PubMed Health⌕ Search

Biomedical subjects

L M Kunkel

Publications and source records attributed to L M Kunkel.

At least 55 records · Page 3Linked to original sources
PubMed1997

Dystrobrevin and dystrophin: an interaction through coiled-coil motifs.

Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction. Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex. Although the nature of dystrobrevin's association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins. Using the yeast two-hybrid system to identify protein-protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin. The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin. We further refined this site of interaction to these proteins' homologous coiled-coil motifs that flank their respective syntrophin-binding sites. We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction. From the accumulated evidence of protein-protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.

Amino Acid Sequence↗
PubMed1997

Dp140: alternatively spliced isoforms in brain and kidney.

Dp140, a protein composed of the distal rod domain and carboxy-terminal domain of dystrophin, is expressed only in brain and kidney; transcription is initiated at a unique first exon located in dystrophin intron 44. Both tissues express specific isoforms with distinct alternative splicing of exons 71-74 and 78. The carboxy-terminal domain of Dp140 is identical to that of full-length 427-kDa dystrophin, and it is this region that is associated, with the "dystrophin-associated protein" complex in skeletal muscle. Alternative splicing encodes domains that have been shown to be protein-binding regions; thus this alternative splicing may regulate the association of Dp140 with integral membrane proteins. The 5' flanking region of genomic DNA adjacent to the Dp140 first exon contains a variety of transcription factor-binding motifs, some of which could regulate neuroglial-specific gene expression.

Alternative Splicing↗
PubMed1997

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 alpha-actinin and spectrin. On the basis of its sequence similarity with alpha-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 alpha-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 alpha-actinin and spectrin.

Blotting, Western↗
PubMed1997

Molecular characterization of Br-cadherin, a developmentally regulated, brain-specific cadherin.

Cadherins are a family of transmembrane proteins that play a crucial role in cell adhesion and in morphogenesis. Several of the cadherins are expressed in the nervous system, but none is neuron-specific. We characterize a new member of the cadherin family, Br-cadherin, which is present exclusively in the central nervous system. Although the Br-cadherin protein is confined to the central nervous system, its mRNA is present in several additional tissues, suggesting that there is posttranscriptional control of this gene's expression. Within the central nervous system, Br-cadherin appears to be expressed specifically by neurons. In the mouse, its expression becomes detectable during the first postnatal week, which corresponds temporally to the onset of synaptogenesis and dendrite outgrowth in the brain. This pattern of expression is consistent with a role for Br-cadherin in neuronal development, perhaps specifically with synaptogenesis.

Aging↗
PubMed1997

The genomic organization of human dystrobrevin.

Dystrophin-related and dystrophin-associated proteins (DAPs) are thought to play an important role in the stability and maintenance of the plasma membrane during muscle contraction and relaxation. Studies conducted on the electric organ of Torpedo californica have shown that some of the DAPs are also involved in the formation and maintenance of neuromuscular junctions (NMJs). In addition, dystrophin and several DAPs have been shown to be the primary genetic defect in a number of phenotypically similar muscular dystrophies. We previously reported the identification and characterization of human dystrobrevin, a protein which is unique in being both homologous to dystrophin and a dystrophin-associated protein. Here we describe the genomic organization of the human dystrobrevin gene. It is encoded by 23 exons spanning at least 180 kb of chromosome 18q12. Three different C-termini of dystrobrevin are generated by the mutually exclusive mRNA splicing of three exons. Two alternatively spliced exons (exons 11A and 12) are utilized exclusively in striated muscles. A comparison between the genomic organization of dystrophin and human dystrobrevin shows that the two genes have significant similarities in their genomic structure, implying an ancestral or evolutionary relationship. Based on intronic sequence, a primer set was designed to specifically amplify each exon of dystrobrevin to screen for mutations by SSCP in patients with neuromuscular diseases for which dystrobrevin could be a candidate.

Arrhythmogenic Right Ventricular Dysplasia↗
PubMed1997

The fate of individual myoblasts after transplantation into muscles of DMD patients.

Muscle biopsies from six patients with Duchenne muscular dystrophy (DMD) participating in a myoblast transplantation clinical trial were reexamined using a fluorescence in situ hybridization (FISH)-based method. Donor nuclei were detected in all biopsies analyzed, including nine where no donor myoblasts were previously thought to be present. In three patients, more than 10% of the original number of donor cells were calculated as present 6 months after implantation. Half of the detected donor nuclei were fused into host myofibers, and of these, nearly 50% produced dystrophin. These findings demonstrate that although donor myoblasts have persisted after injection, their microenvironment influences whether they fuse and express dystrophin. Our methodology could be used for developing new approaches to improve myoblast transfer efficacy and for the analysis of future gene- and/or cell-based therapies of numerous genetic disorders.

Cell Count↗
PubMed1997

A multicopy transcription-repair gene, BTF2p44, maps to the SMA region and demonstrates SMA associated deletions.

