RESUMEN
About two thirds of Duchenne muscular dystrophy (DMD) patients have either gene deletions or duplications. The other DMD cases are most likely the result of point mutations that cannot be easily identified by current strategies. Utilizing a heteroduplex technique and direct sequencing of amplified products, we screened our nondeletion/duplication DMD population for point mutations. We now describe what we believe to be the first dystrophin missense mutation in a DMD patient. The mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in DMD patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.
Asunto(s)
Distrofina/genética , Distrofias Musculares/genética , Mutación Puntual , Secuencia de Aminoácidos , Secuencia de Bases , Niño , ADN/genética , Exones , Femenino , Eliminación de Gen , Humanos , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , Ácidos Nucleicos Heterodúplex/genética , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.
Asunto(s)
Atrofia Muscular Espinal/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Glutatión Transferasa/genética , Células HeLa , Humanos , Atrofia Muscular Espinal/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Complejo SMN , Índice de Severidad de la Enfermedad , Proteína 1 para la Supervivencia de la Neurona MotoraRESUMEN
Spinal muscular atrophy (SMA) is the second most common lethal, autosomal recessive disease in Caucasians (after cystic fibrosis). Childhood SMAs are divided into three groups (type I, II and III), which are allelic variants of the same locus in a region of approximately 850 kb in chromosome 5q12-q13, containing multiple copies of a novel, chromosome 5-specific repeat as well as many atypical pseudogenes. This has hampered the identification of candidate genes. We have identified several coding sequences unique to the SMA region. A genomic fragment detected by one cDNA is homozygously deleted in 17/29 (58%) of type I SMA patients. Of 235 unaffected individuals examined, only two showed the deletion and both are carriers of SMA. Our results suggest that deletion of at least part of this novel gene is directly related to the phenotype of SMA.
Asunto(s)
ADN Complementario/genética , Atrofia Muscular Espinal/genética , Eliminación de Secuencia , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Exones , Homocigoto , Humanos , Datos de Secuencia Molecular , Atrofia Muscular Espinal/clasificación , Fenotipo , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
Duchenne muscular dystrophy (DMD) and its less severe form Becker muscular dystrophy (BMD) are allelic disorders. It has been suggested that in the mutations involving BMD, the translational reading frame of messenger RNA is maintained and a smaller, though partially functional, protein is produced. In order to test this, the exon-intron boundaries of the first ten exons of the DMD gene were determined, and 29 patients were analyzed. In a number of BMD patients (mild and severe BMD), the reading frame of messenger RNA was not maintained. On the basis of these findings, a model for reinitiation from an internal start codon is suggested.
Asunto(s)
Proteínas Musculares/genética , Distrofias Musculares/genética , Cromosoma X , Secuencia de Bases , Southern Blotting , Deleción Cromosómica , Sondas de ADN , Distrofina , Exones , Genes , Humanos , Mutación , FenotipoRESUMEN
A probe for the 5' end of the Duchenne muscular dystrophy (DMD) gene was used to study expression of the gene in normal human muscle, myogenic cell cultures, and muscle from patients with DMD. Expression was found in RNA from normal fetal muscle, adult cardiac and skeletal muscle, and cultured muscle after myoblast fusion. In DMD muscle, expression of this portion of the gene was also revealed by in situ RNA hybridization, particularly in regenerating muscle fibers.
