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1.
Carbohydr Polym ; 272: 118444, 2021 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-34420709

RESUMEN

In this study, a fully aligned microfibrous structure fabricated using fibrin-assisted alginate bioink and electrohydrodynamic direct-printing was proposed for skeletal muscle tissue engineering. To safely construct the aligned alginate/fibrin microfibrous structure laden with myoblasts or endothelial cells, various printing conditions, such as an applied electric field, distance between the nozzle and target, and nozzle moving speed, were selected appropriately. Furthermore, to accelerate the formation of myotubes more efficiently, the alginate/fibrin bioink with vascular endothelial cells was co-printed into a spatially patterned structure within a myoblast-laden structure. The myoblast-laden structure co-cultured with endothelial cells presented fully aligned myotube formation and significantly greater myogenic differentiation compared to the myoblast-laden structure without the endothelial cells owing to the more abundant secretion of angiogenic cytokines. Also, when adipose stem cell- and endothelial cell-laden fibrous structure was implanted in a mouse volumetric muscle loss model, accelerated volumetric muscle repair was observed compared to the defect model. Based on the results, this study demonstrates an alginate-based bioink and new bio-fabricating method to obtain microfibrous cell-laden alginate/fibrin structures with mechanically stable and topographical cues. The proposed method can provide a myoblast/endothelial cell-laden fibrous alginate structure to efficiently induce engineering of skeletal muscle tissue, which could be used in muscle-on-a-chip or recovering structures of volumetric muscle defects.


Asunto(s)
Alginatos/química , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tejido Adiposo/metabolismo , Animales , Bioimpresión/métodos , Diferenciación Celular , Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Femenino , Fibrina/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Tinta , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/química , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/química , Células Madre/metabolismo
2.
J Biomed Mater Res A ; 106(6): 1543-1551, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29368451

RESUMEN

Skeletal muscle has a well-organized tissue structure comprised of aligned myofibers and an encasing extracellular matrix (ECM) sheath or lamina, within which reside satellite cells. We hypothesize that the organization of skeletal muscle tissues in culture can affect both the structure of the deposited ECM and the differentiation potential of developing myotubes. Furthermore, we posit that cellular and ECM cues can be a strong determinant of myoblast fusion and morphology in 3D tissue culture environments. To test these, we utilized a thermoresponsive nanofabricated substratum to engineer anisotropic sheets of myoblasts which could then be transferred and stacked into multilayered tissues. Within such engineered tissues, we found that myoblasts rapidly sense topography and deposit structurally organized ECM proteins. Furthermore, the initial tissue structure was found to exert significant control over myoblast fusion and eventual myotube organization. These results highlight the importance of ECM structure on myoblast fusion and organization, and provide insights into substrate-mediated control of myotube formation in the development of novel, more effective, engineered skeletal muscle tissues. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1543-1551, 2018.


Asunto(s)
Matriz Extracelular/química , Fibras Musculares Esqueléticas/citología , Mioblastos Esqueléticos/citología , Nanoestructuras/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Anisotropía , Adhesión Celular , Diferenciación Celular , Fusión Celular , Línea Celular , Ratones , Desarrollo de Músculos , Fibras Musculares Esqueléticas/química , Mioblastos Esqueléticos/química , Propiedades de Superficie , Temperatura
3.
Glycobiology ; 27(12): 1134-1143, 2017 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-28973355

RESUMEN

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.


Asunto(s)
Glicoproteínas/metabolismo , Proteínas Musculares/metabolismo , Distrofias Musculares/metabolismo , Mioblastos Esqueléticos/metabolismo , Narcissus/química , Lectinas de Plantas/farmacología , Línea Celular Transformada , Glicoproteínas/química , Humanos , Proteínas Musculares/química , Distrofias Musculares/patología , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/patología , Lectinas de Plantas/química
4.
Proc Natl Acad Sci U S A ; 113(8): 2116-21, 2016 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-26858401

RESUMEN

During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.


