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Objectives: We intend to describe early experience using a new, commercially available Micro Plug Set for preterm neonate and infant transcatheter patent ductus arteriosus (PDA) occlusion., Background: Transcatheter PDA occlusion in premature neonates and small infants is safe and effective. The procedure is early in its evolution., Methods: Procedural and short-term outcomes of preterm neonates and infants undergoing transcatheter PDA occlusion with a new, commercially available device were reviewed., Results: Eight preterm neonates and infants born at median 27 weeks gestation (23-36 weeks) underwent transcatheter PDA device closure with the Micro Plug Set. The device is short (2.5 mm) with a range of diameters (3, 4, 5, 6 mm) and delivered through a microcatheter. Procedures were performed at median 41 days of age (12-88 days) and at 1690 g (760-3,310 g). Transvenous PDA device occlusion was performed with fluoroscopic and echocardiography guidance. All procedures were successful with complete PDA occlusion. There were no procedural or short-term adverse events., Conclusions: Preterm neonate and infant transcatheter PDA device closure with a new, commercially available short and microcatheter delivered device (Micro Plug Set) was safe and effective in a small, early series of patients., (© 2020 Wiley Periodicals LLC.)

Background: Patent ductus arteriosus (PDA) stenting is evolving as an alternative to surgical aorto-pulmonary shunts for infants with ductal-dependent pulmonary blood flow. Given anatomical proximity, the PDA can compress the ipsilateral bronchus. We report a case series of four patients with bronchial compression by a tortuous PDA who underwent PDA stenting., Methods: Our four patients received PDA stents for ductal-dependent pulmonary blood flow despite preprocedure imaging evidence of bronchial compression. We reviewed the cross-sectional chest imaging to assess the degree of bronchial compression and the variables that affect it, namely PDA size, PDA tortuosity, and the anatomical relationship between the compressed bronchus and the PDA., Results: Three out of the four patients had postprocedure imaging, and all showed relief of the previously seen bronchial compression. Post-PDA stenting patients had a smaller and straight PDA with significant lateralization away from the compressed bronchus. None of the four patients developed symptoms of bronchial compression poststenting., Conclusions: Our study suggests that pre-existing bronchial compression does not preclude PDA stenting. Stent placement in an engorged and tortuous PDA led to significant improvement in pre-existing bronchial compression. Improvement may be attributed to PDA shrinkage, straightening, and lateralization. Further studies are needed to confirm our findings., (© 2020 Wiley Periodicals LLC.)

Introduction: We postulate a relationship between a transcutaneous hepatic NIRS measurement and a directly obtained hepatic vein saturation. If true, hepatic NIRS monitoring (in conjunction with the current dual-site cerebral-renal NIRS paradigm) might increase the sensitivity for detecting shock since regional oxygen delivery changes in the splanchnic circulation before the kidney or brain. We explored a reliable technique for hepatic NIRS monitoring as a prelude to rigorously testing this hypothesis. This proof-of-concept study aimed to validate hepatic NIRS monitoring by comparing hepatic NIRS measurements to direct hepatic vein samples obtained during cardiac catheterization. Method: IRB-approved prospective pilot study of hepatic NIRS monitoring involving 10 patients without liver disease who were already undergoing elective cardiac catheterization. We placed a NIRS monitor on the skin overlying liver during catheterization. Direct measurement of hepatic vein oxygen saturation during the case compared with simultaneous hepatic NIRS measurement. Results: There was no correlation between the Hepatic NIRS values and the directly measured hepatic vein saturation ( R = -0.035; P = 0.9238). However, the Hepatic NIRS values correlated with the cardiac output ( R = 0.808; P = 0.0047), the systolic arterial blood pressure ( R = 0.739; P = 0.0146), and the diastolic arterial blood pressure ( R = 0.7548; P = 0.0116). Conclusions: Using the technique described, hepatic NIRS does not correlate well with the hepatic vein saturation. Further optimization of the technique might provide a better measurement. Hepatic NIRS does correlate with cardiac output and thus may still provide a valuable additional piece of hemodynamic information when combined with other non-invasive monitoring., (Copyright © 2020 Guyon, Karamlou, Ratnayaka, El-Said, Moore and Rao.)