The childhood-onset spinal muscular atrophies are a clinically heterogeneous group of autosomal recessive disorders characterized by selective degeneration of the anterior horn cells with subsequent weakness and atrophy of limb muscles. The disease locus has been mapped to a region of chromosome 5q13 characterized by genetic instability and DNA duplication. Among the duplicated genes in this region, SMNT (telomeric copy; survival motor neuron) is thought to be the major disease determining gene since it is missing in the majority of SMA patients and since small, intragenic mutations in the gene have been associated with the disorder. Approximately half of the severely affected SMA I patients are also missing both homologues of a neighboring gene, the neuronal apoptosis inhibitory protein (NAIP). These data indicate that loss of NAIP may affect disease severity and further, that the molecular events underlying the childhood-onset SMAs are complex, possibly involving multiple genes. We report a third multicopy gene in the SMA region, encoding the p44 subunit of basal transcription factor II (BTF2p44). One copy of this transcription-repair gene is deleted in at least 15% of all SMA cases.

Chromosome Mapping↗
PubMed1997

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.

Adult↗
PubMed1997

Dystrophies and heart disease.

The muscular dystrophies are a clinically and genetically heterogeneous group of skeletal muscle-wasting diseases that differ widely in their frequency and pattern of cardiac involvement. Myocardial disease manifesting predominantly as cardiomyopathy and congestive heart failure is characteristic of Duchenne and Becker muscular dystrophies and X-linked dilated cardiomyopathy, whereas conduction system abnormalities that cause heart block, arrhythmias, and sudden death are more commonly seen in limb-girdle type 1B, myotonic, and Emery-Dreifuss muscular dystrophies. Primary defects in the mechanical stabilization of the plasma membrane and signal transduction may underlie these two groups of muscular dystrophies. The identification of several new disease genes has yielded additional insights into the pathophysiology of muscular dystrophy. Molecular genetic and biochemical analyses of patient samples now permit accurate diagnosis and genotype-phenotype correlations. Ultimately, this knowledge will provide the foundation for etiology-specific gene therapy.

Cardiomyopathies↗
PubMed1996

The mouse region syntenic for human spinal muscular atrophy lies within the Lgn1 critical interval and contains multiple copies of Naip exon 5.

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval for Lgn1, a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacterium Legionella pneumophila. We have generated a mouse YAC contig across the Lgn1/Sma interval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to the Lgn1 critical region, making these two genes candidates for the Lgn1 phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy of Naip exon 5. Identification of the Lgn1 gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.

Animals↗
PubMed1996

The three human syntrophin genes are expressed in diverse tissues, have distinct chromosomal locations, and each bind to dystrophin and its relatives.

The syntrophins are a biochemically heterogeneous group of 58-kDa intracellular membrane-associated dystrophin-binding proteins. We have cloned and characterized human acidic (alpha 1-) syntrophin and a second isoform of human basic (beta 2-) syntrophin. Comparison of the deduced amino acid structure of the three human isoforms of syntrophin (together with the previously reported human beta 1-syntrophin) demonstrates their overall similarity. The deduced amino acid sequences of human alpha 1- and beta 2-syntrophin are nearly identical to their homologues in mouse, suggesting a strong functional conservation among the individual isoforms, Much like beta 1-syntrophin, human beta 2-syntrophin has multiple transcript classes and is expressed widely, although in a distinct pattern of relative abundance. In contrast, human alpha 1-syntrophin is most abundant in heart and skeletal muscle, and less so in other tissues. Somatic cell hybrids and fluorescent in situ hybridization were both used to determine their chromosomal locations: beta 2-syntrophin to chromosome 16q22-23 and alpha 1-syntrophin to chromosome 20q11.2. Finally, we used in vitro translated proteins in an immunoprecipitation assay to show that, like beta 1-syntrophin, both beta 2- and alpha 1-syntrophin interact with peptides encoding the syntrophin-binding region of dystrophin, utrophin/dystrophin related protein, and the Torpedo 87K protein.

Adult↗
PubMed1996

A method to codetect introduced genes and their products in gene therapy protocols.

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.

Adult↗
PubMed1996

Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy.

Recently, mutations in the genes encoding several of the dystrophin-associated proteins have been identified that produce phenotypes ranging from severe Duchenne-like autosomal recessive muscular dystrophy to the milder limb-girdle muscular dystrophies (LGMDs). LGMD type 2C is generally associated with a more severe clinical course and is prevalent in northern Africa. A previous study identified a single base pair deletion in the gene encoding the dystrophin-associated protein gamma-sarcoglycan in a number of Tunisian muscular dystrophy patients. To investigate whether gamma-sarcoglycan gene mutations cause autosomal recessive muscular dystrophy in other populations, we studied 50 muscular dystrophy patients from the United States and Italy. The muscle biopsies from these 50 patients showed no abnormality of dystrophin but did show diminished immunostaining for the dystrophin-associated protein alpha-sarcoglycan. Four patients with a severe muscular dystrophy phenotype were identified with homozygous, frameshifting mutations in gamma-sarcoglycan. Two of the four have microdeletions that disrupt the distal carboxyl-terminus of gamma-sarcoglycan yet result in a complete absence of gamma-and beta-sarcoglycan suggesting the importance of this region for stability of the sarcoglycan complex. This region of gamma-sarcoglycan, like beta-sarcoglycan, has a number of cysteine residues similar to those in epidermal growth factor cysteine-rich regions.

