lill, Roland; Berndt, Carsten; Rouhier, Nicolas; Schwarzländer, Markus; Meyer, Andreas J.

    2015-01-01

    The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Δgrx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15K83/A, led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins. PMID:26483494

  9. Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates.

    PubMed

    Rodgers, Buel D; Weber, Gregory M; Kelley, Kevin M; Levine, Michael A

    2003-05-01

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indexes in adults, skeletal muscle myostatin mRNA levels were unaffected. By contrast, larval myostatin mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting. These effects were specific for myostatin, as mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase were unchanged. Cortisol levels were elevated in fasted larvae with reduced myostatin mRNA, whereas in addition immersion of larvae in 1 ppm (2.8 microM) cortisol reduced myostatin mRNA in a time-dependent fashion. These results suggest that larval myostatin mRNA levels may initially rise but ultimately fall during a prolonged fast. The reduction is likely mediated by fasting-induced hypercortisolemia, indicating divergent evolutionary mechanisms of glucocorticoid regulation of myostatin mRNA, since these steroids upregulate myostatin gene expression in mammals.

  10. The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD.

    PubMed

    Spiller, Michael P; Kambadur, Ravi; Jeanplong, Ferenc; Thomas, Mark; Martyn, Julie K; Bass, John J; Sharma, Mridula

    2002-10-01

    Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.

  11. Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition.

    PubMed

    Condon, Jennifer C; Jeyasuria, Pancharatnam; Faust, Julie M; Mendelson, Carole R

    2004-04-06

    Parturition is timed to begin only after the developing embryo is sufficiently mature to survive outside the womb. It has been postulated that the signal for the initiation of parturition arises from the fetus although the nature and source of this signal remain obscure. Herein, we provide evidence that this signal originates from the maturing fetal lung. In the mouse, secretion of the major lung surfactant protein, surfactant protein A (SP-A), was first detected in amniotic fluid (AF) at 17 days postcoitum, rising progressively to term (19 days postcoitum). Expression of IL-1beta in AF macrophages and activation of NF-kappaB in the maternal uterus increased with the gestational increase in SP-A. SP-A stimulated IL-1beta and NF-kappaB expression in cultured AF macrophages. Studies using Rosa 26 Lac-Z (B6;129S-Gt(rosa)26Sor) (Lac-Z) mice revealed that fetal AF macrophages migrate to the uterus with the gestational increase in AF SP-A. Intraamniotic (i.a.) injection of SP-A caused preterm delivery of fetuses within 6-24 h. By contrast, injection of an SP-A antibody or NF-kappaB inhibitor into AF delayed labor by >24 h. We propose that augmented production of SP-A by the fetal lung near term causes activation and migration of fetal AF macrophages to the maternal uterus, where increased production of IL-1beta activates NF-kappaB, leading to labor. We have revealed a response pathway that ties augmented surfactant production by the maturing fetal lung to the initiation of labor. We suggest that SP-A secreted by the fetal lung serves as a hormone of parturition.

  12. Structure of the Dimerization Interface in the Mature HIV-1 Capsid Protein Lattice from Solid State NMR of Tubular Assemblies.

    PubMed

    Bayro, Marvin J; Tycko, Robert

    2016-07-13

    The HIV-1 capsid protein (CA) forms the capsid shell that encloses RNA within a mature HIV-1 virion. Previous studies by electron microscopy have shown that the capsid shell is primarily a triangular lattice of CA hexamers, with variable curvature that destroys the ideal symmetry of a planar lattice. The mature CA lattice depends on CA dimerization, which occurs through interactions between helix 9 segments of the C-terminal domain (CTD) of CA. Several high-resolution structures of the CTD-CTD dimerization interface have been reported, based on X-ray crystallography and multidimensional solution nuclear magnetic resonance (NMR), with significant differences in amino acid side chain conformations and helix 9-helix 9 orientations. In a structural model for tubular CA assemblies based on cryogenic electron microscopy (cryoEM) [Zhao et al. Nature, 2013, 497, 643-646], the dimerization interface is substantially disordered. The dimerization interface structure in noncrystalline CA assemblies and the extent to which this interface is structurally ordered within a curved lattice have therefore been unclear. Here we describe solid state NMR measurements on the dimerization interface in tubular CA assemblies, which contain the curved triangular lattice of a mature virion, including quantitative measurements of intermolecular and intramolecular distances using dipolar recoupling techniques, solid state NMR chemical shifts, and long-range side chain-side chain contacts. When combined with restraints on the distance and orientation between helix 9 segments from the cryoEM study, the solid state NMR data lead to a unique high-resolution structure for the dimerization interface in the noncrystalline lattice of CA tubes. These results demonstrate that CA lattice curvature is not dependent on disorder or variability in the dimerization interface. This work also demonstrates the feasibility of local structure determination within large noncrystalline assemblies formed by high

  13. Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

    PubMed

    Romero-Aguirregomezcorta, Jon; Santa, Ángela Patricia; García-Vázquez, Francisco Alberto; Coy, Pilar; Matás, Carmen

    2014-01-01

    Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

  14. Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons.

    PubMed

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Ya-Jean; Chen, Yun-Hsiang; Wang, Dan-Yen; Chen, Linyi; Chen, Chia-Hsiang; Chen, Hwei-Hsien; Chiu, Ing-Ming

    2014-06-01

    Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

  15. Ribosomal protein L11 is related to brain maturation during the adult phase in Apis cerana cerana (Hymenoptera, Apidae)

    NASA Astrophysics Data System (ADS)

    Meng, Fei; Lu, Wenjing; Yu, Feifei; Kang, Mingjiang; Guo, Xingqi; Xu, Baohua

    2012-05-01

    Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 ( RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in ds AccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

  16. Signaling Adaptor Protein SH2B1 Enhances Neurite Outgrowth and Accelerates the Maturation of Human Induced Neurons

    PubMed Central

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Ya-Jean; Chen, Yun-Hsiang; Wang, Dan-Yen; Chen, Linyi; Chen, Chia-Hsiang; Chen, Hwei-Hsien

    2014-01-01

    Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling. PMID:24736401

  17. XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans

    PubMed Central

    Dewal, Mahender B.; DiChiara, Andrew S.; Antonopoulos, Aristotelis; Taylor, Rebecca J.; Harmon, Chyleigh J.; Haslam, Stuart M.; Dell, Anne; Shoulders, Matthew D.

    2015-01-01

    SUMMARY The molecular architecture of the mature N-glycome is dynamic, with consequences for both normal and pathologic processes. Elucidating cellular mechanisms that modulate the N-linked glycome is, therefore, crucial. The unfolded protein response (UPR) is classically responsible for maintaining proteostasis in the secretory pathway by defining levels of chaperones and quality control proteins. Here, we employ chemical biology methods for UPR regulation to show that stress-independent activation of the UPR’s XBP1s transcription factor also induces a panel of N-glycan maturation-related enzymes. The downstream consequence is a distinctive shift towards specific hybrid and complex N-glycans on N-glycoproteins produced from XBP1s-activated cells, which we characterize by mass spectrometry. Pulse-chase studies attribute this shift specifically to altered N-glycan processing, rather than to changes in degradation or secretion rates. Our findings implicate XBP1s in a new role for N-glycoprotein biosynthesis, unveiling an important link between intracellular stress responses and the molecular architecture of extracellular N-glycoproteins. PMID:26496683

  18. Ribosomal protein L11 is related to brain maturation during the adult phase in Apis cerana cerana (Hymenoptera, Apidae).

    PubMed

    Meng, Fei; Lu, Wenjing; Yu, Feifei; Kang, Mingjiang; Guo, Xingqi; Xu, Baohua

    2012-05-01

    Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 (RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in dsAccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

  19. Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation

    PubMed Central

    Hamlett, Isla; Draper, Julia; Strouboulis, John; Iborra, Francisco; Porcher, Catherine

    2008-01-01

    The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL. PMID:18625887

  20. Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase.

    PubMed

    Krauss, Sharon Wald; Spence, Jeffrey R; Bahmanyar, Shirin; Barth, Angela I M; Go, Minjoung M; Czerwinski, Debra; Meyer, Adam J

    2008-04-01

    Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.

  1. VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes.

    PubMed

    Bem, Danai; Smith, Holly; Banushi, Blerida; Burden, Jemima J; White, Ian J; Hanley, Joanna; Jeremiah, Nadia; Rieux-Laucat, Frédéric; Bettels, Ruth; Ariceta, Gema; Mumford, Andrew D; Thomas, Steven G; Watson, Steve P; Gissen, Paul

    2015-07-09

    Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.

  2. VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes

    PubMed Central

    Bem, Danai; Smith, Holly; Banushi, Blerida; Burden, Jemima J.; White, Ian J.; Hanley, Joanna; Jeremiah, Nadia; Rieux-Laucat, Frédéric; Bettels, Ruth; Ariceta, Gema; Mumford, Andrew D.; Thomas, Steven G.; Watson, Steve P.

    2015-01-01

    Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33bfl/fl-ERT2, and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33bfl/fl-ERT2 platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33bfl/fl-ERT2 platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33bfl/fl-ERT2 mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33bfl/fl-ERT2 mouse and ARC patients. PMID:25947942

  3. Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin

    PubMed Central

    Winbanks, Catherine E.; Weeks, Kate L.; Thomson, Rachel E.; Sepulveda, Patricio V.; Beyer, Claudia; Qian, Hongwei; Chen, Justin L.; Allen, James M.; Lancaster, Graeme I.; Febbraio, Mark A.; Harrison, Craig A.; McMullen, Julie R.; Chamberlain, Jeffrey S.

    2012-01-01

    Follistatin is essential for skeletal muscle development and growth, but the intracellular signaling networks that regulate follistatin-mediated effects are not well defined. We show here that the administration of an adeno-associated viral vector expressing follistatin-288aa (rAAV6:Fst-288) markedly increased muscle mass and force-producing capacity concomitant with increased protein synthesis and mammalian target of rapamycin (mTOR) activation. These effects were attenuated by inhibition of mTOR or deletion of S6K1/2. Furthermore, we identify Smad3 as the critical intracellular link that mediates the effects of follistatin on mTOR signaling. Expression of constitutively active Smad3 not only markedly prevented skeletal muscle growth induced by follistatin but also potently suppressed follistatin-induced Akt/mTOR/S6K signaling. Importantly, the regulation of Smad3- and mTOR-dependent events by follistatin occurred independently of overexpression or knockout of myostatin, a key repressor of muscle development that can regulate Smad3 and mTOR signaling and that is itself inhibited by follistatin. These findings identify a critical role of Smad3/Akt/mTOR/S6K/S6RP signaling in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms. PMID:22711699

  4. An evolutionarily conserved Myostatin proximal promoter/enhancer confers basal levels of transcription and spatial specificity in vivo.

    PubMed

    Grade, Carla Vermeulen Carvalho; Salerno, Mônica Senna; Schubert, Frank R; Dietrich, Susanne; Alvares, Lúcia Elvira

    2009-10-01

    Myostatin (Mstn) is a negative regulator of skeletal muscle mass, and Mstn mutations are responsible for the double muscling phenotype observed in many animal species. Moreover, Mstn is a positive regulator of adult muscle stem cell (satellite cell) quiescence, and hence, Mstn is being targeted in therapeutic approaches to muscle diseases. In order to better understand the mechanisms underlying Mstn regulation, we searched for the gene's proximal enhancer and promoter elements, using an evolutionary approach. We identified a 260-bp-long, evolutionary conserved region upstream of tetrapod Mstn and teleost mstn b genes. This region contains binding sites for TATA binding protein, Meis1, NF-Y, and for CREB family members, suggesting the involvement of cAMP in Myostatin regulation. The conserved fragment was able to drive reporter gene expression in C2C12 cells in vitro and in chicken somites in vivo; both normally express Mstn. In contrast, the reporter construct remained silent in the avian neural tube that normally does not express Mstn. This suggests that the identified element serves as a minimal promoter, harboring some spatial specificity. Finally, using bioinformatic approaches, we identified additional genes in the human genome associated with sequences similar to the Mstn proximal promoter/enhancer. Among them are genes important for myogenesis. This suggests that Mstn and these genes may form a synexpression group, regulated by a common signaling pathway.

