text:
G2Cdb::Gene report
- Gene id
- Gene symbol
- Species
- Homo sapiens
- Description
- dystrobrevin, alpha
- Orthologue
- G00000326 (Mus musculus)
Databases (8)
- Curated Gene
- OTTHUMG00000068849 (Vega human gene)
- Gene
- ENSG00000134769 (Ensembl human gene)
- 1837 (Entrez Gene)
- 670 (G2Cdb plasticity & disease)
- DTNA (GeneCards)
- Literature
- Marker Symbol
- Protein Sequence
- Q9Y4J8 (UniProt)
Synonyms (6)
Literature (40)
Pubmed - other
Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat.
Division of Medical & Molecular Genetics, King's College London, London, UK. email@example.com
Background: The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain) is its distant relative, alpha-dystrobrevin. The alpha-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands) in the dystrophin glycoprotein complex.
Results: We show here that, contrary to the literature, most alpha-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse alpha-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms alpha-dystrobrevin-4 and -5) are present in most mammals but specifically ablated in mouse and rat.
Conclusion: Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the alpha-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the alpha-dystrobrevins (and therefore the dystrophin glycoprotein complexes) of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.
Funded by: Biotechnology and Biological Sciences Research Council: S16843
BMC biology 2009;7;85
Defining the human deubiquitinating enzyme interaction landscape.
Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.
Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945
Cell 2009;138;2;389-403
PUBMED: 19615732; PMC: 2716422; DOI: 10.1016/j.cell.2009.04.042
Aberrantly spliced alpha-dystrobrevin alters alpha-syntrophin binding in myotonic dystrophy type 1.
Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan.
Background: Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms in DM1, but the cause of muscle wasting is still unknown. By contrast, many forms of muscular dystrophy are caused by abnormalities of the dystrophin-glycoprotein complex (DGC). alpha-Dystrobrevin is a key component of the DGC in striated muscle and plays important roles in maturation and signal transduction by interacting with alpha-syntrophin. The goal of this study was to investigate alternative splicing of alpha-dystrobrevin in DM1 and examine alpha-syntrophin binding of different alpha-dystrobrevin splice isoforms.
Methods: Splicing patterns of alpha-dystrobrevin in DM1 muscle were studied by reverse-transcriptase PCR. Expression of the variant splice isoform was examined by immunoblotting and immunohistochemistry. Alternatively spliced isoforms were expressed in cultured cells to investigate interaction with alpha-syntrophin. alpha-Syntrophin expression was examined by immunoblotting.
Results: alpha-Dystrobrevin mRNA including exons 11A and 12 was increased in both skeletal and cardiac muscle of DM1 patients. The aberrantly spliced alpha-dystrobrevin isoform was localized to the sarcolemma, and showed increased binding with alpha-syntrophin. Furthermore, levels of alpha-syntrophin associated with the DGC were increased in DM1 muscle.
Conclusion: Alternative splicing of alpha-dystrobrevin is dysregulated in myotonic dystrophy type 1 (DM1) muscle, resulting in changes in alpha-syntrophin binding. These results raise the possibility that effects on alpha-dystrobrevin splicing may influence signaling in DM1 muscle cells.
Neurology 2008;70;9;677-85
PUBMED: 18299519; DOI: 10.1212/01.wnl.0000302174.08951.cf
A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Many human inherited neurodegenerative disorders are characterized by loss of balance due to cerebellar Purkinje cell (PC) degeneration. Although the disease-causing mutations have been identified for a number of these disorders, the normal functions of the proteins involved remain, in many cases, unknown. To gain insight into the function of proteins involved in PC degeneration, we developed an interaction network for 54 proteins involved in 23 inherited ataxias and expanded the network by incorporating literature-curated and evolutionarily conserved interactions. We identified 770 mostly novel protein-protein interactions using a stringent yeast two-hybrid screen; of 75 pairs tested, 83% of the interactions were verified in mammalian cells. Many ataxia-causing proteins share interacting partners, a subset of which have been found to modify neurodegeneration in animal models. This interactome thus provides a tool for understanding pathogenic mechanisms common for this class of neurodegenerative disorders and for identifying candidate genes for inherited ataxias.
Funded by: NICHD NIH HHS: HD24064; NINDS NIH HHS: NS27699
Cell 2006;125;4;801-14
PUBMED: 16713569; DOI: 10.1016/j.cell.2006.03.032
Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.
Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.
By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.
Genome research 2006;16;1;55-65
PUBMED: 16344560; PMC: 1356129; DOI: 10.1101/gr.4039406
The syntrophin-dystrobrevin subcomplex in human neuromuscular disorders.
Institute for Neuromuscular Research, Children's Hospital at Westmead, NSW, Australia.
The syntrophins and alpha-dystrobrevin form a subcomplex with dystrophin at the skeletal muscle membrane, and are also highly concentrated at the neuromuscular synapse. Here we demonstrate that the different syntrophins and alpha-dystrobrevin isoforms have distinct expression patterns during human skeletal muscle development, and are differentially affected by loss of dystrophin anchorage and denervation in human neuromuscular disease. During normal fetal development, and in Duchenne muscular dystrophy and denervation disorders, alpha1-syntrophin and alpha-dystrobrevin are absent or markedly reduced at the sarcolemmal membrane. beta1-Syntrophin is the predominant syntrophin isoform expressed at the muscle membrane during development, and it undergoes upregulation in response to loss of alpha1-syntrophin in Duchenne muscular dystrophy and in denervation. Upregulation of beta1-syntrophin in neuromuscular disorders is associated with re-expression of the fetal nicotinic acetylcholine receptor gamma-subunit, cardiac actin, and neonatal myosin, suggesting reversion of muscle fibers to an immature phenotype. We show that denervation specifically affects expression of the syntrophin-dystrobrevin subcomplex and does not affect levels or localization of other members of the dystrophin-associated protein complex. Our results confirm that dystrophin is required for anchorage of the syntrophin-dystrobrevin subcomplex and suggest that expression of the syntrophin-dystrobrevin complex may be independently regulated through neuromuscular transmission.
Funded by: NINDS NIH HHS: R01 NS033145
Journal of neuropathology and experimental neurology 2005;64;4;350-61
PUBMED: 15835271; DOI: 10.1093/jnen/64.4.350
Association of alpha-dystrobrevin with reorganizing tight junctions.
Division of Medical Microbiology, Department of Molecular and Clinical Medicine, Faculty of Health Sciences, Linköping University, Linköping, 581 85, Sweden. email@example.com
Alpha-dystrobrevin (alpha-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of alpha-DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, alpha-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of alpha-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-alpha-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of alpha-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of alpha-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of alpha-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization.
The Journal of membrane biology 2005;203;1;21-30
PUBMED: 15834686; DOI: 10.1007/s00232-004-0728-1
Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins.
Department of Molecular, Cellular, University of California, Santa Barbara, California 93106, USA.
Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.
Funded by: NINDS NIH HHS: NS33145, NS43377
The Journal of biological chemistry 2004;279;21;22331-46
PUBMED: 15024025; DOI: 10.1074/jbc.M400285200
DAMAGE, a novel alpha-dystrobrevin-associated MAGE protein in dystrophin complexes.
Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195-7290, USA.
Mice rendered null for alpha-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220) Thus, alpha-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity. We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve. In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation. DAMAGE associates directly with alpha-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain. This co-immunoprecipitation is dependent on the presence of alpha-dystrobrevin but not beta-dystrobrevin. The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains. The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288). DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons. In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei. DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin. These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease.
The Journal of biological chemistry 2004;279;8;7014-23
PUBMED: 14623885; DOI: 10.1074/jbc.M312205200
Complete sequencing and characterization of 21,243 full-length human cDNAs.
Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.
As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.
Nature genetics 2004;36;1;40-5
PUBMED: 14702039; DOI: 10.1038/ng1285
Deficiency of the syntrophins and alpha-dystrobrevin in patients with inherited myopathy.
Institute for Neuromuscular Research, The Children's Hospital at Westmead, Locked Bag 4001, NSW 2145, Westmead, Australia.
The syntrophins and dystrobrevins are members of the dystrophin-associated protein complex, and are thought to function as modular adaptors for signalling proteins recruited to the sarcolemmal membrane. We have characterised the expression of the syntrophins (alpha-, beta1-, and beta2-) and alpha-dystrobrevin by immunohistochemistry in normal human muscle and in biopsies from 162 patients with myopathies of unknown aetiology (with normal staining for dystrophin and other dystrophin-associated proteins). Unlike mice, beta2-syntrophin is expressed at the sarcolemma in post-natal human skeletal muscle. Deficiency of alpha-dystrobrevin +/- beta2-syntrophin was present in 16/162 (10%) patients, compared to age-matched controls. All patients presented with congenital-onset hypotonia and weakness, although there was variability in clinical severity. Two major clinical patterns emerged: patients with deficiency of beta2-syntrophin and alpha-dystrobrevin presented with severe congenital weakness and died in the first year of life, and two patients with deficiency of alpha-dystrobrevin had congenital muscular dystrophy with complete external ophthalmoplegia. We have sequenced the coding regions of alpha-dystrobrevin and beta2-syntrophin in these patients, and identified a new isoform of dystrobrevin, but have not identified any mutations. This suggests that disease causing mutations occur outside the coding region of these genes, in gene(s) encoding other components of the syntrophin-dystrobrevin subcomplex, or in gene(s) responsible for their post-translational modification and normal localisation.
Funded by: NINDS NIH HHS: NS33145
Neuromuscular disorders : NMD 2003;13;6;456-67
Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions.
Dept. of Pediatrics, Washington University School of Medicine, Pediatric Research Bldg., St. Louis, MO 63110, USA. email@example.com
alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.
The Journal of cell biology 2003;160;5;741-52
PUBMED: 12604589; PMC: 2173352; DOI: 10.1083/jcb.200209045
Characterization of human alpha-dystrobrevin isoforms in HL-60 human promyelocytic leukemia cells undergoing granulocytic differentiation.
Division of Medical Microbiology, Linköpings Universitet, SE-581 85 Linköping, Sweden. firstname.lastname@example.com
The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.
Molecular biology of the cell 2002;13;12;4195-205
PUBMED: 12475945; PMC: 138626; DOI: 10.1091/mbc.e02-03-0128
Dystrobrevin dynamics in muscle-cell signalling: a possible target for therapeutic intervention in Duchenne muscular dystrophy?
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. firstname.lastname@example.com
The dystrophin-protein complex forms one of the connections between the extracellular matrix and the cytoskeleton of muscle. This link is disrupted in patients with Duchenne and Becker muscular dystrophies. Dystrobrevin is a component of the dystrophin-protein complex that binds to the C-terminus of dystrophin and also to syntrophin. As its name suggests, dystrobrevin is a relative of dystrophin participating in similar intermolecular interactions. Dystrobrevin-deficient mice have a form of muscular dystrophy that leaves the sarcolemma and dystrophin-protein complex intact but affects an as yet unidentified signalling pathway in muscle. Given that the up-regulation of several genes has a beneficial effect on the muscle in some dystrophic mouse models, alpha-dystrobrevin has a number of properties that might be protective in muscular dystrophy. This article discusses the function of dystrobrevin in muscle and reviews its suitability as a therapeutic target for treating patients with Duchenne and Becker muscular dystrophies.
Neuromuscular disorders : NMD 2002;12 Suppl 1;S110-7
Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors.
Department of Neurochemistry, Brain Research Institute, University of Zürich, CH-8057, Switzerland.
Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn -/- myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR beta and delta subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.
The Journal of cell biology 2002;157;5;883-95
PUBMED: 12034776; PMC: 2173406; DOI: 10.1083/jcb.200202110
The expression of alpha-dystrobrevin and dystrophin during skeletal muscle regeneration.
Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, Japan.
The expression of alpha-dystrobrevin and dystrophin in rat tibialis anterior muscles was chronologically evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. In immunohistochemical studies, alpha-dystrobrevin and dystrophin were first stained weakly at the sarcolemma of some regenerating muscle fibers on day 5. On day 7, alpha-dystrobrevin was still stained weakly, whereas dystrophin was stained conspicuously. After day 10, alpha-dystrobrevin and dystrophin were both stained conspicuously on almost all regenerating muscle fibers. In the Western blot analysis, alpha-dystrobrevin and dystrophin were first detected as visible bands on days 5 and 7, respectively. The bands of alpha-dystrobrevin and dystrophin both darkened sequentially up to day 10. The protein levels based on the densitometrical analysis of the bands on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. The sequential line based on these data showed that alpha-dystrobrevin and dystrophin reached 50% of the protein level on day 28 by 6.6 and 5.3 days, respectively. These data provide evidence that alpha-dystrobrevin regenerates more slowly than dystrophin in skeletal muscle.
Journal of muscle research and cell motility 2002;23;2;131-8
Dysbindin, a novel coiled-coil-containing protein that interacts with the dystrobrevins in muscle and brain.
Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom.
The dystrophin-associated protein complex (DPC) is required for the maintenance of muscle integrity during the mechanical stresses of contraction and relaxation. In addition to providing a membrane scaffold, members of the DPC such as the alpha-dystrobrevin protein family are thought to play an important role in intracellular signal transduction. To gain additional insights into the function of the DPC, we performed a yeast two-hybrid screen for dystrobrevin-interacting proteins. Here we describe the identification of a dysbindin, a novel dystrobrevin-binding protein. Dysbindin is an evolutionary conserved 40-kDa coiled-coil-containing protein that binds to alpha- and beta-dystrobrevin in muscle and brain. Dystrophin and alpha-dystrobrevin are co-immunoprecipitated with dysbindin, indicating that dysbindin is DPC-associated in muscle. Dysbindin co-localizes with alpha-dystrobrevin at the sarcolemma and is up-regulated in dystrophin-deficient muscle. In the brain, dysbindin is found primarily in axon bundles and especially in certain axon terminals, notably mossy fiber synaptic terminals in the cerebellum and hippocampus. These findings have implications for the molecular pathology of Duchenne muscular dystrophy and may provide an alternative route for anchoring dystrobrevin and the DPC to the muscle membrane.