Amino Acid Sequence↗
PubMed1996

Genomic screening for beta-sarcoglycan gene mutations: missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E).

Autosomal recessive limb-girdle muscular dystrophies (LGMDs) are genetically heterogeneous. A subgroup of these disorders is caused by mutations in the dystrophin-associated sarcoglycan complex. Truncating mutations in the 43 kDa beta-sarcoglycan gene (LGMD 2E) were originally identified in a sporadic case of Duchenne-like muscular dystrophy, and a common missense mutation (T151R) was identified independently in Indiana Amish pedigrees with a milder form of LGMD. To facilitate mutational analysis of larger numbers of patients directly from genomic DNA, as opposed to reverse transcribed RNA from muscle biopsies, we have determined the genomic structure of the beta-sarcoglycan gene. The open reading frame of the beta-sarcoglycan coding region extends over six exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single strand conformation polymorphism (SSCP) analysis. We screened 15 patients from the Brazilian LGMD patient population, 13 of whom followed a severe course. Most of the patients had been assessed previously for deficiency of alpha-sarcoglycan immunofluorescence on muscle biopsy sections as a marker for disease of the sarcoglycan complex. Novel mutations in two familial and two sporadic cases of severe childhood-onset LGMD were identified. Only one of these patients carried a truncating mutation (homozygous 2 bp deletion, FS164TER), while the other three carried missense mutations (homozygous R91P, homozygous M100K, heterozygous recessive L108R; only one allele could be identified in this family). All three missense mutations occurred in exon 3, coding for the immediate extracellular domain. Complete absence for all three of the known sarcoglycans was noted by immunohistochemistry on muscle biopsy sections of the patients.

Amino Acid Sequence↗
PubMed1996

The sarcoglycan complex in the six autosomal recessive limb-girdle muscular dystrophies.

To enhance our understanding of the autosomal recessive limb-girdle muscular dystrophy (LGMD), patients from six genetically distinct forms (LGMD2A to LGMD2F) were studied with antibodies directed against four sarcoglycan subunits (alpha-, beta-, gamma-, delta-SG), dystrophin, beta-dystroglycan (beta-DG) and merosin. All patients with LGMD2A and 2B had a mild clinical course while those with a primary sarcoglycan mutation (LGMD2C to 2F) had a range of clinical severity. Dystrophin and merosin immunofluorescence pattern was positive in patients with all six AR LGMDs. The majority of patients with a severe Duchenne-like phenotype presented total absence of the SG complex. However, some exceptions were found in 13q linked patients, indicating that the presence of a certain labelling for components of the SG may not be prognostic for a milder phenotype. The observation that the primary absence of alpha-SG results in the total absence of beta- and delta-SG but not of gamma-SG suggests that the alpha-, beta- and delta-subunits of sarcoglycan may be more closely associated. A secondary reduction in dystrophin amount was seen in patients with primary sarcoglycan mutations, which was most marked in patients with primary beta-, gamma- and delta-SG deficiencies. In contrast, beta-DG staining was retained in all patients, suggesting that the association between SG and DG subcomplexes is not so strong. Based on the above findings, we have refined the model for the interaction among the known glycoproteins of the sarcoglycan complex, within the DGC.

Adolescent↗
PubMed1996

Cloning and characterization of the human homologue of a dystrophin related phosphoprotein found at the Torpedo electric organ post-synaptic membrane.

Dystrophin is the protein product which is absent in Duchenne muscular dystrophy (DMD). In mammalian skeletal muscle, dystrophin is found in association with several integral and peripheral membrane proteins, forming a complex known as the dystrophin glycoprotein complex (DGC). In an expressed sequence tag (EST) database search to identify new dystrophin related genes, we isolated EST00891 which showed 57% homology to the cysteine-rich domain of dystrophin and localized to 18q12.1-12.2. This EST is also highly homologous (90%) to the Torpedo californica post-synaptic 87 kDa phosphoprotein. Screening human adult brain and skeletal muscle cDNA libraries with this EST resulted in cloning multiple cDNAs which encode several splice forms all homologous to the C-terminal domain of dystrophin. The largest open reading frame isolated shows 94% homology (86% identity) to the Torpedo 87 kDa protein and 50% homology to the cysteine-rich and carboxy-terminal domains of dystrophin. The other cDNAs isolated encode smaller splice forms of this gene which we have named dystrobrevin. The tissue distribution of dystrobrevin mRNA shows five distinct transcripts which are preferentially expressed between different tissues. In addition, antibodies against either the Torpedo 87 kDa protein or human dystrobrevin demonstrate that at least three of the splice forms are translated as proteins in human brain tissue extracts.

Alternative Splicing↗