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Sample records for mature myostatin protein

  1. Skeletal muscle gene expression after myostatin knockout in mature mice Address for reprint requests and other correspondence: S. Welle, Univ. of Rochester Medical Center, 601 Elmwood Ave., Box 693, Rochester, NY 14642 (e-mail: stephen_welle@urmc.rochester.edu).

    PubMed Central

    Welle, Stephen; Cardillo, Andrew; Zanche, Michelle; Tawil, Rabi

    2009-01-01

    There is much interest in developing anti-myostatin agents to reverse or prevent muscle atrophy in adults, so it is important to characterize the effects of reducing myostatin activity after normal muscle development. For assessment of the effect of loss of myostatin signaling on gene expression in muscle, RNA from mice with postdevelopmental myostatin knockout was analyzed with oligonucleotide microarrays. Myostatin was undetectable in muscle within 2 wk after Cre recombinase activation in 4-month-old male mice with floxed myostatin genes. Three months after myostatin depletion, muscle mass had increased 26% (vs. 2% after induction of Cre activity in mice with normal myostatin genes), at which time the expression of several hundred genes differed in knockout and control mice at nominal P < 0.01. In contrast to previously reported effects of constitutive myostatin knockout, postdevelopmental knockout did not downregulate expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins involved in energy metabolism. Several collagen genes were expressed at 20–50% lower levels in the myostatin-deficient muscles, which had ∼25% less collagen than normal muscles as reflected by hydroxyproline content. Most of the other genes affected by myostatin depletion have not been previously linked to myostatin signaling. Gene set enrichment analysis suggested that Smads are not the only transcription factors with reduced activity after myostatin depletion. These data reinforce other evidence that myostatin regulates collagen production in muscle and demonstrate that many of the previously reported effects of constitutive myostatin deficiency do not occur when myostatin is knocked out in mature muscles. PMID:19509079

  2. Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2.

    PubMed

    Szláma, György; Trexler, Mária; Patthy, László

    2013-08-01

    Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677-23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2. PMID:23829672

  3. 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. PMID:27148972

  4. 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

  5. Cytotoxic Aggregation and Amyloid Formation by the Myostatin Precursor Protein

    PubMed Central

    Starck, Carlene S.; Sutherland-Smith, Andrew J.

    2010-01-01

    Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer's and Parkinson's diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer's amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by β-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis. PMID:20161792

  6. 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.

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

    PubMed

    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. PMID:20036643

  8. 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.

  9. 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.

    PubMed

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

    2013-10-18

    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 IP3/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. PMID:24064350

  10. 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.

  11. Fibronectin-based scaffold domain proteins that bind myostatin: a patent evaluation of WO2014043344.

    PubMed

    Walker, Ryan G; Thompson, Thomas B

    2015-05-01

    Muscular dystrophies (MD) are commonly characterized by progressive loss of muscle mass and function. It is hypothesized that therapeutic blockade of the TGF-β ligand myostatin, a negative regulator of muscle mass, will stimulate muscle growth and restore muscle function. Although many anti-myostatin targets are currently being pursued in the clinical setting, the efficacies of the tested molecules have shown mixed results. The patent WO2014043344 describes a novel approach for myostatin inhibition using a modified fibronectin type III domain that could potentially be used to treat MD and other muscle-related pathologies. PMID:25632990

  12. Muscle protein synthesis, mTORC1/MAPK/Hippo signaling, and capillary density are altered by blocking of myostatin and activins.

    PubMed

    Hulmi, Juha J; Oliveira, Bernardo M; Silvennoinen, Mika; Hoogaars, Willem M H; Ma, Hongqiang; Pierre, Philippe; Pasternack, Arja; Kainulainen, Heikki; Ritvos, Olli

    2013-01-01

    Loss of muscle mass and function occurs in various diseases. Myostatin blocking can attenuate muscle loss, but downstream signaling is not well known. Therefore, to elucidate associated signaling pathways, we used the soluble activin receptor IIb (sActRIIB-Fc) to block myostatin and activins in mice. Within 2 wk, the treatment rapidly increased muscle size as expected but decreased capillary density per area. sActRIIB-Fc increased muscle protein synthesis 1-2 days after the treatment correlating with enhanced mTORC1 signaling (phosphorylated rpS6 and S6K1, r = 0.8). Concurrently, increased REDD1 and eIF2Bε protein contents and phosphorylation of 4E-BP1 and AMPK was observed. In contrast, proangiogenic MAPK signaling and VEGF-A protein decreased. Hippo signaling has been characterized recently as a regulator of organ size and an important regulator of myogenesis in vitro. The phosphorylation of YAP (Yes-associated protein), a readout of activated Hippo signaling, increased after short- and longer-term myostatin and activin blocking and in exercised muscle. Moreover, dystrophic mdx mice had elevated phosphorylated and especially total YAP protein content. These results show that the blocking of myostatin and activins induce rapid skeletal muscle growth. This is associated with increased protein synthesis and mTORC1 signaling but decreased capillary density and proangiogenic signaling. It is also shown for the first time that Hippo signaling is activated in skeletal muscle after myostatin blocking and exercise and also in dystrophic muscle. This suggests that Hippo signaling may have a role in skeletal muscle in various circumstances. PMID:23115080

  13. 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

    2015-01-01

    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. PMID:26305245

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

    PubMed Central

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

    2015-01-01

    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. PMID:26305245

  15. 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

  16. Alternative binding modes identified for growth and differentiation factor-associated serum protein (GASP) family antagonism of myostatin.

    PubMed

    Walker, Ryan G; Angerman, Elizabeth B; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B

    2015-03-20

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  17. Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin*

    PubMed Central

    Walker, Ryan G.; Angerman, Elizabeth B.; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B.

    2015-01-01

    Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

  18. Protein syntehsis during soybean seed maturation

    SciTech Connect

    Rosenberg, L.A.; Rinne, R.W.

    1987-04-01

    The authors previous work has demonstrated that physiological and biochemical changes specifically associated with soybean seed maturation can be separated from events associated with seed development. The objective of this study was to determine if soybean seed metabolism is altered during maturation drying at the level of protein synthesis. Seed harvested 35 days after flowering (0% seedling growth) were induced to mature (100% seedling growth) through controlled dehydration. Proteins labeled with (/sup 35/S)-methionine were extracted and analyzed by 1-D PAGE coupled with autoradiography and densitometry. Results show a 31 kD and 128 kD polypeptide synthesized de novo during dehydration and precocious maturation. The same two polypeptides are synthesized during natural dehydration and maturation (>60 days after flowering). Furthermore, these polypeptides persist during rehydration and germination of both precociously and naturally matured seed, but specifically disappear during early seedling growth. The authors are currently investigating the role of protein synthesis during soybean seed maturation and if it is required for establishment of a soybean seedling.

  19. Protein Supplementation Increases Postexercise Plasma Myostatin Concentration After 8 Weeks of Resistance Training in Young Physically Active Subjects

    PubMed Central

    Pacelli, Quirico F.; Neri, Marco; Toniolo, Luana; Cancellara, Pasqua; Canato, Marta; Moro, Tatiana; Quadrelli, Marco; Morra, Aldo; Faggian, Diego; Plebani, Mario; Bianco, Antonino; Reggiani, Carlo

    2015-01-01

    Abstract Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a counterintuitive positive correlation between MSTN and muscle mass (MM). Our aim was to investigate the influence of 2 months of resistance training (RT) and diets with different protein contents on plasma MSTN, interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and insulin-like growth factor 1 (IGF-1). Eighteen healthy volunteers were randomly divided in two groups: high protein (HP) and normal protein (NP) groups. Different protein diet contents were 1.8 and 0.85 g of protein·kg bw−1·day−1 for HP and NP, respectively. Subjects underwent 8 weeks of standardized progressive RT. MSTN, IGF-1, IL-1β, IL-6, and TNF-α were analyzed before and after the first and the last training sessions. Lean body mass, MM, upper-limb muscle area, and strength were measured. Plasma MSTN showed a significant increase (P<.001) after the last training in the HP group compared with NP group and with starting value. IGF-1 plasma concentration showed a positive correlation with MSTN in HP after the last training (r2=0.6456; P=.0295). No significant differences were found between NP and HP for IL-1β, IL-6, TNF-α, and strength and MM or area. These findings suggest a “paradoxical” postexercise increase of plasma MSTN after 8 weeks of RT and HP diets. This MSTN elevation correlates positively with IGF-1 plasma level. This double increase of opposite (catabolic/anabolic) mediators could explain the substantial overlapping of MM increases in the two groups. PMID:25133710

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

    PubMed

    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

  1. 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.

    PubMed

    Kamanga-Sollo, E; Pampusch, M S; White, M E; Hathaway, M R; Dayton, W R

    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-beta1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-beta1 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-beta1 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-beta1 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-beta1 or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-beta1 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. PMID:16214131

  2. 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.

  3. Myostatin and the skeletal muscle atrophy and hypertrophy signaling pathways.

    PubMed

    Rodriguez, J; Vernus, B; Chelh, I; Cassar-Malek, I; Gabillard, J C; Hadj Sassi, A; Seiliez, I; Picard, B; Bonnieu, A

    2014-11-01

    Myostatin, a member of the transforming growth factor-β superfamily, is a potent negative regulator of skeletal muscle growth and is conserved in many species, from rodents to humans. Myostatin inactivation can induce skeletal muscle hypertrophy, while its overexpression or systemic administration causes muscle atrophy. As it represents a potential target for stimulating muscle growth and/or preventing muscle wasting, myostatin regulation and functions in the control of muscle mass have been extensively studied. A wealth of data strongly suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression. Moreover, myostatin plays a central role in integrating/mediating anabolic and catabolic responses. Myostatin negatively regulates the activity of the Akt pathway, which promotes protein synthesis, and increases the activity of the ubiquitin-proteasome system to induce atrophy. Several new studies have brought new information on how myostatin may affect both ribosomal biogenesis and translation efficiency of specific mRNA subclasses. In addition, although myostatin has been identified as a modulator of the major catabolic pathways, including the ubiquitin-proteasome and the autophagy-lysosome systems, the underlying mechanisms are only partially understood. The goal of this review is to highlight outstanding questions about myostatin-mediated regulation of the anabolic and catabolic signaling pathways in skeletal muscle. Particular emphasis has been placed on (1) the cross-regulation between myostatin, the growth-promoting pathways and the proteolytic systems; (2) how myostatin inhibition leads to muscle hypertrophy; and (3) the regulation of translation by myostatin. PMID:25080109

  4. Expression pattern of myostatin in gastrocnemius muscle of rats after sciatic nerve crush injury.

    PubMed

    Liu, Mei; Zhang, Donglei; Shao, Chenxin; Liu, Jie; Ding, Fei; Gu, Xiaosong

    2007-05-01

    Myostatin is a strong inhibitor of skeletal muscle growth. The purpose of this study was to investigate myostatin expression profiles during denervation-induced muscle atrophy in order to understand the relationship between myostatin expression and muscle atrophy. We constructed a sciatic nerve crush model, undertook morphometric analyses of rat gastrocnemius muscle to evaluate the degree of muscle atrophy, and utilized a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis to measure myostatin mRNA and protein expression levels, respectively, in the gastrocnemius at different time-points after nerve injury. Muscle atrophy changed in a parabola-like manner from day 1 to day 28 after nerve injury, with a maximum value at day 14. During this time, myostatin expression changed in the reverse manner, with myostatin mRNA or protein expression gradually increasing from days 1-14, and then gradually declining to day 28, when the normal level was reached. Statistical analyses further provided evidence for a significant negative linear correlation between myostatin expression and muscle atrophy within a 28-day period after nerve injury. Our study thus describes the expression pattern of myostatin in response to a specific type of muscle atrophy and raises the possibility of developing myostatin as a therapeutic target for future clinical applications. PMID:17326119

  5. 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

  6. 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.

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

    PubMed

    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 Myostatin(Ln/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

  8. 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

  9. The effect of myostatin silencing by lentiviral-mediated RNA interference on goat fetal fibroblasts.

    PubMed

    Lu, Jian; Wei, Caihong; Zhang, Xiaoning; Xu, Lingyang; Zhang, Shifang; Liu, Jiasen; Cao, Jiaxue; Zhao, Fuping; Zhang, Li; Li, Bichun; Du, Lixin

    2013-06-01

    Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may promote muscle growth, we used RNA interference mediated by a lentiviral vector to knockdown myostatin in goat fetal fibroblast cells. We also investigated the expression changes in relevant myogenic regulatory factors (MRFs) and adipogenic regulatory factors in the absence of myostatin in goat fetal fibroblasts. Quantitative RT-PCR revealed that myostatin transcripts were significantly reduced by 75 % (P < 0.01). Western blot showed that myostatin protein expression was reduced by 95 % (P < 0.01). We also found that the mRNA expression of activin receptor IIB (ACVR2B) significantly increased by 350 % (P < 0.01), and p21 increased 172 % (P < 0.01). Furthermore, myostatin inhibition decreased Myf5 and increased MEF2C mRNA expression in goat fetal fibroblasts, suggesting that myostatin regulates MRFs differently in fibroblasts compared to muscle. In addition, the expression of adipocyte marker genes peroxisome proliferator-activated receptor (PPAR) γ and leptin, but not CCAAT/enhance-binding protein (C/EBP) α and C/EBPβ, were upregulated at the transcript level after myostatin silencing. These results suggest that we have generated a novel way to block myostatin in vitro, which could be used to improve livestock meat production and gene therapy of musculoskeletal diseases. This also suggests that myostatin plays a negative role in regulating the expression of adipogenesis related genes in goat fetal fibroblasts. PMID:23604693

  10. Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells.

    PubMed

    Zhou, Zheng-Rong; Zhong, Bu-Shuai; Jia, Ruo-Xin; Wan, Yong-Jie; Zhang, Yan-Li; Fan, Yi-Xuan; Wang, Li-Zhong; You, Ji-Hao; Wang, Zi-Yu; Wang, Feng

    2013-01-15

    Myostatin, a member of the transforming growth factor-β family, acts as a negative regulator of skeletal muscle mass. In this study, myostatin-targeted caprine fibroblasts were obtained and subjected to SCNT to determine whether myostatin-knockout goats could be created. Fibroblasts from a 2-mo-old goat were transfected with a myostatin-targeted vector to prepare transgenic donor cells for nuclear transfer. After serum-starvation (for synchronization of the cell cycle), the percentage of transgenic fibroblasts in the G(0)/G(1) phase increased (66.2% vs. 82.9%; P < 0.05) compared with that in the control group, whereas the apoptosis rate and mitochondrial membrane potential were unaffected (P > 0.05). There were no significant differences between in vivo- and in vitro-matured oocytes as recipient cytoplasts for rates of fusion (86.5% vs. 78.4%), pregnancy (21.6% vs. 16.7%), or kidding (2.7% vs. 0%). One female kid from an in vivo-matured oocyte was born, but died a few hours later. Microsatellite analysis and polymerase chain reaction identification confirmed that this kid was genetically identical to the donor cells. Based on Western blot analysis, myostatin of the cloned kid was not expressed compared with that of nontransgenic kids. In conclusion, SCNT using myostatin-targeted 2-mo-old goat fibroblasts as donors has potential as a method for producing myostatin-targeted goats. PMID:23174778

  11. 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. PMID:16287620

  12. Myostatin gene inactivation prevents skeletal muscle wasting in cancer.

    PubMed

    Gallot, Yann S; Durieux, Anne-Cécile; Castells, Josiane; Desgeorges, Marine M; Vernus, Barbara; Plantureux, Léa; Rémond, Didier; Jahnke, Vanessa E; Lefai, Etienne; Dardevet, Dominique; Nemoz, Georges; Schaeffer, Laurent; Bonnieu, Anne; Freyssenet, Damien G

    2014-12-15

    Cachexia is a muscle-wasting syndrome that contributes significantly to morbidity and mortality of many patients with advanced cancers. However, little is understood about how the severe loss of skeletal muscle characterizing this condition occurs. In the current study, we tested the hypothesis that the muscle protein myostatin is involved in mediating the pathogenesis of cachexia-induced muscle wasting in tumor-bearing mice. Myostatin gene inactivation prevented the severe loss of skeletal muscle mass induced in mice engrafted with Lewis lung carcinoma (LLC) cells or in Apc(Min) (/+) mice, an established model of colorectal cancer and cachexia. Mechanistically, myostatin loss attenuated the activation of muscle fiber proteolytic pathways by inhibiting the expression of atrophy-related genes, MuRF1 and MAFbx/Atrogin-1, along with autophagy-related genes. Notably, myostatin loss also impeded the growth of LLC tumors, the number and the size of intestinal polyps in Apc(Min) (/+) mice, thus strongly increasing survival in both models. Gene expression analysis in the LLC model showed this phenotype to be associated with reduced expression of genes involved in tumor metabolism, activin signaling, and apoptosis. Taken together, our results reveal an essential role for myostatin in the pathogenesis of cancer cachexia and link this condition to tumor growth, with implications for furthering understanding of cancer as a systemic disease. PMID:25336187

  13. Myostatin inhibition by a follistatin-derived peptide ameliorates the pathophysiology of muscular dystrophy model mice

    PubMed Central

    Tsuchida, K

    2008-01-01

    Summary 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-β 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. PMID:19108572

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

    PubMed

    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

  15. 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

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

    PubMed

    Li, Naisi; Yang, Qiyuan; Walker, Ryan G; Thompson, Thomas B; Du, Min; Rodgers, Buel D

    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

  17. 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.

  18. Myostatin/activin pathway antagonism: molecular basis and therapeutic potential.

    PubMed

    Han, H Q; Zhou, Xiaolan; Mitch, William E; Goldberg, Alfred L

    2013-10-01

    Muscle wasting is associated with a wide range of catabolic diseases. This debilitating loss of muscle mass and functional capacity reduces the quality of life and increases the risks of morbidity and mortality. Major progress has been made in understanding the biochemical mechanisms and signaling pathways regulating muscle protein balance under normal conditions and the enhanced protein loss in atrophying muscles. It is now clear that activation of myostatin/activin signaling is critical in triggering the accelerated muscle catabolism that causes muscle loss in multiple disease states. Binding of myostatin and activin to the ActRIIB receptor complex on muscle cell membrane leads to activation of Smad2/3-mediated transcription, which in turn stimulates FoxO-dependent transcription and enhanced muscle protein breakdown via ubiquitin-proteasome system and autophagy. In addition, Smad activation inhibits muscle protein synthesis by suppressing Akt signaling. Pharmacological blockade of the myostatin/activin-ActRIIB pathway has been shown to prevent or reverse the loss of muscle mass and strength in various disease models including cancer cachexia and renal failure. Moreover, it can markedly prolong the lifespan of animals with cancer-associated muscle loss. Furthermore, inhibiting myostatin/activin actions also improves insulin sensitivity, reduces excessive adiposity, attenuates systemic inflammation, and accelerates bone fracture healing in disease models. Based on these exciting advances, the potential therapeutic benefits of myostatin/activin antagonism are now being tested in multiple clinical settings. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. PMID:23721881

  19. 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

  20. Recovery and identification of mature enamel proteins in ancient teeth.

    PubMed

    Porto, Isabel M; Laure, Helen J; Tykot, Robert H; de Sousa, Frederico B; Rosa, Jose C; Gerlach, Raquel F

    2011-12-01

    Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science. PMID:22243232

  1. Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

    PubMed

    Lee, Sang Beum; Kim, Jeong Hwan; Jin, Deuk-Hee; Jin, Hyung-Joo; Kim, Yong Soo

    2016-01-01

    Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth, and its activity is suppressed by MSTN propeptide (MSTNpro), the N-terminal part of MSTN precursor cleaved during post-translational MSTN processing. The current study examined which region of flatfish (Paralichthys olivaceus) MSTN-1 propeptide (MSTN1pro) is critical for MSTN inhibition. Six different truncated forms of MSTN1pro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in Escherichia coli, and partially purified by an affinity chromatography for MSTN-inhibitory activity examination. Peptides covering different regions of flatfish MSTN1pro were also synthesized for MSTN-inhibitory activity examination. A MBP-fused MSTN1pro region consisting of residues 45-100 had the same MSTN-inhibitory potency as the full sequence flatfish MSTN1pro (residues 23-265), indicating that the region of flatfish MSTN1pro consisting of residues 45-100 is sufficient to maintain the full MSTN-inhibitory capacity. A MBP-fused MSTN1pro region consisting of residues 45-80 (Pro45-80) also showed MSTN-inhibitory activity with a lower potency, and the Pro45-80 demonstrated its MSTN binding capacity in a pull-down assay, indicating that the MSTN-inhibitory capacity of Pro45-80 is due to its binding to MSTN. Flatfish MSTN1pro synthetic peptides covering residues 45-65, 45-70, and 45-80 demonstrated MSTN-inhibitory activities, but not the synthetic peptide covering residues 45-54, indicating that residues 45-65 of flatfish MSTN1pro are essential for MSTN inhibition. In conclusion, current study show that like the mammalian MSTNpro, the MSTN-inhibitory region of flatfish MSTN1pro resides near its N-terminus, and imply that smaller sizes of MSTNpro can be effectively used in various applications designed for MSTN inhibition. PMID:26827850

  2. Genetics Home Reference: myostatin-related muscle hypertrophy

    MedlinePlus

    ... Conditions myostatin-related muscle hypertrophy myostatin-related muscle hypertrophy Enable Javascript to view the expand/collapse boxes. ... Open All Close All Description Myostatin-related muscle hypertrophy is a rare condition characterized by reduced body ...

  3. Myostatin expression during development and chronic stress in zebrafish (Danio rerio).

    PubMed

    Vianello, S; Brazzoduro, L; Dalla Valle, L; Belvedere, P; Colombo, L

    2003-01-01

    Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle mass in mammals. We have studied myostatin expression during embryonic and post-hatching development in zebrafish by semiquantitative RT-PCR. The transcript is present in just-fertilized eggs and declines at 8 h post-fertilization (hpf), suggesting a maternal origin. A secondary rise occurs at 16 hpf, indicating the onset of embryonic transcription at the time of muscle cell differentiation. The level of myostatin mRNA decreases slightly at 24 hpf, when somitogenesis is almost concluded, and rises again at and after hatching, during the period of limited muscle hyperplastic growth that is typical of slow-growing, small fish. In the adult muscle, we found the highest expression of myostatin mRNA and protein, which were detectable by Northern and Western blot analyses respectively. Although only the precursor protein form was revealed in the adult lateral muscle, we demonstrated that zebrafish myostatin is proteolytically processed and secreted in cultured cells, as is its mammalian counterpart. These results suggest that myostatin may play an important regulatory role during myogenesis and muscle growth in fish, as it does in mammals. In chronically stressed fish, grown from 16 days post-fertilization to adulthood in an overcrowded environment, we observed both depression of body growth and a diminished level of myostatin mRNA in the adult muscle, as compared with controls. We propose that chronic stunting in fish brings about a general depression of muscle protein synthesis which does not spare myostatin. PMID:12525249

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

    PubMed

    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

  5. 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. PMID:16873457

  6. Inhibition of Myostatin Signaling through Notch Activation following Acute Resistance Exercise

    PubMed Central

    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 (R2 = 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. PMID:23844238

  7. Transforming growth factor-beta1 upregulates myostatin expression in mouse C2C12 myoblasts.

    PubMed

    Budasz-Rwiderska, M; Jank, M; Motyl, T

    2005-06-01

    Myostatin (MSTN) and transforming growth factor-beta1 (TGF-beta1) belong to the same TGF-beta superfamily of proteins. They are involved in regulation of skeletal muscle growth and development as well as muscle catabolism. The aim of the present study was to investigate the relationship between MSTN and TGF-beta1 expression in proliferating and differentiating mouse C2C12 myoblasts cultured in normal and catabolic conditions and to evaluate the effect of exogenous TGF-beta1 as well as "knock down" of TGF-beta1 receptor type II on MSTN expression in proliferating and differentiating myogenic cells. The direct effect of TGF-beta1 on myostatin was also examined. Myostatin expression increased gradually with cell confluency in proliferating cultures, while the level of TGF-beta1, detected in the form of a 100 kDa small latent complex diminished. Myostatin expression was accompanied by a partial cell cycle arrest. Three forms of myostatin were found: a 52 kDa precursor, a 40 kDa latency associated propeptide, and a 26 kDa active peptide. A decrease in myostatin and TGF-beta1 levels was observed during the first three days of differentiation, which was subsequently followed by significant increase of their expression during next three to four days of differentiation. Catabolic state induced by dexamethasone significantly increased the level of all forms of myostatin as well as latent (100 kDa) and active (25 kDa) forms of TGF-beta1 in differentiating myoblasts in a dose dependent manner. Exogenous TGF-beta1 (2 ng/ml) significantly increased myostatin levels both in proliferating and differentiating C2C12 myoblasts, whereas silencing of the TGF-beta1 receptor II gene significantly lowered myostatin level in examined cells. The presented results indicate that TGF-beta1 may control myostatin-related regulation of myogenesis through up-regulation of myostatin, predominantly in the course of terminal differentiation and glucocorticoid-dependent catabolic stimulation. PMID

  8. Myostatin inhibits proliferation and insulin-stimulated glucose uptake in mouse liver cells.

    PubMed

    Watts, Rani; Ghozlan, Mostafa; Hughey, Curtis C; Johnsen, Virginia L; Shearer, Jane; Hittel, Dustin S

    2014-06-01

    Although myostatin functions primarily as a negative regulator of skeletal muscle growth and development, accumulating biological and epidemiological evidence indicates an important contributing role in liver disease. In this study, we demonstrate that myostatin suppresses the proliferation of mouse Hepa-1c1c7 murine-derived liver cells (50%; p < 0.001) in part by reducing the expression of the cyclins and cyclin-dependent kinases that elicit G1-S phase transition of the cell cycle (p < 0.001). Furthermore, real-time PCR-based quantification of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (Malat1), recently identified as a myostatin-responsive transcript in skeletal muscle, revealed a significant downregulation (25% and 50%, respectively; p < 0.05) in the livers of myostatin-treated mice and liver cells. The importance of Malat1 in liver cell proliferation was confirmed via arrested liver cell proliferation (p < 0.05) in response to partial Malat1 siRNA-mediated knockdown. Myostatin also significantly blunted insulin-stimulated glucose uptake and Akt phosphorylation in liver cells while increasing the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS), a protein that is essential for cancer cell proliferation and insulin-stimulated glucose transport. Together, these findings reveal a plausible mechanism by which circulating myostatin contributes to the diminished regenerative capacity of the liver and diseases characterized by liver insulin resistance. PMID:24882465

  9. Propeptide-mediated inhibition of myostatin increases muscle mass through inhibiting proteolytic pathways in aged mice.

    PubMed

    Collins-Hooper, Henry; Sartori, Roberta; Macharia, Raymond; Visanuvimol, Korntip; Foster, Keith; Matsakas, Antonios; Flasskamp, Hannah; Ray, Steve; Dash, Philip R; Sandri, Marco; Patel, Ketan

    2014-09-01

    Mammalian aging is accompanied by a progressive loss of skeletal muscle, a process called sarcopenia. Myostatin, a secreted member of the transforming growth factor-β family of signaling molecules, has been shown to be a potent inhibitor of muscle growth. Here, we examined whether muscle growth could be promoted in aged animals by antagonizing the activity of myostatin through the neutralizing activity of the myostatin propeptide. We show that a single injection of an AAV8 virus expressing the myostatin propeptide induced an increase in whole body weights and all muscles examined within 7 weeks of treatment. Our cellular studies demonstrate that muscle enlargement was due to selective fiber type hypertrophy, which was accompanied by a shift toward a glycolytic phenotype. Our molecular investigations elucidate the mechanism underpinning muscle hypertrophy by showing a decrease in the expression of key genes that control ubiquitin-mediated protein breakdown. Most importantly, we show that the hypertrophic muscle that develops as a consequence of myostatin propeptide in aged mice has normal contractile properties. We suggest that attenuating myostatin signaling could be a very attractive strategy to halt and possibly reverse age-related muscle loss. PMID:24414825

  10. 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.

  11. 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

  12. A myostatin and activin decoy receptor enhances bone formation in mice.

    PubMed

    Bialek, P; Parkington, J; Li, X; Gavin, D; Wallace, C; Zhang, J; Root, A; Yan, G; Warner, L; Seeherman, H J; Yaworsky, P J

    2014-03-01

    Myostatin is a member of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) super-family of secreted differentiation factors. Myostatin is a negative regulator of muscle mass as shown by increased muscle mass in myostatin deficient mice. Interestingly, these mice also exhibit increased bone mass suggesting that myostatin may also play a role in regulating bone mass. To investigate the role of myostatin in bone, young adult mice were administered with either a myostatin neutralizing antibody (Mstn-mAb), a soluble myostatin decoy receptor (ActRIIB-Fc) or vehicle. While both myostatin inhibitors increased muscle mass, only ActRIIB-Fc increased bone mass. Bone volume fraction (BV/TV), as determined by microCT, was increased by 132% and 27% in the distal femur and lumbar vertebrae, respectively. Histological evaluation demonstrated that increased BV/TV in both locations was attributed to increased trabecular thickness, trabecular number and bone formation rate. Increased BV/TV resulted in enhanced vertebral maximum compressive force compared to untreated animals. The fact that ActRIIB-Fc, but not Mstn-mAb, increased bone volume suggested that this soluble decoy receptor may be binding a ligand other than myostatin, that plays a role in regulating bone mass. This was confirmed by the significant increase in BV/TV in myostatin deficient mice treated with ActRIIB-Fc. Of the other known ActRIIB-Fc ligands, BMP3 has been identified as a negative regulator of bone mass. However, BMP3 deficient mice treated with ActRIIB-Fc showed similar increases in BV/TV as wild type (WT) littermates treated with ActRIIB-Fc. This result suggests that BMP3 neutralization is not the mechanism responsible for increased bone mass. The results of this study demonstrate that ActRIIB-Fc increases both muscle and bone mass in mice. Therefore, a therapeutic that has this dual activity represents a potential approach for the treatment of frailty. PMID:24333131

  13. The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy.

    PubMed

    Ohsawa, Yutaka; Takayama, Kentaro; Nishimatsu, Shin-ichiro; Okada, Tadashi; Fujino, Masahiro; Fukai, Yuta; Murakami, Tatsufumi; Hagiwara, Hiroki; Itoh, Fumiko; Tsuchida, Kunihiro; Hayashi, Yoshio; Sunada, Yoshihide

    2015-01-01

    Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings. PMID:26226340

  14. The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy

    PubMed Central

    Ohsawa, Yutaka; Takayama, Kentaro; Nishimatsu, Shin-ichiro; Okada, Tadashi; Fujino, Masahiro; Fukai, Yuta; Murakami, Tatsufumi; Hagiwara, Hiroki; Itoh, Fumiko; Tsuchida, Kunihiro; Hayashi, Yoshio; Sunada, Yoshihide

    2015-01-01

    Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings. PMID:26226340

  15. Factors Associated with the Serum Myostatin Level in Patients Undergoing Peritoneal Dialysis: Potential Effects of Skeletal Muscle Mass and Vitamin D Receptor Activator Use.