Atlantic bluefin tuna are a symbol of both the conflict between preservationist and utilitarian views of top ocean predators, and the struggle to reach international consensus on the management of migratory species. Currently, Atlantic bluefin tuna are managed as an early-maturing eastern stock, which spawns in the Mediterranean Sea, and a late-maturing western stock, which spawns in the Gulf of Mexico. However, electronic tagging studies show that many bluefin tuna, assumed to be of a mature size, do not visit either spawning ground during the spawning season. Whether these fish are spawning in an alternate location, skip-spawning, or not spawning until an older age affects how vulnerable this species is to anthropogenic stressors including exploitation. We use larval collections to demonstrate a bluefin tuna spawning ground in the Slope Sea, between the Gulf Stream and northeast United States continental shelf. We contend that western Atlantic bluefin tuna have a differential spawning migration, with larger individuals spawning in the Gulf of Mexico, and smaller individuals spawning in the Slope Sea. The current life history model, which assumes only Gulf of Mexico spawning, overestimates age at maturity for the western stock. Furthermore, individual tuna occupy both the Slope Sea and Mediterranean Sea in separate years, contrary to the prevailing view that individuals exhibit complete spawning-site fidelity. Overall, this complexity of spawning migrations questions whether there is complete independence in the dynamics of eastern and western Atlantic bluefin tuna and leads to lower estimates of the vulnerability of this species to exploitation and other anthropogenic stressors.

α-Actinins are actin-binding proteins that can be broadly divided into Ca(2+)-sensitive cytoskeletal and Ca(2+)-insensitive sarcomeric isoforms. To date, little is known about functional differences between the isoforms due to their indistinguishable activities in most in vitro assays. To identify functional differences in vivo between sarcomeric isoforms, we employed computational and molecular approaches to characterize the zebrafish (Danio rerio) genome, which contains orthologoues of each human α-actinin gene, including duplicated copies of actn3. Each isoform exhibits a distinct and unique pattern of gene expression as assessed by mRNA in situ hybridization, largely sharing similar expression profiles as seen in humans. The spatial conservation of expression of these genes from lower invertebrates to humans suggests that regulation and subsequent functions of these genes are conserved during evolution. Morpholino-based knockdown of the sarcomeric isoform, actn2, leads to skeletal muscle, cardiac, and ocular defects evident over the first week of development. Remarkably, despite the high degree of sequence conservation between actn2 and actn3, the phenotypes of α-actinin-2 deficient zebrafish can be rescued by overexpression of α-actinin-2 but not by α-actinin-3 mRNAs from zebrafish or human. These data provide functional evidence that the primary sequences of α-actinin-2 and α-actinin-3 evolved differences to optimize their functions.

Introduction: Over the past 10 years, the use of zebrafish for scientific research in the area of muscle development has increased dramatically. Although several protocols exist for the isolation of adult myoblast progenitors from larger fish, no standardized protocol exists for the isolation of myogenic progenitors from adult zebrafish muscle., Methods: Using a variant of a mammalian myoblast isolation protocol, zebrafish muscle progenitors have been isolated from the total dorsal myotome. These zebrafish myoblast progenitors can be cultured for several passages and then differentiated into multinucleated, mature myotubes., Results: Transcriptome analysis of these cells during myogenic differentiation revealed a strong downregulation of pluripotency genes, while, conversely, showing an upregulation of myogenic signaling and structural genes., Conclusions: Together these studies provide a simple, yet detailed method for the isolation and culture of myogenic progenitors from adult zebrafish, while further promoting their therapeutic potential for the study of muscle disease and drug screening., (Copyright © 2011 Wiley Periodicals, Inc.)

Two known zebrafish dystrophin mutants, sapje and sapje-like (sap(c/100)), represent excellent small-animal models of human muscular dystrophy. Using these dystrophin-null zebrafish, we have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Three of seven candidate chemicals restored normal birefringence and increased survival of dystrophin-null fish. One chemical, aminophylline, which is known to be a nonselective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and up-regulate the cAMP-dependent PKA pathway in treated dystrophin-deficient fish. Moreover, other PDE inhibitors also reduced the percentage of affected sapje fish. The identification of compounds, especially PDE inhibitors, that moderate the muscle phenotype in these dystrophin-null zebrafish validates the screening protocol described here and may lead to candidate molecules to be used as therapeutic interventions in human muscular dystrophy.