  5. NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.

    PubMed

    Bastow, Emma L; Bych, Katrine; Crack, Jason C; Le Brun, Nick E; Balk, Janneke

    2017-02-01

    Proteins of the cytosolic pathway for iron-sulphur (FeS) cluster assembly are conserved, except that plants lack a gene for CFD1 (Cytosolic FeS cluster Deficient 1). This poses the question of how NBP35 (Nucleotide-Binding Protein 35 kDa), the heteromeric partner of CFD1 in metazoa, functions on its own in plants. Firstly, we created viable mutant alleles of NBP35 in Arabidopsis to overcome embryo lethality of previously reported knockout mutations. RNAi knockdown lines with less than 30% NBP35 protein surprisingly showed no developmental or biochemical differences to wild-type. Substitution of Cys14 to Ala, which destabilized the N-terminal Fe4 S4 cluster in vitro, caused mild growth defects and a significant decrease in the activity of cytosolic FeS enzymes such as aconitase and aldehyde oxidases. The DNA glycosylase ROS1 was only partially decreased in activity and xanthine dehydrogenase not at all. Plants with strongly depleted NBP35 protein in combination with Cys14 to Ala substitution had distorted leaf development and decreased FeS enzyme activities. To find protein interaction partners of NBP35, a yeast-two-hybrid screen was carried out that identified NBP35 and DRE2 (Derepressed for Ribosomal protein S14 Expression). NBP35 is known to form a dimer, and DRE2 acts upstream in the cytosolic FeS protein assembly pathway. The NBP35-DRE2 interaction was not disrupted by Cys14 to Ala substitution. Our results show that NBP35 has a function in the maturation of FeS proteins that is conserved in plants, and is closely allied to the function of DRE2. © 2016 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  6. Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIVmac can coassemble into mature cores of infectious viruses.

    PubMed

    Chen, Jianbo; Pathak, Vinay K; Peng, Weiqun; Hu, Wei-Shau

    2008-09-01

    We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5alpha protein from the rhesus monkey (rhTRIM5alpha) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIV(mac) CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5alpha. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5alpha would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.

  7. EXTRACTS OF IRRADIATED MATURE HUMAN TOOTH CROWNS CONTAIN MMP-20 PROTEIN AND ACTIVITY

    PubMed Central

    MCGUIRE, J.D.; MOUSA, A.A.; ZHANG, BO J.; TODOKI, L.S.; HUFFMAN, N.T.; CHANDRABABU, K.B.; MORADIAN-OLDAK, J.; KEIGHTLEY, A.; WANG, Y.; WALKER, M.P.; GORSKI, J.P.

    2014-01-01

    Objectives We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. Materials and Methods Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0 to 6 months at 37°C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. Results Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentin and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr=43, 41, 24 and 22 kDa and was immunolocalized predominantly to the morphological dentin enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70 G, subsequent incubation at 37°C for 3–6 months with or without prior irradiation caused the proportion of Mr=24–22 kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70 Gy radiation also contained relatively more 24 and 22 kDa MMP-20 than those of healthy age-related teeth. Conclusion MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentin-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy. PMID:24607847

  8. Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function.

    PubMed

    Howard, Marybeth; Fischer, Horst; Roux, Jeremie; Santos, Bento C; Gullans, Steven R; Yancey, Paul H; Welch, William J

    2003-09-12

    In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to

  9. The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures

    PubMed Central

    Yen, Edwin H. K.; Sodek, Jaro; Melcher, Antony H.

    1979-01-01

    Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO2 (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [3H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO2 [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [3H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO2 [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO2 for the first 48h were unable to respond to a subsequent increase in pO2 during the last 24h. Analysis of pepsin-solubilized collagen α-chains labelled with [14C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO2. Type-III collagen comprised 20–30% of the radioactivity in α-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO2 was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo. PMID:454369

  10. Characterization of two paralogous myostatin genes and evidence for positive selection in Tibet fish: Gymnocypris przewalskii.

    PubMed

    Tong, Chao; Zhang, Cunfang; Shi, Jianquan; Qi, Hongfang; Zhang, Renyi; Tang, Yongtao; Li, Guogang; Feng, Chenguang; Zhao, Kai

    2015-07-10

    Myostatin (mstn) is an important member of TGF-β superfamily, a muscle growth inhibitor. Though mstn has been identified in many organisms, little is known about this gene in highland fish, Gymnocypris przewalskii endemic to the Qinghai-Tibetan Plateau. In this study, we first cloned two paralogous mstn genes (mstn1 and mstn2) from G. przewalskii through homologue cloning. The 3D structures of both Mstn proteins varied in the numbers of β-sheets and conformations of α-helices. The branch-site model showed that mstn1 has undergone positive selection, and two positively selected sites (107M and 181T) were located on the random coils of the 3D protein structure. Expression patterns indicated that the mstn1 expressed widely, while the mstn2 only expressed in the muscle and brain. During the early stage of embryo development, the expression levels of both mstn paralogous genes showed different increasing trends. These results suggest that it is diverging in two mstn paralogues of G. przewalskii via specific differences in gene structure, protein structure, selection pressure and gene expression patterns. Taken together, this study provides novel contribution on the research topics of growth related gene function and mechanism of highland fish in extreme aquatic environment on the Qinghai-Tibetan Plateau.

  11. Proteomics analysis in mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritive, and allergenic proteins

    USDA-ARS?s Scientific Manuscript database

    Protein profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano electrospray ionization liquid chromatography tandem mass ...

  12. Ribosomal protein L3 bound to 23S precursor rRNA stimulates its maturation by Mini-III ribonuclease.

    PubMed

    Redko, Yulia; Condon, Ciarán

    2009-03-01

    Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.

  13. Changes in skeletal muscle and tendon structure and function following genetic inactivation of myostatin in rats.

    PubMed

    Mendias, Christopher L; Lynch, Evan B; Gumucio, Jonathan P; Flood, Michael D; Rittman, Danielle S; Van Pelt, Douglas W; Roche, Stuart M; Davis, Carol S

    2015-04-15

    Myostatin is a negative regulator of skeletal muscle and tendon mass. Myostatin deficiency has been well studied in mice, but limited data are available on how myostatin regulates the structure and function of muscles and tendons of larger animals. We hypothesized that, in comparison to wild-type (MSTN(+/+) ) rats, rats in which zinc finger nucleases were used to genetically inactivate myostatin (MSTN(Δ/Δ) ) would exhibit an increase in muscle mass and total force production, a reduction in specific force, an accumulation of type II fibres and a decrease and stiffening of connective tissue. Overall, the muscle and tendon phenotype of myostatin-deficient rats was markedly different from that of myostatin-deficient mice, which have impaired contractility and pathological changes to fibres and their extracellular matrix. Extensor digitorum longus and soleus muscles of MSTN(Δ/Δ) rats demonstrated 20-33% increases in mass, 35-45% increases in fibre number, 20-57% increases in isometric force and no differences in specific force. The insulin-like growth factor-1 pathway was activated to a greater extent in MSTN(Δ/Δ) muscles, but no substantial differences in atrophy-related genes were observed. Tendons of MSTN(Δ/Δ) rats had a 20% reduction in peak strain, with no differences in mass, peak stress or stiffness. The general morphology and gene expression patterns were similar between tendons of both genotypes. This large rodent model of myostatin deficiency did not have the negative consequences to muscle fibres and extracellular matrix observed in mouse models, and suggests that the greatest impact of myostatin in the regulation of muscle mass may not be to induce atrophy directly, but rather to block hypertrophy signalling.

  14. Changes in skeletal muscle and tendon structure and function following genetic inactivation of myostatin in rats

    PubMed Central

    Mendias, Christopher L; Lynch, Evan B; Gumucio, Jonathan P; Flood, Michael D; Rittman, Danielle S; Van Pelt, Douglas W; Roche, Stuart M; Davis, Carol S

    2015-01-01

    Myostatin is a negative regulator of skeletal muscle and tendon mass. Myostatin deficiency has been well studied in mice, but limited data are available on how myostatin regulates the structure and function of muscles and tendons of larger animals. We hypothesized that, in comparison to wild-type (MSTN+/+) rats, rats in which zinc finger nucleases were used to genetically inactivate myostatin (MSTNΔ/Δ) would exhibit an increase in muscle mass and total force production, a reduction in specific force, an accumulation of type II fibres and a decrease and stiffening of connective tissue. Overall, the muscle and tendon phenotype of myostatin-deficient rats was markedly different from that of myostatin-deficient mice, which have impaired contractility and pathological changes to fibres and their extracellular matrix. Extensor digitorum longus and soleus muscles of MSTNΔ/Δ rats demonstrated 20–33% increases in mass, 35–45% increases in fibre number, 20–57% increases in isometric force and no differences in specific force. The insulin-like growth factor-1 pathway was activated to a greater extent in MSTNΔ/Δ muscles, but no substantial differences in atrophy-related genes were observed. Tendons of MSTNΔ/Δ rats had a 20% reduction in peak strain, with no differences in mass, peak stress or stiffness. The general morphology and gene expression patterns were similar between tendons of both genotypes. This large rodent model of myostatin deficiency did not have the negative consequences to muscle fibres and extracellular matrix observed in mouse models, and suggests that the greatest impact of myostatin in the regulation of muscle mass may not be to induce atrophy directly, but rather to block hypertrophy signalling. PMID:25640143

  15. Proteomic Analysis of the Protein Expression Profile in the Mature Nigella sativa (Black Seed).

    PubMed

    Alanazi, Ibrahim O; Benabdelkamel, Hicham; Alfadda, Assim A; AlYahya, Sami A; Alghamdi, Waleed M; Aljohi, Hasan A; Almalik, Abdulaziz; Masood, Afshan

    2016-08-01

    Nigella sativa (N. sativa) seed has been used as an important nutritional flavoring agent and in traditional medicine for treating many illnesses since ancient times. Understanding the proteomic component of the seed may lead to enhance the understanding of its structural and biological functional complexity. In this study, we have analyzed its proteome profile based on gel-based proteome mapping technique that includes one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy. We have not come across any such studies that have been performed in N. sativa seeds up to date. A total of 277 proteins were identified, and their functional, metabolic, and location-wise annotations were carried out using the UniProt database. The majority of proteins identified in the proteome dataset based on their function were those involved in enzyme catalytic activity, nucleotide binding, and protein binding while the major cellular processes included regulation of biological process followed by regulation of secondary biological process, cell organization and biogenesis, protein metabolism, and transport. The identified proteome was localized mainly to the nucleus then to the cytoplasm, plasma membrane, mitochondria, plastid, and others. A majority of the proteins were involved in biochemical pathways involving carbohydrate metabolism, amino acid and shikimate pathway, lipid metabolism, nucleotide, cell organization and biogenesis, transport, and defense processes. The identified proteins in the dataset help to improve our understanding of the pathways involved in N. sativa seed metabolism and its biochemical features and detail out useful information that may help to utilize these proteins. This study could thus pave a way for future further high-throughput studies using a more targeted proteomic approach.

  16. Inhibition of myostatin in mice improves insulin sensitivity via irisin-mediated cross talk between muscle and adipose tissues.

    PubMed

    Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Chen, Fang; Mitch, William E; Zhang, Liping

    2016-03-01

    In mice, a high-fat diet (HFD) induces obesity, insulin resistance and myostatin production. We tested whether inhibition of myostatin in mice can reverse these HFD-induced abnormalities. C57BL/6 mice were fed a HFD for 16 weeks including the final 4 weeks some mice were treated with an anti-myostatin peptibody. Body composition, the respiratory exchange ratio plus glucose and insulin tolerance tests were examined. Myostatin knock down in C2C12 cells was performed using small hairpin RNA lentivirus. Adipose tissue-derived stem cells were cultured to measure their responses to conditioned media from C2C12 cells lacking myostatin, or to recombinant myostatin or irisin. Isolated peritoneal macrophages were treated with myostatin or irisin to determine whether myostatin or irisin induce inflammatory mechanisms. In HFD-fed mice, peptibody treatment stimulated muscle growth and improved insulin resistance. The improved glucose and insulin tolerances were confirmed when we found increased muscle expression of p-Akt and the glucose transporter, Glut4. In HFD-fed mice, the peptibody suppressed macrophage infiltration and the expression of proinflammatory cytokines in both the muscle and adipocytes. Inhibition of myostatin caused the conversion of white (WAT) to brown adipose tissue, whereas stimulating fatty acid oxidation and increasing energy expenditure. The related mechanism is a muscle-to-fat cross talk mediated by irisin. Myostatin inhibition increased peroxisome proliferator-activated receptor gamma, coactivator 1α expression and irisin production in the muscle. Irisin then stimulated WAT browning. Irisin also suppresses inflammation and stimulates macrophage polarization from M1 to M2 types. These results uncover a metabolic pathway from an increase in myostatin that suppresses irisin leading to the activation of inflammatory cytokines and insulin resistance. Thus, myostatin is a potential therapeutic target to treat insulin resistance of type II diabetes as well

  17. Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration after Injury

    DTIC Science & Technology

    2011-03-01

    07-1-0181 TITLE: Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and...Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration after Injury Dr. Nestor Gonzalez-Cadavid Charles R. Drew...combat fibrosis and lipofibrotic degeneration, and stimulate MDSC fusion with myofibers. Myostatin, muscle dystrophy, stem cells, myogenesis, Oct-4

  18. NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase

    PubMed Central

    Kostyrko, Vitaly A.; Bertsova, Yulia V.; Serebryakova, Marina V.; Baykov, Alexander A.