The Journal of biological chemistry 2001;276;26;24232-41
PUBMED: 11316798; DOI: 10.1074/jbc.M010418200
Desmuslin, an intermediate filament protein that interacts with alpha -dystrobrevin and desmin.
Howard Hughes Medical Institute/Division of Genetics, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, alpha1-syntrophin, and the sarcoglycan complex. The precise role of alpha-dystrobrevin in skeletal muscle has not yet been determined. To study alpha-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and alpha-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.
Proceedings of the National Academy of Sciences of the United States of America 2001;98;11;6156-61
PUBMED: 11353857; PMC: 33438; DOI: 10.1073/pnas.111153298
Novel gene mutations in patients with left ventricular noncompaction or Barth syndrome.
Department of Pediatrics, Toyama Medical and Pharmaceutical University, Toyama, Japan.
Background: Mutations in the gene G4.5 result in a wide spectrum of severe infantile cardiomyopathic phenotypes, including isolated left ventricular noncompaction (LVNC), as well as Barth syndrome (BTHS) with dilated cardiomyopathy (DCM). The purpose of this study was to investigate patients with LVNC or BTHS for mutations in G4.5 or other novel genes.
DNA was isolated from 2 families and 3 individuals with isolated LVNC or LVNC with congenital heart disease (CHD), as well as 4 families with BTHS associated with LVNC or DCM, and screened for mutations by single-strand DNA conformation polymorphism analysis and DNA sequencing. In 1 family with LVNC and CHD, a C-->T mutation was identified at nucleotide 362 of alpha-dystrobrevin, changing a proline to leucine (P121L). Mutations in G4.5 were identified in 2 families with isolated LVNC: a missense mutation in exon 4 (C118R) in 1 and a splice donor mutation (IVS10+2T-->A) in intron 10 in the other. In a family with cardiomyopathies ranging from BTHS or fatal infantile cardiomyopathy to asymptomatic DCM, a splice acceptor mutation in exon 2 of G4.5 (398-2 A-->G) was identified, and a 1-bp deletion in exon 2 of G4.5, resulting in a stop codon after amino acid 41, was identified in a sporadic case of BTHS.
Conclusions: These data demonstrate genetic heterogeneity in LVNC, with mutation of a novel gene, alpha-dystrobrevin, identified in LVNC associated with CHD. In addition, these results confirm that mutations in G4.5 result in a wide phenotypic spectrum of cardiomyopathies.
Circulation 2001;103;9;1256-63
Syncoilin, a novel member of the intermediate filament superfamily that interacts with alpha-dystrobrevin in skeletal muscle.
Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom.
Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function. A key component of the dystrophin protein complex is alpha-dystrobrevin, a dystrophin-associated protein whose absence results in neuromuscular junction defects and muscular dystrophy. To gain further insights into the role of alpha-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel alpha-dystrobrevin-binding partner called syncoilin. Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with alpha-dystrobrevin-1. Expression studies in mammalian cells demonstrate that, while alpha-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments. Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is up-regulated in dystrophin-deficient muscle. These data suggest that alpha-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse.
The Journal of biological chemistry 2001;276;9;6645-55
PUBMED: 11053421; DOI: 10.1074/jbc.M008305200
Dystrobrevin localization in photoreceptor axon terminals and at blood-ocular barrier sites.
Departments of Anatomy and. Ophthalmology, Yamanashi Medical University, Japan. email@example.com
Purpose: Dystrobrevin is a newly discovered dystrophin-associated protein with multiple sites for phosphorylation on tyrosine residues. In the present study, the cellular distribution and subcellular localization of dystrobrevin were examined in the adult rat retina, cornea, lens, iris, ciliary body, and cultured Müller cells.
Methods: Immunoblot analysis, confocal laser scanning microscopy, and immunoelectron microscopy were used to examine dystrobrevin expression.
Results: Immunoblot analysis showed that an approximately 87-kDa band was expressed predominantly in the lens, retina, iris and ciliary body, whereas an approximately 60-kDa band was expressed in cultured Müller cells, cornea, retina, iris, and ciliary body. Confocal microscopy demonstrated dystrobrevin in the inner limiting membrane, outer plexiform layer, and retinal pigment epithelium and around blood vessels in the retina. At the ultrastructural level, dystrobrevin was localized under cell membranes of rod spherules and cone pedicles of photoreceptor cell terminals but often was found in the cytoplasm of endothelial cells and Müller cells. Furthermore, dystrobrevin was colocalized with beta-dystroglycan in corneal endothelium; lens, iris, and ciliary epithelia; and cultured Müller cells.
Conclusions: The present study demonstrates that dystrobrevin is expressed in neurons, glia, and endothelial cells in the rat retina. In addition, dystrobrevin is localized at the blood-ocular barrier sites in extraocular tissue. These data suggest that dystrobrevin plays an important role in visual function.
Investigative ophthalmology & visual science 2000;41;12;3908-14
Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex.
Department of Human Anatomy and Genetics, University of Oxford, UK.
Dystrophin coordinates the assembly of a complex of structural and signalling proteins that is required for normal muscle function. A key component of the dystrophin-associated protein complex (DPC) is alpha-dystrobrevin, a dystrophin-related and -associated protein whose absence results in muscular dystrophy and neuromuscular junction defects [1,2]. The current model of the DPC predicts that dystrophin and dystrobrevin each bind a single syntrophin molecule [3]. The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC [4]. Here we show, using yeast two-hybrid analysis and biochemical binding studies, that alpha-dystrobrevin in fact contains two independent syntrophin-binding sites in tandem. The previously undescribed binding site is situated within an alternatively spliced exon of alpha-dystrobrevin, termed the variable region-3 (vr3) sequence, which is specifically expressed in skeletal and cardiac muscle [5,6]. Analysis of the syntrophin-binding region of dystrobrevin reveals a tandem pair of predicted alpha helices with significant sequence similarity. These alpha helices, each termed a syntrophin-binding motif, are also highly conserved in dystrophin and utrophin. Together these data show that there are four potential syntrophin-binding sites per dystrophin complex in skeletal muscle: two on dystrobrevin and two on dystrophin or utrophin. Furthermore, alternative splicing of dystrobrevin provides a mechanism for regulating the stoichiometry of syntrophin association with the DPC. This is likely to have important consequences for the recruitment of specific signalling molecules to the DPC and ultimately for its function.
Current biology : CB 2000;10;20;1295-8
Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells.
Istituto di Patologia Generale ed Oncologia, Facoltà di Medicina, Seconda Università degli Studi di Napoli, 80138 Napoli, Italy.
Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins. The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.
The Journal of biological chemistry 2000;275;21;15851-60
PUBMED: 10747910; DOI: 10.1074/jbc.M000439200
Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex.
Department of Biology 'Enrico Calef', University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy.
The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy-terminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in beta-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to beta-dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin beta-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca(2+)-dependent binding of calmodulin. The remaining two motifs found in the carboxy-terminus (H1-H2) mediate the formation of utrophin-dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.