    PubMed

    Yamada, Shunsuke; Tsuruya, Kazuhiko; Yoshida, Hisako; Tokumoto, Masanori; Ueki, Kenji; Ooboshi, Hiroaki; Kitazono, Takanari

    2016-07-01

    Myostatin is a member of the transforming growth factor-β family, which regulates synthesis and degradation of skeletal muscle proteins and is associated with the development of sarcopenia. It is up-regulated in the skeletal muscle of chronic kidney disease patients and is considered to be involved in the development of uremic sarcopenia. However, serum myostatin levels have rarely been determined, and the relationship between serum myostatin levels with clinical and metabolic factors remains unknown. This cross-sectional study investigated the association between serum myostatin level and clinical factors in 69 outpatients undergoing peritoneal dialysis. Serum myostatin level was determined by commercially available enzyme-linked immunosorbent assay (ELISA). Univariable and multivariable analysis were conducted to determine factors associated with serum myostatin levels. The factors included age, sex, diabetes mellitus, dialysis history, body mass index, residual kidney function, peritoneal dialysate volume, serum biochemistries, and the use of vitamin D receptor activators (VDRAs). Mean serum myostatin level was 7.59 ± 3.37 ng/mL. There was no association between serum myostatin level and residual kidney function. Serum myostatin levels were significantly and positively associated with lean body mass measured by the creatinine kinetic method and negatively associated with the use of VDRAs after adjustment for potential confounding factors. Our study indicated that serum myostatin levels are associated with skeletal muscle mass and are lower in patients treated with VDRAs. Further studies are necessary to determine the significance of measuring serum myostatin level in patients undergoing peritoneal dialysis. PMID:26895008

  16. Myostatin-induced inhibition of the long noncoding RNA Malat1 is associated with decreased myogenesis.

    PubMed

    Watts, Rani; Johnsen, Virginia L; Shearer, Jane; Hittel, Dustin S

    2013-05-15

    Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily of secreted proteins, is a potent negative regulator of myogenesis. Free myostatin induces the phosphorylation of the Smad family of transcription factors, which, in turn, regulates gene expression, via the canonical TGF-β signaling pathway. There is, however, emerging evidence that myostatin can regulate gene expression independent of Smad signaling. As such, we acquired global gene expression data from the gastrocnemius muscle of C57BL/6 mice following a 6-day treatment with recombinant myostatin compared with vehicle-treated animals. Of the many differentially expressed genes, the myostatin-associated decrease (-11.20-fold; P < 0.05) in the noncoding metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was the most significant and the most intriguing because of numerous reports describing its novel role in regulating cell growth. We therefore sought to further characterize the role of Malat1 expression in skeletal muscle myogenesis. RT-PCR-based quantification of C2C12 and primary human skeletal muscle cells revealed a significant and persistent upregulation (4- to 7-fold; P < 0.05) of Malat1 mRNA during the differentiation of myoblasts into myotubes. Conversely, targeted knockdown of Malat1 using siRNA suppressed myoblast proliferation by arresting cell growth in the G(0)/G(1) phase. These results reveal Malat1 as novel downstream target of myostatin with a considerable ability to regulate myogenesis. The identification of new targets of myostatin will have important repercussions for regenerative biology through inhibition and/or reversal of muscle atrophy and wasting diseases. PMID:23485710

  17. Oligodendroglial maturation is dependent on intracellular protein shuttling.

    PubMed

    Göttle, Peter; Sabo, Jennifer K; Heinen, André; Venables, Gene; Torres, Klintsy; Tzekova, Nevena; Parras, Carlos M; Kremer, David; Hartung, Hans-Peter; Cate, Holly S; Küry, Patrick

    2015-01-21

    Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches. PMID:25609610

  18. Identification of the minimum peptide from mouse myostatin prodomain for human myostatin inhibition.

    PubMed

    Takayama, Kentaro; Noguchi, Yuri; Aoki, Shin; Takayama, Shota; Yoshida, Momoko; Asari, Tomo; Yakushiji, Fumika; Nishimatsu, Shin-ichiro; Ohsawa, Yutaka; Itoh, Fumiko; Negishi, Yoichi; Sunada, Yoshihide; Hayashi, Yoshio

    2015-02-12

    Myostatin, an endogenous negative regulator of skeletal muscle mass, is a therapeutic target for muscle atrophic disorders. Here, we identified minimum peptides 2 and 7 to effectively inhibit myostatin activity, which consist of 24 and 23 amino acids, respectively, derived from mouse myostatin prodomain. These peptides, which had the propensity to form α-helix structure, interacted to myostatin with KD values of 30-36 nM. Moreover, peptide 2 significantly increased muscle mass in Duchenne muscular dystrophy model mice. PMID:25569186

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  20. Oral administration of myostatin-specific recombinant Saccharomyces cerevisiae vaccine in rabbit.

    PubMed

    Liu, Zhongtian; Zhou, Gang; Ren, Chonghua; Xu, Kun; Yan, Qiang; Li, Xinyi; Zhang, Tingting; Zhang, Zhiying

    2016-04-29

    Yeast is considered as a simple and cost-effective host for protein expression, and our previous studies have proved that Saccharomyces cerevisiae can deliver recombinant protein and DNA into mouse dendritic cells and can further induce immune responses as novel vaccines. In order to know whether similar immune responses can be induced in rabbit by oral administration of such recombinant S. cerevisiae vaccine, we orally fed the rabbits with heat-inactivated myostatin-recombinant S. cerevisiae for 5 weeks, and then myostatin-specific antibody in serum was detected successfully by western blotting and ELISA assay. The rabbits treated with myostatin-recombinant S. cerevisiae vaccine grew faster and their muscles were much heavier than that of the control group. As a common experimental animal and a meat livestock with great economic value, rabbit was proved to be the second animal species that have been successfully orally immunized by recombinant S. cerevisiae vaccine after mice. PMID:27005809

  1. Hormone therapy and maximal eccentric exercise alters myostatin-related gene expression in postmenopausal women.

    PubMed

    Dieli-Conwright, Christina M; Spektor, Tanya M; Rice, Judd C; Sattler, Fred R; Schroeder, E Todd

    2012-05-01

    We sought to evaluate baseline mRNA values and changes in gene expression of myostatin-related factors in postmenopausal women taking hormone therapy (HT) and not taking HT after eccentric exercise. Fourteen postmenopausal women participated including 6 controls not using HT (59 ± 4 years, 63 ± 17 kg) and 8 women using HT (59 ± 4 years, 89 ± 24 kg). The participants performed 10 sets of 10 maximal eccentric repetitions of single-leg extension on a dynamometer. Muscle biopsies from the vastus lateralis were obtained from the exercised leg at baseline and 4 hours after the exercise bout. Gene expression was determined using reverse transcriptase polymerase chain reaction for myostatin, activin receptor IIb (ActRIIb), follistatin, follistatin-related gene (FLRG), follistatin-like-3 (FSTL3), and GDF serum-associated protein-1 (GASP-1). In response to the exercise bout, myostatin and ActRIIb significantly decreased (p < 0.05), and follistatin, FLRG, FSTL3, and GASP-1 significantly increased in both groups (p < 0.05). Significantly greater changes in gene expression of all genes occurred in the HT group than in the control group after the acute eccentric exercise bout (p < 0.05). These data suggest that postmenopausal women using HT express greater myostatin-related gene expression, which may reflect a mechanism by which estrogen influences the preservation of muscle mass. Further, postmenopausal women using HT experienced a profoundly greater myostatin-related response to maximal eccentric exercise. PMID:22395277

  2. 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. PMID:24848520

  3. Characterization of maturation-dependent extrinsic proteins of the rat sperm surface

    SciTech Connect

    Rifkin, J.M.; Olson, G.E.

    1985-05-01

    Mammalian spermatozoa must mature in the epididymis before they can fertilize an egg. It is known that modification of the protein composition of the sperm surface is an important part of the maturation process. In this paper, the authors present data on two related glycoproteins that can be extracted from mature but not immature spermatozoa. Cell surface radioiodination has shown that these proteins are on the sperm surface, and immunofluorescence microscopy, by use of monospecific antibodies to the proteins, has indicated that their localization is restricted to the periacrosomal region of the sperm head. The authors have also shown that in vitro, these proteins will bind to the identical region of immature sperm. Immunohistochemical localization of the proteins in the epididymis shows that they are produced and secreted by the cauda region. The significance of the addition of these proteins to the sperm surface in both maturation and fertilization is discussed.

  4. Dickkopf-Related Protein 1 Inhibits the WNT Signaling Pathway and Improves Pig Oocyte Maturation

    PubMed Central

    Spate, Lee D.; Brown, Alana N.; Redel, Bethany K.; Whitworth, Kristin M.; Murphy, Clifton N.; Prather, Randall S.

    2014-01-01

    The ability to mature oocytes in vitro provides a tool for creating embryos by parthenogenesis, fertilization, and cloning. Unfortunately the quality of oocytes matured in vitro falls behind that of in vivo matured oocytes. To address this difference, transcriptional profiling by deep sequencing was conducted on pig oocytes that were either matured in vitro or in vivo. Alignment of over 18 million reads identified 1,316 transcripts that were differentially represented. One pathway that was overrepresented in the oocytes matured in vitro was for Wingless-type MMTV integration site (WNT) signaling. In an attempt to inhibit the WNT pathway, Dickkopf-related protein 1 was added to the in vitro maturation medium. Addition of Dickkopf-related protein 1 improved the percentage of oocytes that matured to the metaphase II stage, increased the number of nuclei in the resulting blastocyst stage embryos, and reduced the amount of disheveled segment polarity protein 1 protein in oocytes. It is concluded that transcriptional profiling is a powerful method for detecting differences between in vitro and in vivo matured oocytes, and that the WNT signaling pathway is important for proper oocyte maturation. PMID:24739947

  5. 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

  6. 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.

  7. Paracrine and endocrine modes of myostatin action

    PubMed Central

    Lee, Yun-Sil; Huynh, Thanh V.

    2016-01-01

    Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit muscle mass. In adult animals, MSTN is made almost exclusively by skeletal muscle and circulates in the blood. A critical question is whether this circulating MSTN protein can enter the active pool to regulate muscle growth or whether all of the activity of MSTN results from locally produced protein. Here, we addressed this question in mice by using a Cdx2-Cre transgene in conjunction with a conditional Mstn-flox allele to generate mice in which Mstn was targeted in a regionally restricted manner. Specifically, we generated mosaic mice in which MSTN production was eliminated in posteriorly located muscles but not in anteriorly located muscles, resulting in mice in which circulating levels of MSTN were reduced roughly by half. Analysis of posteriorly located vs. anteriorly located muscles of these mice revealed clear differential effects indicative of an important paracrine role for MSTN in regulating muscle mass. Significant, albeit more subtle, effects consistent with an endocrine mode of MSTN action were also seen in these mice. These findings have important implications not only for the understanding of the physiological control of muscle mass but also for therapeutic strategies to target MSTN to treat patients with muscle loss. PMID:26769954

  8. 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. PMID:24072591

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Preliminary Investigation into a Potential Role for Myostatin and Its Receptor (ActRIIB) in Lean and Obese Horses and Ponies

    PubMed Central

    Morrison, Philippa K.; Bing, Chen; Harris, Patricia A.; Maltin, Charlotte A.; Grove-White, Dai; Argo, Caroline McG.

    2014-01-01

    Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R2 = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies. PMID:25390640

  11. Preliminary investigation into a potential role for myostatin and its receptor (ActRIIB) in lean and obese horses and ponies.

    PubMed

    Morrison, Philippa K; Bing, Chen; Harris, Patricia A; Maltin, Charlotte A; Grove-White, Dai; Argo, Caroline McG

    2014-01-01

    Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R2 = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies. PMID:25390640

  12. 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

  13. Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

    PubMed

    Mirzaei, Reza; Saei, Azad; Torkashvand, Fatemeh; Azarian, Bahareh; Jalili, Ahmad; Noorbakhsh, Farshid; Vaziri, Behrouz; Hadjati, Jamshid

    2016-08-01

    Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation. PMID:26886282

  14. Myostatin deficiency but not anti-myostatin blockade induces marked proteomic changes in mouse skeletal muscle.

    PubMed

    Salzler, Robert R; Shah, Darshit; Doré, Anthony; Bauerlein, Roy; Miloscio, Lawrence; Latres, Esther; Papadopoulos, Nicholas J; Olson, William C; MacDonald, Douglas; Duan, Xunbao

    2016-07-01

    Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post-developmental Mstn blockade. Using high-resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn(-/-) ) and mice treated for 2-weeks with REGN1033, an anti-Mstn antibody. Relative to wild-type animals, Mstn(-/-) mice had a two-fold greater muscle mass and a >1.5-fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5-fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn(-/-) mice corroborates the mutiple physiological changes including slow-to-fast fiber type switch. Thus, the proteome-wide protein expression differs between Mstn(-/-) mice and mice subjected to specific Mstn blockade post-developmentally, providing molecular-level insights to inform mechanistic hypotheses to explain the observed functional differences. PMID:27214824

  15. The intriguing realm of protein biogenesis: Facing the green co-translational protein maturation networks.

    PubMed

    Breiman, Adina; Fieulaine, Sonia; Meinnel, Thierry; Giglione, Carmela

    2016-05-01

    The ribosome is the cell's protein-making factory, a huge protein-RNA complex, that is essential to life. Determining the high-resolution structures of the stable "core" of this factory was among the major breakthroughs of the past decades, and was awarded the Nobel Prize in 2009. Now that the mysteries of the ribosome appear to be more traceable, detailed understanding of the mechanisms that regulate protein synthesis includes not only the well-known steps of initiation, elongation, and termination but also the less comprehended features of the co-translational events associated with the maturation of the nascent chains. The ribosome is a platform for co-translational events affecting the nascent polypeptide, including protein modifications, folding, targeting to various cellular compartments for integration into membrane or translocation, and proteolysis. These events are orchestrated by ribosome-associated protein biogenesis factors (RPBs), a group of a dozen or more factors that act as the "welcoming committee" for the nascent chain as it emerges from the ribosome. In plants these factors have evolved to fit the specificity of different cellular compartments: cytoplasm, mitochondria and chloroplast. This review focuses on the current state of knowledge of these factors and their interaction around the exit tunnel of dedicated ribosomes. Particular attention has been accorded to the plant system, highlighting the similarities and differences with other organisms. PMID:26555180

  16. Proteomic analysis and candidate allergenic proteins in Populus deltoides CL. "2KEN8" mature pollen.

    PubMed

    Zhang, Jin; Wu, Li-Shuan; Fan, Wei; Zhang, Xiao-Ling; Jia, Hui-Xia; Li, Yu; Yin, Ya-Fang; Hu, Jian-Jun; Lu, Meng-Zhu

    2015-01-01

    Proteomic analysis was used to generate a map of Populus deltoides CL. "2KEN8" mature pollen proteins. By applying 2-D electrophoresis, we resolved 403 protein spots from mature pollen. Using the matrix-assisted laser desorption/ionization time time-of-flight/time-of-flight tandem mass spectrometry method, we identified 178 distinct proteins from 218 protein spots expressed in mature pollen. Moreover, out of these, 28 proteins were identified as putative allergens. The expression patterns of these putative allergen genes indicate that several of these genes are highly expressed in pollen. In addition, the members of profilin allergen family were analyzed and their expression patterns were compared with their homologous genes in Arabidopsis and rice. Knowledge of these identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with poplar pollen allergy. PMID:26284084

  17. Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

    PubMed

    Song, Hye-Won; Cauffman, Karen; Chan, Agnes P; Zhou, Yi; King, Mary Lou; Etkin, Laurence D; Kloc, Malgorzata

    2007-07-01

    The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs. PMID:17309605

  18. 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

  19. Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion.

    PubMed

    Duplay, P; Hofnung, M

    1988-10-01

    Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm. PMID:3049532

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

    PubMed

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

    2003-04-01

    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. PMID:12670177

  1. 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

  2. NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse

    PubMed Central

    PENG, Hui; LIN, Xiujiao; LIU, Fang; WANG, Cheng; ZHANG, Wenchang

    2015-01-01

    Nlrp9a, Nlrp9b and Nlrp9c are preferentially expressed in oocytes and early embryos in the mouse. Simultaneous genetic ablation of Nlrp9a and Nlrp9c does not affect early embryonic development, but the function of Nlrp9b in the process of oocyte maturation and embryonic development has not been elucidated. Here we show that both Nlrp9b mRNA and its protein are expressed in ovaries and the small intestine. Moreover, the NLRP9B protein was restricted to oocytes in the ovary and declined with oocyte aging. After ovulation and fertilization, NLRP9B protein was found in preimplantation embryos. Confocal microscopy demonstrated that it was mainly localized in the cytoplasm in the oocytes and blastomeres. Thus, this protein might play a role in oocyte maturation and early embryonic development. However, knockdown of Nlrp9b expression in GV-stage oocytes using RNA interference did not affect oocyte maturation or subsequent parthenogenetic development after Nlrp9b-deficient oocytes were activated. Furthermore, Nlrp9b knockdown zygotes could reach the blastocyst stage after being cultured for 3.5 days in vitro. These results provide the first evidence that the NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse. PMID:26411641

  3. 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.

  4. 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.

  5. A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species.

    PubMed

    Shin, Sangsu; Song, Yan; Ahn, Jinsoo; Kim, Eunsoo; Chen, Paula; Yang, Shujin; Suh, Yeunsu; Lee, Kichoon

    2015-11-15

    Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species. PMID:26354750

  6. Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

    PubMed

    Wazen, Rima M; Viegas-Costa, Luiz C; Fouillen, Aurélien; Moffatt, Pierre; Adair-Kirk, Tracy L; Senior, Robert M; Nanci, Antonio

    2016-01-01

    The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation. PMID:26956061

  7. 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

  8. 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.

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

    PubMed Central

    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. PMID:27089221

  10. Role for a Novel Usher Protein Complex in Hair Cell Synaptic Maturation

    PubMed Central

    Zallocchi, Marisa; Meehan, Daniel T.; Delimont, Duane; Rutledge, Joseph; Gratton, Michael Anne; Flannery, John; Cosgrove, Dominic

    2012-01-01

    The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1−/− mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzerav3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well. PMID:22363448

  11. 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

    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

  12. 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

  13. G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

    PubMed

    Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

    2015-01-15

    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. PMID:25485460

  14. A proposed nomenclature consensus for the myostatin gene family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since its discovery in 1997, myostatin and its negative effects on skeletal muscle mass have understandably captivated many biomedical, agricultural and comparative biologists as the gains in muscle mass associated with the myostatin null phenotype have never been reproduced by the administration of...

  15. 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.

  16. Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.

    PubMed

    Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

    2016-08-01

    Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. PMID:27215607

  17. Allopregnanolone increases mature excitatory synapses along dendrites via protein kinase A signaling.

    PubMed

    Shimizu, H; Ishizuka, Y; Yamazaki, H; Shirao, T

    2015-10-01

    Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is synthesized in both the periphery and central nervous system and is known to be a potent positive allosteric modulator of the GABAA receptor. Because APα was suggested to improve the symptoms of depression and Alzheimer's disease (AD), which involve synaptic dysfunction and loss, we examined whether APα affects excitatory synapses. Drebrin, which is an actin-binding protein, forms a unique stable actin structure in dendritic spines, and drebrin levels correlate positively with cognitive levels in AD and mild cognitive impairment. We investigated whether APα increases excitatory synapse density along dendrites of mature hippocampal neurons using drebrin-imaging-based evaluation of mature synapses. We prepared primary cultures of hippocampal neurons and either transfected them with GFP or immunostained them against drebrin. Morphological analysis of GFP-transfected neurons revealed that a 24-h exposure to 0.3 or 1 μM APα significantly increased dendritic spine density without any morphological changes to spines. Drebrin cluster density was also increased by 0.3 and 1 μM APα. The protein kinase A (PKA) inhibitor H-89 inhibited the APα-induced increase in drebrin cluster density. These data demonstrate that APα increases mature excitatory synapses via activation of PKA. Therefore, the PKA-cAMP response element-binding protein (CREB) signaling pathway is likely to be involved in the APα-induced increase of mature excitatory synapses. Another possibility is that the PKA-dependent increase in AMPA receptors at dendritic spines mediates the APα function. In conclusion, our study indicates that APα may improve neuropsychiatric disorder outcomes via increasing the numbers of mature excitatory synapses. PMID:26241343

  18. 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

  19. Myostatin stimulates, not inihibits, C2C12 myoblast proliferation.

    PubMed

    Rodgers, Buel D; Wiedeback, Benjamin D; Hoversten, Knut E; Jackson, Melissa F; Walker, Ryan G; Thompson, Thomas B

    2014-03-01

    The immortal C2C12 cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary myosatellite cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary myosatellite cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary myosatellite cells and recombinant myostatin generated in eukaryotes. PMID:24424069

  20. Embryonic poly(A)-binding protein (ePAB) phosphorylation is required for Xenopus oocyte maturation.

    PubMed

    Friend, Kyle; Brook, Matthew; Bezirci, F Betül; Sheets, Michael D; Gray, Nicola K; Seli, Emre

    2012-07-01

    Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP and PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In the present study we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation, but not for ePAB's inherent ability to promote translation. Our results provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo. PMID:22497250

  1. Effects of heat shock protein gp96 on human dendritic cell maturation and CTL expansion.

    PubMed

    Zhang, Yuxia; Zan, Yanlu; Shan, Ming; Liu, Changmei; Shi, Ming; Li, Wei; Zhang, Zhixin; Liu, Na; Wang, Fusheng; Zhong, Weidong; Liao, Fulian; Gao, George F; Tien, Po

    2006-06-01

    We reported previously that heat shock protein gp96 and its N-terminal fragment were able to stimulate CTL expansion specific for a HBV peptide (SYVNTNMGL) in BALB/c mice. Here we characterized the adjuvant effects of gp96 on human HLA-A2 restricted T cells. Full-length gp96 isolated from healthy human liver and recombinant fragments both from prokaryotic cells and eukaryotic cells were analyzed for their ability to stimulate maturation of human dendritic cells. It was found that in vitro these proteins were capable of maturating human monocyte-derived dendritic cells (MDDC) isolated from healthy donors as well as from HBV-positive, hepatocellular carcinoma (HCC) patients. In HLA-A2.1/Kb transgenic mice, gp96 and the recombinant fragments were found to augment CTL response specific for the HBcAg(18-27) FLPSDFFPSV peptide of hepatitis B virus. PMID:16630554

  2. Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins.

    PubMed

    Kimmins, S; MacRae, T H

    2000-04-01

    The selective modulation of transcription exerted by steroids depends upon recognition of signalling molecules by properly folded cytoplasmic receptors and their subsequent translocation into the nucleus. These events require a sequential and dynamic series of protein-protein interactions in order to fashion receptors that bind stably to steroids. Central to receptor maturation, therefore, are several molecular chaperones and their accessory proteins; Hsp70, Hsp40, and hip modulate the 3-dimensional conformation of steroid receptors, permitting reaction via hop with Hsp90, arguably the central protein in the process. Binding to Hsp90 leads to dissociation of some proteins from the receptor complex while others are recruited. Notably, p23 stabilizes receptors in a steroid binding state, and the immunophilins, principally CyP40 and Hsp56, arrive late in receptor complex assembly. In this review, the functions of molecular chaperones during steroid receptor maturation are explored, leading to a general mechanistic model indicative of chaperone cooperation in protein folding. PMID:11147968

  3. 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.

  4. 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. PMID:22362769

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

    PubMed

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

    2015-08-01

    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

  6. 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

  7. 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

  8. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

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

    2011-01-01

    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. PMID:21715334

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

    PubMed

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

    2015-10-26

    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, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), 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

  10. 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

  11. 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

  12. Caveolin-3 regulates myostatin signaling. Mini-review.

    PubMed

    Ohsawa, Y; Okada, T; Kuga, A; Hayashi, S; Murakami, T; Tsuchida, K; Noji, S; Sunada, Y

    2008-07-01

    Caveolins, components of the uncoated invaginations of plasma membrane, regulate signal transduction and vesicular trafflicking. Loss of caveolin-3, resulting from dominant negative mutations of caveolin-3 causes autosomal dominant limb-girdle muscular dystrophy (LGMD) 1C and autosomal dominant rippling muscle disease (AD-RMD). Myostatin, a member of the muscle-specific transforming growth factor (TGF)-beta superfamily, negatively regulates skeletal muscle volume. Herein we review caveolin-3 suppressing of activation of type I myostatin receptor, thereby inhibiting subsequent intracellular signaling. In addition, a mouse model of LGMD1C has shown atrophic myopathy with enhanced myostatin signaling. Myostatin inhibition ameliorates muscular phenotype in the model mouse, accompanied by normalized myostatin signaling. Enhanced myostatin signaling by caveolin-3 mutation in human may contribute to the pathogenesis of LGMD1C. Therefore, myostatin inhibition therapy may be a promising treatment for patients with LGMD1C. More recent studies concerning regulation of TGF-beta superfamily signaling by caveolins have provided new insights into the pathogenesis of several human diseases. PMID:19108573

  13. Caveolin-3 regulates myostatin signaling. Mini-review

    PubMed Central

    Ohsawa, Y; Okada, T; Kuga, A; Hayashi, S; Murakami, T; Tsuchida, K; Noji, S; Sunada, Y

    2008-01-01

    Summary Caveolins, components of the uncoated invaginations of plasma membrane, regulate signal transduction and vesicular trafficking. Loss of caveolin-3, resulting from dominant negative mutations of caveolin-3 causes autosomal dominant limb-girdle muscular dystrophy (LGMD) 1C and autosomal dominant rippling muscle disease (AD-RMD). Myostatin, a member of the muscle-specific transforming growth factor (TGF)-β superfamily, negatively regulates skeletal muscle volume. Herein we review caveolin-3 suppressing of activation of type I myostatin receptor, thereby inhibiting subsequent intracellular signaling. In addition, a mouse model of LGMD1C has shown atrophic myopathy with enhanced myostatin signaling. Myostatin inhibition ameliorates muscular phenotype in the model mouse, accompanied by normalized myostatin signaling. Enhanced myostatin signaling by caveolin-3 mutation in human may contribute to the pathogenesis of LGMD1C. Therefore, myostatin inhibition therapy may be a promising treatment for patients with LGMD1C. More recent studies concerning regulation of TGF-β superfamily signaling by caveolins have provided new insights into the pathogenesis of several human diseases. PMID:19108573

  14. 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

  15. The Src Homology 2 Domain-Containing Adapter Protein B (SHB) Regulates Mouse Oocyte Maturation

    PubMed Central

    Calounova, Gabriela; Livera, Gabriel; Zhang, Xiao-Qun; Liu, Kui; Gosden, Roger G.; Welsh, Michael

    2010-01-01

    SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb −/− mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/− and −/− mice revealed impaired early embryo development in the −/− embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development. PMID:20585392

  16. Myostatin/activin blocking combined with exercise reconditions skeletal muscle expression profile of mdx mice.

    PubMed

    Kainulainen, Heikki; Papaioannou, Konstantinos G; Silvennoinen, Mika; Autio, Reija; Saarela, Janne; Oliveira, Bernardo M; Nyqvist, Miro; Pasternack, Arja; 't Hoen, Peter A C; Kujala, Urho M; Ritvos, Olli; Hulmi, Juha J

    2015-01-01

    Duchenne muscular dystrophy is characterized by muscle wasting and decreased aerobic metabolism. Exercise and blocking of myostatin/activin signaling may independently or combined counteract muscle wasting and dystrophies. The effects of myostatin/activin blocking using soluble activin receptor-Fc (sActRIIB-Fc) administration and wheel running were tested alone or in combination for 7 weeks in dystrophic mdx mice. Expression microarray analysis revealed decreased aerobic metabolism in the gastrocnemius muscle of mdx mice compared to healthy mice. This was not due to reduced home-cage physical activity, and was further downregulated upon sActRIIB-Fc treatment in enlarged muscles. However, exercise activated pathways of aerobic metabolism and counteracted the negative effects of sActRIIB-Fc. Exercise and sActRIIB-Fc synergistically increased expression of major urinary protein, but exercise blocked sActRIIB-Fc induced phosphorylation of STAT5 in gastrocnemius muscle. In conclusion, exercise alone or in combination with myostatin/activin blocking corrects aerobic gene expression profiles of dystrophic muscle toward healthy wild type mice profiles. PMID:25304272

  17. Local overexpression of the myostatin propeptide increases glucose transporter expression and enhances skeletal muscle glucose disposal

    PubMed Central

    Jarmin, S.; Eilers, W.; Elashry, M.; Andersen, D. K.; Dickson, G.; Foster, K.

    2014-01-01

    Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes. PMID:24473441

  18. Stat3 activation links a C/EBPδ to myostatin pathway to stimulate loss of muscle mass.

    PubMed

    Zhang, Liping; Pan, Jenny; Dong, Yanjun; Tweardy, David J; Dong, Yanlan; Garibotto, Giacomo; Mitch, William E

    2013-09-01

    Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models with CKD or acute diabetes. A small-molecule that inhibits Stat3 activation produced similar responses, suggesting a potential for translation strategies. Using CCAAT/enhancer-binding protein δ (C/EBPδ) KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ, and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting. PMID:24011072

  19. Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

    PubMed Central

    Gehmert, Sebastian; Nerlich, Michael; Gosau, Martin; Klein, Silvan; Schreml, Stephan; Prantl, Lukas

    2014-01-01

    Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases. PMID:24575400

  20. 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.

  1. 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

  2. Streptococcus mutans wall-associated protein A promotes TLR4-induced dendritic cell maturation.

    PubMed

    Li, H; Wang, D

    2014-08-01

    Dendritic cells orchestrate innate and adaptive immune responses, which are central to establishing efficient responses to vaccination. Wall-associated protein A (WapA) of Streptococcus mutans was previously used as a vaccine in animal studies for immunization against dental caries. However, as a cell surface protein, whether WapA activates innate immune responses and the effects of WapA on DCs remain unclear. In this study, WapA was cloned into the GST fusion vector pEBG, which can be expressed efficiently in mammalian cells. We found that when added before stimulation with LPS, purified WapA-GST protein increased TLR4-induced NF-κB and MAPK signalling pathway activation. Pretreatment with WapA-GST also increased LPS-induced proinflammatory cytokine production by DCs, including IL-12, IL-6 and TNF-α. Furthermore, expression of the DC maturation markers CD80/86, CD40 and MHC II was also increased by WapA pretreatment. These data indicate that WapA is recognized by DCs and promotes DC maturation. PMID:24846569

  3. 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

  4. Proteomic analysis and candidate allergenic proteins in Populus deltoides CL. “2KEN8” mature pollen

    PubMed Central

    Zhang, Jin; Wu, Li-Shuan; Fan, Wei; Zhang, Xiao-Ling; Jia, Hui-Xia; Li, Yu; Yin, Ya-Fang; Hu, Jian-Jun; Lu, Meng-Zhu

    2015-01-01

    Proteomic analysis was used to generate a map of Populus deltoides CL. “2KEN8” mature pollen proteins. By applying 2-D electrophoresis, we resolved 403 protein spots from mature pollen. Using the matrix-assisted laser desorption/ionization time time-of-flight/time-of-flight tandem mass spectrometry method, we identified 178 distinct proteins from 218 protein spots expressed in mature pollen. Moreover, out of these, 28 proteins were identified as putative allergens. The expression patterns of these putative allergen genes indicate that several of these genes are highly expressed in pollen. In addition, the members of profilin allergen family were analyzed and their expression patterns were compared with their homologous genes in Arabidopsis and rice. Knowledge of these identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with poplar pollen allergy. PMID:26284084

  5. Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

    PubMed Central

    Dominguez, I; Diaz-Meco, M T; Municio, M M; Berra, E; García de Herreros, A; Cornet, M E; Sanz, L; Moscat, J

    1992-01-01

    A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways. Images PMID:1508183

  6. Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins.

    PubMed

    Lin, Yuan; Protter, David S W; Rosen, Michael K; Parker, Roy

    2015-10-15

    Eukaryotic cells possess numerous dynamic membrane-less organelles, RNP granules, enriched in RNA and RNA-binding proteins containing disordered regions. We demonstrate that the disordered regions of key RNP granule components and the full-length granule protein hnRNPA1 can phase separate in vitro, producing dynamic liquid droplets. Phase separation is promoted by low salt concentrations or RNA. Over time, the droplets mature to more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with formation of fibrous structures. Different disordered domains can co-assemble into phase-separated droplets. These biophysical properties demonstrate a plausible mechanism by which interactions between disordered regions, coupled with RNA binding, could contribute to RNP granule assembly in vivo through promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation could contribute to diseases involving aberrant RNA granules. PMID:26412307

  7. Molecular profiles of Quadriceps muscle in myostatin-null mice reveal PI3K and apoptotic pathways as myostatin targets

    PubMed Central

    Chelh, Ilham; Meunier, Bruno; Picard, Brigitte; Reecy, Mark James; Chevalier, Catherine; Hocquette, Jean-François; Cassar-Malek, Isabelle

    2009-01-01

    Background Myostatin (MSTN), a member of the TGF-β superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass. Results Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed. Conclusion All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3ε protein, TCTP/GSK-3β). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo. PMID:19397818

  8. Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zhang, Hui; Cao, Xudong; Qiao, Jun; Sheng, Jinliang; Guo, Fei; Chen, Chuangfu

    2011-10-01

    Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P<0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning. PMID:21698446

  9. Mature adipocyte proteome reveals differentially altered protein abundances between lean, overweight and morbidly obese human subjects.

    PubMed

    Benabdelkamel, Hicham; Masood, Afshan; Almidani, Ghaith M; Alsadhan, Abdulmajeed A; Bassas, Abdulelah F; Duncan, Mark W; Alfadda, Assim A

    2015-02-01

    Overweight (OW) and obese individuals are considered to be graded parts of the scale having increasing weight as a common feature. They may not, however, be part of the same continuum and may differ metabolically. In this study we applied an untargeted proteomic approach to compare protein abundances in mature adipocytes derived from the subcutaneous adipose tissue of overweight and morbidly obese female subjects to those of lean age matched controls. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both lean (L; n = 7, 23.3 ± 0.4 kg/m(2); mean BMI ± SD), overweight (OW; n = 8, 27.9 ± 0.6 kg/m(2); mean BMI ± SD) and morbidly obese (MOB; n = 7, 44.8 ± 3.8 kg/m(2); mean BMI ± SD) individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). One hundred and ten differentially expressed protein spots (i.e., fitting the statistical criteria ANOVA test, p < 0.05; fold-change ≥1.5) were detected, and of these, 89 were identified by MALDI-TOF mass spectrometry. Of these, 66 protein spots were common to both groups whereas 23 were unique to the MOB group. Significant differences were evident in the abundances of key proteins involved in glucose and lipid metabolism, energy regulation, cytoskeletal structure and redox control signaling pathways. Differences in the abundance of some chaperones were also evident. The differentially abundant proteins were investigated using Ingenuity Pathway Analysis (IPA) to establish their associations with known biological functions. The network identified in the OW group with the highest score relates to-: cell-to-cell signaling and interaction; in contrast, in the MOB group the major interacting pathways are associated with lipid metabolism, small molecule biochemistry and cancer. The differences in abundance of the differentially regulated proteins were validated by

  10. 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

  11. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. PROLONGED FASTING AND CORTISOL REDUCE MYOSTATIN MRNA LEVELS IN TILAPIA LARVAE, SHORT-TERM FASTING ELEVATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 indices in adults, skeletal muscle myostatin mRNA...