    2015-01-01

    ABSTRACT Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na+ translocation across the membrane. Na+-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na+-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na+-NQR, resulted in an enzyme incapable of Na+-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na+-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na+-NQR, which could be recovered by an nqrM-containing plasmid. The Na+-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na+-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na+-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na+-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE. IMPORTANCE Na+-translocating NADH:quinone oxidoreductase complex (Na+-NQR) is a unique primary Na+ pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio

  19. NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase.

    PubMed

    Kostyrko, Vitaly A; Bertsova, Yulia V; Serebryakova, Marina V; Baykov, Alexander A; Bogachev, Alexander V

    2015-12-07

    Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na(+) translocation across the membrane. Na(+)-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na(+)-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na(+)-NQR, resulted in an enzyme incapable of Na(+)-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na(+)-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na(+)-NQR, which could be recovered by an nqrM-containing plasmid. The Na(+)-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na(+)-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na(+)-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na(+)-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE. Na(+)-translocating NADH:quinone oxidoreductase complex (Na(+)-NQR) is a unique primary Na(+) pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae

  20. Elevation of myostatin and FOXOs in prolonged muscular impairment induced by eccentric contractions in rat medial gastrocnemius muscle.

    PubMed

    Ochi, Eisuke; Hirose, Tatsuro; Hiranuma, Kenji; Min, Seok-Ki; Ishii, Naokata; Nakazato, Koichi

    2010-02-01

    This study aimed to investigate torque deficit and activation of protein synthesis and/or protein degradation signaling pathways during the early and recovery phase after high- and low-velocity eccentric contractions (ECs). Male Wistar rats (n = 36) were randomly divided into fast angular velocity ECs group (FAST; 180 degrees/s; n = 12), slow ECs group (SLOW; 30 degrees/s; n = 12), and control group (control; n = 12). ECs comprised four sets of five forced dorsiflexions combined with electrical stimulation of the plantar flexors. Isometric tetanic torque was measured before and after ECs. Tissue contents of Akt(P) (P, phosphorylated), mammalian target of rapamycin (mTOR)(P), 70-kDa ribosomal protein S6 kinase (P70S6k), P70S6k(P), forkhead transcription factor 1 of the O class (FOXO1), FOXO1(P), FOXO3, FOXO3(P), myostatin, and activin receptor type IIB (ActRIIB) were measured. The isometric tetanic torque after ECs was significantly lower in FAST than in SLOW (days 1, 3, and 5, P < 0.05; day 2, P < 0.01). The ratio of P70S6k(P) against total P70S6k on days 2 and 7 was significantly higher in SLOW than in the control. The ratio of FOXO1 against total FOXO1, the ratio of FOXO3a against total FOXO3a, and myostatin on days 2 and 7 were significantly higher in FAST than in the control, while that of ActRIIB on day 7 was significantly lower in SLOW than in the other two groups. These results suggest that EC intensity plays a key role in impairment of muscular function and activation of protein synthesis and/or protein degradation signaling pathways.

  1. Comparative analysis of myostatin gene and promoter sequences of Qinchuan and Red Angus cattle.

    PubMed

    He, Y L; Wu, Y H; Quan, F S; Liu, Y G; Zhang, Y

    2013-09-04

    To better understand the function of the myostatin gene and its promoter region in bovine, we amplified and sequenced the myostatin gene and promoter from the blood of Qinchuan and Red Angus cattle by using polymerase chain reaction. The sequences of Qinchuan and Red Angus cattle were compared with those of other cattle breeds available in GenBank. Exon splice sites were confirmed by mRNA sequencing. Compared to the published sequence (GenBank accession No. AF320998), 69 single nucleotide polymorphisms (SNPs) were identified in the Qinchuan myostatin gene, only one of which was an insertion mutation in Qinchuan cattle. There was a 16-bp insertion in the first 705-bp intron in 3 Qinchuan cattle. A total of 7 SNPs were identified in exon 3, in which the mutation occurred in the third base of the codon and was synonymous. On comparing the Qinchuan myostatin gene sequence to that of Red Angus cattle, a total of 50 SNPs were identified in the first and third exons. In addition, there were 18 SNPs identified in the Qinchuan cattle promoter region compared with those of other cattle compared to the Red Angus cattle myostatin promoter region. breeds (GenBank accession No. AF348479), but only 14 SNPs when compared to the Red Angus cattle myostatin promoter region.

  2. Characterization of 5'-regulatory region of human myostatin gene: regulation by dexamethasone in vitro.

    PubMed

    Ma, K; Mallidis, C; Artaza, J; Taylor, W; Gonzalez-Cadavid, N; Bhasin, S

    2001-12-01

    We cloned and characterized a 3.3-kb fragment containing the 5'-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-kappaB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C(2)C(12) and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.

  3. Comparative analysis of the pig BAC sequence involved in the regulation of myostatin gene.

    PubMed

    Yu, Zhengquan; Li, Yan; Meng, Qingyong; Yuan, Jing; Zhao, Zhihui; Li, Wei; Hu, Xiaoxiang; Yan, Bingxue; Fan, Baoliang; Yu, Shuyang; Li, Ning

    2005-04-01

    Myostatin (GDF8, MSTN) is a member of the transforming growth factor beta superfamily that is essential for proper regulation of skeletal muscle mass. In order to study its expression and regulatory mechanism deeply, we have presented a comparative analysis of about 170-kb pig BAC sequence containing the myostatin gene among pig, human and mouse. The genomic region is characterized by high interspersed repeats and low G+C content. As for the myostatin gene, a higher sequence similarity is found between human and pig than between these species and the mouse. One striking feature is that the structure of two TATA-boxes in the nearby downstream of CCAAT-box is identified in the promoter. Further analysis reveals that the TATA-box1 is responsible for the transcription in pig and human, but the TATA-box2 acts on the transcription in mouse. The other interesting feature is that two polyadenylation signal sequences (AATAAA) exist in 3'UTR of the pig myostatin gene. Moreover, a large number of potential transcription factor-binding sites are also identified in evolutionary conserved regions (ECRs), which may be associated with the regulation of myostatin. Many putative transcription factors play an important role in the muscle development, and the complex interaction between myostatin and these factors may be required for proper muscle development.

  4. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  5. Post-Mortem Stability of RNA in Skeletal Muscle and Adipose Tissue and the Tissue-Specific Expression of Myostatin, Perilipin and Associated Factors in the Horse

    PubMed Central

    Morrison, Philippa K.; Bing, Chen; Harris, Patricia A.; Maltin, Charlotte A.; Grove-White, Dai; Argo, Caroline McG.

    2014-01-01

    Obesity, a major concern for equine welfare, is highly prevalent in the leisure horse population. Skeletal-muscle and adipose tissues are important determinants of maintenance energy requirements. The myostatin and perilipin pathways play key roles in the regulation of muscle mass and lipolysis respectively and have both been associated with obesity predisposition in other mammalian species. High quality samples, suitable for molecular biology, are an essential prerequisite for detailed investigations of gene and protein expression. Hence, this study has evaluated a) the post-mortem stability of RNA extracted from skeletal-muscle and adipose-tissues collected under commercial conditions and b) the tissue-specific presence of myostatin, the moystatin receptor (activin receptor IIB, ActRIIB), follistatin and perilipin, genes and proteins across a range of equine tissues. Objectives were addressed using tissues from 7 Thoroughbred horses presented for slaughter at a commercial abattoir; a) samples were collected at 7 time-points from Masseter muscle and perirenal adipose from 5 minutes to 6 hours post-mortem. Extracted RN was appraised by Optical Density analysis and agarose-gel electrophoresis. b) Quantitative real time PCR and Western Blotting were used to evaluate gene and protein expression in anatomically-defined samples collected from 17 tissues (6 organs, 4 skeletal muscles and 7 discrete adipose depots). The results indicate that, under the present collection conditions, intact, good quality RNA could be extracted from skeletal-muscle for up to 2 hours post-mortem. However, RNA from adipose tissue may be more susceptible to degradation/contamination and samples should be collected no later than 30 minutes post-mortem. The data also show that myostatin and ActRIIB genes and proteins were almost exclusively expressed in skeletal muscle. The follistatin gene showed a more diverse gene expression profile, with expression evident in several organs, adipose tissue

  6. Post-mortem stability of RNA in skeletal muscle and adipose tissue and the tissue-specific expression of myostatin, perilipin and associated factors in the horse.

    PubMed

    Morrison, Philippa K; Bing, Chen; Harris, Patricia A; Maltin, Charlotte A; Grove-White, Dai; Argo, Caroline McG

    2014-01-01

    Obesity, a major concern for equine welfare, is highly prevalent in the leisure horse population. Skeletal-muscle and adipose tissues are important determinants of maintenance energy requirements. The myostatin and perilipin pathways play key roles in the regulation of muscle mass and lipolysis respectively and have both been associated with obesity predisposition in other mammalian species. High quality samples, suitable for molecular biology, are an essential prerequisite for detailed investigations of gene and protein expression. Hence, this study has evaluated a) the post-mortem stability of RNA extracted from skeletal-muscle and adipose-tissues collected under commercial conditions and b) the tissue-specific presence of myostatin, the moystatin receptor (activin receptor IIB, ActRIIB), follistatin and perilipin, genes and proteins across a range of equine tissues. Objectives were addressed using tissues from 7 Thoroughbred horses presented for slaughter at a commercial abattoir; a) samples were collected at 7 time-points from Masseter muscle and perirenal adipose from 5 minutes to 6 hours post-mortem. Extracted RN was appraised by Optical Density analysis and agarose-gel electrophoresis. b) Quantitative real time PCR and Western Blotting were used to evaluate gene and protein expression in anatomically-defined samples collected from 17 tissues (6 organs, 4 skeletal muscles and 7 discrete adipose depots). The results indicate that, under the present collection conditions, intact, good quality RNA could be extracted from skeletal-muscle for up to 2 hours post-mortem. However, RNA from adipose tissue may be more susceptible to degradation/contamination and samples should be collected no later than 30 minutes post-mortem. The data also show that myostatin and ActRIIB genes and proteins were almost exclusively expressed in skeletal muscle. The follistatin gene showed a more diverse gene expression profile, with expression evident in several organs, adipose tissue

  7. A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins.

    PubMed

    Zhang, Siyan; Long, Angel; Link, A James

    2012-03-16

    The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-x(L) and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L) and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L) and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L) and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.

  8. The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins.

    PubMed

    Lill, Roland; Dutkiewicz, Rafal; Freibert, Sven A; Heidenreich, Torsten; Mascarenhas, Judita; Netz, Daili J; Paul, Viktoria D; Pierik, Antonio J; Richter, Nadine; Stümpfig, Martin; Srinivasan, Vasundara; Stehling, Oliver; Mühlenhoff, Ulrich

    2015-01-01

    Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron-sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron-sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins.

  9. The Significance of Protein Maturation by Plastidic Type I Signal Peptidase 1 for Thylakoid Development in Arabidopsis Chloroplasts1[C][W][OA

    PubMed Central

    Shipman-Roston, Rebecca L.; Ruppel, Nicholas J.; Damoc, Catalina; Phinney, Brett S.; Inoue, Kentaro

    2010-01-01

    Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here, we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids. PMID:20097790

  10. Immunocytochemical localization of allergenic proteins from mature to activated Zygophyllum fabago L. (Zygophyllaceae) pollen grains.

    PubMed

    Castells, Teresa; Arcalís, Elsa; Moreno-Grau, Stella; Bayo, Javier; Elvira-Rendueles, Belen; Belchí, Juan; Seoane-Camba, Juan A; Suárez-Cervera, María

    2002-02-01

    Zygophyllum fabago L. (Zygophyllaceae) can be found in the Middle East, in North Africa and in the arid zones of the Mediterranean region. It easily establishes itself in new regions, and is considered an invasive plant. They undergo ambophilous pollination, as there is a relationship between this type of pollination and its allergenic incidence. A combination of transmission electron microscopy with immunocytochemical methods was used to localize allergenic proteins during hydration and activation processes. Germination was induced in vitro for 1,2,4,6, and 30 min. The activated proteins reacting with antibodies present in human sera from allergenic patients are found in the cytoplasm, intine, exine and exudates from the pollen grains. The activation time plays an important role on the labelling intensity. Labelling of allergenic proteins was abundant at 1 and 2 min of activation, and decreased at 4 and 6 min. The rapid activation and release of the allergenic proteins appears to be the main cause of allergenic activity of Z. fabago pollen grains.