Funded by: NCI NIH HHS: CA01605; Telethon: 902
FEBS letters 2000;471;2-3;229-34
Biochemical evidence for association of dystrobrevin with the sarcoglycan-sarcospan complex as a basis for understanding sarcoglycanopathy.
Department of Cell Biology, National Institute of Neuroscience, NCNP, Ogawahigashi-chou, Kodaira, Tokyo 187-8502, Japan. email@example.com
The sarcoglycan complex is composed of four membrane-spanning dystrophin-associated proteins (DAPs) and is essential for skeletal muscle survival, since the absence or markedly reduced expression of this complex due to mutation of any one of the sarcoglycan genes causes a group of muscular dystrophies, collectively termed sarcoglycanopathy. Although one of the putative functions of the sarcoglycan complex is its participation in signaling processes, detailed studies have been scarce. Very recently, it was shown that gene knockout mice for a DAP, alpha-dystrobrevin, exhibit a dystrophic phenotype, possibly due to defects in muscle cell signaling. To clarify the putative function of the sarcoglycan complex, it is essential to determine whether or not there is a link between it and the intracellular signaling molecules. To elucidate this, we developed new methods for preparing various DAP complexes containing the sarcoglycan complex from the purified dystrophin-DAP complex. It was suggested from one of the complexes prepared that the sarco-glycan-sarcospan complex (the sarcoglycan complex associated with sarcospan) is associated with syntrophin and/or dystrobrevin. Further analysis of this complex revealed that the N-terminal half of dystrobrevin participates in this association. It is thus considered that the sarcoglycan-sarcospan complex is linked to the signaling protein neuronal nitric oxide synthase via alpha-syntrophin associated with dystrobrevin.
Human molecular genetics 2000;9;7;1033-40
PUBMED: 10767327; DOI: 10.1093/hmg/9.7.1033
Different dystrophin-like complexes are expressed in neurons and glia.
Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom. firstname.lastname@example.com
Duchenne muscular dystrophy is a fatal muscle disease that is often associated with cognitive impairment. Accordingly, dystrophin is found at the muscle sarcolemma and at postsynaptic sites in neurons. In muscle, dystrophin forms part of a membrane-spanning complex, the dystrophin-associated protein complex (DPC). Whereas the composition of the DPC in muscle is well documented, the existence of a similar complex in brain remains largely unknown. To determine the composition of DPC-like complexes in brain, we have examined the molecular associations and distribution of the dystrobrevins, a widely expressed family of dystrophin-associated proteins, some of which are components of the muscle DPC. beta-Dystrobrevin is found in neurons and is highly enriched in postsynaptic densities (PSDs). Furthermore, beta-dystrobrevin forms a specific complex with dystrophin and syntrophin. By contrast, alpha-dystrobrevin-1 is found in perivascular astrocytes and Bergmann glia, and is not PSD-enriched. alpha-Dystrobrevin-1 is associated with Dp71, utrophin, and syntrophin. In the brains of mice that lack dystrophin and Dp71, the dystrobrevin-syntrophin complexes are still formed, whereas in dystrophin-deficient muscle, the assembly of the DPC is disrupted. Thus, despite the similarity in primary sequence, alpha- and beta-dystrobrevin are differentially distributed in the brain where they form separate DPC-like complexes.
Funded by: Wellcome Trust
The Journal of cell biology 1999;147;3;645-58
PUBMED: 10545507; PMC: 2151186; DOI: 10.1083/jcb.147.3.645
Characterisation of alpha-dystrobrevin in muscle.
Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of alpha-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce alpha-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct alpha-dystrobrevin isoforms. In myotubes, alpha-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, alpha-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of alpha-dystrobrevin-1 by agrin. Furthermore, we show that alpha-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus alpha-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that alpha-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.
Funded by: Wellcome Trust
Journal of cell science 1998;111 ( Pt 17);2595-605
From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy.
National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan.
Duchenne and Becker muscular dystrophies are collectively termed dystrophinopathy. Dystrophinopathy and severe childhood autosomal recessive muscular dystrophy (SCARMD) are clinically very similar and had not been distinguished in the early 20th century. SCARMD was first classified separately from dystrophinopathy due to differences in the mode of inheritance. Studies performed several years ago clarified some immunohistochemical and genetic characteristics of SCARMD, but many remained to be clarified. In 1994, the sarcoglycan complex was discovered among dystrophin-associated proteins. Subsequently, on the basis of our immunohistochemical findings which indicated that all components of the sarcoglycan complex are absent in SCARMD muscles, and the previous genetic findings, we proposed that a mutation of any one of the sarcoglycan genes leads to SCARMD. This hypothesis explained and predicted various characteristics of SCARMD at the molecular level, most of which have been verified by subsequent discoveries in our own as well as various other laboratories. SCARMD is now called sarcoglycanopathy, which is caused by a defect of any one of four different sarcoglycan genes, and thus far mutations in sarcoglycan genes have been documented in the SCARMD patients. In this review, the evolution of the concept of sarcoglycanopathy separate from that of dystrophinopathy is explained by comparing studies on these diseases.
Muscle & nerve 1998;21;4;421-38
beta-dystrobrevin, a member of the dystrophin-related protein family.
Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom. email@example.com
The importance of dystrophin and its associated proteins in normal muscle function is now well established. Many of these proteins are expressed in nonmuscle tissues, particularly the brain. Here we describe the characterization of beta-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. beta-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, beta-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, beta-dystrobrevin coimmunoprecipitates with the dystrophin isoforms Dp71 and Dp140. These data provide evidence that the composition of the dystrophin-associated protein complex in the brain differs from that in muscle. This finding may be relevant to the cognitive dysfunction affecting many patients with Duchenne muscular dystrophy.
Funded by: Wellcome Trust
Proceedings of the National Academy of Sciences of the United States of America 1998;95;1;241-6
PUBMED: 9419360; PMC: 18188; DOI: 10.1073/pnas.95.1.241
Dystrobrevin and dystrophin: an interaction through coiled-coil motifs.
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
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.
Funded by: NINDS NIH HHS: NS23740
Proceedings of the National Academy of Sciences of the United States of America 1997;94;23;12413-8
PUBMED: 9356463; PMC: 24974; DOI: 10.1073/pnas.94.23.12413
Dystrobrevin deficiency at the sarcolemma of patients with muscular dystrophy.
Department of Biochemistry, University of Oxford, UK.
Mutations in the genes encoding dystrophin or dystrophin-associated proteins are responsible for Duchenne muscular dystrophy or various forms of limb-girdle muscular dystrophies respectively. We have recently cloned the gene for the murine 87 kDa postsynaptic protein dystrobrevin, a dystrophin-associated protein. Anti-dystrobrevin antibodies stain the sarcolemma in normal skeletal muscle indicating that dystrobrevin co-localises with dystrophin and the dystrophin-associated protein complex. By contrast, dystrobrevin membrane staining is severely reduced in muscles of Duchenne muscular dystrophy patients, consistent with dystrobrevin being a dystrophin-associated protein. Interestingly, dystrobrevin staining at the sarcolemma is dramatically reduced in patients with limb-girdle muscular dystrophy arising from the loss of one or all of the sarcoglycan components. Normal dystrobrevin staining is observed in patients with other forms of limb-girdle muscular dystrophy where dystrophin and the rest of the dystrophin-associated protein complex are normally expressed and in other neuromuscular disorders. Our results show that dystrobrevin-deficiency is a generic feature of dystrophies linked to dystrophin and the dystrophin-associated proteins. This is the first indication that a cytoplasmic component of the dystrophin-associated protein complex may be involved in the pathogenesis of limb-girdle muscular dystrophy.
