Science.gov

Sample records for mature myostatin protein

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

    PubMed

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

    2017-04-01

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

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

    PubMed

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

    2010-04-01

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

  3. Laminin binds to myostatin and attenuates its signaling.

    PubMed

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

    2013-09-01

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

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

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

    PubMed

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

    2001-02-01

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

  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. 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.

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

    PubMed

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

    2005-08-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2007-09-01

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

  11. 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.

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

    PubMed

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

    2005-07-01

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

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

    PubMed

    Roberts, Steven B; Goetz, Frederick W

    2003-11-28

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

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

    PubMed

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

    2007-04-01

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

  15. Myostatin: expanding horizons.

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-08-24

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

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

    PubMed

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

    2003-11-01

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

  18. Regulation of myostatin activity and muscle growth.

    PubMed

    Lee, S J; McPherron, A C

    2001-07-31

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

  19. Reduced serum myostatin concentrations associated with genetic muscle disease progression.

    PubMed

    Burch, Peter M; Pogoryelova, Oksana; Palandra, Joe; Goldstein, Richard; Bennett, Donald; Fitz, Lori; Guglieri, Michela; Bettolo, Chiara Marini; Straub, Volker; Evangelista, Teresinha; Neubert, Hendrik; Lochmüller, Hanns; Morris, Carl

    2017-01-10

    Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC-MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.

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

    PubMed

    Walsh, F S; Celeste, A J

    2005-12-01

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

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

    PubMed

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

    2015-10-23

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

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

    PubMed Central

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

    2015-01-01

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

  3. 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.

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

    PubMed Central

    Lee, Sang Beum; Park, Sung Kwon

    2017-01-01

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

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

    PubMed

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

    2002-12-20

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

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

    PubMed

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

    2007-10-01

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

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

    PubMed

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

    2003-11-01

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

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

    PubMed

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

    2004-10-01

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

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

    PubMed

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

    2011-07-01

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

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

    PubMed

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

    2011-01-30

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

  11. Myostatin DNA vaccine increases skeletal muscle mass and endurance in mice.

    PubMed

    Tang, Liang; Yan, Zhen; Wan, Yi; Han, Wei; Zhang, Yingqi

    2007-09-01

    Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle growth. In mice, genetic disruption of the myostatin gene leads to a marked increase in body weight and muscle mass. Similarly, pharmacological interference with myostatin in vivo in mdx knockout mice results in a functional improvement of the dystrophic phenotype. Consequently, myostatin is an important therapeutic target for treatment of diseases associated with muscle wasting. To construct a therapeutic DNA vaccine against myostatin, we coupled the foreign, immunodominant T-helper epitope of tetanus toxin to the N terminus of myostatin, and BALB/c mice were immunized with the recombinant vector. Sera from vaccinated mice showed the presence of specific antibodies against the recombinant protein. In addition, body weight, muscle mass, and grip endurance of vaccinated mice were significantly increased. Our study provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting.

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

    PubMed Central

    Hitachi, Keisuke; Tsuchida, Kunihiro

    2017-01-01

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

  13. 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.

  14. 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

  15. 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

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

    PubMed

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

    2013-07-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

    2010-01-01

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

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

    PubMed

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

    2010-05-20

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

  20. Sequence analysis of myostatin promoter in cattle.

    PubMed

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

    2003-01-01

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

  1. 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.

  2. Fluorescent proteins: maturation, photochemistry and photophysics.

    PubMed

    Remington, S James

    2006-12-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2008-08-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Walsh, Frank S; Rutkowski, Julia Lynn

    2012-11-01

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

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

    PubMed

    Tsuchida, K

    2008-07-01

    Gene-targeted therapies, such as adeno-associated viral vector (AAV)-mediated gene therapy and cell-mediated therapy using myogenic stem cells, are hopeful molecular strategies for muscular dystrophy. In addition, drug therapies based on the pathophysiology of muscular dystrophy patients are desirable. Multidisciplinary approaches to drug design would offer promising therapeutic strategies. Myostatin, a member of the transforming growth factor-beta superfamily, is predominantly produced by skeletal muscle and negatively regulates the growth and differentiation of cells of the skeletal muscle lineage. Myostatin inhibition would increase the skeletal muscle mass and prevent muscle degeneration, regardless of the type of muscular dystrophy. Myostatin inhibitors include myostatin antibodies, myostatin propeptide, follistatin and follistatin-related protein. Although follistatin possesses potent myostatin-inhibiting activity, it works as an efficient inhibitor of activins. Unlike myostatin, activins regulate the growth and differentiation of nearly all cell types, including cells of the gonads, pituitary gland and skeletal muscle. We have developed a myostatin-specific inhibitor derived from follistatin, designated FS I-I. Transgenic mice expressing this myostatin-inhibiting peptide under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. mdx mice were crossed with FS I-I transgenic mice and any improvement of the pathological signs was investigated. The resulting mdx/FS I-I mice exhibited increased skeletal muscle mass and reduced cell infiltration in muscles. Muscle strength was also recovered in mdx/FS I-I mice. Our data indicate that myostatin inhibition by this follistatin-derived peptide has therapeutic potential for muscular dystrophy.

  8. 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

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

    PubMed

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

    2014-10-08

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

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

    PubMed Central

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

    2016-01-01

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

  11. Protein Composition of the Vaccinia Virus Mature Virion

    SciTech Connect

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

    2007-02-05

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

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

    PubMed

    Kessler, D S; Melton, D A

    1995-07-01

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

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

    PubMed

    Hitachi, Keisuke; Nakatani, Masashi; Tsuchida, Kunihiro

    2014-02-01

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

  14. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2008-02-01

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

  17. 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 ...

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2003-10-01

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

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

    PubMed

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

    2010-01-01

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

  2. 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.

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

    PubMed Central

    Smith, Rosamund C.; Lin, Boris K.

    2013-01-01

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

  4. 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

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

    DTIC Science & Technology

    2008-03-01

    Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced...whether long-term treatment with pioglitazone reduces vascular and renal fibrosis in an animal model of type 2 diabetes 8. Pilot grant (Bathia/Ho...1), will be subjected to anti-myostatin treatments and tested for differentiation as in Task 1: a) anti-myostatin antibody; b) follistatin; c

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

    PubMed

    Chen, P R; Lee, K

    2016-08-01

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

  7. Muscle regeneration in the prolonged absence of myostatin.

    PubMed

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

    2005-02-15

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

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

    PubMed

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

    2000-05-26

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

  9. 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 ...

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

    PubMed

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

    2016-03-08

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

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

    PubMed Central

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

    2016-01-01

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

  12. 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.

  13. 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...

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

    PubMed

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

    2017-05-01

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

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2004-08-01

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

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

    PubMed

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

    1988-03-01

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

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

    PubMed

    Latronico, Ana Claudia; Hochberg, Ze'ev

    2010-10-12

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

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

    PubMed

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

    2007-07-31

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

  1. Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

    PubMed Central

    Zasłona, Zbigniew; Serezani, Carlos H.; Okunishi, Katsuhide; Aronoff, David M.

    2012-01-01

    Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation. PMID:22234697

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2012-08-01

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

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

    PubMed

    Matsakas, A; Diel, P

    2005-03-01

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

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

    PubMed

    Dschietzig, Thomas Bernd

    2014-06-10

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

  6. 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.

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

    PubMed

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

    2015-07-28

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

  8. 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.

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

    PubMed Central

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

    2007-01-01

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

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

    SciTech Connect

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

    2015-08-21

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

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

    PubMed Central

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

    2009-01-01

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

  12. 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

  13. 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

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

    PubMed

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

    2007-11-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2006-10-01

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

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

    SciTech Connect

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

    2010-01-01

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

  18. 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.

  19. 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

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

    PubMed

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

    2012-04-13

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

  1. 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

  2. Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination

    DTIC Science & Technology

    2009-12-01

    Bacillus thuringiensis and the nonpathogenic bac- teria Bacillus megaterium and Bacillus odysseyi, have an addi- tional structure called the...exosporium. J. Bacte- riol. 185:3373–3378. 47. Vary, P. S. 1994. Prime time for Bacillus megaterium . Microbiology 140:1001– 1013. 48. Weaver, J., T. J...Microbiology. All Rights Reserved. Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination Kari M. Severson,1

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

    PubMed

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

    2009-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2001-08-01

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

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

    PubMed

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

    2011-11-01

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

  8. 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

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

    PubMed

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

    2012-05-18

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

  10. 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

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

    PubMed

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

    2011-09-01

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

  13. 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

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

    PubMed

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

    2014-06-01

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

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

    PubMed

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

    2004-07-01

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

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

    PubMed Central

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

    1995-01-01

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

  17. 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.

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

    PubMed

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

    2017-03-17

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

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

    PubMed

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

    2011-02-01

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

  20. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2006-07-07

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

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Small RNA-Mediated Epigenetic Myostatin Silencing.

    PubMed

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

    2012-05-15

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

  6. 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

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

    PubMed

    Thomas, Sandhya S; Mitch, William E

    2013-04-01

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

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

    PubMed

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

    2012-07-01

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

  9. 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...

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

    PubMed

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

    2016-01-01

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

  11. 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

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

    PubMed

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

    2012-05-18

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

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

    PubMed

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

    2003-11-13

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

  14. Functional improvement of dystrophic muscle by myostatin blockade.

    PubMed

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

    2002-11-28

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

  15. MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

    PubMed Central

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

    2014-01-01

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

  16. 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.

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

    PubMed

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

    PubMed

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

    2012-04-01

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

  19. 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

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

    PubMed

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

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

  1. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2010-09-01

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

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

    PubMed

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

    2006-04-01

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

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

    PubMed

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

    2017-02-13

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

  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

    2014-02-07

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

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

    PubMed

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

    2010-03-08

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

  9. 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

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

    PubMed

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

    2009-10-16

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

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

    PubMed

    Allen, David L; Unterman, Terry G

    2007-01-01

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

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

    PubMed

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

    2007-10-01

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

  13. 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

  14. 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.

  15. 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.

  16. 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.

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-03-01

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

  20. 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.

  1. 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.

  2. 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.

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

    PubMed Central

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

    2015-01-01

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

  4. 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...

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

    PubMed

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

    2002-04-26

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

  6. 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.

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

    PubMed

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

    2016-10-02

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

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

    PubMed

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

    2014-08-15

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

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

    PubMed

    Luff, Stephanie A; Papoutsakis, Eleftherios T

    2016-04-08

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

    Thomas, Sandhya S.; Mitch, William E.

    2013-01-01

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

  12. 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

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

    PubMed

    Wani, Na; Wernery, U

    2010-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. 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

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

    PubMed Central

    2016-01-01

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2013-02-01

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

  19. [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.

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

    PubMed

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

    2014-04-01

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

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

    SciTech Connect

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

    2008-01-25

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

  2. Inhibition of HIV-1 Maturation via Small Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

    PubMed

    Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

    2017-02-15

    The HIV-1 capsid protein is an attractive therapeutic target owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsid to HIV-1 infectivity. To date, small molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BI compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor Bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant crosslinks in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here we show that one such compound, Compound 1, interferes with assembly of the conical viral capsid during virion maturation, and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  6. 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.

  7. 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 ...

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2008-11-01

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

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

    PubMed Central

    Boyartchuk, V L; Rine, J

    1998-01-01

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

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

    PubMed

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

    2014-04-01

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

  12. 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

  13. 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.

  14. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    PubMed Central

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

    2017-01-01

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein. PMID:28134243

  15. Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

    PubMed Central

    Mullins, Chris; Bonifacino, Juan S.

    2001-01-01

    The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of α-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and α-factor maturation and identify structural determinants that are critical for these functions. PMID:11689690

  16. 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

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

    PubMed

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

    2010-12-01

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

  18. 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.

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

    PubMed

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

    2006-09-01

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

  20. 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.

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

    PubMed

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

    2012-05-01

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

  2. 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

  3. 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

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

    PubMed

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

    2014-03-14

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

  5. Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

    PubMed

    Romero-Aguirregomezcorta, Jon; Santa, Ángela Patricia; García-Vázquez, Francisco Alberto; Coy, Pilar; Matás, Carmen

    2014-01-01

    Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

  6. 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

  7. Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons.

    PubMed

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Ya-Jean; Chen, Yun-Hsiang; Wang, Dan-Yen; Chen, Linyi; Chen, Chia-Hsiang; Chen, Hwei-Hsien; Chiu, Ing-Ming

    2014-06-01

    Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

  8. XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans

    PubMed Central

    Dewal, Mahender B.; DiChiara, Andrew S.; Antonopoulos, Aristotelis; Taylor, Rebecca J.; Harmon, Chyleigh J.; Haslam, Stuart M.; Dell, Anne; Shoulders, Matthew D.

    2015-01-01

    SUMMARY The molecular architecture of the mature N-glycome is dynamic, with consequences for both normal and pathologic processes. Elucidating cellular mechanisms that modulate the N-linked glycome is, therefore, crucial. The unfolded protein response (UPR) is classically responsible for maintaining proteostasis in the secretory pathway by defining levels of chaperones and quality control proteins. Here, we employ chemical biology methods for UPR regulation to show that stress-independent activation of the UPR’s XBP1s transcription factor also induces a panel of N-glycan maturation-related enzymes. The downstream consequence is a distinctive shift towards specific hybrid and complex N-glycans on N-glycoproteins produced from XBP1s-activated cells, which we characterize by mass spectrometry. Pulse-chase studies attribute this shift specifically to altered N-glycan processing, rather than to changes in degradation or secretion rates. Our findings implicate XBP1s in a new role for N-glycoprotein biosynthesis, unveiling an important link between intracellular stress responses and the molecular architecture of extracellular N-glycoproteins. PMID:26496683

  9. Ribosomal protein L11 is related to brain maturation during the adult phase in Apis cerana cerana (Hymenoptera, Apidae).

    PubMed

    Meng, Fei; Lu, Wenjing; Yu, Feifei; Kang, Mingjiang; Guo, Xingqi; Xu, Baohua

    2012-05-01

    Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 (RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in dsAccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

  10. 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.

  11. VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes.

    PubMed

    Bem, Danai; Smith, Holly; Banushi, Blerida; Burden, Jemima J; White, Ian J; Hanley, Joanna; Jeremiah, Nadia; Rieux-Laucat, Frédéric; Bettels, Ruth; Ariceta, Gema; Mumford, Andrew D; Thomas, Steven G; Watson, Steve P; Gissen, Paul

    2015-07-09

    Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.

  12. 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

  13. 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.

  14. Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIVmac can coassemble into mature cores of infectious viruses.

    PubMed

    Chen, Jianbo; Pathak, Vinay K; Peng, Weiqun; Hu, Wei-Shau

    2008-09-01

    We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5alpha protein from the rhesus monkey (rhTRIM5alpha) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIV(mac) CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5alpha. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5alpha would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.

  15. Bone architecture and disc degeneration in the lumbar spine of mice lacking GDF-8 (myostatin).

    PubMed

    Hamrick, Mark W; Pennington, Catherine; Byron, Craig D

    2003-11-01

    GDF-8, also known as myostatin, is a member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle. Myostatin functions as a negative regulator of skeletal muscle growth and myostatin null mice show a doubling of muscle mass compared to normal mice. We describe here morphology of the lumbar spine in myostatin knockout (Mstn(-/-)) mice using histological and densitometric techniques. The Mstn(-/-) mice examined in this study weigh approximately 10% more than controls (p<0.001) but the iliopsoas muscle is over 50% larger in the knockout mice than in wild-type mice (p<0.001). Peripheral quantitative computed tomography (pQCT) data from the fifth lumbar vertebra show that mice lacking myostatin have approximately 50% greater trabecular bone mineral density (p=0.001) and significantly greater cortical bone mineral content than normal mice. Toluidine blue staining of the intervertebral disc between L4-L5 reveals loss of proteoglycan staining in the hyaline end plates and inner annulus fibrosus of the knockout mice. Loss of cartilage staining in the caudal end plate of L4 is due to ossification of the end plate in the myostatin-deficient animals. Results from this study suggest that increased muscle mass in mice lacking myostatin is associated with increased bone mass as well as degenerative changes in the intervertebral disc.

  16. Myostatin signals through Pax7 to regulate satellite cell self-renewal

    SciTech Connect

    McFarlane, Craig; Hennebry, Alex; Thomas, Mark; Plummer, Erin; Ling, Nicholas; Sharma, Mridula; Kambadur, Ravi

    2008-01-15

    Myostatin, a Transforming Growth Factor-beta (TGF-{beta}) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.

  17. 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.

  18. EXTRACTS OF IRRADIATED MATURE HUMAN TOOTH CROWNS CONTAIN MMP-20 PROTEIN AND ACTIVITY

    PubMed Central

    MCGUIRE, J.D.; MOUSA, A.A.; ZHANG, BO J.; TODOKI, L.S.; HUFFMAN, N.T.; CHANDRABABU, K.B.; MORADIAN-OLDAK, J.; KEIGHTLEY, A.; WANG, Y.; WALKER, M.P.; GORSKI, J.P.

    2014-01-01

    Objectives We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. Materials and Methods Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0 to 6 months at 37°C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. Results Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentin and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr=43, 41, 24 and 22 kDa and was immunolocalized predominantly to the morphological dentin enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70 G, subsequent incubation at 37°C for 3–6 months with or without prior irradiation caused the proportion of Mr=24–22 kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70 Gy radiation also contained relatively more 24 and 22 kDa MMP-20 than those of healthy age-related teeth. Conclusion MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentin-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy. PMID:24607847

  19. Insulin-like growth factor-1 and myostatin mRNA expression in muscle: comparison between 62-77 and 21-31 yr old men.

    PubMed

    Welle, Stephen; Bhatt, Kirti; Shah, Bharati; Thornton, Charles

    2002-06-01

    The present study was done to determine the effect of age on muscle concentrations of mRNAs encoding two growth factors that are thought to be important regulators of muscle mass: insulin-like growth factor-1 (IGF-1) and myostatin. Quantitative RT-PCR assays indicated that the mean IGF-1 mRNA concentration in older muscle (62-77 yr, n=15 men) was approximately 25% less, per ng total RNA (P<0.005), than in young adult muscle (21-31 yr, n=12 men). One third of the older men had IGF-1 mRNA levels below the lowest concentration observed in young muscle. Myostatin mRNA concentrations were similar in young and old muscle. Muscle mass and myofibrillar protein synthesis rates among eight older men did not correlate with either IGF-1 or myostatin mRNA levels. We conclude that IGF-1 gene expression in muscle tends to decline with normal aging. The functional significance is uncertain.

  20. 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.

  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. Ribosomal protein L3 bound to 23S precursor rRNA stimulates its maturation by Mini-III ribonuclease.

    PubMed

    Redko, Yulia; Condon, Ciarán

    2009-03-01

    Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.

  3. 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.

  4. The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD.

    PubMed

    Spiller, Michael P; Kambadur, Ravi; Jeanplong, Ferenc; Thomas, Mark; Martyn, Julie K; Bass, John J; Sharma, Mridula

    2002-10-01

    Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.

  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. 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.

  7. The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins.

    PubMed

    Lill, Roland; Dutkiewicz, Rafal; Freibert, Sven A; Heidenreich, Torsten; Mascarenhas, Judita; Netz, Daili J; Paul, Viktoria D; Pierik, Antonio J; Richter, Nadine; Stümpfig, Martin; Srinivasan, Vasundara; Stehling, Oliver; Mühlenhoff, Ulrich

    2015-01-01

    Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron-sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron-sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins.

  8. A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins.

    PubMed

    Zhang, Siyan; Long, Angel; Link, A James

    2012-03-16

    The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-x(L) and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L) and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L) and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L) and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.

  9. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  10. An evolutionarily conserved Myostatin proximal promoter/enhancer confers basal levels of transcription and spatial specificity in vivo.