  11. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  12. The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction.

    PubMed

    Zhang, Lin; Trncik, Christian; Andrade, Susana L A; Einsle, Oliver

    2017-02-01

    The copper-containing enzyme nitrous oxide reductase (N2OR) catalyzes the transformation of nitrous oxide (N2O) to dinitrogen (N2) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites CuA and CuZ, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N2O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg(2+) that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.

  13. Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins.

    PubMed

    Van Damme, Daniël; Coutuer, Silvie; De Rycke, Riet; Bouget, Francois-Yves; Inzé, Dirk; Geelen, Danny

    2006-12-01

    TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco (Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and beta-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position.

  14. Stage-specific regulation of Solanum lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX proteins.

    PubMed

    Shani, Eilon; Burko, Yogev; Ben-Yaakov, Lilach; Berger, Yael; Amsellem, Ziva; Goldshmidt, Alexander; Sharon, Eran; Ori, Naomi

    2009-10-01

    Class 1 KNOTTED1-LIKE HOMEOBOX (KNOXI) genes encode transcription factors that are expressed in the shoot apical meristem (SAM) and are essential for SAM maintenance. In some species with compound leaves, including tomato (Solanum lycopersicum), KNOXI genes are also expressed during leaf development and affect leaf morphology. To dissect the role of KNOXI proteins in leaf patterning, we expressed in tomato leaves a fusion of the tomato KNOXI gene Tkn2 with a sequence encoding a repressor domain, expected to repress common targets of tomato KNOXI proteins. This resulted in the formation of small, narrow, and simple leaves due to accelerated differentiation. Overexpression of the wild-type form of Tkn1 or Tkn2 in young leaves also resulted in narrow and simple leaves, but in this case, leaf development was blocked at the initiation stage. Expression of Tkn1 or Tkn2 during a series of spatial and temporal windows in leaf development identified leaf initiation and primary morphogenesis as specific developmental contexts at which the tomato leaf is responsive to KNOXI activity. Arabidopsis thaliana leaves responded to overexpression of Arabidopsis or tomato KNOXI genes during the morphogenetic stage but were largely insensitive to their overexpression during leaf initiation. These results imply that KNOXI proteins act at specific stages within the compound-leaf development program to delay maturation and enable leaflet formation, rather than set the compound leaf route.

  15. Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.

    PubMed

    Luo, Junjie; Song, Zhiyuan; Yu, Shengli; Cui, Dan; Wang, Benli; Ding, Fangrong; Li, Song; Dai, Yunping; Li, Ning

    2014-01-01

    Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.

  16. Growing backwards: an inverted role for the shrimp ortholog of vertebrate myostatin and GDF11.

    PubMed

    De Santis, Christian; Wade, Nicholas M; Jerry, Dean R; Preston, Nigel P; Glencross, Brett D; Sellars, Melony J

    2011-08-15

    Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins involved in muscle cell growth and differentiation as well as neurogenesis of vertebrates. Both MSTN and GDF11 negatively regulate their functions. Invertebrates possess a single ortholog of the MSTN/GDF11 family. In order to understand the role of MSTN/GDF11 in crustaceans, the gene ortholog was identified and characterized in the penaeid shrimp Penaeus monodon. The overall protein sequence and specific functional sites were highly conserved with other members of the MSTN/GDF11 family. Gene transcripts of pmMstn/Gdf11, assessed by real-time PCR, were detected in a variety of tissue types and were actively regulated in muscle across the moult cycle. To assess phenotypic function in shrimp, pmMstn/Gdf11 gene expression was downregulated by tail-muscle injection of sequence-specific double-stranded RNA. Shrimp with reduced levels of pmMstn/Gdf11 transcripts displayed a dramatic slowing in growth rate compared with control groups. Findings from this study place the MSTN/GDF11 gene at the centre of growth regulation in shrimp, but suggest that, compared with higher vertebrates, this gene has an opposite role in invertebrates such as shrimp, where levels of gene expression may positively regulate growth.

  17. Regulation of GDF-11 and myostatin activity by GASP-1 and GASP-2

    PubMed Central

    Lee, Yun-Sil; Lee, Se-Jin

    2013-01-01

    Myostatin (MSTN) and growth and differentiation factor-11 (GDF-11) are highly related TGF-β family members that have distinct biological functions. MSTN is expressed primarily in skeletal muscle and acts to limit muscle growth. GDF-11 is expressed more widely and plays multiple roles, including regulating axial skeletal patterning during development. Several MSTN and GDF-11 binding proteins have been identified, including GDF-associated serum protein-1 (GASP-1) and GASP-2, which are capable of inhibiting the activities of these ligands. Here, we show that GASP-1 and GASP-2 act by blocking the initial signaling event (namely, the binding of the ligand to the type II receptor). Moreover, we show that mice lacking Gasp1 and Gasp2 have phenotypes consistent with overactivity of MSTN and GDF-11. Specifically, we show that Gasp2−/− mice have posteriorly directed transformations of the axial skeleton, which contrast with the anteriorly directed transformations seen in Gdf11−/− mice. We also show that both Gasp1−/− and Gasp2−/− mice have reductions in muscle weights, a shift in fiber type from fast glycolytic type IIb fibers to fast oxidative type IIa fibers, and impaired muscle regeneration ability, which are the reverse of what are seen in Mstn−/− mice. All of these findings suggest that both GASP-1 and GASP-2 are important modulators of GDF-11 and MSTN activity in vivo. PMID:24019467

  18. Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

    PubMed Central

    Yu, Shengli; Cui, Dan; Wang, Benli; Ding, Fangrong; Li, Song; Dai, Yunping; Li, Ning

    2014-01-01

    Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle. PMID:24743319

  19. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection

    PubMed Central

    Bird, Brian H.; Albariño, Cesar G.; Nichol, Stuart T.

    2007-01-01

    Rift Valley fever (RVF) virus belongs to the Bunyaviridae family of segmented negative-strand RNA viruses and causes mosquito-borne disease in sub-Saharan Africa. We report the development of a T7 RNA polymerase driven plasmid-based genetic system for the virulent Egyptian isolate, ZH501. We have used this system to rescue a virus that has a 387 nucleotide deletion on the genomic M segment that eliminates the coding region for two non-structural proteins known as NSm. This virus, ΔNSm rZH501, is indistinguishable from the parental ZH501 strain with respect to expression of structural proteins and growth in cultured mammalian cells. PMID:17070883

  20. Role of Endoproteolytic Dibasic Proprotein Processing in Maturation of Secretory Proteins in Trichoderma reesei

    PubMed Central

    Goller, Sabine P.; Schoisswohl, Doris; Baron, Michel; Parriche, Martine; Kubicek, Christian P.

    1998-01-01

    Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R-↓-R-↓-A-) and xylanase II (-K-R-↓-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R-↓-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A-↓-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different

  1. Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

    PubMed Central

    Pérez-Berná, Ana J.; Mangel, Walter F.; McGrath, William J.; Graziano, Vito; Flint, Jane

    2014-01-01

    Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed. PMID:24227847

  2. The effect of myostatin on proliferation and lipid accumulation in 3T3-L1 preadipocytes.

    PubMed

    Zhu, Hui Juan; Pan, Hui; Zhang, Xu Zhe; Li, Nai Shi; Wang, Lin Jie; Yang, Hong Bo; Gong, Feng Ying

    2015-06-01

    Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Pparγ, Cebpα, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22-76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Pparγ and Cebpα expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes. © 2015 Society for Endocrinology.

  3. 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

  4. Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein, production of the viral particle and expression of host proteins involved in pathogenicity.

    PubMed

    Otoguro, Teruhime; Tanaka, Tomohisa; Kasai, Hirotake; Yamashita, Atsuya; Moriishi, Kohji

    2016-11-01

    Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C-terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin-like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti-HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti-HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP-dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV-infected cells was significantly impaired by LY411575 in a dose-dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir-dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV-related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.

  5. [Lipid- and protein-degrading processes during the maturation of ham].

    PubMed

    López Bote, C; Córdoba, J J; Antequera, T

    1993-02-01

    In the present work we review the main degradative pathways for lipids and proteins along the ripening of dry cured hams, with special emphasis on Iberian pig hams. Maximum proteolytic activity is found around the first stages (salting) and specially at the dryer. Lipolytic activity seems to be also higher in this stage. During the steps that follow the post-salting period the oxidation seems to be activated. The products from proteolytic and lipolytic processes might react among each other during the final steps in the cellar.

  6. EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples.

    PubMed

    Bienvenut, Willy Vincent; Scarpelli, Jean-Pierre; Dumestier, Johan; Meinnel, Thierry; Giglione, Carmela

    2017-03-20

    Characterization of mature protein N-termini by large scale proteomics is challenging. This is especially true for proteins undergoing cleavage of transit peptides when they are targeted to specific organelles, such as mitochondria or chloroplast. Protein neo-N-termini can be located up to 100-150 amino acids downstream from the initiator methionine and are not easily predictable. Although some bioinformatics tools are available, they usually require extensive manual validation to identify the exact N-terminal position. The situation becomes even more complex when post-translational modifications take place at the neo-N-terminus. Although N-terminal acetylation occurs mostly in the cytosol, it is also observed in some organelles such as chloroplast. To date, no bioinformatics tool is available to define mature protein starting positions, the associated N-terminus acetylation status and/or yield for each proteoform. In this context, we have developed the EnCOUNTer tool (i) to score all characterized peptides using discriminating parameters to identify bona fide mature protein N-termini and (ii) to determine the N-terminus acetylation yield of the most reliable ones. Based on large scale proteomics analyses using the SILProNAQ methodology, tandem mass spectrometry favoured the characterization of thousands of peptides. Data processing using the EnCOUNTer tool provided an efficient and rapid way to extract the most reliable mature protein N-termini. Selected peptides were subjected to N-terminus acetylation yield determination. In an A. thaliana cell lysate, 1232 distinct proteotypic N-termini were characterized of which 648 were located at the predicted protein N-terminus (position 1/2) and 584 were located further downstream (starting at position > 2). A large number of these N-termini were associated with various well-defined maturation processes occurring on organelle-targeted proteins (mitochondria, chloroplast and peroxisome), secreted proteins or membrane

  7. The Repeat Region of the Circumsporozoite Protein is Critical for Sporozoite Formation and Maturation in Plasmodium

    PubMed Central

    Patzewitz, Eva-Maria; Wall, Richard J.; Hopp, Christine S.; Poulin, Benoit; Mohmmed, Asif; Malhotra, Pawan; Coppi, Alida; Sinnis, Photini; Tewari, Rita

    2014-01-01

    The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective. PMID:25438048

  8. Myostatin in relation to physical activity and dysglycaemia and its effect on energy metabolism in human skeletal muscle cells.

    PubMed

    Hjorth, M; Pourteymour, S; Görgens, S W; Langleite, T M; Lee, S; Holen, T; Gulseth, H L; Birkeland, K I; Jensen, J; Drevon, C A; Norheim, F

    2016-05-01

    Some health benefits of exercise may be explained by an altered secretion of myokines. Because previous focus has been on upregulated myokines, we screened for downregulated myokines and identified myostatin. We studied the expression of myostatin in relation to exercise and dysglycaemia in skeletal muscle, adipose tissue and plasma. We further examined some effects of myostatin on energy metabolism in primary human muscle cells and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. Sedentary men with or without dysglycaemia underwent a 45-min acute bicycle test before and after 12 weeks of combined endurance and strength training. Blood samples and biopsies from m. vastus lateralis and adipose tissue were collected. Myostatin mRNA expression was reduced in skeletal muscle after acute as well as long-term exercise and was even further downregulated by acute exercise on top of 12-week training. Furthermore, the expression of myostatin at baseline correlated negatively with insulin sensitivity. Myostatin expression in the adipose tissue increased after 12 weeks of training and correlated positively with insulin sensitivity markers. In cultured muscle cells but not in SGBS cells, myostatin promoted an insulin-independent increase in glucose uptake. Furthermore, muscle cells incubated with myostatin had an enhanced rate of glucose oxidation and lactate production. Myostatin was differentially expressed in the muscle and adipose tissue in relation to physical activity and dysglycaemia. Recombinant myostatin increased the consumption of glucose in human skeletal muscle cells, suggesting a complex regulatory role of myostatin in skeletal muscle homeostasis. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  9. Protein kinase A inhibition of macrophage maturation is accompanied by an increase in DNA methylation of the colony-stimulating factor 1 receptor gene.