Funded by: Wellcome Trust
Human molecular genetics 1997;6;7;1185-91
PUBMED: 9215691; DOI: 10.1093/hmg/6.7.1185
The genomic organization of human dystrobrevin.
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
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.
Funded by: NINDS NIH HHS: 5 R01 NS 23740-11
Neurogenetics 1997;1;1;37-42
Muscular dystrophies and the dystrophin-glycoprotein complex.
Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242, USA.
Efforts to understand the function of dystrophin, the protein product for the Duchenne muscular dystrophy gene, resulted in the purification of the dystrophin-glycoprotein complex. Over the past year several novel components of this complex have been identified. Recent studies have extended the number of muscular dystrophies associated with the oligomeric complex to six genetically distinct diseases, including three new forms of limb-girdle muscular dystrophy and one form of congenital muscular dystrophy.
Current opinion in neurology 1997;10;2;168-75
Genomic organization of the mouse dystrobrevin gene: comparative analysis with the dystrophin gene.
Department of Biochemistry, University of Oxford, United Kingdom.
Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical.
Genomics 1997;39;3;359-69
PUBMED: 9119373; DOI: 10.1006/geno.1996.4515
Cloning and characterization of the human homologue of a dystrophin related phosphoprotein found at the Torpedo electric organ post-synaptic membrane.
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
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.
Funded by: NINDS NIH HHS: 5 R01 NS 23740-10, NS29343
Human molecular genetics 1996;5;4;489-96
PUBMED: 8845841; DOI: 10.1093/hmg/5.4.489
Isoform diversity of dystrobrevin, the murine 87-kDa postsynaptic protein.
Molecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom.
Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins.
The Journal of biological chemistry 1996;271;13;7802-10
PUBMED: 8631824; DOI: 10.1074/jbc.271.13.7802
The three human syntrophin genes are expressed in diverse tissues, have distinct chromosomal locations, and each bind to dystrophin and its relatives.
Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, USA.
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.
The Journal of biological chemistry 1996;271;5;2724-30
PUBMED: 8576247; DOI: 10.1074/jbc.271.5.2724
Syntrophin binds to an alternatively spliced exon of dystrophin.
Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is a protein of the membrane cytoskeleton that associates with a complex of integral and membrane-associated proteins. Of these, the 58-kD intracellular membrane-associated protein, syntrophin, was recently shown to consist of a family of three related but distinct genes. We expressed the cDNA of human beta 1-syntrophin and the COOH terminus of human dystrophin in reticulocyte lysates using an in vitro transcription/translation system. Using antibodies to dystrophin we immunoprecipitated these two interacting proteins in a variety of salt and detergent conditions. We demonstrate that the 53 amino acids encoded on exon 74 of dystrophin, an alternatively spliced exon, are necessary and sufficient for interaction with translated beta 1-syntrophin in our assay. On the basis of its alternative splicing, dystrophin may thus be present in two functionally distinct populations. In this recombinant expression system, the dystrophin relatives, human dystrophin related protein (DRP or utrophin) and the 87K postsynaptic protein from Torpedo electric organ, also bind to translated beta 1-syntrophin. We have found a COOH-terminal 37-kD fragment of beta 1-syntrophin sufficient to interact with translated dystrophin and its homologues, suggesting that the dystrophin binding site on beta 1-syntrophin occurs on a region that is conserved among the three syntrophin homologues.
Funded by: NICHD NIH HHS: HD18658; NIGMS NIH HHS: 2T32GM07753, T32 GM007753
The Journal of cell biology 1995;128;3;363-71
PUBMED: 7844150; PMC: 2120343; DOI: 10.1083/jcb.128.3.363
(CA) repeat polymorphism in the chromosome 18 encoded dystrophin-like protein.
Division of Genetics, Harvard Medical School, Boston, MA 02115.
Funded by: NICHD NIH HHS: HD 18658; NINDS NIH HHS: NS 23740
Human molecular genetics 1994;3;5;841
PUBMED: 8081380; DOI: 10.1093/hmg/3.5.841-a
Gene lists (4)
|Gene List
|Gene count
|Homo sapiens
|Human PSD
|Human orthologues of mouse PSD adapted from Collins et al (2006)
|Homo sapiens
|Human PSP
|Human orthologues of mouse PSP adapted from Collins et al (2006)
|Homo sapiens
|Human cortex biopsy PSD full list
|Homo sapiens
|Mouse cortex PSD full list (ortho)


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Arnold-Based Business Celebrates 20th Anniversary
Dusty windowsills, grimy countertops and soup-splattered microwaves are often the byproducts of busy schedules, but for 20 years, Molly Maid of Central Anne Arundel County has kept houses clean when residents simply haven't have the time.
Donna Reilly, owner of Molly Maid of Central Anne Arundel County, opened the Arnold-based franchise because she saw a glaring need in the cleaning industry. "We care," Donna said when asked what sets Molly Maid apart. "When a customer is not happy, we do whatever we can to make them happy.
"As we become a busier society, the kids these days have sports programs and other activities," Reilly added. "People have decided there are services they can pay for to make their lives easier and housecleaning is one of them."
For two decades, Donna and her staff have worked hard to exceed customer expectations. The local franchise now has 28 employees — 24 cleaners, one manager and three front-office staff, which includes Donna and her son, Mike, who serves as the general manager and partner in the business. Particularly impressive is the dedication and longevity of Donna's staff; many have been with her for more than 10 years.
Mike has taken on more of the day-to-day operations in his 10 years with Molly Maid, and he is constantly adapting its services to meet customer expectations.
"He comes from a more tech-oriented background," Donna said. "The internet and social media are important ways we connect with our customers."
About two and a half years ago, Molly Maid moved its offices from the Bay Hills shopping center to a spot next to the YWCA of Annapolis & Anne Arundel County, a strategic move, Reilly said, because paying rent to the YWCA is just one more way Molly Maid can support the community and the YWCA's primary cause: addressing domestic abuse.
Through its Ms. Molly Foundation, Molly Maid raises awareness and supports programs to address the needs of victims of domestic abuse. During National Domestic Violence Awareness Month in October each year, Molly Maid holds its Making a Difference Drive. Molly Maid offices nationwide collect funds and personal care items to donate to local community safe houses and agencies.
With Mike playing a larger role in the daily operations, Donna now has the time to become an even bigger supporter of the cause by serving on the board of directors for the YWCA for the last six years.
"It's surprising to me the number of employees who have come through our doors who have struggled with domestic abuse," Donna said. "What is more surprising is how widespread it is, affecting every aspect of our community and society."