  14. Engineering protein therapeutics: predictive performances of a structure-based virtual affinity maturation protocol.

    PubMed

    Oberlin, Michael; Kroemer, Romano; Mikol, Vincent; Minoux, Hervé; Tastan, Erdogan; Baurin, Nicolas

    2012-08-27

    The implementation of a structure based virtual affinity maturation protocol and evaluation of its predictivity are presented. The in silico protocol is based on conformational sampling of the interface residues (using the Dead End Elimination/A* algorithm), followed by the estimation of the change of free energy of binding due to a point mutation, applying MM/PBSA calculations. Several implementations of the protocol have been evaluated for 173 mutations in 7 different protein complexes for which experimental data were available: the use of the Boltzamnn averaged predictor based on the free energy of binding (ΔΔG(*)) combined with the one based on its polar component only (ΔΔE(pol*)) led to the proposal of a subset of mutations out of which 45% would have successfully enhanced the binding. When focusing on those mutations that are less likely to be introduced by natural in vivo maturation methods (99 mutations with at least two base changes in the codon), the success rate is increased to 63%. In another evaluation, focusing on 56 alanine scanning mutations, the in silico protocol was able to detect 89% of the hot-spots. PMID:22788756

  15. 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. PMID:24018114

  16. 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

  17. 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.

  18. Properties of proteins and the glassy matrix in maturation-defective mutant seeds of Arabidopsis thaliana.

    PubMed

    Wolkers, W F; Alberda, M; Koornneef, M; Léon-Kloosterziel, K M; Hoekstra, F A

    1998-10-01

    In situ Fourier transform infrared microspectroscopy was used to study the heat stability of proteins and hydrogen bonding interactions in dry maturation-defective mutant seeds of Arabidopsis thaliana. alpha-Helical, turn and beta-sheet conformations were the major protein secondary structures in all of these seeds. On heating, intermolecular extended beta-sheet structures, typical of protein denaturation, were formed in abscisic acid-insensitive (abi3) and leafy cotyledon (lec) mutant seeds. Proteins in dry wild-type seeds did not denature up to 150 degrees C, but those in dry desiccation-sensitive, lec1-1, lec1-3 and abi3-5 seeds did at 68, 89 and 87 degrees C, respectively. In the desiccation-tolerant abi3-7 and abi3-1 seeds, denaturation commenced above 120 and 135 degrees C, respectively. Seeds of the aba1-1 abi3-1 double mutant showed signs of denaturation already upon drying. The molecular packing in the seeds was studied by observing the shift in the position of the OH-stretching vibration band with temperature. The maximal rate of change of this band with temperature was much higher in the desiccation-sensitive abi3-5, aba1-1 abi3-1, lec1-1, and lec1-3 mutant seeds than in the desiccation-tolerant wild-type, abi3-1, abi3-7, and lec2-1 seeds. We interpret this to mean that the molecular packing density is higher in dry desiccation-tolerant than in dry desiccation-sensitive seeds, which is associated with a higher or lower protein denaturation temperature, respectively. The results are discussed in relation to the physiological and biochemical characteristics of these mutant seeds. PMID:9839460

  19. Functional verification of a porcine myostatin propeptide mutant.

    PubMed

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

    2015-10-01

    Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells. PMID:26174475

  20. 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

  1. Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility.

    PubMed

    Krutskikh, Anton; Poliandri, Ariel; Cabrera-Sharp, Victoria; Dacheux, Jean Louis; Poutanen, Matti; Huhtaniemi, Ilpo

    2012-10-01

    Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction. PMID:22750516

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

    PubMed

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

    2016-09-01

    The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) 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 the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications. PMID:27428511

  3. 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

  4. Immunohistochemical expression of SOX9 protein in immature, mature, and neoplastic canine Sertoli cells.

    PubMed

    Banco, Barbara; Palmieri, Chiara; Sironi, Giuseppe; Fantinato, Eleonora; Veronesi, Maria C; Groppetti, Debora; Giudice, Chiara; Martignoni, Benedetta; Grieco, Valeria

    2016-05-01

    Sex-determining region Y box9 gene (SOX9) protein plays a pivotal role in male sexual development. It regulates the transcription of the anti-Müllerian hormone gene promoting development of testis cords, multiplication, and maturation of Sertoli cells (SCs) and maintenance of spermatogenesis in adult testis. The immunohistochemical expression of SOX9 in normal testes has been reported in humans, mice, and rats. The present study aimed to investigate the expression of SOX9 in canine SCs during testicular maturation and neoplastic transformation. Canine testicular samples derived from three fetuses, four newborns, four prepubertal puppies, five adult dogs, 31 Sertoli cell tumors (SCTs) (one metastasizing), and five Leydig cell tumors (LCTs) were selected from departmental archive and tested immunohistochemically with a polyclonal antibody against SOX9 (1:150). All SCs from fetal, neonatal, and adult testes had a strong and exclusively nuclear labeling for SOX9. In SCs from prepubertal testes, SOX9 staining was highly variable with one negative sample (one of four), two samples with exclusively nuclear staining (two of four), and one with both nuclear and cytoplasmic labeling (one of four). Leydig cells (LCs) and LCTs were always negative. All 31 SCTs were positive for SOX9. The expression of SOX9 was nuclear, nuclear and cytoplasmic, and exclusively cytoplasmic in 18 of 31, 11 of 31, and two of 31 SCTs, respectively. This first report on the immunohistochemical expression of SOX9 in canine testes reports that in normal SCs from fetal, neonatal, and adult testes SOX9 labeled the nucleus, as in humans and laboratory animals. The cytoplasmic labeling observed in one prepubertal pairs of testes and in 11 SCTs could reflect SC immaturity or dedifferentiation, paralleling results observed in rat testes. The expression of SOX9 in SCs and SCTs and its absence in LCs and LCTs suggests that SOX9 is a reliable diagnostic marker for both normal and neoplastic SCs. PMID:26777558

  5. Myostatin and sarcopenia: opportunities and challenges - a mini-review.

    PubMed

    White, Thomas A; LeBrasseur, Nathan K

    2014-01-01

    The progressive loss of skeletal muscle mass, strength and/or function with advancing age, termed sarcopenia, poses a major threat to independence and quality of life. Therefore, there is significant merit in better understanding the biology of sarcopenia and developing therapeutic interventions to prevent, slow or reverse its progression. Since the discovery of myostatin, a potent negative regulator of growth that is highly enriched in skeletal muscle, there has been great interest in it as a potential mediator of sarcopenia as well as a therapeutic target. The complex biology of myostatin, the promise of myostatin inhibition as an effective means to counter sarcopenia, and the challenges facing its clinical translation are reviewed herein. PMID:24457615

  6. 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. PMID

  7. 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

    2014-02-01

    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. PMID:24341390

  8. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein

    PubMed Central

    He, Lihua; Kota, Pradeep; Aleksandrov, Andrei A.; Cui, Liying; Jensen, Tim; Dokholyan, Nikolay V.; Riordan, John R.

    2013-01-01

    Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T1/2∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2–5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein. PMID:23104983

  9. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

    PubMed

    He, Lihua; Kota, Pradeep; Aleksandrov, Andrei A; Cui, Liying; Jensen, Tim; Dokholyan, Nikolay V; Riordan, John R

    2013-02-01

    Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T(1/2)∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein. PMID:23104983

  10. 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

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

    PubMed

    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

  12. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion.

    PubMed

    Curwin, Amy J; Brouwers, Nathalie; Alonso Y Adell, Manuel; Teis, David; Turacchio, Gabriele; Parashuraman, Seetharaman; Ronchi, Paolo; Malhotra, Vivek

    2016-01-01

    The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior. PMID:27115345

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

    PubMed

    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

  14. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion

    PubMed Central

    Curwin, Amy J; Brouwers, Nathalie; Alonso Y Adell, Manuel; Teis, David; Turacchio, Gabriele; Parashuraman, Seetharaman; Ronchi, Paolo; Malhotra, Vivek

    2016-01-01

    The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior. DOI: http://dx.doi.org/10.7554/eLife.16299.001 PMID:27115345

  15. 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

  16. The Protein Dendrite Arborization and Synapse Maturation 1 (Dasm-1) Is Dispensable for Dendrite Arborization▿ †

    PubMed Central

    Mishra, Archana; Knerr, Boris; Paixão, Sónia; Kramer, Edgar R.; Klein, Rüdiger

    2008-01-01

    The development of a highly branched dendritic tree is essential for the establishment of functional neuronal connections. The evolutionarily conserved immunoglobulin superfamily member, the protein dendrite arborization and synapse maturation 1 (Dasm-1) is thought to play a critical role in dendrite formation of dissociated hippocampal neurons. RNA interference-mediated Dasm-1 knockdown was previously shown to impair dendrite, but not axonal, outgrowth and branching (S. H. Shi, D. N. Cox, D. Wang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA 101:13341-13345, 2004). Here, we report the generation and analysis of Dasm-1 null mice. We find that genetic ablation of Dasm-1 does not interfere with hippocampal dendrite growth and branching in vitro and in vivo. Moreover, the absence of Dasm-1 does not affect the modulation of dendritic outgrowth induced by brain-derived neurotrophic factor. Importantly, the previously observed impairment in dendrite growth after Dasm-1 knockdown is also observed when the Dasm-1 knockdown is performed in cultured hippocampal neurons from Dasm-1 null mice. These findings indicate that the dendrite arborization phenotype was caused by off-target effects and that Dasm-1 is dispensable for hippocampal dendrite arborization. PMID:18268009

  17. 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. PMID:26673448

  18. 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. PMID:24047702

  19. 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.

  20. Mitochondrial protein synthesis in rainbow trout (Oncorhynchus mykiss) heart is enhanced in sexually mature males but impaired by low temperature

    PubMed

    West; Driedzic

    1999-09-01

    Throughout the life cycle of the rainbow trout (Oncorhynchus mykiss), the heart exhibits periods of enhanced growth. Two such instances are cardiac enlargement associated with sexual maturity in males and heart growth at seasonally low environmental temperatures. Heart growth includes a parallel increase in the number of mitochondria. These natural models of heart growth have been exploited to study protein synthesis directed by the mitochondrial genome. Methods were developed to assess protein synthesis in mitochondria isolated from the heart of rainbow trout. Protein synthesis was assessed by tracking the incorporation of l-[2,6-(3)H]phenylalanine into trichloracetic-acid-precipitable protein. Amino acid incorporation into mitochondrial protein was linear with respect to time and was inhibited by chloramphenicol. Radiolabel was selectively enhanced in molecular mass fractions over the same size range as polypeptides known to be encoded by the mitochondrial genome. Protein synthesis was measured in mitochondria isolated from sexually mature animals and from animals subjected to different thermal regimes. The relative ventricular mass of sexually mature male rainbow trout was significantly greater than that of sexually mature females (0. 104+/-0.004 versus 0.087+/-0.002; mean +/- s.e.m.). Mitochondria isolated from the heart of males synthesized protein at a faster rate than mitochondria isolated from the heart of females (0.22+/-0. 02 versus 0.11+/-0.02 pmol phenylalanine mg(-)(1 )protein min(-)(1)). That is, 'male' mitochondria are inherently predisposed to synthesize protein at faster rates. We speculate that the difference may result from higher levels of mitochondrial RNA in males than in females. Mitochondria isolated from the heart of sexually immature rainbow trout acclimated to 13 degrees C synthesized protein at the same rate at 25 degrees C (0.456+/-0.075 pmolphenylalanine mg(-)(1 )protein min(-)(1)) and 15 degrees C (0.455+/-0.027 pmol phenylalanine mg

  1. Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex.

    PubMed

    James, H E; Bartling, D; Musgrove, J E; Kirwin, P M; Herrmann, R G; Robinson, C

    1989-11-25

    The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases. PMID:2684958

  2. Sensitive and Quantitative Three-Color Protein Imaging in Fission Yeast Using Spectrally Diverse, Recoded Fluorescent Proteins with Experimentally-Characterized In Vivo Maturation Kinetics

    PubMed Central

    Al-Sady, Bassem; Greenstein, Rachel A.; El-Samad, Hana J.; Braun, Sigurd; Madhani, Hiten D.

    2016-01-01

    Schizosaccharomyces pombe is an outstanding model organism for cell biological investigations, yet the range of useful and well-characterized fluorescent proteins (XFPs) is limited. We generated and characterized three recoded fluorescent proteins for 3-color analysis in S.pombe, Super-folder GFP, monomeric Kusabira Orange 2 and E2Crimson. Upon optimization and expression in S. pombe, the three proteins enabled sensitive simultaneous 3-color detection capability. Furthermore, we describe a strategy that combines a pulse-chase approach and mathematical modeling to quantify the maturation kinetics of these proteins in vivo. We observed maturation kinetics in S. pombe that are expected from those described for these proteins in vitro and/or in other cell types, but also unpredicted behaviors. Our studies provide a kinetically-characterized, integrated three-color XFP toolbox for S. pombe. PMID:27479698

  3. Body composition of transgenic pigs expressing the myostatin pro domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous results have shown that male mice expressing a myostatin pro domain construct (MLC-Pro) have increased body weight, more total body lean mass, and lower percentage of total body fat. Founder transgenic (TG) pigs were generated by standard pronuclear microinjection techniques using the sam...

  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. PMID:27069062

  5. 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. PMID:24334117

  6. 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. PMID:23986966

  7. Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin.

    PubMed

    Yasmin, Cut; Otoi, Takeshige; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani

    2015-03-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission. PMID:25655418

  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. PMID:26206886

  9. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

    PubMed

    Juvale, Parijat S; Wagner, Ryan L; Spalding, Martin H

    2016-04-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  10. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation

    PubMed Central

    Juvale, Parijat S.; Wagner, Ryan L.; Spalding, Martin H.

    2016-01-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide ‘spacer’ region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  11. Olfactory marker protein is critical for functional maturation of olfactory sensory neurons and development of mother preference

    PubMed Central

    Lee, Anderson C.; He, Jiwei; Ma, Minghong

    2011-01-01

    Survival of many altricial animals critically depends on the sense of smell. Curiously, the olfactory system is rather immature at birth and undergoes a maturation process, which is poorly understood. Using patch clamp technique on mouse olfactory sensory neurons (OSNs) with a defined odorant receptor (OR), we demonstrate that OSNs exhibit functional maturation during the first month of postnatal life by developing faster response kinetics, higher sensitivity, and most intriguingly, higher selectivity. OSNs expressing the receptor MOR23 are relatively broadly tuned in neonates and become selective detectors for the cognate odorant within two weeks. Remarkably, these changes are prevented by genetic ablation of olfactory marker protein (OMP), which is exclusively expressed in mature OSNs. Biochemical and pharmacological evidence supports that alteration in odorant-induced phosphorylation of signaling proteins underlie some of the OMP−/− phenotypes. Furthermore, in a novel behavioral assay in which the mouse pups are given a choice between the biological mother and another unfamiliar lactating female, wild-type pups prefer the biological mother, while OMP knockout pups fail to show preference. These results reveal that OSNs undergo an OMP-dependant functional maturation process that coincides with early development of the smell function, which is essential for pups to form preference for their mother. PMID:21414919

  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. PMID:26488807

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

    PubMed

    El Shafey, Nelly; Guesnon, Mickaël; Simon, Françoise; Deprez, Eric; Cosette, Jérémie; Stockholm, Daniel; Scherman, Daniel; Bigey, Pascal; Kichler, Antoine

    2016-02-15

    Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family. PMID:26844629

  15. 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.

  16. Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism*

    PubMed Central

    Kakoi, Hironori; Maeda, Shingo; Shinohara, Naohiro; Matsuyama, Kanehiro; Imamura, Katsuyuki; Kawamura, Ichiro; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-01-01

    Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C2-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C2-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism. PMID:24505141

  17. Dynamic expression of TrkB receptor protein on proliferating and maturing cells in the adult mouse dentate gyrus

    PubMed Central

    Donovan, Michael H.; Yamaguchi, Masahiro; Eisch, Amelia J.

    2008-01-01

    Brain-derived neurotrophic factor (BDNF) is implicated in regulation of adult hippocampal neurogenesis, presumably via its primary receptor, TrkB, but controversy exists about how BDNF affects neurogenesis (e.g. proliferation vs. survival/differentiation). This controversy arises, in part, due to the lack of information about if and when TrkB is expressed on adult neural precursors in vivo. Using multiple methods to analyze proliferating and maturing cells in the adult mouse subgranular zone (SGZ), we find that the proportion of proliferating cells that are TrkB-IR is low and it remains low for at least one week following BrdU labeling, but increases as neuroblasts mature. Use of the nestin-GFP transgenic mouse revealed the likelihood of being TrkB-IR increased with presumed maturity of the cell type. Stem-like cells, which rarely divide, were likely to express TrkB. However, early progenitors and late progenitors, which are still in the cell cycle had rare TrkB expression. Immature neuroblasts, however, were more likely to express TrkB, especially as their morphology became more mature. Taken together, these findings emphasize that expression of TrkB protein is closely linked to progression towards neuronal maturity. This provides evidence that maturing cells but not proliferating cells in the adult mouse SGZ have the molecular machinery necessary to respond directly to BDNF. Furthermore, these findings lay critical groundwork for further exploration of the role of BDNF-TrkB signaling in regulation of adult hippocampal neurogenesis. PMID:18240316

  18. 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

  19. Muscular atrophy of caveolin-3-deficient mice is rescued by myostatin inhibition.

    PubMed

    Ohsawa, Yutaka; Hagiwara, Hiroki; Nakatani, Masashi; Yasue, Akihiro; Moriyama, Keiji; Murakami, Tatsufumi; Tsuchida, Kunihiro; Noji, Sumihare; Sunada, Yoshihide

    2006-11-01

    Caveolin-3, the muscle-specific isoform of caveolins, plays important roles in signal transduction. Dominant-negative mutations of the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy 1C (LGMD1C) with loss of caveolin-3. However, identification of the precise molecular mechanism leading to muscular atrophy in caveolin-3-deficient muscle has remained elusive. Myostatin, a member of the muscle-specific TGF-beta superfamily, negatively regulates skeletal muscle volume. Here we report that caveolin-3 inhibited myostatin signaling by suppressing activation of its type I receptor; this was followed by hypophosphorylation of an intracellular effector, Mad homolog 2 (Smad2), and decreased downstream transcriptional activity. Loss of caveolin-3 in P104L mutant caveolin-3 transgenic mice caused muscular atrophy with increase in phosphorylated Smad2 (p-Smad2) as well as p21 (also known as Cdkn1a), a myostatin target gene. Introduction of the myostatin prodomain, an inhibitor of myostatin, by genetic crossing or intraperitoneal administration of the soluble type II myostatin receptor, another inhibitor, ameliorated muscular atrophy of the mutant caveolin-3 transgenic mice with suppression of p-Smad2 and p21 levels. These findings suggest that caveolin-3 normally suppresses the myostatin-mediated signal, thereby preventing muscular atrophy, and that hyperactivation of myostatin signaling participates in the pathogenesis of muscular atrophy in a mouse model of LGMD1C. Myostatin inhibition may be a promising therapy for LGMD1C patients. PMID:17039257

  20. 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

  1. Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

    PubMed

    Fan, Heng-Yu; Huo, Li-Jun; Meng, Xiao-Qian; Zhong, Zhi-Sheng; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2003-11-01

    Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins

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

    PubMed

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

    2012-08-15

    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

  3. 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

  4. 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

  5. [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. PMID:11475895

  6. Effect of Postnatal Myostatin Inhibition on Bite Mechanics in Mice.

    PubMed

    Williams, Susan H; Lozier, Nicholas R; Montuelle, Stéphane J; de Lacalle, Sonsoles

    2015-01-01

    As a negative regulator of muscle size, myostatin (Mstn) impacts the force-production capabilities of skeletal muscles. In the masticatory system, measures of temporalis-stimulated bite forces in constitutive myostatin KOs suggest an absolute, but not relative, increase in jaw-muscle force. Here, we assess the phenotypic and physiologic impact of postnatal myostatin inhibition on bite mechanics using an inducible conditional KO mouse in which myostatin is inhibited with doxycycline (DOX). Given the increased control over the timing of gene inactivation in this model, it may be more clinically-relevant for developing interventions for age-associated changes in the musculoskeletal system. DOX was administered for 12 weeks starting at age 4 months, during which time food intake was monitored. Sex, age and strain-matched controls were given the same food without DOX. Bite forces were recorded just prior to euthanasia after which muscle and skeletal data were collected. Food intake did not differ between control or DOX animals within each sex. DOX males were significantly larger and had significantly larger masseters than controls, but DOX and control females did not differ. Although there was a tendency towards higher absolute bite forces in DOX animals, this was not significant, and bite forces normalized to masseter mass did not differ. Mechanical advantage for incisor biting increased in the DOX group due to longer masseter moment arms, likely due to a more anteriorly-placed masseter insertion. Despite only a moderate increase in bite force in DOX males and none in DOX females, the increase in masseter mass in males indicates a potentially positive impact on jaw muscles. Our data suggest a sexual dimorphism in the role of mstn, and as such investigations into the sex-specific outcomes is warranted. PMID:26252892

  7. Effect of Postnatal Myostatin Inhibition on Bite Mechanics in Mice

    PubMed Central

    Williams, Susan H.; Lozier, Nicholas R.; Montuelle, Stéphane J.; de Lacalle, Sonsoles

    2015-01-01

    As a negative regulator of muscle size, myostatin (Mstn) impacts the force-production capabilities of skeletal muscles. In the masticatory system, measures of temporalis-stimulated bite forces in constitutive myostatin KOs suggest an absolute, but not relative, increase in jaw-muscle force. Here, we assess the phenotypic and physiologic impact of postnatal myostatin inhibition on bite mechanics using an inducible conditional KO mouse in which myostatin is inhibited with doxycycline (DOX). Given the increased control over the timing of gene inactivation in this model, it may be more clinically-relevant for developing interventions for age-associated changes in the musculoskeletal system. DOX was administered for 12 weeks starting at age 4 months, during which time food intake was monitored. Sex, age and strain-matched controls were given the same food without DOX. Bite forces were recorded just prior to euthanasia after which muscle and skeletal data were collected. Food intake did not differ between control or DOX animals within each sex. DOX males were significantly larger and had significantly larger masseters than controls, but DOX and control females did not differ. Although there was a tendency towards higher absolute bite forces in DOX animals, this was not significant, and bite forces normalized to masseter mass did not differ. Mechanical advantage for incisor biting increased in the DOX group due to longer masseter moment arms, likely due to a more anteriorly-placed masseter insertion. Despite only a moderate increase in bite force in DOX males and none in DOX females, the increase in masseter mass in males indicates a potentially positive impact on jaw muscles. Our data suggest a sexual dimorphism in the role of mstn, and as such investigations into the sex-specific outcomes is warranted. PMID:26252892

  8. Clinical, Agricultural, and Evolutionary Biology of Myostatin: A Comparative Review

    PubMed Central

    Rodgers, Buel D.; Garikipati, Dilip K.

    2008-01-01

    The discovery of myostatin and our introduction to the “Mighty Mouse” over a decade ago spurred both basic and applied research and impacted popular culture as well. The myostatin-null genotype produces “double muscling” in mice and livestock and was recently described in a child. The field’s rapid growth is by no means surprising considering the potential benefits of enhancing muscle growth in clinical and agricultural settings. Indeed, several recent studies suggest that blocking myostatin’s inhibitory effects could improve the clinical treatment of several muscle growth disorders, whereas comparative studies suggest that these actions are at least partly conserved. Thus, neutralizing myostatin’s effects could also have agricultural significance. Extrapolating between studies that use different vertebrate models, particularly fish and mammals, is somewhat confusing because whole genome duplication events have resulted in the production and retention of up to four unique myostatin genes in some fish species. Such comparisons, however, suggest that myostatin’s actions may not be limited to skeletal muscle per se, but may additionally influence other tissues including cardiac muscle, adipocytes, and the brain. Thus, therapeutic intervention in the clinic or on the farm must consider the potential of alternative side effects that could impact these or other tissues. In addition, the presence of multiple and actively diversifying myostatin genes in most fish species provides a unique opportunity to study adaptive molecular evolution. It may also provide insight into myostatin’s nonmuscle actions as results from these and other comparative studies gain visibility in biomedical fields. PMID:18591260

  9. Immunomodulatory Effects of Four Leishmania infantum Potentially Excreted/Secreted Proteins on Human Dendritic Cells Differentiation and Maturation

    PubMed Central

    Markikou-Ouni, Wafa; Drini, Sima; Bahi-Jaber, Narges; Chenik, Mehdi; Meddeb-Garnaoui, Amel

    2015-01-01

    Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vitro differentiation and maturation of human DCs and on cytokine production by DCs and monocytes. During DCs differentiation, LipESP led to a significant decrease in CD1a. LiP23 and LiEF-1α, induced a decrease of HLA-DR and an increase of CD86 surface expression, respectively. During maturation, an up-regulation of HLA-DR and CD80 was found in response to LiP15, LiP23 and LiAAA-ATPase, while an increase of CD40 expression was only observed in response to LiP15. All LipESP induced an over-expression of CD86 with significant differences between proteins. These proteins also induced significant IL-12p70 levels in immature DCs but not in monocytes. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. TNF-α and IL-10 were induced in DCs and monocytes with higher levels observed for LiP15 and LiAAA-ATPase. However, LPS-induced cytokine production during DC maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment. Our findings suggest that LipESP strongly interfere with DCs differentiation suggesting a possible involvement in mechanisms established by the parasite for its survival. These proteins also induce DCs maturation by up-regulating several costimulatory molecules and by inducing the production of proinflammatory cytokines, which is a prerequisite for T cell activation. However, the reduced ability of LipESP-stimulated DCs and monocytes to respond to lipopolysaccharide (LPS

  10. Immunomodulatory Effects of Four Leishmania infantum Potentially Excreted/Secreted Proteins on Human Dendritic Cells Differentiation and Maturation.

    PubMed

    Markikou-Ouni, Wafa; Drini, Sima; Bahi-Jaber, Narges; Chenik, Mehdi; Meddeb-Garnaoui, Amel

    2015-01-01

    Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vitro differentiation and maturation of human DCs and on cytokine production by DCs and monocytes. During DCs differentiation, LipESP led to a significant decrease in CD1a. LiP23 and LiEF-1α, induced a decrease of HLA-DR and an increase of CD86 surface expression, respectively. During maturation, an up-regulation of HLA-DR and CD80 was found in response to LiP15, LiP23 and LiAAA-ATPase, while an increase of CD40 expression was only observed in response to LiP15. All LipESP induced an over-expression of CD86 with significant differences between proteins. These proteins also induced significant IL-12p70 levels in immature DCs but not in monocytes. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. TNF-α and IL-10 were induced in DCs and monocytes with higher levels observed for LiP15 and LiAAA-ATPase. However, LPS-induced cytokine production during DC maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment. Our findings suggest that LipESP strongly interfere with DCs differentiation suggesting a possible involvement in mechanisms established by the parasite for its survival. These proteins also induce DCs maturation by up-regulating several costimulatory molecules and by inducing the production of proinflammatory cytokines, which is a prerequisite for T cell activation. However, the reduced ability of LipESP-stimulated DCs and monocytes to respond to lipopolysaccharide (LPS

  11. Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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), ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  13. 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.

  14. 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. PMID:27457983

  15. Membrane Binding Properties and Terminal Residues of the Mature Hepatitis C Virus Capsid Protein in Insect Cells

    PubMed Central

    Ogino, Tomoaki; Fukuda, Hiroyuki; Imajoh-Ohmi, Shinobu; Kohara, Michinori; Nomoto, Akio

    2004-01-01

    The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with α-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group. PMID:15479818

  16. Polymorphism of the myostatin gene and its association with growth traits in chicken.

    PubMed

    Bhattacharya, T K; Chatterjee, R N

    2013-04-01

    An experiment was carried out on myostatin gene with the objectives of identification of polymorphism in the myostatin gene and estimation of the effect of polymorphism on growth traits in chickens. Single-stranded conformation polymorphism followed by sequencing was performed to reveal polymorphism of the gene. A total of 13 haplotypes were observed across 3 chicken lines (PB-1 and CB as broiler lines and IWI as the layer line). Myostatin haplogroups had a significant effect on BW at 28, 42, and 49 d of age in the PB-1 line. The significant association of haplogroups was observed with BW at d 14 and 49 in the CB line. In the IWI layer line, the myostatin gene was polymorphic but had no significant association with growth traits. It is concluded that the myostatin gene was polymorphic and had a significant effect on growth traits in broiler chickens. PMID:23472013

  17. 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

  18. Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation.

    PubMed

    Van, Bo; Nishi, Miyuki; Komazaki, Shinji; Ichimura, Atsuhiko; Kakizawa, Sho; Nakanaga, Keita; Aoki, Junken; Park, Ki-Ho; Ma, Jianjie; Ueyama, Tomomi; Ogata, Takehiro; Maruyama, Naoki; Takeshima, Hiroshi

    2015-04-28

    Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation. PMID:25841338

  19. Developmental patterns of free and protein-bound biotin during maturation and germination of seeds of Pisum sativum: characterization of a novel seed-specific biotinylated protein.

    PubMed

    Duval, M; Job, C; Alban, C; Douce, R; Job, D

    1994-04-01

    Mature dry pea seeds contain three major biotinylated proteins. Two of these of subunit molecular mass about 75 kDa and 200 kDa are associated with 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) and acetyl-CoA carboxylase activities (EC 6.4.1.2) respectively. The third does not exhibit any of the biotin-dependent carboxylase activities found in higher organisms and represents the major part of the total protein-bound biotin in the seeds. This novel protein has been purified from a whole pea seed extract. Because in SDS/polyacrylamide gels the protein migrates with an apparent molecular mass of about 65 kDa, it is referred to as SBP65, for 65 kDa seed biotinylated protein. The molecular mass of native SBP65 is greater than 400 kDa, suggesting that the native protein assumes a polymeric structure, resulting from the association of six to eight identical subunits. The results of CNBr cleavage experiments suggest that biotin is covalently bound to the protein. The stoichiometry is 1 mol of biotin per 1 mol of 65 kDa polypeptide. The temporal and spatial pattern of expression of SBP65 is described. SBP65 is specifically expressed in the seeds, being absent from leaf, root, stem, pod and flower tissues of pea plants. The level of SBP65 increases dramatically during seed development. The protein is not detectable in very young seeds. Its accumulation pattern parallels that for storage proteins, being maximally expressed in the mature dry seeds. SBP65 disappears at a very high rate during seed germination. The level of free biotin has also been evaluated for various organs of pea plants. In all proliferating tissues examined (young developing seeds, leaf, root, stem, pod and flower tissues), free biotin is in excess of protein-bound biotin. Only in the mature dry seeds is protein-bound biotin (i.e. that bound to SBP65) in excess of free biotin. These temporal expression patterns, and the strict organ specificity for expression of SBP65, are discussed with regard to the

  20. Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

    PubMed Central

    Nebreda, A R; Gannon, J V; Hunt, T

    1995-01-01

    The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone. Images PMID:8521817

  1. 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 Central

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

    2010-01-01

    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

  2. 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

  3. 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

  4. 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.

  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 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 Central

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

    2014-01-01

    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. PMID:24451368

  7. 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

  8. Nitric Oxide Synthase (NOS) Inhibition during Porcine In Vitro Maturation Modifies Oocyte Protein S-Nitrosylation and In Vitro Fertilization

    PubMed Central

    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. PMID:25542028

  9. 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

  10. The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle.

    PubMed

    Spletter, Maria L; Barz, Christiane; Yeroslaviz, Assa; Schönbauer, Cornelia; Ferreira, Irene R S; Sarov, Mihail; Gerlach, Daniel; Stark, Alexander; Habermann, Bianca H; Schnorrer, Frank

    2015-02-01

    In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis. PMID:25532219

  11. The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle

    PubMed Central

    Spletter, Maria L; Barz, Christiane; Yeroslaviz, Assa; Schönbauer, Cornelia; Ferreira, Irene R S; Sarov, Mihail; Gerlach, Daniel; Stark, Alexander; Habermann, Bianca H; Schnorrer, Frank

    2015-01-01

    In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis. PMID:25532219

  12. Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation.