    PubMed

    Zasłona, Zbigniew; Scruggs, Anne M; Peters-Golden, Marc; Huang, Steven K

    2016-10-01

    Macrophage colony-stimulating factor 1 (CSF-1) plays a critical role in the differentiation of mononuclear phagocytes from bone marrow precursors, and maturing monocytes and macrophages exhibit increased expression of the CSF-1 receptor, CSF1R. The expression of CSF1R is tightly regulated by transcription factors and epigenetic mechanisms. We previously showed that prostaglandin E2 and subsequent activation of protein kinase A (PKA) inhibited CSF1R expression and macrophage maturation. Here, we examine the DNA methylation changes that occur at the Csf1r locus during macrophage maturation in the presence or absence of activated PKA. Murine bone marrow cells were matured to macrophages by incubating cells with CSF-1-containing conditioned medium for up to 6 days in the presence or absence of the PKA agonist 6-bnz-cAMP. DNA methylation of Csf1r promoter and enhancer regions was assayed by bisulphite pyrosequencing. DNA methylation of Csf1r decreased during normal macrophage maturation in concert with an increase in Csf1r mRNA expression. Treatment with the PKA agonist inhibited Csf1r mRNA and protein expression, and increased DNA methylation at the Csf1r promoter. This was associated with decreased binding of the transcription factor PU.1 to the Csf1r promoter. Treatment with the PKA agonist inhibited the responsiveness of macrophages to CSF-1. Levels of endogenous PKA activity decreased during normal macrophage maturation, suggesting that attenuation of this signalling pathway contributes to the increase in CSF1R expression during macrophage maturation. Together, these results demonstrate that macrophage maturation is accompanied by Csf1r hypomethylation, and illustrates for the first time the ability of PKA to increase Csf1r DNA methylation. © 2016 John Wiley & Sons Ltd.

  10. The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity.

    PubMed

    Ortega, Marcos E; Gaussier, Hélène; Catalano, Carlos E

    2007-11-02

    Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.

  11. The DNA Maturation Domain of gpA, the DNA Packaging Motor Protein of Bacteriophage Lambda, Contains an ATPase Site Associated with Endonuclease Activity

    PubMed Central

    Ortega, Marcos E.; Gaussier, Helene; Catalano, Carlos E.

    2007-01-01

    Summary Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N-terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in E. coli. Biochemical characterization of gpA-ΔN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N-terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A “P-loop” sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme – DNA maturation and DNA packaging – are discussed. PMID:17870092

  12. Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

    PubMed

    Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

    2017-02-20

    Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

  13. Identification of new proteins in follicular fluid from mature human follicles by direct sample rehydration method of two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Lee, Han-Chul; Lee, Sang-Wha; Lee, Kyo Won; Lee, Sook-Whan; Cha, Kwang-Yul; Kim, Kye Hyun; Lee, Suman

    2005-06-01

    Human follicular fluid (HFF) includes various biologically active proteins which can affect follicle growth and oocyte fertilization. Thus far, these proteins from mature follicles in human follicular fluid have been poorly characterized. Here, two-dimensional polyacrylamide gel electrophoresis (2-DE) with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to identify new proteins in HFF. Mature follicular fluids were obtained from five females after oocyte collection during in vitro fertilization (IVF). We directly rehydrated HFF samples, obtained high-resolution 2-DE maps, and processed them for 2-DE and MALDI-MS. One hundred eighty spots were detected and 10 of these spots were identified. By the 2-DE database, six of them had been reported, as proteins already existing in HFF. Hormone sensitive lipase (HSL), Unnamed protein product 1 (UPP1), Unnamed protein product 2 (UPP2), and apolipoprotein A-IV precursor were newly detected. HSL and apolipoprotein A-IV participate in lipid metabolism. UPP1 has a homology with selenocysteine lyase. We found by RT-PCR that these genes are expressed from human primary granulosa cells. The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis, hormone secretion regulation, or oocyte maturation. Further functional analysis of these proteins is necessitated to determine their biological implications.

  14. Yolk proteins during ovary and egg development of mature female freshwater crayfish (Cherax quadricarinatus).

    PubMed

    Serrano-Pinto, Vania; Vazquez-Boucard, Celia; Villarreal-Colmenares, Humberto

    2003-01-01

    Vitellins from ovaries and eggs at different stages of development in freshwater crayfish (Cherax quadricarinatus) were examined by chromatography, PAGE and SDS-PAGE. With these methods, two forms of vitellin (Vt1 and Vt2) were observed in ovaries and eggs (stages I and V). In ovaries in secondary vitellogenesis, native molecular mass was 470 (Vt1) and 440 (Vt2) kDa. The electrophoretic pattern of the eggs proved to be more complex. The protein molecular mass depend on the development stage of the egg: stage I, 650 kDa (Vt1) and 440 kDa (Vt2); stage V, 390 kDa (Vt1) and 340 kDa (Vt2). The identified vitellins appear to be lipo-glycocarotenoprotein. A similar vitellin polypeptide composition was observed in the two forms of vitellin from ovaries and eggs in stage V. In ovaries the SDS-PAGE analysis showed four subunits with molecular weights of approximately 180, 120, 95 and 80 kDa (Vt1 and Vt2). The polypeptide composition in the two forms of vitellins in stage I and stage III eggs were different at 195, 190, 130 and 110 kDa (Vt1) and 116 and 107 kDa (Vt2). On the other hand, in stage V eggs, 110, 95, 87 and 75 kDa (Vt1 and Vt2) were identified. Two antibodies (Ab1 and Ab2) were prepared against the purified proteins of stage V eggs and their specificity was demonstrated by radial immunoprecipitation, and Western blotting analysis. Two forms of vitellins were also found in stage V eggs after chromatography on Sepharose CL-2B column and hydroxylapatite and polyacrylamide gel electrophoresis.

  15. Fruit weight is controlled by Cell Size Regulator encoding a novel protein that is expressed in maturing tomato fruits.

    PubMed

    Mu, Qi; Huang, Zejun; Chakrabarti, Manohar; Illa-Berenguer, Eudald; Liu, Xiaoxi; Wang, Yanping; Ramos, Alexis; van der Knaap, Esther

    2017-08-01

    Increases in fruit weight of cultivated vegetables and fruits accompanied the domestication of these crops. Here we report on the positional cloning of a quantitative trait locus (QTL) controlling fruit weight in tomato. The derived allele of Cell Size Regulator (CSR-D) increases fruit weight predominantly through enlargement of the pericarp areas. The expanded pericarp tissues result from increased mesocarp cell size and not from increased number of cell layers. The effect of CSR on fruit weight and cell size is found across different genetic backgrounds implying a consistent impact of the locus on the trait. In fruits, CSR expression is undetectable early in development from floral meristems to the rapid cell proliferation stage after anthesis. Expression is low but detectable in growing fruit tissues and in or around vascular bundles coinciding with the cell enlargement stage of the fruit maturation process. CSR encodes an uncharacterized protein whose clade has expanded in the Solanaceae family. The mutant allele is predicted to encode a shorter protein due to a 1.4 kb deletion resulting in a 194 amino-acid truncation. Co-expression analyses and GO term enrichment analyses suggest association of CSR with cell differentiation in fruit tissues and vascular bundles. The derived allele arose in Solanum lycopersicum var cerasiforme and appears completely fixed in many cultivated tomato's market classes. This finding suggests that the selection of this allele was critical to the full domestication of tomato from its intermediate ancestors.

  16. Mycobacterial heat shock protein 70 induces interleukin-10 production: immunomodulation of synovial cell cytokine profile and dendritic cell maturation

    PubMed Central

    DETANICO, T; RODRIGUES, L; SABRITTO, A C; KEISERMANN, M; BAUER, M E; ZWICKEY, H; BONORINO, C

    2004-01-01

    Cytokines are key modulators of the immune responses that take place in the inflamed synovium of arthritis patients. Consequently, substances that can reverse the inflammatory profile of the inflamed joint are potential tools for clinical management of the disease. Mycobacterial heat shock protein 70 (MTBHSP70) has been found to protect rats from experimentally induced arthritis through the induction of interleukin (IL)-10-producing T cells. In this study, we have demonstrated that MTBHSP70 induces IL-10 production in synoviocytes from arthritis patients and peripheral blood monoculear cells (PBMCs) from both patients and healthy controls. IL-10 production was accompanied by a decrease in tumour necrosis factor (TNF)-α production by synovial cells. Separation studies showed that the target cells were mainly monocytes. Accordingly, we observed that MTBHSP70 delayed maturation of murine bone marrow-derived dendritic cells. Our results suggest that MTBHSP may act on antigen-presenting cells (APCs) to modulate the cytokine response in arthritis and support an anti-inflammatory role for this protein, suggesting that it may be of therapeutic use in the modulation of arthritis. PMID:14738465

  17. Fruit weight is controlled by Cell Size Regulator encoding a novel protein that is expressed in maturing tomato fruits

    PubMed Central

    Chakrabarti, Manohar; Liu, Xiaoxi; Wang, Yanping; Ramos, Alexis

    2017-01-01

    Increases in fruit weight of cultivated vegetables and fruits accompanied the domestication of these crops. Here we report on the positional cloning of a quantitative trait locus (QTL) controlling fruit weight in tomato. The derived allele of Cell Size Regulator (CSR-D) increases fruit weight predominantly through enlargement of the pericarp areas. The expanded pericarp tissues result from increased mesocarp cell size and not from increased number of cell layers. The effect of CSR on fruit weight and cell size is found across different genetic backgrounds implying a consistent impact of the locus on the trait. In fruits, CSR expression is undetectable early in development from floral meristems to the rapid cell proliferation stage after anthesis. Expression is low but detectable in growing fruit tissues and in or around vascular bundles coinciding with the cell enlargement stage of the fruit maturation process. CSR encodes an uncharacterized protein whose clade has expanded in the Solanaceae family. The mutant allele is predicted to encode a shorter protein due to a 1.4 kb deletion resulting in a 194 amino-acid truncation. Co-expression analyses and GO term enrichment analyses suggest association of CSR with cell differentiation in fruit tissues and vascular bundles. The derived allele arose in Solanum lycopersicum var cerasiforme and appears completely fixed in many cultivated tomato’s market classes. This finding suggests that the selection of this allele was critical to the full domestication of tomato from its intermediate ancestors. PMID:28817560

  18. Germinal Center B-Cell-Associated Nuclear Protein (GANP) Involved in RNA Metabolism for B Cell Maturation.

    PubMed

    Sakaguchi, N; Maeda, K

    2016-01-01

    Germinal center B-cell-associated nuclear protein (GANP) is upregulated in germinal center B cells against T-cell-dependent antigens in mice and humans. In mice, GANP depletion in B cells impairs antibody affinity maturation. Conversely, its transgenic overexpression augments the generation of high-affinity antigen-specific B cells. GANP associates with AID in the cytoplasm, shepherds AID into the nucleus, and augments its access to the rearranged immunoglobulin (Ig) variable (V) region of the genome in B cells, thereby precipitating the somatic hypermutation of V region genes. GANP is also upregulated in human CD4(+) T cells and is associated with APOBEC3G (A3G). GANP interacts with A3G and escorts it to the virion cores to potentiate its antiretroviral activity by inactivating HIV-1 genomic cDNA. Thus, GANP is characterized as a cofactor associated with AID/APOBEC cytidine deaminase family molecules in generating diversity of the IgV region of the genome and genetic alterations of exogenously introduced viral targets. GANP, encoded by human chromosome 21, as well as its mouse equivalent on chromosome 10, contains a region homologous to Saccharomyces Sac3 that was characterized as a component of the transcription/export 2 (TREX-2) complex and was predicted to be involved in RNA export and metabolism in mammalian cells. The metabolism of RNA during its maturation, from the transcription site at the chromosome within the nucleus to the cytoplasmic translation apparatus, needs to be elaborated with regard to acquired and innate immunity. In this review, we summarize the current knowledge on GANP as a component of TREX-2 in mammalian cells.

  19. Characterization and expression of proprotein convertases in CHO cells: Efficient proteolytic maturation of human bone morphogenetic protein-7.