This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus., (© 2010 Blackwell Publishing Ltd.)

Various muscular dystrophies are associated with the defective glycosylation of alpha-dystroglycan and are known to result from mutations in genes encoding glycosyltransferases. Fukutin-related protein (FKRP) was identified as a homolog of fukutin, the defective protein in Fukuyama-type congenital muscular dystrophy (FCMD), that is thought to function as a glycosyltransferase. Mutations in FKRP have been linked to a variety of phenotypes including Walker-Warburg syndrome (WWS), limb girdle muscular dystrophy (LGMD) 2I and congenital muscular dystrophy 1C (MDC1C). Zebrafish are a useful animal model to reveal the mechanism of these diseases caused by mutations in FKRP gene. Downregulating FKRP expression in zebrafish by two different morpholinos resulted in embryos which had developmental defects similar to those observed in human muscular dystrophies associated with mutations in FKRP. The FKRP morphants showed phenotypes involving alterations in somitic structure and muscle fiber organization, as well as defects in developing eye morphology. Additionally, they were found to have a reduction in alpha-dystroglycan glycosylation and a shortened myofiber length. Moreover, co-injection of fish or human FKRP mRNA along with the morpholino restored normal development, alpha-dystroglycan glycosylation and laminin binding activity of alpha-dystroglycan in the morphants. Co-injection of the human FKRP mRNA containing causative mutations found in human patients of WWS, MDC1C and LGMD2I could not restore their phenotypes significantly. Interestingly, these morphant fish having human FKRP mutations showed a wide phenotypic range similar to that seen in humans.

beta-Synemin was previously identified as an alpha-dystrobrevin-interacting protein in muscle. To better understand its function in neural tissue, in situ and immunohistochemical analyses were performed to identify where the synemin isoforms are expressed in the spinal cord of C57BL/6 and dystrophin-deficient (mdx) C57BL/10 mice. These analyses show that synemin transcript and its encoded protein colocalize in the anterior horn cells and that no differences in synemin expression were found in nerve tissue from C57BL/6 or mdx mice. The expression of synemin mRNA and protein predominantly in the anterior horn cells suggests that synemin performs an essential function in those cells. Because synemin is more highly expressed in the midbrain and pons, its function in neurological cells was further pursued by identifying coexpressed proteins in cells from those regions of the brain. These results show that neurons that express synemin also express tryptophan hydroxylase-1, a marker of serotoninergic nerve fibers. Muscle Nerve, 2009.

Sapje-like (sap(cl100)) was one of eight potential zebrafish muscle mutants isolated as part of an early-pressure screen of 500 families. This mutant shows a muscle tearing phenotype similar to sapje (dys-/-) and both mutants fail to genetically complement suggesting they have a mutation in the same gene. Protein analysis confirms a lack of dystrophin in developing sapje-like embryos. Sequence analysis of the sapje-like dystrophin mRNA shows that exon 62 is missing in the dystrophin transcript causing exon 63 to be translated out of frame terminating translation at a premature stop codon at the end of exon 63. Sequence analysis of sapje-like genomic DNA identified a mutation in the donor splice junction at the end of dystrophin exon 62. This mutation is similar to splicing mutations associated with human forms of Duchenne Muscular Dystrophy. Sapje-like is the first zebrafish dystrophin splicing mutant identified to date and represents a novel disease model which can be used in future studies to identify therapeutic compounds for treating diseases caused by splicing defects.

beta-Synemin has been identified as an alpha-dystrobrevin-interacting protein in human muscle, although at least two synemin transcripts are expressed in brain. To understand synemin's function in neural tissue, in situ and immunohistochemical analyses were performed to identify where alpha- and beta-synemin are expressed in the brain of C57BL/6 and mdx (dystrophin null) mice. This analysis shows that the alpha- and beta-synemin transcripts and their encoded proteins colocalize in neurons, especially in the midbrain and pons. Since alpha-dystrobrevin-1 and synemin do not colocalize in brain as in muscle, this suggests that another member of the dystrophin-associated protein complex might interact with synemin in brain. In support of this, synemin mRNA expression was decreased in mdx mice, suggesting that synemin transcription is linked to dystrophin expression. Our findings show where synemin is expressed in brain and allow one to speculate with regard to its function in neural tissue.