    PubMed

    Hamlett, Isla; Draper, Julia; Strouboulis, John; Iborra, Francisco; Porcher, Catherine; Vyas, Paresh

    2008-10-01

    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

  13. Unh1, an Ustilago maydis Ndt80-like protein, controls completion of tumor maturation, teliospore development, and meiosis.

    PubMed

    Doyle, Colleen E; Kitty Cheung, H Y; Spence, Kelsey L; Saville, Barry J

    2016-09-01

    In this study, Ustilago maydis Ndt80 homolog one, unh1, of the obligate sexual pathogen U. maydis,is described. Unh1 is the sole Ndt80-like DNA-binding protein inU. maydis. In this model basidiomycete, Unh1 plays a role in sexual development, influencing tumor maturation, teliospore development and subsequent meiotic completion. Teliospore formation was reduced in deletion mutants, and those that did form had unpigmented, hyaline cell walls, and germinated without completing meiosis. Constitutively expressing unh1 in haploid cells resulted in abnormal pigmentation, when grown in both potato dextrose broth and minimal medium, suggesting that pigmentation may be triggered by unh1 in U. maydis. The function of Unh1 in sexual development and pigment production depends on the presence of the Ndt80-like DNA-binding domain, identified within Unh1. In the absence of this domain, or when the binding domain was altered with targeted amino acid changes, ectopic expression of Unh1 failed to complement the unh1 deletion with regards to pigment production and sexual development. An investigation of U. maydis genes with upstream motifs similar to Ndt80 recognition sequences revealed that some have altered transcript levels in Δunh1 strains. We propose that the first characterized Ndt80-like DNA-binding protein in a basidiomycete, Unh1, acts as a transcription factor that is required for teliospore maturation and the completion of meiosis in U. maydis. PMID:27397931

  14. 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.

  15. 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. PMID:18212055

  16. Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase▿

    PubMed Central

    Krauss, Sharon Wald; Spence, Jeffrey R.; Bahmanyar, Shirin; Barth, Angela I. M.; Go, Minjoung M.; Czerwinski, Debra; Meyer, Adam J.

    2008-01-01

    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 G1 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. PMID:18212055

  17. 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

    2015-07-01

    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, 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. PMID:25947942

  18. 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

  19. Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin–radixin–moesin (ERM) proteins

    PubMed Central

    Erwig, Lars-Peter; McPhilips, Kathleen A.; Wynes, Murray W.; Ivetic, Alexander; Ridley, Anne J.; Henson, Peter M.

    2006-01-01

    Deletion of apoptotic cells from tissues involves their phagocytosis by macrophages, dendritic cells, and tissue cells. Although much attention has been focused on the participating ligands, receptors, and mechanisms of uptake, little is known of the disposition of the ingested cell within the phagosome. Here we show that uptake of apoptotic cells by macrophages or fibroblasts results in rapid phagosome maturation, whereas macrophage phagosomes containing Ig-opsonized target cells mature at a slower rate. The early maturation was shown to depend on activation of Rho acting through Rho kinase on ezrin–radixin–moesin proteins. Blockade of Rho signaling or inhibition of moesin both delayed maturation rates to those seen with opsonized targets. By contrast, phagosome maturation in dendritic cells was slower, similar between apoptotic and opsonized target cells, and unaffected by Rho inhibition. These observations have direct implications for the clearance of dying cells and the roles played by different phagocytes in antigen digestion and presentation. PMID:16908865

  20. 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

  1. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  2. 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.

  3. Adenomatous Polyposis Coli Protein Deletion in Efferent Olivocochlear Neurons Perturbs Afferent Synaptic Maturation and Reduces the Dynamic Range of Hearing

    PubMed Central

    Hickman, Tyler T.; Liberman, M. Charles

    2015-01-01

    Normal hearing requires proper differentiation of afferent ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) that carry acoustic information to the brain. Within individual IHCs, presynaptic ribbons show a size gradient with larger ribbons on the modiolar face and smaller ribbons on the pillar face. This structural gradient is associated with a gradient of spontaneous rates and threshold sensitivity, which is essential for a wide dynamic range of hearing. Despite their importance for hearing, mechanisms that direct ribbon differentiation are poorly defined. We recently identified adenomatous polyposis coli protein (APC) as a key regulator of interneuronal synapse maturation. Here, we show that APC is required for ribbon size heterogeneity and normal cochlear function. Compared with wild-type littermates, APC conditional knock-out (cKO) mice exhibit decreased auditory brainstem responses. The IHC ribbon size gradient is also perturbed. Whereas the normal-developing IHCs display ribbon size gradients before hearing onset, ribbon sizes are aberrant in APC cKOs from neonatal ages on. Reporter expression studies show that the CaMKII-Cre used to delete the floxed APC gene is present in efferent olivocochlear (OC) neurons, not IHCs or SGNs. APC loss led to increased volumes and numbers of OC inhibitory dopaminergic boutons on neonatal SGN fibers. Our findings identify APC in efferent OC neurons as essential for regulating ribbon heterogeneity, dopaminergic terminal differentiation, and cochlear sensitivity. This APC effect on auditory epithelial cell synapses resembles interneuronal and nerve–muscle synapses, thereby defining a global role for APC in synaptic maturation in diverse cell types. Significance Statement This study identifies novel molecules and cellular interactions that are essential for the proper maturation of afferent ribbon synapses in sensory cells of the inner ear, and for normal hearing. PMID:26085645

  4. 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. PMID:27020565

  5. 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

  6. 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. PMID:12676749

  7. 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. PMID:24862193

  8. ARC Syndrome-Linked Vps33B Protein Is Required for Inflammatory Endosomal Maturation and Signal Termination.

    PubMed

    Akbar, Mohammed Ali; Mandraju, Rajakumar; Tracy, Charles; Hu, Wei; Pasare, Chandrashekhar; Krämer, Helmut

    2016-08-16

    Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. Here, we report that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of ARC syndrome, a disease linked to loss of Vps33B. PMID:27496733

  9. PRL-3 mediates the protein maturation of ULBP2 by regulating the tyrosine phosphorylation of HSP60

    PubMed Central

    Leung, Wai-Hang; Vong, Queenie P.; Lin, Wenwei; Bouck, David; Wendt, Susanne; Sullivan, Erin; Li, Ying; Bari, Rafijul; Chen, Taosheng; Leung, Wing

    2015-01-01

    Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by natural killer cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors however is limited because of adverse side effects. Using high throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding but rather suppressed post-translational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3. PMID:25687758

  10. BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells.

    PubMed

    Iguchi, Tomohiro; Aoki, Kazuhisa; Ikawa, Tomokatsu; Taoka, Masato; Taya, Choji; Yoshitani, Hiroshi; Toma-Hirano, Makiko; Koiwai, Osamu; Isobe, Toshiaki; Kawamoto, Hiroshi; Masai, Hisao; Miyatake, Shoichiro

    2015-08-01

    Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1). PMID:26136427

  11. The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export.

    PubMed

    Gannon, P M; Li, P; Kumamoto, C A

    1989-02-01

    The product of the secB gene is required for export of a subset of secreted proteins to the outer membrane and periplasm of Escherichia coli. Precursor maltose-binding protein (MBP) accumulates in the cytoplasm of secB-carrying mutants, but export of alkaline phosphatase is only minimally affected by secB mutations. When export of MBP-alkaline phosphatase hybrid proteins was analyzed in wild-type and secB-carrying mutant strains, the first third of mature MBP was sufficient to render export of the hybrid proteins dependent on SecB. Substitution of a signal sequence from a SecB-independent protein had no effect on SecB-dependent export. These findings show that the first third of mature MBP is capable of conferring export incompetence on an otherwise competent protein. PMID:2644237

  12. Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation*

    PubMed Central

    Fernández-Pevida, Antonio; Rodríguez-Galán, Olga; Díaz-Quintana, Antonio; Kressler, Dieter; de la Cruz, Jesús

    2012-01-01

    Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process. PMID:22995916

  13. New Insights on the Mechanism of Cyclization in Chromophore Maturation of Wild-Type Green Fluorescence Protein: A Computational Study.

    PubMed

    Ma, Yingying; Zhang, Hao; Sun, Qiao; Smith, Sean C

    2016-06-23

    Cyclization is the first step in the chromophore maturation process of the green fluorescent protein (GFP). In our previous paper [J. Phys. Chem. B 2012, 116, 1426-1436], the results of molecular dynamics simulation suggested the possibility that the amide nitrogen atom of Gly67 attacks the carbonyl carbon of Ser65 directly to complete the cyclization process (one-step mechanism). In this paper, density functional theory (DFT) and quantum mechanical/molecular mechanical (QM/MM) calculations were undertaken to study this step reaction in detail. Three cluster model systems (model A, model B, and model C) and large protein system were set up to investigate the cyclization process. Our results indicate that the one-step mechanism only exists in the two minimum models. However, in model C and the large protein system, the cyclization mechanism involves two steps: the first step is proton of Gly67 amide nitrogen transferring to carbonyl oxygen of Ser65, generating protonated amide, which is stabilized by a hydrogen bond interaction with a crystallographic water molecule, and the second step is Gly67 amide nitrogen attacking the carbonyl carbon of Ser65. Arg96 plays an important role in promoting the cyclization. The energy of cyclized product relative to reactant is about 10.0 kcal/mol endothermic, which is in line with the experimental results. PMID:27232642

  14. Differential maturation of circadian rhythms in clock gene proteins in the suprachiasmatic nucleus and the pars tuberalis during mouse ontogeny

    PubMed Central

    Ansari, Nariman; Agathagelidis, Manuel; Lee, Choogon; Korf, Horst-Werner; von Gall, Charlotte

    2009-01-01

    Circadian rhythms of many body functions in mammals are controlled by a master pacemaker residing in the hypothalamic suprachiasmatic nucleus (SCN) that synchronises peripheral oscillators. The SCN and peripheral oscillators share several components of the molecular clockwork and comprise transcriptional activators (BMAL1 and CLOCK/NPAS2) and inhibitors (mPER1/2 and mCRY1/2). Here we compared the ontogenetic maturation of the clockwork in the SCN and pars tuberalis (PT). The PT is a peripheral oscillator that strongly depends on rhythmic melatonin signals. Immunoreactions for clock gene proteins were determined in the SCN and PT at four different timepoints during four differential stages of mouse ontogeny: foetal (embryonic day 18), newborn (2-day-old), infantile (10-day-old), and adult. In the foetal SCN levels of immunoreactions of all clock proteins were significantly lower as compared to adult levels except for BMAL1. In the newborn SCN the clock protein immunoreactions had not yet reached adult levels, but the infantile SCN showed similar levels of immunreactions as the adult. In contrast, immunoreactions for all clock gene proteins in the foetal PT were as intense as in newborn, infantile, and adult and showed the same phase. As the foetal pineal gland is not yet capable of rhythmic melatonin production, the rhythms in clock gene proteins in the foetal PT are presumably dependent on the maternal melatonin signal. Thus, our data provide the first evidence that maternal melatonin is important for establishing and maintaining circadian rhythms in a foetal peripheral oscillator. PMID:19222558

  15. Differential maturation of circadian rhythms in clock gene proteins in the suprachiasmatic nucleus and the pars tuberalis during mouse ontogeny.

    PubMed

    Ansari, Nariman; Agathagelidis, Manuel; Lee, Choogon; Korf, Horst-Werner; von Gall, Charlotte

    2009-02-01

    Circadian rhythms of many body functions in mammals are controlled by a master pacemaker, residing in the hypothalamic suprachiasmatic nucleus (SCN), which synchronises peripheral oscillators. The SCN and peripheral oscillators share several components of the molecular clockwork and comprise transcriptional activators (BMAL1 and CLOCK/NPAS2) and inhibitors (mPER1/2 and mCRY1/2). Here we compared the ontogenetic maturation of the clockwork in the SCN and pars tuberalis (PT). The PT is a peripheral oscillator that strongly depends on rhythmic melatonin signals. Immunoreactions for clock gene proteins were determined in the SCN and PT at four different timepoints during four differential stages of mouse ontogeny: foetal (embryonic day 18), newborn (2-day-old), infantile (10-day-old), and adult. In the foetal SCN, levels of immunoreactions of all clock proteins were significantly lower than adult levels except for BMAL1. In the newborn SCN the clock protein immunoreactions had not yet reached adult levels, but the infantile SCN showed similar levels of immunoreactions as the adult. In contrast, immunoreactions for all clock gene proteins in the foetal PT were as intense as in newborn, infantile and adult, and showed the same phase. As the foetal pineal gland is not yet capable of rhythmic melatonin production, the rhythms in clock gene proteins in the foetal PT are presumably dependent on the maternal melatonin signal. Thus, our data provide the first evidence that maternal melatonin is important for establishing and maintaining circadian rhythms in a foetal peripheral oscillator. PMID:19222558

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

    PubMed

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

    2014-11-01

    Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO = 0.60 ± 0.11, control = 1.07 ± 0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO = 0.95 ± 0.14, control = 1.05 ± 0.16) and TNF-α (rhEPO = 0.73 ± 0.20, control = 1.01 ± 0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle. PMID:25296358

  17. Uncovering RNA binding proteins associated with age and gender during liver maturation

    PubMed Central

    Chaturvedi, Praneet; Neelamraju, Yaseswini; Arif, Waqar; Kalsotra, Auinash; Janga, Sarath Chandra

    2015-01-01

    In the present study, we perform an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in mice confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are expressed higher in males than females. Altogether, these observations show that several of these RBPs are important and conserved regulators in maintaining liver function. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in aging liver and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes. PMID:25824884

  18. 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

  19. 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. PMID:25861868

  20. Gene Expression and Polymorphism of Myostatin Gene and its Association with Growth Traits in Chicken.

    PubMed

    Dushyanth, K; Bhattacharya, T K; Shukla, R; Chatterjee, R N; Sitaramamma, T; Paswan, C; Guru Vishnu, P

    2016-10-01

    Myostatin is a member of TGF-β super family and is directly involved in regulation of body growth through limiting muscular growth. A study was carried out in three chicken lines to identify the polymorphism in the coding region of the myostatin gene through SSCP and DNA sequencing. A total of 12 haplotypes were observed in myostatin coding region of chicken. Significant associations between haplogroups with body weight at day 1, 14, 28, and 42 days, and carcass traits at 42 days were observed across the lines. It is concluded that the coding region of myostatin gene was polymorphic, with varied levels of expression among lines and had significant effects on growth traits. The expression of MSTN gene varied during embryonic and post hatch development stage. PMID:27565871

  1. 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

  2. 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

  3. 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

  4. Muscle-specific transgenic expression of porcine myostatin propeptide enhances muscle growth in mice.

    PubMed

    Wang, Kaiyun; Li, Zicong; Li, Yang; Zeng, Jinyong; He, Chang; Yang, Jinzeng; Liu, Dewu; Wu, Zhenfang

    2013-10-01

    Myostatin is a well-known negative regulator of skeletal muscle growth. Inhibition of myostatin activity results in increased muscle mass. Myostatin propeptide, as a myostatin antagonist, could be applied to promote meat production in livestock such as pigs. In this study, we generated a transgenic mouse model expressing porcine myostatin propeptide under the control of muscle-specific regulatory elements. The mean body weight of transgenic mice from a line expressing the highest level of porcine myostatin propeptide was increased by 5.4 % (P = 0.023) and 3.2 % (P = 0.031) in males and females, respectively, at 8 weeks of age. Weight of carcass, fore limb and hind limb was respectively increased by 6.0 % (P = 0.038), 9.0 % (P = 0.014), 8.7 % (P = 0.036) in transgenic male mice, compared to wild-type male controls at the age of 9 weeks. Similarly, carcass, fore limb and hind limb of transgenic female mice was 11.4 % (P = 0.002), 14.5 % (P = 0.006) and 14.5 % (P = 0.03) respectively heavier than that of wild-type female mice. The mean cross-section area of muscle fiber was increased by 17 % (P = 0.002) in transgenic mice, in comparison with wild-type controls. These results demonstrated that porcine myostatin propeptide is effective in enhancement of muscle growth. The present study provided useful information for future study on generation of transgenic pigs overexpressing porcine myostatin propeptide for improvement of muscle mass. PMID:23543410

  5. Higher Plasma Myostatin Levels in Cor Pulmonale Secondary to Chronic Obstructive Pulmonary Disease

    PubMed Central

    Ju, Chun-rong; Chen, Miao; Zhang, Jian-heng; Lin, Zhi-ya; Chen, Rong-chang

    2016-01-01

    Objective To analyze plasma myostatin levels and investigate their relationship with right ventricular (RV) function in patients with cor pulmonale secondary to chronic obstructive pulmonary disease (COPD). Methods The study recruited 81 patients with advanced COPD and 40 age-matched controls. The patients were divided into two groups: those with cor pulmonale and those without. Echocardiography was used to evaluate RV function and morphology, and the value of tricuspid annular plane systolic excursion (TAPSE) less than 16 mm was considered RV dysfunction. Plasma myostatin levels were analyzed by enzyme-linked immunosorbent assay, and B-type natriuretic peptide (BNP) levels were analyzed as a comparison of myostatin. Results The data detected cor pulmonale in 39/81 patients, with the mean value of TAPSE of 14.3 mm. Plasma myostatin levels (ng/mL) were significantly higher in patients with cor pulmonale (16.68 ± 2.95) than in those without (13.56 ± 3.09), and much higher than in controls (8.79±2.79), with each p<0.01. Significant differences were also found in plasma BNP levels among the three groups (p<0.05). Multivariate regression analysis suggested that myostatin levels were significantly correlated with the values of TAPSE and RV myocardium performance index among the COPD patients, and that BNP levels were significantly correlated only with systolic pulmonary arterial pressure, with each p<0.05. Conclusions Plasma myostatin levels are increased in COPD patients who have cor pulmonale. Stronger correlations of plasma myostatin levels with echocardiographic indexes of the right heart suggest that myostatin might be superior to BNP in the early diagnosis of cor pulmonale in COPD. PMID:26998756

  6. 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

  7. Myostatin represses physiological hypertrophy of the heart and excitation–contraction coupling

    PubMed Central

    Rodgers, Buel D; Interlichia, Jillian P; Garikipati, Dilip K; Mamidi, Ranganath; Chandra, Murali; Nelson, O Lynne; Murry, Charles E; Santana, Luis F

    2009-01-01

    Although myostatin negatively regulates skeletal muscle growth, its function in heart is virtually unknown. Herein we demonstrate that it inhibits basal and IGF-stimulated proliferation and differentiation and also modulates cardiac excitation–contraction (EC) coupling. Loss of myostatin induced eccentric hypertrophy and enhanced cardiac responsiveness to β-adrenergic stimulation in vivo. This was due to myostatin null ventricular myocytes having larger [Ca2+]i transients and contractions and responding more strongly to β-adrenergic stimulation than wild-type cells. Enhanced cardiac output and β-adrenergic responsiveness of myostatin null mice was therefore due to increased SR Ca2+ release during EC coupling and to physiological hypertrophy, but not to enhanced myofilament function as determined by simultaneous measurement of force and ATPase activity. Our studies support the novel concept that myostatin is a repressor of physiological cardiac muscle growth and function. Thus, the controlled inhibition of myostatin action could potentially help repair damaged cardiac muscle by inducing physiological hypertrophy. PMID:19736304

  8. 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. PMID:27235683

  9. 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

  10. Dual roles of palladin protein in in vitro myogenesis: inhibition of early induction but promotion of myotube maturation.

    PubMed

    Nguyen, Ngoc-Uyen-Nhi; Wang, Hao-Ven

    2015-01-01

    Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro. PMID:25875253

  11. 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

  12. Regulation of GDF-11 and myostatin activity by GASP-1 and GASP-2.

    PubMed

    Lee, Yun-Sil; Lee, Se-Jin

    2013-09-24

    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

  13. 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

  14. 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

  15. P22 coat protein structures reveal a novel mechanism for capsid maturation: Stability without auxiliary proteins or chemical cross-links

    PubMed Central

    Parent, Kristin N.; Khayat, Reza; Tu, Long H.; Suhanovsky, Margaret M.; Cortines, Juliana R.; Teschke, Carolyn M.; Johnson, John E.; Baker, Timothy S.

    2010-01-01

    Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical cross-links (unique to HK97), or auxiliary proteins (lambda, T4, ϕ29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold-labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudo-atomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from inter-subunit interactions among N-terminal helices and an extensive “P-loop”, which obviate the need for cross-links or auxiliary proteins. P22 CP also has a telokin-like, Ig domain that stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like ϕ29 and HSV-1. PMID:20223221

  16. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    PubMed Central

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  17. [Role of Activin A and Myostatin in cancer cachexia].

    PubMed

    Thissen, Jean-Paul; Loumaye, Audrey

    2013-05-01

    Recent works suggest that Activin A (ActA) and Myostatin (Mstn), two members of the TGFβ superfamily, could contribute to skeletal muscle atrophy observed in some cancers. It is known that several human tumoral cell lines synthesize and secrete ActA and Mstn. In addition, systemic treatment with ActA and Mstn in mice induce muscle atrophy. Likewise, Inhibin-α knock-out mice, which are characterized by elevated circulating levels of ActA, exhibit muscle atrophy and die of cachexia. Finally, administration of ActA and Mstn antagonists prevents muscular atrophy and mortality induced by some animal tumors. Collectively, these findings suggest that ActA or Mstn production by several cancers could contribute to cachexia and thus to mortality associated with some cancers in human. This hypothesis is very interesting since new molecules that are able to inhibit ActA and Mstn, in particularly the sActRIIB, are under development. PMID:23566617

  18. 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. PMID:25878062

  19. Association of myostatin on early calf mortality, growth, and carcass composition traits in crossbred cattle.

    PubMed

    Casas, E; Bennett, G L; Smith, T P L; Cundiff, L V

    2004-10-01

    The objective of this study was to investigate a potential association of an inactive myostatin allele with early calf mortality, and evaluate its effect on growth and carcass traits in a crossbred population. Animals were obtained by mating F1 cows to F1 (Belgian Blue x British Breed) or Charolais sires. Cows were obtained from mating Hereford, Angus, and MARC III (1/4 Hereford, 1/4 Angus, 1/4 Pinzgauer, and 1/4 Red Poll) dams to Hereford, Angus, Tuli, Boran, Brahman, or Belgian Blue sires. Belgian Blue was the source of the inactive myostatin allele. Myostatin genotypes were determined for all animals including those that died before weaning. Early calf mortality was examined in the F2 subpopulation (n = 154), derived from the F1 sires mated to F1 cows from Belgian Blue sires, to evaluate animals with zero, one, or two copies of inactive myostatin allele. An overall 1:2:1 ratio (homozygous active myostatin allele:heterozygous:homozygous inactive myostatin allele) was observed in the population; however, a comparison between calves dying before weaning and those alive at slaughter showed an unequal distribution across genotypes (P < 0.01). Calves with two copies of the inactive allele were more likely (P < 0.01) to die before weaning. Postweaning growth traits were evaluated in the surviving animals (n = 1,370), including birth, weaning, and live weight at slaughter, and postweaning ADG. Carcass composition traits analyzed were hot carcass weight, fat thickness, LM area, marbling score, USDA yield grade, estimated kidney, pelvic, and heart fat, retail product yield and weight, fat yield and weight, bone yield and weight, and percentage of carcasses classified as Choice. Charolais lack the inactive myostatin allele segregating in Belgian Blue; thus, in the population sired by Charolais (n = 645), only animals with zero or one copy of the inactive myostatin allele were evaluated. Animals carrying one copy were heavier at birth and at weaning, and their carcasses

  20. 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

  1. Alcohol-induced decrease in muscle protein synthesis associated with increased binding of mTOR and raptor: Comparable effects in young and mature rats

    PubMed Central

    Lang, Charles H; Pruznak, Anne M; Nystrom, Gerald J; Vary, Thomas C

    2009-01-01

    Background Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication. Methods Male F344 rats were studied at approximately 3 (young) or 12 (mature) months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [3H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation. Results Blood alcohol levels (BALs) were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL). However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL). EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E·eIF4G and inactive eIF4E·4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6 and eIF4G was decreased

  2. Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

    PubMed Central

    2011-01-01

    Background Enzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses. Results An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such as N,N-dimethyl-4

  3. Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

    PubMed Central

    Long, K S; Cedervall, T; Walch-Solimena, C; Noe, D A; Huddleston, M J; Annan, R S; Wolin, S L

    2001-01-01

    An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein. PMID:11720288

  4. Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes.

    PubMed

    Demond, Hannah; Trapphoff, Tom; Dankert, Deborah; Heiligentag, Martyna; Grümmer, Ruth; Horsthemke, Bernhard; Eichenlaub-Ritter, Ursula

    2016-01-01

    Delayed ovulation and delayed fertilization can lead to reduced developmental competence of the oocyte. In contrast to the consequences of postovulatory aging of the oocyte, hardly anything is known about the molecular processes occurring during oocyte maturation if ovulation is delayed (preovulatory aging). We investigated several aspects of oocyte maturation in two models of preovulatory aging: an in vitro follicle culture and an in vivo mouse model in which ovulation was postponed using the GnRH antagonist cetrorelix. Both models showed significantly reduced oocyte maturation rates after aging. Furthermore, in vitro preovulatory aging deregulated the protein abundance of the maternal effect genes Smarca4 and Nlrp5, decreased the levels of histone H3K9 trimethylation and caused major deterioration of chromosome alignment and spindle conformation. Protein abundance of YBX2, an important regulator of mRNA stability, storage and recruitment in the oocyte, was not affected by in vitro aging. In contrast, in vivo preovulatory aging led to reduction in Ybx2 transcript and YBX2 protein abundance. Taken together, preovulatory aging seems to affect various processes in the oocyte, which could explain the low maturation rates and the previously described failures in fertilization and embryonic development. PMID:27611906

  5. Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm.

    PubMed Central

    Chiba, K; Kontani, K; Tadenuma, H; Katada, T; Hoshi, M

    1993-01-01

    beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase. Images PMID:8298189

  6. 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. PMID:16162390

  7. 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

  8. Depletion of Myostatin b Promotes Somatic Growth and Lipid Metabolism in Zebrafish.

    PubMed

    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

  9. 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

  10. 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

  11. Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth.

    PubMed

    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

  12. 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.

  13. Myostatin antibody (LY2495655) in older weak fallers: a proof-of-concept, randomised, phase 2 trial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Myostatin suppression of Akirin1 mediates glucocorticoid-induced satellite cell dysfunction.

    PubMed

    Dong, Yanjun; Pan, Jenny S; Zhang, Liping

    2013-01-01

    Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production. PMID:23516508

  15. Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

    PubMed Central

    Dong, Yanjun; Pan, Jenny S.; Zhang, Liping

    2013-01-01

    Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production. PMID:23516508

  16. Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration.

    PubMed

    Ramachandran, Mohanraj; Jin, Chuan; Yu, Di; Eriksson, Fredrik; Essand, Magnus

    2014-09-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. Both HP-NAP protein and oncolytic viruses encoding HP-NAP have been suggested as immunotherapeutic anticancer agents and adjuvants for vaccination but with little known about its mode of action to activate adaptive immunity. Dendritic cells (DCs) are key players in bridging innate and adaptive immune responses, and in this study we aim to evaluate the effect of HP-NAP on DC maturation, migration, and induction of adaptive immune response. Maturation markers CD83, CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and relatively low IL-10 secretion, and migrated toward CCL19. Ag-specific T cells were efficiently expanded by Ag-presenting HP-NAP-activated DCs, which is an important property of functionally mature DCs. Furthermore, intradermal injections of HP-NAP-encoding adenovirus in C57BL/6 mice enhanced resident DC migration to draining lymph nodes, which was verified by imaging lymph nodes by two-photon microscopy and by phenotyping migrating cells by flow cytometry. In conclusion, therapeutic effects of HP-NAP are mediated by maturation of DCs and subsequent activation of Ag-specific T cells in addition to provoking innate immunity. PMID:25049358

  17. Ablation of the cellular prion protein, PrPC, specifically on follicular dendritic cells has no effect on their maturation or function.

    PubMed

    McCulloch, Laura; Brown, Karen L; Mabbott, Neil A

    2013-03-01

    Follicular dendritic cells (FDC) are situated in the primary follicles of lymphoid tissues where they maintain the structural integrity of the B-lymphocyte follicle, and help to drive immunoglobulin class-switch recombination, somatic hypermutation and affinity maturation during the germinal centre response. FDC can also provide a reservoir for pathogens that infect germinal centres including HIV and prions. FDC express high levels of the normal cellular form of the prion protein (PrP(C) ), which makes them susceptible to prion infection. The function of PrP(C) is uncertain and it is not known why FDC require such high levels of expression of a protein that is found mainly on cells of the central nervous system. In this study, the function of FDC was assessed in mice that had PrP(C) ablated specifically in their FDC. In mice with FDC-specific PrP(C) ablation, our analysis revealed no observable deficits in lymphoid follicle microarchitecture and FDC status. No effects on FDC ability to trap immune complexes or drive antigen-specific antibody responses and affinity maturation in B lymphocytes were observed. These data clearly demonstrate that PrP(C) expression is dispensable for the functional maturation of FDC and their ability to maintain antigen-specific antibody responses and affinity maturation. PMID:23121447

  18. Effects of threonine supplementation on whole-body protein synthesis and plasma metabolites in growing and mature horses.

    PubMed

    Mastellar, S L; Moffet, A; Harris, P A; Urschel, K L

    2016-01-01

    Current equine threonine requirement estimates do not account for probable use of threonine to maintain gut health and mucin synthesis. The objective of this study was to determine if threonine supplementation (+Thr) would increase whole-body protein synthesis (WBPS) in weanling colts (Study 1) and adult mares (Study 2). Both studies used a crossover design, where each of six animals was studied twice while receiving the isonitrogenous diets. The basal diets contained lower threonine levels (Basal) than the threonine (+Thr) supplemented diets. Threonine intakes in mg/kg BW/day were as follows: 79 (Basal) and 162 (+Thr) for Study 1 and 58 (Basal) and 119 (+Thr) for Study 2, in comparison to the NRC estimated requirements of 81 and 33 mg/kg BW/day for weanling and mature horses, respectively. Following 5 days of adaptation, blood samples were taken before and 90 min after the morning concentrate meal. The next day, whole-body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C]sodium bicarbonate followed by a 4 h primed, constant infusion of [1-(13)C]phenylalanine. Most plasma amino acid (AA) concentrations were elevated post-feeding (P < 0.01). Lysine and valine plasma concentrations were lower (P <0.10), while methionine, threonine, and glycine plasma concentrations were greater (P <0.10) 90 min post concentrate meal feeding with +Thr in both studies. Phenylalanine flux, intake, oxidation and non-oxidative disposal were similar between treatments (P > 0.05). These findings suggest that supplementation of a single AA can affect the metabolism of several AAs and threonine was not a limiting AA in these diets. PMID:26670331

  19. The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma.

    PubMed

    Li, Bingzong; Fu, Jinxiang; Chen, Ping; Ge, Xueping; Li, Yali; Kuiatse, Isere; Wang, Hua; Wang, Huihan; Zhang, Xingding; Orlowski, Robert Z

    2015-12-11

    Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor. PMID:26483548

  20. Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice.

    PubMed

    Dankbar, Berno; Fennen, Michelle; Brunert, Daniela; Hayer, Silvia; Frank, Svetlana; Wehmeyer, Corinna; Beckmann, Denise; Paruzel, Peter; Bertrand, Jessica; Redlich, Kurt; Koers-Wunrau, Christina; Stratis, Athanasios; Korb-Pap, Adelheid; Pap, Thomas

    2015-09-01

    Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA. PMID:26236992

  1. 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

  2. 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. PMID:26950874

  3. 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

  4. Controlled expression of nif and isc iron-sulfur protein maturation components reveals target specificity and limited functional replacement between the two systems.