Asunto(s)
Regulación de la Expresión Génica , Músculos/metabolismo , Distrofias Musculares/genética , Células Cultivadas , ADN/genética , ADN Recombinante , Humanos , Músculos/embriología , Miocardio/metabolismo , Hibridación de Ácido Nucleico , ARN/metabolismo , ARN Mensajero/metabolismo , Regeneración , Transcripción GenéticaRESUMEN
Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values. To identify a putative enzyme mutant in tissue extracts, aldolase B was purified from human liver by affinity chromatography and monospecific antibodies were prepared from antiserum raised in sheep. Immunodiffusion gels showed a single precipitin line common to pure enzyme and extracts of normal liver and intestine, but no reaction with extracts of brain, muscle, or HFI liver. However, weak positive staining for aldolase in hepatocyte and enterocyte cytosol was demonstrated by indirect immunofluorescence of HFI tissues. This was abolished by pretreatment with pure enzyme protein. Accordingly, a specific radioimmunoassay (detection limit 7.5 ng) was established to quantify immunoreactive aldolase B in human biopsy specimens. Extracts of tissue from affected patients gave 10-25% immunoreactive enzyme in control samples; immunoreactive aldolase in intestinal extracts from four heterozygotes was reduced (to 55%) when compared with seven samples from normal control subjects (P < 0.05). In extracts of HFI tissues, there was a sevenfold reduction in apparent absolute specific activity (1.02 vs. 8.82 U/mg) of immunoreactive fructose-l-phosphate aldolase B, but the apparent specific activity in heterozygotes (7.71 U/mg) was only slightly impaired. Displacement radioimmunotitration of aldolase B in liver supernatants showed a significant (P < 0.005) decrease in antibody avidity for immunoreactive protein in HFI tissue when compared with the pure enzyme or extract of normal control liver. Immunoaffinity chromatography on antialdolase B-Sepharose facilitated isolation and purification of enzyme from liver biopsy specimens. Active aldolase in normal liver, with substrate activity ratios and Michaelis constants identical to biochemically purified human enzyme, could be recovered from antibody columns. Chromatography on monospecific Fab' antialdolase B enabled pure enzyme protein to be retrieved quantitatively from normal control and HFI liver: direct chemical assay showed 1.88 and 1.15 mg aldolase protein/g of tissue, respectively. This confirmed that the catalytic properties of the HFI aldolase were profoundly impaired with specific activities of fructose-l-phosphate cleavage of 7.21 and 0.07 U/mg, respectively. Radioimmunoassay gave estimates of 7.66 and 1.18 U/mg, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that immunopurified aldolase from HFI liver possessed a single subunit size similar to material from control liver extracts: M(r) 39,100 vs. 37,900+/-700 (SD) D, respectively. Electrofocusing under denaturing conditions of aldolase isolated in parallel from control and HFI liver revealed the same complement of subunits and, despite qualitative differences in distribution of bands during degradation, no additional charged species. Fructose phosphate aldolase deficiency in hereditary fructose intolerance is attended by the synthesis of an immunoreactive, but functionally and structurally modified enzyme variant that results from a restricted genetic mutation.
Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Intolerancia a la Fructosa/enzimología , Fructosa-Bifosfato Aldolasa/metabolismo , Isoenzimas/metabolismo , Hígado/enzimología , Adolescente , Adulto , Fructosa-Bifosfato Aldolasa/genética , Fructosadifosfatos/metabolismo , Humanos , Técnicas Inmunológicas , Intestinos/enzimología , Isoenzimas/genética , Cinética , Masculino , Persona de Mediana Edad , Mutación , RadioinmunoensayoRESUMEN
The absence of dystrophin causes the drastic reduction of the dystrophin-associated proteins (DAPs) in the sarcolemma and the loss of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in Duchenne muscular dystrophy (DMD) skeletal muscle. Here, we report a mild reduction of the DAPs in the unique Becker muscular dystrophy patients with huge deletions in the rod domain of dystrophin and a moderate reduction of the DAPs in patients with huge deletions that involve both the NH2-terminal and rod domains of dystrophin. The phenotype of the latter patients was more severe than that of the former. In both cases, however, the reduction in the DAPs was milder than in typical DMD patients or DMD patients lacking the COOH-terminal domains of dystrophin. Our results suggest that (a) the NH2-terminal and rod domains of dystrophin may not be essential for the interaction with the sarcolemmal glycoprotein complex; and (b) defects in the actin binding activity of dystrophin may cause disruption of the anchorage of the dystrophin-glycoprotein complex to the subsarcolemmal cytoskeleton, which may render muscle fibers susceptible to degeneration.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Distrofina/genética , Proteínas de la Membrana , Músculos/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Eliminación de Secuencia , Adolescente , Adulto , Biopsia , Niño , Preescolar , Proteínas del Citoesqueleto/análisis , Distrofina/metabolismo , Exones , Femenino , Ligamiento Genético , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Modelos Genéticos , Peso Molecular , Músculos/patología , Distrofias Musculares/patología , UtrofinaRESUMEN
Proximal spinal muscular atrophy (SMA) is caused by mutations in the telomeric (SMNT), but not centromeric (SMNC), survival motor neuron gene. Here we have identified and analyzed the two SMN promoters. We show that a 750-bp 5'-flanking fragment from each is capable of driving expression from a reporter construct. Within this fragment, we define a approximately 200-bp element that results in high expression in a motor neuron cell line. Sequence comparison of a 3. 4-kb upstream fragment from each gene shows minimal differences. Although these differences produce a 2-fold difference in reporter activity between the two promoters, this is not sufficiently high to explain why SMNT, but not SMNC, is the disease determining gene. Our data thus demonstrate, for the first time, almost complete equivalence between the SMN promoters and rule out the important possibility that differences in them might explain why mutations in only the telomeric SMN gene cause SMA.