    PubMed

    Grade, Carla Vermeulen Carvalho; Salerno, Mônica Senna; Schubert, Frank R; Dietrich, Susanne; Alvares, Lúcia Elvira

    2009-10-01

    Myostatin (Mstn) is a negative regulator of skeletal muscle mass, and Mstn mutations are responsible for the double muscling phenotype observed in many animal species. Moreover, Mstn is a positive regulator of adult muscle stem cell (satellite cell) quiescence, and hence, Mstn is being targeted in therapeutic approaches to muscle diseases. In order to better understand the mechanisms underlying Mstn regulation, we searched for the gene's proximal enhancer and promoter elements, using an evolutionary approach. We identified a 260-bp-long, evolutionary conserved region upstream of tetrapod Mstn and teleost mstn b genes. This region contains binding sites for TATA binding protein, Meis1, NF-Y, and for CREB family members, suggesting the involvement of cAMP in Myostatin regulation. The conserved fragment was able to drive reporter gene expression in C2C12 cells in vitro and in chicken somites in vivo; both normally express Mstn. In contrast, the reporter construct remained silent in the avian neural tube that normally does not express Mstn. This suggests that the identified element serves as a minimal promoter, harboring some spatial specificity. Finally, using bioinformatic approaches, we identified additional genes in the human genome associated with sequences similar to the Mstn proximal promoter/enhancer. Among them are genes important for myogenesis. This suggests that Mstn and these genes may form a synexpression group, regulated by a common signaling pathway.

  11. The Significance of Protein Maturation by Plastidic Type I Signal Peptidase 1 for Thylakoid Development in Arabidopsis Chloroplasts1[C][W][OA

    PubMed Central

    Shipman-Roston, Rebecca L.; Ruppel, Nicholas J.; Damoc, Catalina; Phinney, Brett S.; Inoue, Kentaro

    2010-01-01

    Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here, we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids. PMID:20097790

  12. The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction.

    PubMed

    Zhang, Lin; Trncik, Christian; Andrade, Susana L A; Einsle, Oliver

    2017-02-01

    The copper-containing enzyme nitrous oxide reductase (N2OR) catalyzes the transformation of nitrous oxide (N2O) to dinitrogen (N2) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites CuA and CuZ, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N2O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg(2+) that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.

  13. Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins.

    PubMed

    Van Damme, Daniël; Coutuer, Silvie; De Rycke, Riet; Bouget, Francois-Yves; Inzé, Dirk; Geelen, Danny

    2006-12-01

    TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco (Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and beta-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position.

  14. The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection

    PubMed Central

    Bird, Brian H.; Albariño, Cesar G.; Nichol, Stuart T.

    2007-01-01

    Rift Valley fever (RVF) virus belongs to the Bunyaviridae family of segmented negative-strand RNA viruses and causes mosquito-borne disease in sub-Saharan Africa. We report the development of a T7 RNA polymerase driven plasmid-based genetic system for the virulent Egyptian isolate, ZH501. We have used this system to rescue a virus that has a 387 nucleotide deletion on the genomic M segment that eliminates the coding region for two non-structural proteins known as NSm. This virus, ΔNSm rZH501, is indistinguishable from the parental ZH501 strain with respect to expression of structural proteins and growth in cultured mammalian cells. PMID:17070883

  15. Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

    PubMed Central

    Pérez-Berná, Ana J.; Mangel, Walter F.; McGrath, William J.; Graziano, Vito; Flint, Jane

    2014-01-01

    Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed. PMID:24227847

  16. 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.

  17. Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein, production of the viral particle and expression of host proteins involved in pathogenicity.

    PubMed

    Otoguro, Teruhime; Tanaka, Tomohisa; Kasai, Hirotake; Yamashita, Atsuya; Moriishi, Kohji

    2016-11-01

    Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C-terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin-like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti-HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti-HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP-dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV-infected cells was significantly impaired by LY411575 in a dose-dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir-dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV-related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.

  18. Proteasomal Activity Is Required to Initiate and to Sustain Translational Activation of Messenger RNA Encoding the Stem-Loop-Binding Protein During Meiotic Maturation in Mice1

    PubMed Central

    Yang, Qin; Allard, Patrick; Huang, Michael; Zhang, Wenling; Clarke, Hugh J.

    2009-01-01

    Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3′-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3′-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation. PMID:19759367

  19. [Lipid- and protein-degrading processes during the maturation of ham].

    PubMed

    López Bote, C; Córdoba, J J; Antequera, T

    1993-02-01

    In the present work we review the main degradative pathways for lipids and proteins along the ripening of dry cured hams, with special emphasis on Iberian pig hams. Maximum proteolytic activity is found around the first stages (salting) and specially at the dryer. Lipolytic activity seems to be also higher in this stage. During the steps that follow the post-salting period the oxidation seems to be activated. The products from proteolytic and lipolytic processes might react among each other during the final steps in the cellar.

  20. 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

  1. Changes in skeletal muscle and tendon structure and function following genetic inactivation of myostatin in rats.

    PubMed

    Mendias, Christopher L; Lynch, Evan B; Gumucio, Jonathan P; Flood, Michael D; Rittman, Danielle S; Van Pelt, Douglas W; Roche, Stuart M; Davis, Carol S

    2015-04-15

    Myostatin is a negative regulator of skeletal muscle and tendon mass. Myostatin deficiency has been well studied in mice, but limited data are available on how myostatin regulates the structure and function of muscles and tendons of larger animals. We hypothesized that, in comparison to wild-type (MSTN(+/+) ) rats, rats in which zinc finger nucleases were used to genetically inactivate myostatin (MSTN(Δ/Δ) ) would exhibit an increase in muscle mass and total force production, a reduction in specific force, an accumulation of type II fibres and a decrease and stiffening of connective tissue. Overall, the muscle and tendon phenotype of myostatin-deficient rats was markedly different from that of myostatin-deficient mice, which have impaired contractility and pathological changes to fibres and their extracellular matrix. Extensor digitorum longus and soleus muscles of MSTN(Δ/Δ) rats demonstrated 20-33% increases in mass, 35-45% increases in fibre number, 20-57% increases in isometric force and no differences in specific force. The insulin-like growth factor-1 pathway was activated to a greater extent in MSTN(Δ/Δ) muscles, but no substantial differences in atrophy-related genes were observed. Tendons of MSTN(Δ/Δ) rats had a 20% reduction in peak strain, with no differences in mass, peak stress or stiffness. The general morphology and gene expression patterns were similar between tendons of both genotypes. This large rodent model of myostatin deficiency did not have the negative consequences to muscle fibres and extracellular matrix observed in mouse models, and suggests that the greatest impact of myostatin in the regulation of muscle mass may not be to induce atrophy directly, but rather to block hypertrophy signalling.

  2. Elevation of myostatin and FOXOs in prolonged muscular impairment induced by eccentric contractions in rat medial gastrocnemius muscle.

    PubMed

    Ochi, Eisuke; Hirose, Tatsuro; Hiranuma, Kenji; Min, Seok-Ki; Ishii, Naokata; Nakazato, Koichi

    2010-02-01

    This study aimed to investigate torque deficit and activation of protein synthesis and/or protein degradation signaling pathways during the early and recovery phase after high- and low-velocity eccentric contractions (ECs). Male Wistar rats (n = 36) were randomly divided into fast angular velocity ECs group (FAST; 180 degrees/s; n = 12), slow ECs group (SLOW; 30 degrees/s; n = 12), and control group (control; n = 12). ECs comprised four sets of five forced dorsiflexions combined with electrical stimulation of the plantar flexors. Isometric tetanic torque was measured before and after ECs. Tissue contents of Akt(P) (P, phosphorylated), mammalian target of rapamycin (mTOR)(P), 70-kDa ribosomal protein S6 kinase (P70S6k), P70S6k(P), forkhead transcription factor 1 of the O class (FOXO1), FOXO1(P), FOXO3, FOXO3(P), myostatin, and activin receptor type IIB (ActRIIB) were measured. The isometric tetanic torque after ECs was significantly lower in FAST than in SLOW (days 1, 3, and 5, P < 0.05; day 2, P < 0.01). The ratio of P70S6k(P) against total P70S6k on days 2 and 7 was significantly higher in SLOW than in the control. The ratio of FOXO1 against total FOXO1, the ratio of FOXO3a against total FOXO3a, and myostatin on days 2 and 7 were significantly higher in FAST than in the control, while that of ActRIIB on day 7 was significantly lower in SLOW than in the other two groups. These results suggest that EC intensity plays a key role in impairment of muscular function and activation of protein synthesis and/or protein degradation signaling pathways.

  3. Characterization of 5'-regulatory region of human myostatin gene: regulation by dexamethasone in vitro.

    PubMed

    Ma, K; Mallidis, C; Artaza, J; Taylor, W; Gonzalez-Cadavid, N; Bhasin, S

    2001-12-01

    We cloned and characterized a 3.3-kb fragment containing the 5'-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-kappaB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C(2)C(12) and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.

  4. Comparative analysis of the pig BAC sequence involved in the regulation of myostatin gene.

    PubMed

    Yu, Zhengquan; Li, Yan; Meng, Qingyong; Yuan, Jing; Zhao, Zhihui; Li, Wei; Hu, Xiaoxiang; Yan, Bingxue; Fan, Baoliang; Yu, Shuyang; Li, Ning

    2005-04-01

    Myostatin (GDF8, MSTN) is a member of the transforming growth factor beta superfamily that is essential for proper regulation of skeletal muscle mass. In order to study its expression and regulatory mechanism deeply, we have presented a comparative analysis of about 170-kb pig BAC sequence containing the myostatin gene among pig, human and mouse. The genomic region is characterized by high interspersed repeats and low G+C content. As for the myostatin gene, a higher sequence similarity is found between human and pig than between these species and the mouse. One striking feature is that the structure of two TATA-boxes in the nearby downstream of CCAAT-box is identified in the promoter. Further analysis reveals that the TATA-box1 is responsible for the transcription in pig and human, but the TATA-box2 acts on the transcription in mouse. The other interesting feature is that two polyadenylation signal sequences (AATAAA) exist in 3'UTR of the pig myostatin gene. Moreover, a large number of potential transcription factor-binding sites are also identified in evolutionary conserved regions (ECRs), which may be associated with the regulation of myostatin. Many putative transcription factors play an important role in the muscle development, and the complex interaction between myostatin and these factors may be required for proper muscle development.

  5. Yolk proteins during ovary and egg development of mature female freshwater crayfish (Cherax quadricarinatus).

    PubMed

    Serrano-Pinto, Vania; Vazquez-Boucard, Celia; Villarreal-Colmenares, Humberto

    2003-01-01

    Vitellins from ovaries and eggs at different stages of development in freshwater crayfish (Cherax quadricarinatus) were examined by chromatography, PAGE and SDS-PAGE. With these methods, two forms of vitellin (Vt1 and Vt2) were observed in ovaries and eggs (stages I and V). In ovaries in secondary vitellogenesis, native molecular mass was 470 (Vt1) and 440 (Vt2) kDa. The electrophoretic pattern of the eggs proved to be more complex. The protein molecular mass depend on the development stage of the egg: stage I, 650 kDa (Vt1) and 440 kDa (Vt2); stage V, 390 kDa (Vt1) and 340 kDa (Vt2). The identified vitellins appear to be lipo-glycocarotenoprotein. A similar vitellin polypeptide composition was observed in the two forms of vitellin from ovaries and eggs in stage V. In ovaries the SDS-PAGE analysis showed four subunits with molecular weights of approximately 180, 120, 95 and 80 kDa (Vt1 and Vt2). The polypeptide composition in the two forms of vitellins in stage I and stage III eggs were different at 195, 190, 130 and 110 kDa (Vt1) and 116 and 107 kDa (Vt2). On the other hand, in stage V eggs, 110, 95, 87 and 75 kDa (Vt1 and Vt2) were identified. Two antibodies (Ab1 and Ab2) were prepared against the purified proteins of stage V eggs and their specificity was demonstrated by radial immunoprecipitation, and Western blotting analysis. Two forms of vitellins were also found in stage V eggs after chromatography on Sepharose CL-2B column and hydroxylapatite and polyacrylamide gel electrophoresis.

  6. Identification of new proteins in follicular fluid from mature human follicles by direct sample rehydration method of two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Lee, Han-Chul; Lee, Sang-Wha; Lee, Kyo Won; Lee, Sook-Whan; Cha, Kwang-Yul; Kim, Kye Hyun; Lee, Suman

    2005-06-01

    Human follicular fluid (HFF) includes various biologically active proteins which can affect follicle growth and oocyte fertilization. Thus far, these proteins from mature follicles in human follicular fluid have been poorly characterized. Here, two-dimensional polyacrylamide gel electrophoresis (2-DE) with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to identify new proteins in HFF. Mature follicular fluids were obtained from five females after oocyte collection during in vitro fertilization (IVF). We directly rehydrated HFF samples, obtained high-resolution 2-DE maps, and processed them for 2-DE and MALDI-MS. One hundred eighty spots were detected and 10 of these spots were identified. By the 2-DE database, six of them had been reported, as proteins already existing in HFF. Hormone sensitive lipase (HSL), Unnamed protein product 1 (UPP1), Unnamed protein product 2 (UPP2), and apolipoprotein A-IV precursor were newly detected. HSL and apolipoprotein A-IV participate in lipid metabolism. UPP1 has a homology with selenocysteine lyase. We found by RT-PCR that these genes are expressed from human primary granulosa cells. The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis, hormone secretion regulation, or oocyte maturation. Further functional analysis of these proteins is necessitated to determine their biological implications.

  7. Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

    PubMed

    Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

    2017-02-20

    Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

  8. Mycobacterial heat shock protein 70 induces interleukin-10 production: immunomodulation of synovial cell cytokine profile and dendritic cell maturation

    PubMed Central

    DETANICO, T; RODRIGUES, L; SABRITTO, A C; KEISERMANN, M; BAUER, M E; ZWICKEY, H; BONORINO, C

    2004-01-01

    Cytokines are key modulators of the immune responses that take place in the inflamed synovium of arthritis patients. Consequently, substances that can reverse the inflammatory profile of the inflamed joint are potential tools for clinical management of the disease. Mycobacterial heat shock protein 70 (MTBHSP70) has been found to protect rats from experimentally induced arthritis through the induction of interleukin (IL)-10-producing T cells. In this study, we have demonstrated that MTBHSP70 induces IL-10 production in synoviocytes from arthritis patients and peripheral blood monoculear cells (PBMCs) from both patients and healthy controls. IL-10 production was accompanied by a decrease in tumour necrosis factor (TNF)-α production by synovial cells. Separation studies showed that the target cells were mainly monocytes. Accordingly, we observed that MTBHSP70 delayed maturation of murine bone marrow-derived dendritic cells. Our results suggest that MTBHSP may act on antigen-presenting cells (APCs) to modulate the cytokine response in arthritis and support an anti-inflammatory role for this protein, suggesting that it may be of therapeutic use in the modulation of arthritis. PMID:14738465

  9. 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.

  10. Characterization and expression of proprotein convertases in CHO cells: Efficient proteolytic maturation of human bone morphogenetic protein-7.

    PubMed

    Sathyamurthy, Madhavi; Kim, Che Lin; Bang, You Lim; Kim, Young Sik; Jang, Ju Woong; Lee, Gyun Min

    2015-03-01

    Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.

  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. Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.

    PubMed

    Luo, Junjie; Song, Zhiyuan; Yu, Shengli; Cui, Dan; Wang, Benli; Ding, Fangrong; Li, Song; Dai, Yunping; Li, Ning

    2014-01-01

    Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.

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

    PubMed Central

    Lee, Yun-Sil; Lee, Se-Jin

    2013-01-01

    Myostatin (MSTN) and growth and differentiation factor-11 (GDF-11) are highly related TGF-β family members that have distinct biological functions. MSTN is expressed primarily in skeletal muscle and acts to limit muscle growth. GDF-11 is expressed more widely and plays multiple roles, including regulating axial skeletal patterning during development. Several MSTN and GDF-11 binding proteins have been identified, including GDF-associated serum protein-1 (GASP-1) and GASP-2, which are capable of inhibiting the activities of these ligands. Here, we show that GASP-1 and GASP-2 act by blocking the initial signaling event (namely, the binding of the ligand to the type II receptor). Moreover, we show that mice lacking Gasp1 and Gasp2 have phenotypes consistent with overactivity of MSTN and GDF-11. Specifically, we show that Gasp2−/− mice have posteriorly directed transformations of the axial skeleton, which contrast with the anteriorly directed transformations seen in Gdf11−/− mice. We also show that both Gasp1−/− and Gasp2−/− mice have reductions in muscle weights, a shift in fiber type from fast glycolytic type IIb fibers to fast oxidative type IIa fibers, and impaired muscle regeneration ability, which are the reverse of what are seen in Mstn−/− mice. All of these findings suggest that both GASP-1 and GASP-2 are important modulators of GDF-11 and MSTN activity in vivo. PMID:24019467

  14. Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

    PubMed Central

    Yu, Shengli; Cui, Dan; Wang, Benli; Ding, Fangrong; Li, Song; Dai, Yunping; Li, Ning

    2014-01-01

    Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle. PMID:24743319

  15. A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation, results in delayed degradation of maternal proteins and embryonic lethality.

    PubMed

    Lin, Rueyling

    2003-06-01

    In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.

  16. 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

  17. A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation.

    PubMed

    Sedlik, Christine; Orbach, Daniel; Veron, Philippe; Schweighoffer, Edina; Colucci, Francesco; Gamberale, Romina; Ioan-Facsinay, Andrea; Verbeek, Sjef; Ricciardi-Castagnoli, Paola; Bonnerot, Christian; Tybulewicz, Victor L J; Di Santo, James; Amigorena, Sebastian

    2003-01-15

    Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.

  18. Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH.

    PubMed

    Peaucellier, G; Picard, A; Robert, J J; Capony, J P; Labbe, J C; Doree, M

    1988-01-01

    An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also

  19. 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

  20. Evaluation of ARG protein expression in mature B cell lymphomas compared to non-neoplastic reactive lymph node.

    PubMed

    Kabiri, Zahra; Salehi, Mansoor; Mokarian, Fariborz; Mohajeri, Mohammad Reza; Mahmoodi, Farzaneh; Keyhanian, Kianoosh; Doostan, Iman; Ataollahi, Mohammad Reza; Modarressi, Mohammad Hossein

    2009-01-01

    The participation of Abl-Related Gene (ARG) is demonstrated in pathogenesis of different human malignancies. However there is no conclusive evidence on ARG expression level in mature B cell lymphomas. In this study we evaluated ARG protein expression in Follicular Lymphoma (FL), Burkitt's Lymphoma (BL) and Diffused Large B Cell Lymphoma (DLBCL) in comparison with non-neoplastic lymph nodes. Semi-quantitative fluorescent ImmunoHistoChemistry was applied on 14, 7 and 4 patients with DLBCL, FL and BL respectively, adding to 4 normal and 4 reactive lymph nodes. The mean ratio of ARG/GAPDH expression was significantly different (p<0.00) between lymphomas and control samples, with DLBCL having the highest ARG expression amongst all. Over expression of ARG was seen in FL and BL, with FL expressing statistically more ARG than BL. Moreover, the ARG/GAPDH expression ratio increased from DLBCL stage I towards stage VI, all showing significantly more ARG expression than FL and BL (in all cases p<0.00).

  1. CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation.

    PubMed

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Sun, Qing-Yuan; Fan, Heng-Yu

    2015-08-18

    Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility.

  2. 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

  3. 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

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

    PubMed Central

    Khan, Tayeba; Weber, Hans; DiMuzio, Jillian; Matter, Andrea; Dogdas, Belma; Shah, Tosha; Thankappan, Anil; Disa, Jyoti; Jadhav, Vasant; Lubbers, Laura; Sepp-Lorenzino, Laura; Strapps, Walter R; Tadin-Strapps, Marija

    2016-01-01

    Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85–95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes. PMID:27483025

  5. Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

    PubMed Central

    Li, Kui; Xu, Zhiqiang; Liang, Dong; Li, Jingyun; Li, Junbo; Jia, Wenshuang; Li, Yuehua; Dong, Xiaohua; Cao, Shasha; Wang, Xiaoxiao; Pan, Jianlin; Zhao, Qingshun

    2011-01-01

    Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish. PMID:22194943

  6. 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

  7. Suppression of a signal sequence mutation by an amino acid substitution in the mature portion of the maltose-binding protein.