    PubMed

    Dos Santos, Patricia C; Johnson, Deborah C; Ragle, Brook E; Unciuleac, Mihaela-Carmen; Dean, Dennis R

    2007-04-01

    The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS. PMID:17237162

  5. Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

    PubMed Central

    Kuznetsov, G; Bush, K T; Zhang, P L; Nigam, S K

    1996-01-01

    The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment

  6. High-level soluble expression of bioactive porcine myostatin propeptide in E. coli.

    PubMed

    Haq, Wing Yeung; Kang, Sang Kee; Lee, Sang Beum; Kang, Hee Chul; Choi, Yun Jaie; Lee, Chin Nyean; Kim, Yong Soo

    2013-10-01

    Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal part of unprocessed MSTN that is cleaved off during posttranslational MSTN processing. Easy availability of MSTNPro would help to investigate the potential of the protein as an agent to enhance muscle growth in agricultural animal species. Thus, this study was designed to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the BMP-1/TLD proteolytic cleavage site (pMSTNProM) in Escherichia coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene and were transformed and expressed in soluble forms in E. coli. For each milliliter of cell culture, about 40 μg of soluble MBP-pMSTNProW and MBP-pMSTNProM proteins were purified by amylose resin affinity chromatography. Further purification by anion exchange chromatography of the affinity-purified fractions yielded about 10 μg/mL culture of MBP-pMSTNProW and MBP-pMSTNProM proteins. Factor Xa protease cleaved the fusion partner MBP from MBP-pMSTNPro proteins, and approximately 4.2 μg of pMSTNProW and pMSTNProM proteins were purified per milliliter of culture. MBP-pMSTNProM was resistant to digestion by BMP-1 metalloproteinase, while MBP-pMSTNProW was cleaved into two fragments by BMP-1. Both MBP-pMSTNProW and MBP-pMSTNProM demonstrated their MSTN binding affinities in a pulldown assay. In an in vitro gene reporter assay, both proteins inhibited MSTN bioactivity without a significant difference in their inhibitory capacities, indicating that the cell culture-based gene reporter assay has limitation in detecting the true in vivo biological potencies of mutant forms of MSTNPro proteins at the BMP-1/TLD cleavage site. Current results show that a high-level production of bioactive porcine MSTNpro is possible in E. coli, and it remains to be investigated

  7. 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. PMID:25695746

  8. Epidermal growth factor-mediated mitogen-activated protein kinase3/1 pathway is conducive to in vitro maturation of sheep oocytes.

    PubMed

    Ni, Hemin; Sheng, Xihui; Cui, Xu; Gu, Meichao; Liu, Yunhai; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2015-01-01

    Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo's capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10-6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes. PMID:25799554

  9. Epidermal Growth Factor-Mediated Mitogen-Activated Protein Kinase3/1 Pathway Is Conducive to In Vitro Maturation of Sheep Oocytes

    PubMed Central

    Cui, Xu; Gu, Meichao; Liu, Yunhai; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2015-01-01

    Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10−4 mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10−6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10−4 mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10−6 mol/l U0126 group and 42.6% in 10−4 mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10−4 mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes. PMID:25799554

  10. A Catalog of Proteins Expressed in the AG Secreted Fluid during the Mature Phase of the Chinese Mitten Crabs (Eriocheir sinensis)

    PubMed Central

    He, Lin; Li, Qing; Liu, Lihua; Wang, Yuanli; Xie, Jing; Yang, Hongdan; Wang, Qun

    2015-01-01

    The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database (http://www.ncbi.nlm.nih.gov/) and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction. PMID:26305468

  11. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  12. Highly Specific Detection of Myostatin Prodomain by an Immunoradiometric Sandwich Assay in Serum of Healthy Individuals and Patients

    PubMed Central

    Widera, Christian; Gottlieb, Jens; Vogel, Arndt; Schmidt, Sebastian; Brandes, Gudrun; Heuft, Hans-Gert; Lichtinghagen, Ralf; Kempf, Tibor; Wollert, Kai C.; Bauersachs, Johann; Heineke, Joerg

    2013-01-01

    Background Myostatin is a muscle derived factor that functions as a negative regulator of skeletal muscle growth. Induction of myostatin expression was observed in rodent models of muscle wasting and in cachectic patients with cancer or pulmonary disease. Therefore, there is an increasing interest to use serum myostatin as a biomarker. Methods We established an immunoradiometric sandwich assay (IRMA), which uses a commercially available chicken polyclonal, affinity purified antibody directed against human myostatin prodomain. We determined the serum concentrations of myostatin prodomain in 249 healthy individuals as well as 169 patients with heart failure, 53 patients with cancer and 44 patients with chronic pulmonary disease. Results The IRMA had a detection limit of 0.7ng/ml, an intraassay imprecision of ≤14.1% and an interassay imprecision of ≤ 18.9%. The specificity of our assay was demonstrated by size exclusion chromatography, detection of myostatin by Western-blotting and a SMAD-dependent transcriptional-reporter assay in the signal-rich serum fractions, as well as lack of interference by unspecific substances like albumin, hemoglobin or lipids. Myostatin prodomain was stable at room temperature and resistant to freeze-thaw cycles. Apparently healthy individuals over the age of 55 had a median myostatin prodomain serum concentration of 3.9ng/ml (25th-75th percentiles, 2-7ng/ml) and we could not detect increased levels in patients with stable chronic heart failure or cancer related weight loss. In contrast, we found strongly elevated concentrations of myostatin prodomain (median 26.9ng/ml, 25th-75th percentiles, 7-100ng/ml) in the serum of underweight patients with chronic pulmonary disease. Conclusions We established a highly specific IRMA for the quantification of myostatin prodomain concentration in human serum. Our assay could be useful to study myostatin as a biomarker for example in patients with chronic pulmonary disease, as we detected highly

  13. The critical role of myostatin in differentiation of sheep myoblasts

    SciTech Connect

    Liu, Chenxi; Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng; Ge, Yubin; Liu, Mingjun

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Identification of the effective and specific shRNA to knockdown MSTN. Black-Right-Pointing-Pointer Overexpression of MSTN reversibly suppressed myogenic differentiation. Black-Right-Pointing-Pointer shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. Black-Right-Pointing-Pointer MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. Black-Right-Pointing-Pointer Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  14. Endocranial and masticatory muscle volumes in myostatin-deficient mice

    PubMed Central

    Jeffery, Nathan; Mendias, Christopher

    2014-01-01

    Structural and functional trade-offs are integral to the evolution of the mammalian skull and its development. This paper examines the potential for enlargement of the masticatory musculature to limit the size of the endocranial cavity by studying a myostatin-deficient mouse model of hypermuscularity (MSTN−/−). The study tests the null prediction that the larger MSTN−/− mice have larger brains compared with wild-type (WT) mice in order to service the larger muscles. Eleven post-mortem MSTN−/− mice and 12 WT mice were imaged at high resolution using contrast enhanced micro-CT. Masticatory muscle volumes (temporalis, masseter, internal and external pterygoids) and endocranial volumes were measured on the basis of two-dimensional manual tracings and the Cavalieri principle. Volumes were compared using Kruskal–Wallis and Student's t-tests. Results showed that the masticatory muscles of the MSTN−/− mice were significantly larger than in the WT mice. Increases were in the region of 17–36% depending on the muscle. Muscles increased in proportion to each other, maintaining percentages in the region of 5, 10, 21 and 62% of total muscle volume for the external ptyergoid, internal pterygoid, temporalis and masseter, respectively. Kruskal–Wallis and t-tests demonstrated that the endocranial volume was significantly larger in the WT mice, approximately 16% larger on average than that seen in the MSTN−/− mice. This comparative reduction of MSTN−/− endocranial size could not be explained in terms of observer bias, ageing, sexual dimorphism or body size scaling. That the results showed a reduction of brain size associated with an increase of muscle size falsifies the null prediction and lends tentative support to the view that the musculature influences brain growth. It remains to be determined whether the observed effect is primarily physical, nutritional, metabolic or molecular in nature. PMID:26064569

  15. Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

    PubMed

    Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

    2016-04-19

    Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. PMID:26954624

  16. Extreme muscle development in sheep heterozygous for both myostatin and callipyge mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two mutations causing increased muscle size and decreased fat content in sheep have been described. The callipyge (CLPG) syndrome is only exhibited after 4 to 6 weeks of age in animals inheriting the mutation solely from their sire. In contrast, a mutation of the myostatin gene (MSTN) in the Texel...

  17. Age Related Changes in Craniofacial Morphology in GDF-8 (Myostatin) Deficient Mice

    PubMed Central

    Vecchione, Lisa; Miller, Jeffrey; Byron, Craig; Cooper, Gregory M.; Barbano, Timothy; Cray, James; Losee, Joseph E.; Hamrick, Mark W.; Sciote, James J.; Mooney, Mark P.

    2011-01-01

    It is well recognized that masticatory muscle function helps determine morphology, although the extent of function on final form is still debated. GDF-8 (myostatin), a transcription factor is a negative regulator of skeletal muscle growth. A recent study has shown that mice homozygous for the myostatin mutation had increased muscle mass and craniofacial dysmorphology in adulthood. However, it is unclear whether such dysmorphology is present at birth. This study examines the onset and relationship between hypermuscularity and craniofacial morphology in neonatal and adult mice with GDF-8 deficiency. Fifteen (8 wild-type and 7 GDF-8 −/−), 1 day old and 16 (9 wt and 7 GDF-8 −/−), 180 day old male CD-1 mice were used. Standardized radiographs were taken of each head, scanned, traced, and cephalometric landmarks identified. Significant mean differences were assessed using a group × age, two-way ANOVA. Myostatin-deficient mice had significantly (p<0.01) smaller body and masseter muscle weights and craniofacial skeletons at 1 day of age and significantly greater body and masseter muscle weights at 180 days of age compared to controls. Myostatin-deficient mice showed significantly (p<0.001) longer and “rocker-shaped” mandibles and shorter and wider crania compared to controls at 180 days. Significant correlations were noted between masseter muscle weight and all cephalometric measurements in 180 day old Myostatin-deficient mice. Results suggest in this mouse model, there may be both early systemic skeletal growth deficiencies and later compensatory changes from hypermuscularity. These findings reiterate the role that masticatory muscle function plays on the ontogeny of the cranial vault, base, and most notably the mandible. PMID:19899116

  18. Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebrafish via inactivation of the SMAD signaling pathway.

    PubMed

    Fuentes, Eduardo N; Pino, Katherine; Navarro, Cristina; Delgado, Iselys; Valdés, Juan Antonio; Molina, Alfredo

    2013-12-01

    Myostatin (MSTN) is the main negative regulator of muscle growth and development in vertebrates. In fish, little is known about the molecular mechanisms behind how MSTN inactivation triggers skeletal muscle enhancement, particularly regarding the signaling pathways involved in this process. Moreover, there have not been reports on the biotechnological applications of MSTN and its signal transduction. In this context, zebrafish underwent compensatory growth using fasting and refeeding trials, and MSTN activity was inactivated with dominant negative LAPD76A recombinant proteins during the refeeding period, when a rapid, compensatory muscle growth was observed. Treated fish displayed an overcompensation of growth characterized by higher muscle hypertrophy and growth performance than constantly fed, control fish. Treatment with LAPD76A recombinant proteins triggered inactivation of the SMAD signaling pathway in skeletal muscle, the main signal transduction used by MSTN to achieve its biological actions. Therefore, transient inactivation of MSTN during the compensatory growth of zebrafish led to a decrease in the SMAD signaling pathway in muscle, triggering muscle hypertrophy and finally improving growth performance, thus, zebrafish achieved an overcompensation of growth. The present study shows an attractive strategy for improving muscle growth in a fish species by mixing a classical strategy, such as compensatory growth, and a biotechnological approach, such as the use of recombinant proteins for inhibiting the biological actions of MSTN. The mix of both strategies may represent a method that could be applied in order to improve growth in commercial fish of interest for aquaculture. PMID:24184273

  19. Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

    USGS Publications Warehouse

    Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

    2002-01-01

    Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

  20. Loss of Retinoschisin (RS1) Cell Surface Protein in Maturing Mouse Rod Photoreceptors Elevates the Luminance Threshold for Light-Driven Translocation of Transducin But Not Arrestin

    PubMed Central

    Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A.

    2012-01-01

    Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1–KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1–KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1–KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1–KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1–KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1–KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1–KO mice at P21 but not at P60. Expression of transducin was 15–30% lower in P21 Rs1–KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1–KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1–KO photoreceptors. PMID:22993419

  1. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  2. Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins in human and bovine colostrum and mature milk samples through iTRAQ labeling.

    PubMed

    Yang, Mei; Cong, Min; Peng, Xiuming; Wu, Junrui; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing

    2016-05-18

    Milk fat globule membrane (MFGM) proteins have many functions. To explore the different proteomics of human and bovine MFGM, MFGM proteins were separated from human and bovine colostrum and mature milk, and analyzed by the iTRAQ proteomic approach. A total of 411 proteins were recognized and quantified. Among these, 232 kinds of differentially expressed proteins were identified. These differentially expressed proteins were analyzed based on multivariate analysis, gene ontology (GO) annotation and KEGG pathway. Biological processes involved were response to stimulus, localization, establishment of localization, and the immune system process. Cellular components engaged were the extracellular space, extracellular region parts, cell fractions, and vesicles. Molecular functions touched upon were protein binding, nucleotide binding, and enzyme inhibitor activity. The KEGG pathway analysis showed several pathways, including regulation of the actin cytoskeleton, focal adhesion, neurotrophin signaling pathway, leukocyte transendothelial migration, tight junction, complement and coagulation cascades, vascular endothelial growth factor signaling pathway, and adherens junction. These results enhance our understanding of different proteomes of human and bovine MFGM across different lactation phases, which could provide important information and potential directions for the infant milk powder and functional food industries. PMID:27159491

  3. Development of a small-molecule screening method for inhibitors of cellular response to myostatin and activin A.

    PubMed

    Cash, Jennifer N; Angerman, Elizabeth B; Kirby, R Jason; Merck, Lisa; Seibel, William L; Wortman, Matthew D; Papoian, Ruben; Nelson, Sandra; Thompson, Thomas B

    2013-08-01

    Myostatin, a member of the transforming growth factor (TGF)-β family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-β family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-β family. PMID:23543431

  4. Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

    SciTech Connect

    Yin Huquan; Kim, Youn-Chul; Chung, Young-Suk; Kim, Young-Chul; Shin, Young-Kee; Lee, Byung-Hoon

    2009-04-01

    Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber-DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-{alpha} levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-{alpha}, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c.

  5. Constitutive nuclear factor-kappaB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteins.

    PubMed

    Bureau, Fabrice; Vanderplasschen, Alain; Jaspar, Fabrice; Minner, Frédéric; Pastoret, Paul-Pierre; Merville, Marie-Paule; Bours, Vincent; Lekeux, Pierre

    2002-05-15

    Constitutive nuclear factor kappaB (NF-kappaB) activity protects quiescent mature immune cells from spontaneous apoptosis. Here, we examined whether NF-kappaB exerts its antiapoptotic function in these cells through the control of Bcl-2 family proteins. Specific pharmacologic inhibitors of NF-kappaB were used to achieve total NF-kappaB inactivation in quiescent human blood lymphocytes, granulocytes, and monocytes. NF-kappaB inhibition induced drastic lymphocyte and granulocyte apoptosis, but only moderate monocyte apoptosis. T- and B-cell apoptosis was slow and associated with a gradual down-regulation of the prosurvival Bcl-2 family proteins Bcl-x(L) and Bcl-2, respectively. By contrast, granulocyte apoptosis was fast and accompanied by a rapid cellular accumulation of Bcl-x(S), the proapoptotic Bcl-x isoform that is generated from alternative splicing of the bcl-x pre-mRNA. Finally, antisense bcl-x(L) and bcl-2 knockdown in T and B cells, respectively, and induction of Bcl-x(S) expression in granulocytes through antisense oligonucleotide-mediated redirection of bcl-x pre-mRNA splicing were sufficient to induce significant apoptosis in these cells. Taken together, these results reveal that basal NF-kappaB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes through regulation of distinct members of the Bcl-2 family. This study sheds light on the constitutive mechanisms by which NF-kappaB maintains defense integrity. PMID:11986224

  6. Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

    PubMed

    Chen, Wei; Bao, Yige; Chen, Xuerong; Burton, Jeremy; Gong, Xueli; Gu, Dongqing; Mi, Youjun; Bao, Lang

    2016-04-01

    Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance. PMID:26318856

  7. Preparation of the cortical reaction: maturation-dependent migration of SNARE proteins, clathrin, and complexin to the porcine oocyte's surface blocks membrane traffic until fertilization.

    PubMed

    Tsai, Pei-Shiue; van Haeften, Theo; Gadella, Bart M

    2011-02-01

    The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles. PMID:20944080

  8. The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus.

    PubMed

    Mashruwala, Ameya A; Bhatt, Shiven; Poudel, Saroj; Boyd, Eric S; Boyd, Jeffrey M

    2016-08-01

    Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins. PMID:27517714

  9. The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus

    PubMed Central

    Bhatt, Shiven; Poudel, Saroj; Boyd, Eric S.; Boyd, Jeffrey M.

    2016-01-01

    Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins. PMID:27517714

  10. Role of a redox-based methylation switch in mRNA life cycle (pre- and post-transcriptional maturation) and protein turnover: implications in neurological disorders.

    PubMed

    Trivedi, Malav S; Deth, Richard C

    2012-01-01

    Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation, and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition, and behavior (Cajigas et al., 2010). Thus a regulatory switch, which controls the lifespan, maturation, and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at (1) the pre-transcription level, by regulating precursor-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and (2) the post-transcription level by modulating the regulatory functions of ribonucleoproteins and RNA binding proteins in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of

  11. Role of a Redox-Based Methylation Switch in mRNA Life Cycle (Pre- and Post-Transcriptional Maturation) and Protein Turnover: Implications in Neurological Disorders

    PubMed Central

    Trivedi, Malav S.; Deth, Richard C.

    2012-01-01

    Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation, and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition, and behavior (Cajigas et al., 2010). Thus a regulatory switch, which controls the lifespan, maturation, and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at (1) the pre-transcription level, by regulating precursor-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and (2) the post-transcription level by modulating the regulatory functions of ribonucleoproteins and RNA binding proteins in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of

  12. In Silico Survey of the Mitochondrial Protein Uptake and Maturation Systems in the Brown Alga Ectocarpus siliculosus

    PubMed Central

    Delage, Ludovic; Leblanc, Catherine; Nyvall Collén, Pi; Gschloessl, Bernhard; Oudot, Marie-Pierre; Sterck, Lieven; Poulain, Julie; Aury, Jean-Marc; Cock, J. Mark

    2011-01-01

    The acquisition of mitochondria was a key event in eukaryote evolution. The aim of this study was to identify homologues of the components of the mitochondrial protein import machinery in the brown alga Ectocarpus and to use this information to investigate the evolutionary history of this fundamental cellular process. Detailed searches were carried out both for components of the protein import system and for related peptidases. Comparative and phylogenetic analyses were used to investigate the evolution of mitochondrial proteins during eukaryote diversification. Key observations include phylogenetic evidence for very ancient origins for many protein import components (Tim21, Tim50, for example) and indications of differences between the outer membrane receptors that recognize the mitochondrial targeting signals, suggesting replacement, rearrangement and/or emergence of new components across the major eukaryotic lineages. Overall, the mitochondrial protein import components analysed in this study confirmed a high level of conservation during evolution, indicating that most are derived from very ancient, ancestral proteins. Several of the protein import components identified in Ectocarpus, such as Tim21, Tim50 and metaxin, have also been found in other stramenopiles and this study suggests an early origin during the evolution of the eukaryotes. PMID:21611166

  13. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-09-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16023367

  14. Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

    PubMed

    Roux, Sylvie; Colasante, Cesare; Saint Cloment, Cécile; Barbier, Julien; Curie, Thomas; Girard, Emmanuelle; Molgó, Jordi; Brûlet, Philippe

    2005-12-01

    The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP)and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immuno-gold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated indifferent axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos. PMID:16456925

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

    PubMed Central

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

    2016-01-01

    Here we provide data from shot-gun proteomics, using filtered-aided sample preparation (FASP), dimethyl labeling and LC–MS/MS, to quantify the changes in the repertoire of human milk proteins over lactation. Milk serum proteins were analyzed at week 1, 2, 3 4, 8, 16, and 24 in milk from four individual mothers. A total of 247 proteins were identified, of which 200 proteins were quantified. The data supplied in this article supports the accompanying publication (Zhang et al., 2006) [1]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2016) [2] via the PRIDE partner repository with the dataset identifier PXD003465. PMID:26977438

  16. 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

    2016-06-01

    Here we provide data from shot-gun proteomics, using filtered-aided sample preparation (FASP), dimethyl labeling and LC-MS/MS, to quantify the changes in the repertoire of human milk proteins over lactation. Milk serum proteins were analyzed at week 1, 2, 3 4, 8, 16, and 24 in milk from four individual mothers. A total of 247 proteins were identified, of which 200 proteins were quantified. The data supplied in this article supports the accompanying publication (Zhang et al., 2006) [1]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2016) [2] via the PRIDE partner repository with the dataset identifier PXD003465. PMID:26977438

  17. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.

    PubMed

    Yilmaz, Ozlem; Prat, Francisco; Ibáñez, A Jose; Köksoy, Sadi; Amano, Haruna; Sullivan, Craig V

    2016-01-01

    Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment. PMID:26643259

  18. An Essential Pentatricopeptide Repeat Protein Facilitates 5′ Maturation and Translation Initiation of rps3 mRNA in Maize Mitochondria[C][W

    PubMed Central

    Manavski, Nikolay; Guyon, Virginie; Meurer, Jörg; Wienand, Udo; Brettschneider, Reinhold

    2012-01-01

    Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5′ untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5′ ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5′ maturation and translation initiation of rps3 mRNA. PMID:22773745

  19. Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans

    PubMed Central

    Solinger, Jachen A.; Spang, Anne

    2014-01-01

    The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, whereas in HOPS knockdowns, RAB-5 was found also on membrane structures close to the cell center, indicating the RAB conversion might be impaired in the absence of these tethering complexes. Unlike in yeast, metazoans have two VPS33 homologues, which are Sec1/Munc18 (SM)-family proteins involved in the regulation of membrane fusion. We assume that in wild type, each tethering complex contains a specific SM protein but that they may be able to substitute for each other in case of absence of the other. Of importance, knockdown of both SM proteins allowed bypass of the endosome maturation block in sand-1 mutants. We propose a model in which the SM proteins in tethering complexes are required for coordinated flux of material through the endosomal system. PMID:25273556

  20. Maturation of Rhizobium leguminosarum Hydrogenase in the Presence of Oxygen Requires the Interaction of the Chaperone HypC and the Scaffolding Protein HupK*

    PubMed Central

    Albareda, Marta; Pacios, Luis F.; Manyani, Hamid; Rey, Luis; Brito, Belén; Imperial, Juan; Ruiz-Argüeso, Tomás; Palacios, Jose M.

    2014-01-01

    [NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O2) and bacteroids from legume nodules (∼10–100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen. PMID:24942742

  1. AgRP Neurons Control Systemic Insulin Sensitivity via Myostatin Expression in Brown Adipose Tissue.

    PubMed

    Steculorum, Sophie M; Ruud, Johan; Karakasilioti, Ismene; Backes, Heiko; Engström Ruud, Linda; Timper, Katharina; Hess, Martin E; Tsaousidou, Eva; Mauer, Jan; Vogt, Merly C; Paeger, Lars; Bremser, Stephan; Klein, Andreas C; Morgan, Donald A; Frommolt, Peter; Brinkkötter, Paul T; Hammerschmidt, Philipp; Benzing, Thomas; Rahmouni, Kamal; Wunderlich, F Thomas; Kloppenburg, Peter; Brüning, Jens C

    2016-03-24

    Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis. PMID:27015310

  2. Identification of protein N-termini in Cyanophora paradoxa cyanelles: transit peptide composition and sequence determinants for precursor maturation

    PubMed Central

    Köhler, Daniel; Dobritzsch, Dirk; Hoehenwarter, Wolfgang; Helm, Stefan; Steiner, Jürgen M.; Baginsky, Sacha

    2015-01-01

    Glaucophyta, rhodophyta, and chloroplastida represent the three main evolutionary lineages that diverged from a common ancestor after primary endosymbiosis. Comparative analyses between members of these three lineages are a rich source of information on ancestral plastid features. We analyzed the composition and the cleavage site of cyanelle transit peptides from the glaucophyte Cyanophora paradoxa by terminal amine labeling of substrates (TAILS), and compared their characteristics to those of representatives of the chloroplastida. Our data show that transit peptide architecture is similar between members of these two lineages. This entails a comparable modular structure, an overrepresentation of serine or alanine and similarities in the amino acid composition around the processing peptidase cleavage site. The most distinctive difference is the overrepresentation of phenylalanine in the N-terminal 1–10 amino acids of cyanelle transit peptides. A quantitative proteome analysis with periplasm-free cyanelles identified 42 out of 262 proteins without the N-terminal phenylalanine, suggesting that the requirement for phenylalanine in the N-terminal region is not absolute. Proteins in this set are on average of low abundance, suggesting that either alternative import pathways are operating specifically for low abundance proteins or that the gene model annotation is incorrect for proteins with fewer EST sequences. We discuss these two possibilities and provide examples for both interpretations. PMID:26257763

  3. An activin-A/C chimera exhibits activin and myostatin antagonistic properties.

    PubMed

    Muenster, Uwe; Harrison, Craig A; Donaldson, Cynthia; Vale, Wylie; Fischer, Wolfgang H

    2005-11-01

    Activins are involved in many physiological and pathological processes and, like other members of the transforming growth factor-beta superfamily, signal via type II and I receptor serine kinases. Ligand residues involved in type II receptor binding are located in the two anti-parallel beta strands of the TGF-beta proteins, also known as the fingers. Activin-A mutants able to bind ActRII but unable to bind the activin type I receptor ALK4 define ligand residues involved in ALK4 binding and could potentially act as antagonists. Therefore, a series of FLAG-tagged activin-A/C chimeras were constructed, in each of which eight residues in the wrist loop and helix region (A/C 46-53, 54-61, 62-69, and 70-78) were replaced. Additionally, a chimera was generated in which the entire wrist region (A/C 46-78) was changed from activin-A to activin-C. The chimeras were assessed for ActRII binding, activin bioactivity, as well as antagonistic properties. All five chimeras retained high affinity for mouse ActRII. Of these, only A/C 46-78 was devoid of significant activin bioactivity in an A3 Lux reporter assay in 293T cells at concentrations up to 40 nM. A/C 46-53, 54-61, 62-69, and 70-78 showed activity comparable with wild type activin-A. When tested for the ability to antagonize ligands that signal via activin type II receptors, such as activin-A and myostatin, only the A/C 46-78 chimera showed antagonism (IC(50), 1-10 nM). Additionally, A/C 46-78 decreased follicle-stimulating hormone release from the LbetaT2 cell line and rat anterior pituitary cells in primary culture in a concentration-dependent manner. These data indicate that activin residues in the wrist are involved in ALK4-mediated signaling. The activin antagonist A/C 46-78 may be useful for the study and modulation of activin-dependent processes. PMID:16129674

  4. Expression of porcine myostatin prodomain genomic sequence leads to a decrease in muscle growth, but significant intramuscular fat accretion in transgenic pigs.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myostatin, a member of TGF-beta superfamily, is a dominant inhibitor of skeletal muscle development and growth. Previously, skeletal muscle-specific over-expression of myostatin prodomain cDNA (5’-region 886 nucleotide) dramatically increased growth performance and muscle mass in transgenic mice. I...

  5. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence

    SciTech Connect

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. ); Chapline, C.; Crabb, J.W. )

    1991-10-22

    Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

  6. L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation

    PubMed Central

    Kummer, Mirko; Mühl-Zürbes, Petra; Drassner, Christina; Daniel, Christoph; Klewer, Monika; Steinkasserer, Alexander

    2015-01-01

    ABSTRACT Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. IMPORTANCE HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such

  7. Market maturity

    SciTech Connect

    Meade, B.; Bowden, S.; Ellis, M

    1995-02-01

    The power sector in the Philipines provides one of the most mature independent power markets in Asia. Over the past five years, National Power Corp. (NPC), the government owned utility, has actively invited the power sector into power generation. Distribution has remained in the hands of private and rural cooperative utilities. Private utilities have been operating as full requirements customers of NPC while the growth in capacity additions by independent power producers (IPPs) has outpaced NPC`s for the second year in a row. With a recovering economy and regulatory reform proceeding, the outlook for independent power remains strong through the end of the decade. The Philipine Congress is now reviewing draft legislation that will decentralize NPC and begin the process of privatization and market-based reforms throughout the country`s power sector.

  8. Developmental regulation of molecular signalling in fetal and neonatal diaphragm protein metabolism.

    PubMed

    Song, Yong; Pillow, J Jane

    2013-08-01

    Structural and functional immaturity of the preterm diaphragm predisposes the preterm baby to respiratory muscle weakness and consequent impaired efficiency of spontaneous respiration, potentially necessitating mechanical respiratory support. The ontogeny of several proteolytic genes (calpain, caspase-3, MAFbx and MuRF-1) changes dynamically with gestational and early postnatal development. We aimed to define the molecular signal cascades and triggers responsible for the dynamic changes in the proteolytic pathways during in utero and early postnatal development. Costal diaphragm was obtained immediately following euthanasia of fetal and newborn lambs from 75 to 200 days postconceptional age (term = 150 days). Gene expression of insulin-like growth factor 1 (IGF-1), tumour necrosis factor α (TNF-α) and myostatin decreased steadily in utero from 75 to 145 days (P < 0.05) and the transcripts increased again after birth except of myostatin. Rapid activation of the fork-head transcriptional factors of the O class (FOXO1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways was observed at 24 h of postnatal age. Diaphragm reactive oxygen species (ROS) production increased over 29-fold at 24 h postnatal age, compared with the 145 days fetus (P < 0.01). Local (diaphragmatic) ROS accumulation occurred earlier and was more predominant than systemic (plasma) ROS. There were positive correlations between signalling transduction molecules (FOXO1 and NF-κB) and antioxidant gene expression (superoxide dismutase and glutathione peroxidase 1). We conclude that anabolic (IGF-1) and catabolic (TNF-α and myostatin) factors have a similar developmental pattern with a decreasing trend toward full term. This may reflect in utero integration of cellular events into low protein metabolism as the diaphragm matures in late gestation. On initiation of spontaneous breathing, ROS accumulated and potentially activated cascade of FOXO and NF

  9. Effects of hepatocyte nuclear factor-1A and -4A on pancreatic stone protein/regenerating protein and C-reactive protein gene expression: implications for maturity-onset diabetes of the young

    PubMed Central

    2013-01-01

    Background There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A. Methods Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use. Results Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P = 0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P = 0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects. Conclusion Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects. PMID:23803251

  10. Low Density Lipoprotein-Receptor Related Protein 1 Is Differentially Expressed by Neuronal and Glial Populations in the Developing and Mature Mouse Central Nervous System

    PubMed Central

    Auderset, Loic; Cullen, Carlie L.; Young, Kaylene M.

    2016-01-01

    The low density lipoprotein-receptor related protein 1 (LRP1) is a large endocytic cell surface receptor that is known to interact with a variety of ligands, intracellular adaptor proteins and other cell surface receptors to regulate cellular behaviours ranging from proliferation to cell fate specification, migration, axon guidance, and lipid metabolism. A number of studies have demonstrated that LRP1 is expressed in the brain, yet it is unclear which central nervous system cell types express LRP1 during development and in adulthood. Herein we undertake a detailed study of LRP1 expression within the mouse brain and spinal cord, examining a number of developmental stages ranging from embryonic day 13.5 to postnatal day 60. We report that LRP1 expression in the brain peaks during postnatal development. On a cellular level, LRP1 is expressed by radial glia, neuroblasts, microglia, oligodendrocyte progenitor cells (OPCs), astrocytes and neurons, with the exception of parvalbumin+ interneurons in the cortex. Most cell populations exhibit stable expression of LRP1 throughout development; however, the proportion of OPCs that express LRP1 increases significantly from ~69% at E15.5 to ~99% in adulthood. We also report that LRP1 expression is rapidly lost as OPCs differentiate, and is absent from all oligodendrocytes, including newborn oligodendrocytes. While LRP1 function has been primarily examined in mature neurons, these expression data suggest it plays a more critical role in glial cell regulation–where expression levels are much higher. PMID:27280679

  11. Low Density Lipoprotein-Receptor Related Protein 1 Is Differentially Expressed by Neuronal and Glial Populations in the Developing and Mature Mouse Central Nervous System.

    PubMed

    Auderset, Loic; Cullen, Carlie L; Young, Kaylene M

    2016-01-01

    The low density lipoprotein-receptor related protein 1 (LRP1) is a large endocytic cell surface receptor that is known to interact with a variety of ligands, intracellular adaptor proteins and other cell surface receptors to regulate cellular behaviours ranging from proliferation to cell fate specification, migration, axon guidance, and lipid metabolism. A number of studies have demonstrated that LRP1 is expressed in the brain, yet it is unclear which central nervous system cell types express LRP1 during development and in adulthood. Herein we undertake a detailed study of LRP1 expression within the mouse brain and spinal cord, examining a number of developmental stages ranging from embryonic day 13.5 to postnatal day 60. We report that LRP1 expression in the brain peaks during postnatal development. On a cellular level, LRP1 is expressed by radial glia, neuroblasts, microglia, oligodendrocyte progenitor cells (OPCs), astrocytes and neurons, with the exception of parvalbumin+ interneurons in the cortex. Most cell populations exhibit stable expression of LRP1 throughout development; however, the proportion of OPCs that express LRP1 increases significantly from ~69% at E15.5 to ~99% in adulthood. We also report that LRP1 expression is rapidly lost as OPCs differentiate, and is absent from all oligodendrocytes, including newborn oligodendrocytes. While LRP1 function has been primarily examined in mature neurons, these expression data suggest it plays a more critical role in glial cell regulation-where expression levels are much higher. PMID:27280679

  12. Embryonic Corneal Schwann Cells Express Some Schwann Cell Marker mRNAs, but No Mature Schwann Cell Marker Proteins

    PubMed Central

    Conrad, Abigail H.; Albrecht, Michael; Pettit-Scott, Maya; Conrad, Gary W.

