Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026850 (muscular dystrophy)
 5,870 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to understand the mechanism of defective myofibrilogenesis in muscular dystrophy, we have used the genomic cloned DNA specific for myosin light chain 2A (MLC 2A) to check its expression. The fusion of a partial digest of lambda LC5, containing the upstream sequence of MLC 2A gene with the expression vector of PSVOCAT has already been reported. Using this CAT-fused recombinant containing 1.6 kb of MLC 2A gene, we were able to detect the promoter activity in normal heart cells, H9C2 cell line whereas a restricted expression of MLC 2A gene was noticed in muscular dystrophic muscle cells from heart and skeletal. We have also measured the transient transfection efficiency by contransfecting with the plasmid LacZ. Simultaneous assay of beta-galactosidase and CAT in the cell extract was performed. With beta-galactosidase as control, we confirmed that the promoter activity of MLC 2A gene is inhibited in muscular dystrophy though there is a normal rate of transfection occurred.
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PMID:Restricted expression of cardiac myosin light chain 2A gene in muscular dystrophic condition. 190 50

The first three exons of the human muscle dystrophin gene were expressed as a beta-galactosidase fusion protein. This protein was then used to prepare two monoclonal antibodies (mAbs) which react with native dystrophin on frozen muscle sections and with denatured dystrophin on western blots but which do not cross-react with the dystrophin-related protein, utrophin. Both mAbs recognized dystrophin in muscular dystrophy (MD) patients with deletions of exon 3, and further mapping with 11 overlapping synthetic peptides showed that they both recognize an epitope encoded by the muscle-specific exon 1. Neither mAb recognizes the brain dystrophin isoform, confirming the prediction from mRNA data that this has a different N-terminus. One Becker MD patient with a frameshift deletion of exons 3-7 is shown to produce dystrophin which reacts with the N-terminal mAbs, as well as with mAbs which bind on the C-terminal side of the deletion. The data suggest that transcription begins at the normal muscle dystrophin promoter and that the normal reading frame is restored after the deletion. A number of mechanisms have been proposed for restoration of the reading frame after deletion of exons 3-7, but those which predict dystrophin with an abnormal N-terminus do not appear to be major mechanisms in this patient.
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PMID:Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of exons 3-7. 831 78

Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the beta-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506-immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.
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PMID:Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation. 860 7

Golden retriever muscular dystrophy (GRMD) is an excellent model for the study of the efficacy of gene therapy in dystrophin deficient myopathies for there are many similarities between affected dogs and Duchenne muscular dystrophy (DMD) in boys. GRMD is not caused by deletion mutation but results from a point mutation in the consensus splice acceptor in intron 6 of the canine dystrophin gene. As a result exon 7 is skipped during processing of the GRMD dystrophin messenger RNA. We have developed a rapid test which makes direct use of exon 7 specific genomic PCR products. We have undertaken preliminary experiments on gene therapy using the mini-gene and the full length gene alone and in combination with lipofectin and/or the bacterial beta-galactosidase reporter gene Lac Z. Following direct injection of the Lac Z plasmid, either alone or with lipofectin, about 50% of the sites showed expression when biopsied some 14 days later. The beta-galactosidase activity was present in muscle and granulation tissue but was never abundant. Pups injected intraperitoneally with Lac Z were found to have positive material in their mesenteric lymph nodes, liver and spleen. Those injected with Lac Z and lipofectin also had positive material in the diaphragm, intercostal muscles and abdominal muscles, but again only a small amount of positive material was present at any of the sites. In animals directly injected into the muscle with the dystrophin mini-gene, half had positive staining for dystrophin in biopsies taken 14 days later. Of the 6 sites in the muscles of animals given the mini-gene and lipofectin only one had fibres positive for dystrophin when examined 14 days later. Six pups were injected directly with full-length gene construct and when biopsies were taken 10 days later two of the animals had strongly stained peripheries to a small number of fibres.
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PMID:Use of the dog model for Duchenne muscular dystrophy in gene therapy trials. 926 46