Titin (also called connectin) acts as a scaffold for signaling proteins in muscle and is responsible for establishing and maintaining the structure and elasticity of sarcomeres in striated muscle. Several human muscular dystrophies and cardiomyopathies have previously been linked to mutations in the titin gene. This study reports linkage of the runzel homozygous lethal muscular dystrophy in the zebrafish Danio rerio to a genomic interval containing the titin gene. Analysis of the genomic sequence suggests that zebrafish contain two adjacent titin loci. One titin locus lies within the genetic linkage interval and its expression is significantly reduced in runzel mutants by both immunofluorescence and protein electrophoresis. Morpholino downregulation of this same titin locus in wild-type embryos results in decreased muscle organization and mobility, phenocopying runzel mutants. Additional protein analysis demonstrates that, in wild-type zebrafish, titin isoform sizes are rapidly altered during the development of striated muscle, likely requiring a previously unrecognized need for vertebrate sarcomere remodeling to incorporate developmentally regulated titin isoforms. Decreases of affected titin isoforms in runzel mutants during this time correlate with a progressive loss of sarcomeric organization and suggest that the unaffected titin proteins are capable of sarcomerogenesis but not sarcomere maintenance. In addition, microarray analysis of the ruz transcriptome suggests a novel mechanism of dystrophy pathogenesis, involving mild increases in calpain-3 expression and upregulation of heat shock proteins. These studies should lead to a better understanding of titin's role in normal and diseased muscle.

Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells.

Background: Beta-synemin was originally identified in humans as an alpha-dystrobrevin-binding protein through a yeast two-hybrid screen using an amino acid sequence derived from exons 1 through 16 of alpha-dystrobrevin, a region common to both alpha-dystrobrevin-1 and -2. alpha-Dystrobrevin-1 and -2 are both expressed in muscle and co-localization experiments have determined which isoform preferentially functions with beta-synemin in vivo. The aim of our study is to show whether each alpha-dystrobrevin isoform has the same affinity for beta-synemin or whether one of the isoforms preferentially functions with beta-synemin in muscle., Methods: The two alpha-dystrobrevin isoforms (-1 and -2) and beta-synemin were localized in regenerating rat tibialis anterior muscle using immunoprecipitation, immunohistochemical and immunoblot analyses. Immunoprecipitation and co-localization studies for alpha-dystrobrevin and beta-synemin were performed in regenerating muscle following cardiotoxin injection. Protein expression was then compared to that of developing rat muscle using immunoblot analysis., Results: With an anti-alpha-dystrobrevin antibody, beta-synemin co-immunoprecipitated with alpha-dystrobrevin whereas with an anti-beta-synemin antibody, alpha-dystrobrevin-1 (rather than the -2 isoform) preferentially co-immunoprecipitated with beta-synemin. Immunohistochemical experiments show that beta-synemin and alpha-dystrobrevin co-localize in rat skeletal muscle. In regenerating muscle, beta-synemin is first expressed at the sarcolemma and in the cytoplasm at day 5 following cardiotoxin injection. Similarly, beta-synemin and alpha-dystrobrevin-1 are detected by immunoblot analysis as weak bands by day 7. In contrast, immunoblot analysis shows that alpha-dystrobrevin-2 is expressed as early as 1 day post-injection in regenerating muscle. These results are similar to that of developing muscle. For example, in embryonic rats, immunoblot analysis shows that beta-synemin and alpha-dystrobevin-1 are weakly expressed in developing lower limb muscle at 5 days post-birth, while alpha-dystrobrevin-2 is detectable before birth in 20-day post-fertilization embryos., Conclusion: Our results clearly show that beta-synemin expression correlates with that of alpha-dystrobrevin-1, suggesting that beta-synemin preferentially functions with alpha-dystrobrevin-1 in vivo and that these proteins are likely to function coordinately to play a vital role in developing and regenerating muscle.