Donna called that work with YWCA her biggest accomplishment over her last two decades with Molly Maid.
"As a business owner, it has been great to go from zero to 600 customers, many of which have been with me for 10 or more years and bridge from generation to generation, she said. "But it has also afforded me the opportunity to do something impactful."
As for the future, the Reillys hope to keep expanding, with Mike eventually taking over as owner. When that happens, Mike can count on the advice of Donna and Molly Maid franchisees near and far.
"What sets Molly Maid apart from many services is that the network of 250-plus owners nationwide are really open to talking to one another and helping to solve challenges and continuously improve the service," Mike said. "They can talk about some of the problems they have seen. It's a family situation."
For more information about Molly Maid of Central Anne Arundel County, call 410-349-8080 or visit www.mollymaid.com.
"Mike and I are grateful to the employees, those who are long-term and those who just joined us," Donna said. "And the customers. When you watch the customers' children grow and years later they're calling on the same phone to have their house cleaned, you feel like you've done something right."


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JEAN AND DENIM HARMONY'24 September 2018
Our 2018-19 WSS Menswear Collection is just presenting for your opinion in our online shopping website www.wessi.com . This year there is a category which has denim pants title is so fit your daily combinations. Denim fabric or jeans are so suitable and matchable with every look in your wardrobe. Theese menswear products are flexiable and comfortable because of that's fabric. You can find in this article about denim chicness and jeans harmony with your combinations. Just enjoy with article and find your own path with denim fabrics and jean pants in menswear trendy look.


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FBN Architects
|Architectural practice specialising on housing associations, house builders and public sector clients in Central Scotland
|A solution that minimises the administrative work and certificate creation integral to the business. All the project information is to hand and certificates and letters are created with a couple of clicks saving valuable time and ensuring accuracy of information.
|Key Features
|Automated Project Document creation and filing, editable templates
The Story
A familar story, FBN Architects had been using a FileMaker database created for them from an original created by one of the partners in FileMaker Pro 3. Unfortunately although it was an improvement it was still awkward to use and provided no tangible benefits.
After discussing the requirements with the practice administrator and creator of the original database a new solution was developed that simultaneously simplified the user experience and added better functionality and additional features.
The solution now includes management of the complex task of Drawing Issues, Project Planning and the ability to create and edit templates for letters.


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Since her last album – 2014's Are We There – Sharon Van Etten has dropped off music's radar. She studied counselling, prompted by fans who have turned to this intense singer-songwriter in distress. Van Etten also became a successful actor, in the Netflix drama The OA. She had a son. Her latest album rings these changes, mostly in the album's textures. Piano, the Jupiter 4 synthesiser and some elegant, spacious production courtesy of John Congleton replace Van Etten's previous surging indie rock guitars.
And yet Van Etten remains resolutely herself: possessed of a slow-burning seethe that builds to swirling crescendos, she is a consummate surgeon of relationships, keen on Bruce Springsteen. Named after the option to postpone a computer update, the album starts with the drama of a short story.
"Sitting at the bar I told you everything/You said, 'Holy shit, you almost died'," runs I Told You Everything, hooking the listener in, as a faint imprint of the Rolling Stones' Sympathy for the Devil hovers in the piano chords. Comeback Kid channels gothic atmospherics and Seventeen is this assured album's big pop hit, a tender and agonised song to Van Etten's teenage self.


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Wrong policemen captured 6.000 EUR
Last night on the motorway close to the city of Ried, two criminals dressed as policemen were able to capture 6.000 EUR from one car driver.
The 56-year-old man from Slovakia wanted to buy a car in Germany and passed the motorway exit of Ried, as a supposed police car with blue lights appeared and forced him to stop.
One wrong policeman stayed in the car, the other one came to his window and just told him in short words: "money and mobile phone". The Slovak handed him his 6.000 EUR and the mobile phone. Not enough the wrong policeman applied cuffs to the hands of his victim on the steering wheel.
The Slovak was able to continue his trip to Germany with cuffs on the steering wheel. The German police freed him from the handcuffs.


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These coefficients are part of a more general phenomenon called the Savitzky-Golay filter in numerical analysis. In fact the traditional five-point 'stencil' reads:where . You can derive this result simply by playing around with the Taylor series for with . It's a little tedious, but you can see the derivation here: https://en.wikipedia.org/wiki/Five-poin … he_formula
There are analogous formulae for higher order derivatives too, and several published papers about the error term.
That is very informative, zeta. Thanks for the reply.
I wonder if there is a way to calculate the midpoint differentials by 4 points around it:
dy/dx ]x=0
ddy/dx/dx ]x=0
given y[-0.5], y[0.5], y[-1.5] & y[1.5]]]>
There are analogous formulae for higher order derivatives too, and several published papers about the error term.]]>
The simplest way to calculate numerical derivative is three-point formula:
dy/dx = (y[1]-y[-1]) /2/dx
ddy/dx/dx = (y[1]+y[-1]-2*y[0]) /dx/dx
But I found this one on five points :
dy/dx = ( y[-2] -8*y[-1] +8*y[1] -y[2]) / (12*dx)
ddy/dx/dx = (-y[-2] +16*y[-1] -30*y[0]+ 16*y[1] -y[2]) / (12*dx^2)
It is surprisingly accurate when I test the derivative on exp(3) using exp(2) exp(2.5) exp(3) exp(3.5) exp(4)
the result of 1st numerical derivative and 2nd are
20.04243 & 20.07127
remember the correct answer are both exp(3) = 20.08554
Can anyone prove how this works?
4th polynomial?


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Fantastic Danish Golden Age painting, oil on canvas, early 19th C. Original silver frame. Probably by one of the really important Danish Golden Age painters.
Ref. 12570-1D
H. 17 W. 30 cm.
H. 6.6 W. 11.8 in.
Price: € 2.400
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We also ship to the rest of the world, please contact us for more estimates, or gathered shipping quote if you wish to purchase several items.
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The UN Security Council on Thursday unanimously adopted a resolution to extend for a year the mandate of the United Nations Stabilization Mission in Haiti ( MINUSTAH).
The 15-nation Security Council decided to extend the mandate of MINUSTAH until Oct. 15, 2011, "with the intention of further renewal," the resolution said.
The Security Council also decided to "maintain the current mission overall force levels, which consists of a military component of up to 8,940 troops of all ranks and of a police component of up to 4,391 police," the resolution said.
"I would like to extend my gratitude and that of the government of Haiti as well as its people for this resolution just adopted by the council," said Leo Merores, Haitian ambassador to the UN, after the resolution passed.
The renewal of MINUSTAH, which would have expired Friday, holds particular significance because of Haiti's upcoming legislative and presidential elections on Nov. 28.
"It is therefore clear that the renewal of the mandate of the UN force, the mandate of MINUSTAH, will serve as a strong signal aimed at the people of Haiti to show to them that the Security Council, and beyond this council the whole international community stands side-by-side with Haiti to assist Haiti on the process that will take us to free, fair, and transparent as well as democratic elections," Merores told the Security Council.