    PubMed Central

    Cover, W H; Ryan, J P; Bassford, P J; Walsh, K A; Bollinger, J; Randall, L L

    1987-01-01

    An unusual spontaneous pseudorevertant of an Escherichia coli strain carrying the signal sequence point mutation malE14-1 was characterized. The suppressor mutation, malE2261, resulted in a single substitution of an aspartyl residue for a tyrosyl residue at position 283 in the sequence of the mature maltose-binding protein. The precursor retained the malE14-1 point mutation in the signal sequence. The pseudorevertant carrying both malE14-1 and malE2261 exported twice the amount of maltose-binding protein as that of the mutant carrying the malE14-1 allele alone but only 18% of the amount exported by a strain producing wild-type maltose-binding protein. A strain carrying the suppressor allele malE2261 in combination with a wild-type signal sequence exported normal quantities of maltose-binding protein to the periplasm. Mature MalE2261 had a Kd for maltose of 27 microM, compared with 3.6 microM for mature wild-type maltose-binding protein. The precursor species than contained both changes resulting from malE14-1 and malE2261 was significantly less stable in the cytoplasm than was the precursor containing only the change encoded by malE14-1. Images PMID:3553148

  8. 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.

  9. 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...

  10. 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.

  11. Oral administration of myostatin-specific whole recombinant yeast Saccharomyces cerevisiae vaccine increases body weight and muscle composition in mice.

    PubMed

    Zhang, Tingting; Yang, Hanjiang; Wang, Rui; Xu, Kun; Xin, Ying; Ren, Gang; Zhou, Gang; Zhang, Cunfang; Wang, Ling; Zhang, Zhiying

    2011-10-26

    Myostatin negatively regulates skeletal muscle growth. It was found that active immunization with myostatin-specific vaccine blocked myostatin function in vivo, which resulted in increase of body weight and muscle composition in mice. However, traditional vaccine and its administration method are expensive and laborious. In this study, we investigated the possibility of using heat-killed whole recombinant yeast Saccharomyces cerevisiae vaccine to modulate myostatin function in mice. The CDS of myostatin was obtained from a pig genome by PCR and subcloned into a yeast expression vector, which was driven by a copper-inducible promoter. Expression of recombinant myostatin was induced by CuSO(4) and confirmed by western blot. We vaccinated mice by oral feeding and subcutaneous injection as comparison. We found that oral feeding resulted in the similar effective immune response than injection, which was measured by the presence of myostatin-specific antibodies in mouse serum. Interestingly, animals vaccinated by both methods demonstrated enhanced growth performance compared to control. All animals were healthy looking throughout the course of experiment, suggesting that whole recombinant yeast vaccine is nontoxic and therefore safe to use. Given the simplicity of its nature, heat-killed myostatin-specific whole recombinant yeast vaccine holds a promise to treat human muscle-wasting diseases in the future.

  12. 207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM.

    PubMed

    Pinzon, C A; Snyder, M; Pryor, J; Thompson, B; Golding, M; Long, C

    2016-01-01

    The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of -1577, -689, -555, and -116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1=sgRNA 1-4; Set 2=sgRNA 2-3). Cells were exposed to puromycin (0.2µgmL(-1)) for 72h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes

  13. Statins reduce amyloid β-peptide production by modulating amyloid precursor protein maturation and phosphorylation through a cholesterol-independent mechanism in cultured neurons.

    PubMed

    Hosaka, Ai; Araki, Wataru; Oda, Akiko; Tomidokoro, Yasushi; Tamaoka, Akira

    2013-03-01

    Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2-2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.

  14. Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes.

    PubMed

    Enders, Anselm; Short, Alanna; Miosge, Lisa A; Bergmann, Hannes; Sontani, Yovina; Bertram, Edward M; Whittle, Belinda; Balakishnan, Bhavani; Yoshida, Kaoru; Sjollema, Geoff; Field, Matthew A; Andrews, T Daniel; Hagiwara, Hiromi; Goodnow, Christopher C

    2014-03-25

    IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.

  15. Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases.

    PubMed

    Xu, Li; Zhao, Piming; Mariano, Andrew; Han, Renzhi

    2013-07-30

    Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.Molecular Therapy-Nucleic Acids (2013) 2, e112; doi:10.1038/mtna.2013.39; published online 30 July 2013.

  16. Short bouts of stretching increase myo-D, myostatin and atrogin-1 in rat soleus muscle.

    PubMed

    Peviani, Sabrina Messa; Gomes, Anna Raquel Silveira; Moreira, Roberta Fátima Carreira; Moriscot, Anselmo Sigari; Salvini, Tania Fátima

    2007-03-01

    Stretching is widely used in rehabilitation and sports activities to improve joint range-of-motion and flexibility in humans, but the effect of stretching on the gene expression of skeletal muscle is poorly understood. We evaluated the effect of short bouts of passive stretching of rat soleus muscle on myo-D, myostatin, and atrogin-1 gene expressions. Six groups of animals were submitted to a single session of stretching (10 stretches of 1 minute with 30 seconds of rest between them, performed manually) and were evaluated immediately (I), and 8, 24, 48, 72, and 168 hours after the session. To evaluate the effect of repetitive sessions of stretching on the soleus muscle over 1 week, three groups of animals received a single session per day of stretching and the muscle was evaluated immediately after 2, 3, and 7 sessions. The mRNA levels of myo-D, myostatin, and atrogin-1 were determined by real-time polymerase chain reaction. A single session of stretching increased the mRNA levels of myo-D (after 24 h), myostatin (I, and 168 h later), and atrogin-1 (after 48 h). Repeated daily session of stretching over 1 week increased myostatin (after 7 sessions) and atrogin-1 expression (after 2, 3, and 7 sessions). Thus, short bouts of passive stretching are able to increase the gene expression of factors associated with muscle growth (myo-D), negative regulation of muscle mass (myostatin), and atrophy (atrogin-1), indicating muscle remodeling through different pathways.

  17. Relationship between myostatin and irisin in type 2 diabetes mellitus: a compensatory mechanism to an unfavourable metabolic state?

    PubMed

    García-Fontana, Beatriz; Reyes-García, Rebeca; Morales-Santana, Sonia; Ávila-Rubio, Verónica; Muñoz-Garach, Araceli; Rozas-Moreno, Pedro; Muñoz-Torres, Manuel

    2016-04-01

    Myostatin and irisin are two myokines related to energy metabolism, acting on skeletal muscle and recently suggested on adipose tissue in mice. However, the exact role of these myokines in humans has not been fully established. Our aim was to evaluate the relationship between serum levels of myostatin and irisin in type 2 diabetes mellitus patients and non-diabetic controls and to explore its links with metabolic parameters. Case-control study including 73 type 2 diabetes mellitus patients and 55 non-diabetic subjects as control group. Circulating myostatin and irisin levels were measured by enzyme-linked immunosorbent assays. Type 2 diabetes mellitus patients showed significantly lower myostatin levels (p = 0.001) and higher irisin levels (p = 0.036) than controls. An inverse relationship was observed between myostatin and irisin levels (p = 0.002). Moreover, in type 2 diabetes mellitus patients, after adjusting by confounder factors, myostatin was negatively related to fasting plasma glucose (p = 0.005) and to triglyceride levels (p = 0.028) while irisin showed a positive association with these variables (p = 0.017 and p = 0.006 respectively). A linear regression analysis showed that irisin and fasting plasma glucose levels were independently associated to myostatin levels and that myostatin and triglyceride levels were independently associated to irisin concentrations in type 2 diabetes mellitus patients. Our results suggest that serum levels of myostatin and irisin are related in patients with type 2 diabetes. Triglyceride and glucose levels could modulate myostatin and irisin concentrations as a compensatory mechanism to improve the metabolic state in these patients although further studies are needed to elucidate whether the action of these myokines represents an adaptative response.

  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.

  19. Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain.

    PubMed

    Ferland, Russell J; Cherry, Timothy J; Preware, Patricia O; Morrisey, Edward E; Walsh, Christopher A

    2003-05-26

    Foxp2 and Foxp1 are recently identified members of the Fox family of winged-helix/forkhead transcription factor genes. A recent study has found that mutations in human FOXP2 produce a severe language disorder. Since Foxp2 appears to be important in language, we wanted to explore the expression of this gene and a homologous gene, Foxp1, in the developing brain. In the present study, we investigated the time course and localization of Foxp2 and Foxp1 mRNA and protein expression in the developing and adult mouse using in situ hybridization and immunohistochemistry. Foxp2 and Foxp1 are expressed as early as E12.5 and persist into adulthood. Foxp2 and Foxp1 were most highly expressed in the developing and mature basal ganglia. Expression of Foxp2 was also observed in the cerebral cortex (layer 6), cerebellum (Purkinje neurons), and thalamus. Foxp1 expression was observed in the cerebral cortex (layers 3-5), hippocampus (CA1), and thalamus. Very little ventricular zone expression was observed for Foxp2 and Foxp1 and the expression of both of these genes occurred following neuronal migration, suggesting a role for these genes in postmigratory neuronal differentiation. Furthermore, we demonstrated the expression of FOXP2 in human fetal brain by RT-PCR, in the perisylvian area of the left and right cerebral hemispheres, as well as in the frontal and occipital cortices. Overall, the widespread expression of Foxp2 in the developing brain makes it difficult to draw specific conclusions about which areas of Foxp2 expression are critical to human language function.

  20. Network-based characterization of the synaptic proteome reveals that removal of epigenetic regulator Prmt8 restricts proteins associated with synaptic maturation.

    PubMed

    Lee, Patrick Kia Ming; Goh, Wilson Wen Bin; Sng, Judy Chia Ghee

    2017-02-01

    The brain adapts to dynamic environmental conditions by altering its epigenetic state, thereby influencing neuronal transcriptional programs. An example of an epigenetic modification is protein methylation, catalyzed by protein arginine methyltransferases (PRMT). One member, Prmt8, is selectively expressed in the central nervous system during a crucial phase of early development, but little else is known regarding its function. We hypothesize Prmt8 plays a role in synaptic maturation during development. To evaluate this, we used a proteome-wide approach to characterize the synaptic proteome of Prmt8 knockout versus wild-type mice. Through comparative network-based analyses, proteins and functional clusters related to neurite development were identified to be differentially regulated between the two genotypes. One interesting protein that was differentially regulated was tenascin-R (TNR). Chromatin immunoprecipitation demonstrated binding of PRMT8 to the tenascin-r (Tnr) promoter. TNR, a component of perineuronal nets, preserves structural integrity of synaptic connections within neuronal networks during the development of visual-somatosensory cortices. On closer inspection, Prmt8 removal increased net formation and decreased inhibitory parvalbumin-positive (PV+) puncta on pyramidal neurons, thereby hindering the maturation of circuits. Consequently, visual acuity of the knockout mice was reduced. Our results demonstrated Prmt8's involvement in synaptic maturation and its prospect as an epigenetic modulator of developmental neuroplasticity by regulating structural elements such as the perineuronal nets.

  1. Identification of YbeY-Protein Interactions Involved in 16S rRNA Maturation and Stress Regulation in Escherichia coli

    PubMed Central

    Vercruysse, Maarten; Köhrer, Caroline; Shen, Yang; Proulx, Sandra; Ghosal, Anubrata; Davies, Bryan W.; RajBhandary, Uttam L.

    2016-01-01

    ABSTRACT YbeY is part of a core set of RNases in Escherichia coli and other bacteria. This highly conserved endoribonuclease has been implicated in several important processes such as 16S rRNA 3′ end maturation, 70S ribosome quality control, and regulation of mRNAs and small noncoding RNAs, thereby affecting cellular viability, stress tolerance, and pathogenic and symbiotic behavior of bacteria. Thus, YbeY likely interacts with numerous protein or RNA partners that are involved in various aspects of cellular physiology. Using a bacterial two-hybrid system, we identified several proteins that interact with YbeY, including ribosomal protein S11, the ribosome-associated GTPases Era and Der, YbeZ, and SpoT. In particular, the interaction of YbeY with S11 and Era provides insight into YbeY’s involvement in the 16S rRNA maturation process. The three-way association between YbeY, S11, and Era suggests that YbeY is recruited to the ribosome where it could cleave the 17S rRNA precursor endonucleolytically at or near the 3′ end maturation site. Analysis of YbeY missense mutants shows that a highly conserved beta-sheet in YbeY—and not amino acids known to be important for YbeY’s RNase activity—functions as the interface between YbeY and S11. This protein-interacting interface of YbeY is needed for correct rRNA maturation and stress regulation, as missense mutants show significant phenotypic defects. Additionally, structure-based in silico prediction of putative interactions between YbeY and the Era-30S complex through protein docking agrees well with the in vivo results. PMID:27834201

  2. Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.

    PubMed

    Lessard, Julie; Pelletier, Mélissa; Biertho, Laurent; Biron, Simon; Marceau, Simon; Hould, Frédéric-Simon; Lebel, Stéfane; Moustarah, Fady; Lescelleur, Odette; Marceau, Picard; Tchernof, André

    2015-01-01

    Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin) was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1) were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity.

  3. Characterization of Dedifferentiating Human Mature Adipocytes from the Visceral and Subcutaneous Fat Compartments: Fibroblast-Activation Protein Alpha and Dipeptidyl Peptidase 4 as Major Components of Matrix Remodeling

    PubMed Central

    Lessard, Julie; Pelletier, Mélissa; Biertho, Laurent; Biron, Simon; Marceau, Simon; Hould, Frédéric-Simon; Lebel, Stéfane; Moustarah, Fady; Lescelleur, Odette; Marceau, Picard; Tchernof, André

    2015-01-01

    Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin) was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1) were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity. PMID:25816202

  4. Myostatin signaling through Smad2, Smad3 and Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism.

    PubMed

    Zhu, Xiangyang; Topouzis, Stavros; Liang, Li-Fang; Stotish, Ronald L

    2004-06-21

    As a member of the TGF-beta superfamily, myostatin is a specific negative regulator of skeletal muscle mass. To identify the downstream components in the myostatin signal transduction pathway, we used a luciferase reporter assay to elucidate myostatin-induced activity. The myostatin-induced transcription requires the participation of regulatory Smads (Smad2/3) and Co-Smads (Smad4). Conversely, inhibitory Smad7, but not Smad6, dramatically reduces the myostatin-induced transcription. This Smad7 inhibition is enhanced by co-expression of Smurf1. We have also shown that Smad7 expression is stimulated by myostatin via the interaction between Smad2, Smad3, Smad4 and the SBE (Smad binding element) in the Smad7 promoter. These results suggest that the myostatin signal transduction pathway is regulated by Smad7 through a negative feedback mechanism.

  5. cDNA cloning and expression analysis of myostatin/GDF11 in shrimp, Litopenaeus vannamei.

    PubMed

    Qian, Zhaoying; Mi, Xiao; Wang, Xianzong; He, Shulin; Liu, Yongjie; Hou, Fujun; Liu, Qiao; Liu, Xiaolin

    2013-05-01

    Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins belonging to the transforming growth factor-β (TGF-β) superfamily. In vertebrates, MSTN is known to negatively regulate skeletal muscle growth, and GDF11 is found to inhibit neurogenesis. In invertebrates, only one ortholog of vertebrate MSTN and GDF11 (MSTN/GDF11) existed. Little attention has been paid on its role to date. In this study, the cDNA that encodes a 422-amino-acid MSTN/GDF11 protein (LvMSTN/GDF11) was characterized from a crustacean species, the Pacific white shrimp (Litopenaeus vannamei). Sequence analysis revealed that the overall protein sequence and specific functional sites of LvMSTN/GDF11 were highly conserved with those in other crustacean species. Expression analysis by quantitative real-time reverse transcription polymerase chain reaction technique demonstrated its tissue-specific, larval developmental stage-specific, and molt stage-specific expression pattern, respectively. After in vivo injections of 20 hydroxyecdysone (20E), LvMSTN/GDF11 transcripts were declined in the abdominal (A) and pleopod (P1) muscles, increased in the pereiopod (P2) muscle, and not affected in the thoracic (T) muscle. The observed expression profiles suggest multiple functions of LvMSTN/GDF11 in L. vannamei and its role differs during the larval development and natural molt cycle. The different responses of LvMSTN/GDF11 to acute increases of 20E in the A, P1, P2 and T muscles may indicate that LvMSTN/GDF11 is transcriptionally regulated via ecdysteroids to coincide with its specific roles in the former three muscles, while its role may be independent of 20E regulation in the T muscle.

  6. 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.

  7. 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

  8. Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice.

    PubMed

    Camporez, João-Paulo G; Petersen, Max C; Abudukadier, Abulizi; Moreira, Gabriela V; Jurczak, Michael J; Friedman, Glenn; Haqq, Christopher M; Petersen, Kitt Falk; Shulman, Gerald I

    2016-02-23

    Sarcopenia, or skeletal muscle atrophy, is a debilitating comorbidity of many physiological and pathophysiological processes, including normal aging. There are no approved therapies for sarcopenia, but the antihypertrophic myokine myostatin is a potential therapeutic target. Here, we show that treatment of young and old mice with an anti-myostatin antibody (ATA 842) for 4 wk increased muscle mass and muscle strength in both groups. Furthermore, ATA 842 treatment also increased insulin-stimulated whole body glucose metabolism in old mice, which could be attributed to increased insulin-stimulated skeletal muscle glucose uptake as measured by a hyperinsulinemic-euglycemic clamp. Taken together, these studies provide support for pharmacological inhibition of myostatin as a potential therapeutic approach for age-related sarcopenia and metabolic disease.

  9. 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

  10. Knock down of the myostatin gene by RNA interference increased body weight in chicken.

    PubMed

    Bhattacharya, T K; Shukla, R; Chatterjee, R N; Dushyanth, K

    2017-01-10

    Myostatin is a negative regulator of muscular growth in poultry and other animals. Of several approaches, knocking down the negative regulator is an important aspect to augment muscular growth in chicken. Knock down of myostatin gene has been performed by shRNA acting against the expression of gene in animals. Two methods of knock down of gene in chicken such as embryo manipulation and sperm mediated method have been performed. The hatching percentage in embryo manipulation and sperm mediated method of knock down was 58.0 and 41.5%, respectively. The shRNA in knock down chicken enhanced body weight at 6 weeks by 26.9%. The dressing percentage and serum biochemical parameters such as SGPT and alkaline phosphatase differed significantly (P<0.05) between knock down and control birds. It is concluded that knocking down the myostatin gene successfully augmented growth in chicken.

  11. Cutting edge: FYCO1 recruitment to dectin-1 phagosomes is accelerated by light chain 3 protein and regulates phagosome maturation and reactive oxygen production.

    PubMed

    Ma, Jun; Becker, Courtney; Reyes, Christopher; Underhill, David M

    2014-02-15

    L chain 3 (LC3)-associated phagocytosis is a process in which LC3, a protein canonically involved in engulfing intracellular materials (autophagy), is recruited to traditional phagosomes during internalization of extracellular payloads. LC3's association with phagosomes has been implicated in regulating microbial killing, Ag processing, and phagosome maturation; however, the mechanism by which LC3 influences these processes has not been clear. In this study, we report that FYVE and coiled-coil domain containing 1 (FYCO1), a protein previously implicated in autophagosome trafficking, is recruited directly by LC3 to Dectin-1 phagosomes. During LC3-associated phagocytosis, FYCO1 recruitment facilitates maturation of early p40phox(+) phagosomes into late LAMP1(+) phagosomes. When FYCO1 is lacking, phagosomes stay p40phox(+) longer and produce more reactive oxygen.

  12. Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-γ priming.

    PubMed

    Huang, Ying; Zhou, Xiangmei; Bai, Yu; Yang, Lifeng; Yin, Xiaomin; Wang, Zhigang; Zhao, Deming

    2012-11-09

    Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1β in a dose- and time-dependent. We identified that IFN-γ priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1β production both that the protein and mRNA levels and further activated the NF-κB pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection.

  13. Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1/2 MAPKs.

    PubMed

    Lee, H-Y; Kim, J; Noh, H J; Kim, H-P; Park, S-J

    2014-12-01

    Much remains unknown about the mammalian immune response to Giardia lamblia, a protozoan pathogen that causes diarrhoeal outbreaks. We fractionated protein extracts of G. lamblia trophozoites by Viva-spin centrifugation, DEAE ion exchange and gel filtration chromatography. Resultant fractions were screened for antigenic molecules by western blots analysis using anti-G. lamblia antibodies (Abs), resulting in identification of G. lamblia binding immunoglobulin protein (GlBiP). Maturation of mouse dendritic cells (DCs) in response to recombinant GlBiP (rGlBiP) was detected by increased expression of surface molecules such as CD80, CD86 and MHC class II; these mature DCs, produced pro-inflammatory cytokines (TNF-α, IL-12 and IL-6). Especially, the truncated rGlBiP containing the heat-shock protein 70 domain-induced cytokine production from mouse DCs. rGlBiP-induced DC activation was initiated by TLR4 in a MyD88-dependent way and occurred through activation of p38 and ERK1/2 MAPKs as well as increased activity of NF-κB and AP-1. Moreover, CD4(+) T cells stimulated with rGlBiP-treated DCs produced high levels of IL-2 and IFN-γ. Together, our results suggest that GlBiP contributes to maturation of DCs via activation of TLR4-MyD88-p38, ERK1/2 MAPK, NF-κB and AP-1.