    2009-01-01

    Purpose Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas. Methods Fertile chicken eggs were incubated from E0 at 38°C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression. Results QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/IT-GNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium. Conclusions Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression. PMID:19387082

  13. Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity.

    PubMed

    Sindhu, Anoop; Langewisch, Tiffany; Olek, Anna; Multani, Dilbag S; McCann, Maureen C; Vermerris, Wilfred; Carpita, Nicholas C; Johal, Gurmukh

    2007-12-01

    The maize (Zea mays) brittle stalk2 (bk2) is a recessive mutant, the aerial parts of which are easily broken. The bk2 phenotype is developmentally regulated and appears 4 weeks after planting, at about the fifth-leaf stage. Before this time, mutants are indistinguishable from wild-type siblings. Afterward, all organs of the bk2 mutants turn brittle, even the preexisting ones, and they remain brittle throughout the life of the plant. Leaf tension assays and bend tests of the internodes show that the brittle phenotype does not result from loss of tensile strength but from loss in flexibility that causes the tissues to snap instead of bend. The Bk2 gene was cloned by a combination of transposon tagging and a candidate gene approach and found to encode a COBRA-like protein similar to rice (Oryza sativa) BC1 and Arabidopsis (Arabidopsis thaliana) COBRA-LIKE4. The outer periphery of the stalk has fewer vascular bundles, and the sclerids underlying the epidermis possess thinner secondary walls. Relative cellulose content is not strictly correlated with the brittle phenotype. Cellulose content in mature zones of bk2 mature stems is lowered by 40% but is about the same as wild type in developing stems. Although relative cellulose content is lowered in leaves after the onset of the brittle phenotype, total wall mass as a proportion of dry mass is either unchanged or slightly increased, indicating a compensatory increase in noncellulosic carbohydrate mass. Fourier transform infrared spectra indicated an increase in phenolic ester content in the walls of bk2 leaves and stems. Total content of lignin is unaffected in bk2 juvenile leaves before or after appearance of the brittle phenotype, but bk2 mature and developing stems are markedly enriched in lignin compared to wild-type stems. Despite increased lignin in bk2 stems, loss of staining with phloroglucinol and ultraviolet autofluorescence is observed in vascular bundles and sclerid layers. Consistent with the infrared

  14. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

    PubMed

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K; Kline, Douglas W; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  15. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  16. Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2) identify key functional domains and coincide with protein structural elements in each of the mature proteins

    PubMed Central

    Lang, Dorothy M

    2007-01-01

    Background A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs) having ≥ 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements – helices, β sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein – to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. Results There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs (≥ 33%) are related to cleavage positions and processes. Conclusion The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The distribution of IMRs and their

  17. Salicylic acid signaling controls the maturation and localization of the arabidopsis defense protein ACCELERATED CELL DEATH6.

    PubMed

    Zhang, Zhongqin; Shrestha, Jay; Tateda, Chika; Greenberg, Jean T

    2014-08-01

    ACCELERATED CELL DEATH6 (ACD6) is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid (SA). This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum (ER)- and plasma membrane (PM)-localized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses. PMID:24923602

  18. Gentiana manshurica Kitagawa reverses acute alcohol-induced liver steatosis through blocking sterol regulatory element-binding protein-1 maturation.

    PubMed

    Lian, Li-Hua; Wu, Yan-Ling; Song, Shun-Zong; Wan, Ying; Xie, Wen-Xue; Li, Xin; Bai, Ting; Ouyang, Bing-Qing; Nan, Ji-Xing

    2010-12-22

    This study was undertaken to investigate the protective effects of Gentiana manshurica Kitagawa (GM) on acute alcohol-induced fatty liver. Mice were treated with ethanol (5 g/kg of body weight) by gavage every 12 h for a total of three doses to induce acute fatty liver. Methanol extract of GM (50, 100, or 200 mg/kg) or silymarin (100 mg/kg) was gavaged simultaneously with ethanol for three doses. GM administration significantly reduced the increases in serum ALT and AST levels, the serum and hepatic triglyceride levels, at 4 h after the last ethanol administration. GM was also found to prevent ethanol-induced hepatic steatosis and necrosis, as indicated by liver histopathological studies. Additionally, GM suppressed the elevation of malondialdehyde (MDA) levels, restored the glutathione (GSH) levels, and enhanced the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities. The concurrent administration of GM efficaciously abrogated cytochrome P450 2E1 (CYP2E1) induction. Moreover, GM significantly reduced the nuclear translocation of sterol regulatory element-binding protein-1 (nSREBP-1) in ethanol-treated mice. These data indicated that GM possessed the ability to prevent ethanol-induced acute liver steatosis, possibly through blocking CYP2E1-mediated free radical scavenging effects and SREBP-1-regulated fatty acid synthesis. Especially, GM may be developed as a potential therapeutic candidate for ethanol-induced oxidative damage in liver. PMID:21105651

  19. Late-stage maturation of the Rieske Fe/S protein: Mzm1 stabilizes Rip1 but does not facilitate its translocation by the AAA ATPase Bcs1.

    PubMed

    Cui, Tie-Zhong; Smith, Pamela M; Fox, Jennifer L; Khalimonchuk, Oleh; Winge, Dennis R

    2012-11-01

    The final step in the assembly of the ubiquinol-cytochrome c reductase or bc(1) complex involves the insertion of the Rieske Fe/S cluster protein, Rip1. Maturation of Rip1 occurs within the mitochondrial matrix prior to its translocation across the inner membrane (IM) in a process mediated by the Bcs1 ATPase and subsequent insertion into the bc(1) complex. Here we show that the matrix protein Mzm1 functions as a Rip1 chaperone, stabilizing Rip1 prior to the translocation step. In the absence of Mzm1, Rip1 is prone to either proteolytic degradation or temperature-induced aggregation. A series of Rip1 truncations were engineered to probe motifs necessary for Mzm1 interaction and Bcs1-mediated translocation of Rip1. The Mzm1 interaction with Rip1 persists in Rip1 variants lacking its transmembrane domain or containing only its C-terminal globular Fe/S domain. Replacement of the globular domain of Rip1 with that of the heterologous folded protein Grx3 abrogated Mzm1 interaction; however, appending the C-terminal 30 residues of Rip1 to the Rip1-Grx3 chimera restored Mzm1 interaction. The Rip1-Grx3 chimera and a Rip1 truncation containing only the N-terminal 92 residues each induced stabilization of the bc(1):cytochrome oxidase supercomplex in a Bcs1-dependent manner. However, the Rip1 variants were not stably associated with the supercomplex. The induced supercomplex stabilization by the Rip1 N terminus was independent of Mzm1. PMID:22927643

  20. Structure of [NiFe] Hydrogenase Maturation Protein HypE from Escherichia coli and its Interaction with HypF

    SciTech Connect

    Rangarajan,E.; Asinas, A.; Proteau, A.; Munger, C.; Baardsnes, j.; Iannuzzi, P.; Matte, A.; Cygler, m.

    2008-01-01

    Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H2 as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0- Angstroms resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a Kd (dissociation constant) of {approx}400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]2 heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.

  1. Reorganization of the cortical actin cytoskeleton during maturation division in the Tubifex egg: possible involvement of protein kinase C.

    PubMed

    Shimizu, T

    1997-08-01

    Tubifex eggs undergo a drastic reorganization of the cortical actin cytoskeleton during metaphase of the second meiosis. At the end of the first meiosis, the egg cortex displays only scattered actin filaments and tiny dots of F-actin; during the following 90 min, cortical F-actin gradually increases in amount, becomes organized into foci that are interlinked by actin bundles, and generates a geodesic dome-like organization. In this study, we have characterized this reorganization of the cortical actin cytoskeleton. In living eggs injected with rhodamine-phalloidin at the beginning of the second meiosis, cortical actin assembly (i.e., formation of actin foci and bundles) proceeds normally, but labeled F-actin is not found to be included significantly in the formed cortical actin network, suggesting that the increase in cortical F-actin is not simply ascribable to the recruitment of preexisting actin filaments. Cortical actin assembly can be induced precociously not only by calcium ionophore A23187 but also by a phorbol ester PMA, an agonist of protein kinase C (PKC). Conversely, the formation of actin foci and bundles is inhibited by PKC antagonists, although cortical F-actin increases to some extent in the presence of these inhibitors. Similar inhibition of the cortical reorganization is elicited in eggs whose intracellular free calcium level ([Ca2+]i) has been clamped low by microinjection of a calcium chelator BAPTA. The treatment of BAPTA-injected eggs with PMA results in the formation of actin foci and bundles. An experiment with eggs injected with fluo-3 shows that [Ca2+]i increases during metaphase of the second meiosis. These results suggest that the reorganization of cortical actin during metaphase of the second meiosis requires activation of PKC, which depends on increases in [Ca2+]i. PMID:9245516

  2. Myostatin dysfunction is associated with reduction in overload induced hypertrophy of soleus muscle in mice.

    PubMed

    Minderis, P; Kilikevicius, A; Baltusnikas, J; Alhindi, Y; Venckunas, T; Bunger, L; Lionikas, A; Ratkevicius, A

    2016-08-01

    The aim of the study was to investigate if myostatin dysfunction would promote the gain in muscle mass and peak isometric force (P0 ) of soleus muscle (SOL) in response to functional overloading (FO) after ablation of the gastrocnemius muscle. Fifteen male Berlin high (BEH) mice homozygous for the compact mutation causing dysfunction of myostatin and 17 mice with the corresponding wild-type allele (BEH+/+) were subjected to FO of SOL for 28 days at the age of 14 weeks. Compared with BEH+/+ mice, SOL of BEH was heavier (mean ± SD, 13.5 ± 1.5 vs 21.4 ± 1.8 mg, respectively, P < 0.001). After FO, SOL mass increased relatively more in BEH+/+ than BEH strain (34.9 ± 11.5 vs 17.7 ± 11.9%, respectively, P < 0.01). P0 fell (P < 0.01) only in BEH strain, which also showed an increase (P < 0.01) in optimal muscle length. Specific P0 became even more depressed in BEH compared with BEH+/+ strain (8.4 ± 1.4 vs 10.8 ± 1.3 N/g, respectively, P < 0.001). Phosphorylation p70 S6 kinase did not differ between the strains. In summary, myostatin dysfunction impairs adaptation of SOL muscle to high functional demands. PMID:26304113

  3. A myostatin inhibitor (propeptide-Fc) increases muscle mass and muscle fiber size in aged mice but does not increase bone density or bone strength.

    PubMed

    Arounleut, Phonepasong; Bialek, Peter; Liang, Li-Fang; Upadhyay, Sunil; Fulzele, Sadanand; Johnson, Maribeth; Elsalanty, Mohammed; Isales, Carlos M; Hamrick, Mark W

    2013-09-01

    Loss of muscle and bone mass with age are significant contributors to falls and fractures among the elderly. Myostatin deficiency is associated with increased muscle mass in mice, dogs, cows, sheep and humans, and mice lacking myostatin have been observed to show increased bone density in the limb, spine, and jaw. Transgenic overexpression of myostatin propeptide, which binds to and inhibits the active myostatin ligand, also increases muscle mass and bone density in mice. We therefore sought to test the hypothesis that in vivo inhibition of myostatin using an injectable myostatin propeptide (GDF8 propeptide-Fc) would increase both muscle mass and bone density in aged (24 mo) mice. Male mice were injected weekly (20 mg/kg body weight) with recombinant myostatin propeptide-Fc (PRO) or vehicle (VEH; saline) for four weeks. There was no difference in body weight between the two groups at the end of the treatment period, but PRO treatment significantly increased mass of the tibialis anterior muscle (+ 7%) and increased muscle fiber diameter of the extensor digitorum longus (+ 16%) and soleus (+ 6%) muscles compared to VEH treatment. Bone volume relative to total volume (BV/TV) of the femur calculated by microCT did not differ significantly between PRO- and VEH-treated mice, and ultimate force (Fu), stiffness (S), toughness (U) measured from three-point bending tests also did not differ significantly between groups. Histomorphometric assays also revealed no differences in bone formation or resorption in response to PRO treatment. These data suggest that while developmental perturbation of myostatin signaling through either gene knockout or transgenic inhibition may alter both muscle and bone mass in mice, pharmacological inhibition of myostatin in aged mice has a more pronounced effect on skeletal muscle than on bone. PMID:23832079

  4. The Large Ribosomal Subunit Protein L9 Enables the Growth of EF-P Deficient Cells and Enhances Small Subunit Maturation

    PubMed Central

    Naganathan, Anusha; Wood, Matthew P.; Moore, Sean D.

    2015-01-01

    The loss of the large ribosomal protein L9 causes a reduction in translation fidelity by an unknown mechanism. To identify pathways affected by L9, we identified mutants of E. coli that require L9 for fitness. In a prior study, we characterized L9-dependent mutations in the essential GTPase Der (EngA). Here, we describe a second class of L9-dependent mutations that either compromise or inactivate elongation factor P (EF-P, eIF5A in eukaryotes). Without L9, Δefp cells are practically inviable. Cell fractionation studies revealed that, in both the Der and EF-P mutant cases, L9's activity reduces immature 16S rRNA in 30S particles and partially restores the abundance of monosomes. Inspired by these findings, we discovered that L9 also enhances 16S maturation in wild-type cells. Surprisingly, although the amount of immature 16S in 30S particles was found to be elevated in ΔrplI cells, the amount in polysomes was low and inversely correlated with the immature 16S abundance. These findings provide an explanation for the observed fitness increases afforded by L9 in these mutants and reveal particular physiological conditions in which L9 becomes critical. Additionally, L9 may affect the partitioning of small subunits containing immature 16S rRNA. PMID:25879934

  5. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

    PubMed Central

    Choi, T; Fukasawa, K; Zhou, R; Tessarollo, L; Borror, K; Resau, J; Vande Woude, G F

    1996-01-01

    Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692939

  6. Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    Frías, José E.

    2015-01-01

    ABSTRACT Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. IMPORTANCE Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many

  7. Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

    PubMed Central

    Moussatche, Nissin; Condit, Richard C.

    2014-01-01

    ABSTRACT Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCE The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the

  8. Binding immunoglobulin protein-treated peripheral blood monocyte-derived dendritic cells are refractory to maturation and induce regulatory T-cell development.

    PubMed

    Corrigall, Valerie M; Vittecoq, Olivier; Panayi, Gabriel S

    2009-10-01

    Binding immunoglobulin protein (BiP) has been shown previously to have immunomodulatory functions. Herein we investigated whether BiP could affect the differentiation of monocytes into dendritic cells (DCs) and thence the development of regulatory T cells. Peripheral blood monocyte-derived DCs were matured with lipopolysaccharide in the presence or absence of BiP. DC development and T-cell changes were monitored by flow cytometry and regulatory T-cell function was measured by uptake of tritiated thymidine. More BiP-treated DCs (DC((BiP))s) expressed amounts of intracellular indoleamine 2,3-dioxygenase (IDO) and cell surface leucocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), retained CD14 expression but down-regulated expression of human leucocyte antigen (HLA)-DR and CD86, and produced copious amounts of interleukin (IL)-10, when compared with control DCs. T cells co-cultured with DC((BiP))s developed regulatory function with increased surface expression of CD4(+) CD25(hi) CD27(hi) but with no concomitant increase in forkhead box P3 (Foxp3). These T cells also showed significantly higher levels of intracellular cytotoxic T-lymphocyte antigen (CTLA)-4. The latter could be inhibited by the presence of the IDO inhibitor 1 methyl tryptophan. The addition of neutralizing anti-IL-10 antibody or the specific mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 reversed the inhibition of DC differentiation by BiP. In conclusion, BiP is an immunomodulator able to arrest inflammation through induction of tolerogenic DCs and subsequent generation of T regulatory cells. PMID:19740378

  9. Increases in mature brain-derived neurotrophic factor protein in the frontal cortex and basal forebrain during chronic sleep restriction in rats: possible role in initiating allostatic adaptation.

    PubMed

    Wallingford, J K; Deurveilher, S; Currie, R W; Fawcett, J P; Semba, K

    2014-09-26

    Chronic sleep restriction (CSR) has various negative consequences on cognitive performance and health. Using a rat model of CSR that uses alternating cycles of 3h of sleep deprivation (using slowly rotating activity wheels) and 1h of sleep opportunity continuously for 4 days ('3/1' protocol), we previously observed not only homeostatic but also allostatic (adaptive) sleep responses to CSR. In particular, non-rapid eye movement sleep (NREMS) electroencephalogram (EEG) delta power, an index of sleep intensity, increased initially and then declined gradually during CSR, with no rebound during a 2-day recovery period. To study underlying mechanisms of these allostatic responses, we examined the levels of brain-derived neurotrophic factor (BDNF), which is known to regulate NREMS EEG delta activity, during the same CSR protocol. Mature BDNF protein levels were measured in the frontal cortex and basal forebrain, two brain regions involved in sleep and EEG regulation, and the hippocampus, using Western blot analysis. Adult male Wistar rats were housed in motorized activity wheels, and underwent the 3/1 CSR protocol for 27 h, for 99 h, or for 99 h followed by 24h of recovery. Additional rats were housed in either locked wheels (locked wheel controls [LWCs]) or unlocked wheels that rats could rotate freely (wheel-running controls [WRCs]). BDNF levels did not differ between WRC and LWC groups. BDNF levels were increased, compared to the control levels, in all three brain regions after 27 h, and were increased less strongly after 99 h, of CSR. After 24h of recovery, BDNF levels were at the control levels. This time course of BDNF levels parallels the previously reported changes in NREMS delta power during the same CSR protocol. Changes in BDNF protein levels in the cortex and basal forebrain may be part of the molecular mechanisms underlying allostatic sleep responses to CSR. PMID:25010399

  10. Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

    PubMed Central

    Choi, T; Rulong, S; Resau, J; Fukasawa, K; Matten, W; Kuriyama, R; Mansour, S; Ahn, N; Vande Woude, G F

    1996-01-01

    Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643471

  11. TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system.

    PubMed

    Chiang, Yi-An; Kinoshita, Masato; Maekawa, Shun; Kulkarni, Amod; Lo, Chu-Fang; Yoshiura, Yasutoshi; Wang, Han-Ching; Aoki, Takashi

    2016-01-01

    Although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. In this study, we used a popular genome editing tool with high efficiency and target specificity (TALENs; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (MSTN) in medaka (Oryzias latipes). After the TALEN pair targeting OlMyostatin was injected into fertilized medaka eggs, mutant G0 fish carrying different TALENs-induced frameshifts in the OlMSTN coding sequence were mated together in order to transmit the mutant sequences to the F1 generation. Two F1 mutants with frameshifted myostatin alleles were then mated to produce the F2 generation, and these F2 OlMSTN null (MSTN(-/-)) medaka were evaluated for growth performance. The F2 fish showed significantly increased body length and weight compared to the wild type fish at the juvenile and post-juvenile stages. At the post-juvenile stage, the average body weight of the MSTN(-/-) medaka was ∼25% greater than the wild type. However, we also found that when the F3 generation were challenged with red spotted grouper nervous necrosis virus (RGNNV), the expression levels of the interferon-stimulated genes were lower than in the wild type, and the virus copy number was maintained at a high level. We therefore conclude that although the MSTN(-/-) medaka had a larger phenotype, their immune system appeared to be at least partially suppressed or undeveloped. PMID:26578247

  12. The effects of selecting for the myostatin F94L polymorphism on reproductive traits in pubertal heifers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The myostatin F94L polymorphism influences carcass traits in steers; however, the influence of this polymorphism on female reproductive performance should be characterized as part of using it for marker assisted selection. Results from USMARC indicate that heifers that are homozygous for the L allel...

  13. Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a & -2b): genomic organization, differential expression and pseudogenization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myostatin is an extremely potent negative regulator of vertebrate skeletal muscle development. A phylogenetic analysis suggests that salmonids should possess four distinct genes, although only MSTN-1 orthologs have been characterized. Described herein are the rainbow trout (rt) MSTN-2a and -2b genes...

  14. Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat

    PubMed Central

    Khichar, Jai Prakash; Gahlot, Gyan Chand; Agrawal, Vijay Kumar; Kiran; Dewna, Ajay Singh; Prakash; Ashraf, Mohammad

    2016-01-01

    Aim: To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats. Materials and Methods: A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan), India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd.) as per manufacturer’s protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN) gene (Genebank accession no. DQ167575) was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP) pattern. Results: The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively. Conclusion: Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan. PMID:27397994

  15. Polymorphisms in the Myostatin - 1 gene and their association with growth traits in Ancherythroculter nigrocauda

    NASA Astrophysics Data System (ADS)

    Sun, Yanhong; Li, Qing; Wang, Guiying; Zhu, Dongmei; Chen, Jian; Li, Pei; Tong, Jingou

    2016-05-01

    Myostatin (MSTN) is a member of the transforming growth factor-β gene superfamily that negatively regulates skeletal muscle development and growth. In the present study, partial genomic fragments of Myostatin-1 (MSTN-1) in two commercial hatchery populations of Ancherythroculter nigrocauda, an economically important freshwater fish, were screened for single nucleotide polymorphisms (SNPs) and then genotyped by direct sequencing of PCR products. Five SNPs were identified in intron 1 and exon 2, including a non-synonymous mutation causing an amino acid change (Val to Ile) at position 180. Association analyses based on 300 individuals revealed that the g.1129T>C SNP locus was significantly associated with total length (TL), body length (BL), body height (BH) and body weight (BW) in 6- and 18-month-old populations, while the g.1289G>A locus was significantly associated with BH and BW in the 6-month-old population. Haplotype analyses revealed that fish with the genotype combinations TC/TC or TC/GA showed better growth performance. Our results suggest that g.1129T>C and g.1289G>A have positive effects on growth traits and may be candidate gene markers for marker-assisted selection in A. nigrocauda.

  16. Sterol regulatory element binding protein-1 (SREBP-1)c promoter: Characterization and transcriptional regulation by mature SREBP-1 and liver X receptor α in goat mammary epithelial cells.

    PubMed

    Xu, H F; Luo, J; Wang, H P; Wang, H; Zhang, T Y; Tian, H B; Yao, D W; Loor, J J

    2016-02-01

    Sterol regulatory element binding protein-1 (SREBP-1) is a key transcription factor that regulates lipogenesis in rodent liver. Two isoforms (SREBP-1a and SREBP-1c) of SREBP-1 are transcribed by an alternative promoter on the same gene (SREBF1), and the isoforms differ only in their first exon. Although the regulatory effects of SREBP-1 on lipid and milk fat synthesis have received much attention in ruminants, SREBP-1c promoter and its regulatory mechanisms have not been characterized in the goat. In the present study, we cloned and sequenced a 2,012-bp fragment of the SREBP-1c 5'-flanking region from goat genomic DNA. A luciferase reporter assay revealed that SREBP-1c is transcriptionally activated by the liver X receptor α (LXRα) agonist T0901317, and is decreased by SREBP-1 small interfering (si)RNA. A 5' deletion analysis revealed a core promoter region located -395 to +1 bp upstream of the transcriptional start site (TSS). Site-directed mutagenesis of LXRα binding elements (LXRE1 and LXRE2) and sterol regulatory elements (SRE1 and SRE2) revealed that the full effects of T 4506585 require the presence of both LXRE and SRE. We also characterized a new SRE (SRE1) and demonstrated a direct role of SREBP-1 (auto-loop regulation) in maintaining its basal transcription activity. Results suggest that goat SREBP-1c gene is transcriptionally regulated by mature SREBP-1 (auto-loop circuit regulation) and LXRα in goat mammary epithelial cells. PMID:26709176

  17. Bone Morphogenetic Protein Signaling and Olig1/2 Interact to Regulate the Differentiation and Maturation of Adult Oligodendrocyte Precursor Cells

    PubMed Central

    Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R.; Cao, Qilin

    2009-01-01

    Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. PMID:17872503

  18. GASP/WFIKKN proteins: evolutionary aspects of their functions.

    PubMed

    Monestier, Olivier; Brun, Caroline; Cocquempot, Olivier; Petit, Daniel; Blanquet, Véronique

    2012-01-01

    Growth and differentiation factor Associated Serum Protein (GASP) 1 and 2 are proteins known to be involved in the control of myostatin activity at least in vitro. Most deuterostome GASPs share a modular organization including WAP, follistatin/kazal, IGc2, two kunitz, and NTR domains. Based on an exon shuffling model, we performed independent phylogenetic analyses on these modules and assessed that papilin is probably a sister sequence to GASP with a divergence date estimated from the last common ancestor to bilateria. The final organization was acquired by the addition of the FS domain in early deuterostomes. Our study revealed that Gasp genes diverged during the first round of genome duplication in early vertebrates. By evaluating the substitution rate at different sites on the proteins, we showed a better conservation of the follistatin/kazal domain of GASP1 than GASP2 in mammals, suggesting a stronger interaction with myostatin. We also observed a progressive increase in the conservation of follistatin and kunitz domains from the ancestor of Ciona to early vertebrates. In situ hybridization performed on mouse embryos showed a weak Gasp1 expression in the formed somites at 10.5 dpc and in limb buds from embryonic E10.0 to E12.5. Similar results were obtained for zebrafish embryos. We propose a synthetic view showing possible interactions between GASP1 and myostatin and highlighting the role of the second kunitz domain in preventing myostatin proteolysis. PMID:22937083

  19. Influence of pregnancy in mid-to-late gestation on circulating metabolites, visceral organ mass, and abundance of proteins relating to energy metabolism in mature beef cows.

    PubMed

    Wood, K M; Awda, B J; Fitzsimmons, C; Miller, S P; McBride, B W; Swanson, K C

    2013-12-01

    In mid-to-late gestation, nutrient demand increases to meet the growth requirements of the conceptus and cows may alter metabolism in response to energy demands of pregnancy. By better understanding the metabolic role of pregnancy, there may be opportunities to better understand maintenance energy costs and improve overall feed efficiency. Eighteen mature Simmental/Angus crossbred cows, pregnant (PREG; n = 9) and nonpregnant (OPEN; n = 9), were used to investigate the effect of pregnancy on BW change, carcass traits, visceral organ mass, and circulating serum metabolites. Cows were blocked by day of expected parturition such that each block was slaughtered 4 to 5 wk before parturition. Cows were individually fed for ad libitum intake using Calan gates for 89 to 105 d. Cows were weighed, ultrasounded for rib (over the 12th and 13th rib) and rump fat, and a serum sample obtained at d 1, 56, and 3 to 5 d before slaughter. At slaughter, organs were removed, trimmed of fat, and weighed. Serum was analyzed for β-hydroxybutyrate (BHBA), NEFA, glucose, urea, total cholesterol, and triiodothyronine (T3). Tissue samples from liver, kidney, sternomandibularis muscle, ruminal papillae, pancreas, and small intestinal mucosa were collected at slaughter and snap frozen in liquid N. Western blots were conducted to quantify abundance of: proliferating cell nuclear antigen (PCNA), ATP synthase, ubiquitin, and Na(+)/K+ ATPase for all tissues; PPARγ, PPARγ coactivator 1α (PGC1-α), 5'-adenosine monophosphate-activated protein kinase (AMPK) and phosphorylated-AMPK (pAMPK) for liver, muscle, and rumen; phosphoenolpyruvate carboxykinase (PEPCK) for liver and kidney; and uncoupling protein 2 (UCP2) for liver. Data were analyzed using PROC MIXED in SAS as a replicated randomized complete block. Liver weights (actual, relative to BW, relative to HCW) were heavier (P ≤ 0.02) in OPEN. Rumen mass and kidney fat weight, both relative to BW, were also greater (P ≤ 0.04) in OPEN. On d 56

  20. Human CIA2A (FAM96A) and CIA2B (FAM96B) integrate maturation of different subsets of cytosolic-nuclear iron-sulfur proteins and iron homeostasis

    PubMed Central

    Stehling, Oliver; Mascarenhas, Judita; Vashisht, Ajay A.; Sheftel, Alex D.; Niggemeyer, Brigitte; Rösser, Ralf; Pierik, Antonio J.; Wohlschlegel, James A.; Lill, Roland

    2013-01-01

    SUMMARY Numerous cytosolic and nuclear proteins involved in metabolism, DNA maintenance, protein translation, or iron homeostasis depend on iron-sulfur (Fe/S) cofactors, yet their assembly is poorly defined. Here, we identify and characterize human CIA2A (FAM96A), CIA2B (FAM96B), and CIA1 (CIAO1) as components of the cytosolic Fe/S protein assembly (CIA) machinery. CIA1 associates with either CIA2A or CIA2B and the CIA targeting factor MMS19. The CIA2B-CIA1-MMS19 complex binds to and facilitates assembly of most cytosolic-nuclear Fe/S proteins. In contrast, CIA2A specifically matures iron regulatory protein (IRP) 1 which is critical for cellular iron homeostasis. Surprisingly, a second layer of iron regulation involves the stabilization of IRP2 by CIA2A binding or upon depletion of CIA2B or MMS19, even though IRP2 lacks a Fe/S cluster. In summary, CIA2B-CIA1-MMS19 and CIA2A-CIA1 assist different branches of Fe/S protein assembly, and intimately link this process to cellular iron regulation via IRP1 Fe/S cluster maturation and IRP2 stabilization. PMID:23891004

  1. Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling

    PubMed Central

    Bodnár, Dóra; Geyer, Nikolett; Ruzsnavszky, Olga; Oláh, Tamás; Hegyi, Bence; Sztretye, Mónika; Fodor, János; Dienes, Beatrix; Balogh, Ágnes; Papp, Zoltán; Szabó, László; Müller, Géza; Csernoch, László; Szentesi, Péter

    2014-01-01

    Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation–contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (MstnCmpt-dl1Abc or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa–force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca2+ concentration ([Ca2+]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nm, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nm, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mMs−1) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mm s−1) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals. PMID:24445322

  2. The effect of myostatin genotype on body temperature during extreme temperature events.

    PubMed

    Howard, J T; Kachman, S D; Nielsen, M K; Mader, T L; Spangler, M L

    2013-07-01

    Extreme heat and cold events can create deleterious physiological changes in cattle as they attempt to cope. The genetic background of animals can influence their response to these events. The objective of the current study was to determine the impact of myostatin genotype (MG) on body temperature during periods of heat and cold stress. Two groups of crossbred steers and heifers of unknown pedigree and breed fraction with varying percentages of Angus, Simmental, and Piedmontese were placed in a feedlot over 2 summers and 2 winters. Before arrival, animals were genotyped for the Piedmontese-derived myostatin mutation (C313Y) to determine their MG as either homozygous normal (0 copy; n = 84), heterozygous (1 copy; n = 96), or homozygous for inactive myostatin (2 copy; n = 59). Hourly tympanic and vaginal temperature measurements were collected for steers and heifers, respectively, for 5 d during times of anticipated heat and cold stress. Mean (±SD) ambient temperature for summer and winter stress events were 24.4 (±4.64) and -1.80 (±11.71), respectively. A trigonometric function (sine + cosine) with periods of 12 and 24 h was used to describe the diurnal cyclical pattern. Hourly body temperature was analyzed within a season, and fixed effects included MG, group, trigonometric functions nested within group, and interaction of MG with trigonometric functions nested within group; random effects were animal and residual (Model [I]). A combined analysis of season and group was also investigated with the inclusion of season as a main effect and the nesting of effects within both group and season (Model [C]). In both models, the residual was fitted using an autoregressive covariance structure. A 3-way interaction of MG, season, and trigonometric function periodicities of 24 h (P < 0.001) and 12 h (P < 0.02) for Model [C] indicate that a genotype × environment interaction exists for MG. For MG during summer stress events the additive estimate was 0.10°C (P < 0.01) and

  3. Quantification of GDF11 and Myostatin in Human Aging and Cardiovascular Disease.

    PubMed

    Schafer, Marissa J; Atkinson, Elizabeth J; Vanderboom, Patrick M; Kotajarvi, Brian; White, Thomas A; Moore, Matthew M; Bruce, Charles J; Greason, Kevin L; Suri, Rakesh M; Khosla, Sundeep; Miller, Jordan D; Bergen, H Robert; LeBrasseur, Nathan K

    2016-06-14

    Growth and differentiation factor 11 (GDF11) is a transforming growth factor β superfamily member with a controversial role in aging processes. We have developed a highly specific LC-MS/MS assay to quantify GDF11, resolved from its homolog, myostatin (MSTN), based on unique amino acid sequence features. Here, we demonstrate that MSTN, but not GDF11, declines in healthy men throughout aging. Neither GDF11 nor MSTN levels differ as a function of age in healthy women. In an independent cohort of older adults with severe aortic stenosis, we show that individuals with higher GDF11 were more likely to be frail and have diabetes or prior cardiac conditions. Following valve replacement surgery, higher GDF11 at surgical baseline was associated with rehospitalization and multiple adverse events. Cumulatively, our results show that GDF11 levels do not decline throughout aging but are associated with comorbidity, frailty, and greater operative risk in older adults with cardiovascular disease. PMID:27304512

  4. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos.