    PubMed

    Sathyamurthy, Madhavi; Kim, Che Lin; Bang, You Lim; Kim, Young Sik; Jang, Ju Woong; Lee, Gyun Min

    2015-03-01

    Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.

  20. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    PubMed Central

    Lum, Michelle A.; Balaburski, Gregor M.; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Although alterations in stimulus-induced degradation of PKC have been implicated in disease, mechanistic understanding of this process remains limited. Evidence supports the existence of both proteasomal and lysosomal mechanisms of PKC processing. An established pathway involves rate-limiting priming site dephosphorylation of the activated enzyme and proteasomal clearance of the dephosphorylated protein. However, here we show that agonists promote down-regulation of endogenous PKCα with minimal accumulation of a nonphosphorylated species in multiple cell types. Furthermore, proteasome and lysosome inhibitors predominantly protect fully phosphorylated PKCα, pointing to this form as a substrate for degradation. Failure to detect substantive dephosphorylation of activated PKCα was not due to rephosphorylation because inhibition of Hsp70/Hsc70, which is required for re-priming, had only a minor effect on agonist-induced accumulation of nonphosphorylated protein. Thus, PKC degradation can occur in the absence of dephosphorylation. Further analysis revealed novel functions for Hsp70/Hsc70 and Hsp90 in the control of agonist-induced PKCα processing. These chaperones help to maintain phosphorylation of activated PKCα but have opposing effects on degradation of the phosphorylated protein; Hsp90 is protective, whereas Hsp70/Hsc70 activity is required for proteasomal processing of this species. Notably, down-regulation of nonphosphorylated PKCα shows little Hsp70/Hsc70 dependence, arguing that phosphorylated and nonphosphorylated species are differentially targeted for proteasomal degradation. Finally, lysosomal processing of activated PKCα is not regulated by phosphorylation or Hsps. Collectively, these data demonstrate that phosphorylated PKCα is a direct target for agonist-induced proteasomal degradation via an Hsp-regulated mechanism, and highlight the existence of a novel pathway of PKC desensitization in cells. PMID:23900841

  1. Centrosomal protein CP110 controls maturation of the mother centriole during cilia biogenesis

    PubMed Central

    Yadav, Sharda Prasad; Sharma, Neel Kamal; Liu, Chunqiao; Dong, Lijin; Li, Tiansen; Swaroop, Anand

    2016-01-01

    ABSTRACT Defects in cilia centrosomal genes cause pleiotropic clinical phenotypes, collectively called ciliopathies. Cilia biogenesis is initiated by the interaction of positive and negative regulators. Centriolar coiled coil protein 110 (CP110) caps the distal end of the mother centriole and is known to act as a suppressor to control the timing of ciliogenesis. Here, we demonstrate that CP110 promotes cilia formation in vivo, in contrast to findings in cultured cells. Cp110−/− mice die shortly after birth owing to organogenesis defects as in ciliopathies. Shh signaling is impaired in null embryos and primary cilia are reduced in multiple tissues. We show that CP110 is required for anchoring of basal bodies to the membrane during cilia formation. CP110 loss resulted in an abnormal distribution of core components of subdistal appendages (SDAs) and of recycling endosomes, which may be associated with premature extension of axonemal microtubules. Our data implicate CP110 in SDA assembly and ciliary vesicle docking, two requisite early steps in cilia formation. We suggest that CP110 has unique context-dependent functions, acting as both a suppressor and a promoter of ciliogenesis. PMID:26965371

  2. Generation of Myostatin Gene-Edited Channel Catfish (Ictalurus punctatus) via Zygote Injection of CRISPR/Cas9 System.

    PubMed

    Khalil, Karim; Elayat, Medhat; Khalifa, Elsayed; Daghash, Samer; Elaswad, Ahmed; Miller, Michael; Abdelrahman, Hisham; Ye, Zhi; Odin, Ramjie; Drescher, David; Vo, Khoi; Gosh, Kamal; Bugg, William; Robinson, Dalton; Dunham, Rex

    2017-08-04

    The myostatin (MSTN) gene is important because of its role in regulation of skeletal muscle growth in all vertebrates. In this study, CRISPR/Cas9 was utilized to successfully target the channel catfish, Ictalurus punctatus, muscle suppressor gene MSTN. CRISPR/Cas9 induced high rates (88-100%) of mutagenesis in the target protein-encoding sites of MSTN. MSTN-edited fry had more muscle cells (p < 0.001) than controls, and the mean body weight of gene-edited fry increased by 29.7%. The nucleic acid alignment of the mutated sequences against the wild-type sequence revealed multiple insertions and deletions. These results demonstrate that CRISPR/Cas9 is a highly efficient tool for editing the channel catfish genome, and opens ways for facilitating channel catfish genetic enhancement and functional genomics. This approach may produce growth-enhanced channel catfish and increase productivity.

  3. Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth

    PubMed Central

    Khan, Tayeba; Weber, Hans; DiMuzio, Jillian; Matter, Andrea; Dogdas, Belma; Shah, Tosha; Thankappan, Anil; Disa, Jyoti; Jadhav, Vasant; Lubbers, Laura; Sepp-Lorenzino, Laura; Strapps, Walter R; Tadin-Strapps, Marija

    2016-01-01

    Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85–95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes. PMID:27483025

  4. Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System

    PubMed Central

    Wang, Kankan; Ouyang, Hongsheng; Xie, Zicong; Yao, Chaogang; Guo, Nannan; Li, Mengjing; Jiao, Huping; Pang, Daxin

    2015-01-01

    Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety. PMID:26564781

  5. Depletion of Myostatin b Promotes Somatic Growth and Lipid Metabolism in Zebrafish

    PubMed Central

    Gao, Yanping; Dai, Ziru; Shi, Chuang; Zhai, Gang; Jin, Xia; He, Jiangyan; Lou, Qiyong; Yin, Zhan

    2016-01-01

    Myostatin (MSTN) is a negative regulator of myogenesis in vertebrates. Depletion of mstn resulted in elevated muscle growth in several animal species. However, the report on the complete ablation of mstn in teleost fish has not yet become available. In this study, two independent mstnb-deficient mutant lines in zebrafish were generated with the TALENs technique. In the mstnb-deficient zebrafish, enhanced muscle growth with muscle fiber hyperplasia was achieved. Beginning at the adult stage (80 days postfertilization), the mstnb-deficient zebrafish exhibited increased circumferences and body weights compared with the wild-type sibling control fish. Although the overall total lipid/body weight ratios remained similar between the mstnb-deficient zebrafish and the control fish, the distribution of lipids was altered. The size of the visceral adipose tissues became smaller while more lipids accumulated in skeletal muscle in the mstnb-deficient zebrafish than in the wild-type control fish. Based on the transcriptional expression profiles, our results revealed that lipid metabolism, including lipolysis and lipogenesis processes, was highly activated in the mstnb-deficient zebrafish, which indicated the transition of energy metabolism from protein-dependent to lipid-dependent in mstnb-deficient zebrafish. Our mstnb-deficient model could be valuable in understanding not only the growth trait regulation in teleosts but also the mechanisms of teleost energy metabolism. PMID:27458428

  6. Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

    PubMed Central

    Li, Kui; Xu, Zhiqiang; Liang, Dong; Li, Jingyun; Li, Junbo; Jia, Wenshuang; Li, Yuehua; Dong, Xiaohua; Cao, Shasha; Wang, Xiaoxiao; Pan, Jianlin; Zhao, Qingshun

    2011-01-01

    Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish. PMID:22194943

  7. The regulator of MAT2 (ROM2) protein binds to early maturation promoters and represses PvALF-activated transcription.

    PubMed

    Chern, M S; Bobb, A J; Bustos, M M

    1996-02-01

    The regulation of maturation (MAT)- and late embryogenesis (LEA)-specific gene expression in dicots involves factors related to ABI3, a seed-specific component of the abscisic acid signal transduction pathways from Arabidopsis. In French bean (Phaseolus vulgaris), the ABI3-like factor, PvALF, activates transcription from MAT promoters of phytohemagglutinin (DLEC2) and beta-phaseolin (PHS beta) genes. We describe the regulator of MAT2 (ROM2) as a basic leucine zipper (bZIP) DNA binding protein that recognizes motifs with symmetric (ACGT) and asymmetric (ACCT) core elements present in both MAT promoters. ROM2 antagonizes trans-activation of the DLEC2 promoter by PvALF in transient expression assays. Repression was abolished by mutations that prevented binding of ROM2 to the DLEC2 seed enhancer region. Moreover, a hybrid protein composed of a PvALF activation domain and the DNA binding and dimerization domain of ROM2 activated gene expression, indicating that ROM2 recognizes the DLEC2 enhancer in vivo; consequently, ROM2 functions as a DNA binding site-dependent repressor. Supershift analysis of nuclear proteins, using a ROM2-specific antibody, revealed an increase in ROM2 DNA binding activity during seed desiccation. A corresponding increase in ROM2 mRNA coincided with the period when DLEC2 mRNA levels declined in embryos. These results demonstrate developmental regulation of the ROM2 repressor and point to a role for this factor in silencing DLEC2 transcription during late embryogenesis.

  8. The regulator of MAT2 (ROM2) protein binds to early maturation promoters and represses PvALF-activated transcription.

    PubMed Central

    Chern, M S; Bobb, A J; Bustos, M M

    1996-01-01

    The regulation of maturation (MAT)- and late embryogenesis (LEA)-specific gene expression in dicots involves factors related to ABI3, a seed-specific component of the abscisic acid signal transduction pathways from Arabidopsis. In French bean (Phaseolus vulgaris), the ABI3-like factor, PvALF, activates transcription from MAT promoters of phytohemagglutinin (DLEC2) and beta-phaseolin (PHS beta) genes. We describe the regulator of MAT2 (ROM2) as a basic leucine zipper (bZIP) DNA binding protein that recognizes motifs with symmetric (ACGT) and asymmetric (ACCT) core elements present in both MAT promoters. ROM2 antagonizes trans-activation of the DLEC2 promoter by PvALF in transient expression assays. Repression was abolished by mutations that prevented binding of ROM2 to the DLEC2 seed enhancer region. Moreover, a hybrid protein composed of a PvALF activation domain and the DNA binding and dimerization domain of ROM2 activated gene expression, indicating that ROM2 recognizes the DLEC2 enhancer in vivo; consequently, ROM2 functions as a DNA binding site-dependent repressor. Supershift analysis of nuclear proteins, using a ROM2-specific antibody, revealed an increase in ROM2 DNA binding activity during seed desiccation. A corresponding increase in ROM2 mRNA coincided with the period when DLEC2 mRNA levels declined in embryos. These results demonstrate developmental regulation of the ROM2 repressor and point to a role for this factor in silencing DLEC2 transcription during late embryogenesis. PMID:8742714

  9. A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation, results in delayed degradation of maternal proteins and embryonic lethality.

    PubMed

    Lin, Rueyling

    2003-06-01

    In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.

  10. Myostatin antibody (LY2495655) in older weak fallers: a proof-of-concept, randomised, phase 2 trial

    USDA-ARS?s Scientific Manuscript database

    BACKGROUND: Myostatin inhibits skeletal muscle growth. The humanised monoclonal antibody LY2495655 (LY) binds and neutralises myostatin. We aimed to test whether LY increases appendicular lean body mass (aLBM) and improves physical performance in older individuals who have had recent falls and low m...

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

    SciTech Connect

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi; Yamaguchi, Takahiro

    2010-03-19

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

  12. Oral administration of myostatin-specific whole recombinant yeast Saccharomyces cerevisiae vaccine increases body weight and muscle composition in mice.

    PubMed

    Zhang, Tingting; Yang, Hanjiang; Wang, Rui; Xu, Kun; Xin, Ying; Ren, Gang; Zhou, Gang; Zhang, Cunfang; Wang, Ling; Zhang, Zhiying

    2011-10-26

    Myostatin negatively regulates skeletal muscle growth. It was found that active immunization with myostatin-specific vaccine blocked myostatin function in vivo, which resulted in increase of body weight and muscle composition in mice. However, traditional vaccine and its administration method are expensive and laborious. In this study, we investigated the possibility of using heat-killed whole recombinant yeast Saccharomyces cerevisiae vaccine to modulate myostatin function in mice. The CDS of myostatin was obtained from a pig genome by PCR and subcloned into a yeast expression vector, which was driven by a copper-inducible promoter. Expression of recombinant myostatin was induced by CuSO(4) and confirmed by western blot. We vaccinated mice by oral feeding and subcutaneous injection as comparison. We found that oral feeding resulted in the similar effective immune response than injection, which was measured by the presence of myostatin-specific antibodies in mouse serum. Interestingly, animals vaccinated by both methods demonstrated enhanced growth performance compared to control. All animals were healthy looking throughout the course of experiment, suggesting that whole recombinant yeast vaccine is nontoxic and therefore safe to use. Given the simplicity of its nature, heat-killed myostatin-specific whole recombinant yeast vaccine holds a promise to treat human muscle-wasting diseases in the future.