Asunto(s)
Membrana Celular , Proteínas de la Membrana , Desarrollo de Músculos/fisiología , Proteínas Musculares , Mioblastos Esqueléticos , Animales , Fusión Celular , Línea Celular , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad
5.
J Histochem Cytochem ; 55(6): 607-18, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17312016

RESUMEN

Adult skeletal muscle possesses remarkable regenerative capacity that has conventionally been attributed to the satellite cells. These precursor cells were thought to contain distinct populations with varying myogenic potential. Recently, the identification of multipotent stem cells capable of new myofiber formation has expanded the general view on the muscle regenerative process. Here we examined the characteristics of turkey skeletal muscle-derived cell (MDC) populations that were separated according to their adhesion abilities. We sought to determine whether these abilities could be a potential tool for separating cells with different myogenic commitment. Using the preplate technique, we showed that MDCs display a wide range of adhesion ability, allowing us to isolate a marginal fraction with initial adhesion defect. Methodological investigations revealed that this defect represents an intrinsic and well-established biological feature for these cells. In vitro behavioral and morphological analyses showed that late adherent cells (LACs) share several primitive cell characteristics. Phenotypic assessment indicated that LACs contain early stage myogenic cells and immature progenitors of satellite cells, whereas early adherent cells consist mainly of fully committed precursors. Overall, our findings demonstrate for the first time in an avian model that differential MDC adhesion properties could be used to efficiently purify cells with varying myogenic commitment, including immature progenitor cells. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.


Asunto(s)
Diferenciación Celular , Mioblastos Esqueléticos/citología , Animales , Cadherinas/análisis , Adhesión Celular , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Desmina/análisis , Fibroblastos/citología , Inmunohistoquímica , Microscopía Confocal , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/fisiología , Cadenas Pesadas de Miosina/análisis , Factor de Transcripción PAX7/análisis , Factores de Tiempo , Pavos
6.
Cell Tissue Res ; 327(2): 343-51, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17036228

RESUMEN

The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.


Asunto(s)
Caveolina 3/metabolismo , Desmina/metabolismo , Células Musculares/metabolismo , Desarrollo de Músculos/fisiología , Animales , Caveolas/química , Caveolas/metabolismo , Caveolina 3/análisis , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Desmina/análisis , Inmunoprecipitación , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Células Musculares/química , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/metabolismo , Unión Proteica
7.
Cells Tissues Organs ; 183(2): 87-98, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17053325

RESUMEN

The dystrophin-glycoprotein complex together with the vinculin-talin-integrin complex plays an important role in muscle function; in fact the mutations of their elements lead to diverse forms of muscular dystrophies. The relationship between the elements of dystrophin-glycoprotein complex and vinculin-talin-integrin and the time course of their formation are still not known in detail. In order to better understand this relationship we studied their expression during development in normal human skeletal muscle culture. Using a standardized muscle cell culture procedure, this study was performed to analyze the timing, appearance and the localization of some proteins of the dystrophin-glycoprotein complex and vinculin-talin-integrin complex during cellular proliferation (myoblast) and differentiation (4, 7, 15 and 21 days). The indirect immunofluorescence technique was used and cells were examined using a Meta Zeiss LSM510 confocal laser scanning inverted microscope. We examined the progressive appearance of the following proteins: alpha, beta, gamma, delta-sarcoglycans, beta-dystroglycan, dystrophin, talin, vinculin and integrin isoform alpha7/beta1. Immunofluorescence of these proteins, in satellite cells entering myogenic differentiation, revealed different patterns of localization depending on the time of culture. We showed that nondifferentiated cultures of human myoblasts expressed a perinuclear distribution of all proteins tested. During myoblast differentiation into myotubes (4 days) immunofluorescence gradually increased and was located in the whole cytoplasm. Subsequently, at day 7, a strong and homogeneous cytoplasmic labelling of all proteins was seen. At 15 days the distribution of the proteins was on the membrane. At this time some myotubes displayed a significant degree of precostameric banding pattern. As fusion proceeded at 21 days, the cytodistribution progressively changed and appeared along fibrillar longitudinal structures, and myotubes showed a clear periodic distribution (costameres). In conclusion, in normal human muscle cultures DGC and vinculin-talin-integrin proteins are first localized in the perinuclear region, then they diffuse in the cytoplasm and finally form at the plasma membrane into typical rib-like structures that are sarcolemma-associated.