    PubMed

    Zhang, Xuemei; Wang, Liqin; Wu, Yangsheng; Li, Wenrong; An, Jing; Zhang, Fuchun; Liu, Mingjun

    2016-10-01

    Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos. PMID:27189642

  5. Conformation Traits and Gaits in the Icelandic Horse are Associated with Genetic Variants in Myostatin (MSTN).

    PubMed

    François, Liesbeth; Jäderkvist Fegraeus, Kim; Eriksson, Susanne; Andersson, Lisa S; Tesfayonas, Yohannes G; Viluma, Agnese; Imsland, Freyja; Buys, Nadine; Mikko, Sofia; Lindgren, Gabriella; Velie, Brandon D

    2016-09-01

    Many genes are known to have an influence on conformation and performance traits; however, the role of one gene, Myostatin (MSTN), has been highlighted in recent studies on horses. Myostatin acts as a repressor in the development and regulation of differentiation and proliferative growth of skeletal muscle. Several studies have examined the link between MSTN, conformation, and performance in racing breeds, but no studies have investigated the relationship in Icelandic horses. Icelandic horses, a highly unique breed, are known both for their robust and compact conformation as well as their additional gaits tölt and pace. Three SNPs (g.65868604G>T [PR8604], g.66493737C>T [PR3737], and g.66495826A>G [PR5826]) flanking or within equine MSTN were genotyped in 195 Icelandic horses. The SNPs and haplotypes were analyzed for association with official estimated breeding values (EBV) for conformation traits (n = 11) and gaits (n = 5). The EBV for neck, withers, and shoulders was significantly associated with both PR8604 and PR3737 (P < 0.05). PR8604 was also associated with EBV for total conformation (P = 0.05). These associations were all supported by the haplotype analysis. However, while SNP PR5826 showed a significant association with EBVs for leg stance and hooves (P < 0.05), haplotype analyses for these traits failed to fully support these associations. This study demonstrates the possible role of MSTN on both the form and function of horses from non-racing breeds. Further analysis of Icelandic horses as well as other non-racing breeds would be beneficial and likely help to completely understand the influence of MSTN on conformation and performance in horses. PMID:27208149

  6. Masticatory HyperMuscularity is not related to Reduced Cranial Volume in Myostatin-Knockout Mice

    PubMed Central

    Cray, James; Kneib, Jared; Vecchione, Lisa; Byron, Craig; Cooper, Gregory M.; Losee, Joseph E.; Siegel, Michael I.; Hamrick, Mark W.; Sciote, James J.; Mooney, Mark P.

    2013-01-01

    It has been suggested recently that masticatory muscle size reduction in humans resulted in greater encephalization through decreased compressive forces on the cranial vault. Following this logic, if masticatory muscle size were increased, then a reduction in brain growth should also occur. The present study was designed to test this hypothesis using a myostatin (GDF-8) knockout mouse model. Myostatin is a negative regulator of skeletal muscle growth, and individuals lacking this gene show significant hypermuscularity. Sixty-two (32 wild-type and 30 GDF-8 −/− knockout), 1, 28, 56 and 180 day old CD-1 mice were used. Body and masseter muscle weights were collected following dissection and standardized lateral and dorsoventral cephalographs were obtained. Cephalometric landmarks were identified on the radiographs and cranial volume was calculated. Mean differences were assessed using a two-way ANOVA. KO mice had significantly greater body and masseter weights beginning at 28 days compared to WT controls. No significant differences in cranial volumes were noted between KO and WT. Muscle weight was not significantly correlated with cranial volume in 1, 28, or 180 day old mice. Muscle weights exhibited a positive correlation with cranial volume at 56 days. Results demonstrate that masticatory hypermuscularity is not associated with reduced cranial volume. In contrast, there is abundant data demonstrating the opposite, brain growth determines cranial vault growth and masticatory apparatus only affects ectocranial morphology. The results presented here do not support the hypothesis that a reduction in masticatory musculature relaxed compressive forces on the cranial vault allowing for greater encephalization. PMID:21618442

  7. Improved muscle healing through enhanced regeneration and reduced fibrosis in myostatin-null mice.

    PubMed

    McCroskery, Seumas; Thomas, Mark; Platt, Leanne; Hennebry, Alex; Nishimura, Takanori; McLeay, Lance; Sharma, Mridula; Kambadur, Ravi

    2005-08-01

    Numerous stimulatory growth factors that can influence muscle regeneration are known. Recently, it has been demonstrated that neutralization of muscle growth inhibitory factors, such as myostatin (Mstn; also known as growth differentiation factor 8, Gdf8), also leads to increased muscle regeneration in mdx mice that are known to have cycles of degeneration. However, the precise mechanism by which Mstn regulates muscle regeneration has not yet been fully determined. To investigate the role of Mstn in adult skeletal muscle regeneration, wild-type and myostatin-null (Mstn-/-) mice were injured with notexin. Forty-eight hours after injury, accelerated migration and enhanced accretion of myogenic cells (MyoD1+) and macrophages (Mac-1+) was observed at the site of regeneration in Mstn-/- muscle as compared with wild-type muscle. Inflammatory cell numbers decreased more rapidly in the Mstn-/- muscle, indicating that the whole process of inflammatory cell response is accelerated in Mstn-/- mice. Consistent with this result, the addition of recombinant Mstn reduced the activation of satellite cells (SCs) and chemotactic movements of both myoblasts and macrophages ex vivo. Examination of regenerated muscle (28 days after injury) also revealed that Mstn-/- mice showed increased expression of decorin mRNA, reduced fibrosis and improved healing as compared with wild-type mice. On the basis of these results, we propose that Mstn negatively regulates muscle regeneration not only by controlling SC activation but also by regulating the migration of myoblasts and macrophages to the site of injury. Thus, antagonists of Mstn could potentially be useful as pharmacological agents for the treatment of disorders of overt degeneration and regeneration. PMID:16079293

  8. Double-muscled and conventional cattle have the same net energy requirements if these are related to mature and current body protein mass, and to gain composition.

    PubMed

    Schiavon, S; Bittante, G

    2012-11-01

    The hypothesis tested in this paper is that double-muscled (DBM) and conventional cattle, considerably differing in body composition, have similar NE requirements when: a) NE(m) is scaled as a function of current (P(i)) and adult (P(m)) protein mass; and b) ME for gain (ME(g)) is estimated from protein (Pr) and lipid (Lr) retention and their partial ME use efficiencies, the k(p) and k(l) values, respectively. First, 2 databases were examined: 1 was developed combining well known literature information from comparative slaughter trials conducted on British beef steers; the other was based on a trial conducted using extremely lean DBM Piemontese bulls. From the first database, NE(m) was calculated to be 1.625 × P(i) ÷ P(m) × P(m)(0.73) (MJ/kg(0.73)). From the second database, the daily ME(g) was determined as 22.8 MJ × Pr ÷ k(p) + 38.74 MJ × Lr ÷ k(l), assuming (from prior reports) that k(p) = 0.20 and k(l) = 0.75. Thereafter, ME(m) was defined as ME intake minus ME(g), and, hence, NE(m) was predicted as 1.625 × P(i) ÷ P(m) × P(m)(0.73) (where 1.625 was the value obtained from the first dataset). The resulting k(m) (NE(m)/ME(m)) averaged 0.67. This k(m) value did not differ from that (0.65; P = 0.12) predicted by Garrett's equation, which uses dietary ME content as the only predictive variable. Second, the procedure was tested for the ability to detect effects on k(m) caused by increasing BW and dietary factors not estimable from the dietary ME content only. Data were gathered from a trial involving 48 DBM Piemontese bulls divided into 4 groups fed 1 of 4 diets differing in CP content (145 or 108 g/kg DM), with or without addition of 80 g/d of rumen-protected CLA (rpCLA). Bulls were examined at 3 consecutive periods of growth, corresponding to 365, 512 and 631 kg of average BW. All energy balance items were influenced by increasing BW, except k(m) (P = 0.61), in agreement with the expectation that NE(m) requirement depends on the degree of maturity (P

  9. Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

    PubMed Central

    Lange Consiglio, Anna; Dell'Aquila, Maria Elena; Fiandanese, Nadia; Ambruosi, Barbara; Cho, Yoon S; Bosi, Giampaolo; Arrighi, Silvana; Lacalandra, Giovanni M; Cremonesi, Fausto

    2009-01-01

    Background The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI). Methods Compact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml) of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R) was investigated by immunocytochemical staining. Results In expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P < 0.05) and fertilization after ICSI (56% vs 23% for 10 ng/ml leptin-treated and control groups, respectively; P < 0.05). However, the developmental rate and quality of 8-cell stage embryos derived from leptin-treated oocytes (100 ng/ml) was significantly reduced, in contrast to previous data in other species where leptin increased embryo cleavage. Ob and Ob-R proteins were detected up to the 8-cell stage with cortical and cytoplasmic granule-like distribution pattern in each blastomere. Conclusion Leptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin. PMID:19835605

  10. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  11. RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide.

    PubMed

    Miao, Yuanxin; Yang, Jinzeng; Xu, Zhong; Jing, Lu; Zhao, Shuhong; Li, Xinyun

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition. PMID:25860951

  12. Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition.

    PubMed

    MacDonald, Elizabeth M; Andres-Mateos, Eva; Mejias, Rebeca; Simmers, Jessica L; Mi, Ruifa; Park, Jae-Sung; Ying, Stephanie; Hoke, Ahmet; Lee, Se-Jin; Cohn, Ronald D

    2014-04-01

    The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy. PMID:24504412

  13. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  14. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  15. Structure, function and dynamics in adenovirus maturation

    DOE PAGESBeta

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  16. Alteration of energy metabolism gene expression in cumulus cells affects oocyte maturation via MOS-mitogen-activated protein kinase pathway in dairy cows with an unfavorable "Fertil-" haplotype of one female fertility quantitative trait locus.

    PubMed

    Brisard, Daphné; Desmarchais, Alice; Touzé, Jean-Luc; Lardic, Lionel; Freret, Sandrine; Elis, Sebastien; Nuttinck, Fabienne; Camous, Sylvaine; Dupont, Joelle; Uzbekova, Svetlana

    2014-03-01

    Prim'Holstein heifers selected for the "Fertil-" homozygous haplotype of QTL-Female-Fert ility-BTA3 showed a greater rate of early pregnancy failure and slower embryo development after IVM suggesting lower oocyte quality than those selected for "Fertile+". We aimed to ascertain intrafollicular factors related to lower oocyte quality in "Fertil-" cows. Analysis of individual oocytes showed meiotic progression delay in "Fertil-" compared with "Fertil+" dairy cows after in vivo maturation and IVM (P < 0.05). Expression of several genes localized to QTL-F-Fert-BTA3 or related to meiosis and mitogen-activated protein kinase pathway was analyzed in individual metaphase-II oocytes using reverse transcription- real-time polymerase chain reaction. Energy metabolism, apoptosis, extracellular matrix, and QTL-F-Fert-BTA3 genes were analyzed in surrounding cumulus cells (CC). In vivo, a significant decrease in prostaglandin synthase PTGES1 and PTGS2 expression coupled with lower PTGS2 protein abundance in CC and reduced expression of MOS in enclosed metaphase-II oocytes from "Fertil-" cows was observed. IVM strongly deregulated gene expression in CC and in oocytes compared with in vivo; nevertheless, differential expression of several genes including PEX19, NAMPT and MOS was observed between the two haplotypes. During IVM, PTGS2 activity inhibitor NS398 (50 μM) led to lower expression of fatty acid synthase (FASN) in CC and of MOS in treated metaphase-II oocytes. Using immunofluorescence, MOS protein was localized to a midbody-like contractile ring separating the polar body from the ooplasm, suggesting a role in the terminal stage of oocyte maturation. Our results suggest that factors involved in prostaglandin synthesis and lipid metabolism in CC could impair oocyte maturation, and might be involved in the reduced fertility of "Fertil-" cows. PMID:24377862

  17. Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

    PubMed Central

    Miyoshi, Kazuchika; Rzucidlo, S Jacek; Gibbons, John R; Arat, Sezen; Stice, Steven L

    2001-01-01

    Background Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. Results Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. Conclusions Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells. PMID:11476669

  18. Essential amino acids increase microRNA-499, -208b, and -23a and downregulate myostatin and myocyte enhancer factor 2C mRNA expression in human skeletal muscle.

    PubMed

    Drummond, Micah J; Glynn, Erin L; Fry, Christopher S; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2009-12-01

    Essential amino acids (EAA) stimulate muscle protein synthesis in humans. However, little is known about whether microRNAs (miRNA) and genes associated with muscle growth are expressed differently following EAA ingestion. Our purpose in this experiment was to determine whether miRNA and growth-related mRNA expressed in skeletal muscle are up- or downregulated in humans following the ingestion of EAA. We hypothesized that EAA would alter miRNA expression in skeletal muscle as well as select growth-related genes. Muscle biopsies were obtained from the vastus lateralis of 7 young adult participants (3 male, 4 female) before and 3 h after ingesting 10 g of EAA. Muscle samples were analyzed for muscle miRNA (miR-499, -208b, -23a, -1, -133a, and -206) and muscle-growth related genes [MyoD1, myogenin, myostatin, myocyte enhancer factor C (MEF2C), follistatin-like-1 (FSTL1), histone deacytylase 4, and serum response factor mRNA] before and after EAA ingestion using real-time PCR. Following EAA ingestion, miR-499, -208b, -23a, -1, and pri-miR-206 expression increased (P < 0.05). The muscle-growth genes MyoD1 and FSTL1 mRNA expression increased (P < 0.05), and myostatin and MEF2C mRNA were downregulated following EAA ingestion (P < 0.05). We conclude that miRNA and growth-related genes expressed in skeletal muscle are rapidly altered within hours following EAA ingestion. Further work is needed to determine whether these miRNA are post-transcriptional regulators of growth-related genes following an anabolic stimulus. PMID:19828686

  19. Knockdown of myostatin expression by RNAi enhances muscle growth in transgenic sheep.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zi, Ha; Qiao, Jun; Wang, Pengyang; Sheng, Jinliang; Chen, Chuangfu

    2013-01-01

    Myostatin (MSTN) has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT) technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production. PMID:23526994

  20. Association of myostatin variants with growth traits of Zhikong scallop ( Chlamys farreri)

    NASA Astrophysics Data System (ADS)

    Fu, Qiang; Guo, Huihui; Feng, Liying; Li, Xue; Zhang, Lingling; Wang, Shi; Hu, Xiaoli; Bao, Zhenmin

    2016-02-01

    Scallop is a popular sea food and an important aquaculture shellfish. Identification of genes and genetic variants relating to scallop growth could benefit high-yielding scallop breeding. Myostatin ( MSTN) is a conservative regulator of muscle growth, and has become one of the most important target genes for genetic improvement of the production of farmed animals. In this study, four single nucleotide polymorphisms (SNPs) were identified in the 5' flanking region of MSTN gene ( CfMSTN) in Zhikong scallop ( Chlamys farreri). The association of these SNPs with scallop growth traits, including shell length, shell height, body weight and striated muscle weight was analyzed. The SNP g-1162G

  1. Endurance training prevents TWEAK but not myostatin-mediated cardiac remodelling in cancer cachexia.

    PubMed

    Padrão, Ana Isabel; Moreira-Gonçalves, Daniel; Oliveira, Paula A; Teixeira, Catarina; Faustino-Rocha, Ana I; Helguero, Luísa; Vitorino, Rui; Santos, Lúcio Lara; Amado, Francisco; Duarte, José Alberto; Ferreira, Rita

    2015-02-01

    Strategies to prevent tumour burden-induced cardiac remodelling that might progress to heart failure are necessary to improve patients' health outcomes and tolerability to cancer therapies. Exercise has been suggested as a measure to prevent cardiac damage; however, its effectiveness on regulating cardiac remodelling secondary to cancer was never addressed. Using an animal model of mammary tumorigenesis, we studied the impact of 35weeks of endurance training on heart, focusing on the signalling pathways modulated by pro-inflammatory and wasting cytokines. The cardiac fibrosis and myofiber disorganization induced by tumour burden was paralleled by the increase of myostatin and TWEAK with the activation of signalling pathways involving Smad-3, NF-κB, TRAF-6 and atrogin-1. The activation of Akt/mTOR was observed in heart from rats with tumours, for which contributed the extracellular matrix. Endurance training prevented the increase of serum and cardiac TWEAK promoted by cancer, as well as the activation of NF-κB, TRAF6, atrogin-1 and p70S6K in heart. Data highlight the impact of exercise in the modulation of signalling pathways activated by wasting cytokines and the resulting outcomes on heart adaptation. Future studies focused on the cellular pathways underlying cardiac remodelling will assist in the development of exercise programs targeting cancer-related cardiac alterations. PMID:25575785

  2. Protective effect of myostatin gene deletion on aging-related muscle metabolic decline.

    PubMed

    Chabi, B; Pauly, M; Carillon, J; Carnac, G; Favier, F B; Fouret, G; Bonafos, B; Vanterpool, F; Vernus, B; Coudray, C; Feillet-Coudray, C; Bonnieu, A; Lacan, D; Koechlin-Ramonatxo, C

    2016-06-01

    While myostatin gene deletion is a promising therapy to fight muscle loss during aging, this approach induces also skeletal muscle metabolic changes such as mitochondrial deficits, redox alteration and increased fatigability. In the present study, we evaluated the effects of aging on these features in aged wild-type (WT) and mstn knockout (KO) mice. Moreover, to determine whether an enriched-antioxidant diet may be useful to prevent age-related disorders, we orally administered to the two genotypes a melon concentrate rich in superoxide dismutase for 12 weeks. We reported that mitochondrial functional abnormalities persisted (decreased state 3 and 4 of respiration; p<0.05) in skeletal muscle from aged KO mice; however, differences with WT mice were attenuated at old age in line with reduced difference on running endurance between the two genotypes. Interestingly, we showed an increase in glutathione levels, associated with lower lipid peroxidation levels in KO muscle. Enriched antioxidant diet reduced the aging-related negative effects on maximal aerobic velocity and running limit time (p<0.05) in both groups, with systemic adaptations on body weight. The redox status and the hypertrophic phenotype appeared to be beneficial to KO mice, mitigating the effect of aging on the skeletal muscle metabolic remodeling. PMID:26944368

  3. Using "Mighty Mouse" to understand masticatory plasticity: myostatin-deficient mice and musculoskeletal function.

    PubMed

    Ravosa, Matthew J; López, Elisabeth K; Menegaz, Rachel A; Stock, Stuart R; Stack, M Sharon; Hamrick, Mark W

    2008-09-01

    Knockout mice lacking myostatin (Mstn), a negative regulator of the growth of skeletal muscle, develop significant increases in the relative mass of masticatory muscles as well as the ability to generate higher maximal muscle forces. Wild-type and Mstn-deficient mice were compared to investigate the postnatal influence of elevated masticatory loads due to increased jaw-adductor and bite forces on the biomineralization of mandibular articular and cortical bone, the internal structure of the jaw joints, and the composition of temporomandibular joint (TMJ) articular cartilage. To provide an interspecific perspective on the long-term responses of mammalian jaw joints to altered loading conditions, the findings on mice were compared to similar data for growing rabbits subjected to long-term dietary manipulation. Statistically significant differences in joint proportions and bone mineral density between normal and Mstn-deficient mice, which are similar to those observed between rabbit loading cohorts, underscore the need for a comprehensive analysis of masticatory tissue plasticity vis-à-vis altered mechanical loads, one in which variation in external and internal structure are considered. Differences in the expression of proteoglycans and type-II collagen in TMJ articular cartilage between the mouse and rabbit comparisons suggest that the duration and magnitude of the loading stimulus will significantly affect patterns of adaptive and degradative responses. These data on mammals subjected to long-term loading conditions offer novel insights regarding variation in ontogeny, life history, and the ecomorphology of the feeding apparatus. PMID:21669797

  4. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9

    PubMed Central

    Lv, Qingyan; Yuan, Lin; Deng, Jichao; Chen, Mao; Wang, Yong; Zeng, Jian; Li, Zhanjun; Lai, Liangxue

    2016-01-01

    CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases. PMID:27113799

  5. Cone Early Maturity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hop cone early maturity is thought to be caused by diffuse infections of cone, just prior to harvest, by Podosphaera macularis. The disease is best managed by limiting the amount of leaf infection by P. macularis prior to bloom. The yield and quality reductions associated with Hop cone early matur...

  6. Sperm Proteome Maturation in the Mouse Epididymis

    PubMed Central

    Skerget, Sheri; Rosenow, Matthew A.; Petritis, Konstantinos; Karr, Timothy L.

    2015-01-01

    In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm. PMID:26556802

  7. Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

    PubMed

    Masci, Stefania; Laino, Paolo; Janni, Michela; Botticella, Ermelinda; Di Carli, Mariasole; Benvenuto, Eugenio; Danieli, Pier Paolo; Lilley, Kathryn S; Lafiandra, Domenico; D'Ovidio, Renato

    2015-04-22

    Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters. PMID:25823882

  8. Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF

    PubMed Central

    Nicke, Tristan; Schnitzer, Tobias; Münch, Karin; Adamczack, Julia; Haufschildt, Kristin; Buchmeier, Sabine; Kucklick, Martin; Felgenträger, Undine; Jänsch, Lothar; Riedel, Katharina; Layer, Gunhild

    2013-01-01

    The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS. PMID:23683062

  9. Viral and host control of cytomegalovirus maturation

    PubMed Central

    Tandon, Ritesh; Mocarski, Edward S.

    2012-01-01

    Maturation in herpesviruses initiates in the nucleus of the infected cell with encapsidation of viral DNA to form nucleocapsids and concludes with envelopment in the cytoplasm to form infectious virions that egress the cell. The entire process of virus maturation is orchestrated by protein-protein interactions and enzymatic activities of viral and host origin. Viral tegument proteins play important roles in maintaining the structural stability of capsids and directing the acquisition of virus envelope. Envelopment occurs at modified host membranes and exploits host vesicular trafficking. In this review, we summarize the current knowledge and concepts in human cytomegalovirus (HCMV) maturation and their parallels in other herpesviruses with an emphasis on viral and host factors regulating this process. PMID:22633075

  10. Signaling through C/EBP homologous protein and death receptor 5 and calpain activation differentially regulate THP-1 cell maturation-dependent apoptosis induced by Shiga toxin type 1.

    PubMed

    Lee, Moo-Seung; Cherla, Rama P; Lentz, Erin K; Leyva-Illades, Dinorah; Tesh, Vernon L

    2010-08-01

    Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity. PMID:20515924

  11. Brain-derived neurotrophic factor facilitates in vivo internalization of tetanus neurotoxin C-terminal fragment fusion proteins in mature mouse motor nerve terminals.

    PubMed

    Roux, Sylvie; Saint Cloment, Cécile; Curie, Thomas; Girard, Emmanuelle; Miana Mena, Francisco-Javier; Barbier, Julien; Osta, Rosario; Molgó, Jordi; Brûlet, Philippe

    2006-09-01

    In a previous study it was reported that fusion proteins composed of the atoxic C-terminal fragment of tetanus toxin (TTC) and green fluorescent protein or beta-galactosidase (GFP-TTC and beta-gal-TTC, respectively) rapidly cluster at motor nerve terminals of the mouse neuromuscular junction (NMJ). Because this traffic involves presynaptic activity, probably via the secretion of active molecules, we examined whether it is affected by brain-derived neurotrophic factor (BDNF). Quantitative confocal microscopy and a fluorimetric assay for beta-gal activity revealed that co-injecting BDNF and the fusion proteins significantly increased the kinetics and amount of the proteins' localization at the NMJ and their internalization by motor nerve terminals. The observed increases were independent of synaptic vesicle recycling because BDNF did not affect spontaneous quantal acetylcholine release. In addition, injecting anti-BDNF antibody shortly before injecting GFP-TTC, and before co-injecting GFP-TTC and BDNF, significantly reduced the fusion protein's localization at the NMJ. Co-injecting GFP-TTC with neurotrophin-4 (NT-4) or glial-derived neurotrophic factor (GDNF), but not with nerve growth factor, neurotrophin-3 or ciliary neurotrophic factor, also significantly increased the fusion protein's localization at the NMJ. Thus, TTC probes may use for their neuronal internalization endocytic pathways normally stimulated by BDNF, NT-4 and GDNF binding. Different tyrosine kinase receptors with similar signalling pathways are activated by BDNF/NT-4 and GDNF binding. Thus, activated components of these signalling pathways may be involved in the TTC probes' internalization, perhaps by facilitating localization of receptors of TTC in specific membrane microdomains or by recruiting various factors needed for internalization of TTC. PMID:17004918

  12. The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions

    PubMed Central

    Gershburg, Svetlana; Geltz, Joshua; Peterson, Karin E.; Halford, William P.; Gershburg, Edward

    2015-01-01

    Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells. PMID:26115119

  13. Sirtuin 1 promotes the proliferation of C2C12 myoblast cells via the myostatin signaling pathway.

    PubMed

    Wang, Liang; Zhang, Ting; Xi, Yongyong; Yang, Cuili; Sun, Chengcao; Li, Dejia

    2016-08-01

    Accumulating evidence suggests that Sirtuin (Sirt)1 serves a significant role in proliferation and differentiation of myoblast cells; however the signaling mechanisms involved remain to be established. Myostatin (MSTN), a member of transforming growth factor‑β family, is an vital regulator of myoblast, fibroblast growth and differentiation. To determine if MSTN is involved in the regulation of myoblast cell proliferation by Sirt1, the present study administrated the Sirt1 activator resveratrol, inhibitor nicotinamide (NAM) and MSTN inhibitor SB431542 to C2C12 myoblast cells. It was demonstrated that the Sirt1 activator, resveratrol, repressed, whereas the Sirt1 inhibitor, NAM, enhanced C2C12 myoblast cells proliferation in a Sirt1‑dependent manner. SB431542 promoted the proliferation of C2C12 myoblast cells and reversed the inhibition effect of NAM on C2C12 myoblast cell proliferation. Additionally, resveratrol upregulated the mRNA expression of MyoD, but inhibited the expression of MSTN. Additionally, NAM significantly repressed the expression of MyoD and the phosphorylation of P107 (p‑P107), but enhanced the expression of MSTN and the protein expression of P107. SB431542 significantly mitigated the effect of NAM on the expression of MyoD, P107 and p‑P107. Taken together, these results indicated that Sirt1 promotes the proliferation of C2C12 myoblast cells via the MSTN signaling pathway. PMID:27279047

  14. Data Product Maturity

    Atmospheric Science Data Center

    2013-03-25

    ... document, maturity levels are provided separately for each scientific data set (SDS) included with the data files. The data product ... indiscriminate use of these data products as the basis for research findings, journal publications, and/or presentations.   ...

  15. Effect of moderate dietary restriction on visceral organ weight, hepatic oxygen consumption, and metabolic proteins associated with energy balance in mature pregnant beef cows.

    PubMed

    Wood, K M; Awda, B J; Fitzsimmons, C; Miller, S P; McBride, B W; Swanson, K C

    2013-09-01

    Twenty-two nonlactating multiparous pregnant beef cows (639 ± 68 kg) were used to investigate the effect of dietary restriction on the abundance of selected proteins regulating cellular energy metabolism. Cows were fed at either 85% (n = 11; LOW) or 140% (n = 11; HIGH) of total NE requirements. The diet consisted of a haylage-based total mixed ration containing 20% wheat straw. Cows were slaughtered by block (predicted date of parturition), beginning 83 d after the initiation of dietary treatments and every week thereafter for 6 wk, such that each block was slaughtered at approximately 250 d of gestation. Tissue samples from liver, kidney, sternomandibularis muscle, ruminal papilli (ventral sac), pancreas, and small intestinal muscosa were collected at slaughter and snap frozen in liquid N2. Western blots were conducted to quantify abundance of proliferating cell nuclear antigen (PCNA), ATP synthase, ubiquitin, and Na/K+ ATPase for all tissues; PPARγ, PPARγ coactivator 1 α (PGC-1α), and 5´-adenosine monophosphate-activated protein kinase (AMPK) and the activated form phosphorylated-AMPK (pAMPK) for liver, muscle, and rumen; phosphoenolpyruvate carboxykinase (PEPCK) for liver and kidney; and uncoupling protein 2 (UCP2) for liver. Statistical analysis was conducted using Proc Mixed in SAS and included the fixed effects of dietary treatment, cow age, block, and the random effect of pen. Dietary treatments resulted in cows fed HIGH having greater (P ≤ 0.04) ADG and final BW than cows fed LOW. Abundance of ubiquitin in muscle was greater (P = 0.009) in cows fed LOW, and PCG-1 α in liver was greater (P = 0.03) in cows fed HIGH. Hepatic O2 consumption was greater in HIGH (P ≤ 0.04). Feed intake can influence the abundance of important metabolic proteins and suggest that protein degradation may increase in muscle from moderately nutrient restricted cows and that energy metabolism in liver increases in cows fed above NE requirements. PMID:23893975

  16. Real-time PCR genotyping and frequency of the myostatin F94L mutation in beef cattle breeds.

    PubMed

    Vankan, D M; Waine, D R; Fortes, M R S

    2010-04-01

    This research developed two real-time PCR assays, employing high-resolution melt and allele-specific analysis to accurately genotype the F94L mutation in cattle. This mutation (g.433C > A) in the growth differentiation factor 8 or myostatin gene has recently been shown to be functionally associated with increased muscle mass and carcass yield in cattle. The F94L mutation is not, like other myostatin mutations, associated with reduced fertility and dystocia. It is therefore a candidate for introgression into other breeds to improve retail beef yield and the development of a simple and accurate test to genotype this specific mutation is warranted. Variations in the efficiency of enzyme cleavage compromised the accuracy of genotyping by published methods, potentially resulting in an overestimation of the frequency of the mutant allele. The frequency of the F94L mutation was determined by real-time PCR in 1140 animals from 15 breeds of cattle in Australia. The mutation was present in Simmental (0.8%), Piedmontese (2%), Droughtmaster (4%) and Limousin (94.2%) but not found in Salers, Angus, Poll Hereford, Hereford, Gelbvieh, Charolais, Jersey, Brahman, Holstein, Shorthorn or Maine Anjou. The low prevalence of F94L in all beef breeds except Limousin indicates the significant potential for this mutation to improve retail yield in Australian beef cattle. PMID:22444040

  17. Dopaminergic Toxin 1-Methyl-4-Phenylpyridinium, Proteins α-Synuclein and Glia Maturation Factor Activate Mast Cells and Release Inflammatory Mediators.

    PubMed

    Kempuraj, Duraisamy; Thangavel, Ramasamy; Yang, Evert; Pattani, Sagar; Zaheer, Smita; Santillan, Donna A; Santillan, Mark K; Zaheer, Asgar

    2015-01-01

    Parkinson's disease (PD) is characterized by the presence of Lewy bodies and degeneration of dopaminergic neurons. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Lewy body component α-synuclein activates glia in PD pathogenesis. Mast cells and glia maturation factor (GMF) are implicated in neuroinflammatory conditions including Multiple Sclerosis. However, the role of mast cells in PD is not yet known. We have analyzed the effect of recombinant GMF, MPP+, α-synuclein and interleukin-33 (IL-33) on mouse bone marrow-derived cultured mast cells (BMMCs), human umbilical cord blood-derived cultured mast cells (hCBMCs) and mouse brain-derived cultured astrocytes by quantifying cytokines/chemokines released using ELISA or by detecting the expression of co-stimulatory molecules CD40 and CD40L by flow cytometry. GMF significantly released chemokine (C-C motif) ligand 2 (CCL2) from BMMCs but its release was reduced in BMMCs from GMF knockout mice. GMF, α-synuclein and MPP+ released IL-1β, β-hexosaminidase from BMMCs, and IL-8 from hCBMCs. GMF released CCL5, and IL-33- induced the expression of GMF from hCBMCs. Novel GMF expression was detected in hCBMCs and BMMCs by immunocytochemistry. GMF released tumor necrosis factor-alpha (TNF-α) from mouse astrocytes, and this release was greater in BMMC- astrocyte coculture than in individual cultures. Flow cytometry results showed increased IL-33 expression by GMF and MPP+, and GMF-induced CD40 expression in astrocytes. Proinflammatory mediator release by GMF, MPP+ and α-synuclein, as well as GMF expression by mast cells indicate a potential therapeutic target for neurodegenerative diseases including PD. PMID:26275153

  18. Korean red ginseng extract rejuvenates testicular ineffectiveness and sperm maturation process in aged rats by regulating redox proteins and oxidative defense mechanisms.