  14. 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.

  15. Controlled Expression of nif and isc Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems▿ †

    PubMed Central

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

    2007-01-01

    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

  16. 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.

  17. Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses.

    PubMed

    Li, Ran; Liu, Dong-Hua; Cao, Chun-Na; Wang, Shao-Qiang; Dang, Rui-Hua; Lan, Xian-Yong; Chen, Hong; Zhang, Tao; Liu, Wu-Jun; Lei, Chu-Zhao

    2014-03-15

    The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26T>C, g.156T>C, g.587A>G, g.598C>T, g.1485C>T, g.2115A>G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A>G and g.598C>T residing in the 5'UTR region were novel SNPs identified by this study. The g.2115A>G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.

  18. Functional analysis of pig myostatin gene promoter with some adipogenesis- and myogenesis-related factors.

    PubMed

    Deng, Bing; Wen, Jianghui; Ding, Yi; Gao, Qishuang; Huang, Haijun; Ran, Zhiping; Qian, Yunguo; Peng, Jian; Jiang, Siwen

    2012-04-01

    Myostatin (MSTN) is primarily expressed in muscle and plays an important role in muscle and fat development in pigs. However, there is little information about the regulation of pig MSTN. In order to elucidate whether pig MSTN could be regulated by muscle- and fat-related factors, the porcine MSTN promoter was amplified and cloned into pGL3-basic vector, and transfected into cells to analyze the transcriptional activity of promoter with muscle- and fat-related factors through dual-luciferase reporter assays. 5'-deletion expression showed that there was a negative-regulatory region located between nucleotides -1519 and -1236 bp, and there were some positive-regulatory regions located between -1236 and -568 bp. The longest fragment (1.7 kb) was cotransfected with muscle-related transcription factor myogenic differentiation 1 (MyoD), resulting in promoter transcriptional activity upregulation. The fragment was treated by the adipogenic agents (DIM) including dexamethasone, insulin, and isobutyl-1-methylxanthine (IBMX). We found that MSTN promoter transcriptional activity can be regulated by IBMX, but not by DIM. CCAAT/enhancer binding protein (C/EBP) α and C/EBPβ, two proteins which are induced by DIM during adipogenesis were cotransfected with the 1.7-kb fragment, respectively, resulting in promoter transcriptional activity downregulation. Treating the fragment with rosiglitazone which induce the expression of peroxisome proliferator-activated receptor γ (PPARγ), resulting in promoter transcriptional activity upregulation. Cotransfection experiments confirmed this result. Taken together, we showed that porcine MSTN could be upregulated by IBMX, MyoD, and PPARγ but downregulated by C/EBPα and C/EBPβ.

  19. EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

    PubMed Central

    Qu, Yi; Lu, Danyu; Jiang, Hao; Chi, Xiaochun; Zhang, Hongquan

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes. Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes. However, the role of EZH2 in oocyte meiotic maturation was unknown. Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes. We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion. EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes. Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation. However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly. Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy. Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation. Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation. PMID:27226494

  20. High-fat diet reduces local myostatin-1 paralog expression and alters skeletal muscle lipid content in rainbow trout, Oncorhynchus mykiss.

    PubMed

    Galt, Nicholas J; Froehlich, Jacob Michael; Meyer, Ben M; Barrows, Frederic T; Biga, Peggy R

    2014-06-01

    Muscle growth is an energetically demanding process that is reliant on intramuscular fatty acid depots in most fishes. The complex mechanisms regulating this growth and lipid metabolism are of great interest for human health and aquaculture applications. It is well established that the skeletal muscle chalone, myostatin, plays a role in lipid metabolism and adipogenesis in mammals; however, this function has not been fully assessed in fishes. We therefore examined the interaction between dietary lipid levels and myostatin expression in rainbow trout (Oncorhynchus mykiss). Five weeks of high-fat diet (HFD; 25 % lipid) intake increased white muscle lipid content and decreased circulating glucose levels and hepatosomatic index when compared to low-fat diet (LFD; 10 % lipid) intake. In addition, HFD intake reduced myostatin-1a and myostatin-1b expression in white muscle and myostatin-1b expression in brain tissue. Characterization of the myostatin-1a, myostatin-1b, and myostatin-2a promoters revealed putative binding sites for a subset of transcription factors associated with lipid metabolism. Taken together, these data suggest that HFD may regulate myostatin expression through cis-regulatory elements sensitive to increased lipid intake. Further, these findings provide a framework for future investigations of mechanisms describing the relationships between myostatin and lipid metabolism in fish.

  1. 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

  2. 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

  3. Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana.

    PubMed

    Lange, Heike; Holec, Sarah; Cognat, Valérie; Pieuchot, Laurent; Le Ret, Monique; Canaday, Jean; Gagliardi, Dominique

    2008-05-01

    Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

  4. 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

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

    PubMed

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

    2016-07-14

    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, Rab5(GTP)-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.

  6. 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...

  7. Exercise does not influence myostatin and follistatin messenger RNA expression in young women.

    PubMed

    Jensky, Nicole E; Sims, Jennifer K; Dieli-Conwright, Christina M; Sattler, Fred R; Rice, Judd C; Schroeder, E Todd

    2010-02-01

    We evaluated changes in myostatin, follistatin, and MyoD messenger RNA (mRNA) gene expression using eccentric exercise (EE) and concentric exercise (CE) as probes to better understand the mechanisms of muscle hypertrophy in young women. Twelve women performed single-leg maximal eccentric (n = 6, 25 +/- 1 years, 59 +/- 7 kg) or concentric (n = 6, 24 +/- 1 years, 65 +/- 7 kg) isokinetic knee extension exercise for 7 sessions. Muscle biopsies were taken from the vastus lateralis at baseline, 8 hours after the first exercise session, and 8 hours after the seventh exercise session. In the EE group, there were no changes in myostatin and follistatin (p > or = 0.17); however, MyoD expression increased after 1 exercise bout (p = 0.02). In the CE group, there were no changes in myostatin, follistatin, or MyoD mRNA gene expression (p > or = 0.07). Differences between the EE and CE groups were not significant (p > or = 0.05). These data suggest that a single bout or multiple bouts of maximal EE or CE may not significantly alter myostatin or follistatin mRNA gene expression in young women. However, MyoD mRNA expression seems to increase only after EE.

  8. Joint dysfunction and functional decline in middle age myostatin null mice.

    PubMed

    Guo, Wen; Miller, Andrew D; Pencina, Karol; Wong, Siu; Lee, Amanda; Yee, Michael; Toraldo, Gianluca; Jasuja, Ravi; Bhasin, Shalender

    2016-02-01

    Since its discovery as a potent inhibitor for muscle development, myostatin has been actively pursued as a drug target for age- and disease-related muscle loss. However, potential adverse effects of long-term myostatin deficiency have not been thoroughly investigated. We report herein that male myostatin null mice (mstn(-/-)), in spite of their greater muscle mass compared to wild-type (wt) mice, displayed more significant functional decline from young (3-6months) to middle age (12-15months) than age-matched wt mice, measured as gripping strength and treadmill endurance. Mstn(-/-) mice displayed markedly restricted ankle mobility and degenerative changes of the ankle joints, including disorganization of bone, tendon and peri-articular connective tissue, as well as synovial thickening with inflammatory cell infiltration. Messenger RNA expression of several pro-osteogenic genes was higher in the Achilles tendon-bone insertion in mstn(-/-) mice than wt mice, even at the neonatal age. At middle age, higher plasma concentrations of growth factors characteristic of excessive bone remodeling were found in mstn(-/-) mice than wt controls. These data collectively indicate that myostatin may play an important role in maintaining ankle and wrist joint health, possibly through negative regulation of the pro-osteogenic WNT/BMP pathway.

  9. Single nucleotide polymorphisms in the upstream regulatory region alter the expression of myostatin.

    PubMed

    Hu, Wei; Chen, Songyu; Zhang, Ran; Lin, Yushuang

    2013-06-01

    The expression of the gene encoding myostatin (MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin.

  10. Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells.

    PubMed

    Fan, Huitao; Zhang, Rui; Tesfaye, Dawit; Tholen, Ernst; Looft, Christian; Hölker, Michael; Schellander, Karl; Cinar, Mehmet Ulas

    2012-12-01

    Satellite cells function as skeletal muscle stem cells to support postnatal muscle growth and regeneration following injury or disease. There is great promise for the improvement of muscle performance in livestock and for the therapy of muscle pathologies in humans by the targeting of myostatin (MSTN) in this cell population. Human diet contains many histone deacetylase (HDAC) inhibitors, such as the bioactive component sulforaphane (SFN), whose epigenetic effects on MSTN gene in satellite cells are unknown. Therefore, we aimed to investigate the epigenetic influences of SFN on the MSTN gene in satellite cells. The present work provides the first evidence, which is distinct from the effects of trichostatin A (TSA), that SFN supplementation in vitro not only acts as a HDAC inhibitor but also as a DNA methyltransferase (DNMT) inhibitor in porcine satellite cells. Compared with TSA and 5-aza-2'-deoxycytidine (5-aza-dC), SFN treatment significantly represses MSTN expression, accompanied by strongly attenuated expression of negative feedback inhibitors of the MSTN signaling pathway. miRNAs targeting MSTN are not implicated in posttranscriptional regulation of MSTN. Nevertheless, a weakly enriched myoblast determination (MyoD) protein associated with diminished histone acetylation in the MyoD binding site located in the MSTN promoter region may contribute to the transcriptional repression of MSTN by SFN. These findings reveal a new mode of epigenetic repression of MSTN by the bioactive compound SFN. This novel pharmacological, biological activity of SFN in satellite cells may thus allow for the development of novel approaches to weaken the MSTN signaling pathway, both for therapies of human skeletal muscle disorders and for livestock production improvement.

  11. Molecular characterization of Myostatin gene from Zhikong scallop Chlamys farreri (Jones et Preston 1904).

    PubMed

    Hu, Xiaoli; Guo, Huihui; He, Yan; Wang, Shan; Zhang, Lingling; Wang, Shi; Huang, Xiaoting; Roy, Scott William; Lu, Wei; Hu, Jingjie; Bao, Zhenmin

    2010-01-01

    The scallop is an economically important sea food prized for its large and delicious adductor muscle. Studying the molecular basis of scallop muscle growth is important for both scallop breeding and our understanding of muscle mass regulation in bivalve. Myostatin (MSTN) is a conserved negative regulator of muscle growth and development. Here we report the MSTN gene from Zhikong scallop (Chlamys farreri Jones et Preston 1904). The C. farreri MSTN consists of 11651 nucleotides encoding 457 amino acids. The gene has a 3-exon/2-intron structure that is conserved with vertebrate homologs. The exons are 586, 380 and 408 bp in length, respectively, and separated by introns of 5086 and 1518 bp. The protein sequence contains characteristic conserved residues including a cleavage motif of proteolysis (RXXR) and nine cysteines. Three transcription initiation sites were found at 62, 146, and 296 bp upstream of the translation start codon ATG. In silico analysis of the promoter region identified a TATA-box and several muscle-specific regulatory elements including COMP, MEF2s, MTBFs and E-boxes. Minisatellite DNA was found in intron 1. By fluorescence in situ hybridization (FISH), the gene was mapped to the long arm of a pair of middle subtelocentric chromosome. Quantitative analysis of MSTN transcripts in embryos/larvae indicated high expression level in gastrulae and limited expression at other stages. In adult scallops, MSTN is predominantly expressed in striated muscle, with different expression levels in other tissues. Our data provide valuable genomic and expression information which will aid the further study on scallop MSTN function and MSTN evolution.

  12. 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.

  13. Binding Characteristics of Small Molecules that Mimic Nucleocapsid Protein-induced Maturation of Stem-loop-1 of HIV-1 RNA†

    PubMed Central

    Chung, Janet; Ulyanov, Nikolai B.; Guilbert, Christophe; Mujeeb, Anwer; James, Thomas L.

    2010-01-01

    As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5’-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. Based on the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only an order of magnitude tighter than the best small molecule ligand tested. The study presented here provides guidelines for design of superior small molecule binders to the SL1 RNA that have the potential of being developed as an antiviral by either interfering with SL1-NCp7 interaction at the packaging and/or maturation stages. PMID:20565056

  14. 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

  15. Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

    PubMed Central

    Condezo, Gabriela N.; Marabini, Roberto; Ayora, Silvia; Carazo, José M.; Alba, Raúl; Chillón, Miguel

    2015-01-01

    ABSTRACT Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCE Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in

  16. Myostatin inhibition in muscle, but not adipose tissue, decreases fat mass and improves insulin sensitivity.

    PubMed

    Guo, Tingqing; Jou, William; Chanturiya, Tatyana; Portas, Jennifer; Gavrilova, Oksana; McPherron, Alexandra C

    2009-01-01

    Myostatin (Mstn) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Mstn(-/-) mice have a dramatic increase in muscle mass, reduction in fat mass, and resistance to diet-induced and genetic obesity. To determine how Mstn deletion causes reduced adiposity and resistance to obesity, we analyzed substrate utilization and insulin sensitivity in Mstn(-/-) mice fed a standard chow. Despite reduced lipid oxidation in skeletal muscle, Mstn(-/-) mice had no change in the rate of whole body lipid oxidation. In contrast, Mstn(-/-) mice had increased glucose utilization and insulin sensitivity as measured by indirect calorimetry, glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamp. To determine whether these metabolic effects were due primarily to the loss of myostatin signaling in muscle or adipose tissue, we compared two transgenic mouse lines carrying a dominant negative activin IIB receptor expressed specifically in adipocytes or skeletal muscle. We found that inhibition of myostatin signaling in adipose tissue had no effect on body composition, weight gain, or glucose and insulin tolerance in mice fed a standard diet or a high-fat diet. In contrast, inhibition of myostatin signaling in skeletal muscle, like Mstn deletion, resulted in increased lean mass, decreased fat mass, improved glucose metabolism on standard and high-fat diets, and resistance to diet-induced obesity. Our results demonstrate that Mstn(-/-) mice have an increase in insulin sensitivity and glucose uptake, and that the reduction in adipose tissue mass in Mstn(-/-) mice is an indirect result of metabolic changes in skeletal muscle. These data suggest that increasing muscle mass by administration of myostatin antagonists may be a promising therapeutic target for treating patients with obesity or diabetes.

  17. A comparative examination of cortisol effects on muscle myostatin and HSP90 gene expression in salmonids.

    PubMed

    Galt, Nicholas J; McCormick, Stephen D; Froehlich, Jacob Michael; Biga, Peggy R

    2016-10-01

    Cortisol, the primary corticosteroid in teleost fishes, is released in response to stressors to elicit local functions, however little is understood regarding muscle-specific responses to cortisol in these fishes. In mammals, glucocorticoids strongly regulate the muscle growth inhibitor, myostatin, via glucocorticoid response elements (GREs) leading to muscle atrophy. Bioinformatics methods suggest that this regulatory mechanism is conserved among vertebrates, however recent evidence suggests some fishes exhibit divergent regulation. Therefore, the aim of this study was to evaluate the conserved actions of cortisol on myostatin and hsp90 expression to determine if variations in cortisol interactions have emerged in salmonid species. Representative salmonids; Chinook salmon (Oncorhynchus tshawytscha), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar); were injected intraperitoneally with a cortisol implant (50μg/g body weight) and muscle gene expression was quantified after 48h. Plasma glucose and cortisol levels were significantly elevated by cortisol in all species, demonstrating physiological effectiveness of the treatment. HSP90 mRNA levels were elevated by cortisol in brook trout, Chinook salmon, and Atlantic salmon, but were decreased in cutthroat trout. Myostatin mRNA levels were affected in a species, tissue (muscle type), and paralog specific manner. Cortisol treatment increased myostatin expression in brook trout (Salvelinus) and Atlantic salmon (Salmo), but not in Chinook salmon (Oncorhynchus) or cutthroat trout (Oncorhynchus). Interestingly, the VC alone increased myostatin mRNA expression in Chinook and Atlantic salmon, while the addition of cortisol blocked the response. Taken together, these results suggest that cortisol affects muscle-specific gene expression in species-specific manners, with unique Oncorhynchus-specific divergence observed, that are not predictive solely based upon

  18. Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

    PubMed Central

    Gorzelnik, Karl V.; Cui, Zhicheng; Reed, Catrina A.; Jakana, Joanita; Young, Ry; Zhang, Junjie

    2016-01-01

    Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA–coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly. PMID:27671640

  19. Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease.

    PubMed

    Johansson, Bente B; Torsvik, Janniche; Bjørkhaug, Lise; Vesterhus, Mette; Ragvin, Anja; Tjora, Erling; Fjeld, Karianne; Hoem, Dag; Johansson, Stefan; Ræder, Helge; Lindquist, Susanne; Hernell, Olle; Cnop, Miriam; Saraste, Jaakko; Flatmark, Torgeir; Molven, Anders; Njølstad, Pål R

    2011-10-07

    CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.

  20. Extraction and proteome analysis of starch granule-associated proteins in mature wheat kernel (Triticum aestivum L.).

    PubMed

    Bancel, Emmanuelle; Rogniaux, Hélène; Debiton, Clément; Chambon, Christophe; Branlard, Gérard

    2010-06-04

    Starch consists of the two glucose polymers, amylose and amylopectin, and is deposited as semicrystalline granules inside plastids. The starch granule proteome is particularly challenging to study due to the amount of interfering compounds (sugars, storage proteins), the very low starch granule-associated protein content and also the dynamic range of abundant proteins. Here we present the protocol for extraction and 2-DE of wheat starch granule-associated proteins whose most important steps are: (i) washing and sonication to remove interfering compounds (storage proteins) from the surface of the granules, (ii) scanning electron microscopy (SEM) observations to monitor purification and granules swelling, (iii) appropriate protein extraction and solubilization to obtain enough proteins for Coomassie blue staining and proteomic analysis. Our objective was to minimize the amount of contamination by storage proteins and to preserve the structure of the starch and of starch-associated proteins and to maximize the number of polypeptides that can be resolved. For quantitative proteomic analysis of proteins associated with wheat starch granules, we developed a two-step protein extraction protocol including TCA/acetone precipitation and phenol extraction. With this protocol, proteins were extracted from wheat starch granules and solubilized and satisfactory blue-stained 2-DE protein maps were obtained. The majority of the spots associated with starch granules were identified by peptide mass fingerprinting and MS/MS and functionally classified into carbohydrate metabolism and stress defense.

  1. Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

    PubMed Central

    Copela, Laura A.; Fernandez, Cesar F.; Sherrer, R. Lynn; Wolin, Sandra L.

    2008-01-01

    Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3′ ends. The La protein binds the 3′ ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Δ cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Δ cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Δ cells. Our experiments reveal that one consequence of Rex1p-dependent 3′ trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP. PMID:18456844

  2. Expression of the nuclear RING finger protein SNURF/RNF4 during rat testis development suggests a role in spermatid maturation.

    PubMed

    Yan, Wei; Hirvonen-Santti, Sirpa J; Palvimo, Jorma J; Toppari, Jorma; Jänne, Olli A

    2002-10-01

    A small nuclear RING finger protein, termed SNURF (or RNF4), is a coregulator of androgen receptor-dependent transcription. To elucidate the physiological role of SNURF in vivo, cell type-specific localization and changes in SNURF mRNA and protein accumulation were followed during testicular development and spermatogenesis of the rat. Two SNURF transcripts, approximately 3.0 and 1.6 kb in size, were detected in adult rat testis. Both mRNA species are capable of encoding full-length SNURF protein. The 3.0 kb SNURF mRNA is persistently expressed in Sertoli cells of both immature and mature testes, whereas the expression of the 1.6 kb transcript appears after day 30 of postnatal life and is restricted to step 4-11 spermatids. Increased accumulation of SNURF in step 4-11 spermatids, which do not express the androgen receptor, indicates that SNURF action is not restricted to the regulation of androgen signaling. Germ cell expression of SNURF coincides with the last transcriptional activity of the haploid genome and alterations in chromatin structure, suggesting that SNURF is involved in the regulation of processes required for late steps of spermatid maturation.