During a gene trap screen, an insertion of the gene trap vector into the dystrophin gene, creating a new allele for the Dmd gene, has been discovered. Because the ROSA beta geo vector was used, the new allele is called Dmd(mdx-beta geo). The insertion occurred 3' of exon 63 of the dystrophin gene, resulting in a mutation that affects all presently known dystrophin isoforms. In contrast to spontaneous or ENU-induced alleles, Dmd(mdx-beta geo) can be used to follow dystrophin expression by staining for beta-galactosidase activity. The high sensitivity of this method revealed additional and earlier expression of dystrophin during embryogenesis than that seen previously with other methods. Dystrophin promoters are active predominantly in the dermamyotome, limb buds, telencephalon, floor plate, eye, liver, pancreas anlagen, and cardiovascular system. Adult Dmd(mdx-beta geo) mice show reporter gene expression in brain, eye, liver, pancreas, and lung. In skeletal and heart muscle, beta-galactosidase activity is not detectable, confirming Western blot data that indicate the absence of the mutant full-length protein in these tissues. Hemizygous Dmd(mdx-beta geo) mice show muscular dystrophy with degenerating muscle fibers, cellular infiltration, and regenerated muscle fibers that have centrally located nuclei. Some mutant animals develop a dilated esophagus, probably due to constriction by the hypertrophic crura of the diaphragm.
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PMID:Dmd(mdx-beta geo): a new allele for the mouse dystrophin gene. 962 97

To monitor the presence of introduced genes and the distribution of the encoded proteins in host tissues after gene transfer, we combined fluorescence in situ hybridization (FISH) and immunohistochemistry in two separate gene therapy paradigms. In brain tissue sections from animals injected with pHSVlac vector, we localized nuclei containing vector DNA both in cells expressing and not expressing beta-galactosidase (beta-gal). This suggests that the efficiency of gene transfer is affected not only by gene delivery, but also by cellular controls on gene expression. In a second paradigm, following myoblast transplantation, we detected donor nuclei in the muscle of a patient with Duchenne's muscular dystrophy. The donor nuclei were either surrounded by host nuclei or apparently fused in the patient's muscle fiber producing dystrophin. The combined FISH and immunohistochemistry assay offers greater sensitivity and more information than currently used polymerase chain reaction and protein detection methods.
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PMID:A method to codetect introduced genes and their products in gene therapy protocols. 963 Oct 42

Mutations in laminin alpha2, a subunit of the basement membrane protein laminin-2/merosin, cause merosin-deficient congenital muscular dystrophy. To gain insight into the molecular mechanism of disease, we generated and used a mutant mouse, dyW, in which the lacZ gene was inserted into the lama2 gene so that beta-galactosidase would be expressed in place of laminin alpha2. Heterozygous and homozygous mutant mice are normal at birth, but homozygous mice develop muscular dystrophy at 2 to 3 weeks of age. The lama2/lacZ gene was highly expressed in muscle in the early stages of embryonic myogenesis, but was down-regulated at later stages in both heterozygous and homozygous mice. No beta-galactosidase activity was detected in skeletal muscle after birth in adult heterozygous mice. In contrast, high beta-galactosidase activity was detected in postnatal homozygous mice. Induction of injury in heterozygous mice resulted in intense reexpression of beta-galactosidase in the injured muscle early in regeneration, with a decline in enzyme activity as repair of the tissue progressed. Although the initial response to injury was similar in heterozygous and homozygous mice with abundant beta-galactosidase-positive, mononucleated cells in the injured area, repair was rarely completed in the homozygous mice, evidently caused by excessive death of cells associated with immature myofibers. The defect in muscle repair was very efficiently corrected in homozygous dyW mice expressing a human LAMA2 transgene in skeletal muscle. The data show the importance of laminin alpha2 in muscle regeneration and suggest that a major contributor to disease in muscular dystrophy is abortive regeneration.
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PMID:Activation of the lama2 gene in muscle regeneration: abortive regeneration in laminin alpha2-deficiency. 1061 10

Muscle tissue from Duchenne muscular dystrophy patients and the Dmd(mdx/mdx) (hereafter referred to as mdx) mouse is characterized by an abundance of necrotic myofibers and infiltrating macrophages. Both features may provide additional stimulus to the immune response directed against novel antigens, such as those delivered by gene therapy vectors. It has previously been shown that the immune evasion achieved by adeno-associated virus in healthy muscle fails in one model of muscular dystrophy. Here, we examined the immune response to adenoviral vectors and their transgenes in normal and mdx mice. We found that mdx mouse muscles contain 20 times more macrophages and 7 times more dendritic cells than healthy muscles. This higher professional antigen-presenting cell content results in a stronger immune response to antigens that can be directly presented by those cells, including viral antigens and constitutively expressed transgene products. However, we did not detect a significant immune response to beta-galactosidase expressed specifically in muscle, even at high expression levels. This result suggests that cross-presentation is not more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle.
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PMID:Immune evasion by muscle-specific gene expression in dystrophic muscle. 1173 36

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.
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PMID:Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice. 1173 59

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.
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PMID:Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system. 1662 76


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