Asunto(s)
Centrómero/genética , Neuronas Motoras/metabolismo , Regiones Promotoras Genéticas , Telómero/genética , Secuencia de Bases , Línea Celular , Datos de Secuencia Molecular , Distrofias Musculares/genéticaRESUMEN
BACKGROUND: Receptor protein tyrosine phosphatase rho (RPTPrho, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPrho gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPrho expression is restricted to the central nervous system. RESULTS: The cloning of the mouse cDNA, identification of alternatively spliced exons, detection of an 8 kb 3'-UTR, and the genomic organization of human and mouse RPTPrho genes are described. The two genes are comprised of at least 33 exons. Both RPTPrho genes span over 1 Mbp and are the largest RPTP genes characterized. Exons encoding the extracellular segment through the intracellular juxtamembrane 'wedge' region are widely spaced, with introns ranging from 9.7 to 303.7 kb. In contrast, exons encoding the two phosphatase domains are more tightly clustered, with 15 exons spanning approximately 60 kb, and introns ranging in size from 0.6 kb to 13.1 kb. Phase 0 introns predominate in the intracellular, and phase 1 in the extracellular segment. CONCLUSIONS: We report the first genomic characterization of a RPTP type IIB gene. Alternatively spliced variants may result in different RPTPrho isoforms. Our findings suggest that RPTPrho extracellular and intracellular segments originated as separate modular proteins that fused into a single transmembrane molecule during a later evolutionary period.
RESUMEN
Autosomal recessive childhood onset spinal muscular atrophy (SMA) is a leading cause of infant mortality caused by mutations in the survival motor neuron (SMN) gene. The SMN protein is involved in RNA processing and is localised in structures called GEMs in the nucleus. Nothing is yet understood about why mutations in SMN gene result in the selective motor neuron loss observed in patients. The SMN protein domains conserved across several species may indicate functionally significant regions. Exon 3 of SMN contains homology to a tudor domain, where a Type I SMA patient has been reported to harbour a missense mutation. We have generated missense mutants in this region of SMN and have tested their ability to form GEMs when transfected into HeLa cells. Our results show such mutant SMN proteins still localise to GEMs. Furthermore, exon 7 deleted SMN protein appears to exert a dominant negative effect on localisation of endogenous SMN protein. However, exon 3 mutant protein and exon 5 deleted protein exert no such effect.
Asunto(s)
Atrofia Muscular Espinal/genética , Mutación , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Homología de Secuencia de AminoácidoRESUMEN
The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557. We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA. The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557. We tested all our key recombinant families with these markers. In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823. Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA. Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 5/genética , ADN Satélite/genética , Atrofia Muscular Espinal/genética , Secuencia de Bases , Cromosomas Artificiales de Levadura/genética , Consanguinidad , Sondas de ADN/química , Sondas de ADN/genética , Femenino , Dosificación de Gen , Ligamiento Genético , Marcadores Genéticos/genética , Heterocigoto , Homocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
We have identified a novel receptor-like protein tyrosine phosphatase (RPTPrho) transcript whose expression in the cerebellar cortex is restricted to the granule cell layer of lobules 1-6. Acidic fibroblast growth factor (FGF-1) mRNA follows a similar cerebellar expression pattern. Together, the two markers define a sharp boundary in lobule 6, slightly caudal to the primary fissure. Anterior and posterior compartments became discernible only during postnatal weeks two and six, for RPTPrho and FGF-1, respectively. A rostrocaudal boundary in lobule 6 of the murine cerebellar cortex has also been identified morphologically by the effects of the meander tail mutation. The position of the RPTPrho and FGF-1 boundary on the rostrocaudal axis of the cerebellar cortex was close to, but not coincident with, the caudal extent of the disorganized anterior lobe of meander tail and the rostral extent of Otx-2 expression. The restricted pattern of FGF-1 and RPTPrho implies that these molecules may have specific signaling roles in the tyrosine phosphorylation/dephosphorylation pathway in the anterior compartment of the adult cerebellar cortex.