  3. Identification of the Mycobacterium tuberculosis protein PE-PGRS62 as a novel effector that functions to block phagosome maturation and inhibit iNOS expression.

    PubMed

    Thi, Emily P; Hong, Chris Joon Ho; Sanghera, Gaganjit; Reiner, Neil E

    2013-05-01

    Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.

  4. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  5. 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.

  6. BCR-crosslinking induces a transcription of protein phosphatase component G5PR that is required for mature B-cell survival

    SciTech Connect

    Huq Ronny, Faisal Mahmudul; Igarashi, Hideya; Sakaguchi, Nobuo . E-mail: nobusaka@kaiju.medic.kumamoto-u.ac.jp

    2006-02-03

    BCR-crosslinking triggers activation-induced cell death (AICD) selectively in the restricted stage of B-cell differentiation. We examined the transcription of a protein phosphatase subunit G5PR in immature and mature B-cells, because absence of this factor augmented cell sensitivity to AICD, associated with increased activation of JNK and Bim. BCR-crosslinking-induced G5pr transcription in AICD-resistant mature splenic IgM{sup lo}IgD{sup hi} B-cells but not in AICD susceptible immature IgM{sup hi}IgD{sup lo} B-cells. Thus, G5pr induction correlated with the prevention of AICD; High in mature splenic CD23{sup hi} B-cells but low in immature B-cells of neonatal mice, sub-lethally irradiated mice, or xid mice. Lack of G5pr upregulation was associated with the prolonged activation of JNK. The G5pr cDNA transfection protected an immature B-cell line WEHI-231 from BCR-mediated AICD. The differential expression of G5PR might be responsible for the antigen-dependent selection of B-cells.

  7. 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

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

    PubMed

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

    2015-07-01

    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 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) 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.

  9. A critical role for the protein kinase PKK in the maintenance of recirculating mature B cells and the development of B1 cells.

    PubMed

    Chen, Luojing; Oleksyn, David; Pulvino, Mary; Sanz, Ignacio; Ryan, Daniel; Ryan, Charlotte; Lin, Chyuan-Sheng; Poligone, Brian; Pentland, Alice P; Ritchlin, Christopher; Zhao, Jiyong

    2016-04-01

    Protein kinase C associated kinase (PKK) regulates NF-κB activation and is required for the survival of certain lymphoma cells. Mice lacking PKK die soon after birth, and previous studies suggest that the role of PKK in B cell development might be context dependent. We have generated a mouse strain harboring conditional null alleles for PKK and a Cre-recombinase transgene under the control of the endogenous CD19 promoter. In the present study, we show that knockout of PKK in B cells results in the reduction of long-lived recirculating mature B cell population in lymph nodes and bone marrow as well as a decrease in peritoneal B1 cells, while PKK deficiency has no apparent effect on early B cell development in bone marrow or the development of follicular and marginal zone B cells in the spleen. In addition, we demonstrate that PKK-deficient B cells display defective proliferation and survival responses to stimulation of B cell receptor (BCR), which may underlie the reduction of recirculating mature B cells in PKK mutant mice. Consistently, BCR-mediated NF-κB activation, known to be required for the survival of activated but not resting B cells, is attenuated in PKK-deficient B cells. Thus, our results reveal a critical role of PKK in the maintenance of recirculating mature B cells as well as the development of B1 cells in mice.

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

    DTIC Science & Technology

    2011-03-01

    07-1-0181 TITLE: Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and...views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army...Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration after Injury Dr. Nestor Gonzalez-Cadavid Charles R. Drew

  11. Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme.

    PubMed

    Akama, Kuniko; Horikoshi, Tomoe; Sugiyama, Atsushi; Nakahata, Satoko; Akitsu, Aoi; Niwa, Nobuyoshi; Intoh, Atsushi; Kakui, Yasutaka; Sugaya, Michiko; Takei, Kazuo; Imaizumi, Noriaki; Sato, Takaya; Matsumoto, Rena; Iwahashi, Hitoshi; Kashiwabara, Shin-ichi; Baba, Tadashi; Nakamura, Megumi; Toda, Tosifusa

    2010-06-01

    In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.

  12. 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.

  13. 170 SUPPLEMENT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE DURING IN VITRO MATURATION ACTIVATES SMAD2 AND cAMP RESPONSIVE ELEMENT BINDING PROTEIN SIGNALING.

    PubMed

    Yoon, J D; Lee, E; Hyun, S-H

    2016-01-01

    Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated reactive oxygen species (ROS) levels, specific gene transcription levels in oocytes and cumulus cells (CC) after IVM, and specific protein expression and activation levels in matured CC by Western blotting. Each concentration (0, 1, 10, and 100ngmL(-1)) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analysed by ANOVA followed by Duncan using SPSS (IBM Corp., Armonk, NY). Data are presented as the mean (replicate numbers) and differences were considered significant at P<0.05. After 44h of IVM, oocytes are mechanically denuded from CC with 0.1% hyaluronidase and only metaphase II stage oocytes are counted as nuclear matured oocytes. Each group of matured oocytes are stained by 2',7'-dichlorodihydrofluorescein diacetate and the fluorescence was captured as graphic files under an epifluorescence microscope. The fluorescence intensities of oocytes were measured using Image J software (National Institutes for Health, Bethesda, MD). The groups treated with 10 and 100ngmL(-1) of GDF8 showed significantly more than 10% decrease in intracellular ROS levels compared with other groups (5 times). To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, real-time PCR was performed. In matured oocytes, the developmental competence marker POU5F1, antioxidant enzymes regulator NRF2, and antiapoptosis gene BCL-2 mRNA transcription levels were significantly increased in the 10ngmL(-1) treatment group compared with control (4 times). In CC, the 10ngmL(-1) treatment groups showed significantly higher PCNA and NRF2 mRNA levels, and the 1 and 10ngmL(-1) treatment groups observed significantly increased cumulus expansion

  14. 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.

  15. 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.

  16. Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

    SciTech Connect

    Schaehs, Philipp; Weidinger, Petra; Probst, Olivia C.; Svoboda, Barbara; Stadlmann, Johannes; Beug, Hartmut; Waerner, Thomas; Mach, Lukas

    2008-10-01

    Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.

  17. 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.

  18. Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation

    PubMed Central

    Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

    2017-01-01

    Active cellular transporters of harmful agents—multidrug resistance (mdr) proteins—are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins—MDR1, BCRP, MRP1, MRP4 and MRP5—in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific. PMID:28212450

  19. 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

  20. RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

    PubMed

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-02-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

  1. In silico survey of the mitochondrial protein uptake and maturation systems in the brown alga Ectocarpus siliculosus.

    PubMed

    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.

  2. Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism.

    PubMed

    McFarlane, Craig; Plummer, Erin; Thomas, Mark; Hennebry, Alex; Ashby, Murray; Ling, Nicholas; Smith, Heather; Sharma, Mridula; Kambadur, Ravi

    2006-11-01

    Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.

  3. Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

    PubMed Central

    Christopher, D A; Hallick, R B

    1990-01-01

    We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases. PMID:2136640

  4. 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.

  5. 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

  6. Chromodomain, Helicase and DNA-binding CHD1 protein, CHR5, are involved in establishing active chromatin state of seed maturation genes.

    PubMed

    Shen, Yuan; Devic, Martine; Lepiniec, Loïc; Zhou, Dao-Xiu

    2015-08-01

    Chromatin modification and remodelling are the basis for epigenetic regulation of gene expression. LEAFY COTYLEDON 1 (LEC1), LEAFY COTYLEDON 2 (LEC2), ABSCISIC ACID-INSENSITIVE 3 (ABI3) and FUSCA3 (FUS3) are key regulators of embryo development and are repressed after seed maturation. The chromatin remodelling CHD3 protein PICKLE (PKL) is involved in the epigenetic silencing of the genes. However, the chromatin mechanism that establishes the active state of these genes during early embryo development is not clear. We show that the Arabidopsis CHD1-related gene, CHR5, is activated during embryo development. Mutation of the gene reduced expression of LEC1, ABI3 and FUS3 in developing embryo and accumulation of seed storage proteins. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL in embryo gene expression and seed storage protein accumulation, which likely acted on the promoter region of the genes. CHR5 was shown to be associated with the promoters of ABI3 and FUS3 and to be required to reduce nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in establishing the active state of embryo regulatory genes by reducing nucleosomal barrier, which may be exploited to enhance seed protein production.

  7. Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System*

    PubMed Central

    Ozer, Hatice K.; Dlouhy, Adrienne C.; Thornton, Jeremy D.; Hu, Jingjing; Liu, Yilin; Barycki, Joseph J.; Balk, Janneke; Outten, Caryn E.

    2015-01-01

    The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways. PMID:26396185

  8. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    PubMed

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  9. Inbred strains of zebrafish exhibit variation in growth performance and myostatin expression following fasting.

    PubMed

    Meyer, Ben M; Froehlich, Jacob M; Galt, Nicholas J; Biga, Peggy R

    2013-01-01

    Although the zebrafish (Danio rerio) has been widely utilized as a model organism for several decades, there is little information available on physiological variation underlying genetic variation among the most commonly used inbred strains. This study evaluated growth performance using physiological and molecular markers of growth in response to fasting in six commonly used zebrafish strains [AB, TU, TL, SJA, WIK, and petstore (PET) zebrafish]. Fasting resulted in a standard decrease in whole blood glucose levels, a typical vertebrate glucose metabolism pattern, in AB, PET, TL, and TU zebrafish strains. Alternatively, fasting did not affect glucose levels in SJA and WIK zebrafish strains. Similarly, fasting had no effect on myostatin mRNA levels in AB, PET, TU, and WIK zebrafish strains, but decreased myostatin-1 and -2 mRNA levels in SJA zebrafish. Consistent with previous work, fasting increased myostatin-2 mRNA levels in TL zebrafish. These data demonstrate that variation is present in growth performance between commonly used inbred strains of zebrafish. These data can help future research endeavors by highlighting the attributes of each strain with regard to growth performance so that the most fitting strain may be utilized.

  10. 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.

  11. Inbred strains of zebrafish exhibit variation in growth performance and myostatin expression following fasting

    PubMed Central

    Meyer, Ben M.; Froehlich, Jacob M.; Galt, Nicholas J.; Biga, Peggy R.

    2012-01-01

    Although the zebrafish (Danio rerio) has been widely utilized as a model organism for several decades, there is little information available on physiological variation underlying genetic variation among the most commonly used inbred strains. This study evaluated growth performance using physiological and molecular markers of growth in response to fasting in six commonly used zebrafish strains [AB, TU, TL, SJA, WIK, and petstore (PET) zebrafish]. Fasting resulted in a standard decrease in whole blood glucose levels, typical vertebrate glucose metabolism pattern, in AB, PET, TL, and TU zebrafish strains. Alternatively, fasting did not affect glucose levels in SJA and WIK zebrafish strains. Similarly, fasting had no effect on myostatin mRNA levels in AB, PET, TU, and WIK zebrafish strains, but decreased myostatin-1 and -2 mRNA levels in SJA zebrafish. Consistent with previous work, fasting increased myostatin-2 mRNA levels in TL zebrafish. These data demonstrate that variation is present in growth performance between commonly used inbred strains of zebrafish. These data can help future research endeavors by highlighting the attributes of each strain with regard to growth performance so that the most fitting strain may be utilized. PMID:23047051

  12. The LIM homeodomain protein Lhx6 regulates maturation of interneurons and network excitability in the mammalian cortex.

    PubMed

    Neves, Guilherme; Shah, Mala M; Liodis, Petros; Achimastou, Angeliki; Denaxa, Myrto; Roalfe, Grant; Sesay, Abdul; Walker, Matthew C; Pachnis, Vassilis

    2013-08-01

    Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

  13. 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

  14. 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...

  15. Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris.

    PubMed

    Aramini, James M; Hamilton, Keith; Rossi, Paolo; Ertekin, Asli; Lee, Hsiau-Wei; Lemak, Alexander; Wang, Huang; Xiao, Rong; Acton, Thomas B; Everett, John K; Montelione, Gaetano T

    2012-05-08

    Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro.

  16. PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Jang, Jichan; Wang, Li; Jeanjean, Robert; Zhang, Cheng-Cai

    2007-04-01

    Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and heterocysts. Under diazotrophic conditions, the mutant DeltaprpJ (S20) did not grow, lacked only one of the two heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglE(A) and nifH, two genes necessary for the formation of the glycolipid layer of heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for heterocyst maturation because it regulates the synthesis of only one of the two heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both heterocyst glycolipids.

  17. ADP-ribosylation factor 1 protein regulates trypsinogen activation via organellar trafficking of procathepsin B protein and autophagic maturation in acute pancreatitis.

    PubMed

    Orlichenko, Lidiya; Stolz, Donna B; Noel, Pawan; Behari, Jaideep; Liu, Shiguang; Singh, Vijay P

    2012-07-13

    Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

  18. 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.

  19. Calcineurin B homologous protein 3 promotes the biosynthetic maturation, cell surface stability, and optimal transport of the Na+/H+ exchanger NHE1 isoform.

    PubMed

    Zaun, Hans C; Shrier, Alvin; Orlowski, John

    2008-05-02

    Calcineurin B homologous protein (CHP) 1 and 2 are Ca(2+)-binding proteins that modulate several cellular processes, including cytoplasmic pH by positively regulating plasma membrane-type Na(+)/H(+) exchangers (NHEs). Recently another CHP-related protein, termed tescalcin or CHP3, was also shown to interact with the ubiquitous NHE1 isoform, but seemingly suppressed its activity. However, the precise physical and functional nature of this association was not examined in detail. In this study, biochemical and cellular studies were undertaken to further delineate this relationship. Glutathione S-transferase-NHE1 fusion protein pulldown assays revealed that full-length CHP3 binds directly to the proximal juxtamembrane C-terminal region (amino acids 505-571) of rat NHE1 in the same region that binds CHP1 and CHP2. The interaction was further validated by coimmunoprecipitation and coimmunolocalization experiments using full-length CHP3 and wild-type NHE1 in transfected Chinese hamster ovary AP-1 cells. Simultaneous mutation of four hydrophobic residues within this region ((530)FLDHLL(535)) to either Ala, Gln, or Arg (FL-A, FL-Q, or FL-R) abrogated this interaction both in vitro and in intact cells. The NHE1 mutants were sorted properly to the cell surface but showed markedly reduced (FL-A) or minimal (FL-R and FL-Q) activity. Interestingly, and contrary to an earlier finding, ectopic coexpression of CHP3 up-regulated the cell surface activity of wild-type NHE1. This stimulation was not observed with the CHP3 binding-defective mutants. Mechanistically, overexpression of CHP3 did not alter the H(+) sensitivity of wild-type NHE1 but rather promoted its biosynthetic maturation and half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein.

  20. NfuA, a new factor required for maturing Fe/S proteins in Escherichia coli under oxidative stress and iron starvation conditions.

    PubMed

    Angelini, Sandra; Gerez, Catherine; Ollagnier-de Choudens, Sandrine; Sanakis, Yiannis; Fontecave, Marc; Barras, Frédéric; Py, Béatrice

    2008-05-16

    Iron/sulfur (Fe/S) proteins are central to the functioning of cells in both prokaryotes and eukaryotes. Here, we show that the yhgI gene, which we renamed nfuA, encodes a two-domain protein that is required for Fe/S biogenesis in Escherichia coli. The N-terminal domain resembles the so-called Fe/S A-type scaffold but, curiously, has lost the functionally important Cys residues. The C-terminal domain shares sequence identity with Nfu proteins. Mössbauer and UV-visible spectroscopic analyses revealed that, upon reconstitution, NfuA binds a [4Fe-4S] cluster. Moreover, NfuA can transfer this cluster to apo-aconitase. Mutagenesis studies indicated that the N- and C-terminal domains are important for NfuA function in vivo. Similarly, the functional importance of Cys residues present in the Nfu-like domain was demonstrated in vivo by introducing Cys-->Ser mutations. In vivo investigations revealed that the nfuA gene is important for E. coli to sustain oxidative stress and iron starvation. Also, combining nfuA with either isc or suf mutations led to additive phenotypic deficiencies, indicating that NfuA is a bona fide new player in Isc- and Suf-dependent Fe/S biogenesis pathways. Taken together, these data demonstrate that NfuA intervenes in the maturation of apoproteins in E. coli, allowing them to acquire Fe/S clusters. By taking into account results from numerous previous transcriptomic studies that had suggested a link between NfuA and protein misfolding, we discuss the possibility that NfuA could act as a scaffold/chaperone for damaged Fe/S proteins.

  1. Theoretical studies of chromophore maturation in the wild-type green fluorescent protein: ONIOM(DFT:MM) investigation of the mechanism of cyclization.

    PubMed

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

    2012-02-02

    The availability of a gene encoding green fluorescence immediately stimulates interest in the puzzle of autocatalytic formation of the green fluorescent protein (GFP) chromophore. Numerous experimental and theoretical studies have indicated that cyclization is the first and most important step in the maturation process of the GFP. In our previous paper based on cluster models [J. Phys. Chem. B2010, 114, 9698-9705], two possible mechanisms have been investigated with the conclusion that the backbone condensation initiated by deprotonation of the Gly67 amide nitrogen is easier than deprotonation of the Tyr66 α-carbon. However, the impact of the protein environment on the reaction mechanism remains to be explored. In this paper, we investigated the two possible mechanisms with inclusion of protein environmental effects by using molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) calculations. Our calculations reveal no hydrogen bonding network that would facilitate deprotonation of the amide nitrogen of Gly67, although it is the lower energy pathway in the cluster model system. Contrastingly, there is a hydrogen bonding network between Tyr66 α-carbon and Glu222, which is in good agreement with X-ray data. The ONIOM studies show that proton transfer from Tyr66 α-carbon to Glu222 is a long-distance charge transfer process. The charge distribution of the MM region has a significant perturbation to the wave function for the QM region, with the QM energy for the proton transfer product being increased under the influence of the electrostatic protein environment. The barrier for the rate-limiting step in cyclization is quite high, about 40.0 kcal/mol in the case of ONIOM-EE.

  2. New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration

    PubMed Central

    Pasteuning-Vuhman, Svitlana; Boertje-van der Meulen, Johanna W.; van Putten, Maaike; Overzier, Maurice; ten Dijke, Peter; Kiełbasa, Szymon M.; Arindrarto, Wibowo; Wolterbeek, Ron; Lezhnina, Ksenia V.; Ozerov, Ivan V.; Aliper, Aleksandr M.; Hoogaars, Willem M.; Aartsma-Rus, Annemieke; Loomans, Cindy J. M.

    2017-01-01

    Skeletal muscle fibrosis and impaired muscle regeneration are major contributors to muscle wasting in Duchenne muscular dystrophy (DMD). Muscle growth is negatively regulated by myostatin (MSTN) and activins. Blockage of these pathways may improve muscle quality and function in DMD. Antisense oligonucleotides (AONs) were designed specifically to block the function of ALK4, a key receptor for the MSTN/activin pathway in skeletal muscle. AON-induced exon skipping resulted in specific Alk4 down-regulation, inhibition of MSTN activity, and increased myoblast differentiation in vitro. Unexpectedly, a marked decrease in muscle mass (10%) was found after Alk4 AON treatment in mdx mice. In line with in vitro results, muscle regeneration was stimulated, and muscle fiber size decreased markedly. Notably, when Alk4 was down-regulated in adult wild-type mice, muscle mass decreased even more. RNAseq analysis revealed dysregulated metabolic functions and signs of muscle atrophy. We conclude that ALK4 inhibition increases myogenesis but also regulates the tight balance of protein synthesis and degradation. Therefore, caution must be used when developing therapies that interfere with MSTN/activin pathways.—Pasteuning-Vuhman, S., Boertje-van der Meulen, J. W., van Putten, M., Overzier, M., ten Dijke, P., Kiełbasa, S. M., Arindrarto, W., Wolterbeek, R., Lezhnina, K. V., Ozerov, I. V., Aliper, A. M., Hoogaars, W. M., Aartsma-Rus, A., Loomans, C. J. M. New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration. PMID:27733450

  3. The byssus of the zebra mussel, Dreissena polymorpha. I: Morphology and in situ protein processing during maturation.

    PubMed

    Rzepecki, L M; Waite, J H

    1993-10-01

    The zebra mussel, Dreissena polymorpha, owes its notoriety as a biofouler to its adhesive skills and opportunism. Adhesion by the adult mussel to hard substrata is mediated by a nonliving extracorporeal structure called the byssus, which is superficially similar to the byssus of marine mussels in that it consists of a tight bundle of sclerotized threads tipped by adhesive plaques. Juvenile zebra mussels secrete a homologous structure on settlement, but they also employ an elongated belaying byssus while climbing that consists of an elastic, mucous filament anchored at irregular intervals by a byssal thread and plaque. This multiply anchored belaying line can be 20 to 30 times the mussel length. Histochemical tests show that the thread and plaque of both kinds of byssus contains a complex distribution of proteins that are subject to chemical processing after secretion. This processing may result from the formation of crosslinks following the catecholoxidase-catalyzed oxidation of peptidyl 3,4-dihydroxyphenylalanine during sclerotization.