  13. Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

    PubMed Central

    Elhag, G A; Thomas, F J; McCreery, T P; Bourque, D P

    1992-01-01

    Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco. Images PMID:1542565

  14. Short bouts of stretching increase myo-D, myostatin and atrogin-1 in rat soleus muscle.

    PubMed

    Peviani, Sabrina Messa; Gomes, Anna Raquel Silveira; Moreira, Roberta Fátima Carreira; Moriscot, Anselmo Sigari; Salvini, Tania Fátima

    2007-03-01

    Stretching is widely used in rehabilitation and sports activities to improve joint range-of-motion and flexibility in humans, but the effect of stretching on the gene expression of skeletal muscle is poorly understood. We evaluated the effect of short bouts of passive stretching of rat soleus muscle on myo-D, myostatin, and atrogin-1 gene expressions. Six groups of animals were submitted to a single session of stretching (10 stretches of 1 minute with 30 seconds of rest between them, performed manually) and were evaluated immediately (I), and 8, 24, 48, 72, and 168 hours after the session. To evaluate the effect of repetitive sessions of stretching on the soleus muscle over 1 week, three groups of animals received a single session per day of stretching and the muscle was evaluated immediately after 2, 3, and 7 sessions. The mRNA levels of myo-D, myostatin, and atrogin-1 were determined by real-time polymerase chain reaction. A single session of stretching increased the mRNA levels of myo-D (after 24 h), myostatin (I, and 168 h later), and atrogin-1 (after 48 h). Repeated daily session of stretching over 1 week increased myostatin (after 7 sessions) and atrogin-1 expression (after 2, 3, and 7 sessions). Thus, short bouts of passive stretching are able to increase the gene expression of factors associated with muscle growth (myo-D), negative regulation of muscle mass (myostatin), and atrophy (atrogin-1), indicating muscle remodeling through different pathways.

  15. Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases.

    PubMed

    Xu, Li; Zhao, Piming; Mariano, Andrew; Han, Renzhi

    2013-07-30

    Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.Molecular Therapy-Nucleic Acids (2013) 2, e112; doi:10.1038/mtna.2013.39; published online 30 July 2013.

  16. 207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM.

    PubMed

    Pinzon, C A; Snyder, M; Pryor, J; Thompson, B; Golding, M; Long, C

    2016-01-01

    The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of -1577, -689, -555, and -116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1=sgRNA 1-4; Set 2=sgRNA 2-3). Cells were exposed to puromycin (0.2µgmL(-1)) for 72h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes

  17. A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation.

    PubMed

    Sedlik, Christine; Orbach, Daniel; Veron, Philippe; Schweighoffer, Edina; Colucci, Francesco; Gamberale, Romina; Ioan-Facsinay, Andrea; Verbeek, Sjef; Ricciardi-Castagnoli, Paola; Bonnerot, Christian; Tybulewicz, Victor L J; Di Santo, James; Amigorena, Sebastian

    2003-01-15

    Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.

  18. Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker

    USGS Publications Warehouse

    Patino, R.; Yoshizaki, G.; Bolamba, D.; Thomas, P.

    2003-01-01

    The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2?? and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquin- olinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 ??M) and PGF2?? (5 ??M) did not induce maturation, and NDGA (10 ??M) did not affect MIH-dependent maturation. However, IM (100 ??M) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 ??g/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 ??M; H7, 50??M) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 ??M) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2?? restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role

  19. Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH.

    PubMed

    Peaucellier, G; Picard, A; Robert, J J; Capony, J P; Labbe, J C; Doree, M

    1988-01-01

    An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also

  20. Relationship between myostatin and irisin in type 2 diabetes mellitus: a compensatory mechanism to an unfavourable metabolic state?

    PubMed

    García-Fontana, Beatriz; Reyes-García, Rebeca; Morales-Santana, Sonia; Ávila-Rubio, Verónica; Muñoz-Garach, Araceli; Rozas-Moreno, Pedro; Muñoz-Torres, Manuel

    2016-04-01

    Myostatin and irisin are two myokines related to energy metabolism, acting on skeletal muscle and recently suggested on adipose tissue in mice. However, the exact role of these myokines in humans has not been fully established. Our aim was to evaluate the relationship between serum levels of myostatin and irisin in type 2 diabetes mellitus patients and non-diabetic controls and to explore its links with metabolic parameters. Case-control study including 73 type 2 diabetes mellitus patients and 55 non-diabetic subjects as control group. Circulating myostatin and irisin levels were measured by enzyme-linked immunosorbent assays. Type 2 diabetes mellitus patients showed significantly lower myostatin levels (p = 0.001) and higher irisin levels (p = 0.036) than controls. An inverse relationship was observed between myostatin and irisin levels (p = 0.002). Moreover, in type 2 diabetes mellitus patients, after adjusting by confounder factors, myostatin was negatively related to fasting plasma glucose (p = 0.005) and to triglyceride levels (p = 0.028) while irisin showed a positive association with these variables (p = 0.017 and p = 0.006 respectively). A linear regression analysis showed that irisin and fasting plasma glucose levels were independently associated to myostatin levels and that myostatin and triglyceride levels were independently associated to irisin concentrations in type 2 diabetes mellitus patients. Our results suggest that serum levels of myostatin and irisin are related in patients with type 2 diabetes. Triglyceride and glucose levels could modulate myostatin and irisin concentrations as a compensatory mechanism to improve the metabolic state in these patients although further studies are needed to elucidate whether the action of these myokines represents an adaptative response.

  1. Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA maturation.

    PubMed

    Huang, Ying; Bayfield, Mark A; Intine, Robert V; Maraia, Richard J

    2006-07-01

    By sequence-specific binding to 3' UUU-OH, the La protein shields precursor (pre)-RNAs from 3' end digestion and is required to protect defective pre-transfer RNAs from decay. Although La is comprised of a La motif and an RNA-recognition motif (RRM), a recent structure indicates that the RRM beta-sheet surface is not involved in UUU-OH recognition, raising questions as to its function. Progressively defective suppressor tRNAs in Schizosaccharomyces pombe reveal differential sensitivities to La and Rrp6p, a 3' exonuclease component of pre-tRNA decay. 3' end protection is compromised by mutations to the La motif but not the RRM surface. The most defective pre-tRNAs require a second activity of La, in addition to 3' protection, that requires an intact RRM surface. The two activities of La in tRNA maturation map to its two conserved RNA-binding surfaces and suggest a modular model that has implications for its other ligands.

  2. Evaluation of ARG protein expression in mature B cell lymphomas compared to non-neoplastic reactive lymph node.

    PubMed

    Kabiri, Zahra; Salehi, Mansoor; Mokarian, Fariborz; Mohajeri, Mohammad Reza; Mahmoodi, Farzaneh; Keyhanian, Kianoosh; Doostan, Iman; Ataollahi, Mohammad Reza; Modarressi, Mohammad Hossein

    2009-01-01

    The participation of Abl-Related Gene (ARG) is demonstrated in pathogenesis of different human malignancies. However there is no conclusive evidence on ARG expression level in mature B cell lymphomas. In this study we evaluated ARG protein expression in Follicular Lymphoma (FL), Burkitt's Lymphoma (BL) and Diffused Large B Cell Lymphoma (DLBCL) in comparison with non-neoplastic lymph nodes. Semi-quantitative fluorescent ImmunoHistoChemistry was applied on 14, 7 and 4 patients with DLBCL, FL and BL respectively, adding to 4 normal and 4 reactive lymph nodes. The mean ratio of ARG/GAPDH expression was significantly different (p<0.00) between lymphomas and control samples, with DLBCL having the highest ARG expression amongst all. Over expression of ARG was seen in FL and BL, with FL expressing statistically more ARG than BL. Moreover, the ARG/GAPDH expression ratio increased from DLBCL stage I towards stage VI, all showing significantly more ARG expression than FL and BL (in all cases p<0.00).

  3. Dynamic Changes of Neuroskeletal Proteins in DRGs Underlie Impaired Axonal Maturation and Progressive Axonal Degeneration in Type 1 Diabetes

    PubMed Central

    Kamiya, Hideki; Zhang, Weixian; Sima, Anders A. F.

    2009-01-01

    We investigated mechanisms underlying progressive axonal dysfunction and structural deficits in type 1 BB/Wor-rats from 1 week to 10 month diabetes duration. Motor and sensory conduction velocities were decreased after 4 and 6 weeks of diabetes and declined further over the remaining 9 months. Myelinated sural nerve fibers showed progressive deficits in fiber numbers and sizes. Structural deficits in unmyelinated axonal size were evident at 2 month and deficits in number were present at 4 mo. These changes were preceded by decreased availability of insulin, C-peptide and IGF-1 and decreased expression of neurofilaments and β-III-tubulin. Upregulation of phosphorylating stress kinases like Cdk5, p-GSK-3β, and p42/44 resulted in increased phosphorylation of neurofilaments. Increasing activity of p-GSK-3β correlated with increasing phosphorylation of NFH, whereas decreasing Cdk5 correlated with diminishing phosphorylation of NFM. The data suggest that impaired neurotrophic support results in sequentially impaired synthesis and postranslational modifications of neuroskeletal proteins, resulting in progressive deficits in axonal function, maturation and size. PMID:19834568

  4. CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation.

    PubMed

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Sun, Qing-Yuan; Fan, Heng-Yu

    2015-08-18

    Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility.

  5. cDNA cloning and expression analysis of myostatin/GDF11 in shrimp, Litopenaeus vannamei.

    PubMed

    Qian, Zhaoying; Mi, Xiao; Wang, Xianzong; He, Shulin; Liu, Yongjie; Hou, Fujun; Liu, Qiao; Liu, Xiaolin

    2013-05-01

    Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins belonging to the transforming growth factor-β (TGF-β) superfamily. In vertebrates, MSTN is known to negatively regulate skeletal muscle growth, and GDF11 is found to inhibit neurogenesis. In invertebrates, only one ortholog of vertebrate MSTN and GDF11 (MSTN/GDF11) existed. Little attention has been paid on its role to date. In this study, the cDNA that encodes a 422-amino-acid MSTN/GDF11 protein (LvMSTN/GDF11) was characterized from a crustacean species, the Pacific white shrimp (Litopenaeus vannamei). Sequence analysis revealed that the overall protein sequence and specific functional sites of LvMSTN/GDF11 were highly conserved with those in other crustacean species. Expression analysis by quantitative real-time reverse transcription polymerase chain reaction technique demonstrated its tissue-specific, larval developmental stage-specific, and molt stage-specific expression pattern, respectively. After in vivo injections of 20 hydroxyecdysone (20E), LvMSTN/GDF11 transcripts were declined in the abdominal (A) and pleopod (P1) muscles, increased in the pereiopod (P2) muscle, and not affected in the thoracic (T) muscle. The observed expression profiles suggest multiple functions of LvMSTN/GDF11 in L. vannamei and its role differs during the larval development and natural molt cycle. The different responses of LvMSTN/GDF11 to acute increases of 20E in the A, P1, P2 and T muscles may indicate that LvMSTN/GDF11 is transcriptionally regulated via ecdysteroids to coincide with its specific roles in the former three muscles, while its role may be independent of 20E regulation in the T muscle.

  6. Myostatin signaling through Smad2, Smad3 and Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism.

    PubMed

    Zhu, Xiangyang; Topouzis, Stavros; Liang, Li-Fang; Stotish, Ronald L

    2004-06-21

    As a member of the TGF-beta superfamily, myostatin is a specific negative regulator of skeletal muscle mass. To identify the downstream components in the myostatin signal transduction pathway, we used a luciferase reporter assay to elucidate myostatin-induced activity. The myostatin-induced transcription requires the participation of regulatory Smads (Smad2/3) and Co-Smads (Smad4). Conversely, inhibitory Smad7, but not Smad6, dramatically reduces the myostatin-induced transcription. This Smad7 inhibition is enhanced by co-expression of Smurf1. We have also shown that Smad7 expression is stimulated by myostatin via the interaction between Smad2, Smad3, Smad4 and the SBE (Smad binding element) in the Smad7 promoter. These results suggest that the myostatin signal transduction pathway is regulated by Smad7 through a negative feedback mechanism.