Background: Human muscular dystrophies are a heterogeneous group of genetic disorders which cause decreased muscle strength and often result in premature death. There is no known cure for muscular dystrophy, nor have all causative genes been identified. Recent work in the small vertebrate zebrafish Danio rerio suggests that mutation or misregulation of zebrafish dystrophy orthologs can also cause muscular degeneration phenotypes in fish. To aid in the identification of new causative genes, this study identifies and maps zebrafish orthologs for all known human muscular dystrophy genes., Results: Zebrafish sequence databases were queried for transcripts orthologous to human dystrophy-causing genes, identifying transcripts for 28 out of 29 genes of interest. In addition, the genomic locations of all 29 genes have been found, allowing rapid candidate gene discovery during genetic mapping of zebrafish dystrophy mutants. 19 genes show conservation of syntenic relationships with humans and at least two genes appear to be duplicated in zebrafish. Significant sequence coverage on one or more BAC clone(s) was also identified for 24 of the genes to provide better local sequence information and easy updating of genomic locations as the zebrafish genome assembly continues to evolve., Conclusion: This resource supports zebrafish as a dystrophy model, suggesting maintenance of all known dystrophy-associated genes in the zebrafish genome. Coupled with the ability to conduct genetic screens and small molecule screens, zebrafish are thus an attractive model organism for isolating new dystrophy-causing genes/pathways and for use in high-throughput therapeutic discovery.

Zebrafish reproduce in large quantities, grow rapidly, and are transparent early in development. For these reasons, zebrafish have been used extensively to model vertebrate development and disease. Like mammals, zebrafish express dystrophin and many of its associated proteins early in development and these proteins have been shown to be vital for zebrafish muscle stability. In dystrophin-null zebrafish, muscle degeneration becomes apparent as early as 3 days post-fertilization (dpf) making the zebrafish an excellent organism for large-scale screens to identify other genes involved in the disease process or drugs capable of correcting the disease phenotype. Being transparent, developing zebrafish are also an ideal experimental model for monitoring the fate of labeled transplanted cells. Although zebrafish dystrophy models are not meant to replace existing mammalian models of disease, experiments requiring large numbers of animals may be best performed in zebrafish. Results garnered from using this model could lead to a better understanding of the pathogenesis of the muscular dystrophies and the development of future therapies.

Synemin is an intermediate filament protein shown previously to interact with alpha-dystrobrevin and desmin. Immunoblot analysis detects a beta-synemin protein of 170 kDa in human skeletal muscle and an alpha-synemin protein of 225 kDa in monkey brain. Low-resolution immunohistochemical analysis localizes beta-synemin within muscle along the sarcolemma, whereas confocal microscopic analysis further refines localization to the costamere and muscle Z-lines. In addition to these locations, beta-synemin is also enriched at the neuromuscular and myotendinous junctions, other regions that undergo high stress during myofiber contraction. Based on its localization and its expression pattern, it is proposed that beta-synemin functions as a structural protein involved in maintaining muscle integrity through its interactions with alpha-dystrobrevin, desmin, and other structural proteins., (Copyright 2004 Wiley Periodicals, Inc.)

Calpain 3 (C3) is the only muscle-specific member of the calcium-dependent protease family. Although neither its physiological function nor its in vivo substrates are known, C3 must be an important protein for normal muscle function as mutations in the C3 gene result in limb-girdle muscular dystrophy type 2A. Previous reports have shown that the ubiquitous calpains (mu and m) proteolyze filamins in nonmuscle cells. This observation suggests that the muscle-specific filamin C (FLNC) is a good candidate substrate for C3. Binding studies using recombinant proteins establish that recombinant C3 and native FLNC can interact. When these two proteins are translated in vitro and incubated together, C3 cleaves the C-terminal portion of FLNC. Cleavage is specific as C3 fails to cleave FLNC lacking its C-terminal hinge and putative dimerization domains. Cotransfection experiments in COS-7 cells confirm that C3 can cleave the C-terminus of FLNC in live cells. The C-terminus of FLNC has been shown to bind the cytoplasmic domains of both delta- and gamma-sarcoglycan. Removal of the last 127 amino acids from FLNC, a protein that mimics FLNC after C3 cleavage, abolishes this interaction with the sarcoglycans. These studies confirm that C3 can cleave FLNC in vitro and suggest that FLNC may be an in vivo substrate for C3, functioning to regulate protein-protein interactions with the sarcoglycans. Thus, calpain-mediated remodeling of cytoskeletal-membrane interactions, such as those that occur during myoblast fusion and muscle repair, may involve regulation of FLNC-sarcoglycan interactions.