  4. Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

    PubMed Central

    Thompson, G A; Scherer, D E; Foxall-Van Aken, S; Kenny, J W; Young, H L; Shintani, D K; Kridl, J C; Knauf, V C

    1991-01-01

    Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme. Images PMID:2006194

  5. Activation of ribosomal protein S6 phosphorylation during meiotic maturation of Xenopus laevis oocytes: in vitro ordered appearance of S6 phosphopeptides.

    PubMed Central

    Martin-Pérez, J; Rudkin, B B; Siegmann, M; Thomas, G

    1986-01-01

    During meiotic maturation of Xenopus laevis stage 6 oocytes into unfertilized eggs, 40S ribosomal protein S6 undergoes multiple phosphorylation. Extracts prepared from unfertilized eggs are up to 10-fold more efficient in phosphorylating S6 than those prepared from immature oocytes. When analyzed by DEAE chromatography the S6 kinase activity elutes as a single peak. If extracts from unfertilized eggs are prepared in the absence of beta-glycerol phosphate, a putative phosphatase inhibitor, there is a severe reduction in recovered S6 kinase activity. Under optimal conditions, incubation of unfertilized egg extracts with 40S ribosomes in the presence of ATP leads to the average incorporation of 3.5 mol of phosphate/mol of S6. Prior incubation of these extracts with the cAMP-dependent protein kinase inhibitor does not inhibit S6 phosphorylation indicating that another kinase is responsible. Analysis of the in vitro phosphorylated peptides demonstrates that they migrate to the equivalent position of those observed previously in vivo and in vitro. More strikingly, if each of the increasingly phosphorylated derivatives of S6 is analyzed independently, it is found that the phosphopeptides appear in a specific order. Images Fig.1. Fig.4. Fig.5. Fig.6. PMID:3709523

  6. Long-Term Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Golden Retriever Muscular Dystrophy

    PubMed Central

    Sleeper, Meg M.; Forbes, Sean C.; Morine, Kevin J.; Reynolds, Caryn; Singletary, Gretchen E.; Trafny, Dennis; Pham, Jennifer; Bogan, Janet; Kornegay, Joe N.; Vandenborne, Krista; Walter, Glenn A.; Sweeney, H. Lee

    2011-01-01

    Abstract Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive disease affecting 1 in 3,500 newborn boys for which there is no effective treatment or cure. One novel strategy that has therapeutic potential for DMD is inhibition of myostatin, a negative regulator of skeletal muscle mass that may also promote fibrosis. Therefore, our goal in this study was to evaluate systemic myostatin inhibition in the golden retriever model of DMD (GRMD). GRMD canines underwent liver-directed gene transfer of a self-complementary adeno-associated virus type 8 vector designed to express a secreted dominant-negative myostatin peptide (n=4) and were compared with age-matched, untreated GRMD controls (n=3). Dogs were followed with serial magnetic resonance imaging (MRI) for 13 months to assess cross-sectional area and volume of skeletal muscle, then euthanized so that tissue could be harvested for morphological and histological analysis. We found that systemic myostatin inhibition resulted in increased muscle mass in GRMD dogs as assessed by MRI and confirmed at tissue harvest. We also found that hypertrophy of type IIA fibers was largely responsible for the increased muscle mass and that reductions in serum creatine kinase and muscle fibrosis were associated with long-term myostatin inhibition in GRMD. This is the first report describing the effects of long-term, systemic myostatin inhibition in a large-animal model of DMD, and we believe that the simple and effective nature of our liver-directed gene-transfer strategy makes it an ideal candidate for evaluation as a novel therapeutic approach for DMD patients. PMID:21787232

  7. 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

  8. The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier.

    PubMed

    Menezes, Michael J; McClenahan, Freyja K; Leiton, Cindy V; Aranmolate, Azeez; Shan, Xiwei; Colognato, Holly

    2014-11-12

    Laminins are major constituents of the gliovascular basal lamina of the blood-brain barrier (BBB); however, the role of laminins in BBB development remains unclear. Here we report that Lama2(-/-) mice, lacking expression of the laminin α2 subunit of the laminin-211 heterotrimer expressed by astrocytes and pericytes, have a defective BBB in which systemically circulated tracer leaks into the brain parenchyma. The Lama2(-/-) vascular endothelium had significant abnormalities, including altered integrity and composition of the endothelial basal lamina, inappropriate expression of embryonic vascular endothelial protein MECA32, substantially reduced pericyte coverage, and tight junction abnormalities. Additionally, astrocytic endfeet were hypertrophic and lacked appropriately polarized aquaporin4 channels. Laminin-211 appears to mediate these effects at least in part by dystroglycan receptor interactions, as preventing dystroglycan expression in neural cells led to a similar set of BBB abnormalities and gliovascular disturbances, which additionally included perturbed vascular endothelial glucose transporter-1 localization. These findings provide insight into the cell and molecular changes that occur in congenital muscular dystrophies caused by Lama2 mutations or inappropriate dystroglycan post-translational modifications, which have accompanying brain abnormalities, including seizures. Our results indicate a novel role for laminin-dystroglycan interactions in the cooperative integration of astrocytes, endothelial cells, and pericytes in regulating the BBB.

  9. 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.

  10. The Major Immediate-Early Protein IE2 of Human Cytomegalovirus Is Sufficient to Induce Proteasomal Degradation of CD83 on Mature Dendritic Cells

    PubMed Central

    Heilingloh, Christiane S.; Grosche, Linda; Kummer, Mirko; Mühl-Zürbes, Petra; Kamm, Lisa; Scherer, Myriam; Latzko, Melanie; Stamminger, Thomas; Steinkasserer, Alexander

    2017-01-01

    Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation. PMID:28203230

  11. High-fat diet reduces local myostatin-1 paralog expression and alters skeletal muscle lipid content in rainbow trout, Oncorhynchus mykiss

    PubMed Central

    Galt, Nicholas J.; Froehlich, Jacob Michael; Meyer, Ben M.; Barrows, Frederic T.; Biga, Peggy R.

    2014-01-01

    Muscle growth is an energetically demanding process that is reliant on intramuscular fatty acid depots in most fishes. The complex mechanisms regulating this growth and lipid metabolism are of great interest for human health and aquaculture applications. It is well established that the skeletal muscle chalone, myostatin, plays a role in lipid metabolism and adipogenesis in mammals; however, this function has not been fully assessed in fishes. We therefore examined the interaction between dietary lipid levels and myostatin expression in rainbow trout (Oncorhynchus mykiss). Five-weeks of high-fat (HFD; 25% lipid) dietary intake increased white muscle lipid content, and decreased circulating glucose levels and hepatosomatic index when compared to low-fat diet (LFD; 10% lipid) intake. In addition HFD intake reduced myostatin-1a and -1b expression in white muscle and myostatin-1b expression in brain tissue. Characterization of the myostatin-1a, -1b, and -2a promoters revealed putative binding sites for a subset of transcription factors associated with lipid metabolism. Taken together, these data suggest that HFD may regulate myostatin expression through cis-regulatory elements sensitive to increased lipid intake. Further, these findings provide a framework for future investigations of mechanisms describing the relationships between myostatin and lipid metabolism in fish. PMID:24264425

  12. IGF and myostatin pathways are respectively induced during the earlier and the later stages of skeletal muscle hypertrophy induced by clenbuterol, a β₂-adrenergic agonist.

    PubMed

    Abo, Tokuhisa; Iida, Ryo-Hei; Kaneko, Syuhei; Suga, Takeo; Yamada, Hiroyuki; Hamada, Yoshiki; Yamane, Akira

    2012-12-01

    Clenbuterol, a β₂-adrenergic agonist, increases the hypertrophy of skeletal muscle. Insulin-like growth factor (IGF) is reported to work as a potent positive regulator in the clenbuterol-induced hypertrophy of skeletal muscles. However, the precise regulatory mechanism for the hypertrophy of skeletal muscle induced by clenbuterol is unknown. Myostatin, a member of the TGFβ super family, is a negative regulator of muscle growth. The aim of the present study is to elucidate the function of myostatin and IGF in the hypertrophy of rat masseter muscle induced by clenbuterol. To investigate the function of myostatin and IGF in regulatory mechanism for the clenbuterol-induced hypertrophy of skeletal muscles, we analysed the expression of myostatin and phosphorylation levels of myostatin and IGF signaling components in the masseter muscle of rat to which clenbuterol was orally administered for 21 days. Hypertrophy of the rat masseter muscle was induced between 3 and 14 days of oral administration of clenbuterol and was terminated at 21 days. The expression of myostatin and the phosphorylation of smad2/3 were elevated at 21 days. The phosphorylation of IGF receptor 1 (IGFR1) and akt1 was elevated at 3 and 7 days. These results suggest that myostatin functions as a negative regulator in the later stages in the hypertrophy of rat masseter muscle induced by clenbuterol, whereas IGF works as a positive regulator in the earlier stages.

  13. Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.

    PubMed

    Lu, Ming-Chi; Yu, Chia-Li; Yu, Hui-Chun; Huang, Hsien-Bin; Koo, Malcolm; Lai, Ning-Sheng

    2016-01-01

    We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage.

  14. Deciphering the hidden informational content of protein sequences: foldability of proinsulin hinges on a flexible arm that is dispensable in the mature hormone.

    PubMed

    Liu, Ming; Hua, Qing-xin; Hu, Shi-Quan; Jia, Wenhua; Yang, Yanwu; Saith, Sunil Evan; Whittaker, Jonathan; Arvan, Peter; Weiss, Michael A

    2010-10-01

    Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe(B1) in placental mammals) is variably positioned within crystal structures and exhibits (1)H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1-B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.

  15. Lower maternal body condition during pregnancy affects skeletal muscle structure and glut-4 protein levels but not glucose tolerance in mature adult sheep.

    PubMed

    Costello, Paula M; Hollis, Lisa J; Cripps, Roselle L; Bearpark, Natasha; Patel, Harnish P; Sayer, Avan Aihie; Cooper, Cyrus; Hanson, Mark A; Ozanne, Susan E; Green, Lucy R

    2013-10-01

    Suboptimal maternal nutrition and body composition are implicated in metabolic disease risk in adult offspring. We hypothesized that modest disruption of glucose homeostasis previously observed in young adult sheep offspring from ewes of a lower body condition score (BCS) would deteriorate with age, due to changes in skeletal muscle structure and insulin signaling mechanisms. Ewes were fed to achieve a lower (LBCS, n = 10) or higher (HBCS, n = 14) BCS before and during pregnancy. Baseline plasma glucose, glucose tolerance and basal glucose uptake into isolated muscle strips were similar in male offspring at 210 ± 4 weeks. Vastus total myofiber density (HBCS, 343 ± 15; LBCS, 294 ± 14 fibers/mm(2), P < .05) and fast myofiber density (HBCS, 226 ± 10; LBCS 194 ± 10 fibers/mm(2), P < .05), capillary to myofiber ratio (HBCS, 1.5 ± 0.1; LBCS 1.2 ± 0.1 capillary:myofiber, P < .05) were lower in LBCS offspring. Vastus protein levels of Akt1 were lower (83% ± 7% of HBCS, P < .05), and total glucose transporter 4 was increased (157% ± 6% of HBCS, P < .001) in LBCS offspring, Despite the reduction in total myofiber density in LBCS offspring, glucose tolerance was normal in mature adult life. However, such adaptations may lead to complications in metabolic control in an overabundant postnatal nutrient environment.

  16. 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.

  17. A Sensitive Assay Using a Native Protein Substrate For Screening HIV-1 Maturation Inhibitors Targeting the Protease Cleavage Site between Matrix and Capsid

    PubMed Central

    Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A.; Janzen, William; Swanstrom, Ronald

    2013-01-01

    The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the current study, a sensitive assay based on fluorescence polarization is described that can monitor cleavage at the MA/CA site in the context of the folded protein substrate. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (Fluorescein Arsenical Hairpin) reagent which binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), significant differences in polarization values were measurable as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 PR inhibitor, which resulted in a gradual decrease in the FP values demonstrating that the assay is sensitive discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z'–value of 0.79 and average coefficient of variation values less than 3%. The robustness and reproducibility of the assay was further validated using the LOPAC1280 compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein. PMID:23763575

  18. Posttranscriptional mechanisms involving microRNA-27a and b contribute to fast-specific and glucocorticoid-mediated myostatin expression in skeletal muscle.

    PubMed

    Allen, David L; Loh, Amanda S

    2011-01-01

    Expression of the antigrowth factor myostatin (MSTN) differs between fast and slow skeletal muscles and is increased in nearly every form of muscle atrophy, but the contribution of transcriptional vs. posttranscriptional mechanisms to its differing expression in these states has not been defined. We show here that levels of mature MSTN mRNA were sixfold greater in fast vs. slow muscle and were increased twofold in fast muscle in response to dexamethasone (Dex) injection in vivo and in C₂C₁₂ myotubes following Dex treatment in vitro, but that levels of MSTN pre-mRNA, a readout of transcription, only minimally and nonsignificantly differed in these states. Moreover, Dex treatment with or without cotransfection with a glucocorticoid receptor expression construct had only modest effects on mouse MSTN promoter activity in C₂C₁₂ myotubes. We therefore explored the potential contribution of posttranscriptional mechanisms, and the role of the microRNAs miR-27a and b in particular, on MSTN expression. The MSTN 3'-untranslated region (UTR) contains a putative recognition sequence for miR-27a and b that is conserved across a wide range of vertebrate species. Cotransfection of a MSTN 3'-UTR-luciferase construct with a miR-27b expression construct significantly attenuated by approximately half while mutation of the miR-27 recognition sequence significantly increased by approximately twofold the activity of a MSTN 3'-UTR construct and decreased mRNA degradation of a luciferase reporter construct in C₂C₁₂ myotubes. Expression of miR-27a and b was almost sixfold greater in slow-twitch than in fast-twitch muscle in vivo, and miR-27a expression was significantly decreased by nearly half by glucocorticoid treatment in vitro. Finally, the miR-27a and b promoters were activated by cotransfection with the slow-specific signaling molecules calcineurin and peroxisome proliferator-activated receptor-γ coactivator-1α. The present data represent the first demonstration

  19. 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

  20. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  1. 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.

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

    PubMed Central

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

    2010-01-01

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

  3. 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...

  4. Metabolic differences in bovine cumulus-oocyte complexes matured in vitro in the presence or absence of follicle-stimulating hormone and bone morphogenetic protein 15.

    PubMed

    Sutton-McDowall, Melanie L; Mottershead, David G; Gardner, David K; Gilchrist, Robert B; Thompson, Jeremy G

    2012-10-01

    Bidirectional communication between cumulus cells and the oocyte is necessary to achieve oocyte developmental competence. The aim of the present study was to examine the effects of recombinant human bone morphogenetic protein 15 (rhBMP15) and follicle-stimulating hormone (FSH) supplementation on bovine cumulus-oocyte complex (COC) metabolism during maturation. Bovine COCs were matured in the presence of absence of FSH, rhBMP15, or both for 23 h. The addition of FSH and rhBMP15 increased blastocyst development (without rhBMP15 and FSH, 28.4% ± 7.4%; with FSH and rhBMP15, 51.5% ± 5.4%; P < 0.05). Glucose uptake and lactate production was significantly increased by greater than 2-fold with FSH (P < 0.05), whereas rhBM15 supplementation did not increase these levels. rhBMP15 supplementation (regardless of FSH) significantly decreased ADP levels in COCs, leading to an increase in ATP:ADP ratios (P < 0.05). Indicators of mitochondrial activity and cellular REDOX, oxidized flavin adenine dinucleotide (FAD(++)) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), levels within the oocyte of COCs were significantly higher with rhBMP15 alone, whereas the presence of FSH diminished the rhBMP15 effect. Regardless of treatment, no changes in REDOX state (FAD(++):NAD(P)H). The significant increase in FAD(++) and NAD(P)H in COCs with rhBMP15 was mediated via cumulus cells, because no differences were found in denuded oocytes cultured in the presence or absence of FSH, rhBMP15, or both. The present study demonstrates that a principal metabolic consequence of FSH supplementation of COCs is to alter the glycolytic rate of cumulus cells, whereas that of rhBMP15 is to regulate oxidative phosphorylation in the oocyte, even though it acts via cumulus cells. These effects are tempered when FSH and rhBMP15 are present together but, nonetheless, yield the best oocyte developmental competence.

  5. Alterations in myostatin expression are associated with changes in cardiac left ventricular mass but not ejection fraction in the mouse.

    PubMed

    Artaza, Jorge N; Reisz-Porszasz, Suzanne; Dow, Joan S; Kloner, Robert A; Tsao, James; Bhasin, Shalender; Gonzalez-Cadavid, Nestor F

    2007-07-01

    Myostatin (Mst) is a negative regulator of skeletal muscle in humans and animals. It is moderately expressed in the heart of sheep and cattle, increasing considerably after infarction. Genetic blockade of Mst expression increases cardiomyocyte growth. We determined whether Mst overexpression in the heart of transgenic mice reduces left ventricular size and function, and inhibits in vitro cardiomyocyte proliferation. Young transgenic mice overexpressing Mst in the heart (Mst transgenic mice (TG) under a muscle creatine kinase (MCK) promoter active in cardiac and skeletal muscle, and Mst knockout (Mst (-/-)) mice were used. Xiscan angiography revealed that the left ventricular ejection fraction did not differ between the Mst TG and the Mst (-/-) mice, when compared with their respective wild-type strains, despite the decrease in whole heart and left ventricular size in Mst TG mice, and their increase in Mst (-/-) animals. The expected changes in cardiac Mst were measured by RT-PCR and western blot. Mst and its receptor (ActRIIb) were detected by RT-PCR in rat H9c2 cardiomyocytes. Transfection of H9c2 with plasmids expressing Mst under muscle-specific creatine kinase promoter, or cytomegalovirus promoter, enhanced p21 and reduced cdk2 expression, when assessed by western blot. A decrease in cell number occurred by incubation with recombinant Mst (formazan assay), without affecting apoptosis or cardiomyocyte size. Anti-Mst antibody increased cardiomyocyte replication, whereas transfection with the Mst-expressing plasmids inhibited it. In conclusion, Mst does not affect cardiac systolic function in mice overexpressing or lacking the active protein, but it reduces cardiac mass and cardiomyocyte proliferation.

  6. Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression.

    PubMed

    Hennebry, Alex; Berry, Carole; Siriett, Victoria; O'Callaghan, Paul; Chau, Linda; Watson, Trevor; Sharma, Mridula; Kambadur, Ravi

    2009-03-01

    Myostatin (Mstn) is a secreted growth factor belonging to the tranforming growth factor (TGF)-beta superfamily. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fiber formation in the biceps femoris (BF) muscle. Using Mstn null ((-/-)) mice, we suggest a possible mechanism behind Mstn-mediated fiber-type diversity. Histological analysis revealed increased type IIB fibers with a concomitant decrease in type IIA and type I fibers in the Mstn(-/-) tibialis anterior and BF muscle. Functional electrical stimulation of Mstn(-/-) BF revealed increased fatigue susceptibility, supporting increased type IIB fiber content. Given the role of myocyte enhancer factor 2 (MEF2) in oxidative type I fiber formation, MEF2 levels in Mstn(-/-) tissue were quantified. Results revealed reduced MEF2C protein in Mstn(-/-) muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in electrophoretic mobility-shift assay using Mstn(-/-) nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn(-/-) muscle. Conversely, Mstn addition was sufficient to directly upregulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibers, we analyzed MyoD levels in the muscle. In contrast to MEF2C, MyoD levels were increased in Mstn(-/-) muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn could regulate fiber-type composition by regulating the expression of MEF2C and MyoD during myogenesis.

