In vitro expressed dystrophin fragments do not associate with each other

Dystrophin, a component of the muscle membrane cytoskeleton, is the protein altered in Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). Dystrophin shares significant homology with other cytoskeletal proteins, such as α-actinin and spectrin. On the basis of its sequence similarity with α-actinin and spectrin, dystrophin has been proposed to function as dimer. However, the existence of both dimers and monomers have been observed by electron microscopy. To address this apparent discrepancy, we expressed dystrophin fragments composed of different domains in an in vitro translation system. The expressed fragments were tested for their ability to interact with each other and full-length dystrophin by both immunoprecipitation and blot overlay assays. These assays were successfully used to demonstrate the dimerization of α-actinin and spectrin, yet failed to detect any interaction between dystrophin fragments. Although these in vitro results do not prove that dystrophin is not a dimer in vivo, they do indicate that this interaction is not like that of the α-actinin and spectrin.     

Mutational analysis of the PEX gene in patients with X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets (HYP) is a dominant disorder characterized by renal phosphate wasting and abnormal vitamin D metabolism. PEX, the gene that is defective in HYP and is located on Xp22.1, is homologous to members of the neutral endopeptidase family. However, the complete coding sequence of the PEX cDNA, the structure of the PEX gene, and the role that PEX plays in phosphate transport remain unknown. We determined the genomic structure of the published PEX gene, which was found to be composed of 18 short exons, and demonstrated that the genomic organization of PEX shares homology to members of the family of neutral endopeptidases. Primer sets were designed from the intron sequence, to amplify each PEX exon from genomic DNA of HYP patients. Mutations in PEX were identified in 9/22 unrelated HYP patients, confirming that defects in PEX are responsible for HYP. The mutations detected included three nonsense mutations, a 1-bp deletion leading to a frameshift, a donor splice-site mutation, and missense mutations in four patients. Although the entire PEX gene has not been identified and some mutations may have been missed, the lack of detection of mutations in the remaining 13 patients, especially in 1 patient who has an apparently balanced, de novo 9;13 translocation, implies that there may be other loci involved in the generation of the HYP phenotype.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

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Modeling human muscle disease in zebrafish

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.     

Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models.

Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF beta were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on <20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-gamma-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-gamma treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-beta synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-beta, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.