    PubMed

    Kopalli, Spandana Rajendra; Hwang, Seock-Yeon; Won, Yu-Jin; Kim, Sung-Won; Cha, Kyu-Min; Han, Chang-Kyun; Hong, Jae-Yup; Kim, Si-Kwan

    2015-09-01

    Distortion of intracellular oxidant and antioxidant balances appears to be a common feature that underlies in age-related male sexual impairment. Therefore regulating oxidative defense mechanisms might be an ideal approach in improving male sexual dysfunctions. In the present study, the effect of Korean red ginseng aqueous extract (KRG) on age-induced testicular dysfunction in rats was investigated. KRG (200mg/kg) mixed with regular pellet diet was administered orally for six months and the morphological, spermatogenic and antioxidant enzyme status in testis of aged rats (18months) were evaluated. Data indicated a significant change in morphology and decrease in spermatogenesis-related parameters in aged rats (AC) compared with young rats (YC). Sperm number, germ cell count, Sertoli cell count and Sertoli cell index were significantly (p<0.05) restored in KRG-treated aged rat groups (G-AC). Further the increased lipid peroxidation as measured by malondialdehyde (p<0.05), and altered enzymatic (superoxide dismutase, glutathione peroxidase, glutathione S-transferase, glutathione reductase and catalase) and non-enzymatic (reduced glutathione, ascorbic acid and α-tocopherol) antioxidants (p<0.05) were attenuated by KRG treatment in aged rats to near normal levels as in YC groups. Furthermore, proteomic analysis demonstrated differential expression of selected proteins such as phosphatidylinositol transfer protein, fatty acid binding protein-9, triosephosphate isomerase-1 and aldehyde (aldose) reductase-1in aged rats was significantly (p<0.05) protected by KRG treatment. In conclusion, long-term administration of KRG restored aging-induced testicular ineffectiveness in rats by modulating redox proteins and oxidative defense mechanisms. PMID:25980653

  19. Bluetongue virus capsid assembly and maturation.

    PubMed

    Mohl, Bjorn-Patrick; Roy, Polly

    2014-08-01

    Maturation is an intrinsic phase of the viral life cycle and is often intertwined with egress. In this review we focus on orbivirus maturation by using Bluetongue virus (BTV) as a representative. BTV, a member of the genus Orbivirus within the family Reoviridae, has over the last three decades been subjected to intense molecular study and is thus one of the best understood viruses. BTV is a non-enveloped virus comprised of two concentric protein shells that encapsidate 10 double-stranded RNA genome segments. Upon cell entry, the outer capsid is shed, releasing the core which does not disassemble into the cytoplasm. The polymerase complex within the core then synthesizes transcripts from each genome segment and extrudes these into the cytoplasm where they act as templates for protein synthesis. Newly synthesized ssRNA then associates with the replicase complex prior to encapsidation by inner and outer protein layers of core within virus-triggered inclusion bodies. Maturation of core occurs outside these inclusion bodies (IBs) via the addition of the outer capsid proteins, which appears to be coupled to a non-lytic, exocytic pathway during early infection. Similar to the enveloped viruses, BTV hijacks the exocytosis and endosomal sorting complex required for trafficking (ESCRT) pathway via a non-structural glycoprotein. This exquisitely detailed understanding is assembled from a broad array of assays, spanning numerous and diverse in vitro and in vivo studies. Presented here are the detailed insights of BTV maturation and egress. PMID:25196482

  20. Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

    PubMed

    Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

    2003-09-01

    The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria. PMID:12730097

  1. [Effects of noopept and cortexin on the behavior of matured rats treated with corticoliberin or 70-kDa heat shock proteins in early ontogeny].

    PubMed

    Shabanov, P D; Lebedev, A A; Stetsenko, V P; Lavrov, N V; Sablina, G V; Gudasheva, T A; Ostrovaskaia, R U

    2007-01-01

    Young Wistar rats aged 4 days were injected intraperitoneally with corticotropin releasing hormone (CRH), which is an agent activating the stress system, or 70-kDa heat shock proteins (HSP-70)--intracellular shaperons, possessing antistress properties. In grown adult rats aged 90-100 days, the effects of nootropic drugs noopept and cortexin (1 mg/kg, i.p.) were assessed. The activation of stress or antistress systems with CRH or HSP-70 significantly altered the drug action. The effects were different in males and females and depended on animal gender. The spectrum of pharmacological activity of noopept and cortexin changed: noopept demonstrated preferable psychoactivating and antiaggressive effects, whereas cortexin showed mild anxiolytic and antidepressant activity. It is suggested that the behavioral effects of nootropes depend on the conditions of the stress system formation in early ontogeny. PMID:17402584

  2. Maturation processing and characterization of streptopain.

    PubMed

    Chen, Chiu-Yueh; Luo, Shih-Chi; Kuo, Chih-Feng; Lin, Yee-Shin; Wu, Jiunn-Jong; Lin, Ming T; Liu, Ching-Chuan; Jeng, Wen-Yih; Chuang, Woei-Jer

    2003-05-01

    Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn(356) might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions. PMID:12621045

  3. L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

    PubMed Central

    2014-01-01

    Background The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. Methods After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). Results MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Conclusions Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty

  4. Jealousy and Moral Maturity.

    ERIC Educational Resources Information Center

    Mathes, Eugene W.; Deuger, Donna J.

    Jealousy may be perceived as either good or bad depending upon the moral maturity of the individual. To investigate this conclusion, a study was conducted testing two hypothesis: a positive relationship exists between conventional moral reasoning (reference to norms and laws) and the endorsement and level of jealousy; and a negative relationship…

  5. In vitro maturation alters gene expression in bovine oocytes.

    PubMed

    Adona, Paulo R; Leal, Cláudia L V; Biase, Fernando H; De Bem, Tiago H; Mesquita, Lígia G; Meirelles, Flávio V; Ferraz, André L; Furlan, Luiz R; Monzani, Paulo S; Guemra, Samuel

    2016-08-01

    Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence. PMID:26885679

  6. Atelocollagen-mediated systemic administration of myostatin-targeting siRNA improves muscular atrophy in caveolin-3-deficient mice.

    PubMed

    Kawakami, Emi; Kinouchi, Nao; Adachi, Taro; Ohsawa, Yutaka; Ishimaru, Naozumi; Ohuchi, Hideyo; Sunada, Yoshihide; Hayashi, Yoshio; Tanaka, Eiji; Noji, Sumihare

    2011-01-01

    Small interfering RNA (siRNA)-mediated silencing of gene expression is rapidly becoming a powerful tool for molecular therapy. However, the rapid degradation of siRNAs and their limited duration of activity require efficient delivery methods. Atelocollagen (ATCOL)-mediated administration of siRNAs is a promising approach to disease treatment, including muscular atrophy. Herein, we report that ATCOL-mediated systemic administration of a myostatin-targeting siRNA into a caveolin-3-deficient mouse model of limb-girdle muscular dystrophy 1C (LGMD1C) induced a marked increase in muscle mass and a significant recovery of contractile force. These results provide evidence that ATCOL-mediated systemic administration of siRNAs may be a powerful therapeutic tool for disease treatment, including muscular atrophy. PMID:21261610

  7. HIV-1 Assembly, Budding, and Maturation

    PubMed Central

    Sundquist, Wesley I.; Kräusslich, Hans-Georg

    2012-01-01

    A defining property of retroviruses is their ability to assemble into particles that can leave producer cells and spread infection to susceptible cells and hosts. Virion morphogenesis can be divided into three stages: assembly, wherein the virion is created and essential components are packaged; budding, wherein the virion crosses the plasma membrane and obtains its lipid envelope; and maturation, wherein the virion changes structure and becomes infectious. All of these stages are coordinated by the Gag polyprotein and its proteolytic maturation products, which function as the major structural proteins of the virus. Here, we review our current understanding of the mechanisms of HIV-1 assembly, budding, and maturation, starting with a general overview and then providing detailed descriptions of each of the different stages of virion morphogenesis. PMID:22762019

  8. A Limousin specific myostatin allele affects longissimus muscle area and fatty acid profiles in a Wagyu-Limousin F2 population.

    PubMed

    Alexander, L J; Kuehn, L A; Smith, T P L; Matukumalli, L K; Mote, B; Koltes, J E; Reecy, J; Geary, T W; Rule, D C; Macneil, M D

    2009-05-01

    A microsatellite-based genome scan of a Wagyu x Limousin F(2) cross population previously demonstrated QTL affecting LM area and fatty acid composition were present in regions near the centromere of BTA2. In this study, we used 70 SNP markers to examine the centromeric 24 megabases (Mb) of BTA2, including the Limousin-specific F94L myostatin allele (AB076403.1; 415C > A) located at approximately 6 Mb on the draft genome sequence of BTA2. A significant effect of the F94L marker was observed (F = 60.17) for LM area, which indicated that myostatin is most likely responsible for the effect. This is consistent with previous reports that the substitution of Leu for Phe at AA 94 of myostatin (caused by the 415C > A transversion) is associated with increased muscle growth. Surprisingly, several fatty acid trait QTL, which affected the amount of unsaturated fats, also mapped to or very near the myostatin marker, including the ratio of C16:1 MUFA to C16:0 saturated fat (F = 16.72), C18:1 to C18:0 (F = 18.88), and total content of MUFA (F = 17.12). In addition, QTL for extent of marbling (F = 14.73) approached significance (P = 0.05), and CLA concentration (F = 9.22) was marginally significant (P = 0.18). We also observed associations of SNP located at 16.3 Mb with KPH (F = 15.00) and for the amount of SFA (F = 12.01). These results provide insight into genetic differences between the Wagyu and Limousin breeds and may lead to a better tasting and healthier product for consumers through improved selection for lipid content of beef. PMID:19213716

  9. Hydrodynamic Limb Vein Injection of Adeno-Associated Virus Serotype 8 Vector Carrying Canine Myostatin Propeptide Gene into Normal Dogs Enhances Muscle Growth

    PubMed Central

    Qiao, Chunping; Li, Juan; Zheng, Hui; Bogan, Janet; Li, Jianbin; Yuan, Zhenhua; Zhang, Cheng; Bogan, Dan; Kornegay, Joe

    2009-01-01

    Abstract Inhibition or blockade of myostatin, a negative growth factor of skeletal muscle, enhances muscle growth and therefore is considered a promising strategy for the treatment of muscle-wasting diseases such as the muscular dystrophies. Previously, we showed that myostatin blockade in both normal and dystrophin-deficient mdx mice by systemic delivery of the myostatin propeptide (MPRO) gene by an adeno-associated virus serotype 8 (AAV8) vector could enhance muscle growth and ameliorate dystrophic lesions. Here, we further investigate whether the muscle growth effect of myostatin blockade can be achieved in dogs by gene transfer. First, we cloned the canine MPRO gene, packaged it in the AAV8 vector, and showed robust muscle-enhancing effects after systemic delivery into neonatal mice. This vector was then further tested in two 3-month-old normal dogs (weighing 9.7 and 6.3 kg). The vector was delivered to one limb by hydrodynamic vein injection, and the contralateral limb served as a control. The delivery procedure was safe, without discernible adverse effects. AAV vector DNA and MPRO gene expression were detected by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining of muscle biopsies. Overexpression of MPRO resulted in enhanced muscle growth without a cytotoxic T lymphocytic immune response, as evidenced by larger myofibers in multiple muscles, increased muscle volume determined by magnetic resonance imaging, and the lack of CD4+ and CD8+ T cell infiltration in the vector-injected limbs. Our preliminary study thus supports further investigation of this therapeutic strategy in the dystrophin-deficient golden retriever muscular dystrophy dog model. PMID:18828709

  10. Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

    PubMed

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-03-11

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  11. Nuclear/Nucleolar GTPase 2 Proteins as a Subfamily of YlqF/YawG GTPases Function in Pre-60S Ribosomal Subunit Maturation of Mono- and Dicotyledonous Plants*

    PubMed Central

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I. Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-01-01

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with kcat values 0.12 ± 0.007 min−1 (n = 6) and 0.087 ± 0.002 min−1 (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  12. Effect of resistance exercise intensity on the expression of PGC-1α isoforms and the anabolic and catabolic signaling mediators, IGF-1 and myostatin, in human skeletal muscle.

    PubMed

    Schwarz, Neil A; McKinley-Barnard, Sarah K; Spillane, Mike B; Andre, Thomas L; Gann, Joshua J; Willoughby, Darryn S

    2016-08-01

    The purpose of this study was to investigate the acute messenger (mRNA) expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) isoforms, insulin-like growth factor-1Ea (IGF-1Ea), and myostatin in response to 2 resistance exercise intensities. In a uniform-balanced, crossover design, 10 participants performed 2 separate testing sessions involving a lower body resistance exercise component consisting of a lower intensity (50% of 1-repetition maximum; 1RM) protocol and a higher intensity (80% of 1RM) protocol of equal volumes. Muscle samples were obtained at before exercise, 45 min, 3 h, 24 h, and 48 h postexercise. Resistance exercise did not alter total PGC-1α mRNA expression; however, distinct responses of each PGC-1α isoform were observed. The response of each isoform was consistent between sessions, suggesting no effect of resistance exercise intensity on the complex transcriptional expression of the PGC-1α gene. IGF-1Ea mRNA expression significantly increased following the higher intensity session compared with pre-exercise and the lower intensity session. Myostatin mRNA expression was significantly reduced compared with pre-exercise values at all time points with no difference between exercise intensity. Further research is needed to determine the effects of the various isoforms of PGC-1α in human skeletal muscle on the translational level as well as their relation to the expression of IGF-1 and myostatin. PMID:27467217

  13. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  14. A Socioanalytic Model of Maturity

    ERIC Educational Resources Information Center

    Hogan, Robert; Roberts, Brent W.

    2004-01-01

    K0 describes a point of view on maturity that departs from earlier treatments in two ways. First, it rejects the popular assumption from humanistic psychology that maturity is a function of self-actualization and stipulates that maturity is related to certain performance capacities--namely, the ability to form lasting relationships and to achieve…

  15. Mechanical Properties of Murine Leukemia Virus Particles: Effect of Maturation

    PubMed Central

    Kol, Nitzan; Gladnikoff, Micha; Barlam, David; Shneck, Roni Z.; Rein, Alan; Rousso, Itay

    2006-01-01

    After budding from the host cell, retroviruses undergo a process of internal reorganization called maturation, which is prerequisite to infectivity. Viral maturation is accompanied by dramatic morphological changes, which are poorly understood in physical/mechanistic terms. Here, we study the mechanical properties of live mature and immature murine leukemia virus particles by indentation-type experiments conducted with an atomic force microscope tip. We find that both mature and immature particles have an elastic shell. Strikingly, the virus shell is twofold stiffer in the immature (0.68 N/m) than the mature (0.31 N/m) form. However, finite-element simulation shows that the average Young's modulus of the immature form is more than fourfold lower than that of the mature form. This finding suggests that per length unit, the protein-protein interactions in the mature shell are stronger than those in the immature shell. We also show that the mature virus shell is brittle, since it can be broken by application of large loading forces, by firm attachment to a substrate, or by repeated application of force. Our results are the first analysis of the mechanical properties of an animal virus, and demonstrate a linkage between virus morphology and mechanical properties. PMID:16632508

  16. Fascin1 promotes cell migration of mature dendritic cells.

    PubMed

    Yamakita, Yoshihiko; Matsumura, Fumio; Lipscomb, Michael W; Chou, Po-chien; Werlen, Guy; Burkhardt, Janis K; Yamashiro, Shigeko

    2011-03-01

    Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes. PMID:21263068

  17. An unexpected twist in viral capsid maturation

    SciTech Connect

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-04-14

    Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 {angstrom} resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 {angstrom}. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and -sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol{sup -1} of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic

  18. IL-1R/TLR2 through MyD88 Divergently Modulates Osteoclastogenesis through Regulation of Nuclear Factor of Activated T Cells c1 (NFATc1) and B Lymphocyte-induced Maturation Protein-1 (Blimp1).

    PubMed

    Chen, Zhihong; Su, Lingkai; Xu, Qingan; Katz, Jenny; Michalek, Suzanne M; Fan, Mingwen; Feng, Xu; Zhang, Ping

    2015-12-11

    Toll-like receptors (TLR) and the receptor for interleukin-1 (IL-1R) signaling play an important role in bacteria-mediated bone loss diseases including periodontitis, rheumatoid arthritis, and osteomyelitis. Recent studies have shown that TLR ligands inhibit the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from un-committed osteoclast precursors, whereas IL-1 potentiates RANKL-induced osteoclast formation. However, IL-1R and TLR belong to the same IL-1R/TLR superfamily, and activate similar intracellular signaling pathways. Here, we investigate the molecular mechanisms underlying the distinct effects of IL-1 and Porphyromonas gingivalis lipopolysaccharide (LPS-PG) on RANKL-induced osteoclast formation. Our results show that LPS-PG and IL-1 differentially regulate RANKL-induced activation of osteoclast genes encoding Car2, Ctsk, MMP9, and TRAP, as well as expression of NFATc1, a master transcription factor of osteoclastogenesis. Regulation of osteoclast genes and NFATc1 by LPS-PG and IL-1 is dependent on MyD88, an important signaling adaptor for both TLR and IL-1R family members. Furthermore, LPS-PG and IL-1 differentially regulate RANKL-costimulatory receptor OSCAR (osteoclast-associated receptor) expression and Ca(2+) oscillations induced by RANKL. Moreover, LPS-PG completely abrogates RANKL-induced gene expression of B lymphocyte-induced maturation protein-1 (Blimp1), a global transcriptional repressor of anti-osteoclastogenic genes encoding Bcl6, IRF8, and MafB. However, IL-1 enhances RANKL-induced blimp1 gene expression but suppresses the gene expression of bcl6, irf8, and mafb. Our study reveals the involvement of multiple signaling molecules in the differential regulation of RANKL-induced osteoclastogenesis by TLR2 and IL-1 signaling. Understanding the signaling cross-talk among TLR, IL-1R, and RANK is critical for identifying therapeutic strategies to control bacteria-mediated bone loss. PMID:26483549

  19. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

    PubMed

    Crispo, M; Mulet, A P; Tesson, L; Barrera, N; Cuadro, F; dos Santos-Neto, P C; Nguyen, T H; Crénéguy, A; Brusselle, L; Anegón, I; Menchaca, A

    2015-01-01

    While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and

  20. Local Applications of Myostatin-siRNA with Atelocollagen Increase Skeletal Muscle Mass and Recovery of Muscle Function

    PubMed Central

    Kawakami, Emi; Kawai, Nobuhiko; Kinouchi, Nao; Mori, Hiroyo; Ohsawa, Yutaka; Ishimaru, Naozumi; Sunada, Yoshihide; Noji, Sumihare; Tanaka, Eiji

    2013-01-01

    Background Growing evidence suggests that small-interfering RNA (siRNA) can promote gene silencing in mammalian cells without induction of interferon synthesis or nonspecific gene suppression. Recently, a number of highly specific siRNAs targeted against disease-causing or disease-promoting genes have been developed. In this study, we evaluate the effectiveness of atelocollagen (ATCOL)-mediated application of siRNA targeting myostatin (Mst), a negative regulator of skeletal muscle growth, into skeletal muscles of muscular dystrophy model mice. Methods and Findings We injected a nanoparticle complex containing myostatin-siRNA and ATCOL (Mst-siRNA/ATCOL) into the masseter muscles of mutant caveolin-3 transgenic (mCAV-3Tg) mice, an animal model for muscular dystrophy. Scrambled (scr) -siRNA/ATCOL complex was injected into the contralateral muscles as a control. Two weeks after injection, the masseter muscles were dissected for histometric analyses. To investigate changes in masseter muscle activity by local administration of Mst-siRNA/ATCOL complex, mouse masseter electromyography (EMG) was measured throughout the experimental period via telemetry. After local application of the Mst-siRNA/ATCOL complex, masseter muscles were enlarged, while no significant change was observed on the contralateral side. Histological analysis showed that myofibrils of masseter muscles treated with the Mst-siRNA/ATCOL complex were significantly larger than those of the control side. Real-time PCR analysis revealed a significant downregulation of Mst expression in the treated masseters of mCAV-3Tg mice. In addition, expression of myogenic transcription factors was upregulated in the Mst-siRNA-treated masseter muscle, while expression of adipogenic transcription factors was significantly downregulated. EMG results indicate that masseter muscle activity in mCAV-3Tg mice was increased by local administration of the Mst-siRNA/ATCOL complex. Conclusion These data suggest local administration of

  1. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

    PubMed Central

    Crispo, M.; Mulet, A. P.; Tesson, L.; Barrera, N.; Cuadro, F.; dos Santos-Neto, P. C.; Nguyen, T. H.; Crénéguy, A.; Brusselle, L.; Anegón, I.; Menchaca, A.

    2015-01-01

    While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and

  2. CFD - Mature Technology?

    NASA Technical Reports Server (NTRS)

    Kwak, Dochan

    2005-01-01

    Over the past 30 years, numerical methods and simulation tools for fluid dynamic problems have advanced as a new discipline, namely, computational fluid dynamics (CFD). Although a wide spectrum of flow regimes are encountered in many areas of science and engineering, simulation of compressible flow has been the major driver for developing computational algorithms and tools. This is probably due to a large demand for predicting the aerodynamic performance characteristics of flight vehicles, such as commercial, military, and space vehicles. As flow analysis is required to be more accurate and computationally efficient for both commercial and mission-oriented applications (such as those encountered in meteorology, aerospace vehicle development, general fluid engineering and biofluid analysis) CFD tools for engineering become increasingly important for predicting safety, performance and cost. This paper presents the author's perspective on the maturity of CFD, especially from an aerospace engineering point of view.

  3. Vaccinia Virus Infection Requires Maturation of Macropinosomes.

    PubMed

    Rizopoulos, Zaira; Balistreri, Giuseppe; Kilcher, Samuel; Martin, Caroline K; Syedbasha, Mohammedyaseen; Helenius, Ari; Mercer, Jason

    2015-08-01

    The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live-cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome-associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow-up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late-penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration. PMID:25869659

  4. Transcriptional Landscape of Cardiomyocyte Maturation

    PubMed Central

    Uosaki, Hideki; Cahan, Patrick; Lee, Dong I.; Wang, Songnan; Miyamoto, Matthew; Fernandez, Laviel; Kass, David A.; Kwon, Chulan

    2015-01-01

    SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation. PMID:26586429

  5. COPI selectively drives maturation of the early Golgi

    DOE PAGESBeta

    Papanikou, Effrosyni; Day, Kasey J.; Austin, Jotham; Glick, Benjamin S.

    2015-12-28

    COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

  6. Two Rab2 interactors regulate dense-core vesicle maturation.

    PubMed

    Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M

    2014-04-01

    Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1, and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

  7. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  8. Small molecules dorsomorphin and LDN-193189 inhibit myostatin/GDF8 signaling and promote functional myoblast differentiation.

    PubMed

    Horbelt, Daniel; Boergermann, Jan H; Chaikuad, Apirat; Alfano, Ivan; Williams, Eleanor; Lukonin, Ilya; Timmel, Tobias; Bullock, Alex N; Knaus, Petra

    2015-02-01

    GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis. PMID:25368322

  9. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system.

    PubMed

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-01-01

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

  10. Expression of myostatin in the spotted rose snapper Lutjanus guttatus during larval and juvenile development under cultured conditions.

    PubMed

    Torres-Velarde, J; Ibarra-Castro, L; Rodríguez-Ibarra, E; Sifuentes-Romero, I; Hernández-Cornejo, R; García-Gasca, A

    2015-11-01

    In this study, the developmental expression pattern of myostatin (mstn) in the spotted rose snapper Lutjanus guttatus under culture conditions is presented. The full coding sequence of mstn from L. guttatus was isolated from muscle tissue, obtaining 1134 nucleotides which encode a peptide of 377 amino acids. The phylogenetic analysis indicated that this sequence corresponds to mstn-1. mstn expression was detected in embryonic stages, and maintained at low levels until 28 days post-hatch, when it showed a significant increase, coinciding with the onset of metamorphosis. After that, expression was fluctuating, coinciding probably with periods of rapid and slow muscle growth or individual growth rates. mstn expression was also analysed by body mass with higher levels detected in smaller animals, irrespective of age. mstn was also expressed in other tissues from L. guttatus, presenting higher levels in brain, eye and gill. In brain for instance, two variants of mstn were isolated, both coding sequences were identical to muscle, except that one of them contained a 75 nucleotide deletion in exon 1, maintaining the reading frame but deleting two conserved cysteine residues. Phylogenetic analysis indicated that this brain variant was also mstn-1. The function of this variant is not clear and needs further investigation. These results indicate that mstn-1 participates in different physiological processes other than muscle growth in fishes. PMID:26376729

  11. Organization and Functional Analysis of the 5′ Flanking Regions of Myostatin-1 and 2 Genes from Larimichthys crocea

    PubMed Central

    Dong, Xiaojing; Zhang, Xiaoju; Diallo, Amadou

    2012-01-01

    Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. There are two types of MSTNs in fish, but little is known about their gene regulation. Here, the 5′ flanking fragments of 1029 bp from MSTN-1 and 643 bp from MSTN-2 were cloned, sequenced, and analyzed in Larimichthys crocea. Both fragments contained CAAT box and several putative cis-regulatory elements. However, putative TATA box, MyoD, MEF3, SP1, USF, and GH-CSE sites were identified only in the L. crocea MSTN-1 (lcMSTN-1) promoter. Transcriptional activities of four fragments (1013, 841, 514, and 261 bp) truncated from lcMSTN-1 upstream region and two fragments (643 and 296 bp) from lcMSTN-2 upstream region were examined in vitro, using transient transfection in CIK and L6 cells. In CIK cells, the promoter activity correlated positively with the length of truncated fragments in both MSTN-1 and 2. The lcMSTN-2 promoter showed a higher activity than lcMSTN-1 in the corresponding region, which was consistent with MSTN gene expression in vivo. In L6 cells, lcMSTN-2 upstream showed an extremely high luciferase activity. These data indicated that both cloned 5′ flanking sequences contained functional promoters, and that transcription regulation of lcMSTN-1 and 2 promoters was significantly different between mammalian and fish cells. PMID:22149889

  12. Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells.

    PubMed

    Hati Boruah, Jyoti Lakshmi; Ranjan, Rakesh; Gogoi, Hamen; Pandey, Saurabh Kumar; Kumar, Dharmendra; Phukan, Amlan Jyoti; Bori, Joygeswar; Sarkhel, Bikash Chandra

    2016-01-01

    Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique. PMID:26690650

  13. Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation*

    PubMed Central

    Horbelt, Daniel; Boergermann, Jan H.; Chaikuad, Apirat; Alfano, Ivan; Williams, Eleanor; Lukonin, Ilya; Timmel, Tobias; Bullock, Alex N.; Knaus, Petra

    2015-01-01

    GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis. PMID:25368322

  14. Effects of concurrent strength and endurance training on genes related to myostatin signaling pathway and muscle fiber responses.

    PubMed

    de Souza, Eduardo O; Tricoli, Valmor; Aoki, Marcelo S; Roschel, Hamilton; Brum, Patrícia C; Bacurau, Aline V N; Silva-Batista, Carla; Wilson, Jacob M; Neves, Manoel; Soares, Antonio G; Ugrinowitsch, Carlos

    2014-11-01

    Concurrent training (CT) seems to impair training-induced muscle hypertrophy. This study compared the effects of CT, strength training (ST) and interval training (IT) on the muscle fiber cross-sectional area (CSA) response, and on the expression of selected genes involved in the myostatin (MSTN) signaling mRNA levels. Thirty-seven physically active men were randomly divided into 4 groups: CT (n = 11), ST (n = 11), IT (n = 8), and control group (C) (n = 7) and underwent an 8-week training period. Vastus lateralis biopsy muscle samples were obtained at baseline and 48 hours after the last training session. Muscle fiber CSA, selected genes expression, and maximum dynamic ST (1 repetition maximum) were evaluated before and after training. Type IIa and type I muscle fiber CSA increased from pre- to posttest only in the ST group (17.08 and 17.9%, respectively). The SMAD-7 gene expression significantly increased at the posttest in the ST (53.9%) and CT groups (39.3%). The MSTN and its regulatory genes ActIIb, FLST-3, FOXO-3a, and GASP-1 mRNA levels remained unchanged across time and groups. One repetition maximum increased from pre- to posttest in both the ST and CT groups (ST = 18.5%; CT = 17.6%). Our findings are suggestive that MSTN and their regulatory genes at transcript level cannot differentiate muscle fiber CSA responses between CT and ST regimens in humans. PMID:24832980

  15. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs

    PubMed Central

    Qian, Lili; Tang, Maoxue; Yang, Jinzeng; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Li, Hegang; Jiang, Ke; Gao, Pengfei; Ma, Dezun; Chen, Yaoxing; An, Xiaorong; Li, Kui; Cui, Wentao

    2015-01-01

    Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN−/−) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases. PMID:26400270

  16. Analysis of Horse Myostatin Gene and Identification of Single Nucleotide Polymorphisms in Breeds of Different Morphological Types

    PubMed Central

    Dall'Olio, Stefania; Fontanesi, Luca; Nanni Costa, Leonardo; Tassinari, Marco; Minieri, Laura; Falaschini, Adalberto

    2010-01-01

    Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds. PMID:20706663

  17. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system

    PubMed Central

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-01-01

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

  18. Transcriptome analysis of embryo maturation in maize

    PubMed Central

    2013-01-01

    Background Maize is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of maize grain (as well as rice, wheat and other grains) will be critical. Maize grain development is understood from the perspective of morphology, hormone responses, and storage reserve accumulation. This includes various studies on gene expression during embryo development and maturation but a global study of gene expression of the embryo has not been possible until recently. Transcriptome analysis is a powerful new tool that can be used to understand the genetic basis of embryo maturation. Results We undertook a transcriptomic analysis of normal maturing embryos at 15, 21 and 27 days after pollination (DAP), of one elite maize germplasm line that was utilized in crosses to transgenic plants. More than 19,000 genes were analyzed by this method and the challenge was to select subsets of genes that are vitally important to embryo development and maturation for the initial analysis. We describe the changes in expression for genes relating to primary metabolic pathways, DNA synthesis, late embryogenesis proteins and embryo storage proteins, shown through transcriptome analysis and confirmed levels of transcription for some genes in the transcriptome using qRT-PCR. Conclusions Numerous genes involved in embryo maturation have been identified, many of which show changes in expression level during the progression from 15 to 27 DAP. An expected array of genes involved in primary metabolism was identified. Moreover, more than 30% of transcripts represented un-annotated genes, leaving many functions to be discovered. Of particular interest are the storage protein genes, globulin-1, globulin-2 and an unidentified cupin family gene. When expressing foreign proteins in maize, the globulin-1 promoter is most often used, but this cupin family gene has much higher expression and may be a

  19. An Unexpected Twist in Viral Capsid Maturation

    PubMed Central

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-01-01

    Lambda-like dsDNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm. of pressure during genome packaging1. The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å2. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and Hydrogen/Deuterium exchange data presented here demonstrates that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy3. We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many ds

  20. The Mature Athlete

    PubMed Central

    McCarthy, Moira M.; Hannafin, Jo A.

    2014-01-01

    Context: Aging changes the biology, healing capacity, and biomechanical function of tendons and ligaments and results in common clinical pathologies that present to orthopedic surgeons, primary care physicians, physical therapists, and athletic trainers. A better understanding of the age-related changes in these connective tissues will allow better patient care. Evidence Acquisition: The PubMed database was searched in December 2012 for English-language articles pertaining to age-related changes in tendons and ligaments. Level of Evidence: Level 5. Results: The mature athlete faces challenges associated with age-dependent changes in the rotator cuff, Achilles tendon, lateral humeral epicondylar tendons, quadriceps tendon, and patellar tendon. The anterior cruciate ligament and the medial collateral ligament are the most studied intra-articular and extra-articular ligaments, and both are associated with age-dependent changes. Conclusion: Tendons and ligaments are highly arranged connective tissue structures that maintain joint motion and joint stability. These structures are subject to vascular and compositional changes with increasing age that alter their mechanotransduction, biology, healing capacity, and biomechanical function. Emerging research into the etiology of age-dependent changes will provide further information to help combat the age-related clinical complications associated with the injuries that occur to tendons and ligaments. PMID:24427441

  1. Career Maturity of Welfare Recipients.

    ERIC Educational Resources Information Center

    Beckman, Carol M.

    To investigate the career maturity of welfare recipients, this thesis examines six independent variables: (1) race; (2) sex; (3) age; (4) level of formal education; (5) general intelligence; and (6) locus of control. Scales taken from the Career Maturity Inventory served as the dependent variables. The sample consisted of 83 welfare recipients who…

  2. XGef Mediates Early CPEB Phosphorylation during Xenopus Oocyte Meiotic Maturation