  7. 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.

  8. Genetic variations in the myostatin gene (MSTN) in New Zealand sheep breeds.

    PubMed

    Han, J; Forrest, R H; Hickford, J G H

    2013-11-01

    Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5'UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3'UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.

  9. Early and Late Loss of the Cytoskeletal Scaffolding Protein, Ankyrin G Reveals its Role in Maturation and Maintenance of Nodes of Ranvier in Myelinated Axons.

    PubMed

    Saifetiarova, Julia; Taylor, Anna M; Bhat, Manzoor A

    2017-02-01

    The mechanisms that govern node of Ranvier organization, stability and long-term maintenance remain to be fully elucidated. One of the molecular components of the node is the cytoskeletal scaffolding protein, Ankyrin G (AnkG), which interacts with multiple members of the nodal complex. The role of AnkG in nodal organization and maintenance is still not clearly defined, as to whether AnkG functions as an initial nodal organizer or whether it functions as a nodal stabilizer after the nodal complex has been assembled. Using a mouse model system, we report here that perinatal and juvenile neuronal ablation of AnkG has differential consequences on nodal stability. Early loss of AnkG creates immature nodes with abnormal morphology, which undergo accelerated destabilization within a month, resulting in rapid NaV channel and βIV Spectrin loss with reduced effects on Neurofascin 186. On the other hand, late ablation of AnkG from established nodal complexes leads to slow but progressive nodal destabilization over 10 months, primarily affecting βIV Spectrin, followed by NaV channels, with modest impact on Neurofascin 186. We also show that Ankyrin R and βI Spectrin are not sufficient to prevent nodal disorganization after AnkG ablation. Additionally, nodal disorganization in both early and late AnkG mutants is accompanied by axonal pathology and neurological dysfunction. Together, our results suggest that AnkG plays an indispensable role in maturation and long-term stabilization of the newly assembled nodal complex, and that loss of AnkG after nodal stabilization does not lead to rapid nodal disassembly but loss of specific nodal components in a time-dependent manner.

  10. GASP/WFIKKN Proteins: Evolutionary Aspects of Their Functions

    PubMed Central

    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

  11. Impact of resistance loading on myostatin expression and cell cycle regulation in young and older men and women.

    PubMed

    Kim, Jeong-su; Cross, James M; Bamman, Marcas M

    2005-06-01

    Myostatin inhibits myoblast proliferation and differentiation in developing muscle. Mounting evidence suggests that myostatin also plays a limiting role in growth/repair/regeneration of differentiated adult muscle by inhibiting satellite cell activation. We tested the hypothesis that myostatin mRNA expression would decrease after resistance loading (RL) with a blunted response in older (O) females (F) who have shown minimal hypertrophy [vs. males (M)] after long-term RL. As myostatin is thought to modulate cell cycle activity, we also studied the response of gene transcripts key to stimulation (cyclin B1 and D1) and inhibition (p21cip and p27kip) of the cell cycle, along with the muscle-specific load-sensitive mitogen mechano-growth factor (MGF). Twenty young (Y; 20-35 yr, 10 YF, 10 YM) and 18 O (60-75 yr, 9 OF, 9 OM) consented to vastus lateralis biopsy before and 24 h after a bout of RL (3 sets x 8-12 repetitions to volitional fatigue of squat, leg press, knee extension). Gene expression levels were determined by relative RT-PCR with 18S as an internal standard and analyzed by age x gender x load repeated-measures ANOVA. A load effect was found for four transcripts (P < 0.005) including myostatin, cyclin D1, p27kip, and MGF as mRNA levels decreased for myostatin (-44%) and p27kip (-16%) and increased for cyclin D1 (34%) and MGF (49%). For myostatin, age x load and gender x load interactions (P < 0.05) were driven by a lack of change in OF, while marked declines were noted in YM (-56%), YF (-48%), and OM (-40%). Higher cyclin D1 levels in OF led to a main age effect (36%, O > Y) and an age x gender interaction (66%, OF > YF vs. 10%, OM > YM; P < 0.05). An age x gender x load interaction (P < 0.05) for cyclin D1 resulted from a 48% increase in OF. Post hoc testing within groups revealed a significant increase in MGF after RL in YM only (91%, P < 0.05). Higher levels of cyclin B1 in O (27%, O > Y) led to a main age effect (P < 0.05). An age x load interaction for

  12. 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

  13. Activation of Natural Killer T Cells by α-Galactosylceramide Rapidly Induces the Full Maturation of Dendritic Cells In Vivo and Thereby Acts as an Adjuvant for Combined CD4 and CD8 T Cell Immunity to a Coadministered Protein

    PubMed Central

    Fujii, Shin-ichiro; Shimizu, Kanako; Smith, Caroline; Bonifaz, Laura; Steinman, Ralph M.

    2003-01-01

    The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of α-galactosylceramide (αGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-γ production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by αGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of αGalCer, mice were given αGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-γ producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, αGalCer, NKT, or NK cells. Therefore a single dose of αGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein. PMID:12874260

  14. Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myostatin.

    PubMed

    Guardiola, Ombretta; Lafuste, Peggy; Brunelli, Silvia; Iaconis, Salvatore; Touvier, Thierry; Mourikis, Philippos; De Bock, Katrien; Lonardo, Enza; Andolfi, Gennaro; Bouché, Ann; Liguori, Giovanna L; Shen, Michael M; Tajbakhsh, Shahragim; Cossu, Giulio; Carmeliet, Peter; Minchiotti, Gabriella

    2012-11-20

    Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-β ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine.

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

    PubMed Central

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

    2016-01-01

    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

  16. Application of myostatin in sheep breeding programs: A review

    PubMed Central

    Miar, Younes; Salehi, Abdolreza; Kolbehdari, Davood; Aleyasin, Seyed Ahmad

    2014-01-01

    Plasma membrane H+-ATPase is a major integral membrane protein with a role in various physiological processes including abiotic stress response. To study the effect of NaCl on the expression pattern of a gene encoding the plasma membrane H+-ATPase, an experiment was carried out in a completely random design with three replications. A pair of specific primers was designed based on the sequence of the gene encoding plasma membrane H+-ATPase in Aeluropus littoralis to amplify a 259 bp fragment from the target gene by PCR. A gene encoding actin was used as reference gene to normalize the expression level of the target gene. A pair of specific primers was designed to amplify a 157 bp fragment from the actin gene by PCR. Plants were treated with different concentrations of NaCl, 0, 50, 100, 150, 200, 250, 500 and 1000 mM, for two days. Our results showed that the expression level of the plasma membrane H+-ATPase gene increased dramatically at 500 mM and then decreased with increasing concentrations of NaCl. The results also indicated that the leaves of plants, were treated with high concentrations of NaCl changed morphologically, but those grown under low concentrations of NaCl as well as the control plants did not show morphological changes in their leaves. Our results suggest a relation between morphological changes of treated plants and the expression level of the plasma membrane H+-ATPase gene in Aeluropus littoralis. PMID:27843975

  17. Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN-1 and -2.

    PubMed

    Ostbye, Tone-Kari K; Wetten, Ola F; Tooming-Klunderud, Ave; Jakobsen, Kjetill S; Yafe, Anat; Etzioni, Shulamit; Moen, Thomas; Andersen, Oivind

    2007-11-15

    Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple

  18. 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

  19. Muscle expressions of MGF, IGF-IEa, and myostatin in intact and hypophysectomized rats: effects of rhGH and testosterone alone or combined.

    PubMed

    Rigamonti, A E; Locatelli, L; Cella, S G; Bonomo, S M; Giunta, M; Molinari, F; Sartorio, A; Müller, E E

    2009-01-01

    Myostatin and mechano-growth factor (MGF), an isoform of insulin-like growth factor-I (IGF-I), are two important regulators of muscle hypertrophy. The aim of the present study was to investigate the effects of recombinant human growth hormone (rhGH) and/or testosterone on muscle MGF/IGF-IEa/myostatin expression in intact and hypophysectomized rats treated for 15 d with 1) saline or rhGH, 2) sesame oil or testosterone, 3) saline+sesame oil, or rhGH+testosterone (first experiment) or for 7 d with saline or rhGH (second experiment). Animals were killed by decapitation 24 h or 4 d after the last injection (first or second experiment, respectively). Muscle expressions of MGF, IGF-IEa, and myostatin were determined by RT-PCR. A significant increase in the weight of gastrocnemius muscle was observed only in hypophysectomized rats treated with rhGH alone or in combination with testosterone. Administration of rhGH to hypophysectomized rats caused a marked increase in both MGF and IGF-IEa muscle mRNA levels (without any change in the muscle expression of myostatin), an effect that was abolished when testosterone was combined with rhGH. Conversely, in intact rats rhGH increased myostatin muscle mRNA levels without affecting those of MGF and IGF-IEa. Testosterone, alone or combined with rhGH, induced an inhibition of myostatin expression in the muscle of intact rats, but did not change muscle paradigms of hypophysectomized rats. In conclusion, rhGH and/or testosterone anabolic effects in the muscle are mediated by a different expression of MGF/IGF-IEa/myostatin, which is related to the pituitary function.

  20. The role of myostatin and activin receptor IIB in the regulation of unloading-induced myofiber type-specific skeletal muscle atrophy.

    PubMed

    Babcock, Lyle W; Knoblauch, Mark; Clarke, Mark S F

    2015-09-15

    Chronic unloading induces decrements in muscle size and strength. This adaptation is governed by a number of molecular factors including myostatin, a potent negative regulator of muscle mass. Myostatin must first be secreted into the circulation and then bind to the membrane-bound activin receptor IIB (actRIIB) to exert its atrophic action. Therefore, we hypothesized that myofiber type-specific atrophy observed after hindlimb suspension (HLS) would be related to myofiber type-specific expression of myostatin and/or actRIIB. Wistar rats underwent HLS for 10 days, after which the tibialis anterior was harvested for frozen cross sectioning. Simultaneous multichannel immunofluorescent staining combined with differential interference contrast imaging was employed to analyze myofiber type-specific expression of myostatin and actRIIB and myofiber type cross-sectional area (CSA) across fiber types, myonuclei, and satellite cells. Hindlimb suspension (HLS) induced significant myofiber type-specific atrophy in myosin heavy chain (MHC) IIx (P < 0.05) and MHC IIb myofibers (P < 0.05). Myostatin staining associated with myonuclei was less in HLS rats compared with controls, while satellite cell staining for myostatin remained unchanged. In contrast, the total number myonuclei and satellite cells per myofiber was reduced in HLS compared with ambulatory control rats (P < 0.01). Sarcoplasmic actRIIB staining differed between myofiber types (I < IIa < IIx < IIb) independent of loading conditions. Myofiber types exhibiting the greatest cytoplasmic staining of actRIIB corresponded to those exhibiting the greatest degree of atrophy following HLS. Our data suggest that differential expression of actRIIB may be responsible for myostatin-induced myofiber type-selective atrophy observed during chronic unloading.

  1. Influenza Virus–induced Dendritic Cell Maturation Is Associated with the Induction of Strong T Cell Immunity to a Coadministered, Normally Nonimmunogenic Protein

    PubMed Central

    Brimnes, Marie K.; Bonifaz, Laura; Steinman, Ralph M.; Moran, Thomas M.

    2003-01-01

    We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-γ, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-γ. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2–3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently. PMID:12847140

  2. 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…

  3. Frequent sequence variation in the human myostatin (GDF8) gene as a marker for analysis of muscle-related phenotypes.

    PubMed

    Ferrell, R E; Conte, V; Lawrence, E C; Roth, S M; Hagberg, J M; Hurley, B F

    1999-12-01

    Myostatin is a recently identified member of the transforming growth factor-beta family of regulatory factors, also known as growth and differentiation factor 8 (GDF8). The nucleotide sequence of human myostatin was determined in 40 individuals. The invariant promoter contains a consensus MyoD binding site, and the coding sequence contains five missense substitutions in conserved amino acid residues (A55T, K153R, E164K, P198A, and I225T). Two of these, A55T in exon 1 and K153R in exon 2, are polymorphic in the general population with significantly different allele frequencies in Caucasians and African Americans (P < 0.001). Neither of the common polymorphisms had a significant impact on muscle mass response to strength training in either Caucasians or African Americans, although skewed allele frequencies preclude detection of small effects. These allelic variants provide markers for examining association between the myostatin gene and interindividual variation in muscle mass and differences in loss of muscle mass with aging.

  4. Structural and functional analysis of myostatin-2 promoter alleles from the marine fish Sparus aurata: evidence for strong muscle-specific promoter activity and post-transcriptional regulation.

    PubMed

    Nadjar-Boger, Elisabeth; Hinits, Yaniv; Funkenstein, Bruria

    2012-09-25

    Myostatin (MSTN) is a negative regulator of skeletal muscle growth. In contrast to mammals, fish possess at least two paralogs of MSTN: MSTN-1 and MSTN-2. In this study, we analyzed the structural-functional features of the four variants of Sparus aurata MSTN-2 5'-flanking region: saMSTN-2a, saMSTN-2as, saMSTN-2b and saMSTN-2c. In silico analysis revealed numerous putative cis regulatory elements including several E-boxes known as binding sites to myogenic transcription factors. Transient transfection experiments using non-muscle and muscle cell lines showed surprisingly high transcriptional activity in muscle cells, suggesting the presence of regulatory elements unique to differentiated myotubes. These observations were confirmed by in situ intramuscular injections of promoter DNA followed by reporter gene assays. Moreover, high promoter activity was found in differentiated neural cell, in agreement with MSTN-2 expression in brain. Progressive 5'-deletion analysis, using reporter gene assays, showed that the core promoter is located within the first -127 bp upstream of the ATG, and suggested the presence of regulatory elements that either repress or induce transcriptional activity. Transient transgenic zebrafish provided evidence for saMSTN-2 promoter ability to direct GFP expression to myofibers. Finally, our data shows that although no mature saMSTN-2 mRNA is observed in muscle; unspliced forms accumulate, confirming high level of transcription. In conclusion, our study shows for the first time that MSTN-2 promoter is a very robust promoter, especially in muscle cells.

  5. 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

    2015-04-09

    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.

  6. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    PubMed Central

    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

  7. Analysis of the 227 bp short interspersed nuclear element (SINE) insertion of the promoter of the myostatin (MSTN) gene in different horse breeds.

    PubMed

    Dall'Olio, Stefania; Scotti, Emilio; Fontanesi, Luca; Tassinari, Marco

    2014-01-01

    The myostatin (MSTN) gene encodes a protein known to be a negative regulator of muscle mass in mammalian species. Different polymorphisms of the horse (Equus caballus) MSTN gene have been identified, including single nucleotide polymorphisms and a short interspersed nuclear element (SINE) insertion of 227 bp within the promoter of the gene. The SINE insertion has been associated with performance traits in Thoroughbred racehorses and it was proposed as a predictor of optimum racing distance. The aims of this study were to perform in silico analysis to identify putative gains or abrogation of transcription-factor binding sites (TFBSs) generated by the SINE allele of the promoter and to analyse the frequency of the SINE insertion in horses used for racing (gallop and trot) and other purposes. The SINE insertion was genotyped in 227 horses from 10 breeds belonging to different morphological types (brachimorphic, mesomorphic, meso-dolichomorphic and dolichomorphic). The presence of the insertion was confirmed in the Quarter Horse (SINE allele frequency of 0.81) and in the Thoroughbred (0.51), whereas the SINE allele did not segregate in any of the other analysed breeds. As the SINE MSTN gene polymorphism may be population or breed specific, it is not a useful marker for association studies in all breeds.

  8. The effect of eight weeks resistance and aerobic training on myostatin and follistatin expression in cardiac muscle of rats

    PubMed Central

    Rashidlamir, Amir; Attarzadeh Hosseini, Seyyed Reza; Hejazi, Keyvan; Motevalli Anberani, Seyyed Mohamad

    2016-01-01

    Introduction: The clinical studies have shown that the myostatin gene expression and its serum density occur more frequently in heart patients than in healthy individuals. The purpose of this study is to investigate the influence of 8-week resistance and aerobic exercise on the myostatin and follistatin gene expression of myocardium muscle of healthy male Wistar rats. Methods: In this experimental study, 20 five-week-old adult Wistar rats (250 ± 26.5 g) were divided into three groups: healthy control group (n = 6), resistance exercise group (n = 7), and aerobic exercise group (n = 7). The resistance and aerobic exercise plan consisted of 8 weeks and 3 sessions per week. The resistance exercise group performed climbing a one-meter 26-stair ladder with a slope of 85 degrees for 3 sets of 5 repetitions per session. The aerobic exercise group performed running at a speed of 12 meters per minute for 30 minutes during the first sessions gradually increasing up to a speed of 30 meters per minute for 60 minutes during the final sessions (equivalent to 70% to 80% of maximum oxygen consumption). The differences between the groups were evaluated using a one-way analysis of variance (ANOVA) test. When appropriate, LSD post-hoc test was used. The significance level for the study was less than 0.05. Results: The results of this study shows that after 8 weeks of exercise, there is no significant difference between myostatin mRNA gene expression levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise (P = 0.172, F = 1.953). However, the mean differences between follistatin mRNA levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise are statistically significant (F = 38.022, P = 0.001). Furthermore, the ratio of follistatin to myostatin mRNA gene expression of the heart muscle (P = 0.001, F = 10.288) shows significant difference among the three groups. Conclusion: Our results indicate

  9. The effect of eight weeks resistance and aerobic training on myostatin and follistatin expression in cardiac muscle of rats.

    PubMed

    Rashidlamir, Amir; Attarzadeh Hosseini, Seyyed Reza; Hejazi, Keyvan; Motevalli Anberani, Seyyed Mohamad

    2016-01-01

    Introduction: The clinical studies have shown that the myostatin gene expression and its serum density occur more frequently in heart patients than in healthy individuals. The purpose of this study is to investigate the influence of 8-week resistance and aerobic exercise on the myostatin and follistatin gene expression of myocardium muscle of healthy male Wistar rats. Methods: In this experimental study, 20 five-week-old adult Wistar rats (250 ± 26.5 g) were divided into three groups: healthy control group (n = 6), resistance exercise group (n = 7), and aerobic exercise group (n = 7). The resistance and aerobic exercise plan consisted of 8 weeks and 3 sessions per week. The resistance exercise group performed climbing a one-meter 26-stair ladder with a slope of 85 degrees for 3 sets of 5 repetitions per session. The aerobic exercise group performed running at a speed of 12 meters per minute for 30 minutes during the first sessions gradually increasing up to a speed of 30 meters per minute for 60 minutes during the final sessions (equivalent to 70% to 80% of maximum oxygen consumption). The differences between the groups were evaluated using a one-way analysis of variance (ANOVA) test. When appropriate, LSD post-hoc test was used. The significance level for the study was less than 0.05. Results: The results of this study shows that after 8 weeks of exercise, there is no significant difference between myostatin mRNA gene expression levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise (P = 0.172, F = 1.953). However, the mean differences between follistatin mRNA levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise are statistically significant (F = 38.022, P = 0.001). Furthermore, the ratio of follistatin to myostatin mRNA gene expression of the heart muscle (P = 0.001, F = 10.288) shows significant difference among the three groups. Conclusion: Our results indicate

  10. Structure, function and dynamics in adenovirus maturation.

    PubMed

    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. Finally, possible roles for maturation of the terminal protein are discussed.

  11. Structure, function and dynamics in adenovirus maturation

    DOE PAGES

    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

  12. 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

  13. 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.

  14. Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy.

    PubMed

    Liu, He-he; Wang, Ji-wen; Yu, Hai-yue; Zhang, Rong-ping; Chen, Xi; Jin, Hai-bo; Dai, Fei; Li, Liang; Xu, Feng

    2012-06-01

    Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.

  15. A pre-microRNA-149 (miR-149) genetic variation affects miR-149 maturation and its ability to regulate the Puma protein in apoptosis.