Asunto(s)
Distrofina/metabolismo , Glicoproteínas/metabolismo , Integrinas/metabolismo , Músculo Esquelético/citología , Mioblastos Esqueléticos/metabolismo , Talina/metabolismo , Vinculina/metabolismo , Adulto , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Separación Inmunomagnética , Masculino , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/fisiología , Factores de Tiempo
8.
Dev Dyn ; 235(11): 3132-43, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16960856

RESUMEN

Present knowledge indicates that fibre recruitment (hyperplasia) in developing teleost fish occurs in three distinct phases. However, the origin and relationship of the myogenic precursors activated during the different phases remains unclear. Here, we address this issue using molecular techniques on embryos and larvae of pearlfish, a large cyprinid species. Results provide comprehensive molecular characterisation of cell recruitment over the three phases of myogenesis, identifying muscle types as they arise. Specifically, we show that the myogenic cells arising during 2nd phase myogenesis are clearly different from the myogenic cells arising during the 3rd phase and that the dermomyotome is a major source of myogenic cells driving 2nd phase hyperplasia. These findings are discussed in relation to their implications for the generality of vertebrate developmental patterns.


Asunto(s)
Cyprinidae/crecimiento & desarrollo , Desarrollo de Músculos , Mioblastos Esqueléticos/fisiología , Animales , Proliferación Celular , Cyprinidae/genética , Cyprinidae/metabolismo , Expresión Génica , Desarrollo de Músculos/genética , Proteína MioD/análisis , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/metabolismo , Factores Reguladores Miogénicos/análisis , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Cadenas Pesadas de Miosina/análisis , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Factor de Transcripción PAX7/análisis , Factor de Transcripción PAX7/metabolismo
9.
Dev Dyn ; 235(11): 2930-9, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16958127

RESUMEN

Pitx2 is a paired-related homeobox gene that has been shown to play a central role during development. In the mouse, there are three isoforms, Pitx2a, b, and c, which differ only in their amino terminal regions. Pitx2 is expressed in myotomes, myoblasts, and myofibers and may be involved in muscle patterning. However, the mechanism by which Pitx2 acts in muscle cell lineages as well as the distinct functions of the individual isoforms have not been investigated. In this study, we used Sol8 myoblasts to investigate the function of Pitx2 in skeletal myogenesis. We found that Pitx2c is the main Pitx2 isoform present in Sol8 myoblasts. Overexpression of Pitx2c in Sol8 myoblasts inhibited myocyte differentiation and myotube formation. Furthermore, Sol8 cells overexpressing Pitx2c maintained high proliferative capacity and a significant up-regulation of the cell cycle genes cyclin D1, cyclin D2, and c-myc. Gene expression analysis for Pax3 and the s MyoD and myogenin showed that Pitx2c-overexpression caused Sol8 cells to remain as myoblasts, in an undifferentiated myogenic state. Furthermore, down-regulation of the muscle-specific genes sTnI and MyHC3 demonstrated that Sol8-overexpressing Pitx2c myoblasts failed to reach terminal differentiation. This study sheds light on previously unknown functions of the Pitx2c isoform in balancing proliferation vs. differentiation in a myogenic cell line.


Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Desarrollo de Músculos/genética , Mioblastos Esqueléticos/citología , Factores de Transcripción/fisiología , Animales , Ciclo Celular/genética , Proliferación Celular , Ciclina D1/genética , Ciclina D2 , Ciclinas/genética , Genes myc/genética , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Ratones , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/metabolismo , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Factores de Transcripción/análisis , Factores de Transcripción/genética , Transcripción Genética , Proteína del Homeodomínio PITX2
10.
Dev Dyn ; 235(11): 3007-15, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16958136

RESUMEN

Chemokines and their receptors play major roles in numerous physiological and pathological processes during development and disease. CXCR4 is the most abundantly expressed chemokine receptor during development. In contrast to other chemokine receptors, CXCR4 binds and is activated exclusively by its ligand stromal derived factor-1 (SDF-1) or CXCL12. SDF-1 signaling has a wide range of effects on CXCR4-expressing cells depending on the cell type ranging from cell growth to adhesion, chemotaxis, and migration. CXCR4 also serves as a co-receptor for HIV-1 entry into T-cells and has been implicated in the pathogenesis of rheumatoid arthritis and cancer growth and invasion. Numerous inhibitors and antagonists of CXCR4 have been produced and are being tested for their efficiency to target its role in pathogenesis. Our initial expression analysis revealed that CXCR4 is expressed by the migrating myogenic and angiogenic precursors in the developing chick limb. In this study, we used the most specific peptidic inhibitors of CXCR4, T140 and its analog TN14003, to analyse the effect of blocking CXCR4/SDF-1 signaling on the undetermined bioptent migratory progenitors in the developing chick limb. Our results point to defects in migration and an altered differentiation program of these CXCR4-expressing progenitor pool in the limb.