Many cases of muscular dystrophy in humans are caused by mutations in members of the dystrophin associated protein complex (DAPC). Zebrafish are small vertebrates whose bodies are composed predominantly of skeletal muscle, making them attractive models for studying mammalian muscle disorders. Potential orthologs to most of the human DAPC proteins have been found in zebrafish by database screening. Expression of the sarcoglycans, dystroglycan and dystrophin has been confirmed by western blotting. Immunohistochemical and biochemical techniques localize these proteins to the muscle cell membrane in adult zebrafish. Morpholino (MO) experiments designed to inhibit the translation of dystrophin mRNA produce juvenile zebrafish that are less active than zebrafish injected with control morpholinos. Western blot analysis of the dystrophin morpholino-injected zebrafish shows concurrent reduction of dystrophin and the sarcoglycans, suggesting that these proteins, like those in mammals, are part of a complex whose integrity is dependent on dystrophin expression. These results indicate that the zebrafish is an excellent animal model in which to approach the study of dystrophin and its associated proteins.

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development.

Chromatin remodeling complexes such as SWI/SNF use the energy of ATP hydrolysis to remodel nucleosomal DNA and increase transcription of nucleosomal templates. Human heat shock factor one (hHSF1) is a tightly regulated activator that stimulates transcriptional initiation and elongation using different portions of its activation domains. Here we demonstrate that hHSF1 associates with BRG1, the ATPase subunit of human SWI/SNF (hSWI/SNF) at endogenous protein concentrations. We also show that hHSF1 activation domains recruit hSWI/SNF to a chromatin template in a purified system. Mutation of hHSF1 residues responsible for activation of transcriptional elongation has the most severe effect on recruitment of SWI/SNF and association of hHSF1 with BRG1, suggesting that recruitment of chromatin remodeling activity might play a role in stimulation of elongation.

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, alpha1-syntrophin, and the sarcoglycan complex. The precise role of alpha-dystrobrevin in skeletal muscle has not yet been determined. To study alpha-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and alpha-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.

SWI-SNF alters DNA-histone interactions within a nucleosome in an ATP-dependent manner. These alterations cause changes in the topology of a closed circular nucleosomal array that persist after removal of ATP from the reaction. We demonstrate here that a remodeled closed circular array will revert toward its original topology when ATP is removed, indicating that the remodeled array has a higher energy than that of the starting state. However, reversion occurs with a half-life measured in hours, implying a high energy barrier between the remodeled and standard states. The addition of competitor DNA accelerates reversion of the remodeled array by more than 10-fold, and we interpret this result to mean that binding of human SWI-SNF (hSWI-SNF), even in the absence of ATP hydrolysis, stabilizes the remodeled state. In addition, we also show that SWI-SNF is able to remodel a closed circular array in the absence of topoisomerase I, demonstrating that hSWI-SNF can induce topological changes even when conditions are highly energetically unfavorable. We conclude that the remodeled state is less stable than the standard state but that the remodeled state is kinetically trapped by the high activation energy barrier separating it from the unremodeled conformation.

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones.

The histone N-terminal tails have been shown previously to be important for chromatin assembly, remodeling, and stability. We have tested the ability of human SWI-SNF (hSWI-SNF) to remodel nucleosomes whose tails have been cleaved through a limited trypsin digestion. We show that hSWI-SNF is able to remodel tailless mononucleosomes and nucleosomal arrays, although hSWI-SNF remodeling of tailless nucleosomes is less effective than remodeling of nucleosomes with tails. Analogous to previous observations with tailed nucleosomal templates, we show both (i) that hSWI-SNF-remodeled trypsinized mononucleosomes and arrays are stable for 30 min in the remodeled conformation after removal of ATP and (ii) that the remodeled tailless mononucleosome can be isolated on a nondenaturing acrylamide gel as a novel species. Thus, nucleosome remodeling by hSWI-SNF can occur via interactions with a tailless nucleosome core.