    PubMed

    Ding, Su-Ling; Wang, Jian-Xun; Jiao, Jian-Qin; Tu, Xin; Wang, Qing; Liu, Fang; Li, Qian; Gao, Jie; Zhou, Qun-Yong; Gu, Dong-Feng; Li, Pei-Feng

    2013-09-13

    MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs that function as negative regulators of gene expression. They are transcribed from endogenous DNA and form hairpin structures (termed as pre-miRNAs) that are processed to form mature miRNAs. It remains largely unknown as to the molecular consequences of the natural genetic variation in pre-miRNAs. Here, we report that an A→G polymorphism (rs71428439) is located in Homo sapiens miR-149 stem-loop region. This polymorphism results in a change in the structure of the miR-149 precursor. Our results showed that the genotype distribution of this polymorphism in myocardial infarction cases was significantly different from that in the control subjects. We examined the biological significance of this polymorphism on the production of mature miR-149, and we observed that the G-allelic miR-149 precursor displayed a lower production of mature miR-149 compared with the A-allelic one. Further investigations disclosed that miR-149 could withstand mitochondrial fission and apoptosis through targeting the pro-apoptotic factor p53-up-regulated modulator of apoptosis (Puma). Enforced expression of miR-149 promoted cell survival, whereas knockdown of miR-149 rendered cells to be sensitive to apoptotic stimulation. Intriguingly, the A to G variation led pre-miR-149 to elicit an attenuated effect on the inhibition of mitochondrial fission and apoptosis. Finally, this polymorphism exerts its influence on cardiac function in the mouse model of myocardial infarction. These data suggest that this polymorphism in the miR-149 precursor may result in important phenotypic traits of myocardial infarction. Our findings warrant further investigations on the relationship between miR-149 polymorphism and myocardial infarction.

  16. The novel gene CpEdi-9 from the resurrection plant C. plantagineum encodes a hydrophilic protein and is expressed in mature seeds as well as in response to dehydration in leaf phloem tissues.

    PubMed

    Rodrigo, Maria Jesus; Bockel, Christine; Blervacq, Anne-Sophie; Bartels, Dorothea

    2004-08-01

    The resurrection plant Craterostigma plantagineum Hochst. is used as an experimental system to investigate desiccation tolerance in higher plants. A search for genes activated during early stages of dehydration identified the gene CpEdi-9, which is expressed in mature seeds and in response to dehydration in the phloem cells of vascular tissues of leaves. Elements for the tissue-specific expression pattern reside in the isolated promoter of the CpEdi-9 gene, as shown through the analysis of transgenic plants. The CpEdi-9 promoter could be a suitable tool for expressing genes in the vascular system of dehydrated plants. CpEdi-9 encodes a small (10 kDa) hydrophilic protein, which does not have significant sequence homologies to known genes. The predicted protein CpEDI-9 shares some physicochemical features with LEA proteins from plants and a nematode. Based on the unique expression pattern and on the nucleotide sequence we propose that CpEdi-9 defines a new class of hydrophilic proteins that are supposed to contribute to cellular protection during dehydration. This group of proteins may have evolved because desiccation tolerance requires the abundant expression of protective proteins during early stages of dehydration in all tissues.

  17. Short-term strength training and the expression of myostatin and IGF-I isoforms in rat muscle and tendon: differential effects of specific contraction types.

    PubMed

    Heinemeier, K M; Olesen, J L; Schjerling, P; Haddad, F; Langberg, H; Baldwin, K M; Kjaer, M

    2007-02-01

    In skeletal muscle, an increased expression of insulin like growth factor-I isoforms IGF-IEa and mechano-growth factor (MGF) combined with downregulation of myostatin is thought to be essential for training-induced hypertrophy. However, the specific effects of different contraction types on regulation of these factors in muscle are still unclear, and in tendon the functions of myostatin, IGF-IEa, and MGF in relation to training are unknown. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric, or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve during general anesthesia. mRNA levels for myostatin, IGF-IEa, and MGF in muscle and Achilles' tendon were measured by real-time RT-PCR. Muscle myostatin mRNA decreased in response to all types of training (2- to 8-fold) (P < 0.05), but the effect of eccentric training was greater than concentric and isometric training (P < 0.05). In tendon, myostatin mRNA was detected, but no changes were seen after exercise. IGF-IEa and MGF increased in muscle (up to 15-fold) and tendon (up to 4-fold) in response to training (P < 0.01). In tendon no difference was seen between training types, but in muscle the effect of eccentric training was greater than concentric training for both IGF-IEa and MGF (P < 0.05), and for IGF-IEa isometric training had greater effect than concentric (P < 0.05). The results indicate a possible role for IGF-IEa and MGF in adaptation of tendon to training, and the combined changes in myostatin and IGF-IEa/MGF expression could explain the important effect of eccentric actions for muscle hypertrophy.

  18. Krüppel-like Factor 11 Regulates the Expression of Metabolic Genes via an Evolutionarily Conserved Protein Interaction Domain Functionally Disrupted in Maturity Onset Diabetes of the Young*

    PubMed Central

    Lomberk, Gwen; Grzenda, Adrienne; Mathison, Angela; Escande, Carlos; Zhang, Jin-San; Calvo, Ezequiel; Miller, Laurence J.; Iovanna, Juan; Chini, Eduardo N.; Fernandez-Zapico, Martin E.; Urrutia, Raul

    2013-01-01

    The function of Krüppel-like factor 11 (KLF11) in the regulation of metabolic pathways is conserved from flies to human. Alterations in KLF11 function result in maturity onset diabetes of the young 7 (MODY7) and neonatal diabetes; however, the mechanisms underlying the role of this protein in metabolic disorders remain unclear. Here, we investigated how the A347S genetic variant, present in MODY7 patients, modulates KLF11 transcriptional activity. A347S affects a previously identified transcriptional regulatory domain 3 (TRD3) for which co-regulators remain unknown. Structure-oriented sequence analyses described here predicted that the KLF11 TRD3 represents an evolutionarily conserved protein domain. Combined yeast two-hybrid and protein array experiments demonstrated that the TRD3 binds WD40, WWI, WWII, and SH3 domain-containing proteins. Using one of these proteins as a model, guanine nucleotide-binding protein β2 (Gβ2), we investigated the functional consequences of KLF11 coupling to a TRD3 binding partner. Combined immunoprecipitation and biomolecular fluorescence complementation assays confirmed that activation of three different metabolic G protein-coupled receptors (β-adrenergic, secretin, and cholecystokinin) induces translocation of Gβ2 to the nucleus where it directly binds KLF11 in a manner that is disrupted by the MODY7 A347S variant. Using genome-wide expression profiles, we identified metabolic gene networks impacted upon TRD3 disruption. Furthermore, A347S disrupted KLF11-mediated increases in basal insulin levels and promoter activity and blunted glucose-stimulated insulin secretion. Thus, this study characterizes a novel protein/protein interaction domain disrupted in a KLF gene variant that associates to MODY7, contributing to our understanding of gene regulation events in complex metabolic diseases. PMID:23589285

  19. Krüppel-like factor 11 regulates the expression of metabolic genes via an evolutionarily conserved protein interaction domain functionally disrupted in maturity onset diabetes of the young.

    PubMed

    Lomberk, Gwen; Grzenda, Adrienne; Mathison, Angela; Escande, Carlos; Zhang, Jin-San; Calvo, Ezequiel; Miller, Laurence J; Iovanna, Juan; Chini, Eduardo N; Fernandez-Zapico, Martin E; Urrutia, Raul

    2013-06-14

    The function of Krüppel-like factor 11 (KLF11) in the regulation of metabolic pathways is conserved from flies to human. Alterations in KLF11 function result in maturity onset diabetes of the young 7 (MODY7) and neonatal diabetes; however, the mechanisms underlying the role of this protein in metabolic disorders remain unclear. Here, we investigated how the A347S genetic variant, present in MODY7 patients, modulates KLF11 transcriptional activity. A347S affects a previously identified transcriptional regulatory domain 3 (TRD3) for which co-regulators remain unknown. Structure-oriented sequence analyses described here predicted that the KLF11 TRD3 represents an evolutionarily conserved protein domain. Combined yeast two-hybrid and protein array experiments demonstrated that the TRD3 binds WD40, WWI, WWII, and SH3 domain-containing proteins. Using one of these proteins as a model, guanine nucleotide-binding protein β2 (Gβ2), we investigated the functional consequences of KLF11 coupling to a TRD3 binding partner. Combined immunoprecipitation and biomolecular fluorescence complementation assays confirmed that activation of three different metabolic G protein-coupled receptors (β-adrenergic, secretin, and cholecystokinin) induces translocation of Gβ2 to the nucleus where it directly binds KLF11 in a manner that is disrupted by the MODY7 A347S variant. Using genome-wide expression profiles, we identified metabolic gene networks impacted upon TRD3 disruption. Furthermore, A347S disrupted KLF11-mediated increases in basal insulin levels and promoter activity and blunted glucose-stimulated insulin secretion. Thus, this study characterizes a novel protein/protein interaction domain disrupted in a KLF gene variant that associates to MODY7, contributing to our understanding of gene regulation events in complex metabolic diseases.

  20. The TadV Protein of Actinobacillus actinomycetemcomitans Is a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins†

    PubMed Central

    Tomich, Mladen; Fine, Daniel H.; Figurski, David H.

    2006-01-01

    The tad locus of Actinobacillus actinomycetemcomitans encodes genes for the biogenesis of Flp pili, which allow the bacterium to adhere tenaciously to surfaces and form strong biofilms. Although tad (tight adherence) loci are widespread among bacterial and archaeal species, very little is known about the functions of the individual components of the Tad secretion apparatus. Here we characterize the mechanism by which the pre-Flp1 prepilin is processed to the mature pilus subunit. We demonstrate that the tadV gene encodes a prepilin peptidase that is both necessary and sufficient for proteolytic maturation of Flp1. TadV was also found to be required for maturation of the TadE and TadF pilin-like proteins, which we term pseudopilins. Using site-directed mutagenesis, we show that processing of pre-Flp1, pre-TadE, and pre-TadF is required for biofilm formation. Mutation of a highly conserved glutamic acid residue at position +5 of Flp1, relative to the cleavage site, resulted in a processed pilin that was blocked in assembly. In contrast, identical mutations in TadE or TadF had no effect on biofilm formation, indicating that the mechanisms by which Flp1 pilin and the pseudopilins function are distinct. We also determined that two conserved aspartic acid residues in TadV are critical for function of the prepilin peptidase. Together, our results indicate that the A. actinomycetemcomitans TadV protein is a member of a novel subclass of nonmethylating aspartic acid prepilin peptidases. PMID:16980493

  1. 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

  2. Characterization and functional analysis of the 5' flanking region of Sparus aurata myostatin-1 gene.

    PubMed

    Funkenstein, Bruria; Balas, Viki; Rebhan, Yanai; Pliatner, Anna

    2009-05-01

    Myostatin (MSTN) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Although several MSTN promoters were described in fish, no functional analysis was reported so far. Here, the 5' flanking region (1372 bp) of the MSTN-1 gene of the marine fish Sparus aurata (saMSTN-1) was cloned, sequenced and characterized. It contains two consensus sequences for TATA box (TATAA), a CAAT box, ten putative E-boxes known as binding sites to myogenic basic helix-loop-helix transcription factors (TFs) and two putative binding sites to TF Myocyte enhancing factor-2 (MEF2). In addition, it has several putative binding sites to TF Pit-1a and several response elements to nuclear receptors (GRE, ERE, PRE, ARE, TRE, RARE and PPARRE) and cAMP-response elements. Transcriptional activity of five genomic fragments (truncated at their upstream region) of 372, 941, 972, 1113 and 1355 bp was studied in vitro, using transient transfection in A204 cells. All constructs directed luciferase activity, with the highest activity obtained by the 1113 bp fragment. These experiments show that all five genomic fragments are functional MSTN promoters and differences in promoter activity might be due to presence of enhancers and/or repressor sequences, regulating MSTN promoter activity.

  3. [Analysis on single nucleotide polymorphisms of porcine myostatin gene in different breeds].

    PubMed

    Jiang, Y L; Li, N; Wu, C X; Du, L X

    2001-01-01

    By PCR-RFLPs and PCR-SSCP approach, three single nucleotide polymorphisms (SNPs) of porcine myostatin gene (MSTN) were analyzed in different breeds including "doubled-muscled" Yorkshire, Yorkshire, Landrace, Hamshire, Duroc, Piteran, Erhualian, Min, Hubei White and some hybrids. The three SNPs were located in the 3' encoding region, 5' promoter region and intronl region respectively. For the SNP in the 3' encoding region, which was caused by C-->T transition, the mutation frequency was relatively low: no TT genotype was detected in 274 individuals of different breeds. For the SNP in the 5' promoter region, 560 pigs were investigated. The allele T dominates in the imported lean-type pig breeds such as Yorkshire, Landrace, Duroc, Hampshire, Piteran and hybrid, however, in Erhualian and Hubei White pigs, the allele A was in majority. Polymorphism showed the similar pattern for the SNP in intron 1 region. G was the dominant allele in Yorkshire, Landrace and their hybrids, while in Erhualian and Hubei White pigs the frequency of A was much higher. Obviously they were not in Hardy-Weinberg equilibrium state. For Min and Yorshire x Erhualian pigs, they were in Hardy-Weinberg equilibrium state for the SNPs in the 5' promoter region and (or) intron 1 region. The frequency for the A alleles of SNPs in the 5' promoter region and intron 1 region was higher for "double-muscled" Yorkshire than for Yorkshire and linkage for these two mutation sites was also observed.

  4. Membrane remodeling during reticulocyte maturation

    PubMed Central

    Liu, Jing; Guo, Xinhua; Mohandas, Narla; Chasis, Joel A.

    2010-01-01

    The transition of reticulocytes into erythrocytes is accompanied by extensive changes in the structure and properties of the plasma membrane. These changes include an increase in shear resistance, loss of surface area, and acquisition of a biconcave shape. The processes by which these changes are effected have remained largely undefined. Here we examine how the expression of 30 distinct membrane proteins and their interactions change during murine reticulocyte maturation. We show that tubulin and cytosolic actin are lost, whereas the membrane content of myosin, tropomyosin, intercellular adhesion molecule-4, glucose transporter-4, Na-K-ATPase, sodium/hydrogen exchanger 1, glycophorin A, CD47, Duffy, and Kell is reduced. The degradation of tubulin and actin is, at least in part, through the ubiquitin-proteasome degradation pathway. In regard to the protein-protein interactions, the formation of membrane-associated spectrin tetramers from dimers is unperturbed, whereas the interactions responsible for the formation of the membrane-skeletal junctions are weaker in reticulocytes, as is the attachment of transmembrane proteins to these structures. This weakness, in part, results from the elevated phosphorylation of 4.1R in reticulocytes, which leads to a decrease in shear resistance by reducing its interaction with spectrin and actin. These observations begin to unravel the mechanistic basis of crucial changes accompanying reticulocyte maturation. PMID:20038785

  5. Myostatin, activin receptor IIb, and follistatin-like-3 gene expression are altered in adipose tissue and skeletal muscle of obese mice.

    PubMed

    Allen, David L; Cleary, Allison S; Speaker, Kristin J; Lindsay, Sarah F; Uyenishi, Jill; Reed, Jason M; Madden, Molly C; Mehan, Ryan S

    2008-05-01

    Myostatin (MSTN) is a secreted growth inhibitor expressed in muscle and adipose. We sought to determine whether expression of MSTN, its receptor activin RIIb (ActRIIb), or its binding protein follistatin-like-3 (FSTL3) are altered in subcutaneous or visceral adipose or in skeletal muscle in response to obesity. MSTN and ActRIIb mRNA levels were low in subcutaneous (SQF) and visceral fat (VF) from wild-type mice but were 50- to 100-fold higher in both SQF and VF from ob/ob compared with wild-type mice. FSTL3 mRNA levels were increased in SQF but decreased in VF in ob/ob compared with wild-type mice. Moreover, MSTN mRNA levels were twofold greater in tibialis anterior (TA) from ob/ob mice, whereas ActRIIb and FSTL3 mRNA levels were unchanged. MSTN mRNA levels were also increased in TA and SQF from mice on a high-fat diet. Injection of ob/ob mice with recombinant leptin caused FSTL3 mRNA levels to decrease in both VF and SQF in ob/ob mice; MSTN and ActRIIb mRNA levels tended to decrease only in VF. Finally, MSTN mRNA levels and promoter activity were low in adipogenic 3T3-L1 cells, but an MSTN promoter-reporter construct was activated in 3T3-L1 cells by cotransfection with the adipogenic transcription factors SREBP-1c, C/EBPalpha, and PPARgamma. These results demonstrate that expression of MSTN and its associated binding proteins can be modulated in adipose tissue and skeletal muscle by chronic obesity and suggest that alterations in their expression may contribute to the changes in growth and metabolism of lean and fat tissues occurring during obesity.

  6. Characterization of a myostatin gene (MSTN1) from spotted halibut (Verasper variegatus) and association between its promoter polymorphism and individual growth performance.

    PubMed

    Li, Hongjun; Fan, Jingfeng; Liu, Shuxi; Yang, Qing; Mu, Guiqiang; He, Chongbo

    2012-04-01

    Myostatin (MSTN) is a member of the transforming growth factor-β superfamily which could play an important role in negatively regulating skeletal muscle growth and development in mammal and non-mammal species. In the present study, a MSTN1 gene (designated as VvMSTN1) was cloned and characterized in one flatfish species, spotted halibut (Verasper variegatus). In the 3078 bp genomic sequence, three exons, two introns and a promoter sequence were identified. Sequence analysis of the promoter region revealed that it contained several cis-regulatory elements such as CAAT-box, TATA-box and E-boxes. The deduced protein sequence included a signal peptide, a TGF-β propeptide in the N-terminal region and the TGF-β active peptide in the C-terminal region. Phylogenetic analysis suggested that VvMSTN1 is an orthologue of teleost MSTN1 proteins which arose along with MSTN2 during a duplication event at the base of teleost evolution. Quantitative real-time PCR analysis revealed that VvMSTN1 mRNA was ubiquitously expressed in all nine tested tissues, with the most transcriptionally abundant in skeletal muscle. A primary assessment of sequence variability revealed five single nucleotide polymorphisms (SNPs) existed in the promoter region, among which three (G-653T, T-355C and G-253A) were genotyped with an advanced melting temperature (T(m))-shift method and tested for their association with growth traits (body length, body depth and total mass). Results indicated that genotype CC of locus T-355C had significantly higher growth traits than genotype TC and TT (P<0.05) in female spotted halibut. These results suggest that V. variegatus MSTN could be selected as a candidate gene for the future molecular breeding of stains with enhanced individual growth performance.

  7. [Adolescent brain maturation].

    PubMed

    Holzer, L; Halfon, O; Thoua, V

    2011-05-01

    Recent progress in neuroscience has yielded major findings regarding brain maturation during adolescence. Unlike the body, which reaches adult size and morphology during this period, the adolescent brain is still maturing. The prefrontal cortex appears to be an important locus of maturational change subserving executive functions that may regulate emotional and motivational issues. The recent expansion of the adolescent period has increased the lag between the onset of emotional and motivational changes activated by puberty and the completion of cognitive development-the maturation of self-regulatory capacities and skills that are continuing to develop long after puberty has occurred. This "disconnect" predicts risk for a broad set of behavioral and emotional problems. Adolescence is a critical period for high-level cognitive functions such as socialization that rely on maturation of the prefrontal cortex. Intervention during the period of adolescent brain development provides opportunities and requires an interdisciplinary approach.

  8. The Presence Of Strange Males' Odor Induces Behavioral Responses And Elevated Levels Of Low Molecular Weight Proteins Excreted In The Urine Of Mature Water Vole Males (Arvicola amphibius L).

    PubMed

    Nazarova, Galina G; Proskurniak, Lyudmila P; Yuzhik, Ekaterina I

    2016-03-01

    We hypothesized that low molecular weight urinary proteins play a role in male-male chemical communication in the water vole, Arvicola ampibius L. We studied the effect of placing soiled litter from strange males into the cage of another sexually mature male on the intensity of its digging and scattering, urination on the litter, and alteration in the levels of low molecular weight proteins (15-25 kDa) excreted in the urine before and after 4 days of exposure as determined by chip electrophoresis. The intensity of digging and scattering was positively correlated with levels of testosterone in serum of males exposed to strange male odors (r = 0.56; P < 0.01), as well as with the concentration of low molecular weight proteins in the donor's urine (r = 0.52, P < 0.05). At the end of the experiment, the level of low molecular weight protein in excreted urine was elevated in the males exposed to the strange male's litter. These results highlight the importance of quantitative inter-individual variation of low molecular weight urinary proteins in the modulation of the physiology and behavior of conspecifics.