Asunto(s)
Movimiento Celular , Embrión de Pollo/citología , Extremidades/embriología , Oligopéptidos/farmacología , Péptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Animales , Apoptosis , Proteínas Aviares/análisis , Proteínas Aviares/genética , Vasos Sanguíneos/citología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inhibidores , Embrión de Pollo/química , Embrión de Pollo/efectos de los fármacos , Extremidades/irrigación sanguínea , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Organogénesis/efectos de los fármacos , Receptores CXCR4/análisis , Receptores CXCR4/genética , Células Madre/química , Células Madre/efectos de los fármacos , Células Madre/fisiología
11.
Muscle Nerve ; 33(2): 254-64, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16281277

RESUMEN

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. Data from the literature indicate that myoblast transfer enhances recovery of muscle function. Thus, we tested the hypothesis that transfer of adult myoblasts improves the properties of reinnervated rabbit tibialis anterior (TA) muscles in both the short term (4 months) and long term (14 months). Two months after transection and immediate suture of the common peroneal nerve, TA muscles were made to degenerate by cardiotoxin injection and then transplanted with adult myoblasts cultured for 13 days. Under these conditions, muscles studied at 4 months were heavier, contained larger fibers, and developed a significantly higher maximal force than muscles that had only been denervated-reinnervated. In the long term, although muscles made to degenerate were heavier and developed a significantly higher maximal force than denervated-reinnervated muscles, myoblast transfer failed to improve these parameters. However, the overall characteristics of long-term operated muscles tended clearly to approach those of the controls. Taken together, these results may have significant implications in certain orthopedic contexts, particularly after immediate or delayed muscle reinnervation.


Asunto(s)
Músculo Esquelético/citología , Músculo Esquelético/inervación , Mioblastos Esqueléticos/trasplante , Animales , Trasplante de Células/métodos , Células Cultivadas , Inmunohistoquímica , Masculino , Contracción Muscular , Músculo Esquelético/química , Músculo Esquelético/fisiología , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/fisiología , Cadenas Pesadas de Miosina/análisis , Conejos , Factores de Tiempo
12.
J Med Genet ; 41(11): 826-36, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15520407

RESUMEN

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.


Asunto(s)
Distrofia Muscular Facioescapulohumeral/genética , Mioblastos Esqueléticos/metabolismo , Proteínas/genética , Activación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 4/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Datos de Secuencia Molecular , Desarrollo de Músculos , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/citología , Proteínas Nucleares , Regiones Promotoras Genéticas , Proteínas/análisis , Proteínas/metabolismo , Regulación hacia Arriba
13.
Biochem J ; 381(Pt 2): 547-59, 2004 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-15084145

RESUMEN

Muskelin is an intracellular protein with a C-terminal kelch-repeat domain that was initially characterized as having functional involvement in cell spreading on the extracellular matrix glycoprotein thrombospondin-1. As one approach to understanding the functional properties of muskelin, we have combined bioinformatic and biochemical studies. Through analysis of a new dataset of eight animal muskelins, we showed that the N-terminal region of the polypeptide corresponds to a predicted discoidin-like domain. This domain architecture is conserved in fungal muskelins and reveals a structural parallel between the muskelins and certain extracellular fungal galactose oxidases, although the phylogeny of the two groups appears distinct. In view of the fact that a number of kelch-repeat proteins have been shown to self-associate, co-immunoprecipitation, protein pull-down assays and studies of cellular localization were carried out with wild-type, deletion mutant and point mutant muskelins to investigate the roles of the discoidin-like and kelch-repeat domains. We obtained evidence for cis- and trans-interactions between the two domains. These studies provide evidence that muskelin self-associates through a head-to-tail mechanism involving the discoidin-like domain.