  7. Knock down of the myostatin gene by RNA interference increased body weight in chicken.

    PubMed

    Bhattacharya, T K; Shukla, R; Chatterjee, R N; Dushyanth, K

    2017-01-10

    Myostatin is a negative regulator of muscular growth in poultry and other animals. Of several approaches, knocking down the negative regulator is an important aspect to augment muscular growth in chicken. Knock down of myostatin gene has been performed by shRNA acting against the expression of gene in animals. Two methods of knock down of gene in chicken such as embryo manipulation and sperm mediated method have been performed. The hatching percentage in embryo manipulation and sperm mediated method of knock down was 58.0 and 41.5%, respectively. The shRNA in knock down chicken enhanced body weight at 6 weeks by 26.9%. The dressing percentage and serum biochemical parameters such as SGPT and alkaline phosphatase differed significantly (P<0.05) between knock down and control birds. It is concluded that knocking down the myostatin gene successfully augmented growth in chicken.

  8. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    PubMed Central

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-01-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals. PMID:26950874

  9. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases.

    PubMed

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-03-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  10. Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice

    PubMed Central

    Camporez, João-Paulo G.; Petersen, Max C.; Abudukadier, Abulizi; Moreira, Gabriela V.; Jurczak, Michael J.; Friedman, Glenn; Haqq, Christopher M.; Petersen, Kitt Falk; Shulman, Gerald I.

    2016-01-01

    Sarcopenia, or skeletal muscle atrophy, is a debilitating comorbidity of many physiological and pathophysiological processes, including normal aging. There are no approved therapies for sarcopenia, but the antihypertrophic myokine myostatin is a potential therapeutic target. Here, we show that treatment of young and old mice with an anti-myostatin antibody (ATA 842) for 4 wk increased muscle mass and muscle strength in both groups. Furthermore, ATA 842 treatment also increased insulin-stimulated whole body glucose metabolism in old mice, which could be attributed to increased insulin-stimulated skeletal muscle glucose uptake as measured by a hyperinsulinemic-euglycemic clamp. Taken together, these studies provide support for pharmacological inhibition of myostatin as a potential therapeutic approach for age-related sarcopenia and metabolic disease. PMID:26858428

  11. Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice.

    PubMed

    Camporez, João-Paulo G; Petersen, Max C; Abudukadier, Abulizi; Moreira, Gabriela V; Jurczak, Michael J; Friedman, Glenn; Haqq, Christopher M; Petersen, Kitt Falk; Shulman, Gerald I

    2016-02-23

    Sarcopenia, or skeletal muscle atrophy, is a debilitating comorbidity of many physiological and pathophysiological processes, including normal aging. There are no approved therapies for sarcopenia, but the antihypertrophic myokine myostatin is a potential therapeutic target. Here, we show that treatment of young and old mice with an anti-myostatin antibody (ATA 842) for 4 wk increased muscle mass and muscle strength in both groups. Furthermore, ATA 842 treatment also increased insulin-stimulated whole body glucose metabolism in old mice, which could be attributed to increased insulin-stimulated skeletal muscle glucose uptake as measured by a hyperinsulinemic-euglycemic clamp. Taken together, these studies provide support for pharmacological inhibition of myostatin as a potential therapeutic approach for age-related sarcopenia and metabolic disease.

  12. Protein quality and utilization of timothy, oat-supplemented timothy, and alfalfa at differing harvest maturities in exercised Arabian horses.

    PubMed

    Woodward, A D; Nielsen, B D; Liesman, J; Lavin, T; Trottier, N L

    2011-12-01

    To evaluate the protein quality and postgut N utilization of full-bloom timothy hay, oat-supplemented timothy-hay diets, and alfalfa hay harvested at different maturities, apparent whole tract N digestibility, urinary N excretion, and serum AA profiles were determined in light to moderately exercised Arabian horses. Six Arabian geldings (16.0 ± 0.3 yr; 467 ± 11 kg of BW) were randomly allocated to a 6 × 6 Latin square design. Diets included full-bloom timothy grass hay (G), G + 0.2% BW oat (G1), G + 0.4% BW oat (G2), mid-bloom alfalfa (A1), early-bloom alfalfa (A2), and early-bud alfalfa hay (A3). Forages were fed at 1.6% of the BW of the horse (as-fed). Each period consisted of an 11-d adaptation period followed by total collection of feces and urine for 3 d. Blood samples were taken on d 11 for analysis of serum AA concentrations. During the 3-d collection period, urine and feces were collected every 8 h and measured and weighed, respectively. Approximately 10% of the total urine volume and fecal weight per period was retained for N analyses. Fecal DM output was less (P < 0.05) in A1, A2, or A3 compared with G, G1, or G2. Apparent whole tract N digestibility was greater (P < 0.01) in A1, A2, and A3 compared with G, G1, or G2, and was greater (P < 0.05) in G1 and G2 compared with G. Nitrogen retention was not different from zero, and there were no differences (P > 0.05) in N retention among diets. Urinary N excretion and total N excretion were greater (P < 0.05) in A1, A2, and A3 compared with G, G1, or G2. Plasma concentrations for the majority of AA increased curvilinearly in response to feeding G, A1, A2, and A3 (quadratic, P < 0.05), with values appearing to maximize 2-h postfeeding. Although alfalfa N digestibility increased with decreasing harvest maturity, N retention did not differ and urinary volume and N excretion increased, indicating that postabsorptive N utilization decreased. In contrast, inclusion of oats at either 0.2 or 0.4% of the BW of the

  13. Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes.

    PubMed

    Lonergan, P; Fair, T; Khatir, H; Cesaroni, G; Mermillod, P

    1998-08-01

    The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final

  14. Suppression of a signal sequence mutation by an amino acid substitution in the mature portion of the maltose-binding protein.

    PubMed Central

    Cover, W H; Ryan, J P; Bassford, P J; Walsh, K A; Bollinger, J; Randall, L L

    1987-01-01

    An unusual spontaneous pseudorevertant of an Escherichia coli strain carrying the signal sequence point mutation malE14-1 was characterized. The suppressor mutation, malE2261, resulted in a single substitution of an aspartyl residue for a tyrosyl residue at position 283 in the sequence of the mature maltose-binding protein. The precursor retained the malE14-1 point mutation in the signal sequence. The pseudorevertant carrying both malE14-1 and malE2261 exported twice the amount of maltose-binding protein as that of the mutant carrying the malE14-1 allele alone but only 18% of the amount exported by a strain producing wild-type maltose-binding protein. A strain carrying the suppressor allele malE2261 in combination with a wild-type signal sequence exported normal quantities of maltose-binding protein to the periplasm. Mature MalE2261 had a Kd for maltose of 27 microM, compared with 3.6 microM for mature wild-type maltose-binding protein. The precursor species than contained both changes resulting from malE14-1 and malE2261 was significantly less stable in the cytoplasm than was the precursor containing only the change encoded by malE14-1. Images PMID:3553148

  15. Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

    PubMed

    Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

    2005-10-20

    The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

  16. Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta

    PubMed Central

    Oestreich, Arin K.; Kamp, William M.; McCray, Marcus G.; Carleton, Stephanie M.; Karasseva, Natalia; Lenz, Kristin L.; Jeong, Youngjae; Daghlas, Salah A.; Yao, Xiaomei; Wang, Yong; Pfeiffer, Ferris M.; Ellersieck, Mark R.; Schulz, Laura C.; Phillips, Charlotte L.

    2016-01-01

    During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta. PMID:27821779

  17. Pharmacological inhibition of myostatin protects against skeletal muscle atrophy and weakness after anterior cruciate ligament tear.

    PubMed

    Wurtzel, Caroline Nw; Gumucio, Jonathan P; Grekin, Jeremy A; Khouri, Roger K; Russell, Alan J; Bedi, Asheesh; Mendias, Christopher L

    2017-02-08

    Anterior cruciate ligament (ACL) tears are among the most frequent knee injuries in sports medicine, with tear rates in the US up to 250,000 per year. Many patients who suffer from ACL tears have persistent atrophy and weakness even after considerable rehabilitation. Myostatin is a cytokine that directly induces muscle atrophy, and previous studies rodent models and patients have demonstrated an upregulation of myostatin after ACL tear. Using a preclinical rat model, our objective was to determine if the use of a bioneutralizing antibody against myostatin could prevent muscle atrophy and weakness after ACL tear. Rats underwent a surgically induced ACL tear and were treated with either a bioneutralizing antibody against myostatin (10B3, GlaxoSmithKline) or a sham antibody (E1-82.15, GlaxoSmithKline). Muscles were harvested at either 7 or 21 days after induction of a tear to measure changes in contractile function, fiber size, and genes involved in muscle atrophy and hypertrophy. These time points were selected to evaluate early and later changes in muscle structure and function. Compared to the sham antibody group, 7 days after ACL tear, myostatin inhibition reduced the expression of proteolytic genes and induced the expression of hypertrophy genes. These early changes in gene expression lead to a 22% increase in muscle fiber cross-sectional area and a 10% improvement in maximum isometric force production that were observed 21 days after ACL tear. Overall, myostatin inhibition lead to several favorable, although modest, changes in molecular biomarkers of muscle regeneration and reduced muscle atrophy and weakness following ACL tear. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Haplotype diversity of the myostatin gene among beef cattle breeds

    PubMed Central

    Dunner, Susana; Miranda, M Eugenia; Amigues, Yves; Cañón, Javier; Georges, Michel; Hanset, Roger; Williams, John; Ménissier, François

    2003-01-01

    A total of 678 individuals from 28 European bovine breeds were both phenotyped and analysed at the myostatin locus by the Single Strand Conformation Polymorphism (SSCP) method. Seven new mutations were identified which contribute to the high polymorphism (1 SNP every 100 bp) present in this small gene; twenty haplotypes were described and a genotyping method was set up using the Oligonucleotide Ligation Assay (OLA) method. Some haplotypes appeared to be exclusive to a particular breed; this was the case for 5 in the Charolaise (involving mutation Q204X) and 7 in the Maine-Anjou (involving mutation E226X). The relationships between the different haplotypes were studied, thus allowing to test the earlier hypothesis on the origin of muscular hypertrophy in Europe: muscular hypertrophy (namely nt821(del11)) was mainly spread in different waves from northern Europe milk purpose populations in most breeds; however, other mutations (mostly disruptive) arose in a single breed, were highly selected and have since scarcely evolved to other populations. PMID:12605853

  19. Myostatin gene mutated mice induced with tale nucleases.

    PubMed

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

    2015-01-01

    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

  20. Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses.

    PubMed

    Li, Ran; Liu, Dong-Hua; Cao, Chun-Na; Wang, Shao-Qiang; Dang, Rui-Hua; Lan, Xian-Yong; Chen, Hong; Zhang, Tao; Liu, Wu-Jun; Lei, Chu-Zhao

    2014-03-15

    The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26T>C, g.156T>C, g.587A>G, g.598C>T, g.1485C>T, g.2115A>G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A>G and g.598C>T residing in the 5'UTR region were novel SNPs identified by this study. The g.2115A>G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.

  1. Bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn.

    PubMed

    Zhang, Lina; Boeren, Sjef; Hageman, Jos A; van Hooijdonk, Toon; Vervoort, Jacques; Hettinga, Kasper

    2015-01-01

    In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.

  2. Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes

    PubMed Central

    Enders, Anselm; Short, Alanna; Miosge, Lisa A.; Bergmann, Hannes; Sontani, Yovina; Bertram, Edward M.; Whittle, Belinda; Balakishnan, Bhavani; Yoshida, Kaoru; Sjollema, Geoff; Field, Matthew A.; Andrews, T. Daniel; Hagiwara, Hiromi; Goodnow, Christopher C.

    2014-01-01

    IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells. PMID:24616512

  3. Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes.

    PubMed

    Enders, Anselm; Short, Alanna; Miosge, Lisa A; Bergmann, Hannes; Sontani, Yovina; Bertram, Edward M; Whittle, Belinda; Balakishnan, Bhavani; Yoshida, Kaoru; Sjollema, Geoff; Field, Matthew A; Andrews, T Daniel; Hagiwara, Hiromi; Goodnow, Christopher C

    2014-03-25

    IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.

  4. Statins reduce amyloid β-peptide production by modulating amyloid precursor protein maturation and phosphorylation through a cholesterol-independent mechanism in cultured neurons.

    PubMed

    Hosaka, Ai; Araki, Wataru; Oda, Akiko; Tomidokoro, Yasushi; Tamaoka, Akira

    2013-03-01

    Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2-2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.