Merores also thanked MINUSTAH staff, troop-contributing countries, and the Group of Friends for Haiti, a diplomatic coalition, for their continuing assistance on the ground in Haiti.
MINUSTAH has been working in Haiti since violence and upheaval broke out in the island country in 2004. Since the devastating January earthquake in Haiti, MINUSTAH has been engaged in efforts to help Haitians with reconstruction and recovery.


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|Team info
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Join Susan and Nancy for two days of fun filled sewing! Bring a project already in process or start something new. Susan and Nancy will be working on projects too but will be available for help and advice. We'll gather each day for show and tell, what's new or demos. Is there a demo you'd like to see? Let us know. Hours are 9:30 – 5:00 but stay and sew until 8:00 PM on Saturday if you wish.
One weekend session; fee: $30.00Fall class registration begins Thursday August 16, 2018Session A: Sat. & Sun. October 20 & 21, 9:30 - 5:00
Session B: Sat. & Sun. November 3 & 4, 9:30 -5:00Session C: Sat. & Sun. November 10 & 11, 9:30 -5:00


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Kiss Before Christmas: A Christmas Romance Novella
An English Girl in New York. As the snow starts to fall on Manhattan, PA Dory Mackenzie is a long way from home, far from the comforts and cheer of her cozy family Christmases back in Liverpool.So when her boss Tyler Alexander offers to pay her pricey ticket home for New Year, Dory just can't say no... even if she has to pretend to be his girlfriend for the holidays!Tyler may be hiding the real reason he needs a girlfriend-of-convenience but that's the least of Dory's worries when she's up against the disapproval of his high society mother, and the suspicions of his gorgeous older brother Lucas, the black sheep of the family.There's no fooling Lucas, and it's not just the truth he wants to get closer to... So before the bells have even rung out for Christmas day, Dory finds herself caught up in the Alexander family's dramas - and sharing more than just a few kisses under the mistletoe!Lose yourself in this gorgeously romantic and heart-warming novella this holiday season.
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Category: Travel Information
Updated on July 31, 2018
Directions to The Carolina Hotel
The Carolina Hotel 80 Carolina Vista Pinehurst, NC The historic Carolina Hotel is located in the Village of Pinehurst. Click here to access Google Maps. Complimentary valet parking is available until full with tipping at your discretion.


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129 Philip IV enforces surrender of papal letters 132 Case of Ger6nimo de Villanueva 133 His connection with the Convent of San Placido . 143 Persistent Resistance of Philip IV 146 Copies of the Papers sent to Rome in 1651 154 Efl Fortstohavethem returned continued until 1660 . Chapter I — The Inquisitor- General and Supreme Council. 179 Itgraduallyintervenesin Sentenceaand Trials ISl Itrequiresmonthly Reports of current Business 183 Centralization becomes complete — The Tribunals are merely ministerial 185 Appellat« Jurisdiction of Inquisitor-general and Suprema . 370 Disposition made the Proceeds 371 Lavish Grants to Favorites 372 Ferdinand's Kindliness 378 Reckless Grants by Charles V 380 Influence of Confiscation 386' Chapter II — Fines and Penances. 442-3) appears to- be the only writer who assumes that the Clementines render episcopal jurisdiction merely consultative. When, in 1583, the Bishop of Tortosa committed the same offence, the Suprenia wrote, January 14, 1584, that the popes had given the Inquisition exclusive jurisdiction over heresy and had prohibited its cognizance by others and that he must not in future intervene in such matters.' Undeterred by this, the Council of Tarragona, in 1591, reasserted the ancient episcopal jurisdiction by ordering all bishops to be vigilant in watching their flocks and, if they found any disseminators of heresy, to see to their condign punishment according to the canons.' How completely justified was the council in this and how false was the assertion of the Suprema, wa.s manifested in 1595, when the Archbishop of Granada complained to Clement VIII that the inquisitors had forbidden him to iasue an edict on the subject of heresy and the pope forthwith wrote to the inquisitor-general that this must not be allowed, for the faculties delegated to inquisitors in no way abridged episcopal jurisdiction.* After this, at least, the Suprema could not plead ignorance and yet it persisted in the assertion that it knew to be false.
Conunis^ons of Officials expire with the Inquisitor-general . 161 Suprema at first merely a Consultative Body 162"' Rapid Increase of its Functions 164 It becomes the ruling Power 167 Its struggle with Inquisitor-general Mendoaa in the Case of Froilan Diaz 169 Mendoza removes the opposing Members 174 Philip V decides in favor of the Suprema and of Froilan Diaz 177 The Suprema reduces the Tribunals to Subordination . 187 Control over all Details of the Tribunals 189 Control over the Finanees 190 Salaries and Perquisites of the Suprema 194 It« Enjfiyment of Bull-fights 198 Its Revenues and Expenses 200 Chapter II — The Tribunal. 293 It becomes a Prerequisite in the Inquisition 294 Verification of Limpieza 295 Number of Generations required — Penitents of the Inquisition . Pecuniary Penance 389 Distinguished from Confiscation 391 Sometimes substituted for Confiscation 394 Its Productiveness 396 Fines as Pimishment 389 Chapter III — Dispensations. Chap, n CLAIM OF EXCLVSIVE JURISDICTION 7 his licence, or a commi Bsion from the inquisitor-general, should have meddled with matters belonging to the Inquisition and have collected certain pecuniary penances, although he had already been forbidden to do so. A savage quarrel broke out in Guatiraala between the bishop, Juan Ramfrez, and the commissioner of the Inquisition, Phelipe Rijiz del Carral, who was also dean of the chapter.
Refrain fivm automated querying Do not send automated queries of any sort to Google's system: If you are conducting research on machine translation, optical character recognition or other areas where access to a large amount of text is helpful, please contact us. Bias Ortiz neghgently told him that there was no necessity of reading the paper; he was not well but, if he were able, he would be present at all the cases; if he did not come he committed his powers to the two inquisitors, or to either of them who was willing to accept the commission.' Apparently Ortiz did not come, for in several sentences rendered this year at Toledo the inquisitors styled themselves "apostolic inquisitors holding the powers of the Ordinary."* ' Eibl.
