Biomedical subjects
Publications and source records attributed to H Stedman.
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Muscle type phosphofructokinase (M-PFK) deficiency is a rare inherited glycogen storage disease in humans that causes exertional myopathy and hemolysis. The molecular basis of canine M-PFK deficiency, the only naturally occurring animal homologue, was investigated. Lack of M-PFK enzyme activity was caused by a nonsense mutation in the penultimate exon of the M-PFK gene, leading to rapid degradation of a truncated (40 amino acids) and therefore unstable M-PFK protein. A polymerase chain reaction-based test was devised to identify M-PFK-deficient and carrier animals. This represents one of only a few inborn errors of metabolism where the molecular defect has been identified in a large animal model which can now be used to develop and assess novel therapeutic strategies.
Analysis of deduced protein sequence and structural motifs of approximately 5500 residues of human fetal skeletal muscle nebulin reveals the design principles of this giant multifunctional protein in the sarcomere. The bulk of the sequence is constructed of approximately 150 tandem copies of approximately 35-residue modules that can be classified into seven types. The majority of these modules form 20 super-repeats, with each super-repeat containing a 7-module set (one of each type in the same order). These super-repeats are further divided into eight segments: with six segments containing adjacent, highly homologous super-repeats, one single repeat segment consisting of 8 nebulin modules of the same type, and a non-repeat segment terminating with a SH3 domain at the C terminus. The interactions of actin, tropomyosin, troponin, and calmodulin with nebulin fragments consisting of either repeating modules or the SH3 domain support its role as a giant actin-binding cofilament of the composite thin filament. Such affinity profiles also suggest that nebulin may bind to tropomyosin and troponin to form a composite calcium-linked regulatory complex on the thin filament. The modular construction, super-repeat structure, and segmental organization of nebulin sequence appear to encode thin filament length, periodicity, insertion, and sarcomere proportion in the resting muscle.
The canine muscle-type-phosphofructokinase-encoding gene (M-PFK) was sequenced by using a combination of cDNA cloning and RT-PCR amplification. The canine M-PFK sequence shares 88 and 90% identity with rabbit and human M-PFK, respectively. The canine ORF was determined to be 6-bp longer than either human or rabbit M-PFK due to a 6-bp insertion at the end of exon 13.
A 3-year-old female American Cocker Spaniel with a chronic hemolytic disorder and hemolytic crises was found to have M-type phosphofructokinase deficiency. This inherited erythroenzymopathy and myopathy is commonly diagnosed in English Springer Spaniels, but the family study of this Cocker Spaniel, although supporting an autosomal recessive mode of inheritance, did not reveal any English Springer Spaniel ancestors. Molecular genetic studies did, however, identify the same mutation in this dog as we previously reported in the English Springer Spaniel breed, suggesting that this mutation originated prior to the separation of these 2 breeds.
Duchenne muscular dystrophy (DMD) is the most common and the most severe of the muscular dystrophies in man. It is inherited as an X-linked recessive trait and is characterized by ongoing necrosis of skeletal muscle fibres with regeneration and eventually fibrosis and fatty infiltration. Although the gene and gene product which are defective in DMD have recently been identified, the pathogenesis of the disease is still poorly understood. A myopathy has been described in the dog which has been shown to be inherited as an X-linked trait and which is therefore a potential model of the human disease. We have studied the phenotypic expression of the disease, canine X-linked muscular dystrophy (CXMD), and have examined the molecular relationship between it and DMD. We report here that dogs with CXMD faithfully mimic the phenotype of Duchenne muscular dystrophy and that they lack the Duchenne gene transcript and its protein product, dystrophin.
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.
Myology has greatly benefited from the recent unification of concepts in molecular, cellular, and developmental biology. The interplay between intrinsic and extrinsic factors in determining the physiologic characteristics of individual myofibers has emerged as an important theme. Of special note is the manner in which the study of contractile protein gene structure and expression has contributed to our understanding of the development and ultimate plasticity of the contractile apparatus. As mechanistic models of normal myogenesis achieve increasing sophistication, the opportunities for understanding the pathogenesis of progressive muscle disfunction improve. In this article we review recent progress in basic myology which will be of interest to clinicians studying the heritable neuromuscular disorders.
The contribution of "reverse genetic" strategies to neuromuscular disease research is evident in the progression of breakthroughs that have recently culminated in the cloning of the Duchenne muscular dystrophy (DMD) cDNA. The resultant improvements in our understanding of the genetic basis of Becker muscular dystrophy (BMD) and DMD serve as models for similar investigation of other heritable disorders. These genetic advances have outpaced concurrent work on the molecular pathogenesis of the dystrophic process, with the curious result that inferences about the DMD protein's amino acid sequence have preceded any information about its function or intracellular localization. In recognition that this foundation sets the stage for the rapid elucidation of the disease's pathogenesis, we review the experimental basis of such advances, with reference to relevant progress in basic myology, pathology, and molecular biology. We conclude with a view towards the ultimate clinical implications of these experimental breakthroughs.
By virtue of the protein's size, myofibrillar localization, and proposed functional role, the gene encoding the giant sarcomere matrix protein nebulin represents a possible site for myopathic mutations. Using polyclonal anti-nebulin antisera to screen a cDNA expression library, we have isolated and characterized two separate human fetal muscle cDNA clones. By recovering fusion polypeptide-bound portions of our polyclonal antiserum and reutilizing them to probe Western blots, we further demonstrate that the expressed cDNAs encode polypeptide epitopes unique to the protein nebulin. Both cDNAs detect a 25-kb skeletal muscle RNA transcript and localize to human chromosome 2. The identification of nebulin cDNA clones enables the complete analysis of this enormous mRNA by transcript walking through muscle cDNA libraries. Here we report a restriction map of the 3' end of the human nebulin transcript, with reference to the genomic fragments identified by the cDNA.