Asunto(s)
Corteza Cerebelosa/química , Factor 1 de Crecimiento de Fibroblastos/genética , Mesencéfalo/química , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Rombencéfalo/química , Animales , Biomarcadores/química , Corteza Cerebelosa/citología , Corteza Cerebelosa/embriología , Desarrollo Embrionario y Fetal/fisiología , Genes Homeobox , Mesencéfalo/embriología , Ratones , Ratones Endogámicos C57BL , Neuronas/química , Rombencéfalo/embriología , Transducción de Señal/fisiologíaRESUMEN
The mechanism by which prednisone improves muscle strength and function in Duchenne muscular dystrophy (DMD) is unknown. We addressed the possibility that clinical improvement was related to prednisone-induced alterations in skeletal muscle dystrophin. We performed muscle biopsies on patients at the conclusion of a randomized, double-blind, 6-month trial of prednisone and analyzed dystrophin content using Western blots and antibody staining of tissue sections. These studies demonstrated no significant differences in dystrophin content between treatment (prednisone 1.5 mg/kg/d, n = 12; prednisone 0.75 mg/kg/d, n = 9) and placebo (n = 12) groups. Of interest, however, was the presence of varying numbers of dystrophin-positive fibers (revertants) occurring individually or in clusters in antibody-stained tissue sections of more than one-half of the Duchenne patients. Mutation analysis revealed that revertants occurred in DMD patients with identifiable deletions half of the Duchenne patients. Mutation analysis revealed that revertants occurred in DMD patients with identifiable deletions or duplications, and in nondeletion patients. Prednisone treatment did not influence the prevalence of revertants. Revertants are most likely due to a second-site mutation occurring in a somatic cell allowing for restoration of the translational reading frame of the dystrophin transcript.
Asunto(s)
Distrofina/genética , Músculos/metabolismo , Distrofias Musculares/genética , Prednisona/uso terapéutico , Adolescente , Niño , Preescolar , Deleción Cromosómica , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Distrofina/análisis , Exones , Humanos , Músculos/patología , Distrofias Musculares/tratamiento farmacológico , Distrofias Musculares/patologíaRESUMEN
The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.
Asunto(s)
Distrofina/genética , Mutación del Sistema de Lectura , Distrofias Musculares/genética , Secuencia de Bases , Niño , Eliminación de Gen , Humanos , Masculino , Datos de Secuencia Molecular , Distrofias Musculares/patologíaRESUMEN
The dystrophin-glycoprotein complex is considered to be a major trans-sarcolemmal structure which provides a linkage between the subsarcolemmal actin cytoskeleton and the extracellular matrix component laminin. Recently, deficiency of the dystrophin-associated proteins has been shown to play an important role in the molecular pathogenesis of several forms of muscular dystrophy. These include Duchenne muscular dystrophy (DMD), symptomatic DMD carriers, Becker muscular dystrophy and severe childhood autosomal recessive muscular dystrophy with DMD-like phenotype prevalent in North Africa. In Fukuyama-type congenital muscular dystrophy (FCMD), the finding of abnormal expression of the dystrophin-associated proteins may provide a clue to its molecular pathogenesis. These recent findings indicate that the linkage between the subsarcolemmal cytoskeleton and extracellular matrix via the dystrophin-glycoprotein complex is critical for maintaining the integrity of muscle cell function.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Distrofina/fisiología , Glicoproteínas de Membrana/fisiología , Distrofias Musculares/fisiopatología , Niño , Proteínas del Citoesqueleto/genética , Distroglicanos , Distrofina/genética , Genes Recesivos , Tamización de Portadores Genéticos , Humanos , Glicoproteínas de Membrana/genética , Distrofias Musculares/clasificación , Distrofias Musculares/genética , Eliminación de SecuenciaRESUMEN
We describe the cloning, chromosomal localization and characterization of RPTPrho, a new member of the RPTPmu/kappa phosphatase subfamily. Receptor tyrosine phosphatases in this subfamily are comprised of a MAM domain near the N-terminal, an immunoglobulin-like domain, four fibronectin type III repeats, a single transmembrane domain, and a large juxtamembrane segment followed by two intracellular phosphatase domains. An alternatively spliced mini-exon was identified in the extracellular segment of RPTPrho, between the fourth fibronectin type III repeat and the transmembrane domain. The RPTPrho gene was mapped to human chromosome 20 and mouse chromosome 2. Northern blot analysis demonstrated that RPTPrho expression was restricted to the central nervous system, and in situ hybridization studies showed that the RPTPrho transcript was distributed throughout the murine brain and spinal cord. Exceptionally high levels of the transcript were present in the cortex and olfactory bulbs during perinatal development, but were down-regulated during postnatal week two. The motifs found in the extracellular segment of type II receptor protein tyrosine phosphatases are commonly found in neural cell adhesion molecules, suggesting that RPTPrho may be involved in both signal transduction and cellular adhesion in the central nervous system.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Receptores de Superficie Celular/metabolismo , Médula Espinal/metabolismo , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , Humanos , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas Tirosina Fosfatasas Clase 2 Similares a ReceptoresAsunto(s)
Síndrome de Bardet-Biedl/genética , Herencia Multifactorial , Poliposis Adenomatosa del Colon/genética , Alelos , Animales , Genes APC , Genes Dominantes , Genes Recesivos , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Proteínas Asociadas a Microtúbulos , Atrofia Muscular Espinal/genética , Mutación , Fenotipo , Proteínas/genéticaRESUMEN
This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. beta-Galactosidase expression was observed in alpha and gamma motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).