Asunto(s)
Secuencia Conservada/fisiología , Lectinas/química , Péptidos/fisiología , Proteínas/química , Proteínas/metabolismo , Proteínas Protozoarias/química , Secuencia de Aminoácidos/genética , Animales , Proteínas Aviares/química , Moléculas de Adhesión Celular , Línea Celular , Ciona intestinalis/genética , Dimerización , Discoidinas , Proteínas de Drosophila/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/química , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Ratones , Datos de Secuencia Molecular , Mioblastos Esqueléticos/química , Péptidos/química , Estructura Terciaria de Proteína/fisiología , Proteínas/genética , Ratas , Secuencias Repetitivas de Aminoácido/fisiología , Alineación de Secuencia/métodos , Transfección/métodos , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
14.
Biochem J ; 381(Pt 3): 743-52, 2004 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-15101819

RESUMEN

Cardiac hypertrophy and remodelling in chagasic disease might be associated with mitochondrial dysfunction. In the present study, we characterized the cardiac metabolic responses to Trypanosoma cruzi infection and progressive disease severity using a custom-designed mitoarray (mitochondrial function-related gene array). Mitoarrays consisting of known, well-characterized mitochondrial function-related cDNAs were hybridized with 32P-labelled cDNA probes generated from the myocardium of mice during immediate early, acute and chronic phases of infection and disease development. The mitoarray successfully identified novel aspects of the T. cruzi-induced alterations in the expression of the genes related to mitochondrial function and biogenesis that were further confirmed by real-time reverse transcriptase-PCRs. Of note is the up-regulation of transcripts essential for fatty acid metabolism associated with repression of the mRNAs for pyruvate dehydrogenase complex in infected hearts. We observed no statistically significant changes in mRNAs for the enzymes of tricarboxylic acid cycle. These results suggest that fatty acid metabolism compensates the pyruvate dehydrogenase complex deficiencies for the supply of acetyl-CoA for a tricarboxylic acid cycle, and chagasic hearts may not be limited in reduced energy (NADH and FADH2). The observation of a decrease in mRNA level for several subunits of the respiratory chain complexes by mitoarray as well as global genome analysis suggests a limitation in mitochondrial oxidative phosphorylation-mediated ATP-generation capacity as the probable basis for cardiac homoeostasis in chagasic disease.


Asunto(s)
Enfermedad de Chagas/enzimología , Enfermedad de Chagas/genética , Perfilación de la Expresión Génica/métodos , Mitocondrias Cardíacas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Línea Celular , Regulación de la Expresión Génica/genética , Genes/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Mitocondrias Cardíacas/enzimología , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/enzimología , Mioblastos Esqueléticos/parasitología , Trypanosoma cruzi/parasitología
15.
Biochem J ; 381(Pt 3): 599-608, 2004 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-15086317

RESUMEN

Sarcolemmal-membrane-associated proteins (SLMAPs) define a new class of coiled-coil tail-anchored membrane proteins generated by alternative splicing mechanisms. An in vivo expression analysis indicated that SLMAPs are present in somites (11 days post-coitum) as well as in fusing myotubes and reside at the level of the sarcoplasmic reticulum and transverse tubules in adult skeletal muscles. Skeletal-muscle myoblasts were found to express a single 5.9 kb transcript, which encodes the full-length approximately 91 kDa SLMAP3 isoform. Myoblast differentiation was accompanied by the stable expression of the approximately 91 kDa SLMAP protein as well as the appearance of an approximately 80 kDa isoform. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Membrane targeting of the de-regulated SLMAPs was not critical for the inhibition of myotube development. Protein-protein interaction assays indicated that SLMAPs are capable of self-assembling, and the de-regulated expression of mutants that were not capable of forming SLMAP homodimers also inhibited myotube formation. These results imply that regulated levels and the temporal induction of SLMAP isoforms are important for normal muscle development.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Músculo Esquelético/embriología , Mioblastos Esqueléticos/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Dimerización , Embrión de Mamíferos/química , Femenino , Proteínas Repetidas Ricas en Leucina , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Peso Molecular , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Embarazo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas/metabolismo , Factores de Tiempo
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