These convictions were part of the mental and moral fibre of the com- munity and were the outcome of the assiduous teachings of the Church for centuries, until it was classed with the primal truths that it was the highest duty of the sovereign to crush out dissi- dence at whatever cost, and that hatred of the heretic was enjoined on every Christian by both divine and human law. Prior to the organization of the Inquisition in the thirteenth century, the cognizance of heresy was a natural attribute of the episcopal office. Maintain attribution Tht Goog Xt "watermark" you see on each file is essential for in forming people about this project and helping them find additional materials through Google Book Search. Keep it legal Whatever your use, remember that you are responsible for ensuring that what you are doing is legal. Importance attached to Unity of Belief Popular Abhorrence of Heresy —Limitations of Inquisitorial Jurisdiction "■Classification of Heresy — Heresy both a sin and a crime . Original Episcopal Jurisdiction The Inquisitio D obtains exclusive Jurisdiction Episcopal Concurrence — ^It is merely formal Jurisdiction over the B'orum of Conscience The Question of Papal Indulgences Exten^on of Exclusive Jurisdiction Chapter II — The Regdlah Orders. It reeogniied the traditional jurisdiction of the bishops and innted their coaperation. I] EXCLUSIVE JURISDICTION ENFORCED 9 ration, evoked to himself all cases connected with heresy and committed them to the inquisitor-general and his deputies, inhibiting all judges and Ordinaries from intervening in them, in conseiijuence of which they have ceased to take cognizance of such matters and have referred to the inquisitors whatever came to their knowledge. In 1536 the tribunal of Valencia created much excitement by including in its Edict of Faith a number of matters of the kind but, on complaint from the vicar-general, the Suprema ordered the omission of every- thing not in the old edicts.' The attempts continued and, in 1552, a decision was required from the Suprema that eating pork on Saturdays was not a case for the Inquisition,' and the Con- cordia of 1568 contains a clause prohibiting inquisitors from entertaining cases belonging to the Ordinaries. Do not assume that just because we believe a book is in the public domain for users in the United States, that the work is also in the public domain for users in other countries. The Regular Clergy claim exemption from the Inquisition . The bishop issued edicts at his discretion and could initiate prosecutions. As the bishop has laid his hand on things beyond liis jurisdiction, he is ordered in future not to meddle with anything touching the Inquisition, as otherwise fitting measures will be taken.' The only foundation for this mendacious asser- tion was, as we shall see hereafter, that, in the struggle made by Ferdinand and Charles V to prevent appeals to Rome from the Inquisition, briefs were sometimes obtained from popes evoking to themselves all cases pending in the tribunals and committing them to the inquisitor-general, with inhibition to any one, inchid- ing cardinals and officials of the curia, to entertain appeals from him. In a carta acordada of November 23, 1612, the Suprema made an attempt to define the boundaries of the rival jurisdictions, in which it allowed to the spiritual courts exclusive jurisdiction only over ecclesiastics in matters touching their duties as pastors, the ministry of the Church, simony and cases relating to Orders, benefices and spiritual affairs, while it admitted cumulative jiuis- diction in usury, gambling and incontinence.* Restricted as were these admissions, the Suprema itself did not observe them. 81 Sentence rendered April 24, 1576 82 Carranza's Death, May 2d — Estimates of him .... It had the audacity to propose to prosecute him, but the Suprema wisely ordered it to take no action against him." ' Arobivo de Simancas, Inquisicion, Lib. 84 Jurisdiction claimed over Bishops 87 Cases of Toro of Oviedo and Queipo of Mechoacan .... Its Effectiveness in stimulating Demmciations 91 Its comprehensive Details 93 Its Anathema 95 Popular Training in Delation 99 Chapter V — ^Appeals to Rome.


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New 2019 Chevrolet Silverado 2500HD Crew Cab Standard Box 4-Wheel Drive LT
Final Price $56,425
Nav System, 4x4, Turbo Charged Engine, Chrome Wheels, CD Player, Onboard Communications System, Back-Up Camera, Satellite Radio, ENGINE, DURAMAX 6.6L TURBO-DIESEL V8... Hitch. BLACK exterior and JET BLACK interior, LT trim.KEY FEATURES INCLUDEBack-Up Camera, Satellite Radio, CD Player, Onboard Communications System, Chrome Wheels 4x4, Privacy Glass, Keyless Entry, Steering Wheel Controls, Electronic Stability Control.OPTION PACKAGESENGINE, DURAMAX 6.6L TURBO-DIESEL V8, B20-DIESEL COMPATIBLE (445 hp [332 kW] @ 2800 rpm, 910 lb-ft of torque [1220 Nm] @ 1600 rpm) Includes capped fuel fill, (K40) exhaust brake and (K05) engine block heater.), CUSTOM SPORT EDITION includes (AQC) Appearance Package, (UD5) front and rear park assist, (TC7) body-color grille with chrome bars, (VB5) body-color front bumper, (VT5) body-color rear bumper, (CGN) spray-on bedliner and (PYU) 20" (50.8 cm) polished aluminum wheels on 1LT. 1LT trim also includes (T3U) fog lamps, (DP9) chrome mirror caps, (B85) chrome body-side moldings and (E20) chrome door handles. LT CONVENIENCE PACKAGE includes (AG1) driver 10-way power seat adjuster when ordered with (AZ3) bench seats only, (BTV) Remote vehicle starter system, (C49) rear-window defogger, (N37) manual tilt and telescoping steering column, (KI4) 110-volt power outlet, (T3U) fog lamps and (CJ2) dual-zone climate control, SEATS, FRONT CLOTH BUCKET with (KA1) heated seat cushions and seat backs. Includes 10-way power driver and 6-way power front passenger seat adjusters, including driver/front passenger power recline and driver/front passenger power lumbar. Also includes adjustable head restraints, (D07) floor console, storage pockets and (K4C) wireless charging. TRAILERING EQUIPMENT Trailering hitch platform 2.5" with a 2.0" insert for HD, 7-wire harness with independent fused trailering circuits mated to a 7-way sealed connector to hook up parking lampsEXPERTS REPORTEdmunds.com's review says "You'll find bigContact us for the most up to date information regarding our vehicles. Market conditions can change quickly and we price our vehicles according to current, local market values, so you can be sure you're getting the best price.***Prices include all rebates and incentives assigned to dealer, plus tax and license.***


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* EYTCC 'Udder Run' for Dove House Hospice
August 11 @ 9:00 am - 4:00 pm
Departing from 10:00 am, this will be a gentle road run of approx. 30 miles driving through beautiful country lanes towards the coast.
The route will be in a descriptive format. Not a Tulip plan. On arrival at the destination, you will spend the afternoon relaxing.
There is a gentle, safe walk down to the beach. Safe for children or pets, off the lead, you can spend an
enjoyable, quiet afternoon.
Road run exploring the East Riding, from Asda Car Park at Bilton HU11 4AL, to Mr Moo's Ice Cream Parlour (min vehicle age 20yr). Directions handed out at start. Only £3 – in aid of Dove House Hospice.
Departing from Asda at Bilton: 10am
*Pre-Booked Members Only – £3
Visiting Public Info
The classic vehicles can be seen briefly at the rear of Bilton's Asda Car Park between 9am-10am. And from around 12pm-3pm at Mr Moo's Ice Cream Parlour and cafe, on the adjacent field. Plenty of parking available, and viewing the vehicles is free!
Please supervise children for safety purposes
and please do no touch any of the vehicles without the owners permission.
Many events are open to all wishing to display vehicles (20 years or older) upon application – the remainder, with an asterisk* or marked accordingly – are for club members only. Some of the larger events may facilitate admission for 15 years or older, for (Members Only) 'future classics' vehicles. A summary list of events, can be seen here.
Event Application Forms
Events Terms & Conditions (opens in a new window)
NOTE: All files are in PDF format. If you can't see the files, you can download a free reader here..
Event Application Forms – can also be requested by post – postal address: EYTCC Events, PO Box 320, Beverley, HU17 0WA. If requesting by post, please include a stamped addressed envelope – A5 or A4 size.