Asunto(s)
Genes , Técnicas Genéticas , Neuronas Motoras/metabolismo , Médula Espinal/citología , Animales , Transporte Biológico , Núcleo Celular/metabolismo , ADN/farmacocinética , Escherichia coli/genética , Vectores Genéticos , Histocitoquímica , Inyecciones , Inyecciones Intramusculares , Operón Lac , Neuronas Motoras/citología , Plásmidos , Ratas , Nervio Ciático , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Erythrocyte ghost membranes have been prepared by two different methods from patients with Duchenne muscular dystrophy (DMD), carriers of DMD, patients with other neuromuscular diseases, and normal individuals. The susceptibility of the membrane Na+,K+-adenosine triphosphatase (ATPase) to the cardiac glycoside, ouabain, has been investigated using various assay conditions. A stimulation of the enzyme has not been detected under any of the conditions employed. Using either a "high salt" (100 mM NaCl, 20 mM KCl) or a "low salt" (1 mM NaCl, 2 mM KCl) assay in the presence of EGTA a reduced susceptibility of the enzyme to ouabain was observed in preparations from patients with DMD compared with those from normal individuals. This behaviour was not manifest in preparations from NAD carriers or from patients with other neuromuscular diseases. The response of the erythrocyte membrane Na+,K+-ATPase activity to changes in temperature has also been investigated. The temperature response of the enzyme from DMD and DMD carrier preparations was indistinguishable from that of normal preparations. In all cases a break in the Arrhenius plot occurred at 21 degrees C.
Asunto(s)
Membrana Eritrocítica/enzimología , Eritrocitos/enzimología , Distrofias Musculares/enzimología , ATPasa Intercambiadora de Sodio-Potasio/sangre , Adolescente , Adulto , ATPasas Transportadoras de Calcio/sangre , Niño , Preescolar , Tamización de Portadores Genéticos , Humanos , Lactante , Magnesio/sangre , Distrofias Musculares/genética , Ouabaína/farmacología , TemperaturaRESUMEN
OBJECTIVE: To evaluate the potential importance of dystrophin, alpha-sarcoglycan (adhalin), and beta-dystroglycan, by use of western blot analysis, in several breeds of dogs with dilated cardiomyopathy. SAMPLE POPULATION: Myocardial samples obtained from 12 dogs were evaluated, including tissues from 7 dogs affected with dilated cardiomyopathy, 4 control dogs with no identifiable heart disease (positive control), and 1 dog affected with Duchenne muscular dystrophy (negative control for dystrophin). Of the affected dogs, 4 breeds were represented (Doberman Pinscher, Dalmatian, Bullmastiff, and Irish Wolfhound). PROCEDURE: Western blot analysis was used for evaluation of myocardial samples obtained from dogs with and without dilated cardiomyopathy for the presence of dystrophin and 2 of its associated glycoproteins, alpha-sarcoglycan and beta-dystroglycan. RESULTS: Detectable differences were not identified between dogs with and without myocardial disease in any of the proteins evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: Abnormalities in dystrophin, alpha-sarcoglycan, and beta-dystroglycan proteins were not associated with the development of dilated cardiomyopathy in the dogs evaluated in this study. In humans, the development of molecular biological techniques has allowed for the identification of specific causes of dilated cardiomyopathy that were once considered to be idiopathic. The use of similar techniques in veterinary medicine may aid in the identification of the cause of idiopathic dilated cardiomyopathy in dogs, and may offer new avenues for therapeutic intervention.